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WO2025088192A1 - Methods for the coupling of di(alkyl)amines to polypeptides - Google Patents

Methods for the coupling of di(alkyl)amines to polypeptides Download PDF

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Publication number
WO2025088192A1
WO2025088192A1 PCT/EP2024/080338 EP2024080338W WO2025088192A1 WO 2025088192 A1 WO2025088192 A1 WO 2025088192A1 EP 2024080338 W EP2024080338 W EP 2024080338W WO 2025088192 A1 WO2025088192 A1 WO 2025088192A1
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Prior art keywords
compound
alkyl
group
mol
lipid
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French (fr)
Inventor
Lars Friedrich
Uli Binder
Mario Gomez
Anne BENEDIKT
Katrin HEITEL
Melanie Liefke
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XL Protein GmbH
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XL Protein GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention provides for inventive means and methods for the coupling of di(alkyl)amines to polypeptides or peptides capable of forming a random coil conformation.
  • the present invention further provides inventive compounds produced by the herein detailed methods, wherein said compounds are characterized by formula (I): A-[B-]bC-D.
  • Said compounds comprise a polypeptide/peptide capable of forming a random coil conformation, an N-terminal protecting group, a di(alkyl)amino group, and optionally a linker.
  • the present invention provides lipid nanoparticles comprising said compounds characterized by formula (I) as well as means and methods for the production of said lipid nanoparticles.
  • Formulations comprising the inventive compounds and/or the lipid nanoparticles of the present invention are also provided. Further, the present invention also relates to uses of the inventive compounds, the lipid nanoparticles, and/or the formulations disclosed herein.
  • Lipid formulations such as lipid nanoparticles (LNPs) are frequently used as drug delivery systems for the delivery of active ingredients and therapeutic agents.
  • Major hurdles of drug delivery via LNPs are plasma half-life, endosomal entrapment, and stability of the LNPs.
  • Coupling of certain polymers, in particular poly(ethylene)glycol (PEG) to the LNPs (or to lipids comprised in such LNPs) has been shown to overcome, or at least reduce these hurdles.
  • PEG poly(ethylene)glycol
  • PEGylated lipids poly(ethylene)glycol
  • the art has in particular provided poly(ethylene)glycol (PEG) coupled to lipids (“PEGylated lipids”) as a means to improve plasma half-life of such lipids or of LNPs.
  • PEGylated lipids were employed, inter alia, in mRNA vaccines against SARS-CoV-2 (including, inter alia, in known and recently employed SARS-CoV-2 vaccines from known sources).
  • SARS-CoV-2 including, inter alia, in known and recently employed SARS-CoV-2 vaccines from known sources.
  • more than one billion doses were administered globally (see, e.g., Ju (2022) ACS Nano 16, 11769-11780).
  • vaccination with such SARS-CoV-2 mRNA vaccines has been shown to induce anti-PEG immunoglobulins (anti-PEG IgM and IgG) by up to 68.5-fold, which may lead to immunogenic reactions towards PEG polymers (i.e., PEG immunity; see, e.g., Ju; loc. cit.).
  • polypeptide mimetics have been provided in the art, some of which are based on naturally occurring amino acid sequences or synthetic amino acid stretches. Most natural amino acid sequences do not behave like an ideal random coil in physiological solution, which constitutes an important characteristic of PEG, because they either tend to adopt a folded conformation (secondary structure) or, if unfolded, they usually are insoluble and form aggregates.
  • PAS Conformationally disordered polypeptides comprising the small residues Pro, Ala, and Ser.
  • P/A- and PAS-polypeptides are hydrophilic, uncharged biological polymers with biophysical properties very similar to polyethylene glycol (PEG). Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in blood plasma.
  • conformationally disordered polypeptides In contrast to the specific interactions with structured proteins, the properties of conformationally disordered polypeptides often pose a challenge for the immune system in the generation of cognate antibodies, a feature that is exploited by pathogens to evade the immune response (Giri (2016) Front. Cell. Infect. Microbiol. 6-144; Goh (2016) Mol. BioSyst. 12, 1881-1891). Accordingly, conformationally disordered P/A- and PAS-polypeptides lack toxicity or immunogenicity in mice and might, thus, also constitute a valid alternative for the PEGylation of lipids and LNPs.
  • the present invention provides for inventive means and methods for the chemical coupling of P/A- and PAS-polypeptides to di(alkyl)amines. Namely, the present invention provides for a method for the production of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group; and b) the purification of a compound of formula (I).
  • PAS-polypeptides as opposed to chemical coupling
  • PAS-polypeptides increases the chances for potential cellular contaminants and/or increases the number of required purification steps.
  • PASylated nanoghosts based on PLGA nanoparticles described by Krishnamurthy (2019, loc. cit.) differ in their structure and composition considerably from typical LNPs and, resultingly, are likely not suitable for use in standard LNP -applications (such as the delivery of mRNA, siRNA, and the like as part of vaccines or therapeutics).
  • the present invention solves these caveats by distinct means and methods involving the coupling of a di(alkyl)amine to a compound comprising a P/A- and/or PAS -polypeptide.
  • the present invention provides for a convenient and/or reliable method for coupling of P/A- and/or PAS-polypeptides to lipids, namely di(alkyl)amines.
  • the present invention is broadly applicable which is illustrated, inter alia, from the vast diversity of different P/A- and/or PAS-polypeptides the inventors have successfully coupled to di(alkyl)amines, demonstrating remarkable and sequence-independent coupling efficiencies using the herein provided inventive means and methods.
  • the remarkable and advantageous purity of the compounds obtained/obtainable by the herein provided means and methods is further evident from the herein provided non-limiting illustrative figures. This is in clear contrast to the non-enabling teachings provided by Zhang (2023, loc. cit ).
  • inventive compounds that are obtained by or obtainable by the means and methods detailed herein.
  • inventive compounds are characterized by formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
  • the herein provided compounds comprising di(alkyl)amines may be, inter alia, less prone to cleavage as compared to e.g., similar compounds comprising phospholipids instead of di(alkyl)amines. Consequently, anchoring of these compounds in, inter alia, LNPs may be advantageously high, as will be further detailed herein below.
  • polypeptides capable of forming a random coil conformation P/A- or PAS-polypeptides
  • inventive compounds/inventive conjugates are, inter alia, even advantageous as compared to herein tested reference PEGylated di(alkyl)amines.
  • the herein provided compounds show advantageous effects on cell viability parameters as compared to said reference PEGylated lipids, as illustrated in the appended examples.
  • lipid nanoparticles comprising the herein provided compounds have not been foreshadowed.
  • the present invention further provides for a method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), wherein the method comprises the steps of a) the provision of the compound of formula (I) in an ethanolic solution; b) the mixing of said ethanolic solution with an aqueous solution, thereby preparing/producing a lipid nanoparticle; and c), optionally the dialysis of said lipid nanoparticle.
  • lipid nanoparticles obtained/obtainable by the means and methods detailed herein and/or lipid nanoparticles that comprise the inventive compound of formula (I) are provided herein.
  • lipid nanoparticle colloidal dispersions comprising the lipid nanoparticles of the present invention are herein provided.
  • a lipid nanoparticle colloidal dispersion refers to a suspension comprising the lipid nanoparticles of the present invention and/or refers to a suspension comprising lipid nanoparticles comprising the inventive compound characterized by formula (I).
  • lipid nanoparticles and lipid nanoparticle dispersions are highly stable, as can been seen from the lack of turbidity and visible precipitation, as compared to the lipid particles provided by Zhang (2023, loc. ci ). Further, the herein provided lipid nanoparticles may show advantageous characteristics, inter alia, with regard to their RNA encapsulation efficiency and LNP shielding capacities as compared to LNPs comprising a reference PEGylated compound. This is, inter alia, evident from the herein provided examples.
  • the present invention provides for means, methods, and uses for the production/preparation of pharmaceutical and non-pharmaceutical formulations comprising the inventive compound of formula (I) and/or the lipid nanoparticle or the lipid nanoparticle colloidal dispersion of the present invention.
  • uses of the pharmaceutical and non-pharmaceutical formulations comprising the herein provided lipid nanoparticles, the herein provided lipid nanoparticle colloidal dispersion, and/or the herein provided inventive compounds of formula (I) are disclosed herein.
  • inventive compound of formula (I) provided in accordance with the present invention and the method for the production/preparation of said compound will be described in greater detail in the following.
  • This detailed description relates to and is applicable to all aspects of the present invention, including not only the compound or the method for the production/preparation of said compound as such but also to the lipid nanoparticles, the lipid nanoparticle suspensions, the pharmaceutical formulations, and the nonpharmaceutical formulations comprising said compound.
  • This detailed description further relates to any uses of the compound the lipid nanoparticles, the lipid nanoparticle suspensions, the pharmaceutical formulations, and the non-pharmaceutical formulations comprising said compound, as well as methods for their production/preparation and methods using the same.
  • the present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, b is 1 or 0, so that B can be present or absent,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group.
  • the compound of formula (I) can be a compound of the formula A-C-D if b is 0, or a compound of formula A-B-C-D if b is 1.
  • A is a di(alkyl)amino group.
  • the compound according to the present invention comprises a di(alkyl)amino group (group A).
  • group A a di(alkyl)amino group
  • the herein detailed means and methods refer to the coupling of a di(alkyl)amine to either compound L-B-C-D or compound L-C-D resultingly preparing/producing a di(alkyl)amino group either coupled to group B or group C in compounds A-B-C-D or A-C-D, respectively.
  • the present invention provides for P/A- and/or PAS-polypeptides coupled to di(alkyl)amino groups.
  • di(alkyl)amino groups comprise two alkyl chains linked by a nitrogen atom, wherein the nitrogen forms a tertiary amine further linking the di(alkyl)amino group to group B or group C in compounds A-B-C-D or A-C-D, respectively.
  • group A comprises two alkyl chains, and said alkyl chains are independently a linear or branched alkyl chain.
  • group A in formula (I) can be illustrated by the formula -N(R A1 )2, wherein the two groups R A1 linked to the nitrogen atom are independently a linear or branched alkyl chain.
  • both alkyl chains are linear.
  • the two alkyl chains may independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
  • group A comprises two linear alkyl chains that each comprise 14 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(tetradecyl)amino group. It will be understood, that for example, when group A is a di(tetradecyl)amino group, the di(alkyl)amine to be coupled to either compound L-B-C-D or compound L-C-D is a di(tetradecyl)amine.
  • group A is not particularly limited.
  • Examples 1 and 2 illustrate the efficient coupling of di(tetradecyl)amine with P/A- and PAS-peptides
  • Example 9 illustrates that di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine and di(octadecyl)amine may be coupled efficiently to e.g., PAS peptides.
  • the two alkyl chains comprised in group A may independently comprise about 8 to about 20 carbon atoms, preferably about 10 to 18, more preferably about 10, about 12, about 14, about 16 or about 18 carbon atoms.
  • group A comprises two linear alkyl chains that each comprise 10 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(decyl)amino group.
  • group A comprises two linear alkyl chains that each comprise 12 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(dodecyl)amino group.
  • group A comprises two linear alkyl chains that each comprise 16 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(hexadecyl)amino group.
  • group A comprises two linear alkyl chains that each comprise 18 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(octadecyl)amino group.
  • group A instead of di(alkyl)amines, also di(alkenyl)amines may be coupled to compound L-B-C-D or compound L-C-D preparing/producing a di(alkenyl)amino group either coupled to group B or group C in compounds A’-B-C-D or A’-C-D, respectively. Accordingly, the present invention also provides for a method for the production/preparation of a compound of the formula (II):
  • A’-[B-] b C-D (II) wherein A’ is a di(alkenyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkenyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (II), wherein L is a leaving group; and b) the purification of a compound of formula (II).
  • inventive compounds characterized by the formula (II):
  • A’-[B-] b C-D (II) wherein A’ is a di(alkenyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
  • group A’ in formula (II) can be illustrated by the formula - N(R A2 ) 2 , wherein the two groups R A2 linked to the nitrogen atom are independently a linear or branched alkenyl chain.
  • two alkenyl chains of the di(alkenyl)amine and/or of group A’ may independently comprise about 8 to about 20 carbon atoms.
  • two alkenyl chains of the di(alkenyl)amine and/or of group A’ may each comprise about 1 or about 2 double bonds.
  • di(alkyl)amines instead of di(alkyl)amines, also di(alkynyl)amines may be coupled to compound L-B-C-D or compound L-C-D preparing/producing a di(alkynyl)amino group either coupled to group B or group C in compounds A”-B-C-D or A”-C-D, respectively. Accordingly, the present invention also provides for a method for the production/preparation of a compound of the formula (III):
  • A”-[B-] b C-D (III) wherein A” is a di(alkynyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkynyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (II), wherein L is a leaving group; and b) the purification of a compound of formula (II).
  • the present invention also provides for inventive compounds characterized by the formula (III):
  • A”-[B-] b C-D (III) wherein A” is a di(alkynyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
  • group A” in formula (III) can be illustrated by the formula -N(R A3 ) 2 , wherein the two groups R A3 linked to the nitrogen atom are independently a linear or branched alkynyl chain.
  • two alkynyl chains of the di(alkynyl)amine and/or of group A” may independently comprise about 8 to about 20 carbon atoms.
  • two alkenyl chains of the di(alkynyl)amine and/or of group A” may each comprise about 1 or about 2 triple bonds.
  • the present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, b is 1 or 0, so that B can be present or absent,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group.
  • B is an optional linker.
  • the inventive compound may comprise an optional linker. Accordingly, group B is either present or absent. It is understood that if group B is absent, group A is directly linked to group C and if group B is present, group A and group C are linked via said linker (i.e., group B), resulting in compound A-C-D or A-B-C-D, respectively.
  • linkers/linker groups that may be deployed in the context of the present invention.
  • linkers/linker groups are provided.
  • Group B may comprise one or more amino acid residues, wherein said one or more amino acid residues may be independently natural or unnatural amino acid(s). Accordingly, group B may comprise a carboxy and an amino terminus (i.e., C- and N-terminus, respectively).
  • group B comprises at least two carbon atoms between the amino group and the carboxy group of group B may provide a distance of at least two carbon atoms between the amino group and the carboxy group of group B (which is the case if, e.g., group B is an co-amino-Cs-is alkanoic acid, such as s-aminohcxanoic acid), or they may provide a distance of only one carbon atom between the amino group and the carboxy group of group B (which is the case if, e.g., group B is alanine).
  • group B can comprise one or more amino acid residues, which again can be natural or unnatural amino acid(s)
  • the hydrocarbyl moiety comprised in said -HN- (C2-12 hydrocarbyl)-C(O)- may be, e.g., an alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl, or any combination thereof (e.g., an alkaryl or an aralkyl, such as benzyl, phenethyl, or methylphenyl).
  • said hydrocarbyl moiety may have 3 to 10 carbon atoms, and preferably 4 to 8 carbon atoms
  • the two points of attachment on the aforementioned cyclic hydrocarbyl groups (such as said aryl or said cycloalkyl; including also any of the specific cyclic groups referred to in the following, such as the phenyl comprised in the -HN-(CH2)o-2-phenyl-(CH2)o-2-C(0)- referred to in the subsequent paragraph) may neither be on the same ring carbon atom nor on adjacent ring carbon atoms; if such a cyclic group has six ring atoms (as in phenyl or cyclohexyl), a 1,4-attachment (para) or a 1,3 -attachment (meta) is preferred, and a 1,4-attachment is particularly preferred.
  • group B is preferably -HN-(C2-i2 hydrocarbyl)-C(O)-.
  • group B provides an amino terminus, such as the group -NH- in -HN-(C2-i2 hydrocarbyl)-C(O)-, the amino terminus typically forms a bond to the polypeptide C.
  • group B provides a carboxy terminus, such as the group -C(O)- in -HN-(C2-i2 hydrocarbyl)-C(O)-, the carboxy terminus typically forms a bond to the di(alkyl)amino group A.
  • the di(alkyl)amino group A and the linker B may form an amide.
  • group B may be selected from the group consisting of -HN-(C2-i2 alkyl)-C(O)-, -HN-(CH2)o 2- phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-.
  • group B may be selected from -HN-CH2-(Ci-n alkyl)-C(O)-, -HN-(Ci-n alkyl)-CH2-C(O)-, -HN-(CH2)o-2-phenyl-(CH2)o-2- C(O)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-.
  • group B may be selected from - HN-CH 2 CH 2 -C(O)-, -HN-CH 2 CH 2 -(CI.IO alkyl)-C(O)-, -HN-(Cnio alkyl)-CH 2 CH 2 -C(O)-, -HN-(CH 2 )o- 2 - phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-.
  • group B may be, e.g., selected from -HN-(CH2)2 i2-C(O)-, -HN-(CH2)o-2-phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o-2-cyclohexyl- (CH2)O-2-C(0)-. Accordingly, group B may be selected from -HN-(CH2)3 io-C(0)-, -HN-phenyl-C(O)-, and -HN-cyclohexyl-C(O)-.
  • group B may be selected from -HN-(CH2)4-C(O)-, -HN-(CH2)5-C(O)-
  • group B may be selected from the group consisting of s-aminohcxanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, para-aminobenzoic acid, and paraaminocyclohexanecarboxylic acid (i.e., 4-aminocyclohexanecarboxylic acid).
  • group B may also be a standard proteinogenic a-amino acid comprising at least two carbon atoms between its amino group and its carboxy group, particularly alanine or proline.
  • group B may also be selected, e.g., from alanine (e.g., L-alanine or D-alanine), proline (e.g., L-proline), P-alanine, y-aminobutyric acid (GABA), 5-aminovaleric acid (Ava), s-aminohcxanoic acid (Ahx), 7-aminoheptanoic acid, 8-aminooctanoic acid (Aoa), 9-aminononanoic acid, para-aminobenzoic acid (Abz), para-aminocyclohexanecarboxylic acid (ACHA; e.g., cis-ACHA or trans-ACHA), and para- (aminomethyl)cyclohexanecarboxylic acid (AMCHA; e.g.
  • alanine e.g., L-alanine or D-alanine
  • proline e.g., L-proline
  • group B may be an L-lysine residue providing an s -amino group in its side chain that is linked via a CO-(Ci-8 hydrocarbyl) group to the N-atom of the di(alkyl)amino group (group A).
  • a linker group B may be obtained/obtainable, for example, by reacting the lysine side chain with iodoacetic acid N- hydroxysuccinimide ester, followed by coupling with group A.
  • iodoacetic acid other activated iodo-carboxylic acids may be used, for example iodopropionic acid, iodobutyric acid, iodovaleric acid, iodocaproic acid, always in their activated form, for example as N-hydroxy succinimide (NHS) ester.
  • NHS N-hydroxy succinimide
  • iodoacetic acid or other activated iodo-carboxylic acids also activated bromo-carboxylic acids or activated chloro-carboxylic acids (e.g. bromoacetic acid-NHS ester and chloroacetic acid-NHS ester) may be used.
  • group B may comprise a cysteine residue providing a thiol group in its side chain that may be linked to a di(alkyl)amine.
  • the present inventors have surprisingly identified means and methods for the coupling of PA-/PAS-peptides each comprising a cysteine residue to di(alkyl)amines. This is illustratively shown in Example 10. The underlying reaction mechanism is illustrated in Figure 14 and the purity of the resulting compounds is shown in Figure 15.
  • the present inventors could surprisingly couple the thiol group of said cysteine residue to di(alkyl)amines (see, Example 10) by employing abispecific crosslinker.
  • the present inventors could surprisingly a compound of formula L-B-C-D to a compound A using an active ester. Accordingly, in the context of the present invention bispecific crosslinkers are active esters.
  • Example 10 illustrates that an organic solvent comprising dichloromethane and methanol and further comprising a bispecific crosslinker (e.g., siiccinimidyl-/ram-4-(N-malcimidylmcthyl)cyclohcxanc- 1 -carboxylate (SMCC)) can be employed in coupling a compound L-B-C-D comprising a Cys residue in B (e.g., an N-terminally protected PAS-Cys polypeptide) to a di(alkyl)amino group (e.g., to di(tetradecyl)amine), resulting in the formation of a compound A-B-C-D (e.g., PAS(20)C-linker- di(tetradecyl)amine) .
  • a bispecific crosslinker e.g., siiccinimidyl-/ram-4-(N-malcimidylmcthyl)cyclohcxanc-
  • the herein provided means and methods may further comprise the coupling of a compound of formula L-B-C-D to a compound A using a bispecific crosslinker.
  • the herein provided methods may further comprise a bispecific crosslinker for the preparation of a compound of the formula (I).
  • Bispecific crosslinkers may be selected from the group consisting of succinimidyl-tra «5-4-(N-maleimidylmethyl)cyclohexane-l- carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP), N-(y- maleimidobutyryloxy)succinimide ester (GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane- 1 -carboxylate (SIAC), succinimidyl-6-((((4- (iodoacety
  • the bispecific crosslinker when employing a bispecific crosslinker such as SMCC, in the coupling of a Cys residue to another molecule, the bispecific crosslinker specifically reacts with the thiol group of said Cys residue (such as via a Michael-addition).
  • the hydrogen i.e., H-
  • the bispecific crosslinker or parts thereof subsequent to the coupling reaction will be comprised in the bispecific crosslinker or parts thereof subsequent to the coupling reaction.
  • the leaving group L comprised in the to-be-coupled compound L-B-C-D may be the H- comprised in the thiol group of said Cys residue, which H- may subsequently, after the coupling reaction, be comprised in said bispecific crosslinker or parts thereof.
  • Said bispecific crosslinker may be comprised in (an) organic solvent(s).
  • said organic solvent(s) may comprise dichloromethane and methanol.
  • said organic solvent(s) comprises a bispecific crosslinker, it may comprise about 0.5 to about 10 molar equivalents of said bispecific crosslinker relative to compound A, preferably about 0.5 to about 5 molar equivalents, preferably about 1 to about 5 molar equivalents, preferably about 1 to about 4 molar equivalents, preferably about 1 to about 3 molar equivalents, preferably about 1 to about 2 molar equivalents, more preferably about 1 molar equivalent.
  • bispecific crosslinker or parts thereof may be comprised in the resulting compound. Accordingly, when coupling a compound L-B-C-D with a compound A, said bispecific crosslinker or parts thereof may be comprised in the resulting compound of A-B-C-D. Such parts of the bispecific crosslinker may be considered to form part of the resulting (then modified) linker B.
  • the skilled person can readily determine which parts of said bispecific crosslinker may be comprised in the resulting compound and which parts of said bispecific crosslinker may be removed during the coupling reaction.
  • Parts of the above-mentioned cross-linkers (such as SMCC) that are removed during the coupling reaction may for example be N- hydroxysuccinimide (NHS) or nitrophenol.
  • NHS N- hydroxysuccinimide
  • nitrophenol N- hydroxysuccinimide
  • the bispecific crosslinker e.g., SMCC
  • the compound L-B-C-D e.g., a PAS(20)C peptide; see left reaction scheme illustrated in Figure 15
  • the compound A e.g., di(tetradecyl)amine; see right reaction scheme illustrated in Figure 15
  • said intermediate reaction products may react with compound A or compound L-B-C-D, respectively.
  • said intermediate reaction products may be isolated and subsequently employed in the herein provided means and methods. Accordingly, in one embodiment the present invention provides for a method for the preparation of a compound of the formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, preferably wherein B comprises a Cys residue, wherein B was previously reacted with a bispecific crosslinker, thereby coupling said bispecific crosslinker or parts thereof to B,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group
  • the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D (or a derivate thereof) to yield a compound of formula (I), wherein L is a leaving group (such as N-hydroxysuccinimide); and b) the purification of a compound of formula (I), wherein said compound of formula (I) comprises said bispecific crosslinker or parts thereof.
  • the present invention provides for a method for the preparation of a compound of the formula (I):
  • A is a di(alkyl)amino group, wherein A was previously reacted with a bispecific crosslinker, thereby coupling said bispecific crosslinker or parts thereof to A,
  • B is a linker, preferably wherein B comprises a Cys residue
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group
  • the method comprises the steps of a) the coupling of a di(alkyl)amine (or a derivate thereof) to a compound L-B-C-D to yield a compound of formula (I), wherein L is a leaving group (such as H- comprised in the thiol group of said Cys residue); and b) the purification of a compound of formula (I), wherein said compound of formula (I) comprises said bispecific crosslinker or parts thereof.
  • group B are comprised in SEQ IDs NO: 1, 2, and 6 and illustrated in the appended Examples.
  • group B may be absent, if b is 0. Accordingly, in one aspect the present invention b is 0 (and accordingly, B is absent).
  • group B applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
  • the present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, b is 1 or 0, so that B can be present or absent,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.
  • Group C which is comprised in the compound of formula (I), is a polypeptide or peptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of either alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.
  • random coil as used herein relates generally to any conformation of a polymeric molecule, including amino acid polymers/amino acid sequences/polypeptides, in which the individual monomeric elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomeric elements while still being chemically bound to said adjacent monomeric elements.
  • a polypeptide, amino acid sequence or amino acid polymer adopting/having/forming/capable of forming a "random coil conformation” substantially lacks a defined secondary and tertiary structure.
  • polypeptides capable of forming a random coil are conformationally disordered polypeptides and are also herein also interchangeably referred to as “random coil polypeptides”.
  • polypeptides forming a random coil conformation and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., W. H. Freeman and Company, New York; Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York; Smith (1996) Fold Des TR95-R106).
  • Such polypeptides are particularly capable of forming a random coil conformation when present in an aqueous environment (e.g., an aqueous solution or an aqueous buffer).
  • CD spectroscopy represents a light absorption spectroscopy method in which the difference in absorbance of right- and left- circularly polarized light by a substance is measured.
  • the secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with a wavelength between approximately 190 and 250 nm. At these wavelengths, the different secondary structures commonly found in polypeptides can be analyzed, since a-helix, parallel and anti-parallel B-sheet, and random coil conformations each give rise to a characteristic shape and magnitude of the CD spectrum.
  • CD spectrometry the skilled artisan is readily capable of determining whether polypeptide (or segment thereof) forms/adopts random coil conformation in aqueous solution or at physiological conditions.
  • Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectrometry, infrared and Raman spectroscopy, measurement of the hydrodynamic volume via size exclusion chromatography, analytical ultracentrifugation or dynamic/static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity (Cantor (1980) loc. cit , Creighton (1993) loc. cit , Smith (1996) loc. cit.).
  • the encoded amino acid sequence/polypeptide may also form random coil conformation when other residues than proline, alanine and, optionally, serine are comprised as a minor constituent in said amino acid sequence/polypeptide.
  • minor constituent means that maximally 5 mol% or maximally 10 mol% amino acid residues are different from proline, alanine, or serine in the encoded random coil polypeptides of this invention. This means that maximally 10 of 100 amino acids may be different from proline, alanine and, optionally, serine, preferably maximally 8 mol%, i.e.
  • maximally 8 of 100 amino acids may be different from proline, alanine and, optionally, serine, more preferably maximally 6 mol%, i.e. maximally 6 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more preferably maximally 5 mol%, i.e. maximally 5 of 100 amino acids may be different from proline, alanine and, optionally, serine, particularly preferably maximally 4 mol%, i.e. maximally 4 of 100 amino acids may be different from proline, alanine and, optionally, serine, more particularly preferably maximally 3 mol%, i.e.
  • maximally 3 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more particularly preferably maximally 2 mol%, i.e. maximally 2 of 100 amino acids may be different from proline, alanine and, optionally, serine and most preferably maximally 1 mol%, i.e. maximally 1 of 100 of the amino acids that are comprised in the random coil polypeptide may be different from proline, alanine and, optionally, serine.
  • Said amino acids different from proline, alanine and, optionally, serine may be selected from the group consisting of Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai, including posttranslationally modified amino acids or non-natural amino acids (see, e.g., Budisa (2004) Angew Chem Int Ed Engl 43:6426-6463; Young (2010) J Biol Chem 285: 11039-11044; Liu (2010) Annu Rev Biochem 79:413-444; Wagner (1983) AngewChem Int Ed Engl 22:816-828; Walsh (2010) Drug Discov Today 15: 773-780.
  • P/A -polypeptides can also comprise Ser as a minor constituent.
  • serine can also be considered as minor constituent.
  • the encoded random coil polypeptide / amino acid sequence may, in particular, consist exclusively of proline, alanine and, optionally, serine residues (i.e., no other amino acid residues are present in the encoded random coil polypeptide or in the amino acid sequence).
  • group C comprises an amino acid sequence consisting of alanine and proline residues (P and A).
  • polypeptides such as group C in this specific embodiment
  • P/A-polypeptides or simply as “P/A”.
  • P/A (or group C, in this specific embodiment) is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from proline and alanine residues.
  • P/A includes at least one proline residue and at least one alanine residue.
  • the proportion of the number of proline residues comprised in the P/A to the total number of amino acid residues comprised in P/A is preferably >10 mol% and ⁇ 70 mol%, more preferably >20 mol% and ⁇ 50 mol%, and even more preferably >25 mol% and ⁇ 40 mol%.
  • 10 mol% to 70 mol% of the total number of amino acid residues in P/A are proline residues; more preferably, 20 mol% to 50 mol% of the total number of amino acid residues comprised in P/A are proline residues; and even more preferably, 25 mol% to 40 mol% (e.g., 25 mol%, 30 mol%, 35 mol% or 40 mol%) of the total number of amino acid residues comprised in P/A are proline residues.
  • P/A does not contain any consecutive proline residues (i.e., that it does not contain any partial PP sequence or multiples thereof).
  • P/A (or group C, in this specific embodiment) comprises no more than 6 identical consecutive amino acid residues (i.e., that it does not, inter alia, contain any partial AAAAAA sequence or multiples thereof, wherein “A” refers to an alanine residue in the context and “AAAAAA” refers to 6 consecutive alanine residues).
  • At least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, yet even more preferably at least 99 mol%, and most preferably 100 mol% of the number of amino acid residues in P/A (or group C, in this specific embodiment) are independently selected from proline and alanine.
  • the remaining amino acid residues in P/A are preferably selected from the 20 standard proteinogenic a-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine. Accordingly, it is preferred that P/A is composed of proline, alanine, glycine, and serine residues (wherein less than 10 mol%, preferably less than 5 mol%, of the number of amino acid residues in P/A are glycine or serine residues), and it is most preferred that P/A is composed of proline and alanine residues, i.e. consists solely of proline and alanine residues. It will be understood that, as specified above, P/A includes at least one proline residue and at least one alanine residue.
  • the number of amino acid residues that P/A (or group C, in this specific embodiment) is composed of is preferably about 10 to about 300 amino acid residues, more preferably about 10 to about 250 amino acid residues, more preferably about 10 to about 200 amino acid residues, even more preferably about 15 to about 150 amino acid residues, more preferably about 10 to about 140 amino acid residues, even more preferably about 10 to about 130 amino acid residues, even more preferably about 15 to about 120 amino acid residues, even more preferably about 15 to about 110 amino acid residues, and yet even more preferably about 20 to about 100 amino acid residues.
  • P/A sequences comprising about 20, about 40 or about 100 amino acid residues may be even more preferred.
  • P/A amino acid sequences include, in particular, such amino acid sequences that comprise (or, more preferably, that consist of): (i) the sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 9; also referred to as “P/A#l”), or (ii) the sequence AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10; also referred to as “P/A#2”), or (iii) the sequence APAAAPAPAAAPAPAAAP (SEQ ID NO: 13; also referred to as “P/A#5”), or (iv) a fragment of any of these sequences, or (v) a combination of two or more of these sequences (which may be the same or different, i.e., any combination of two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the sequences P/A#l, P/A#2 and/or P/A#5; a corresponding example
  • group C comprises an amino acid sequence consisting of alanine, proline, and serine residues (P, A, and S).
  • polypeptides (such as group C in this specific embodiment) comprising an amino acid sequence consisting of alanine, proline, and serine residues may be referred to as “PAS-polypeptides” or simply as “PAS”.
  • PAS is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from alanine, proline, and serine residues.
  • PAS includes at least one proline residue and at least one alanine residue.
  • the encoded amino acid sequence comprises more than about 4 mol%, preferably more than about 6 mol%, more preferably more than about 10 mol%, more preferably more than about 15 mol%, more preferably more than about 20 mol%, more preferably more than about 22 mol%, 23 mol% or 24 mol%, more preferably more than about 26 mol%, 29 mol%, or 30 mol%, more preferably more than about 31 mol%, 32 mol%, 33 mol%, 34 mol% or 35 mol% and most preferably more than about 25 mol% proline residues.
  • the encoded amino acid sequence preferably comprises less than about 40 mol%, more preferably less than 38 mol%, 35 mol%, 30 mol%, 26 mol% proline residues, wherein the lower values are preferred.
  • PAS does not contain any consecutive proline residues (i.e., that it does not contain any partial PP sequence or multiples thereof).
  • PAS (or group C, in this specific embodiment) comprises no more than 6 identical consecutive amino acid residues (i.e., that it does not, inter alia, contain any partial AAAAAA sequence or multiples thereof, wherein “A” refers to an alanine residue in the context and “AAAAAA” refers to 6 consecutive alanine residues).
  • At least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, yet even more preferably at least 99 mol%, and most preferably 100 mol% of the number of amino acid residues in PAS (or group C, in this specific embodiment) are independently selected from proline, alanine, and serine.
  • the remaining amino acid residues in PAS are preferably selected from the 20 standard proteinogenic a-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine.
  • PAS is composed of proline, alanine, glycine, and serine residues (wherein less than 10 mol%, preferably less than 5 mol%, of the number of amino acid residues in PAS are glycine or serine residues), and it is most preferred that PAS is composed of proline and alanine residues, i.e., consists solely of proline, alanine, and serine residues. It will be understood that, as specified above, PAS includes at least one proline residue, at least one alanine residue, and at least one serine residue.
  • the number of amino acid residues that PAS (or group C, in this specific embodiment) is composed of is preferably about 10 to about 300 amino acid residues, more preferably about 10 to about 250 amino acid residues, more preferably about 10 to about 200 amino acid residues, even more preferably about 15 to about 150 amino acid residues, more preferably about 10 to about 140 amino acid residues, even more preferably about 10 to about 130 amino acid residues, even more preferably about 15 to about 120 amino acid residues, even more preferably about 15 to about 110 amino acid residues, and yet even more preferably about 20 to about 100 amino acid residues.
  • PAS sequences comprising about 20, about 40 or about 100 amino acid residues may be even more preferred.
  • Non-limiting examples of preferred PAS amino acid sequences include, in particular, such amino acid sequences that comprise (or, more preferably, that consist of): (i) the sequence
  • ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 16; also referred to as “PAS#1”), or (ii) the sequence
  • AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17; also referred to as “PAS#2”), or (iii) the sequence
  • SSPSAPSPSSPASPSPSSPA SEQ ID NO: 20; also referred to as “PAS#5”
  • PAS#5 SSPSAPSPSSPASPSPSSPA
  • PAS#5 SSPSAPSPSSPASPSPSSPA
  • PAS#5 SSPSAPSPSSPASPSPSSPA
  • PAS#1-PAS#1 PAS#1-PAS#5
  • group C i.e., the P/A and/or PAS polypeptides
  • group C may comprise a combination of two or more of the exemplary and non-limiting sequences detailed in any one of SEQ IDs NO: 9 to 22.
  • Combinations of the same P/A or PAS sequence i.e., multiples of the same P/A or PAS sequence
  • “a combination of two or more” sequences also includes multiples of thereof.
  • the term multiples may refer to, inter alia, 2, 3, 4, 5, 6, 7, 9, or 10 polypeptides (e.g., but not limiting, comprising the amino acid sequence SEQ ID NO: 16; PAS#1) combined into a single polypeptide.
  • SEQ ID NO: 4 comprises the amino acid sequence depicted in SEQ ID NO: 16 (PAS#1) 2-times
  • SEQ ID NO: 5 and 6 each comprise the amino acid sequence depicted in SEQ ID NO: 16 (PAS#1) 5-times.
  • group C may comprise an amino acid sequence selected from any one of SEQ IDs NO: 9 to 22, or multiples thereof. Such examples are further illustratively and non- limitingly provided as SEQ IDs NO: 1 to 8.
  • amino acid residues that group C may have any configuration.
  • each a-amino acid residue comprised in P/A may have the L-configuration or the D-configuration.
  • any proline residue in P/A may be in the form of L-proline or D-proline
  • any alanine residue in P/A may be in the form of L-alanine or D-alanine. It will be understood that not all amino acids have distinct L- and D-configurations; in particular, glycine residues have only one configuration.
  • a-amino acid residues comprised in P/A that can have the L-configuration or the D- configuration, preferably at least 75 mol%, more preferably at least 80 mol%, even more preferably at least 90 mol%, yet even more preferably at least 95 mol%, still more preferably at least 98 mol%, and most preferably 100 mol% of the number of said a-amino acid residues are present in the L-configuration.
  • group C applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
  • the present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, b is 1 or 0, so that B can be present or absent,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group.
  • D is an N-terminal protecting group.
  • the group D in the compound of formula (I) is a protecting group which is attached to the N-terminal amino group, particularly the N-terminal a-amino group, of the polypeptide capable of forming a random coil conformation (group B).
  • group B The terms “protecting group” and “protection group” may be used synonymously in the context of the present invention.
  • group D may be selected from formyl (i.e., -CHO), -CO(Ci-6 alkyl), pyroglutamoyl (i.e., 5-oxopyrrolidin-2-yl-carbonyl), and homopyroglutamoyl (i.e., 6-oxopiperidin-2-yl- carbonyl), wherein the alkyl moiety comprised in said -CO(Ci-6 alkyl) is optionally substituted with one or more groups (e.g., one, two or three groups) independently selected from -OH, -O(Ci-4 alkyl), -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci-4 alkyl) and -COOH.
  • groups e.g., one, two or three groups
  • group D may be selected from formyl, -CO(Ci-4 alkyl), pyroglutamoyl and homopyroglutamoyl, wherein the alkyl moiety comprised in said -CO(Ci-4 alkyl) is optionally substituted with one or two groups independently selected from -OH, -O(Ci-4 alkyl), -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci-4 alkyl) and -COOH.
  • group D may be selected from formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl (i.e., -CO-CH2-COOH), propionyl, 2-hydroxypropionyl, 3 -hydroxypropionyl, 2-methoxypropionyl, 3 -methoxypropionyl, 2-ethoxypropionyl, 3 -ethoxypropionyl, succinyl (i.e., -CO-CH2CH2-COOH; or cyclosuccinyl, i.e.
  • group D is selected from acetyl and pyroglutamoyl, with pyroglutamoyl being especially preferred.
  • group D are comprised in SEQ IDs NO 1 to 8 and illustrated in the appended Examples.
  • group D applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
  • the present invention provides for a method for the production/preparation of a compound of the formula (I):
  • A is a di(alkyl)amino group
  • B is a linker, b is 1 or 0, so that B can be present or absent,
  • C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
  • D is an N-terminal protecting group
  • the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group; and b) the purification of a compound of formula (I).
  • L is a leaving group
  • the leaving group L is replaced by the group A during the coupling of the di(alkyl)amine and the compound L-B-C-D and/or during the coupling of the di(alkyl)amine and compound L-C-D.
  • the leaving group L is replaced by a di(alkyl)amino group, resulting in the production/preparation of compound A-B-C-D or of compound A-C-D.
  • the di(alkyl)amino group A is provided by the di(alkyl)amine which is used in the coupling reaction via the replacement of a hydrogen atom of the di(alkyl)amine by the bond formed between the di(alkyl)amino group and the linking group B, if present, or the polypeptide C, respectively.
  • suitable leaving groups that may be deployed in the context of the present invention. In the following, exemplary and non-limiting examples of such leaving groups are provided.
  • the compound L-B-C-D and/or the compound L-C-D typically comprises a group -C(O)-L, wherein L represents the leaving group and is preferably selected from -OH, -I, -Br, -Cl, -H, and -OR 3 , wherein R 3 is a Ci-Ce alkyl, most preferably -OH.
  • the leaving group may also be the alcohol corresponding to an active ester (e.g. N- hydroxysuccinimide, NHS, or 1 -hydroxybenzotriazole, HOBT), such that the group -C(O)-L represents an active ester group (e.g. a NHS ester group or a HOBT ester group).
  • an active ester e.g. N- hydroxysuccinimide, NHS, or 1 -hydroxybenzotriazole, HOBT
  • Non-limiting examples of such leaving groups are provided in Examples 1 and 2.
  • compound L-C-D comprises a polypeptide capable of forming a random coil conformation (inter alia, a P/A- and/or PAS -polypeptide).
  • the di(alkyl)amino group is typically linked via the nitrogen atom (N) to carbon atom (C) of the carboxyl group of the carboxy terminus of said polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide), such that an amide is formed.
  • the leaving group L can be -OH, which forms part of said C-terminal carboxyl group.
  • the C-terminal carboxyl group may already comprise a leaving group L (namely an -OH).
  • the leaving group L may be -OH forming part of the C-terminal carboxyl group of the polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide).
  • L may be -OH and may, thus, be comprised in the C-terminal carboxyl group of group C.
  • L is preferably selected form -OH, -I, -Br, -Cl and -OR 3 , wherein R 3 is a Ci-Ce alkyl, most preferably -OH.
  • R 3 is a Ci-Ce alkyl.
  • the leaving group may also be the alcohol corresponding to an active ester (e.g. N-hydroxysuccinimide, NHS, or 1- hydroxybenzotriazole, HOBT), such that the group -C(O)-L represents an active ester group (e.g. a NHS ester group or a HOBT ester group).
  • an active ester e.g. N-hydroxysuccinimide, NHS, or 1- hydroxybenzotriazole, HOBT
  • Figure 1 illustratively shows an exemplarily and non-limiting coupling, wherein L is a C-terminal -OH of a polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS- polypeptide).
  • the polypeptide capable of forming a random coil conformation comprises an amino acid sequence consisting of alanine and proline or consisting of alanine, proline, and serine, accordingly, in the context of the present invention, said polypeptides preferably do not comprise any reactive amino acid side chains (i.e., preferably do not comprise any carboxy groups, amino groups, or thiol groups).
  • the N-terminus of said polypeptide is linked to and thereby protected by an N-terminal protecting group.
  • Structural features of said N-terminal protecting group have been disclosed herein above. Consequently, the coupling of the polypeptide capable of forming a random coil structure is preferably occurring via its C-terminal carboxy group or via a linker attached to said C-terminal carboxy group.
  • the present invention provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B- C-D or compound L-C-D each comprise only a single reactive group and wherein the di(alkyl)amine and compound L-B-C-D or compound L-C-D are coupled via said reactive group.
  • the terms “linked”, “coupled”, “attached”, and “connected” may be used interchangeably herein above and below.
  • the present invention provides for means and methods for targeted and/or directional coupling of a single P/A- and/or PAS-polypeptide or a compound comprising a P/A- and/or PAS-polypeptide to the nitrogen atom of a single di(alkyl)amine via either the unprotected C-terminal carboxy group or a linker attached to said C-terminal carboxy group of said P/A- and/or PAS-polypeptide.
  • reaction/coupling reagents and conditions that allow for convenient and reliable production/preparation of the herein disclosed compounds of formula (I), as is, inter alia, illustrated in the appended examples.
  • the P/A- and PAS-polypeptides are capable of forming a random coil conformation when present in an aqueous environment and are, thus, highly polar/hydrophilic/lipophobic.
  • polar protic solvents such as water and methanol
  • polar aprotic solvents such as dimethyl sulfoxide (DMSO) and dimethylformamide are suitable to dissolve P/A- or PAS-polypeptides.
  • di(alkyl)amines for example, but not limiting, di(tetradecyl)amine
  • due their two alkyl chains are particularly nonpolar/hydrophobic/lipophilic.
  • Diethyl ether and dichloromethane may be used to solubilize di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine). Accordingly, the present inventors had to identify a solvent or a combination of solvents that allows for simultaneous solubilization of P/A- or PAS-polypeptides and di(alkyl)amines.
  • the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in (an) organic solvent(s), preferably said organic solvent(s) comprises methanol and di chloromethane.
  • the present invention provides for a method for the production/preparation of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in (an) organic solvent(s), preferably said organic solvent(s) comprises dichloromethane and methanol; and b) the purification of
  • At least one of the di(alkyl)amine, or compound L-B-C-D or compound L-C-D may be provided in an organic solvent comprising dichloromethane and methanol.
  • compound L-B-C-D or compound L-C-D may be provided in an organic solvent comprising dichloromethane and methanol and the di(alkyl)amine may be added to/dissolved in said solution comprising the organic solvent and the compound L-B-C-D or compound L-C-D.
  • compound L-B-C-D or compound L-C- D may be dissolved in methanol and the di(alkyl)amine may be dissolved in dichloromethane. It is evident that both solutions can be mixed to provide both compound L-B-C-D or compound L-C-D and the di(alkyl)amine in an organic solvent comprising dichloromethane and methanol.
  • the present inventors have surprisingly found that performing the coupling of the di(alkyl)amine, and of compound L-B-C-D or compound L-C-D in an organic solvent comprising dichloromethane and methanol is particular useful if the volume ratio of dichloromethane and methanol comprised in said solvent is at least about 3 volume parts dichloromethane and about 1 volume part methanol (volume ratio of about 3 : 1; volume ratio of about 3 to 1) to about 8 volume parts dichloromethane and 1 volume part methanol (volume ratio of about 8 : 1; volume ratio of about 8 to 1).
  • a volume ratio of lower than about 3 to 1 ( ⁇ 3 : 1; ⁇ 3 to 1; less than about 3 volume parts dichloromethane and about 1 volume part methanol) also negatively affects coupling efficiencies of said P/A- and/or PAS-polypeptides to the di(alkyl)amine, as production/preparation of P/A- and/or PAS-polypeptide methyl esters occurs more frequently.
  • the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C- D are comprised in an organic solvent comprising dichloromethane and methanol, wherein said organic solvent comprises about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol.
  • the present invention provides for a method for the production/preparation of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume
  • a volume ratio of e.g., 3 : 1 dichloromethane : methanol (3 to 1; 3 volume parts dichloromethane and 1 volume part methanol) refers to a volume ratio of two solvents (e.g., dichloromethane and methanol), wherein the first solvent (e.g., dichloromethane) is 3 -fold more abundant as compared to the second solvent (e.g., methanol).
  • a volume ratio of 3 : 1 (3 to 1; 3 volume parts dichloromethane and 1 volume part methanol) would correspond to 75 ml dichloromethane and 25 ml methanol in 100 ml total volume of said solvent.
  • a volume ratio of 4 : 1 (4 to 1; 4 volume parts dichloromethane and 1 volume part methanol) would correspond to 80 ml dichloromethane and 20 ml methanol in 100 ml total volume of said solvent.
  • a volume ratio of 5 : 1 (5 to 1; 5 volume parts dichloromethane and 1 volume part methanol) would correspond to approx. 83.33 ml dichloromethane and approx. 16.67 ml methanol in 100 ml total volume of said solvent.
  • the skilled person is able to calculate the respective amounts/volumes of both dichloromethane and methanol to be mixed to obtain a solvent of a certain volume ratio.
  • a volume ratio of about 5 : 1 (about 5 to 1 ; about 5 volume parts dichloromethane and about 1 volume part methanol) is most advantageous for solubilizing P/A- and/or PAS-polypeptides and di(alkyl)amines and for reducing the production/preparation of P/A- and/or PAS- polypeptide methyl esters.
  • the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C- D are comprised in an organic solvent comprising dichloromethane and methanol, wherein said organic solvent comprises about 5 volume parts di chloromethane and about 1 volume part methanol.
  • the present invention provides for a method for the production/preparation of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume
  • present invention provides for a method for the production/preparation of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising dichloromethane and methanol; and b) the purification of a compound of
  • the present invention further provides for a method for the production/preparation of a compound of the formula (I):
  • A-[B-] b C-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts,
  • A-[B-] b C-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising about 5 volume parts dichloromethane and about 1 volume part methanol; and b)
  • the herein above detailed organic solvent that is particular suitable for the coupling of the di(alkyl)amine to the P/A- or PAS-polypeptides may further comprise other/further/additional reagents that are advantageous for said coupling reaction.
  • Such other/further/additional reagents may, inter alia, be a coupling reagent.
  • the inventors identified coupling reagents that allow for the efficient and convenient coupling of P/A- or PAS-polypeptides or compounds comprising the same to di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine).
  • the present invention also provides for 2-(lH-Benzotriazole-l-yl)- 1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) as a highly efficient coupling reagent resulting in approx. 70% coupling rates with the herein disclosed P/A-polypeptides and approx. 40% coupling rates with the herein disclosed PAS-polypeptides.
  • 2-(lH-Benzotriazole-l-yl)- 1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) as a highly efficient coupling reagent resulting in approx. 70% coupling rates with the herein disclosed P/A-polypeptides and approx.
  • the present inventors assayed various coupling reagents (including propylphosphonic anhydride (T3P), pentafluorophenyl trifluoroacetate, Ethyl 2-Cyano-2- (Hydroxyimino)Acetate (OxymaPure®)), and surprisingly found that TBTU resulted in superior coupling rates (2-fold to 50-fold improved coupling rates) as compared to all other tested coupling reagents.
  • T3P propylphosphonic anhydride
  • pentafluorophenyl trifluoroacetate pentafluorophenyl trifluoroacetate
  • Ethyl 2-Cyano-2- (Hydroxyimino)Acetate OxymaPure®
  • the present invention further provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled using a coupling reagent, and wherein said coupling reagent is 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate (TBTU). It was found that the addition of about 1 mol. eq. TBTU to about 3 mol. eq.
  • TBTU to the coupling reaction was beneficial/advantageous for (high) coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine).
  • the addition of more than 5 mol. eq. TBTU to the coupling reaction showed drastically reduced coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine) as compared to e.g., 2 mol. eq. TBTU.
  • the present invention further provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound E-B-C-D or compound L-C-D are coupled using a coupling reagent, and wherein said coupling reagent is 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate (TBTU), and wherein about 2 mol. eq. TBTU are to be added to the reaction.
  • a coupling reagent is 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate (TBTU), and wherein about 2 mol. eq. TBTU are to be added to the reaction.
  • the present invention further relates to a non-nucleophilic base to be used in accordance with the herein detailed methods. Accordingly, the present invention relates to a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are further coupled using a non-nucleophilic base , and wherein said non-nucleophilic base is selected from N,N-Diisopropylethylamine (DIPEA), l,8-Diazabicycloundec-7-ene (DBU), 1,5-Diazabicyclo[4.3.0]non- 5-ene (DBN), triethylamine (TEA), preferably N,N-Diisopropylethy lamin (DIPEA).
  • DIPEA N,N-Diisopropylethylamine
  • DBU l,8-Diazabicycloundec-7-ene
  • the present invention further relates to a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are further coupled using a non- nucleophilic base , wherein said non-nucleophilic base is N,N-Diisopropylethylamin (DIPEA), and wherein about 1.5 mol. eq. are to be added to the reaction.
  • DIPEA N,N-Diisopropylethylamin
  • the present invention further relates to a method for the production/preparation of a compound of formula (I), wherein a di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled in a single reaction.
  • said single reaction may be incubated under an argon cloud for at least about 1 min, for at least about 5 min, for at least about 10 min, for at least about 20 min, for at least about 30 min, for at least about 40 min, for at least about 50 min, for at least about 60 min, for at least about 10 min, preferably for about 60 min.
  • the present invention relates to a method for the production/preparation of a compound of formula (I), wherein a di(alkyl)amine, and compound L-B-C-D or compound L-C-D are reacted for at least about 1 min, for at least about 5 min, for at least about 10 min, for at least about 20 min, for at least about 30 min, for at least about 40 min, for at least about 50 min, for at least about 60 min, for at least about 10 min, preferably for about 60 min.
  • Figure 1 schematically illustrates an herein detailed, yet non-limiting, exemplary reacting scheme for coupling of a PAS40-polypeptide with di(tetradecyl)amine.
  • SEQ IDs NO: 1 Illustrative and non-limiting examples of compound L-B-C-D are found in SEQ IDs NO: 1, 2, and 6, whereas illustrative and non-limiting examples of compound L-C-D are found in SEQ IDs NO: 3 to 5, 7, and 8.
  • SEQ IDs NO: 7 and 8 comprise a lysine residue (K) in position 12.
  • the free amino group on the sidechain of said lysine residue may be used to couple said compound L-C-D to the herein provided di(alkyl)amine, accordingly, preparing/producing compound A-C-D.
  • the herein provided means and methods (inter alia, and non-limiting, the means and methods illustratively show in Example 2) allow the skilled person to perform such coupling reactions.
  • a lysine residue may be readily introduced at any position into any herein disclosed P/A and/or PAS sequences.
  • said lysine residue may be introduced into a central position of a given amino acid sequence encoding the herein provided P/A- and/or PAS-polypeptides.
  • a random coil polypeptide of e.g. 40 amino acid residues it may be preferred that such a lysine residue may be introduced at position 21, resulting in a polypeptide comprising 41 amino acid residues.
  • the present invention further relates to means and methods for the purification of a compound of formula (I).
  • the present invention further relates to the purification of the compound of formula (I), wherein at least some of the reagents are precipitated from the reaction by the addition of a hydrophilic solvent.
  • the present invention further relates to liquid chromatography of the compound of formula (I), wherein liquid chromatography preferably refers to high-pressure liquid chromatography (HPLC) of the compound of formula (I).
  • liquid chromatography such as HPLC
  • HPLC high-pressure liquid chromatography
  • liquid chromatography may be performed on the compound of formula (I) after the herein above detailed precipitation of at least some of the reagents.
  • Figure 2 illustratively shows an exemplary HPLC -chromatogram of PAS40-lipids, clearly demonstrating efficient purification of said compound.
  • the present invention provides for inventive compounds of formula (I) or as obtained/obtainable by the means and methods as provided herein.
  • the present invention also always relates to salts and solution of or comprising the compounds of formula (I), when referring to said compounds.
  • inventive compounds that are characterized by formula (I):
  • A-[B-] b C-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
  • Appended Figures 3 to 8 provide illustrative, non-limiting examples of compounds of formula (I) in accordance with the present invention.
  • exemplary, non-limiting structures, as well as illustrative HPLC-chromatograms and mass-spectrometric analyses of said compounds are provided.
  • inventive compounds may herein also be referred to as e.g., Pga-PAS20- di(tetradecyl)amine (a non-limiting example of compound A-C-D), Pga-P/A20-Ahx-di(tetradecyl)amine (a non-limiting example of compound A-B-C-D), Pga-PAS40-di(tetradecyl)amine (anon-limiting example of compound A-C-D) or Pga-P/A40-Ahx-di(tetradecyl)amine (a non-limiting example of compound A-B-C- D), or Pga-PAS100-K-di(tetradecyl)amine (a non-limiting example of compound A-C-D).
  • Pga-PAS20- di(tetradecyl)amine a non-limiting example of compound A-C-D
  • PEG2k compound (2-[(polyethylene glycol)-2000]-N,N- di(tetradecyl)acetamide) may also be referred to as PEG2k-N,N-di(tetradecyl)acetamide
  • inventive compounds according to formula (I) may in one aspect not comprise the optional linker group B (i.e., and accordingly, be of the general formula A-C-D.
  • optional linker group B i.e., and accordingly, be of the general formula A-C-D.
  • the skilled person is aware that such a compound may, in the context of the present invention, also be represented by the following formula:
  • D is the herein above detailed N-terminal protecting group (group D)
  • -PP-C(O)- corresponds to the polypeptide C (group C) as defined herein above, providing a functional group -C(O)- at its C-terminus for attachment of group A (the herein above detailed di(alkyl)amino group, -NR’R 2 corresponds to the group A (said di(alkyl)amino group)
  • R1 and R2 are independently two linear or branched alkyl chains, preferably said two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
  • inventive compounds according to formula (I) may in one aspect comprise the optional linker group B (i.e., and accordingly, be of the general formula A-B-C-D.
  • linker group B i.e., and accordingly, be of the general formula A-B-C-D.
  • D is the herein above detailed N-terminal protecting group (group D)
  • -PP-C(O)- corresponds to the polypeptide C (group C) as defined herein above, providing a functional group -C(O)- at its C-terminus for attachment of group B (the herein above detailed linker group)
  • -HN-(C2-i2 hydrocarbyl)-C(O)- corresponds to said linker group B
  • -NR’R 2 corresponds to the group A (said di(alkyl)amino group)
  • R1 and R2 are independently two linear or branched alkyl chains, preferably said two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
  • an amide bond is preferably formed between a carboxy group -C(O)- provided by group B and the di(alkyl)amino group A.
  • an amide bond is preferably formed between a carboxy group -C(O)- at the C-terminus of the polypeptide C and the di(alkyl)amino group A.
  • the herein provided compounds of formula (I) cause more than 70% cell viability, when 10 pg/ml thereof are transfected into He La cells and when measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Also, the herein provided compounds of formula (I) cause improved cell viability as compared to a reference PEG2k compound (2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide), when 10 pg/ml thereof are transfected into HeLa cells and measured by MTS assay.
  • the herein provided compounds of formula (I) are clearly beneficial/advantageous with regard to its effects on cell viability as compared to state of the art compounds. That means, inter alia, that the herein provided compounds cause less cytotoxicity as compared to a reference PEG2k compound. That means, inter alia, that the herein provided compounds have low or reduced cytotoxicity/ low or reduced toxicity/ low or reduced negative effects on cell viability as compared to a reference PEG2k compound. In the context of the present invention, it is a desired criterion/characteristic that the herein provided compounds of formula (I) have the herein detailed low or reduced cytotoxicity/ low or reduced toxicity/ low or reduced negative effects on cell viability as compared to a reference PEG2k compound.
  • Example 5 and Table 3 further illustrate the herein detailed beneficial/advantageous effects of the herein provided compounds of formula (I).
  • the present invention further provides for a method for the production/preparation of a lipid nanoparticle comprising one or more compounds of formula (I). Further, the present invention also relates to the use of the compound of formula (I) for the production/preparation of a lipid nanoparticle.
  • the present invention provides for a method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), wherein the method comprises the steps of a) the provision of the compound of formula (I) in an ethanolic solution; b) the mixing of said ethanolic solution with an aqueous solution, thereby preparing/producing a lipid nanoparticle; and c) optionally the dialysis of said lipid nanoparticle.
  • the provision of the compound of formula (I) in an ethanolic solution may also refer to obtaining and/or preparing an ethanolic solution comprising the compound of formula (I).
  • the skilled artisan is aware of means and methods for the production/preparation of lipid nanoparticles. Such means and methods are further detailed herein below and further illustrative, yet non-limiting examples may be found in the examples, namely in Example 3.
  • an ethanolic solution may be used interchangeably with the term “ethanol solution”.
  • an ethanolic solution / an ethanol solution is a non-aqueous solution that consists of ethanol.
  • an aqueous solution is a non-ethanolic solution that consists of water.
  • an ethanolic solution may further comprise a cationic lipid, a non-cationic lipid, and a sterol.
  • Said ethanolic solution may comprise between about 2 mM and about 60 mM total lipid, preferably between about 7.5 mM and about 30 mM, more preferably 9.43 mM total lipid.
  • total lipid comprises any lipid comprised in said ethanolic solution (e.g., the compound of formula (I), the cationic lipid, the noncationic lipid, and the sterol. Accordingly, in the context of the present invention, the compound of formula (I) is a lipid.
  • the herein below detailed mole percentages (mol%) only refer to the mole percentages (mol%) of a certain compound (such as, the compound of formula (I), the cationic lipid, the non-cationic lipid, or the sterol) as compared to the total amount of lipids (total lipids) in said ethanolic solution, without taking into account the ethanol molecules in said ethanolic solution.
  • Said ethanolic solution may comprise between about 1 mol% and about 5 mol%, preferably about 1.5 mol% of the compound of formula (I).
  • said ethanolic solution comprises between about 99 mol% and 95 mol%, preferably about 98.5 mol% of the other herein above mentioned lipids (i.e., the cationic lipid, the non-cationic lipid, and the sterol), respectively.
  • said ethanolic solution may comprise between about 45 mol% and about 55 mol%, preferably about 50 mol% of the cationic lipid.
  • cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a Steroid portion and a connector between these two domains.
  • the cationic lipid carries a net positive charge at about physiological pH.
  • cationic lipids may either refer to lipids with a constitutively positively charged headgroup or to ionizable cationic lipids with apparent pKa values below about 7, that may comprise a neutral or positively charged headgroup.
  • ionizable cationic lipids may be preferred. Accordingly, in the context of the present invention, the cationic lipid may be DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4- (dimethylamino)butanoate) .
  • the present invention further relates to a non-cationic lipid, wherein said non-cationic lipid may be an anionic lipid, preferably wherein said anionic lipid is a phospholipid, more preferably distearoylphosphatidylcholine (DSPC).
  • said ethanolic solution may comprise between about 7 mol% and about 13 mol%, preferably about 10 mol% of the non-cationic lipid.
  • said ethanolic solution may comprise between about 35 mol% and about 42 mol%, preferably about 38.5 mol% of the sterol.
  • said sterol may be cholesterol.
  • the herein disclosed method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I) may comprise the introduction/inclusion of one or more active ingredients into said lipid nanoparticle. Said one or more active ingredients may be comprised in said aqueous solution. Accordingly, mixing of said aqueous solution that comprises said one or more active ingredients with the herein above and below detailed ethanolic solution comprising the compound of formula (I), may result in the production/preparation of a lipid nanoparticle comprising said one or more ingredients and said compound of formula (I).
  • said one or more active ingredients are one or more nucleic acids, one or more polypeptides, one or more proteins, or combinations thereof, preferably one or more nucleic acids.
  • Said one or more nucleic acids may be selected from the group consisting of mRNA, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA for gene editing systems, DNA, plasmids, antisense oligonucleotides, and combinations thereof, preferably mRNA.
  • siRNA small interfering RNA
  • Dicer-substrate dsRNA small hairpin RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • tRNA tRNA
  • rRNA tRNA
  • the aqueous solution comprising the one or more active ingredients may further comprise one or more acids and/or one or more buffers/buffer systems, preferably one or more acids. Accordingly, the aqueous solution may comprise a concentration of between about 5 mM and about 50 mM, preferably between about 10 mM and about 30 mM, more preferably about 11 mM of said one or more acids or of the one or more buffers/buffer systems, preferably the one or more acids.
  • the one or more acids may be selected from acetic acid, HEPES (4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid), and citric acid, preferably acetic acid.
  • the one or more active ingredients and the one or more buffers/buffer systems or acids may be comprised in said aqueous solution at a volume ratio between about 1 : 3, and about 1 : 4, preferably a volume ratio of about 1 : 3.
  • a volume ratio of about 1 : 3 of the one or more active ingredients and the one or more acids (or the one or more buffers/buffer systems) may refer to about one volume part of the one or more active ingredients or of a solution comprising the one or more active ingredients and about three volume parts of the one or more acids, the one or more buffers/buffer systems, or solutions comprising the same.
  • the method for the production/preparation of a lipid nanoparticle may comprise an optional step of dialyzing the lipid nanoparticle.
  • the lipid nanoparticle may not be dialyzed.
  • said lipid nanoparticle may be dialyzed using a buffer, preferably wherein said buffer is selected from the group of phosphate buffer, phosphate buffer saline, and phosphate buffer saline comprising sucrose, more preferably phosphate buffered saline.
  • the term “buffer” may be interchangeably used with the term “buffer system” and may also refer to solution comprising the same. The preparation of such buffers/buffer systems/solutions comprising the same is standard in the art.
  • the present invention further relates to a lipid nanoparticle comprising the compound of formula (I) and/or a lipid nanoparticle obtained/obtainable by the methods for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), detailed herein above. Accordingly, the present invention provides for inventive lipid nanoparticles that comprise the inventive compounds of formula (I) and/or are obtained/obtainable by the inventive methods detailed herein above.
  • said lipid nanoparticles comprising the compound of formula (I) comprises highly beneficial/advantageous characteristics that may be desired in the context of the present invention, yet may have been surprisingly found by the present inventors.
  • the herein detailed inventive lipid nanoparticles have a Zeta-potential of less than 10 mV after about 3 h of dialysis using phosphate buffered saline, when measured by diffraction light scattering (DLS). That means, inter alia, that said lipid nanoparticle has a lower/reduced Zeta-potential as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of formula (I) after about 3 h of dialysis using phosphate buffered saline, when measured by DLS.
  • DLS diffraction light scattering
  • the surface charge of, for example, a lipid nanoparticle may be influenced by molecules (such as, for example, PEG polymers, P/A-polypeptides, or PAS- polypeptides) on the surface of said lipid nanoparticle that may shield the same. Accordingly, the Zetapotential of, for example, a lipid nanoparticle may provide insights into the capacity of molecules (such as, for example, PEG polymers, P/A-polypeptides, or PAS-polypeptides) on the surface of said lipid nanoparticle to shield the surface of said lipid nanoparticle.
  • molecules such as, for example, PEG polymers, P/A-polypeptides, or PAS-polypeptides
  • a low Zeta-potential of lipid nanoparticles comprising the compound of formula (I) may be desired and may indicate successful shielding/protection of said lipid nanoparticle and/or the surface of said lipid nanoparticle.
  • shielding and “protecting” may be used interchangeably.
  • lipid nanoparticles comprising the compound of formula (I) have at least 91% RNA encapsulation efficiency, when measured using RiboGreen Assay. That means, inter alia, that said lipid nanoparticles have improved/increased RNA encapsulation efficiency as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of formula (I), when measured using RiboGreen Assay.
  • Example 7 the herein provided compounds of formula (I), when introduced into lipid nanoparticles are, thus, superior in regard to shielding/protecting said lipid nanoparticle and/or the membrane of said lipid nanoparticle, as compared to a reference PEG2k compound.
  • this may be a desired characteristic or said compound of formula (I) and the lipid nanoparticle comprising said compound of formula (I).
  • the herein provided lipid nanoparticle comprising the compound of formula (I) may have improved/increased transfection rates as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of any one of claims 35 to 38, when transfected into Jurkat, A549, HepG2, and/or C2C12 cells and when measured by luminescence readout.
  • a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of any one of claims 35 to 38, when transfected into Jurkat, A549, HepG2, and/or C2C12 cells and when measured by luminescence readout.
  • High/increased/improved transfection rates may be desired in the context of the present invention.
  • the present invention further provides for a lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion comprising the lipid nanoparticle detailed herein above, wherein said lipid nanoparticle comprises the compound of formula (I). Accordingly, the present invention further provides for a lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion comprising the compound of formula (I).
  • the term “lipid nanoparticle”, as used herein below and above, may also refer to said “lipid nanoparticle suspension” and/or to said “lipid nanoparticle colloidal dispersion” comprising said lipid nanoparticle and/or the compound of formula (I).
  • Said lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion may be stored at about 4 °C for at least about 4 days without signs of turbidity and/or visible precipitation of said lipid nanoparticle suspension or components thereof.
  • the lipid nanoparticle/ the lipid nanoparticle suspension/ the lipid nano particle colloidal dispersion comprising the compound of formula (I) show surprising and/or desired beneficial/advantageous effects, as compared to a reference lipid nanoparticle/ a reference lipid nanoparticle suspension/ a reference lipid nano particle colloidal dispersion that does not comprise the compound of formula (I).
  • lipid nanoparticles/ lipid nanoparticle suspensions/ lipid nano particle colloidal dispersions The only difference between the herein provided lipid nanoparticles/ lipid nanoparticle suspensions/ lipid nano particle colloidal dispersions and the reference lipid nanoparticle/ the reference lipid nanoparticle suspension/ the reference lipid nano particle colloidal dispersion is the presence of the compound of formula (I) in the former and the presence of a reference PEG2k compound (2- [(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide) in the later.
  • any herein above and below detailed technical advantage of the herein provided lipid nanoparticles/ the lipid nanoparticle suspensions/ the lipid nano particle colloidal dispersions may be attributed to the herein provided compound of formula (I) comprised in said lipid nanoparticles/ said lipid nanoparticle suspensions/ said lipid nano particle colloidal dispersions.
  • any technical advantage of said lipid nanoparticles/ said lipid nanoparticle suspensions/ said lipid nano particle colloidal dispersions provided herein further contributes to the inventive character of the herein provided inventive compound of formula (I). This further evident for the appended, non-limiting Examples.
  • inventive compounds may also be employed in the production/preparation of lipid (or lipoid) particles and/or lipid (or lipoid) formulations that defer from the ones exemplarily herein provided.
  • lipid (or lipoid) particles including but not limited to lipid nanoparticles
  • lipid (or lipoid) formulations as described in and/or as produced in accordance with the means and methods described in any of the following (or any combination of the following) are envisaged and, thus, incorporated herein by reference in their entirety: e.g., WO 2022/180213, WO 2010/053572, WO 2012/000104, WO 2010/053572, WO 2014/028487, WO 2015/095351, WO 2018/089540, WO 2017/218704.
  • the present invention further relates to means, methods and uses for the production/preparation of a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound.
  • the present invention relates to the use of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound for the production/preparation of a pharmaceutical composition.
  • the present invention further provides for a method for the production/preparation of a pharmaceutical composition, wherein the method comprises the formulation of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound into a pharmaceutical composition.
  • Said method for the production/preparation of a pharmaceutical composition may comprise the formulation of said compound, said lipid nanoparticle, and/or said lipid nanoparticle suspension using a pharmaceutically acceptable carrier.
  • said pharmaceutical composition may be formulated for intramuscular or intravenous administration. Accordingly, said pharmaceutical composition may be formulated for use as a medicament or for use as a vaccine.
  • the present invention also relates to pharmaceutical compositions comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound. Furthermore, the present invention relates to pharmaceutical compositions obtained/obtainable by the herein above and below detailed means, methods, and uses.
  • the present invention further relates to the use of said pharmaceutical compositions in the treatment of or in a method of treatment of a disease and/or a medical condition.
  • the present invention further relates to means, methods, and uses for the production/preparation of a non- pharmaceutical composition
  • a non- pharmaceutical composition comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound.
  • the present invention relates to the use of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound for the production/preparation of a non-pharmaceutical composition.
  • the present invention further provides for a method for the production/preparation of a non-pharmaceutical composition, wherein the method comprises the formulation of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound into a non-pharmaceutical composition.
  • the present invention also relates to non-pharmaceutical compositions comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound. Furthermore, the present invention relates to non-pharmaceutical compositions obtained/obtainable by the herein above and below detailed means, methods, and uses.
  • the present invention further relates to the use of said non-pharmaceutical compositions.
  • a non-pharmaceutical composition may in the context of the present invention be a cosmetic composition.
  • polypeptide and “peptide” are used herein interchangeably and refer to a polymer of two or more amino acids linked via amide bonds that are formed between an amino group of one amino acid and a carboxy group of another amino acid.
  • the amino acids comprised in the peptide or protein which are also referred to as amino acid residues, may be selected from the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) but also from non-proteinogenic and/or non-standard a-amino acids (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (
  • the amino acid residues comprised in the peptide or protein are selected from a-amino acids, more preferably from the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D-isomer, and are preferably all present as the L-isomer).
  • the peptide or protein may be unmodified or may be modified, e.g., at its N-terminus, at its C-terminus and/or at a functional group in the side chain of any of its amino acid residues (particularly at the side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and/or Arg residues).
  • Such modifications may include, e.g., the attachment of any of the protecting groups described for the corresponding functional groups in: Wuts PGM, Greene’s protective groups in organic synthesis, 5 th edition, John Wiley & Sons, 2014.
  • Such modifications may also include, e.g., the glycosylation and/or the acylation with one or more fatty acids (e.g., one or more Cs-so alkanoic or alkenoic acids; forming a fatty acid acylated peptide or protein).
  • the amino acid residues comprised in the peptide or protein may, e.g., be present as a linear molecular chain (forming a linear peptide or protein) or may form one or more rings (corresponding to a cyclic peptide or protein) or branched structures.
  • the peptide or protein may also form oligomers consisting of two or more identical or different molecules.
  • amino acid refers, in particular, to any one of the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro (also called an imino acid), Ser, Thr, Trp, Tyr, or Vai) but also to a non-proteinogenic and/or non-standard a-amino acid (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group (e.g., a cycloalkyl group,
  • amino acid preferably refers to an a-amino acid, more preferably to any one of the 20 standard proteinogenic a-amino acids (which may be in the form of the L-isomer or the D-isomer but are preferably in the form of the L-isomer).
  • hydrocarbon chain refers to a hydrocarbon group consisting of carbon atoms and hydrogen atoms.
  • hydrocarbyl refers to a monovalent hydrocarbon group which may be acyclic (i.e., non-cyclic) or cyclic, or it may be composed of both acyclic and cyclic groups/subunits.
  • An acyclic hydrocarbyl or an acyclic subunit in a hydrocarbyl may be linear or branched, and may further be saturated or unsaturated.
  • a cyclic hydrocarbyl or a cyclic subunit in a hydrocarbyl may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • a “C2-12 hydrocarbyl” denotes a hydrocarbyl group having 2 to 12 carbon atoms.
  • Exemplary hydrocarbyl groups include, inter alia, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or a composite group composed of two or more of the aforementioned groups (such as, e.g., alkylcycloalkyl, alkylcycloalkenyl, alkylarylalkenyl, arylalkyl, or alkynylaryl).
  • hydrocarbyl group is attached to a parent moiety and is further substituted, e.g., as in the case of a residue H2N-(C2-i2 hydrocarbyl)-COOH, then the corresponding hydrocarbyl group within this residue may also be considered divalent.
  • alkyl refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to- carbon double bond or any carbon-to-carbon triple bond.
  • a “C1.4 alkyl” denotes an alkyl group having 1 to 4 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C 1.4 alkyl.
  • alkenyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • C2-4 alkenyl denotes an alkenyl group having 2 to 4 carbon atoms.
  • Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1- en-l-yl, prop-l-en-2-yl, or prop-2-en-l-yl), butenyl, or butadienyl (e.g., buta-l,3-dien-l-yl or buta-1,3- dien-2-yl).
  • propenyl e.g., prop-1- en-l-yl, prop-l-en-2-yl, or prop-2-en-l-yl
  • butenyl e.g., buta-l,3-dien-l-yl or buta-1,3- dien-2-yl.
  • alkenyl preferably refers to C2-4 alkenyl.
  • alkynyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds.
  • C2-4 alkynyl denotes an alkynyl group having 2 to 4 carbon atoms.
  • Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl.
  • alkynyl preferably refers to C2-4 alkynyl.
  • aryl refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • Aryl may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., IH-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl.
  • dialinyl i.e., 1,2-dihydronaphthyl
  • tetralinyl i.e., 1,2,3,4-tetrahydronaphthyl
  • indanyl e.g., indenyl (e.g., IH-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl.
  • an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
  • heteroaryl refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group).
  • aromatic ring group comprises one or more (such as, e.g., one, two,
  • each heteroatom-containing ring comprised in said aromatic ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heteroaryl may, e.g., refer to thienyl (i.e., thiophenyl), benzo [b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1 -benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., lH-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1H- pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl),
  • heteroaryl preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized;
  • heteroaryl examples include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, IH-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.
  • cycloalkyl refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings).
  • Cycloalkyl may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl.
  • cycloalkyl preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl.
  • a particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members.
  • a particularly preferred example of a “cycloalkyl” is cyclohexyl.
  • cycloalkenyl refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond.
  • Cycloalkenyl may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, “cycloalkenyl” preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl.
  • a particularly preferred “cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
  • nucleic acid(s) refers to a compound(s) containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- or triple -stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of antisense molecules, plasmid DNA (pDNA), linear or circular DNA, PCR products, or vectors.
  • RNA may be in the form of self-amplifying RNA (saRNA) or small hairpin RNA (shRNA), small interfering RNA (siRNA), chemically modified or unmodified messenger RNA (mRNA), antisense RNA, circular RNA (circRNA) comprising at least one coding sequence, micro RNA (miRNA), micRNA, multivalent RNA, transfer RNA (tRNA), single guided RNA (sgRNA), replicating RNA (repRNA), dicer substrate RNA or viral RNA (vRNA), antisense oligonucleotide (ASO), double-stranded RNA (dsRNA) and combinations thereof.
  • saRNA self-amplifying RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • antisense RNA circular RNA (circRNA) comprising at least one coding sequence
  • miRNA micro RNA
  • micRNA multivalent RNA
  • tRNA transfer RNA
  • sgRNA
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • a “cationic lipid” refers to a lipid capable of being positively charged.
  • Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
  • Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH value.
  • Ionizable lipid refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range, e.g., pH ⁇ 3 to pH ⁇ 9. Ionizable lipids may be synthetic or naturally derived.
  • an “effective amount” or “therapeutically effective amount” of an active agent such as a nucleic acid is an amount sufficient to produce the desired effect, e.g., an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid.
  • An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid.
  • an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater.
  • Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.
  • Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence, or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
  • the present invention refers as well to compositions comprising at least one compound of formula (I) according to the present invention and at least one active agent.
  • the at least one active agent is preferably comprised in an effective amount.
  • Active agents include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic. Active agents may be any type of molecule or compound, including e.g., nucleic acids, nucleic acid analogues, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and PrimatizedTM antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.
  • nucleic acids include nucleic acid analogues, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies
  • the active agent is a therapeutic agent, or a salt or derivative thereof.
  • Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification.
  • therapeutic agents include any therapeutically effective agent or drug, such as antiinflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.
  • therapeutically effective agent or drug such as antiinflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.
  • the therapeutic agent is an oncology drug, which may also be referred to as an antitumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like.
  • oncology drugs that may be used according to the invention include, but are not limited to, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, Cytoxan, daunorubicin, dexamethasone, dexrazoxane,
  • the at least one active agent is selected from the group consisting of proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogues, organic molecules having a molecular weight up to 1000 g/mol and combinations thereof.
  • any known protein is in general suitable.
  • exemplary proteins include glycoproteins and apolipoproteins.
  • apolipoprotein or “lipoprotein” refers to apolipoproteins known to those of skill in the art and variants and fragments thereof and to apolipoprotein agonists, analogues, or fragments thereof as well as chimeric construction of an apolipoprotein.
  • Apolipoproteins utilized in the invention also include recombinant, synthetic, semi- synthetic or purified apolipoproteins.
  • peptide in general suitable.
  • the term peptide according to the present invention includes peptidomimetic.
  • the peptide or peptidomimetic can be about 5 to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • a "cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell.
  • a microbial cell-permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a-defensin, -defensin orbactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin).
  • a cell permeation peptide can also include a nuclear localization signal (NLS).
  • a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T.
  • MPG bipartite amphipathic peptide
  • a targeting peptide tethered to an iRNA agent and/or the carrier oligomer can be an amphipathic a-helical peptide.
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D- or L-peptides; a-, fy, or y-peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • carbohydrate Any known carbohydrate is in general suitable.
  • Exemplarily carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid.
  • compositions of the present invention are particularly useful for the delivery of nucleic acids or nucleic acid analogues, including, e.g., siRNA molecules, mRNA molecules, plasmids, micro RNA, antagomirs, aptamers, and ribozymes.
  • nucleic acids or nucleic acid analogues including, e.g., siRNA molecules, mRNA molecules, plasmids, micro RNA, antagomirs, aptamers, and ribozymes.
  • compositions of the present invention may be used to modulate the expression of target genes and proteins both in vitro and in vivo by contacting cells with a composition of the present invention associated with a nucleic acid that reduces target gene expression, e.g., an siRNA or micro RNA, or a nucleic acid that may be used to increase expression of a desired protein, e.g., an mRNA or a plasmid encoding the desired protein.
  • a nucleic acid that reduces target gene expression e.g., an siRNA or micro RNA
  • a nucleic acid that may be used to increase expression of a desired protein e.g., an mRNA or a plasmid encoding the desired protein.
  • nucleic acids and nucleic acid analogues or plasmids are in general suitable. Their methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor or in vitro transcription. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art.
  • Nucleic acids and nucleic acid analogues include polymers containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- or triple-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of linear DNA, circular DNA, plasmid DNA (pDNA), antisense molecules, PCR products, or vectors.
  • RNA may be in the form of chemically modified or unmodified messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA) comprising at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA) and combinations thereof.
  • the nucleic acids may include one or more oligonucleotide modification.
  • Nucleic acids of the present invention may be of various lengths, generally dependent upon the particular form of nucleic acid.
  • plasmids or genes may be from about 1,000 to 100,000 nucleotide residues in length.
  • oligonucleotides may range from about 10 to 100 nucleotides in length.
  • oligonucleotides, single -stranded, doublestranded, and triple-stranded may range in length from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length.
  • the term "circular DNA” includes any DNA that forms a closed loop and has no ends. Examples of circular DNA are plasmid DNA, minicircle DNA and doggybone DNA (dbDNA).
  • plasmid DNA preparation for use with embodiments of this invention commonly utilizes, but is not limited to, expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest.
  • a gene in the plasmid of interest that encodes resistance to a particular antibiotic allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures.
  • Methods of isolating plasmid DNA are widely used and well known in the art.
  • Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.
  • the present invention specifically refers to compositions for the delivery of mRNA or siRNA molecules.
  • RNA molecules For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequencespecific mRNA.
  • In vitro transcription describes a process of template- directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest.
  • Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification.
  • RNA polymerase adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts.
  • rNTPs cytidine ribonucleoside triphosphates
  • In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), as well as with commercially available reagents including RNA polymerases and rNTPs.
  • the methodology for in vitro transcription of mRNA is well known in the art.
  • the desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.).
  • Techniques for the isolation of the mRNA transcripts are well known in the art.
  • Well known procedures include phenol/chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.
  • Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography, silica-based affinity chromatography and polyacrylamide gel electrophoresis.
  • RNA purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3 ’ extension.
  • dsRNA double-stranded RNA
  • Endogenous eukaryotic mRNA typically contain a cap structure on the 5'- end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts.
  • CBP mRNA Cap Binding Protein
  • the 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide.
  • the conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.
  • cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA.
  • 5’-capping of synthetic mRNA can be performed co- transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription).
  • the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine -guanine linkage where one guanine contains an N7 methyl group as well as a 3'-O-methyl group.
  • up to 20% of transcripts remain uncapped during this co- transcriptional process and the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability.
  • synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5'-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 ’-cap which have enhanced binding of cap binding proteins, increased half-life, and reduced susceptibility to 5' endonucleases and/or reduced 5' decapping. Numerous synthetic 5 ’-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.
  • poly-A tail a long chain of adenine nucleotides
  • Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase.
  • the first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template.
  • the latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 ’termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length.
  • a tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (Epicenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.
  • modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity.
  • the modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art.
  • a large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA, e.g., as disclosed in US 2012/0251618.
  • In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.
  • RNA is a self-amplifying RNA.
  • a self-amplifying RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself).
  • a self-amplifying RNA molecule is thus in certain embodiments: a (+) strand molecule that can be directly translated after delivery to a cell, and this translation provides for an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded protein, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein.
  • the overall result of this sequence of transcriptions is an amplification in the number of the introduced self-amplifying RNAs and so the encoded protein becomes a major polypeptide product of the host cells.
  • the RNA is a circular RNA (circRNA) is a type of single-stranded RNA which, unlike linear RNA, forms a covalently closed continuous loop by joining the 3' and 5' ends normally present in an RNA molecule.
  • circRNA can be designed to encode and express proteins.
  • the oligonucleotide (or a strand thereof) of the present invention specifically hybridizes to or is complementary to a target polynucleotide.
  • RNA is a SiRNA.
  • SiRNAs are RNA duplexes normally 16 to 30 nucleotides long that can associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC).
  • RISC RNAi-induced silencing complex
  • siRNA can be designed to knock down protein expression with high specificity.
  • siRNA function through a natural mechanism evolved to control gene expression through non-coding RNA.
  • a "single strand siRNA compound” as used herein, is an siRNA compound which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include a hairpin or pan-handle structure. Single strand siRNA compounds may be antisense with regard to the target molecule.
  • a single strand siRNA compound may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA.
  • a single strand siRNA compound is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.
  • a “double stranded siRNA compound” as used herein, is an siRNA compound which includes more than one, and in some cases two, strands in which interchain hybridization can form a region of duplex structure.
  • the antisense strand of a double stranded siRNA compound may be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
  • antisense strand means the strand of an siRNA compound that is sufficiently complementary to a target molecule, e.g., a target RNA.
  • the sense strand of a double stranded siRNA compound may be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
  • the double strand portion of a double stranded siRNA compound may be equal to or at least, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It may be equal to or less than 200, 100, or 50, nucleotides pairs in length.
  • Ranges may be 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the siRNA compound is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller siRNA compounds, e.g., siRNAs agents.
  • the sense and antisense strands may be chosen such that the double-stranded siRNA compound includes a single strand or unpaired region at one or both ends of the molecule.
  • a double-stranded siRNA compound may contain sense and antisense strands, paired to contain an overhang, e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. Some embodiments will have at least one 3' overhang. In one embodiment, both ends of an siRNA molecule will have a 3' overhang. In some embodiments, the overhang is 2 nucleotides.
  • the length for the duplexed region is between 15 and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA compound range discussed above.
  • siRNA compounds can resemble in length and structure the natural Dicer processed products from long dsiRNAs.
  • Embodiments in which the two strands of the siRNA compound are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and a 3' overhang are also within the invention.
  • the siRNA compounds described herein, including double-stranded siRNA compounds and singlestranded siRNA compounds can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein.
  • mRNA e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced e.g., a transcript of a gene that encodes a protein.
  • a gene e.g., a gene that encodes a protein.
  • the RNA to be silenced is an endogenous gene or a pathogen gene.
  • RNAi refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., a siRNA compound of 21 to 23 nucleotides.
  • an siRNA compound is "sufficiently complementary" to a target RNA, e.g., a. target mRNA, such that the siRNA compound silences production of protein encoded by the target mRNA.
  • the siRNA compound is "exactly complementary" to a target RNA, e.g., the target RNA and the siRNA compound anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity.
  • a "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to atarget RNA.
  • the siRNA compound specifically discriminates a single-nucleotide difference. In this case, the siRNA compound only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.
  • dicer substrate siRNA can be employed as a less immunogenic alternative.
  • DsiRNA is 25 to 30 nucleotides in length, and after cellular uptake is further cleaved and processed by the Dicer enzyme converting it into the active form which then associates with the RISC.
  • Antisense RNA directed to a target polynucleotide.
  • the term "antisense RNA” or simply “antisense” is meant to include RNA that are complementary to a targeted polynucleotide sequence.
  • Antisense RNA are single strands of RNA that are complementary to a chosen sequence, e.g., a target gene mRNA.
  • Antisense RNA are thought to inhibit gene expression by binding to a complementary mRNA. Binding to the target mRNA can lead to inhibition of gene expression either by preventing translation of complementary mRNA strands by binding to it, or by leading to degradation of the target mRNA.
  • antisense RNA contains from about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense RNA that may not be exactly complementary to the desired target gene.
  • miRNAs are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals, but are not translated into protein.
  • Processed miRNAs are single stranded 17 to 25 nucleotide (nt) RNA molecules that become incorporated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation.
  • RISC RNA-induced silencing complex
  • the RNA is transfer RNA (tRNA).
  • Transfer RNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length that serves as the physical link between the mRNA and the amino acid sequence of proteins. Transfer RNA does this by carrying an amino acid to the protein synthesizing machinery of a cell called the ribosome. Complementation of a 3-nucleotide codon in a messenger RNA (mRNA) by a 3-nucleotide anticodon of the tRNA results in protein synthesis based on the mRNA code. As such, tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code.
  • mRNA messenger RNA
  • tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code.
  • the nucleic acid is a single guide RNA applied to direct CRISPR/Cas9 mediated gene editing.
  • the single guide RNA hybridizes with a target sequence in the genome of a cell and complexes with the Cas9 protein at the target site initiating single or double strand breaks.
  • the at least one active agent is selected from antagomirs, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermirs, miRNA mimics, antimir or miRNA inhibitors and UI adaptors.
  • Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-0-methylation of sugar, phosphorothioate backbone and, for example, a cholesterol-moiety at 3'-end.
  • Aptamers are nucleic acid or peptide molecules that bind to a particular molecule of interest with high affinity and specificity.
  • DNA or RNA aptamers have been successfully produced which bind many different entities from large proteins to small organic molecules.
  • Aptamers may be RNA or DNA based, and may include a riboswitch.
  • a riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene's activity.
  • the aptamer may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. Further, as described more fully herein, the term "aptamer” specifically includes "secondary aptamers" containing a consensus sequence derived from comparing two or more known aptamers to a given target.
  • Ribozymes are RNA molecules complexes having specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Nucleic acids associated with lipid particles of the present invention may be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-or double- stranded) capable of inducing an immune response when administered to a subject, which may be a mammal or other patient.
  • ISS immunostimulatory oligonucleotides
  • transcription factors recognize their relatively short binding sequences, even in the absence of surrounding genomic DNA, short oligonucleotides bearing the consensus binding sequence of a specific transcription factor can be used as tools for manipulating gene expression in living cells.
  • This strategy involves the intracellular delivery of such "decoy oligonucleotides", which are then recognized and bound by the target factor. Occupation of the transcription factor's DNA-binding site by the decoy renders the transcription factor incapable of subsequently binding to the promoter regions of target genes.
  • a supermir refers to a single stranded, double stranded or partially double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which has a nucleotide sequence that is substantially identical to a miRNA and that is antisense with respect to its target.
  • This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages and which contain at least one non-naturally-occurring portion which functions similarly.
  • miRNA mimics represent a class of molecules that can be used to imitate the gene silencing ability of one or more miRNAs.
  • miRNA mimic refers to synthetic non-coding RNAs (i.e., the miRNA is not obtained by purification from a source of the endogenous miRNA) that are capable of entering the RNAi pathway and regulating gene expression.
  • miRNA mimics can be designed as mature molecules (e.g., single stranded) or mimic precursors (e.g., pri- or pre-miRNAs).
  • inhibitors are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the ability of specific miRNAs.
  • the inhibitors are nucleic acid or modified nucleic acids in nature including oligonucleotides comprising RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNAs), or any combination of the above.
  • Modifications include 2' modifications and intemucleotide modifications (e.g., phosphorothioate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization, or potency.
  • miRNA inhibitors can comprise conjugates that can affect delivery, intracellular compartmentalization, stability, and/or potency.
  • Inhibitors can adopt a variety of configurations including single stranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpin designs, in general, microRNA inhibitors comprise contain one or more sequences or portions of sequences that are complementary or partially complementary with the mature strand (or strands) of the miRNA to be targeted, in addition, the miRNA inhibitor may also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA.
  • the additional sequences may be the reverse complements of the sequences that are adjacent to the mature miRNA in the pri -miRNA from which the mature miRNA is derived, or the additional sequences may be arbitrary sequences (having a mixture of A, G, C, or U).
  • U1 adaptors inhibit poly A sites and are bifunctional oligonucleotides with a target domain complementary to a site in the target gene's terminal exon and a 'U1 domain' that binds to the U1 smaller nuclear RNA component of the U1 snRNP.
  • U1 snRNP is a ribonucleoprotein complex that functions primarily to direct early steps in spliceosome formation by binding to the pre-mRNA exon- intron boundary.
  • Nucleotides 2- 11 of the 5'end of U1 snRNA base pair bind with the 5'ss of the pre mRNA.
  • oligonucleotides of the invention are U1 adaptors.
  • the at least one active agent is selected from the group consisting of linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA (circRNA) comprising at least one coding sequence; small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA); and combinations thereof.
  • pDNA plasmid DNA
  • saRNA self-amplifying RNA
  • mRNA messenger RNA
  • circRNA circular RNA comprising at least one coding sequence
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • dicer substrate RNA antisense oligonucleotide
  • tRNA transfer RNA
  • sgRNA single guide
  • the at least one active agent is an organic molecule having a molecular weight up to 1000 g/mol, also referred to as small molecule in the pharmaceutical field, preferably the organic molecule is selected from paclitaxel, doxorubicin, irinotecan, vincristine and oxaliplatin.
  • composition according to the present invention can further comprise a compound selected from a lipid, different from the compound of formula (I), like an ionizable lipid, a cationic lipid, a neutral lipid or a structural lipid, a sterol, or a sterol derivative; a buffering agent, a pharmaceutically acceptable salt, a cryoprotectant or any combination thereof.
  • Suitable lipids different to the compound of formula (I), according to the present invention, which can be further present are for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides or mixtures thereof.
  • Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques.
  • lipids containing saturated fatty acids with carbon chain lengths in the range of CIO to C20 are preferred.
  • lipids with mono- or diunsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
  • Preferred lipids are 1,2- Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC) or any related phosphatidylcholine.
  • DOPE 1,2- Dioleoyl-sn-glycero-3 -phosphoethanolamine
  • DSPC 1 ,2-distearoyl-sn-glycero-3 -phosphocholine
  • POPC dipalmitoylphosphatidylcholine
  • lipids are composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol as well as sterols, in particular cholesterol and phytosterols.
  • the further lipid is an ionizable lipid, preferably selected from l,2-distearoyl-3- dimethylammonium -propane, l,2-dipalmitoyl-3-dimethylammonium-propane, l,2-dimyristoyl-3- dimethylammonium -propane, l,2-dioleoyl-3 -dimethylammonium -propane, l,2-dioleyloxy-3- dimethylaminopropane, (6Z,9Z,28Z,3 lZ)-heptatriacont-6, 9, 28, 31 -tetraene- 19-yl 4-
  • the further lipid is an cationic lipid, preferably selected from salts of 1,2-di-O- octadecenyl-3 -trimethylammonium propane, l,2-dioleoyl-3-trimethylammonium-propane, Nl-[2-((lS)-l- [(3 -aminopropyl)amino] -4 - [di (3 -amino-propyl)amino]butylcarboxamido)ethyl] -3 ,4-di [oleyloxy] - benzamide, N4-cholesteryl-spermine, 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, 0,0’- ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine, l,2-dilauroyl-sn-glycero-3- e
  • lipids suitable in the compositions of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
  • the further lipid is selected from phospholipids, aminolipids, and sphingolipids.
  • Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine.
  • phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylglycerols, and [3- acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • the further lipid is selected from polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate), Myq52 (Polyoxyethylene (40) stearate), and Brij S10 (Polyoxyethylene (10) stearyl ether) or combinations thereof.
  • polysorbate 80 also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate
  • Myq52 Polyoxyethylene (40) stearate
  • Brij S10 Polyoxyethylene (10) stearyl ether
  • a cryoprotective agent is an agent that protects a composition from experiencing adverse effects upon freezing and thawing.
  • cryoprotective agents such as polyols and/or carbohydrates, among others, may be added to prevent substantial particle agglomeration.
  • a buffering agent can be included as well. Suitable buffering agents are for example phosphate, acetate, citrate, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid, amino acids, and other organic compounds; antioxidants including ascorbic acid and methionine.
  • At least one of the following additives can be further present in the composition: preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates including monosaccharides, disaccharides, and other sugar compounds like glucose, mannose, or dextrins; preserv
  • composition preferably the lipid nanoparticle, comprises the at least one active agent to the compound of formula (I) in a weight to weight ratio of 1:0.01 to 1: 100.
  • the composition preferably the lipid nanoparticle, comprises at least one further compound selected from one or more lipids, different from the compound of formula (I); a buffering agent; a pharmaceutically acceptable salt, different from the buffering agent; a cryoprotectant or any combination thereof.
  • the composition preferably the lipid nanoparticle, further comprises one or more lipids, different from the compound of formula (I), more preferably further comprises one, two or three additional lipids, different from the compound of formula (I).
  • the composition preferably the lipid nanoparticle, consists of the compound of formula (I), at least one active agent and one or more lipids, different from the compound of formula (I), more preferably one, two or three additional lipids, different from the compound of formula (I).
  • the compound of formula (I) is present in a ratio of about 0.1 to about 10 mol%, based on the total lipid content. In one embodiment the compound of formula (I) is present in a ratio of greater than 10 mol%, based on the total lipid content. In one embodiment the compound of formula (I) is present in a ratio of 0.5 mol% to 5 mol%, based on the total lipid content. In some embodiments the compound of formula (I) is present in a ratio of 1.5 mol%.
  • the cationic lipid is preferably present in a ratio of about 10 to about 80 mol%, based on the total lipid content. In one embodiment the cationic lipid is present in a ratio of about 50 mol%, based on the total lipid content.
  • the ionizable lipid is preferably present in a ratio of about 10 to about 80 mol%, based on the total lipid content. In one embodiment the ionizable lipid is present in a ratio of about 50 mol%, based on the total lipid content.
  • the structural lipid is preferably present in a ratio of about 10 to about 40 mol%, based on the total lipid content. In one embodiment the structural lipid is present in a ratio of about 10 mol%, based on the total lipid content.
  • the composition preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is a sterol such as cholesterol or phytosterols or derivatives thereof
  • the sterol is preferably present in a ratio of about 10 to about 60 mol%, based on the total lipid content. In one embodiment the sterol is present in a ratio of about 35 to about 41 mol%, based on the total lipid content. In one embodiment the sterol is present in a ratio of about 38.5 mol%%, based on the total lipid content.
  • the stabilizing agent is preferably present in a ratio of about 0 to about 10 mol%, based on the total lipid content.
  • the at least one buffering agent is present in a molar concentration of 0. 1 mM to 1000 mM with respect to the total volume of the solution in which the composition is dispersed.
  • compositions of the present invention may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intracistemal injection or infusion techniques.
  • compositions preferably pharmaceutical compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient in some embodiments take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of formula (I) of the present invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art.
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound of formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure.
  • a composition, preferably pharmaceutical composition of the present invention may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalatory administration.
  • composition preferably pharmaceutical composition of the present invention
  • the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the composition preferably pharmaceutical composition
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol or oil.
  • composition preferably pharmaceutical composition, of the present invention may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion, or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to a compound of formula (I), one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included.
  • the liquid composition may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose
  • composition preferably pharmaceutical composition of the present invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • composition preferably pharmaceutical composition, of the present invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • a composition for rectal administration may contain an oleaginous base as a suitable non-irritating excipient.
  • bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
  • composition preferably pharmaceutical composition, of the present invention may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • composition preferably pharmaceutical composition, of the present invention may consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of formula (I) of the present invention may be delivered in single phase, bi-phasic, or tri -phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit.
  • the composition is a lipid nanoparticle.
  • the active agent is encapsulated within an aqueous interior of the lipid nanoparticle.
  • the active agent is present within one or more lipid layers of the lipid nanoparticle.
  • the active agent is bound to the exterior or interior lipid surface of a lipid nanoparticle.
  • Lipid nano particles include, but are not limited to, liposomes.
  • a liposome is a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes may have one or more lipid membranes.
  • Liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar.
  • lipid particles When complexed with nucleic acids, lipid particles may also be lipoplexes, which are composed of cationic lipid bilayers sandwiched between DNA layers.
  • the lipid nanoparticles of present invention may be formulated as a pharmaceutical composition, e.g., which further comprises a pharmaceutically acceptable diluent, excipient, or carrier, such as physiological saline or phosphate buffer, selected in accordance with the route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable diluent, excipient, or carrier such as physiological saline or phosphate buffer, selected in accordance with the route of administration and standard pharmaceutical practice.
  • lipid nanoparticles of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., normal saline will be employed as the pharmaceutically acceptable carrier.
  • suitable carriers include, e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • the carrier is preferably added following lipid particle formation.
  • the compositions can be diluted into pharmaceutically acceptable carriers such as normal saline.
  • the resulting pharmaceutical preparations may be sterilized by conventional, well known sterilization techniques.
  • the aqueous solutions can then be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the lipidic suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free -radical quenchers, such as a-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • lipid nanoparticle refers to particles having at least one dimension on the order of nanometers (e.g, 1-1,000 nm) which include one or more of the compounds of formula (I).
  • lipid nanoparticles comprising at least one compound of formula (I)) are included in a formulation that can be used to deliver a therapeutic agent, such as a nucleic acid (e.g, mRNA) to a target site of interest (e.g, cell, tissue, organ, tumor, and the like).
  • a therapeutic agent such as a nucleic acid (e.g, mRNA)
  • a target site of interest e.g, cell, tissue, organ, tumor, and the like.
  • the lipid nanoparticles comprise a compound of formula (I) and a nucleic acid.
  • the therapeutic agent such as a nucleic acid
  • the therapeutic agent may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.
  • the lipid nanoparticles have a mean diameter of from about 30 run to about 150 nm, from about 40 run to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125
  • Systemic delivery means that a useful, preferably therapeutic, amount of an active agent is exposed to most parts of the body.
  • Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.
  • Local delivery refers to delivery of an active agent directly to a target site within an organism.
  • an agent can be locally delivered by direct injection into a disease site such as a tumour, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like.
  • Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.
  • compositions of the present invention may also be administered simultaneously with, prior to, or after administration of one or more other active agents.
  • combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the present invention and one or more additional active agents, as well as administration of the composition of the present invention and each active agent in its own separate pharmaceutical dosage formulation.
  • a composition of the present invention and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • the compounds of formula (I) of the present invention and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
  • compositions preferably pharmaceutical compositions, of the present invention may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile, distilled water or other carrier so as to form a dispersion.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non- covalently interact with the compound of the disclosure so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • compositions of the present invention are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • the composition of the present invention is a pharmaceutical composition for the treatment of illness in humans.
  • the composition of the present invention is a pharmaceutical composition for the treatment of illness in mammals.
  • a polypeptide comprising proline, alanine and, optionally, serine may refer to a polypeptide that either consists of proline and alanine, or may refer to a polypeptide that consists of proline, alanine, and serine.
  • compositions comprising “a” conjugate of the invention can be interpreted as referring to a composition comprising “one or more” conjugates of the invention.
  • the term “about” preferably refers to ⁇ 10% of the indicated numerical value, more preferably to ⁇ 5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term “about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint ⁇ 10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint ⁇ 5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint.
  • the term “about” is used in connection with the endpoint of an open-ended range, it preferably refers to the corresponding range starting from the lower endpoint -10% or from the upper endpoint +10%, more preferably to the range starting from the lower endpoint -5% or from the upper endpoint +5%, and even more preferably to the open-ended range defined by the exact numerical value of the corresponding endpoint. If the term “about” is used in connection with a parameter that is quantified in integers, such as the number of amino acid residues in a protein, the numbers corresponding to ⁇ 10% or ⁇ 5% of the indicated numerical value are to be rounded to the nearest integer (using the tie-breaking rule “round half up”).
  • the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, ...” In addition thereto, this term also includes the narrower meanings of “consisting essentially of’ and “consisting of’.
  • a comprising B and C has the meaning of “A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., “A containing B, C and D” would also be encompassed), but this term also includes the meaning of “A consisting essentially of B and C” and the meaning of “A consisting of B and C” (i.e., no other components than B and C are comprised in A).
  • production or “producing”, “produces”, “produce”
  • preparation or “preparing”, “prepares”, “prepare”, respectively.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures, by practitioners of the chemical, biological and biophysical arts.
  • the “treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only).
  • the “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.
  • the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease.
  • Such a partial or complete response may be followed by a relapse.
  • a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above).
  • the treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
  • prevention of a disorder or disease is also well known in the art.
  • a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease.
  • the subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition.
  • Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators or biomarkers.
  • a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms).
  • the term “prevention” comprises the use of a conjugate of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
  • the groups (A, B, C, and D) of the compound of formula (I) may herein above and below be referred to as “A”, “B”, “C”, and “D”, also, they may herein above and below further be referred to as “group A”, “group B”, “group C”, and “group D”.
  • group A group A
  • group B group B
  • group C group C
  • group D group D
  • the compounds A-B-C-D, A-C-D, L-B-C-D, and L-C-D may either be referred to as “A-B-C-D”, “A-C-D”, “L-B-C-D,” and “C-D” or as “compound A-B-C-D”, “compound A-C-D”, “compound L-B-C-D”, and “compound L-C-D”, respectively.
  • compound A-B-C-D and compound A-C-D may separately or collectively be referred to as “a compound of formula (I)”.
  • the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments.
  • the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in the P/A peptides and the conjugates according to the invention.
  • FIG. 1 Reaction scheme for the coupling of PAS-polypeptides to di(tetradecyl)amine.
  • DIPEA non-nucleophilic base JV,JV-diisopropylethylamine
  • DCM dichloromethane
  • methanol a mixture of dichloromethane
  • Pga-PAS40 N-terminally protected PAS-polypeptide
  • TBTU 2-( I H-bcnzotriazol- l -yl)- ' ' " "- tetramethyluronium tetrafluoroborate
  • the resulting hydroxybenzotriazole active ester of the peptide subsequently reacts selectively with the single amino group of di(tetradecyl)amine while free 1- Hydroxybenzotriazole (HOBt) is released.
  • DIPEA non-nucleophilic base JV,JV-diisopropylethylamine
  • DIPEA dichloromethane
  • Figure 2 Exemplary chromatogram from the RP-HPLC purification of Pga-PAS40- di(tetradecyl)amine.
  • the flowthrough contains unreacted educts and side products, while Pga-PAS40- di(tetradecyl)amine eluted in a broad single peak during the DCM gradient.
  • Figure 3 Characterization of Pga-PAS20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 4 Characterization of Pga-PAS40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 5 Characterization of Pga-PAS100-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 6 Characterization of Pga-P/A20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 7 Characterization of Pga-P/A40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 8 Characterization of Pga-PAS100-K-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 9 Characterization of RNA encapsulation efficiency of PEG2k-, P/A-, and PAS-LNPs by agarose gel electrophoresis analysis.
  • LNPs were prepared as described in Example 3 and applied to the wells of a 1% agarose gel and run on the E-Gel power snap electrophoresis system by Thermo Fisher Scientific, as described in Example 6.
  • Lane M shows RiboRuler High Range RNAladder
  • lane 1 shows 20 pl of FLux mRNA as positive control
  • lanes 3 to 7 show Entries g to 1 from Example 3, respectively. Only in lane 1 a single clear band, corresponding to FLux mRNA, can be observed.
  • FIG. 10 Characterization of transfection efficiency of PEG2k-, P/A-, and PAS-LNPs by luciferase assay.
  • LNPs were prepared as described in Example 3 and transfected into HeLa, Jurkat, A549, HepG2, C2C12 cell lines according to the methods described in Example 8. jetMessenger and jetPEI were used as positive controls and untreated cells were used as negative control. One day after transfection, transfection efficiency was determined using the Luciferase Assay System (Promega GmbH, Walldorf, Germany). The luminescence signal was quantified with the Infinite 200 PRO multiplate reader. While transformation efficiencies varied for different cell lines, all tested LNPs showed high transformation efficiencies, in parts higher than the positive controls (jetMessenger and jetPEI).
  • Figure 11 Characterization of Pga-PAS40-di(decyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 12 Characterization of Pga-PAS40-di(dodecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 13 Characterization of Pga-PAS40-di(hexadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • Figure 14 Characterization of Pga-PAS40-di(octadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
  • FIG. 15 Reaction scheme for the coupling of PAS-polypeptides with C-terminal cysteine to di(acyl)amines.
  • DCM dichloromethane
  • methanol methanol
  • the resulting SMCC active ester of the peptide reacts selectively with the single secondary amino group of di(tetradecyl)amine while free N-hydroxysuccinimide (NHS) is released (left reaction scheme).
  • said bispecific crosslinker may first react with the single secondary amino group of di(tetradecyl)amine and only then the resulting intermediate reacts with the thiol group of the PAS-Cys polypeptide (right reaction scheme).
  • the resulting compound comprises a PAS-polypeptide linked to a di(acyl)amine via a linker (i.e., /raw.s-4-(N-malcimidylmcthyl)cyclohcxanc- 1 -carboxylate (MCC).
  • a linker i.e., /raw.s-4-(N-malcimidylmcthyl)cyclohcxanc- 1 -carboxylate (MCC).
  • Figure 16 Characterization of Pga-PAS20C-MCC-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom). Examples
  • Example 1 Coupling of di(tetradecyl)amine with P/A- and PAS-peptides via their single carboxy group
  • a 30 mM solution of the N-terminally blocked peptides Pga-P/A20-Ahx (P/A20; SEQ ID No.: 1, Almac group, London, UK), Pga-P/A40-Ahx (P/A40; SEQ ID No.: 2, Almac), Pga-PAS20 (PAS20; SEQ ID No.: 3, XL-protein, Freising, Germany), Pga-PAS40 (PAS40; SEQ ID No.: 4, XL-protein) or Pga-PASIOO (PAS 100; SEQ ID No.: 5, XL-protein) was prepared in a dichloromethane (DCM)Zmethanol mixture (5: 1).
  • DCM dichloromethane
  • the P/A- or PAS-polypeptide was dissolved in methanol alone and a solution of di(tetradecyl)amine in DCM was added.
  • a solution of di(tetradecyl)amine in DCM was added.
  • 8 volumes of a 10: 1 methanol/water mixture were added and the mixture was incubated on ice for 20 min.
  • the precipitate was removed by fdtration (Acrodisc 1 pm glass fiber syringe filter, PALL, Port Washingtom, NY, USA) and the coupling product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a VP 250/10 Nucleodur C18 Gravity column (Macherey-Nagel, Duren, Germany).
  • the mobile phase was 85% (v/v) MeOH, 10% (v/v) H2O, 5% (v/v) DCM, 0.1% (v/v) formic acid and the coupling products were eluted in a 0-100% gradient with MeOH/DCM (1: 1) ( Figure 2).
  • Pga-PAS100-di(tetradecyl)amine 70% MeOH, 30%H20, 0.1% formic acid was used as mobile phase and a gradient with 0-100% with 90% MeOH (v/v), 10% (v/v) DCM and 0. 1% (v/v) formic acid was used for the elution.
  • the elution fractions were pooled, concentrated using a SpeedVac concentrator and diluted 1: 10 in H2O before freezing and lyophilization.
  • Analytical reverse phase-HPLC runs were conducted on aResourceRPC polystyrene/divinylbenzene column (1 ml column volume, Cytiva) using the mobile phases described above for the preparative RP-HPLC runs of the respective P/A- or PAS-polypeptide-di(tetradecyl)amines.
  • the N-terminally protected Pga-PASIOO-K peptide (PAS100-K; SEQ ID No.: 6) was dissolved in 100 mM sodium bicarbonate buffer (pH 8.4) and iodoacetylated at the s-amino group of its C-terminal lysine side chain by incubation with iodoacetic acid N-hydroxy succinimide ester (CAS 39028-27-8, Apollo Scientific, Bredbury, UK) at 25 °C for 1 h. After dialysis against ultra-pure water and lyophilization, the activated PAS100-K peptide was dissolved in MeOH/DCM (1:7).
  • the 10-fold molar amount of di(tetradecyl)amine and the 2-fold molar amount of DIPEA were added and the coupling reaction was allowed to proceed for 24 h at 25 °C.
  • the addition of 8 vol. eq. of H2O/MeOH (1: 10) and incubation on ice for 20 min resulted in the precipitation of unreacted di(alkyl)amine, which was removed by filtration (Acrodisc glass fiber 1 pm syringe filter, PALL).
  • the coupling product was purified by RP-HPLC on a VP 250/10 Nucleodur C18 Gravity column (Macherey-Nagel, Duren, Germany).
  • CleanCap® Flue mRNA was obtained from TriLink BioTechnologies (San Diego, CA, USA).
  • D-Lin-MC3-DMA was obtained from MedCHemExpress (Monmouth Junction, NJ, USA), cholesterol was purchased from Merck KGaA, DSPC was obtained from NOF (White Plains, NY, USA). All cell lines were supplied by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany).
  • aqueous solution (or an aqueous phase) containing 0. 133 g/1 FLuc mRNA and 11 mM acetic acid was mixed with an ethanolic solution (or an ethanolic phase) containing 9.43 mM total lipid (50 mol% DLin- MC3-DMA, 38.5 mol% cholesterol, 10 mol% DSPC, 1.5 mol% PEG2k-compound, P/A-compound or PAS-compound) at a 3: 1 volume ratio by pipetting.
  • the crude colloidal LNP dispersions were dialyzed against phosphate-buffered saline (PBS) for 3 h (3x buffer exchange). Purified colloidal LNP dispersions (i.e., LNPs or purified colloidal dispersions) were stored at 4 °C until further use.
  • PBS phosphate-buffered saline
  • Example 4 Determination of particle diameter (Z-average), polydispersity index (PDI), and Zetapotential of PEG2k-, P/A-, and PAS-LNPs
  • Results are shown in Table 2. Measurements were conducted using a Zetasizer NanoZS from Malvern Instruments GmbH (Herrenberg, Germany). A DTS 1070 clear disposable folded capillary cell from Malvern Panalytical GmbH (Kassel, Germany) was used. For particle diameter measurement, samples were diluted 1: 10 in RNAse free water corresponding to an RNA concentration of 5 ng/pl. For Zeta-potential measurement, purified (after dialysis) and crude (before dialysis) colloidal LNP dispersions from Example 3 were diluted 1:30 in RNAse free water corresponding to an RNA concentration of 1.67 ng/pl.
  • Z-average i.e., mean particle diameter
  • polydispersity index PDI
  • average Zeta-potential values were measured before and after dialysis and calculated from the data of at least 10 runs.
  • Example 5 MTS cell viability assay using PEG2k-, P/A-, and PAS-compounds and -LNPs 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was conducted with He La cells. One day before transfection 10,000 cells were seeded into each well of a
  • 96-well plate in a volume of 100 pl of the respective medium (containing 10% fetal bovine serum (FBS) and 30 pg/ml Gentamicin) and cultured for 24 h at 37 °C and 5% CO2. On day 2, the old medium was removed and 90 pl of fresh medium were added to the cells.
  • FBS fetal bovine serum
  • Gentamicin fetal bovine serum
  • samples were adjusted to a volume of 10 pl.
  • purified colloidal LNP dispersions from Example 3 were adjusted to an mRNA concentration of 5 to 20 ng/pl using RNAse free water for dilution.
  • P/A- and PAS- compounds are less toxic towards human cells than PEG2k-compounds, when 10 pg/ml of the respective compounds are applied to HeLa cells in the MTS assay. It is, therefore, clear that the inventors surprisingly found that P/A- and PAS-compounds are superior for use as pharmaceutical component as compared to PEG2k-compounds . Viability of HeLa cells was barely affected by any tested dosages of any of the tested PEG2k-, P/A-, or PAS-LNPs. Thus, P/A- and PAS-LNPs are clearly non-toxic to human cells.
  • Example 6 Characterization of PEG2k-, P/A-, and PAS-LNPs by agarose gel electrophoresis (AGE) RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3 was characterized by agarose gel electrophoresis. Assay was carried out using the E-Gel power snap electrophoresis system by Thermo Fisher Scientific. For the evaluation of the samples a 1% agarose gel with a volume capacity of 20 pL per well was used. Results are summarized in Figure 9.
  • lane “M” contains RiboRuler High Range RNA Ladder, Thermo Fisher Scientific
  • lane 1 contains mFluc mRNA (negative control)
  • lane 2 contains mRFLuc-MC3 LNP-PEG2k-N,N- di(tetradecyl)acetamide (mRFluc-MC3 LNP l,2-Dimyristoyl-rac-glycero-3 -methoxypoly ethylengly col-2000)
  • lane 3 contains mRFLuc-MC3 LNP-PEG2k-N,N-di(tetradecyl)acetamide
  • lane 4 contains mRFLuc-MC3 LNP-Pga-P/A20- Ahx-di(tetradecyl)amine
  • lane 5 contains mRFLuc-MC3 LNP-Pga-P/A40-Ahx-di(tetradecyl)amine
  • lane 6 contains mRFLuc-MC3
  • RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3 was further characterized by RiboGreen Assay.
  • the Thermo Fischer Quant-iT RiboGreen RNA Assay Kit was used. The procedure was performed according to manufacturer’s protocol with slight adjustments. Samples were diluted to a theoretical RNA concentration of 0.4 pg/ml using either Tris-EDTA (TE)-buffer or Triton- buffer and added to a 96-well plate at a volume of 100 pl. For dissolution of LNPs in the presence of Tritonbuffer the plate was placed into an incubator for 10 minutes at 37 °C and 5% CO2. 100 pl dye solution were added to each well followed by thorough pipetting. Fluorescence signals were measured with the Infinite 200 PRO microplate reader at excitation and emission wavelengths of 480 nm and 520 nm, respectively. All samples and standards were measured in duplicates.
  • RNA encapsulation efficiency of all test P/A- and PAS-LNPs was higher as compared to PEG2k-LNPs, as is evident from Table 5.
  • P/A- and PAS-compounds are, thus, more effective at shielding LNP membranes as compared to PEG2k-LNPs.
  • Example 8 Determination of transfection efficiency of PEG2k-, P/A-, and PAS-LNPs by luciferase assay
  • Luciferase assay was conducted with several immortal cell lines (HeLa, Jurkat, C2C12, HepG2, A549). Cell lines were grown according to standard cell culture conditions.
  • HeLa, C2C12, HepG2, and A549 cells were used as adherent cells.
  • Jurkat cells were used as suspension cells.
  • 50,000 cells per well were seeded into a 96-well plate in a volume of 90 pl of RPMI1640 medium (containing 10% FBS and 30 pg/ml Gentamicin). Samples were adjusted to an mRNA concentration of 10 ng/pl using RNAse free water for dilution. 10 pl of the respective diluted samples were added to the cells equaling an amount of 100 ng mRNA per well in a total volume of 100 pl. Cells were further incubated for 24 h at 37 °C and 5% CO2. On day two, transfection efficiency was determined using the Luciferase Assay System (Promega GmbH). The luminescence signal was quantified with the Infinite 200 PRO multiplate reader.
  • P/A- and PAS-LNPs While transformation efficiencies varied for different cell lines, all tested P/A- and PAS-LNPs showed high transformation efficiencies, partly higher than the positive controls (i.e., jetMessenger and jetPEI). Remarkably, P/A20- and PAS20-LNPs showed highest transformation efficiencies among the P/A- and PAS-LNPs and even higher transformation efficiencies for A549, Jurkat, HepG2, and C2C12 cells as compared to PEG2k-LNPs. Collectively, P/A- and PAS-LNPs can efficiently transfect various human cell lines.
  • Example 9 Coupling of di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine and di(octadecyl)amine with a Pga-PAS40 peptide via its single carboxylate group
  • a 30 mM solution of the N-terminally blocked Pga-PAS40 peptide (PAS40; SEQ ID No.: 4, XL-protein) was prepared in a dichloromethane (DCM)/methanol mixture (5: 1).
  • DCM dichloromethane
  • 1.5 molar equivalents of di(decyl)amine (TCI GmbH, Eschborn, Germany) or di(dodecyl)amine (TCI GmbH) or di(hexadecyl)amine (Ambeed, Arlington Hts, IL, USA) or di(octadecyl)amine (Ambeed, Arlington Hts, IL, USA) was dissolved in the PAS40 solution, followed by addition of 2 molar equivalents of DIPEA base (N,N- diisopropylethylamine; Merck) and 1.5 molar equivalents of TBTU (2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetra
  • PAS-di(alkyl)amines were dissolved in 50 % (v/v) acetonitrile in H2O with 0.1 % (v/v) formic acid and electrospray ionization-mass spectrometry (ESI-MS) was performed using a maXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany) in the positive ion mode. All PAS-di(alkyl)amines were obtained in a uniform composition, as was evident from the presence of single peaks in all analytical RP-HPLC and ESI-MS analyses (shown in Figures 11 to 14).
  • Example 10 Coupling of di(tetradecyl)amine with a Pga-PAS20-C peptide via the thiol group of its C-terminal cysteine residue using a bispecific crosslinker
  • a 20 mM solution of the N-terminally blocked Pga-PAS20-C peptide (PAS20C; SEQ ID NO: 23, XL- protein) was prepared in a dichloromethane (DCM)/methanol mixture (5: 1).
  • PAS-di(alkyl)amines were dissolved in 50 % (v/v) acetonitrile in H2O with 0.1 % (v/v) formic acid and electrospray ionization-mass spectrometry (ESI-MS) was performed using a maXis Q-TOF instrument (Bruker Daltonics) in the positive ion mode.
  • ESI-MS electrospray ionization-mass spectrometry

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Abstract

The present invention provides for inventive means and methods for the coupling of di(alkyl)amines to polypeptides or peptides capable of forming a random coil conformation. The present invention further provides inventive compounds produced by the herein detailed methods, wherein said compounds are characterized by formula (I): A-[B-]bC-D. Said compounds comprise a polypeptide/peptide capable of forming a random coil conformation, an N-terminal protecting group, a di(alkyl)amino group, and optionally a linker. Furthermore, the present invention provides lipid nanoparticles comprising said compounds characterized by formula (I) as well as means and methods for the production of said lipid nanoparticles. Formulations comprising the inventive compounds and/or the lipid nanoparticles of the present invention are also provided. Further, the present invention also relates to uses of the inventive compounds, the lipid nanoparticles, and/or the formulations disclosed herein.

Description

Methods for the coupling of di(alkyl)amines to polypeptides
The present invention provides for inventive means and methods for the coupling of di(alkyl)amines to polypeptides or peptides capable of forming a random coil conformation. The present invention further provides inventive compounds produced by the herein detailed methods, wherein said compounds are characterized by formula (I): A-[B-]bC-D. Said compounds comprise a polypeptide/peptide capable of forming a random coil conformation, an N-terminal protecting group, a di(alkyl)amino group, and optionally a linker. Furthermore, the present invention provides lipid nanoparticles comprising said compounds characterized by formula (I) as well as means and methods for the production of said lipid nanoparticles. Formulations comprising the inventive compounds and/or the lipid nanoparticles of the present invention are also provided. Further, the present invention also relates to uses of the inventive compounds, the lipid nanoparticles, and/or the formulations disclosed herein.
Lipid formulations such as lipid nanoparticles (LNPs) are frequently used as drug delivery systems for the delivery of active ingredients and therapeutic agents. Major hurdles of drug delivery via LNPs are plasma half-life, endosomal entrapment, and stability of the LNPs. Coupling of certain polymers, in particular poly(ethylene)glycol (PEG), to the LNPs (or to lipids comprised in such LNPs) has been shown to overcome, or at least reduce these hurdles. The art has in particular provided poly(ethylene)glycol (PEG) coupled to lipids (“PEGylated lipids”) as a means to improve plasma half-life of such lipids or of LNPs.
PEGylated lipids were employed, inter alia, in mRNA vaccines against SARS-CoV-2 (including, inter alia, in known and recently employed SARS-CoV-2 vaccines from known sources). Here, more than one billion doses were administered globally (see, e.g., Ju (2022) ACS Nano 16, 11769-11780). However, vaccination with such SARS-CoV-2 mRNA vaccines has been shown to induce anti-PEG immunoglobulins (anti-PEG IgM and IgG) by up to 68.5-fold, which may lead to immunogenic reactions towards PEG polymers (i.e., PEG immunity; see, e.g., Ju; loc. cit.). It has been proposed that hospitalizations and death cases in response to SARS-CoV-2 mRNA vaccination arose due to anaphylactic reactions towards poly(ethylene)glycol (PEG) (see, e.g., Moghimi (2021) Molecular Therapy 29(3), 898-900). Moreover, PEG immunity may lead to the accelerated clearance of PEGylated therapeutics and, thus, to reduced therapeutic efficacy (see, e.g., Yang (2015) Wiley Interdiscip Rev Nanomed Nanobiotechnol.7(5), 655-677). Accordingly, there is a need for lipids and LNPs that do not induce unwanted allergic reactions as described for PEGylated lipids/LNPs. In order to overcome some of the drawbacks of PEGylation technology, certain recombinant polypeptide mimetics have been provided in the art, some of which are based on naturally occurring amino acid sequences or synthetic amino acid stretches. Most natural amino acid sequences do not behave like an ideal random coil in physiological solution, which constitutes an important characteristic of PEG, because they either tend to adopt a folded conformation (secondary structure) or, if unfolded, they usually are insoluble and form aggregates.
Novel conformationally disordered polypeptides comprising the small residues Pro, Ala, and Ser, were developed and termed “PAS” polypeptides. (Schlapschy, M., Binder, U., Borger, C., Theobald, I., Wachinger, K., Kisling, S., Haller D. & Skerra, A. (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins by retarding renal filtration. Protein Eng. Des. Sei, 26(8), 489-501; WO 2008/155134, EP-B1 2 173 890, US 8,563,521 and WO 2017/109087). This “PASylation®” technology has been previously deployed to increase the hydrodynamic volume of, inter alia, pharmaceutically active proteins, resulting in increased plasma half- life of said proteins. A similar modification of therapeutic proteins with polypeptides consisting of Pro and Ala (“P/A” polypeptides) has also been proposed (see, e.g., WO 2011/144756, EP-B1 2 571 510, US 9,221,882, and/or WO 2018/234455). P/A- and PAS-polypeptides are hydrophilic, uncharged biological polymers with biophysical properties very similar to polyethylene glycol (PEG). Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in blood plasma.
In contrast to the specific interactions with structured proteins, the properties of conformationally disordered polypeptides often pose a challenge for the immune system in the generation of cognate antibodies, a feature that is exploited by pathogens to evade the immune response (Giri (2016) Front. Cell. Infect. Microbiol. 6-144; Goh (2016) Mol. BioSyst. 12, 1881-1891). Accordingly, conformationally disordered P/A- and PAS-polypeptides lack toxicity or immunogenicity in mice and might, thus, also constitute a valid alternative for the PEGylation of lipids and LNPs.
Krishnamurthy (2019) Nanomedicine: NBM, volume: 18; pages: 169-178) expressed PAS-polypeptides at the external surface of mammalian cell membranes surrounding poly(lactic-co-glycolic acid) (PLGA), thereby creating PASylated lipid-polymer nanoparticles (“nanoghosts”). However, chemical linkage, which may be desired in certain circumstances, was not achieved herein.
Zhang (2023) Nanomedicine: NBM, volume: 47, pages 102-622) attempted chemical coupling of P/A- and PAS-polypeptides to lipids and produced liposomes comprising the same. However, the resulting liposome formulation showed high turbidity and visible precipitation of said liposomes or components thereof. Accordingly, the prior art provides no technical teaching on how P/A- and/or PAS-polypeptides could be successfully coupled to lipids. The technical problem underlying the present invention is therefore the provision of means and methods for the convenient and/or reliable coupling of P/A- and/or PAS- polypeptides with lipids.
The technical problem is solved by provision of the embodiments provided herein below and as characterized in the appended claims.
Accordingly, the present invention provides for inventive means and methods for the chemical coupling of P/A- and PAS-polypeptides to di(alkyl)amines. Namely, the present invention provides for a method for the production of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group; and b) the purification of a compound of formula (I).
As mentioned above, Krishnamurthy (2019, loc. cit.) expressed PAS-polypeptides at the external surface of mammalian cell membranes surrounding poly(lactic-co-glycolic acid) (PLGA), thereby creating PASylated lipid-polymer nanoparticles (“nanoghosts”). However, only recombinant expression of PAS- polypeptides in lipid particles was achieved and thereby no chemical (direct) coupling of P/A- and/or PAS- polypeptides to lipids is taught therein. It is conceivable that recombinant expression of PAS-polypeptides (as opposed to chemical coupling) to lipids or lipid particles increases the chances for potential cellular contaminants and/or increases the number of required purification steps. Furthermore, the PASylated nanoghosts based on PLGA nanoparticles described by Krishnamurthy (2019, loc. cit.) differ in their structure and composition considerably from typical LNPs and, resultingly, are likely not suitable for use in standard LNP -applications (such as the delivery of mRNA, siRNA, and the like as part of vaccines or therapeutics).
Zhang and colleagues (2023, loc. cit.) chemically coupled P/A- or PAS-polypeptides to lipids, resulting in the production/preparation of P/A-lipids comprising either a single C 16 or two Cl 1 hydrocarbon chains or PAS-lipids comprising a single C16 hydrocarbon chain. However, it remains obscure how Zhang (2023, loc. cit. ) performed said chemical coupling and as is evident the method employed by the authors have concrete disadvantages. Namely, PAS-lipids resulted in high turbidity and visible precipitation of liposome formulations comprising the same. Further, double-chained P/A-polypeptides did barely increase liposome blood circulation times compared to liposomes comprising no external modification. The later was likely due to reduced anchoring of the double-chained P/A-lipid and, accordingly, the authors conclude that said double-chained P/A -lipids may not be suitable for stabilizing larger lipid particles of approx. 100 nm or more in diameter. Collectively, this shows the difficulties of using P/A- and PAS-lipids in lipid particles and illustrates a need for the provision of P/A- and PAS-lipids that are suitable for the use in lipid particles.
The present invention solves these caveats by distinct means and methods involving the coupling of a di(alkyl)amine to a compound comprising a P/A- and/or PAS -polypeptide.
Theses caveats have successfully been overcome by the means and methods of the present invention: in particular and as also illustrated in the appended examples, the present invention provides for a convenient and/or reliable method for coupling of P/A- and/or PAS-polypeptides to lipids, namely di(alkyl)amines. The present invention is broadly applicable which is illustrated, inter alia, from the vast diversity of different P/A- and/or PAS-polypeptides the inventors have successfully coupled to di(alkyl)amines, demonstrating remarkable and sequence-independent coupling efficiencies using the herein provided inventive means and methods. The remarkable and advantageous purity of the compounds obtained/obtainable by the herein provided means and methods is further evident from the herein provided non-limiting illustrative figures. This is in clear contrast to the non-enabling teachings provided by Zhang (2023, loc. cit ).
The present invention also provides for inventive compounds that are obtained by or obtainable by the means and methods detailed herein. The inventive compounds are characterized by formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
The herein provided compounds comprising di(alkyl)amines may be, inter alia, less prone to cleavage as compared to e.g., similar compounds comprising phospholipids instead of di(alkyl)amines. Consequently, anchoring of these compounds in, inter alia, LNPs may be advantageously high, as will be further detailed herein below. Herein, we provide polypeptides capable of forming a random coil conformation (P/A- or PAS-polypeptides) coupled to di(alkyl)amines. These inventive conjugates are compared to PEGylates di(alkyl)amines in the appended examples. As is evident from the appended non-limiting examples, the herein provided inventive compounds/inventive conjugates are, inter alia, even advantageous as compared to herein tested reference PEGylated di(alkyl)amines. Namely, the herein provided compounds, inter alia, show advantageous effects on cell viability parameters as compared to said reference PEGylated lipids, as illustrated in the appended examples.
The herein provided compounds comprising di(alkyl)amines and P/A- and/or PAS-polypeptides are not foreshadowed in the prior art. WO 2011/144756 suggested that P/A-polypeptides may be coupled to lipid vesicles, however, did provide a detailed technical teaching regarding concrete means and methods how to couple P/A- and/or PAS-polypeptides to lipids or lipid vesicles. Similarly, lipid nanoparticles (LNPs) comprising the herein provided compounds have not been foreshadowed.
Accordingly, the present invention further provides for a method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), wherein the method comprises the steps of a) the provision of the compound of formula (I) in an ethanolic solution; b) the mixing of said ethanolic solution with an aqueous solution, thereby preparing/producing a lipid nanoparticle; and c), optionally the dialysis of said lipid nanoparticle. Accordingly, also lipid nanoparticles obtained/obtainable by the means and methods detailed herein and/or lipid nanoparticles that comprise the inventive compound of formula (I), are provided herein. Further, also lipid nanoparticle colloidal dispersions comprising the lipid nanoparticles of the present invention are herein provided. In the context of the present invention, a lipid nanoparticle colloidal dispersion refers to a suspension comprising the lipid nanoparticles of the present invention and/or refers to a suspension comprising lipid nanoparticles comprising the inventive compound characterized by formula (I).
The herein provided lipid nanoparticles and lipid nanoparticle dispersions are highly stable, as can been seen from the lack of turbidity and visible precipitation, as compared to the lipid particles provided by Zhang (2023, loc. ci ). Further, the herein provided lipid nanoparticles may show advantageous characteristics, inter alia, with regard to their RNA encapsulation efficiency and LNP shielding capacities as compared to LNPs comprising a reference PEGylated compound. This is, inter alia, evident from the herein provided examples.
Moreover, the present invention provides for means, methods, and uses for the production/preparation of pharmaceutical and non-pharmaceutical formulations comprising the inventive compound of formula (I) and/or the lipid nanoparticle or the lipid nanoparticle colloidal dispersion of the present invention. Furthermore, uses of the pharmaceutical and non-pharmaceutical formulations comprising the herein provided lipid nanoparticles, the herein provided lipid nanoparticle colloidal dispersion, and/or the herein provided inventive compounds of formula (I) are disclosed herein. The inventive compound of formula (I) provided in accordance with the present invention and the method for the production/preparation of said compound will be described in greater detail in the following. This detailed description relates to and is applicable to all aspects of the present invention, including not only the compound or the method for the production/preparation of said compound as such but also to the lipid nanoparticles, the lipid nanoparticle suspensions, the pharmaceutical formulations, and the nonpharmaceutical formulations comprising said compound. This detailed description further relates to any uses of the compound the lipid nanoparticles, the lipid nanoparticle suspensions, the pharmaceutical formulations, and the non-pharmaceutical formulations comprising said compound, as well as methods for their production/preparation and methods using the same.
The present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group.
In line with the above, the compound of formula (I) can be a compound of the formula A-C-D if b is 0, or a compound of formula A-B-C-D if b is 1.
In the above formula (I), A is a di(alkyl)amino group.
The compound according to the present invention comprises a di(alkyl)amino group (group A). Accordingly, the herein detailed means and methods refer to the coupling of a di(alkyl)amine to either compound L-B-C-D or compound L-C-D resultingly preparing/producing a di(alkyl)amino group either coupled to group B or group C in compounds A-B-C-D or A-C-D, respectively. In other words, the present invention provides for P/A- and/or PAS-polypeptides coupled to di(alkyl)amino groups.
The skilled person is aware, that di(alkyl)amino groups comprise two alkyl chains linked by a nitrogen atom, wherein the nitrogen forms a tertiary amine further linking the di(alkyl)amino group to group B or group C in compounds A-B-C-D or A-C-D, respectively. Accordingly, group A comprises two alkyl chains, and said alkyl chains are independently a linear or branched alkyl chain. To that extent, group A in formula (I) can be illustrated by the formula -N(RA1)2, wherein the two groups RA1 linked to the nitrogen atom are independently a linear or branched alkyl chain.
Preferably, both alkyl chains are linear. Further, the two alkyl chains may independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
In a preferred embodiment, group A comprises two linear alkyl chains that each comprise 14 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(tetradecyl)amino group. It will be understood, that for example, when group A is a di(tetradecyl)amino group, the di(alkyl)amine to be coupled to either compound L-B-C-D or compound L-C-D is a di(tetradecyl)amine.
The enclosed Examples illustratively demonstrate that in the context of the herein provided means and methods, group A is not particularly limited. For example, Examples 1 and 2 illustrate the efficient coupling of di(tetradecyl)amine with P/A- and PAS-peptides and Example 9 illustrates that di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine and di(octadecyl)amine may be coupled efficiently to e.g., PAS peptides. Accordingly, the two alkyl chains comprised in group A may independently comprise about 8 to about 20 carbon atoms, preferably about 10 to 18, more preferably about 10, about 12, about 14, about 16 or about 18 carbon atoms.
In another preferred embodiment, group A comprises two linear alkyl chains that each comprise 10 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(decyl)amino group.
In another preferred embodiment, group A comprises two linear alkyl chains that each comprise 12 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(dodecyl)amino group.
In another preferred embodiment, group A comprises two linear alkyl chains that each comprise 16 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(hexadecyl)amino group.
In another preferred embodiment, group A comprises two linear alkyl chains that each comprise 18 carbon atoms. Accordingly, as also illustrated in the appended examples and as provided as a specific embodiment, group A may be in particular a di(octadecyl)amino group. In the context of the present invention, instead of di(alkyl)amines, also di(alkenyl)amines may be coupled to compound L-B-C-D or compound L-C-D preparing/producing a di(alkenyl)amino group either coupled to group B or group C in compounds A’-B-C-D or A’-C-D, respectively. Accordingly, the present invention also provides for a method for the production/preparation of a compound of the formula (II):
A’-[B-]bC-D (II) wherein A’ is a di(alkenyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkenyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (II), wherein L is a leaving group; and b) the purification of a compound of formula (II).
Accordingly, the present invention also provides for inventive compounds characterized by the formula (II):
A’-[B-]bC-D (II) wherein A’ is a di(alkenyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group. To that extent, group A’ in formula (II) can be illustrated by the formula - N(RA2)2, wherein the two groups RA2 linked to the nitrogen atom are independently a linear or branched alkenyl chain. Preferably, two alkenyl chains of the di(alkenyl)amine and/or of group A’ may independently comprise about 8 to about 20 carbon atoms. Preferably, two alkenyl chains of the di(alkenyl)amine and/or of group A’ may each comprise about 1 or about 2 double bonds.
In the context of the present invention, instead of di(alkyl)amines, also di(alkynyl)amines may be coupled to compound L-B-C-D or compound L-C-D preparing/producing a di(alkynyl)amino group either coupled to group B or group C in compounds A”-B-C-D or A”-C-D, respectively. Accordingly, the present invention also provides for a method for the production/preparation of a compound of the formula (III):
A”-[B-]bC-D (III) wherein A” is a di(alkynyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkynyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (II), wherein L is a leaving group; and b) the purification of a compound of formula (II).
Accordingly, the present invention also provides for inventive compounds characterized by the formula (III):
A”-[B-]bC-D (III) wherein A” is a di(alkynyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group. To that extent, group A” in formula (III) can be illustrated by the formula -N(RA3)2, wherein the two groups RA3 linked to the nitrogen atom are independently a linear or branched alkynyl chain. Preferably, two alkynyl chains of the di(alkynyl)amine and/or of group A” may independently comprise about 8 to about 20 carbon atoms. Preferably, two alkenyl chains of the di(alkynyl)amine and/or of group A” may each comprise about 1 or about 2 triple bonds.
The herein provided definition of group A applies mutatis mutandis for all means and methods of the present invention but also for all the herein provided inventive compounds.
The present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group.
In the above formula (I), B is an optional linker. The inventive compound may comprise an optional linker. Accordingly, group B is either present or absent. It is understood that if group B is absent, group A is directly linked to group C and if group B is present, group A and group C are linked via said linker (i.e., group B), resulting in compound A-C-D or A-B-C-D, respectively. The terms “linker”, “linker group” may be used interchangeably within the context of the present invention. It is understood that ifb = 0, B is absent. Accordingly, ifb = 1, B is present.
The skilled person is aware of suitable linkers/linker groups that may be deployed in the context of the present invention. In the following, exemplary and non-limiting examples of such linkers/linker groups are provided.
Group B may comprise one or more amino acid residues, wherein said one or more amino acid residues may be independently natural or unnatural amino acid(s). Accordingly, group B may comprise a carboxy and an amino terminus (i.e., C- and N-terminus, respectively). Further, group B comprises at least two carbon atoms between the amino group and the carboxy group of group B may provide a distance of at least two carbon atoms between the amino group and the carboxy group of group B (which is the case if, e.g., group B is an co-amino-Cs-is alkanoic acid, such as s-aminohcxanoic acid), or they may provide a distance of only one carbon atom between the amino group and the carboxy group of group B (which is the case if, e.g., group B is alanine). Since group B can comprise one or more amino acid residues, which again can be natural or unnatural amino acid(s), group B may be for example -HN-(C2-i2 hydrocarbyl)-C(O)-, wherein optionally one or more -CH2- units in the hydrocarbyl moiety comprised in said -HN-(C2-i2 hydrocarbyl)- C(O)- are each replaced by a group independently selected from -O-, -S-, -NH- and -N(CI-4 alkyl)-, and further wherein optionally one or more =CH- units (if present) in the hydrocarbyl moiety comprised in said -HN-(C2-i2 hydrocarbyl)-C(O)- are each replaced by =N-. The hydrocarbyl moiety comprised in said -HN- (C2-12 hydrocarbyl)-C(O)- may be, e.g., an alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl, or any combination thereof (e.g., an alkaryl or an aralkyl, such as benzyl, phenethyl, or methylphenyl). Moreover, said hydrocarbyl moiety may have 3 to 10 carbon atoms, and preferably 4 to 8 carbon atoms Furthermore the two points of attachment on the aforementioned cyclic hydrocarbyl groups (such as said aryl or said cycloalkyl; including also any of the specific cyclic groups referred to in the following, such as the phenyl comprised in the -HN-(CH2)o-2-phenyl-(CH2)o-2-C(0)- referred to in the subsequent paragraph) may neither be on the same ring carbon atom nor on adjacent ring carbon atoms; if such a cyclic group has six ring atoms (as in phenyl or cyclohexyl), a 1,4-attachment (para) or a 1,3 -attachment (meta) is preferred, and a 1,4-attachment is particularly preferred. Moreover, it is preferred that no -CH2- units and no =CH- units (if present) in the hydrocarbyl moiety comprised in said -HN-(C2-i2 hydrocarbyl)-C(O)- are replaced by the above-mentioned hetero groups (i.e., no -CH2- units are replaced by -O-, -S-, -NH- or -N(CI-4 alkyl)-, and no =CH- units, if present, are replaced by =N-). Accordingly, group B is preferably -HN-(C2-i2 hydrocarbyl)-C(O)-. If group B provides an amino terminus, such as the group -NH- in -HN-(C2-i2 hydrocarbyl)-C(O)-, the amino terminus typically forms a bond to the polypeptide C. If group B provides a carboxy terminus, such as the group -C(O)- in -HN-(C2-i2 hydrocarbyl)-C(O)-, the carboxy terminus typically forms a bond to the di(alkyl)amino group A. Thus, it will be understood by the skilled person that the di(alkyl)amino group A and the linker B may form an amide.
Accordingly, group B may be selected from the group consisting of -HN-(C2-i2 alkyl)-C(O)-, -HN-(CH2)o 2- phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-. Therefore, group B may be selected from -HN-CH2-(Ci-n alkyl)-C(O)-, -HN-(Ci-n alkyl)-CH2-C(O)-, -HN-(CH2)o-2-phenyl-(CH2)o-2- C(O)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-. Accordingly, group B may be selected from - HN-CH2CH2-C(O)-, -HN-CH2CH2-(CI.IO alkyl)-C(O)-, -HN-(Cnio alkyl)-CH2CH2-C(O)-, -HN-(CH2)o-2- phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o 2-(C3-s cycloalkyl)-(CH2)o-2-C(0)-. Thus, group B may be, e.g., selected from -HN-(CH2)2 i2-C(O)-, -HN-(CH2)o-2-phenyl-(CH2)o-2-C(0)-, and -HN-(CH2)o-2-cyclohexyl- (CH2)O-2-C(0)-. Accordingly, group B may be selected from -HN-(CH2)3 io-C(0)-, -HN-phenyl-C(O)-, and -HN-cyclohexyl-C(O)-.
As shown in the appended examples, group B may be selected from -HN-(CH2)4-C(O)-, -HN-(CH2)5-C(O)-
-HN-(CH2)6-C(O)-, -HN-(CH2)7-C(O)-, -HN-(CH2)8-C(O)-,
Figure imgf000012_0001
Accordingly, group B may be selected from the group consisting of s-aminohcxanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, para-aminobenzoic acid, and paraaminocyclohexanecarboxylic acid (i.e., 4-aminocyclohexanecarboxylic acid).
The use of a natural amino acid (that comprises at least two carbon atoms between its amino group and its carboxy group) as group B, particularly a standard proteinogenic a-amino acid such as alanine or proline, can also be advantageous since such amino acids are considered to be safe and well tolerated. Accordingly, group B may also be a standard proteinogenic a-amino acid comprising at least two carbon atoms between its amino group and its carboxy group, particularly alanine or proline.
Thus, group B may also be selected, e.g., from alanine (e.g., L-alanine or D-alanine), proline (e.g., L-proline), P-alanine, y-aminobutyric acid (GABA), 5-aminovaleric acid (Ava), s-aminohcxanoic acid (Ahx), 7-aminoheptanoic acid, 8-aminooctanoic acid (Aoa), 9-aminononanoic acid, para-aminobenzoic acid (Abz), para-aminocyclohexanecarboxylic acid (ACHA; e.g., cis-ACHA or trans-ACHA), and para- (aminomethyl)cyclohexanecarboxylic acid (AMCHA; e.g. cis-AMCHA or trans-AMCHA). Alternatively, group B may be an L-lysine residue providing an s -amino group in its side chain that is linked via a CO-(Ci-8 hydrocarbyl) group to the N-atom of the di(alkyl)amino group (group A). Such a linker group B may be obtained/obtainable, for example, by reacting the lysine side chain with iodoacetic acid N- hydroxysuccinimide ester, followed by coupling with group A. Instead of iodoacetic acid, other activated iodo-carboxylic acids may be used, for example iodopropionic acid, iodobutyric acid, iodovaleric acid, iodocaproic acid, always in their activated form, for example as N-hydroxy succinimide (NHS) ester. Instead of iodoacetic acid or other activated iodo-carboxylic acids, also activated bromo-carboxylic acids or activated chloro-carboxylic acids (e.g. bromoacetic acid-NHS ester and chloroacetic acid-NHS ester) may be used.
Alternatively, group B may comprise a cysteine residue providing a thiol group in its side chain that may be linked to a di(alkyl)amine. The present inventors have surprisingly identified means and methods for the coupling of PA-/PAS-peptides each comprising a cysteine residue to di(alkyl)amines. This is illustratively shown in Example 10. The underlying reaction mechanism is illustrated in Figure 14 and the purity of the resulting compounds is shown in Figure 15. The present inventors could surprisingly couple the thiol group of said cysteine residue to di(alkyl)amines (see, Example 10) by employing abispecific crosslinker. In other words, and as detailed herein, the present inventors could surprisingly a compound of formula L-B-C-D to a compound A using an active ester. Accordingly, in the context of the present invention bispecific crosslinkers are active esters.
In brief, Example 10 illustrates that an organic solvent comprising dichloromethane and methanol and further comprising a bispecific crosslinker (e.g., siiccinimidyl-/ram-4-(N-malcimidylmcthyl)cyclohcxanc- 1 -carboxylate (SMCC)) can be employed in coupling a compound L-B-C-D comprising a Cys residue in B (e.g., an N-terminally protected PAS-Cys polypeptide) to a di(alkyl)amino group (e.g., to di(tetradecyl)amine), resulting in the formation of a compound A-B-C-D (e.g., PAS(20)C-linker- di(tetradecyl)amine) .
Accordingly, in an embodiment of the present invention, it is further envisaged that the herein provided means and methods may further comprise the coupling of a compound of formula L-B-C-D to a compound A using a bispecific crosslinker. In other words, the herein provided methods may further comprise a bispecific crosslinker for the preparation of a compound of the formula (I). Bispecific crosslinkers may be selected from the group consisting of succinimidyl-tra«5-4-(N-maleimidylmethyl)cyclohexane-l- carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP), N-(y- maleimidobutyryloxy)succinimide ester (GMBS), succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane- 1 -carboxylate (SIAC), succinimidyl-6-((((4- (iodoacetyl)amino)methyl)cyclohexane- 1 -carbonyl)amino)hexanoate (SIACX), p-nitrophenyl iodoacetate (NPIA), and iodoacetic acid N-hydroxysuccinimide ester (or one of its analogs), preferably SMCC.
When employing a bispecific crosslinker such as SMCC, in the coupling of a Cys residue to another molecule, the bispecific crosslinker specifically reacts with the thiol group of said Cys residue (such as via a Michael-addition). The skilled person is aware that the hydrogen (i.e., H-) comprised in said thiol group will be comprised in the bispecific crosslinker or parts thereof subsequent to the coupling reaction.
Accordingly, without being bound by theory, the leaving group L comprised in the to-be-coupled compound L-B-C-D (with B comprising a Cys residue) may be the H- comprised in the thiol group of said Cys residue, which H- may subsequently, after the coupling reaction, be comprised in said bispecific crosslinker or parts thereof.
Said bispecific crosslinker may be comprised in (an) organic solvent(s). As mentioned above, said organic solvent(s) may comprise dichloromethane and methanol. If said organic solvent(s) comprises a bispecific crosslinker, it may comprise about 0.5 to about 10 molar equivalents of said bispecific crosslinker relative to compound A, preferably about 0.5 to about 5 molar equivalents, preferably about 1 to about 5 molar equivalents, preferably about 1 to about 4 molar equivalents, preferably about 1 to about 3 molar equivalents, preferably about 1 to about 2 molar equivalents, more preferably about 1 molar equivalent.
The skilled person is aware that when employing bispecific crosslinkers in the coupling of two molecules, said bispecific crosslinker or parts thereof may be comprised in the resulting compound. Accordingly, when coupling a compound L-B-C-D with a compound A, said bispecific crosslinker or parts thereof may be comprised in the resulting compound of A-B-C-D. Such parts of the bispecific crosslinker may be considered to form part of the resulting (then modified) linker B. The skilled person can readily determine which parts of said bispecific crosslinker may be comprised in the resulting compound and which parts of said bispecific crosslinker may be removed during the coupling reaction. Parts of the above-mentioned cross-linkers (such as SMCC) that are removed during the coupling reaction may for example be N- hydroxysuccinimide (NHS) or nitrophenol. This is also illustrated in, e.g., Bioconjugate Techniques, G. Hermanson, 3rd Edition (2013), which is hereby incorporated by reference in its entirety.
As illustratively shown in Figure 15, the bispecific crosslinker (e.g., SMCC) may either in a first reaction step react with the compound L-B-C-D (e.g., a PAS(20)C peptide; see left reaction scheme illustrated in Figure 15) or with the compound A (e.g., di(tetradecyl)amine; see right reaction scheme illustrated in Figure 15), each resulting in a different intermediate reaction product. In a second reaction step, said intermediate reaction products may react with compound A or compound L-B-C-D, respectively. Accordingly, in the context of the present invention it is further envisaged that said intermediate reaction products may be isolated and subsequently employed in the herein provided means and methods. Accordingly, in one embodiment the present invention provides for a method for the preparation of a compound of the formula (I):
A-B-C-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, preferably wherein B comprises a Cys residue, wherein B was previously reacted with a bispecific crosslinker, thereby coupling said bispecific crosslinker or parts thereof to B,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D (or a derivate thereof) to yield a compound of formula (I), wherein L is a leaving group (such as N-hydroxysuccinimide); and b) the purification of a compound of formula (I), wherein said compound of formula (I) comprises said bispecific crosslinker or parts thereof.
In another embodiment the present invention provides for a method for the preparation of a compound of the formula (I):
A-B-C-D (I) wherein
A is a di(alkyl)amino group, wherein A was previously reacted with a bispecific crosslinker, thereby coupling said bispecific crosslinker or parts thereof to A,
B is a linker, preferably wherein B comprises a Cys residue,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine (or a derivate thereof) to a compound L-B-C-D to yield a compound of formula (I), wherein L is a leaving group (such as H- comprised in the thiol group of said Cys residue); and b) the purification of a compound of formula (I), wherein said compound of formula (I) comprises said bispecific crosslinker or parts thereof.
In the context of compound L-B-C-D or compound A that were previously reacted with a bispecific crosslinker, the term “or a derivate thereof’ refers to the above-mentioned intermediate reaction product. Such intermediate reaction products may also be considered as activated compound L-B-C-D or activated compound A.
Illustrative and non-limiting examples of group B are comprised in SEQ IDs NO: 1, 2, and 6 and illustrated in the appended Examples.
As mentioned herein above, group B may be absent, if b is 0. Accordingly, in one aspect the present invention b is 0 (and accordingly, B is absent).
The herein provided definition of group B applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
The present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group. In the above formula (I), C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.
Group C, which is comprised in the compound of formula (I), is a polypeptide or peptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of either alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.
The term "random coil" as used herein relates generally to any conformation of a polymeric molecule, including amino acid polymers/amino acid sequences/polypeptides, in which the individual monomeric elements that form said polymeric structure are essentially randomly oriented towards the adjacent monomeric elements while still being chemically bound to said adjacent monomeric elements. In particular, a polypeptide, amino acid sequence or amino acid polymer adopting/having/forming/capable of forming a "random coil conformation" substantially lacks a defined secondary and tertiary structure. Accordingly, in the context of the present invention, polypeptides capable of forming a random coil are conformationally disordered polypeptides and are also herein also interchangeably referred to as “random coil polypeptides”.
The nature of polypeptides forming a random coil conformation and their methods of experimental identification are known to the person skilled in the art and have been described in the scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., W. H. Freeman and Company, New York; Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., W. H. Freeman and Company, New York; Smith (1996) Fold Des TR95-R106). Such polypeptides are particularly capable of forming a random coil conformation when present in an aqueous environment (e.g., an aqueous solution or an aqueous buffer). The presence of a random coil conformation can be determined using methods known in the art, in particular by means of spectroscopic techniques such as circular dichroism (CD) spectroscopy. CD spectroscopy represents a light absorption spectroscopy method in which the difference in absorbance of right- and left- circularly polarized light by a substance is measured. The secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with a wavelength between approximately 190 and 250 nm. At these wavelengths, the different secondary structures commonly found in polypeptides can be analyzed, since a-helix, parallel and anti-parallel B-sheet, and random coil conformations each give rise to a characteristic shape and magnitude of the CD spectrum. Accordingly, by using CD spectrometry the skilled artisan is readily capable of determining whether polypeptide (or segment thereof) forms/adopts random coil conformation in aqueous solution or at physiological conditions. Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectrometry, infrared and Raman spectroscopy, measurement of the hydrodynamic volume via size exclusion chromatography, analytical ultracentrifugation or dynamic/static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity (Cantor (1980) loc. cit , Creighton (1993) loc. cit , Smith (1996) loc. cit.). In addition to the experimental methods detailed above, theoretical methods for the prediction of secondary structures in proteins have been described. One example of such a theoretical method is the Chou-Fasman method (Chou and Fasman, loc.cit.) which is based on an analysis of the relative frequencies of each amino acid in a-helices, B-sheets, and turns based on known protein structures solved, for example, with X-ray crystallography. However, theoretical prediction of protein secondary structure is known to be unreliable. As exemplified herein below, amino acid sequences expected to adopt an a-helical secondary structure according to the Chou-Fasman method were experimentally found to form a random coil. Accordingly, theoretical methods such as the Chou-Fasman algorithm may only have limited predictive value whether a given polypeptide adopts random coil conformation, as also illustrated in the appended examples and figures. Nonetheless, the above described theoretical prediction is often the first approach in the evaluation of a putative secondary structure of a given polypeptide/amino acid sequence. A theoretical prediction of a random coil structure also often indicates that it might be worthwhile verifying by the above experimental means whether a given polypeptide/amino acid sequence has indeed a random coil conformation.
The skilled person is aware that the encoded amino acid sequence/polypeptide may also form random coil conformation when other residues than proline, alanine and, optionally, serine are comprised as a minor constituent in said amino acid sequence/polypeptide. The term "minor constituent" as used herein means that maximally 5 mol% or maximally 10 mol% amino acid residues are different from proline, alanine, or serine in the encoded random coil polypeptides of this invention. This means that maximally 10 of 100 amino acids may be different from proline, alanine and, optionally, serine, preferably maximally 8 mol%, i.e. maximally 8 of 100 amino acids may be different from proline, alanine and, optionally, serine, more preferably maximally 6 mol%, i.e. maximally 6 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more preferably maximally 5 mol%, i.e. maximally 5 of 100 amino acids may be different from proline, alanine and, optionally, serine, particularly preferably maximally 4 mol%, i.e. maximally 4 of 100 amino acids may be different from proline, alanine and, optionally, serine, more particularly preferably maximally 3 mol%, i.e. maximally 3 of 100 amino acids may be different from proline, alanine and, optionally, serine, even more particularly preferably maximally 2 mol%, i.e. maximally 2 of 100 amino acids may be different from proline, alanine and, optionally, serine and most preferably maximally 1 mol%, i.e. maximally 1 of 100 of the amino acids that are comprised in the random coil polypeptide may be different from proline, alanine and, optionally, serine. Said amino acids different from proline, alanine and, optionally, serine may be selected from the group consisting of Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai, including posttranslationally modified amino acids or non-natural amino acids (see, e.g., Budisa (2004) Angew Chem Int Ed Engl 43:6426-6463; Young (2010) J Biol Chem 285: 11039-11044; Liu (2010) Annu Rev Biochem 79:413-444; Wagner (1983) AngewChem Int Ed Engl 22:816-828; Walsh (2010) Drug Discov Today 15: 773-780. In certain cases P/A -polypeptides can also comprise Ser as a minor constituent. For example, in case the encoded random coil polypeptide consists of proline and alanine, serine can also be considered as minor constituent.
Generally, it is preferred herein that these “minor” amino acids (other than proline, alanine and, optionally, serine) are not present in the encoded random coil polypeptide as described herein or the encoded random coil polypeptide as part/fragment of a fusion protein. In accordance with the invention, the encoded random coil polypeptide / amino acid sequence may, in particular, consist exclusively of proline, alanine and, optionally, serine residues (i.e., no other amino acid residues are present in the encoded random coil polypeptide or in the amino acid sequence).
In one embodiment, group C comprises an amino acid sequence consisting of alanine and proline residues (P and A). Accordingly, in the context of the present invention, polypeptides (such as group C in this specific embodiment) comprising an amino acid sequence consisting of alanine and proline residues may be referred to as “P/A-polypeptides” or simply as “P/A”.
Preferably, P/A (or group C, in this specific embodiment) is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from proline and alanine residues. Preferably, P/A includes at least one proline residue and at least one alanine residue.
More preferably, in P/A (or in group C, in this specific embodiment) the proportion of the number of proline residues comprised in the P/A to the total number of amino acid residues comprised in P/A is preferably >10 mol% and <70 mol%, more preferably >20 mol% and <50 mol%, and even more preferably >25 mol% and <40 mol%. Accordingly, it is preferred that 10 mol% to 70 mol% of the total number of amino acid residues in P/A are proline residues; more preferably, 20 mol% to 50 mol% of the total number of amino acid residues comprised in P/A are proline residues; and even more preferably, 25 mol% to 40 mol% (e.g., 25 mol%, 30 mol%, 35 mol% or 40 mol%) of the total number of amino acid residues comprised in P/A are proline residues. Moreover, it is preferred that P/A does not contain any consecutive proline residues (i.e., that it does not contain any partial PP sequence or multiples thereof). Further, it is preferred that P/A (or group C, in this specific embodiment) comprises no more than 6 identical consecutive amino acid residues (i.e., that it does not, inter alia, contain any partial AAAAAA sequence or multiples thereof, wherein “A” refers to an alanine residue in the context and “AAAAAA” refers to 6 consecutive alanine residues).
As detailed herein above, it is furthermore preferred that at least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, yet even more preferably at least 99 mol%, and most preferably 100 mol% of the number of amino acid residues in P/A (or group C, in this specific embodiment) are independently selected from proline and alanine. The remaining amino acid residues in P/A are preferably selected from the 20 standard proteinogenic a-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine. Accordingly, it is preferred that P/A is composed of proline, alanine, glycine, and serine residues (wherein less than 10 mol%, preferably less than 5 mol%, of the number of amino acid residues in P/A are glycine or serine residues), and it is most preferred that P/A is composed of proline and alanine residues, i.e. consists solely of proline and alanine residues. It will be understood that, as specified above, P/A includes at least one proline residue and at least one alanine residue.
The number of amino acid residues that P/A (or group C, in this specific embodiment) is composed of is preferably about 10 to about 300 amino acid residues, more preferably about 10 to about 250 amino acid residues, more preferably about 10 to about 200 amino acid residues, even more preferably about 15 to about 150 amino acid residues, more preferably about 10 to about 140 amino acid residues, even more preferably about 10 to about 130 amino acid residues, even more preferably about 15 to about 120 amino acid residues, even more preferably about 15 to about 110 amino acid residues, and yet even more preferably about 20 to about 100 amino acid residues. P/A sequences comprising about 20, about 40 or about 100 amino acid residues may be even more preferred.
Examples of preferred P/A amino acid sequences include, in particular, such amino acid sequences that comprise (or, more preferably, that consist of): (i) the sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 9; also referred to as “P/A#l”), or (ii) the sequence AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10; also referred to as “P/A#2”), or (iii) the sequence APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 13; also referred to as “P/A#5”), or (iv) a fragment of any of these sequences, or (v) a combination of two or more of these sequences (which may be the same or different, i.e., any combination of two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the sequences P/A#l, P/A#2 and/or P/A#5; a corresponding example is a dimer of P/A# 1 (“P/A#l- P/A#l”); further examples include P/A#l-P/A#2, P/A#l-P/A#5, P/A#2-P/A#l, P/A#2-P/A#2, P/A#2-P/A#5, P/A#5-P/A#l, P/A#5-P/A#2, P/A#5-P/A#5, P/A# 1 -P/A# 1 -P/A# 1, P/A#l-P/A#l-P/A#2, P/A#l-P/A#l-P/A#5, P/A#l-P/A#2-P/A#l, P/A#l-P/A#2- P/A#2, P/A#l-P/A#2-P/A#5, P/A#l-P/A#5-P/A#l, P/A#l-P/A#5-P/A#2, P/A#l-P/A#5-P/A#5, P/A#2- P/A#l-P/A#l, P/A#2-P/A#l-P/A#2, P/A#2-P/A#l-P/A#5, P/A#2-P/A#2-P/A#l, P/A#2-P/A#2-P/A#2, P/A#2-P/A#2-P/A#5, P/A#2-P/A#5-P/A#l, P/A#2-P/A#5-P/A#2, P/A#2-P/A#5-P/A#5, P/A#5-P/A#l- P/A#l, P/A#5-P/A#l-P/A#2, P/A#5-P/A#l-P/A#5, P/A#5-P/A#2-P/A#l, P/A#5-P/A#2-P/A#2, P/A#5- P/A#2-P/A#5, P/A#5-P/A#5-P/A#l, P/A#5-P/A#5-P/A#2, or P/A#5-P/A#5-P/A#5).
In another embodiment, group C comprises an amino acid sequence consisting of alanine, proline, and serine residues (P, A, and S). Accordingly, in the context of the present invention, polypeptides (such as group C in this specific embodiment) comprising an amino acid sequence consisting of alanine, proline, and serine residues may be referred to as “PAS-polypeptides” or simply as “PAS”.
Preferably, PAS (or group C, in this specific embodiment) is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from alanine, proline, and serine residues. Preferably, PAS includes at least one proline residue and at least one alanine residue.
More preferably, in PAS (or in group C, in this specific embodiment) the encoded amino acid sequence comprises more than about 4 mol%, preferably more than about 6 mol%, more preferably more than about 10 mol%, more preferably more than about 15 mol%, more preferably more than about 20 mol%, more preferably more than about 22 mol%, 23 mol% or 24 mol%, more preferably more than about 26 mol%, 29 mol%, or 30 mol%, more preferably more than about 31 mol%, 32 mol%, 33 mol%, 34 mol% or 35 mol% and most preferably more than about 25 mol% proline residues. The encoded amino acid sequence preferably comprises less than about 40 mol%, more preferably less than 38 mol%, 35 mol%, 30 mol%, 26 mol% proline residues, wherein the lower values are preferred. Moreover, it is preferred that PAS does not contain any consecutive proline residues (i.e., that it does not contain any partial PP sequence or multiples thereof). Further, it is preferred that PAS (or group C, in this specific embodiment) comprises no more than 6 identical consecutive amino acid residues (i.e., that it does not, inter alia, contain any partial AAAAAA sequence or multiples thereof, wherein “A” refers to an alanine residue in the context and “AAAAAA” refers to 6 consecutive alanine residues).
As detailed herein above, it is furthermore preferred that at least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, yet even more preferably at least 99 mol%, and most preferably 100 mol% of the number of amino acid residues in PAS (or group C, in this specific embodiment) are independently selected from proline, alanine, and serine. The remaining amino acid residues in PAS are preferably selected from the 20 standard proteinogenic a-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine. Accordingly, it is preferred that PAS is composed of proline, alanine, glycine, and serine residues (wherein less than 10 mol%, preferably less than 5 mol%, of the number of amino acid residues in PAS are glycine or serine residues), and it is most preferred that PAS is composed of proline and alanine residues, i.e., consists solely of proline, alanine, and serine residues. It will be understood that, as specified above, PAS includes at least one proline residue, at least one alanine residue, and at least one serine residue.
The number of amino acid residues that PAS (or group C, in this specific embodiment) is composed of is preferably about 10 to about 300 amino acid residues, more preferably about 10 to about 250 amino acid residues, more preferably about 10 to about 200 amino acid residues, even more preferably about 15 to about 150 amino acid residues, more preferably about 10 to about 140 amino acid residues, even more preferably about 10 to about 130 amino acid residues, even more preferably about 15 to about 120 amino acid residues, even more preferably about 15 to about 110 amino acid residues, and yet even more preferably about 20 to about 100 amino acid residues. PAS sequences comprising about 20, about 40 or about 100 amino acid residues may be even more preferred.
Non-limiting examples of preferred PAS amino acid sequences include, in particular, such amino acid sequences that comprise (or, more preferably, that consist of): (i) the sequence
ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 16; also referred to as “PAS#1”), or (ii) the sequence
AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17; also referred to as “PAS#2”), or (iii) the sequence
SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 20; also referred to as “PAS#5”), or (iv) a fragment of any of these sequences, or (v) a combination of two or more of these sequences (which may be the same or different, i.e., any combination of two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the sequences PAS#1, PAS#2 and/or PAS#5; a corresponding example is a dimer of PAS#1 (“PAS#1- PAS#1”); further examples include PAS#1-PAS#2, PAS#1-PAS#5, PAS#2-PAS#1, PAS#2- PAS#2, PAS#2-PAS#5, PAS#5-PAS#1, PAS#5-PAS#2, PAS#5-PAS#5, PAS# 1 -PAS# 1 -PAS# 1, PAS#1- PAS#1-PAS#2, PAS#1-PAS#1-PAS#5, PAS#1-PAS#2-PAS#1, PAS#1-PAS#2-PAS#2, PAS#1-PAS#2- PAS#5, PAS#1-PAS#5-PAS#1, PAS#1-PAS#5-PAS#2, PAS#1-PAS#5-PAS#5, PAS#2-PAS#1-PAS#1, PAS#2-PAS#1-PAS#2, PAS#2-PAS#1-PAS#5, PAS#2-PAS#2-PAS#1, PAS#2-PAS#2-PAS#2, PAS#2- PAS#2-PAS#5, PAS#2-PAS#5-PAS#1, PAS#2-PAS#5-PAS#2, PAS#2-PAS#5-PAS#5, PAS#5-PAS#1- PAS#1, PAS#5-PAS#1-PAS#2, PAS#5-PAS#1-PAS#5, PAS#5-PAS#2-PAS#1, PAS#5-PAS#2-PAS#2, PAS#5-PAS#2-PAS#5, PAS#5-PAS#5-PAS#1, PAS#5-PAS#5-PAS#2, or PAS#5-PAS#5-PAS#5).
As detailed herein above, group C (i.e., the P/A and/or PAS polypeptides) may comprise a combination of two or more of the exemplary and non-limiting sequences detailed in any one of SEQ IDs NO: 9 to 22. Combinations of the same P/A or PAS sequence (i.e., multiples of the same P/A or PAS sequence) are most preferred herein. Accordingly, in the context of the present invention, “a combination of two or more” sequences also includes multiples of thereof. The term multiples may refer to, inter alia, 2, 3, 4, 5, 6, 7, 9, or 10 polypeptides (e.g., but not limiting, comprising the amino acid sequence SEQ ID NO: 16; PAS#1) combined into a single polypeptide. Solely for exemplary purposes, SEQ ID NO: 4 comprises the amino acid sequence depicted in SEQ ID NO: 16 (PAS#1) 2-times, whereas SEQ ID NO: 5 and 6 each comprise the amino acid sequence depicted in SEQ ID NO: 16 (PAS#1) 5-times. It is obvious to the person skilled in the art that also any other combination of such P/A and PAS sequence is envisaged herein. Accordingly, in the context of the present invention group C may comprise an amino acid sequence selected from any one of SEQ IDs NO: 9 to 22, or multiples thereof. Such examples are further illustratively and non- limitingly provided as SEQ IDs NO: 1 to 8. Further examples of the herein above (and below) detailed P/A and/or PAS amino acid sequences (or P/A- and/or PAS-polypeptides comprising such amino acid sequences) are, inter alia, illustrated in WO 2008/155134, WO 2011/144756, WO 2017/109087, and WO 2018/234455, which are hereby incorporated by reference in their entirety.
The amino acid residues that group C (i.e., P/A or PAS) is composed of may have any configuration. In particular, each a-amino acid residue comprised in P/A may have the L-configuration or the D-configuration. Thus, any proline residue in P/A may be in the form of L-proline or D-proline, and any alanine residue in P/A may be in the form of L-alanine or D-alanine. It will be understood that not all amino acids have distinct L- and D-configurations; in particular, glycine residues have only one configuration. Among those a-amino acid residues comprised in P/A that can have the L-configuration or the D- configuration, preferably at least 75 mol%, more preferably at least 80 mol%, even more preferably at least 90 mol%, yet even more preferably at least 95 mol%, still more preferably at least 98 mol%, and most preferably 100 mol% of the number of said a-amino acid residues are present in the L-configuration.
The herein provided definition of group C applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
The present invention provides for compounds according to formula (I) and methods for the production/preparation of such compounds of formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group.
In the above formula (I), D is an N-terminal protecting group.
The group D in the compound of formula (I) is a protecting group which is attached to the N-terminal amino group, particularly the N-terminal a-amino group, of the polypeptide capable of forming a random coil conformation (group B). The terms “protecting group” and “protection group” may be used synonymously in the context of the present invention.
The skilled person is aware of suitable protection groups that may be deployed in the context of the present invention. In the following, exemplary and non-limiting examples of such protection groups are provided. In the context of the present invention, group D may be selected from formyl (i.e., -CHO), -CO(Ci-6 alkyl), pyroglutamoyl (i.e., 5-oxopyrrolidin-2-yl-carbonyl), and homopyroglutamoyl (i.e., 6-oxopiperidin-2-yl- carbonyl), wherein the alkyl moiety comprised in said -CO(Ci-6 alkyl) is optionally substituted with one or more groups (e.g., one, two or three groups) independently selected from -OH, -O(Ci-4 alkyl), -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci-4 alkyl) and -COOH. Moreover, group D may be selected from formyl, -CO(Ci-4 alkyl), pyroglutamoyl and homopyroglutamoyl, wherein the alkyl moiety comprised in said -CO(Ci-4 alkyl) is optionally substituted with one or two groups independently selected from -OH, -O(Ci-4 alkyl), -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci-4 alkyl) and -COOH. Accordingly, group D may be selected from formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl (i.e., -CO-CH2-COOH), propionyl, 2-hydroxypropionyl, 3 -hydroxypropionyl, 2-methoxypropionyl, 3 -methoxypropionyl, 2-ethoxypropionyl, 3 -ethoxypropionyl, succinyl (i.e., -CO-CH2CH2-COOH; or cyclosuccinyl, i.e. -CO-CH2CH2-CO-), butyryl, 2-hydroxybutyryl, 3 -hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3 -methoxybutyryl, 4-methoxybutyryl, glycine betainyl (i.e., -CO-CH2-N+(-CH3)3), glutaryl (i.e., -CO-CH2CH2CH2-COOH), pyroglutamoyl, and homopyroglutamoyl. It is preferred that group D is selected from acetyl and pyroglutamoyl, with pyroglutamoyl being especially preferred.
Illustrative and non-limiting examples of group D are comprised in SEQ IDs NO 1 to 8 and illustrated in the appended Examples.
The herein provided definition of group D applies mutatis mutandis to all means and methods of the present invention but also to all the herein provided inventive compounds.
The present invention provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group; and b) the purification of a compound of formula (I).
In the above method, L is a leaving group.
In the context of the present invention, the leaving group L is replaced by the group A during the coupling of the di(alkyl)amine and the compound L-B-C-D and/or during the coupling of the di(alkyl)amine and compound L-C-D. This means in the context of the present invention that when a di(alkyl)amine is coupled to compound L-B-C-D or compound L-C-D, the leaving group L is replaced by a di(alkyl)amino group, resulting in the production/preparation of compound A-B-C-D or of compound A-C-D. As will be appreciated by the skilled person, the di(alkyl)amino group A is provided by the di(alkyl)amine which is used in the coupling reaction via the replacement of a hydrogen atom of the di(alkyl)amine by the bond formed between the di(alkyl)amino group and the linking group B, if present, or the polypeptide C, respectively. The skilled person is aware of suitable leaving groups that may be deployed in the context of the present invention. In the following, exemplary and non-limiting examples of such leaving groups are provided. In the context of the present invention, the compound L-B-C-D and/or the compound L-C-D typically comprises a group -C(O)-L, wherein L represents the leaving group and is preferably selected from -OH, -I, -Br, -Cl, -H, and -OR3, wherein R3 is a Ci-Ce alkyl, most preferably -OH. In another preferred embodiment, the leaving group may also be the alcohol corresponding to an active ester (e.g. N- hydroxysuccinimide, NHS, or 1 -hydroxybenzotriazole, HOBT), such that the group -C(O)-L represents an active ester group (e.g. a NHS ester group or a HOBT ester group). Non-limiting examples of such leaving groups are provided in Examples 1 and 2.
It is evident for the skilled person, that if b is 0, and accordingly B is absent the di(alkyl)amino group of the compound of formula (I) is coupled/linked to group C (i.e., the polypeptide capable of forming a random coil conformation). In other words, if B is absent, the di(alkyl)amine is coupled to compound L-C-D, resulting in the preparation/production of compound A-C-D.
In the context of the present invention and as detailed herein above and below, compound L-C-D comprises a polypeptide capable of forming a random coil conformation (inter alia, a P/A- and/or PAS -polypeptide). As detailed herein above and as illustratively shown in the appended non-limiting examples, with regard to compound A-C-D, the di(alkyl)amino group is typically linked via the nitrogen atom (N) to carbon atom (C) of the carboxyl group of the carboxy terminus of said polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide), such that an amide is formed. When coupling a di(alkyl)amine to the C-terminal carboxyl group of a polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide) the leaving group L can be -OH, which forms part of said C-terminal carboxyl group.
Accordingly, it is evident to the person skilled in the art, that when coupling a di(alkyl)amine to a polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide), the C-terminal carboxyl group may already comprise a leaving group L (namely an -OH). In the context of the present invention, when B is absent, the leaving group L, may be -OH forming part of the C-terminal carboxyl group of the polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide). Accordingly, in the compound L-C-D, L may be -OH and may, thus, be comprised in the C-terminal carboxyl group of group C. As detailed herein above, L is preferably selected form -OH, -I, -Br, -Cl and -OR3, wherein R3 is a Ci-Ce alkyl, most preferably -OH. Accordingly, when B is absent, L may also be selected from -OH, -I, -Br, -Cl, -H, and -OR3, wherein R3 is a Ci-Ce alkyl. In the context of the present invention, when B is absent, -I, -Br, -Cl and -OR3, wherein R3 is a Ci-Ce alkyl, may replace -OH of the C-terminal carboxyl group of the polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS-polypeptide). In another preferred embodiment, the leaving group may also be the alcohol corresponding to an active ester (e.g. N-hydroxysuccinimide, NHS, or 1- hydroxybenzotriazole, HOBT), such that the group -C(O)-L represents an active ester group (e.g. a NHS ester group or a HOBT ester group).
Figure 1 illustratively shows an exemplarily and non-limiting coupling, wherein L is a C-terminal -OH of a polypeptide capable of forming a random coil conformation (inter alia, said P/A- and/or PAS- polypeptide).
As detailed herein above, the prior art does not provide any means and methods that allow for the coupling of polypeptides capable of forming a random coil conformation to di(alkyl)amines. In accordance with the present invention, an inventive method for targeted and/or directional coupling of such polypeptides to di(alkyl)amines was found.
In the context of the present invention it is desirable to obtain compounds that comprise a single polypeptide capable of forming a random coil conformation and a single di(alkyl)amino group via chemical coupling. Accordingly, it is desired to couple a polypeptide that comprises only a single reactive group with a single di(alkyl)amine. As detailed herein above and below, the polypeptide capable of forming a random coil conformation comprises an amino acid sequence consisting of alanine and proline or consisting of alanine, proline, and serine, accordingly, in the context of the present invention, said polypeptides preferably do not comprise any reactive amino acid side chains (i.e., preferably do not comprise any carboxy groups, amino groups, or thiol groups). Further, in the context of the present invention, the N-terminus of said polypeptide is linked to and thereby protected by an N-terminal protecting group. Structural features of said N-terminal protecting group have been disclosed herein above. Consequently, the coupling of the polypeptide capable of forming a random coil structure is preferably occurring via its C-terminal carboxy group or via a linker attached to said C-terminal carboxy group. Accordingly, the present invention provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B- C-D or compound L-C-D each comprise only a single reactive group and wherein the di(alkyl)amine and compound L-B-C-D or compound L-C-D are coupled via said reactive group. In accordance with the present invention, the terms “linked”, “coupled”, “attached”, and “connected” may be used interchangeably herein above and below.
Accordingly, the present invention provides for means and methods for targeted and/or directional coupling of a single P/A- and/or PAS-polypeptide or a compound comprising a P/A- and/or PAS-polypeptide to the nitrogen atom of a single di(alkyl)amine via either the unprotected C-terminal carboxy group or a linker attached to said C-terminal carboxy group of said P/A- and/or PAS-polypeptide. Only with extensive experimental efforts, the present inventors have found reaction/coupling reagents and conditions that allow for convenient and reliable production/preparation of the herein disclosed compounds of formula (I), as is, inter alia, illustrated in the appended examples.
As detailed herein above, the P/A- and PAS-polypeptides are capable of forming a random coil conformation when present in an aqueous environment and are, thus, highly polar/hydrophilic/lipophobic. Only few polar protic solvents, such as water and methanol, and polar aprotic solvents, such as dimethyl sulfoxide (DMSO) and dimethylformamide are suitable to dissolve P/A- or PAS-polypeptides. Contrastingly, di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine), due their two alkyl chains, are particularly nonpolar/hydrophobic/lipophilic. Diethyl ether and dichloromethane may be used to solubilize di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine). Accordingly, the present inventors had to identify a solvent or a combination of solvents that allows for simultaneous solubilization of P/A- or PAS-polypeptides and di(alkyl)amines.
The person skilled in the art is aware of means and methods to assess whether a solvent or a mixture of solvents is capable of solubilizing a compound (i.e., solute) or a combination of compounds. Inter alia, the occurrence of precipitates and/or turbidity may be an indication to the person skilled in the art that a certain combination of solvent and solute is not effectively dissolving and/or solubilizing said solute. The occurrence of precipitates and/or turbidity can be readily assessed by photo spectrometric methods or simply by visual assessment by the person skilled in the art. It was found that none of the herein above-mentioned solvents was able to dissolve both P/A- or PAS- polypeptides (or compounds comprising the same) and di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine). Tetrahydrofuran, dioxan, and l-methoxy-2 -propanol, which are often used to dissolve reactants with very different polarity, all were unable to dissolve both di(tetradecyl)amine and P/A- or PAS- polypeptides or compounds comprising the same. Among all tested combinations of the herein above- mentioned solvents, only a mixture of the polar protic methanol with excess of the aprotic dichloromethane was able to dissolve both reactants. Accordingly, the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in (an) organic solvent(s), preferably said organic solvent(s) comprises methanol and di chloromethane.
Accordingly, the present invention provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in (an) organic solvent(s), preferably said organic solvent(s) comprises dichloromethane and methanol; and b) the purification of a compound of formula (I).
In the context of the present invention at least one of the di(alkyl)amine, or compound L-B-C-D or compound L-C-D may be provided in an organic solvent comprising dichloromethane and methanol. This means, inter alia, that compound L-B-C-D or compound L-C-D may be provided in an organic solvent comprising dichloromethane and methanol and the di(alkyl)amine may be added to/dissolved in said solution comprising the organic solvent and the compound L-B-C-D or compound L-C-D. Alternatively, it is further evident, inter alia, from the non-limiting examples that compound L-B-C-D or compound L-C- D may be dissolved in methanol and the di(alkyl)amine may be dissolved in dichloromethane. It is evident that both solutions can be mixed to provide both compound L-B-C-D or compound L-C-D and the di(alkyl)amine in an organic solvent comprising dichloromethane and methanol.
The present inventors have surprisingly found that performing the coupling of the di(alkyl)amine, and of compound L-B-C-D or compound L-C-D in an organic solvent comprising dichloromethane and methanol is particular useful if the volume ratio of dichloromethane and methanol comprised in said solvent is at least about 3 volume parts dichloromethane and about 1 volume part methanol (volume ratio of about 3 : 1; volume ratio of about 3 to 1) to about 8 volume parts dichloromethane and 1 volume part methanol (volume ratio of about 8 : 1; volume ratio of about 8 to 1). It was found that solvents comprising higher volume ratios of dichloromethane to methanol (>8 : 1 ; >8 to 1 ; more than about 8 volume parts dichloromethane and about 1 volume part methanol) the solubility of the herein provided P/A- and/or PAS-polypeptides may be compromised, consequently negatively affecting coupling efficiencies of said P/A- and/or PAS- polypeptides to the di(alkyl)amine. Further, it was found that a volume ratio of lower than about 3 to 1 (<3 : 1; <3 to 1; less than about 3 volume parts dichloromethane and about 1 volume part methanol) also negatively affects coupling efficiencies of said P/A- and/or PAS-polypeptides to the di(alkyl)amine, as production/preparation of P/A- and/or PAS-polypeptide methyl esters occurs more frequently.
Accordingly, the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C- D are comprised in an organic solvent comprising dichloromethane and methanol, wherein said organic solvent comprises about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol.
Accordingly, the present invention provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol; and b) the purification of a compound of formula (I).
In the context of the present invention, a volume ratio of e.g., 3 : 1 dichloromethane : methanol (3 to 1; 3 volume parts dichloromethane and 1 volume part methanol) refers to a volume ratio of two solvents (e.g., dichloromethane and methanol), wherein the first solvent (e.g., dichloromethane) is 3 -fold more abundant as compared to the second solvent (e.g., methanol). For example, a volume ratio of 3 : 1 (3 to 1; 3 volume parts dichloromethane and 1 volume part methanol) would correspond to 75 ml dichloromethane and 25 ml methanol in 100 ml total volume of said solvent. For example, a volume ratio of 4 : 1 (4 to 1; 4 volume parts dichloromethane and 1 volume part methanol) would correspond to 80 ml dichloromethane and 20 ml methanol in 100 ml total volume of said solvent. For example, a volume ratio of 5 : 1 (5 to 1; 5 volume parts dichloromethane and 1 volume part methanol) would correspond to approx. 83.33 ml dichloromethane and approx. 16.67 ml methanol in 100 ml total volume of said solvent. The skilled person is able to calculate the respective amounts/volumes of both dichloromethane and methanol to be mixed to obtain a solvent of a certain volume ratio.
It was further surprisingly found that a volume ratio of about 5 : 1 (about 5 to 1 ; about 5 volume parts dichloromethane and about 1 volume part methanol) is most advantageous for solubilizing P/A- and/or PAS-polypeptides and di(alkyl)amines and for reducing the production/preparation of P/A- and/or PAS- polypeptide methyl esters.
Accordingly, the present invention provides for a method for the production/preparation of a compound of formula (I), wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C- D are comprised in an organic solvent comprising dichloromethane and methanol, wherein said organic solvent comprises about 5 volume parts di chloromethane and about 1 volume part methanol.
Accordingly, the present invention provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol, preferably about 5 volume parts dichloromethane and 1 volume part methanol; and b) the purification of a compound of formula (I).
In one preferred aspect, present invention provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising dichloromethane and methanol; and b) the purification of a compound of formula (I).
The present invention further provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol, preferably about 5 volume parts dichloromethane and about 1 volume part methanol; and b) the purification of a compound of formula (I). The present invention further provides for a method for the production/preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein A is a di(tetradecyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(tetradecyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group, and wherein during the coupling, the di(tetradecyl)amine, and compound L- B-C-D or compound L-C-D are comprised in an organic solvent comprising about 5 volume parts dichloromethane and about 1 volume part methanol; and b) the purification of a compound of formula (I). In the context of the present invention, the herein above detailed organic solvent that is particular suitable for the coupling of the di(alkyl)amine to the P/A- or PAS-polypeptides may further comprise other/further/additional reagents that are advantageous for said coupling reaction. Such other/further/additional reagents may, inter alia, be a coupling reagent.
Accordingly, the inventors identified coupling reagents that allow for the efficient and convenient coupling of P/A- or PAS-polypeptides or compounds comprising the same to di(alkyl)amines (for example, but not limiting, di(tetradecyl)amine). Thus, the present invention also provides for 2-(lH-Benzotriazole-l-yl)- 1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) as a highly efficient coupling reagent resulting in approx. 70% coupling rates with the herein disclosed P/A-polypeptides and approx. 40% coupling rates with the herein disclosed PAS-polypeptides. The present inventors assayed various coupling reagents (including propylphosphonic anhydride (T3P), pentafluorophenyl trifluoroacetate, Ethyl 2-Cyano-2- (Hydroxyimino)Acetate (OxymaPure®)), and surprisingly found that TBTU resulted in superior coupling rates (2-fold to 50-fold improved coupling rates) as compared to all other tested coupling reagents. Accordingly, the present invention further provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled using a coupling reagent, and wherein said coupling reagent is 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate (TBTU). It was found that the addition of about 1 mol. eq. TBTU to about 3 mol. eq. TBTU to the coupling reaction was beneficial/advantageous for (high) coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine). The addition of more than 5 mol. eq. TBTU to the coupling reaction showed drastically reduced coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine) as compared to e.g., 2 mol. eq. TBTU. The inventor surprisingly found that the addition of about 2 mol. eq. TBTU was particularly beneficial/advantageous for (high) coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine). Accordingly, the present invention further provides for a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound E-B-C-D or compound L-C-D are coupled using a coupling reagent, and wherein said coupling reagent is 2-(lH-Benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate (TBTU), and wherein about 2 mol. eq. TBTU are to be added to the reaction.
The present invention further relates to a non-nucleophilic base to be used in accordance with the herein detailed methods. Accordingly, the present invention relates to a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are further coupled using a non-nucleophilic base , and wherein said non-nucleophilic base is selected from N,N-Diisopropylethylamine (DIPEA), l,8-Diazabicycloundec-7-ene (DBU), 1,5-Diazabicyclo[4.3.0]non- 5-ene (DBN), triethylamine (TEA), preferably N,N-Diisopropylethy lamin (DIPEA). The inventor surprisingly found that the addition of about 1.5 mol. eq. DIPEA was particularly beneficial/advantageous for (high) coupling rates of the P/A- or PAS-polypeptides to the di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine). However, also the addition of 1 mol. eq. DIPEA to 10 mol. eq. DIPEA to the coupling reaction may be envisaged in the context of the present invention. Accordingly, the present invention further relates to a method for the production/preparation of a compound of formula (I), wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are further coupled using a non- nucleophilic base , wherein said non-nucleophilic base is N,N-Diisopropylethylamin (DIPEA), and wherein about 1.5 mol. eq. are to be added to the reaction.
The inventors surprisingly found that the coupling of P/A- or PAS-polypeptides with di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine) may be performed in a single reaction. Accordingly, the present invention further relates to a method for the production/preparation of a compound of formula (I), wherein a di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled in a single reaction.
In accordance with the present invention, said single reaction may be incubated under an argon cloud for at least about 1 min, for at least about 5 min, for at least about 10 min, for at least about 20 min, for at least about 30 min, for at least about 40 min, for at least about 50 min, for at least about 60 min, for at least about 10 min, preferably for about 60 min. Accordingly, the present invention relates to a method for the production/preparation of a compound of formula (I), wherein a di(alkyl)amine, and compound L-B-C-D or compound L-C-D are reacted for at least about 1 min, for at least about 5 min, for at least about 10 min, for at least about 20 min, for at least about 30 min, for at least about 40 min, for at least about 50 min, for at least about 60 min, for at least about 10 min, preferably for about 60 min.
The herein above detailed conditions and using the herein above detailed reagents and inventive combination of solvents are further illustrated in the non-limiting examples and figures. In particular Figure 1 schematically illustrates an herein detailed, yet non-limiting, exemplary reacting scheme for coupling of a PAS40-polypeptide with di(tetradecyl)amine.
Illustrative and non-limiting examples of compound L-B-C-D are found in SEQ IDs NO: 1, 2, and 6, whereas illustrative and non-limiting examples of compound L-C-D are found in SEQ IDs NO: 3 to 5, 7, and 8. SEQ IDs NO: 7 and 8 comprise a lysine residue (K) in position 12. In the context of the present invention, the free amino group on the sidechain of said lysine residue may be used to couple said compound L-C-D to the herein provided di(alkyl)amine, accordingly, preparing/producing compound A-C-D. The herein provided means and methods (inter alia, and non-limiting, the means and methods illustratively show in Example 2) allow the skilled person to perform such coupling reactions. The skilled artisan is aware that such a lysine residue may be readily introduced at any position into any herein disclosed P/A and/or PAS sequences. Preferably said lysine residue may be introduced into a central position of a given amino acid sequence encoding the herein provided P/A- and/or PAS-polypeptides. In other words, in an amino acid sequence encoding a random coil polypeptide of e.g., 40 amino acid residues it may be preferred that such a lysine residue may be introduced at position 21, resulting in a polypeptide comprising 41 amino acid residues. Advantages of coupling compound L-C-D to the herein provided di(alkyl)amine may the production/preparation of a compound A-C-D, which comprises a branched random coil polypeptide. Nonlimiting examples of such branched P/A- and/or PAS-polypeptides are provided as SEQ IDs NO: 7 and 8.
The skilled person is aware that the herein above and/or below provided means and methods for the coupling of a di(alkyl)amine to either a compound L-B-C-D or a compound L-C-D may interchangeably be deployed for the coupling of a di(alkenyl)amine or a di(alkynyl)amine) to either a compound L-B-C-D or a compound L-C-D, as has been detailed herein above.
The present invention further relates to means and methods for the purification of a compound of formula (I).
After coupling of P/A- or PAS-polypeptides with di(alkyl)amine (for example, but not limiting, di(tetradecyl)amine), about 8 vol. eq. of a mcthanol/lLO mixture (about 10 volume parts methanol and about 1 volume part H2O) may be added to precipitate unreacted di(alkyl)amines. further, the reaction may be incubated on ice or at approx. 0 °C for about 20 min. Subsequently, the reaction may be filtered on, for example, but not limiting, a glass fiber filter comprising approx. 1 pm mesh size to remove the precipitate. Accordingly, the present invention further relates to the purification of the compound of formula (I), wherein at least some of the reagents are precipitated from the reaction by the addition of a hydrophilic solvent.
The present invention further relates to liquid chromatography of the compound of formula (I), wherein liquid chromatography preferably refers to high-pressure liquid chromatography (HPLC) of the compound of formula (I). In accordance with the presence invention liquid chromatography (such as HPLC) may be performed on the compound of formula (I) after the herein above detailed precipitation of at least some of the reagents.
The herein detailed methods for purification of a compound of formula (I) are further illustrated in the nonlimiting, examples. Specifically, Figure 2 illustratively shows an exemplary HPLC -chromatogram of PAS40-lipids, clearly demonstrating efficient purification of said compound.
As detailed herein above and below, the present invention provides for inventive compounds of formula (I) or as obtained/obtainable by the means and methods as provided herein. In any of the embodiments detailed herein below or above, the present invention also always relates to salts and solution of or comprising the compounds of formula (I), when referring to said compounds.
Accordingly, the present invention provides for inventive compounds that are characterized by formula (I):
A-[B-]bC-D (I) wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0, so that B can be present or absent, C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group.
Appended Figures 3 to 8 provide illustrative, non-limiting examples of compounds of formula (I) in accordance with the present invention. Here, exemplary, non-limiting structures, as well as illustrative HPLC-chromatograms and mass-spectrometric analyses of said compounds are provided. The skilled person is aware that the inventive compounds may herein also be referred to as e.g., Pga-PAS20- di(tetradecyl)amine (a non-limiting example of compound A-C-D), Pga-P/A20-Ahx-di(tetradecyl)amine (a non-limiting example of compound A-B-C-D), Pga-PAS40-di(tetradecyl)amine (anon-limiting example of compound A-C-D) or Pga-P/A40-Ahx-di(tetradecyl)amine (a non-limiting example of compound A-B-C- D), or Pga-PAS100-K-di(tetradecyl)amine (a non-limiting example of compound A-C-D). The person skilled in the art is further aware that the reference PEG2k compound (2-[(polyethylene glycol)-2000]-N,N- di(tetradecyl)acetamide) may also be referred to as PEG2k-N,N-di(tetradecyl)acetamide
As detailed herein above, the herein provided inventive compounds according to formula (I) may in one aspect not comprise the optional linker group B (i.e., and accordingly, be of the general formula A-C-D. The skilled person is aware that such a compound may, in the context of the present invention, also be represented by the following formula:
D-PP-C(O)-NR’R2
Wherein D is the herein above detailed N-terminal protecting group (group D), -PP-C(O)- corresponds to the polypeptide C (group C) as defined herein above, providing a functional group -C(O)- at its C-terminus for attachment of group A (the herein above detailed di(alkyl)amino group, -NR’R2 corresponds to the group A (said di(alkyl)amino group), wherein R1 and R2 are independently two linear or branched alkyl chains, preferably said two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
As detailed herein above, the herein provided inventive compounds according to formula (I) may in one aspect comprise the optional linker group B (i.e., and accordingly, be of the general formula A-B-C-D. The skilled person is aware that such a compound may, in the context of the present invention, also be represented by the following formula:
D-PP-C(O)-HN-(C2.i2 hydrocarbyl)-C(O)-NR1R2
Wherein D is the herein above detailed N-terminal protecting group (group D), -PP-C(O)- corresponds to the polypeptide C (group C) as defined herein above, providing a functional group -C(O)- at its C-terminus for attachment of group B (the herein above detailed linker group), -HN-(C2-i2 hydrocarbyl)-C(O)- corresponds to said linker group B, and -NR’R2 corresponds to the group A (said di(alkyl)amino group), wherein R1 and R2 are independently two linear or branched alkyl chains, preferably said two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
As has been detailed herein above, in the compounds of formula (I) wherein B is present (i.e., compound A-B-C-D), an amide bond is preferably formed between a carboxy group -C(O)- provided by group B and the di(alkyl)amino group A. As has been detailed herein above, in the compounds of formula (I) wherein B absent (i.e., compound A-C-D), an amide bond is preferably formed between a carboxy group -C(O)- at the C-terminus of the polypeptide C and the di(alkyl)amino group A.
The herein provided compounds of formula (I) cause more than 70% cell viability, when 10 pg/ml thereof are transfected into He La cells and when measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Also, the herein provided compounds of formula (I) cause improved cell viability as compared to a reference PEG2k compound (2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide), when 10 pg/ml thereof are transfected into HeLa cells and measured by MTS assay.
Accordingly, the herein provided compounds of formula (I) are clearly beneficial/advantageous with regard to its effects on cell viability as compared to state of the art compounds. That means, inter alia, that the herein provided compounds cause less cytotoxicity as compared to a reference PEG2k compound. That means, inter alia, that the herein provided compounds have low or reduced cytotoxicity/ low or reduced toxicity/ low or reduced negative effects on cell viability as compared to a reference PEG2k compound. In the context of the present invention, it is a desired criterion/characteristic that the herein provided compounds of formula (I) have the herein detailed low or reduced cytotoxicity/ low or reduced toxicity/ low or reduced negative effects on cell viability as compared to a reference PEG2k compound.
The surprising, yet beneficial/advantageous effects of the herein provided compounds of formula (I) are further detailed in the appended examples. In particular, Example 5 and Table 3 further illustrate the herein detailed beneficial/advantageous effects of the herein provided compounds of formula (I). The present invention further provides for a method for the production/preparation of a lipid nanoparticle comprising one or more compounds of formula (I). Further, the present invention also relates to the use of the compound of formula (I) for the production/preparation of a lipid nanoparticle.
Accordingly, the present invention provides for a method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), wherein the method comprises the steps of a) the provision of the compound of formula (I) in an ethanolic solution; b) the mixing of said ethanolic solution with an aqueous solution, thereby preparing/producing a lipid nanoparticle; and c) optionally the dialysis of said lipid nanoparticle.
In the context of the present invention, the provision of the compound of formula (I) in an ethanolic solution may also refer to obtaining and/or preparing an ethanolic solution comprising the compound of formula (I). The skilled artisan is aware of means and methods for the production/preparation of lipid nanoparticles. Such means and methods are further detailed herein below and further illustrative, yet non-limiting examples may be found in the examples, namely in Example 3.
In the context of the present invention, the term “ethanolic solution” may be used interchangeably with the term “ethanol solution”. As used herein, an ethanolic solution / an ethanol solution is a non-aqueous solution that consists of ethanol. In the context of the present invention, an aqueous solution is a non-ethanolic solution that consists of water. In the context of the present invention, an ethanolic solution may further comprise a cationic lipid, a non-cationic lipid, and a sterol. Said ethanolic solution may comprise between about 2 mM and about 60 mM total lipid, preferably between about 7.5 mM and about 30 mM, more preferably 9.43 mM total lipid. In the context of the present invention, the term “total lipid” comprises any lipid comprised in said ethanolic solution (e.g., the compound of formula (I), the cationic lipid, the noncationic lipid, and the sterol. Accordingly, in the context of the present invention, the compound of formula (I) is a lipid. With regard to the ethanolic solution, the herein below detailed mole percentages (mol%) only refer to the mole percentages (mol%) of a certain compound (such as, the compound of formula (I), the cationic lipid, the non-cationic lipid, or the sterol) as compared to the total amount of lipids (total lipids) in said ethanolic solution, without taking into account the ethanol molecules in said ethanolic solution. Said ethanolic solution may comprise between about 1 mol% and about 5 mol%, preferably about 1.5 mol% of the compound of formula (I). Accordingly, said ethanolic solution comprises between about 99 mol% and 95 mol%, preferably about 98.5 mol% of the other herein above mentioned lipids (i.e., the cationic lipid, the non-cationic lipid, and the sterol), respectively.
In accordance with the present invention, said ethanolic solution may comprise between about 45 mol% and about 55 mol%, preferably about 50 mol% of the cationic lipid. Generally, cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a Steroid portion and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at about physiological pH. In the context of the present invention, cationic lipids may either refer to lipids with a constitutively positively charged headgroup or to ionizable cationic lipids with apparent pKa values below about 7, that may comprise a neutral or positively charged headgroup. In the context of the present invention, ionizable cationic lipids may be preferred. Accordingly, in the context of the present invention, the cationic lipid may be DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4- (dimethylamino)butanoate) .
The present invention further relates to a non-cationic lipid, wherein said non-cationic lipid may be an anionic lipid, preferably wherein said anionic lipid is a phospholipid, more preferably distearoylphosphatidylcholine (DSPC). In accordance with the present invention, said ethanolic solution may comprise between about 7 mol% and about 13 mol%, preferably about 10 mol% of the non-cationic lipid.
Furthermore, said ethanolic solution may comprise between about 35 mol% and about 42 mol%, preferably about 38.5 mol% of the sterol. In the context of the present invention, said sterol may be cholesterol.
In the context of the present invention, the herein disclosed method for the production/preparation of a lipid nanoparticle comprising the compound of formula (I) may comprise the introduction/inclusion of one or more active ingredients into said lipid nanoparticle. Said one or more active ingredients may be comprised in said aqueous solution. Accordingly, mixing of said aqueous solution that comprises said one or more active ingredients with the herein above and below detailed ethanolic solution comprising the compound of formula (I), may result in the production/preparation of a lipid nanoparticle comprising said one or more ingredients and said compound of formula (I). In accordance with the present invention, said one or more active ingredients are one or more nucleic acids, one or more polypeptides, one or more proteins, or combinations thereof, preferably one or more nucleic acids. Said one or more nucleic acids may be selected from the group consisting of mRNA, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA for gene editing systems, DNA, plasmids, antisense oligonucleotides, and combinations thereof, preferably mRNA.
The aqueous solution comprising the one or more active ingredients may further comprise one or more acids and/or one or more buffers/buffer systems, preferably one or more acids. Accordingly, the aqueous solution may comprise a concentration of between about 5 mM and about 50 mM, preferably between about 10 mM and about 30 mM, more preferably about 11 mM of said one or more acids or of the one or more buffers/buffer systems, preferably the one or more acids. The skilled artisan is aware of exemplary buffers/buffer systems suitable for use in a method for the production/preparation of a lipid nanoparticle. Accordingly, the one or more acids may be selected from acetic acid, HEPES (4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid), and citric acid, preferably acetic acid.
In the context of the present invention, the one or more active ingredients and the one or more buffers/buffer systems or acids, preferably one or more acids, may be comprised in said aqueous solution at a volume ratio between about 1 : 3, and about 1 : 4, preferably a volume ratio of about 1 : 3. The person skilled in the art is aware that, for example a volume ratio of about 1 : 3 of the one or more active ingredients and the one or more acids (or the one or more buffers/buffer systems) may refer to about one volume part of the one or more active ingredients or of a solution comprising the one or more active ingredients and about three volume parts of the one or more acids, the one or more buffers/buffer systems, or solutions comprising the same.
As detailed herein above and below, the method for the production/preparation of a lipid nanoparticle may comprise an optional step of dialyzing the lipid nanoparticle. In one aspect of the present invention, the lipid nanoparticle may not be dialyzed. In a further aspect, said lipid nanoparticle may be dialyzed using a buffer, preferably wherein said buffer is selected from the group of phosphate buffer, phosphate buffer saline, and phosphate buffer saline comprising sucrose, more preferably phosphate buffered saline. In the context of the present invention, the term “buffer” may be interchangeably used with the term “buffer system” and may also refer to solution comprising the same. The preparation of such buffers/buffer systems/solutions comprising the same is standard in the art.
The herein above detailed method for the production/preparation of a lipid nanoparticle is furthermore detailed in the appended non-limiting examples, namely in Example 3.
The present invention further relates to a lipid nanoparticle comprising the compound of formula (I) and/or a lipid nanoparticle obtained/obtainable by the methods for the production/preparation of a lipid nanoparticle comprising the compound of formula (I), detailed herein above. Accordingly, the present invention provides for inventive lipid nanoparticles that comprise the inventive compounds of formula (I) and/or are obtained/obtainable by the inventive methods detailed herein above.
As will be detailed herein below, said lipid nanoparticles comprising the compound of formula (I) comprises highly beneficial/advantageous characteristics that may be desired in the context of the present invention, yet may have been surprisingly found by the present inventors.
Accordingly, the herein detailed inventive lipid nanoparticles have a Zeta-potential of less than 10 mV after about 3 h of dialysis using phosphate buffered saline, when measured by diffraction light scattering (DLS). That means, inter alia, that said lipid nanoparticle has a lower/reduced Zeta-potential as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of formula (I) after about 3 h of dialysis using phosphate buffered saline, when measured by DLS. The skilled person is aware that the Zeta-potential of, for example, a lipid nanoparticle provides for insights into said lipid nanoparticles surface charge. The surface charge of, for example, a lipid nanoparticle may be influenced by molecules (such as, for example, PEG polymers, P/A-polypeptides, or PAS- polypeptides) on the surface of said lipid nanoparticle that may shield the same. Accordingly, the Zetapotential of, for example, a lipid nanoparticle may provide insights into the capacity of molecules (such as, for example, PEG polymers, P/A-polypeptides, or PAS-polypeptides) on the surface of said lipid nanoparticle to shield the surface of said lipid nanoparticle. Accordingly, in the context of the present invention, a low Zeta-potential of lipid nanoparticles comprising the compound of formula (I) may be desired and may indicate successful shielding/protection of said lipid nanoparticle and/or the surface of said lipid nanoparticle. This is further evident from the illustrative, yet non-limiting Example 4. In the context of the present invention the terms “shielding” and “protecting” may be used interchangeably.
Further, the herein provided lipid nanoparticles comprising the compound of formula (I) have at least 91% RNA encapsulation efficiency, when measured using RiboGreen Assay. That means, inter alia, that said lipid nanoparticles have improved/increased RNA encapsulation efficiency as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of formula (I), when measured using RiboGreen Assay. As is further evident from the appended illustrative, yet non-limiting, Example 7, the herein provided compounds of formula (I), when introduced into lipid nanoparticles are, thus, superior in regard to shielding/protecting said lipid nanoparticle and/or the membrane of said lipid nanoparticle, as compared to a reference PEG2k compound. In the context of the present invention this may be a desired characteristic or said compound of formula (I) and the lipid nanoparticle comprising said compound of formula (I).
Moreover, the herein provided lipid nanoparticle comprising the compound of formula (I) may have improved/increased transfection rates as compared to a reference lipid nanoparticle comprising equal amounts of the reference PEG2k compound instead of the compound of any one of claims 35 to 38, when transfected into Jurkat, A549, HepG2, and/or C2C12 cells and when measured by luminescence readout. This is further illustrated in the appended, non-limiting Example 8. High/increased/improved transfection rates may be desired in the context of the present invention.
The present invention further provides for a lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion comprising the lipid nanoparticle detailed herein above, wherein said lipid nanoparticle comprises the compound of formula (I). Accordingly, the present invention further provides for a lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion comprising the compound of formula (I). In accordance with the present invention, the term “lipid nanoparticle”, as used herein below and above, may also refer to said “lipid nanoparticle suspension” and/or to said “lipid nanoparticle colloidal dispersion” comprising said lipid nanoparticle and/or the compound of formula (I). Said lipid nanoparticle suspension and/or a lipid nanoparticle colloidal dispersion may be stored at about 4 °C for at least about 4 days without signs of turbidity and/or visible precipitation of said lipid nanoparticle suspension or components thereof.
As detailed herein above, the lipid nanoparticle/ the lipid nanoparticle suspension/ the lipid nano particle colloidal dispersion comprising the compound of formula (I) show surprising and/or desired beneficial/advantageous effects, as compared to a reference lipid nanoparticle/ a reference lipid nanoparticle suspension/ a reference lipid nano particle colloidal dispersion that does not comprise the compound of formula (I). The only difference between the herein provided lipid nanoparticles/ lipid nanoparticle suspensions/ lipid nano particle colloidal dispersions and the reference lipid nanoparticle/ the reference lipid nanoparticle suspension/ the reference lipid nano particle colloidal dispersion is the presence of the compound of formula (I) in the former and the presence of a reference PEG2k compound (2- [(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide) in the later. Accordingly, any herein above and below detailed technical advantage of the herein provided lipid nanoparticles/ the lipid nanoparticle suspensions/ the lipid nano particle colloidal dispersions may be attributed to the herein provided compound of formula (I) comprised in said lipid nanoparticles/ said lipid nanoparticle suspensions/ said lipid nano particle colloidal dispersions. Hence, any technical advantage of said lipid nanoparticles/ said lipid nanoparticle suspensions/ said lipid nano particle colloidal dispersions provided herein further contributes to the inventive character of the herein provided inventive compound of formula (I). This further evident for the appended, non-limiting Examples.
The person skilled in the art is aware that the herein provided inventive compounds may also be employed in the production/preparation of lipid (or lipoid) particles and/or lipid (or lipoid) formulations that defer from the ones exemplarily herein provided. Accordingly, also lipid (or lipoid) particles (including but not limited to lipid nanoparticles) and/or lipid (or lipoid) formulations as described in and/or as produced in accordance with the means and methods described in any of the following (or any combination of the following) are envisaged and, thus, incorporated herein by reference in their entirety: e.g., WO 2022/180213, WO 2010/053572, WO 2012/000104, WO 2010/053572, WO 2014/028487, WO 2015/095351, WO 2018/089540, WO 2017/218704.
The present invention further relates to means, methods and uses for the production/preparation of a pharmaceutical composition comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound.
Accordingly, the present invention relates to the use of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound for the production/preparation of a pharmaceutical composition. The present invention further provides for a method for the production/preparation of a pharmaceutical composition, wherein the method comprises the formulation of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound into a pharmaceutical composition.
Said method for the production/preparation of a pharmaceutical composition may comprise the formulation of said compound, said lipid nanoparticle, and/or said lipid nanoparticle suspension using a pharmaceutically acceptable carrier. In one aspect, said pharmaceutical composition may be formulated for intramuscular or intravenous administration. Accordingly, said pharmaceutical composition may be formulated for use as a medicament or for use as a vaccine.
The present invention also relates to pharmaceutical compositions comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound. Furthermore, the present invention relates to pharmaceutical compositions obtained/obtainable by the herein above and below detailed means, methods, and uses.
The present invention further relates to the use of said pharmaceutical compositions in the treatment of or in a method of treatment of a disease and/or a medical condition.
The present invention further relates to means, methods, and uses for the production/preparation of a non- pharmaceutical composition comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound.
Accordingly, the present invention relates to the use of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound for the production/preparation of a non-pharmaceutical composition. The present invention further provides for a method for the production/preparation of a non-pharmaceutical composition, wherein the method comprises the formulation of the compound of formula (I), a lipid nanoparticle comprising said compound, and/or a lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound into a non-pharmaceutical composition.
The present invention also relates to non-pharmaceutical compositions comprising the compound of formula (I), the lipid nanoparticle comprising said, and/or the lipid nanoparticle suspension comprising said lipid nanoparticle and/or said compound. Furthermore, the present invention relates to non-pharmaceutical compositions obtained/obtainable by the herein above and below detailed means, methods, and uses.
The present invention further relates to the use of said non-pharmaceutical compositions. A non-pharmaceutical composition may in the context of the present invention be a cosmetic composition.
The terms “polypeptide” and “peptide” are used herein interchangeably and refer to a polymer of two or more amino acids linked via amide bonds that are formed between an amino group of one amino acid and a carboxy group of another amino acid. The amino acids comprised in the peptide or protein, which are also referred to as amino acid residues, may be selected from the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) but also from non-proteinogenic and/or non-standard a-amino acids (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group (e.g., a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group) like, e.g., cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine, or phenylglycine) as well as P-amino acids (e.g., -alanine), y-amino acids (e.g., y-aminobutyric acid, isoglutamine, or statine) and 5-amino acids. Preferably, the amino acid residues comprised in the peptide or protein are selected from a-amino acids, more preferably from the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D-isomer, and are preferably all present as the L-isomer). The peptide or protein may be unmodified or may be modified, e.g., at its N-terminus, at its C-terminus and/or at a functional group in the side chain of any of its amino acid residues (particularly at the side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and/or Arg residues). Such modifications may include, e.g., the attachment of any of the protecting groups described for the corresponding functional groups in: Wuts PGM, Greene’s protective groups in organic synthesis, 5th edition, John Wiley & Sons, 2014. Such modifications may also include, e.g., the glycosylation and/or the acylation with one or more fatty acids (e.g., one or more Cs-so alkanoic or alkenoic acids; forming a fatty acid acylated peptide or protein). The amino acid residues comprised in the peptide or protein may, e.g., be present as a linear molecular chain (forming a linear peptide or protein) or may form one or more rings (corresponding to a cyclic peptide or protein) or branched structures. The peptide or protein may also form oligomers consisting of two or more identical or different molecules.
As used herein, the term “amino acid” refers, in particular, to any one of the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro (also called an imino acid), Ser, Thr, Trp, Tyr, or Vai) but also to a non-proteinogenic and/or non-standard a-amino acid (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group (e.g., a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group) like, e.g., cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine, or phenylglycine), or a P-amino acid (e.g., P-alanine), a y-amino acid (e.g., y-aminobutyric acid, isoglutamine, or statine) or a 5-amino acid, or any other compound comprising at least one carboxylic acid group and at least one amino group. Unless defined otherwise, the term “amino acid” preferably refers to an a-amino acid, more preferably to any one of the 20 standard proteinogenic a-amino acids (which may be in the form of the L-isomer or the D-isomer but are preferably in the form of the L-isomer).
The term “hydrocarbon chain” refers to a hydrocarbon group consisting of carbon atoms and hydrogen atoms.
As used herein, the term “hydrocarbyl” refers to a monovalent hydrocarbon group which may be acyclic (i.e., non-cyclic) or cyclic, or it may be composed of both acyclic and cyclic groups/subunits. An acyclic hydrocarbyl or an acyclic subunit in a hydrocarbyl may be linear or branched, and may further be saturated or unsaturated. A cyclic hydrocarbyl or a cyclic subunit in a hydrocarbyl may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. A “C2-12 hydrocarbyl” denotes a hydrocarbyl group having 2 to 12 carbon atoms. Exemplary hydrocarbyl groups include, inter alia, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or a composite group composed of two or more of the aforementioned groups (such as, e.g., alkylcycloalkyl, alkylcycloalkenyl, alkylarylalkenyl, arylalkyl, or alkynylaryl). Notwithstanding the above, it will be understood that if a hydrocarbyl group is attached to a parent moiety and is further substituted, e.g., as in the case of a residue H2N-(C2-i2 hydrocarbyl)-COOH, then the corresponding hydrocarbyl group within this residue may also be considered divalent.
As used herein, the term “alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to- carbon double bond or any carbon-to-carbon triple bond. A “C1.4 alkyl” denotes an alkyl group having 1 to 4 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C 1.4 alkyl.
As used herein, the term “alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term “C2-4 alkenyl” denotes an alkenyl group having 2 to 4 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1- en-l-yl, prop-l-en-2-yl, or prop-2-en-l-yl), butenyl, or butadienyl (e.g., buta-l,3-dien-l-yl or buta-1,3- dien-2-yl). Unless defined otherwise, the term “alkenyl” preferably refers to C2-4 alkenyl.
As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term “C2-4 alkynyl” denotes an alkynyl group having 2 to 4 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term “alkynyl” preferably refers to C2-4 alkynyl.
As used herein, the term “aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). “Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., IH-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl. Unless defined otherwise, an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
As used herein, the term “heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two O atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. “Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo [b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1 -benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., lH-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1H- pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, P-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [l,10]phenanthrolinyl, [l,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[l,5- a]pyrimidinyl (e.g., pyrazolo[l,5-a]pyrimidin-3-yl), l,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo [b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., lH-l,2,3-triazolyl, 2H-l,2,3-triazolyl, lH-l,2,4-triazolyl, or 4H-l,2,4-triazolyl), benzotriazolyl, IH-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl, 1,2,4-triazinyl, or 1,3,5- triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1,3- dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g., imidazo[l,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or 1,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term “heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a “heteroaryl” include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, IH-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.
As used herein, the term “cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). “Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, “cycloalkyl” preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, a particularly preferred example of a “cycloalkyl” is cyclohexyl.
As used herein, the term “cycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. “Cycloalkenyl” may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, “cycloalkenyl” preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl. A particularly preferred “cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds. The term "nucleic acid(s)" as used herein refers to a compound(s) containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- or triple -stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of antisense molecules, plasmid DNA (pDNA), linear or circular DNA, PCR products, or vectors. RNA may be in the form of self-amplifying RNA (saRNA) or small hairpin RNA (shRNA), small interfering RNA (siRNA), chemically modified or unmodified messenger RNA (mRNA), antisense RNA, circular RNA (circRNA) comprising at least one coding sequence, micro RNA (miRNA), micRNA, multivalent RNA, transfer RNA (tRNA), single guided RNA (sgRNA), replicating RNA (repRNA), dicer substrate RNA or viral RNA (vRNA), antisense oligonucleotide (ASO), double-stranded RNA (dsRNA) and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated.
The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.
A "cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions.
The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH value.
The term "ionizable lipid" refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range, e.g., pH ~3 to pH ~9. Ionizable lipids may be synthetic or naturally derived.
An "effective amount" or "therapeutically effective amount" of an active agent such as a nucleic acid is an amount sufficient to produce the desired effect, e.g., an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an in increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence, or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.
The present invention refers as well to compositions comprising at least one compound of formula (I) according to the present invention and at least one active agent. The at least one active agent is preferably comprised in an effective amount.
Active agents, as used herein, include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic. Active agents may be any type of molecule or compound, including e.g., nucleic acids, nucleic acid analogues, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.
In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification.
In one embodiment, therapeutic agents include any therapeutically effective agent or drug, such as antiinflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.
In one embodiment the therapeutic agent is an oncology drug, which may also be referred to as an antitumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like. Examples of oncology drugs that may be used according to the invention include, but are not limited to, adriamycin, alkeran, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin A, cytarabine, cytosine arabinoside, daunorubicin, Cytoxan, daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan (Camptostar, CPT- 111), letrozole, leucovorin, leustatin, leuprolide, levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that may be used according to the invention are ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors and camptothecins.
In a preferred embodiment the at least one active agent is selected from the group consisting of proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogues, organic molecules having a molecular weight up to 1000 g/mol and combinations thereof.
Any known protein is in general suitable. Exemplarily proteins include glycoproteins and apolipoproteins. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins known to those of skill in the art and variants and fragments thereof and to apolipoprotein agonists, analogues, or fragments thereof as well as chimeric construction of an apolipoprotein. Apolipoproteins utilized in the invention also include recombinant, synthetic, semi- synthetic or purified apolipoproteins.
Any known peptide is in general suitable. The term peptide according to the present invention includes peptidomimetic. The peptide or peptidomimetic can be about 5 to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a-defensin, -defensin orbactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T.
In one embodiment, a targeting peptide tethered to an iRNA agent and/or the carrier oligomer can be an amphipathic a-helical peptide. Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D- or L-peptides; a-, fy, or y-peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
Any known carbohydrate is in general suitable. Exemplarily carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid.
As described herein, the compositions of the present invention are particularly useful for the delivery of nucleic acids or nucleic acid analogues, including, e.g., siRNA molecules, mRNA molecules, plasmids, micro RNA, antagomirs, aptamers, and ribozymes. Therefore, the compositions of the present invention may be used to modulate the expression of target genes and proteins both in vitro and in vivo by contacting cells with a composition of the present invention associated with a nucleic acid that reduces target gene expression, e.g., an siRNA or micro RNA, or a nucleic acid that may be used to increase expression of a desired protein, e.g., an mRNA or a plasmid encoding the desired protein.
Any known nucleic acids and nucleic acid analogues or plasmids are in general suitable. Their methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor or in vitro transcription. Methods of synthesizing DNA and RNA nucleotides are widely used and well known in the art.
Nucleic acids and nucleic acid analogues include polymers containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- or triple-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of linear DNA, circular DNA, plasmid DNA (pDNA), antisense molecules, PCR products, or vectors. RNA may be in the form of chemically modified or unmodified messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA) comprising at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA) and combinations thereof. The nucleic acids may include one or more oligonucleotide modification.
Nucleic acids of the present invention may be of various lengths, generally dependent upon the particular form of nucleic acid. For example, in particular embodiments, plasmids or genes may be from about 1,000 to 100,000 nucleotide residues in length. In particular embodiments, oligonucleotides may range from about 10 to 100 nucleotides in length. In various related embodiments, oligonucleotides, single -stranded, doublestranded, and triple-stranded, may range in length from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length. The term "circular DNA" includes any DNA that forms a closed loop and has no ends. Examples of circular DNA are plasmid DNA, minicircle DNA and doggybone DNA (dbDNA).
For plasmid DNA, preparation for use with embodiments of this invention commonly utilizes, but is not limited to, expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art. Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.
In preferred embodiments, the present invention specifically refers to compositions for the delivery of mRNA or siRNA molecules.
For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequencespecific mRNA. In vitro transcription describes a process of template- directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification.
Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art.
The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include phenol/chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography, silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3 ’ extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques to remove these dsRNA contaminants have been developed and are known in the art including but not limited to scale able HPLC purification. HPLC purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo.
A significant variety of modifications have been described in the art which are used to alter specific properties of in vitro transcribed mRNA, and improve its utility. These include, but are not limited to modifications to the 5 ’ and 3 ’ termini of the mRNA. Endogenous eukaryotic mRNA typically contain a cap structure on the 5'- end of a mature molecule which plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation. Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5 '-cap contains a 5 '-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.
Multiple distinct cap structures can be used to generate the 5'-cap of in vitro transcribed synthetic mRNA. 5’-capping of synthetic mRNA can be performed co- transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine -guanine linkage where one guanine contains an N7 methyl group as well as a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this co- transcriptional process and the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5'-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 ’-cap which have enhanced binding of cap binding proteins, increased half-life, and reduced susceptibility to 5' endonucleases and/or reduced 5' decapping. Numerous synthetic 5 ’-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.
On the 3 ’-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly-A tail has been extensively shown to enhance both translational efficiency and stability of mRNA.
Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase. The first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template. The latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 ’termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length. 5 ’-capping and 3 ’-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (Epicenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.
In addition to 5 ’ cap and 3 ’ poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency of translation and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structure and nucleoside modifications since most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus rendering it immunostimulatory which in turn can inhibit effective mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity. The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA, e.g., as disclosed in US 2012/0251618. In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.
Other components of mRNA which can be modified to provide benefit in terms of translatability and stability include the 5' and 3’ untranslated regions (UTR). Optimization of the UTRs (favourable 5’ and 3’ UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA. In one embodiment the RNA is a self-amplifying RNA. A self-amplifying RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself). A self-amplifying RNA molecule is thus in certain embodiments: a (+) strand molecule that can be directly translated after delivery to a cell, and this translation provides for an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded protein, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein. The overall result of this sequence of transcriptions is an amplification in the number of the introduced self-amplifying RNAs and so the encoded protein becomes a major polypeptide product of the host cells.
In one embodiment the RNA is a circular RNA (circRNA) is a type of single-stranded RNA which, unlike linear RNA, forms a covalently closed continuous loop by joining the 3' and 5' ends normally present in an RNA molecule. Like mRNA, circRNA can be designed to encode and express proteins. In particular embodiments, the oligonucleotide (or a strand thereof) of the present invention specifically hybridizes to or is complementary to a target polynucleotide.
In one embodiment the RNA is a hairpin siRNA have a duplex region equal to or at least 17, 18, 19, 29, 21 , 22, 23, 24, or 25 nucleotide pairs. The duplex region will may be equal to or less than 200, 100, or 50, in length. In certain embodiments, ranges for the duplex region are 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may have a single strand overhang or terminal unpaired region. In certain embodiments, the overhangs are 2 to 3 nucleotides in length. In some embodiments, the overhang is at the sense side of the hairpin and in some embodiments on the antisense side of the hairpin.
In one embodiment the RNA is a SiRNA. SiRNAs are RNA duplexes normally 16 to 30 nucleotides long that can associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts, therefore siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA function through a natural mechanism evolved to control gene expression through non-coding RNA.
A "single strand siRNA compound" as used herein, is an siRNA compound which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include a hairpin or pan-handle structure. Single strand siRNA compounds may be antisense with regard to the target molecule.
A single strand siRNA compound may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand siRNA compound is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.
A "double stranded siRNA compound" as used herein, is an siRNA compound which includes more than one, and in some cases two, strands in which interchain hybridization can form a region of duplex structure. The antisense strand of a double stranded siRNA compound may be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length. As used herein, term "antisense strand" means the strand of an siRNA compound that is sufficiently complementary to a target molecule, e.g., a target RNA.
The sense strand of a double stranded siRNA compound may be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50, nucleotides in length. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
The double strand portion of a double stranded siRNA compound may be equal to or at least, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It may be equal to or less than 200, 100, or 50, nucleotides pairs in length.
Ranges may be 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
In many embodiments, the siRNA compound is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller siRNA compounds, e.g., siRNAs agents.
The sense and antisense strands may be chosen such that the double-stranded siRNA compound includes a single strand or unpaired region at one or both ends of the molecule. Thus, a double-stranded siRNA compound may contain sense and antisense strands, paired to contain an overhang, e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. Some embodiments will have at least one 3' overhang. In one embodiment, both ends of an siRNA molecule will have a 3' overhang. In some embodiments, the overhang is 2 nucleotides.
In certain embodiments, the length for the duplexed region is between 15 and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA compound range discussed above. siRNA compounds can resemble in length and structure the natural Dicer processed products from long dsiRNAs. Embodiments in which the two strands of the siRNA compound are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and a 3' overhang are also within the invention.
The siRNA compounds described herein, including double-stranded siRNA compounds and singlestranded siRNA compounds can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted. As used herein, the phrase "mediates RNAi" refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., a siRNA compound of 21 to 23 nucleotides.
In one embodiment, an siRNA compound is "sufficiently complementary" to a target RNA, e.g., a. target mRNA, such that the siRNA compound silences production of protein encoded by the target mRNA. In another embodiment, the siRNA compound is "exactly complementary" to a target RNA, e.g., the target RNA and the siRNA compound anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to atarget RNA. Moreover, in certain embodiments, the siRNA compound specifically discriminates a single-nucleotide difference. In this case, the siRNA compound only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.
In addition to conventional siRNA, dicer substrate siRNA can be employed as a less immunogenic alternative. DsiRNA is 25 to 30 nucleotides in length, and after cellular uptake is further cleaved and processed by the Dicer enzyme converting it into the active form which then associates with the RISC.
Antisense RNA directed to a target polynucleotide. The term "antisense RNA" or simply "antisense" is meant to include RNA that are complementary to a targeted polynucleotide sequence. Antisense RNA are single strands of RNA that are complementary to a chosen sequence, e.g., a target gene mRNA. Antisense RNA are thought to inhibit gene expression by binding to a complementary mRNA. Binding to the target mRNA can lead to inhibition of gene expression either by preventing translation of complementary mRNA strands by binding to it, or by leading to degradation of the target mRNA. In particular embodiments, antisense RNA contains from about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense RNA that may not be exactly complementary to the desired target gene.
Micro RNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals, but are not translated into protein. Processed miRNAs are single stranded 17 to 25 nucleotide (nt) RNA molecules that become incorporated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation.
In one embodiment the RNA is transfer RNA (tRNA). Transfer RNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length that serves as the physical link between the mRNA and the amino acid sequence of proteins. Transfer RNA does this by carrying an amino acid to the protein synthesizing machinery of a cell called the ribosome. Complementation of a 3-nucleotide codon in a messenger RNA (mRNA) by a 3-nucleotide anticodon of the tRNA results in protein synthesis based on the mRNA code. As such, tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code.
In one embodiment, the nucleic acid is a single guide RNA applied to direct CRISPR/Cas9 mediated gene editing. The single guide RNA hybridizes with a target sequence in the genome of a cell and complexes with the Cas9 protein at the target site initiating single or double strand breaks.
In one embodiment the at least one active agent is selected from antagomirs, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermirs, miRNA mimics, antimir or miRNA inhibitors and UI adaptors.
Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-0-methylation of sugar, phosphorothioate backbone and, for example, a cholesterol-moiety at 3'-end.
Aptamers are nucleic acid or peptide molecules that bind to a particular molecule of interest with high affinity and specificity. DNA or RNA aptamers have been successfully produced which bind many different entities from large proteins to small organic molecules. Aptamers may be RNA or DNA based, and may include a riboswitch. A riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene's activity. The aptamer may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. Further, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers" containing a consensus sequence derived from comparing two or more known aptamers to a given target.
Ribozymes are RNA molecules complexes having specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Nucleic acids associated with lipid particles of the present invention may be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-or double- stranded) capable of inducing an immune response when administered to a subject, which may be a mammal or other patient.
Because transcription factors recognize their relatively short binding sequences, even in the absence of surrounding genomic DNA, short oligonucleotides bearing the consensus binding sequence of a specific transcription factor can be used as tools for manipulating gene expression in living cells. This strategy involves the intracellular delivery of such "decoy oligonucleotides", which are then recognized and bound by the target factor. Occupation of the transcription factor's DNA-binding site by the decoy renders the transcription factor incapable of subsequently binding to the promoter regions of target genes.
A supermir refers to a single stranded, double stranded or partially double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or modifications thereof, which has a nucleotide sequence that is substantially identical to a miRNA and that is antisense with respect to its target. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intemucleoside (backbone) linkages and which contain at least one non-naturally-occurring portion which functions similarly. Such modified or substituted oligonucleotides are preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. miRNA mimics represent a class of molecules that can be used to imitate the gene silencing ability of one or more miRNAs. Thus, the term "microRNA mimic" refers to synthetic non-coding RNAs (i.e., the miRNA is not obtained by purification from a source of the endogenous miRNA) that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics can be designed as mature molecules (e.g., single stranded) or mimic precursors (e.g., pri- or pre-miRNAs).
The terms "antimir," "microRNA inhibitor," "miR inhibitor," or "inhibitor," are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the ability of specific miRNAs. In general, the inhibitors are nucleic acid or modified nucleic acids in nature including oligonucleotides comprising RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNAs), or any combination of the above. Modifications include 2' modifications and intemucleotide modifications (e.g., phosphorothioate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization, or potency. In addition, miRNA inhibitors can comprise conjugates that can affect delivery, intracellular compartmentalization, stability, and/or potency. Inhibitors can adopt a variety of configurations including single stranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpin designs, in general, microRNA inhibitors comprise contain one or more sequences or portions of sequences that are complementary or partially complementary with the mature strand (or strands) of the miRNA to be targeted, in addition, the miRNA inhibitor may also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequences may be the reverse complements of the sequences that are adjacent to the mature miRNA in the pri -miRNA from which the mature miRNA is derived, or the additional sequences may be arbitrary sequences (having a mixture of A, G, C, or U).
U1 adaptors inhibit poly A sites and are bifunctional oligonucleotides with a target domain complementary to a site in the target gene's terminal exon and a 'U1 domain' that binds to the U1 smaller nuclear RNA component of the U1 snRNP. U1 snRNP is a ribonucleoprotein complex that functions primarily to direct early steps in spliceosome formation by binding to the pre-mRNA exon- intron boundary. Nucleotides 2- 11 of the 5'end of U1 snRNA base pair bind with the 5'ss of the pre mRNA. In one embodiment, oligonucleotides of the invention are U1 adaptors.
In a preferred embodiment the at least one active agent is selected from the group consisting of linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA (circRNA) comprising at least one coding sequence; small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single guide RNA (sgRNA) or viral RNA (vRNA); and combinations thereof.
In one embodiment the at least one active agent is an organic molecule having a molecular weight up to 1000 g/mol, also referred to as small molecule in the pharmaceutical field, preferably the organic molecule is selected from paclitaxel, doxorubicin, irinotecan, vincristine and oxaliplatin.
The composition according to the present invention can further comprise a compound selected from a lipid, different from the compound of formula (I), like an ionizable lipid, a cationic lipid, a neutral lipid or a structural lipid, a sterol, or a sterol derivative; a buffering agent, a pharmaceutically acceptable salt, a cryoprotectant or any combination thereof.
Suitable lipids, different to the compound of formula (I), according to the present invention, which can be further present are for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides or mixtures thereof. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In one embodiment, lipids containing saturated fatty acids with carbon chain lengths in the range of CIO to C20 are preferred. In one embodiment, lipids with mono- or diunsaturated fatty acids with carbon chain lengths in the range of CIO to C20 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Preferred lipids are 1,2- Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 -palmitoyl -2 -oleoyl-sn-glycero-3 -phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC) or any related phosphatidylcholine.
Further suitable lipids are composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol as well as sterols, in particular cholesterol and phytosterols. In one embodiment the further lipid is an ionizable lipid, preferably selected from l,2-distearoyl-3- dimethylammonium -propane, l,2-dipalmitoyl-3-dimethylammonium-propane, l,2-dimyristoyl-3- dimethylammonium -propane, l,2-dioleoyl-3 -dimethylammonium -propane, l,2-dioleyloxy-3- dimethylaminopropane, (6Z,9Z,28Z,3 lZ)-heptatriacont-6, 9, 28, 31 -tetraene- 19-yl 4-
(dimethylamino)butanoate, 9-Heptadecanyl 8- { (2 -hydroxyethyl) [6-oxo-6-
(undecyloxy)hexyl] amino (octanoate, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l-yl-l,3- dioxolane-4-ethanamine, [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate). In one embodiment the further lipid is an cationic lipid, preferably selected from salts of 1,2-di-O- octadecenyl-3 -trimethylammonium propane, l,2-dioleoyl-3-trimethylammonium-propane, Nl-[2-((lS)-l- [(3 -aminopropyl)amino] -4 - [di (3 -amino-propyl)amino]butylcarboxamido)ethyl] -3 ,4-di [oleyloxy] - benzamide, N4-cholesteryl-spermine, 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, 0,0’- ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine, l,2-dilauroyl-sn-glycero-3- ethylphosphocholine, l,2-dimyristoyl-sn-glycero-3 -ethylphosphocholine, l,2-dipahnitoyl-sn-glycero-3- ethylphosphocholine, 1 ,2-distearoyl-sn-glycero-3 -ethylphosphocholine, 1 ,2-dioleoyl-sn-glycero-3 - ethylphosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoleoyl-sn- glycero-3-ethylphosphocholine, dimethyldioctadecylammonium, l,2-dimyristoyl-3 -trimethylammonium - propane, I,2-dipalmitoyl-3 -trimethylammonium -propane, l,2-stearoyl-3 -trimethylammonium -propane, N- (4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium and 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol. The salt can be any pharmaceutically acceptable salt and is preferably a fluoride or chloride salt.
Further lipids suitable in the compositions of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
In one embodiment the further lipid is selected from phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and [3- acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
In one embodiment the further lipid is selected from polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate), Myq52 (Polyoxyethylene (40) stearate), and Brij S10 (Polyoxyethylene (10) stearyl ether) or combinations thereof. These lipids are known in the field as stabilizing agents and can be present in the compositions of the present invention in addition to compound (I) of the present invention and any further lipid described herein.
A cryoprotective agent is an agent that protects a composition from experiencing adverse effects upon freezing and thawing. For example, in the present invention, cryoprotective agents such as polyols and/or carbohydrates, among others, may be added to prevent substantial particle agglomeration. A buffering agent can be included as well. Suitable buffering agents are for example phosphate, acetate, citrate, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid, amino acids, and other organic compounds; antioxidants including ascorbic acid and methionine.
Additionally, at least one of the following additives can be further present in the composition: preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates including monosaccharides, disaccharides, and other sugar compounds like glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes), vehicles, binders, disintegrants, immunological adjuvants like a cell penetrating peptide, for example human lactoferrin protein or a fragment thereof, Tat, Ant, Rev, FHV, HSV-1 protein VP22, C6, C6M1, PF20, NAP, POD, polyarginine, polylysine, PTD-5, Transportan, MAP, TP10, Pep-7, Azurin p 18, Azurin p28, hCT18-32, Bac 7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, Melittin, Penetratin, EB1, MPG, CADY, Pep4, preferably a human lactoferrin protein or a fragment thereof, fdlers (diluents), lubricants, glidants (flow enhancers), compression aids, colors, sweeteners, suspending/dispersing agents, film formers/coatings, flavors, printing inks.
In one embodiment the composition, preferably the lipid nanoparticle, comprises the at least one active agent to the compound of formula (I) in a weight to weight ratio of 1:0.01 to 1: 100.
In one embodiment the composition, preferably the lipid nanoparticle, comprises at least one further compound selected from one or more lipids, different from the compound of formula (I); a buffering agent; a pharmaceutically acceptable salt, different from the buffering agent; a cryoprotectant or any combination thereof. In a preferred embodiment the composition, preferably the lipid nanoparticle, further comprises one or more lipids, different from the compound of formula (I), more preferably further comprises one, two or three additional lipids, different from the compound of formula (I). In a preferred embodiment the composition, preferably the lipid nanoparticle, consists of the compound of formula (I), at least one active agent and one or more lipids, different from the compound of formula (I), more preferably one, two or three additional lipids, different from the compound of formula (I).
In one embodiment of the composition, preferably a lipid nanoparticle, the compound of formula (I) is present in a ratio of about 0.1 to about 10 mol%, based on the total lipid content. In one embodiment the compound of formula (I) is present in a ratio of greater than 10 mol%, based on the total lipid content. In one embodiment the compound of formula (I) is present in a ratio of 0.5 mol% to 5 mol%, based on the total lipid content. In some embodiments the compound of formula (I) is present in a ratio of 1.5 mol%. In one embodiment of the composition, preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is a cationic lipid, the cationic lipid is preferably present in a ratio of about 10 to about 80 mol%, based on the total lipid content. In one embodiment the cationic lipid is present in a ratio of about 50 mol%, based on the total lipid content.
In one embodiment of the composition, preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is an ionizable lipid, the ionizable lipid is preferably present in a ratio of about 10 to about 80 mol%, based on the total lipid content. In one embodiment the ionizable lipid is present in a ratio of about 50 mol%, based on the total lipid content.
In one embodiment of the composition, preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is a structural lipid, also known as “helper lipid”, with neutral or negative net charge, the structural lipid is preferably present in a ratio of about 10 to about 40 mol%, based on the total lipid content. In one embodiment the structural lipid is present in a ratio of about 10 mol%, based on the total lipid content.
In one embodiment of the composition, preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is a sterol such as cholesterol or phytosterols or derivatives thereof, the sterol is preferably present in a ratio of about 10 to about 60 mol%, based on the total lipid content. In one embodiment the sterol is present in a ratio of about 35 to about 41 mol%, based on the total lipid content. In one embodiment the sterol is present in a ratio of about 38.5 mol%%, based on the total lipid content.
In one embodiment of the composition, preferably a lipid nanoparticle, where a further lipid, different from the compound of formula (I), is contained and is a stabilizing agent, the stabilizing agent is preferably present in a ratio of about 0 to about 10 mol%, based on the total lipid content.
In one embodiment of the composition, preferably a lipid nanoparticle, where at least one buffering agent is present, the at least one buffering agent is present in a molar concentration of 0. 1 mM to 1000 mM with respect to the total volume of the solution in which the composition is dispersed.
In one embodiment of the composition, preferably a lipid nanoparticle, where at least one cryoprotectant is present, the at least one cryoprotectant is present in a mass concentration of 0.1 wt% to 50 wt% with respect to the total volume of the solution in which the composition is dispersed. The compositions of the present invention may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intracistemal injection or infusion techniques.
Compositions, preferably pharmaceutical compositions of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient in some embodiments take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of formula (I) of the present invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. In some embodiments, the composition to be administered will, in any event, contain a therapeutically effective amount of a compound of formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure.
A composition, preferably pharmaceutical composition of the present invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalatory administration.
When intended for oral administration, the composition, preferably pharmaceutical composition of the present invention is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the composition, preferably pharmaceutical composition, may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, or wafer. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition of some embodiments is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
The composition, preferably pharmaceutical composition, of the present invention may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to a compound of formula (I), one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included.
The liquid composition, preferably liquid pharmaceutical compositions of the present invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
The composition, preferably pharmaceutical composition of the present invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
The composition, preferably pharmaceutical composition, of the present invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. A composition for rectal administration may contain an oleaginous base as a suitable non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
The composition, preferably pharmaceutical composition, of the present invention may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
The composition, preferably pharmaceutical composition, of the present invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of formula (I) of the present invention may be delivered in single phase, bi-phasic, or tri -phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit.
In one preferred embodiment the composition is a lipid nanoparticle. In particular embodiments, the active agent is encapsulated within an aqueous interior of the lipid nanoparticle. In other embodiments, the active agent is present within one or more lipid layers of the lipid nanoparticle. In other embodiments, the active agent is bound to the exterior or interior lipid surface of a lipid nanoparticle. Lipid nano particles include, but are not limited to, liposomes. As used herein, a liposome is a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar. When complexed with nucleic acids, lipid particles may also be lipoplexes, which are composed of cationic lipid bilayers sandwiched between DNA layers.
The lipid nanoparticles of present invention may be formulated as a pharmaceutical composition, e.g., which further comprises a pharmaceutically acceptable diluent, excipient, or carrier, such as physiological saline or phosphate buffer, selected in accordance with the route of administration and standard pharmaceutical practice.
In particular embodiments, lipid nanoparticles of the invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Generally, normal saline will be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other salt containing carriers, the carrier is preferably added following lipid particle formation. Thus, after the lipid nanoparticles are formed, the compositions can be diluted into pharmaceutically acceptable carriers such as normal saline.
The resulting pharmaceutical preparations may be sterilized by conventional, well known sterilization techniques. The aqueous solutions can then be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the lipidic suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free -radical quenchers, such as a-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
The term "lipid nanoparticle" refers to particles having at least one dimension on the order of nanometers (e.g, 1-1,000 nm) which include one or more of the compounds of formula (I). In some embodiments, lipid nanoparticles comprising at least one compound of formula (I)) are included in a formulation that can be used to deliver a therapeutic agent, such as a nucleic acid (e.g, mRNA) to a target site of interest (e.g, cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles comprise a compound of formula (I) and a nucleic acid. In some embodiments, the therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.
In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 run to about 150 nm, from about 40 run to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, measured via dynamic light scattering preferably according to ISO 22412:2017, whereby the samples were diluted 1: 10 in RNAse free water corresponding to an RNA concentration of 5 ng/pL. Preferably the measurements can be conducted with a Malvern Zetasizer NanoZS.
Some techniques of administration can lead to the systemic delivery of certain active agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an active agent is exposed to most parts of the body. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.
"Local delivery" as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumour, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude a systemic pharmacological effect.
Compositions of the present invention may also be administered simultaneously with, prior to, or after administration of one or more other active agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the present invention and one or more additional active agents, as well as administration of the composition of the present invention and each active agent in its own separate pharmaceutical dosage formulation. For example, a composition of the present invention and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Where separate dosage formulations are used, the compounds of formula (I) of the present invention and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens.
The compositions, preferably pharmaceutical compositions, of the present invention may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile, distilled water or other carrier so as to form a dispersion. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non- covalently interact with the compound of the disclosure so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
The compositions of the present invention are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic agent employed; the metabolic stability and length of action of the therapeutic agent; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In a preferred embodiment the composition of the present invention is a pharmaceutical composition for the treatment of illness in humans. In a further preferred embodiment, the composition of the present invention is a pharmaceutical composition for the treatment of illness in mammals.
As used herein, the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent. Whenever the term “optional”, “optionally” or “may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. I.e., in the context of the present invention the term “a polypeptide comprising proline, alanine and, optionally, serine” may refer to a polypeptide that either consists of proline and alanine, or may refer to a polypeptide that consists of proline, alanine, and serine.
As used herein, and unless explicitly indicated otherwise or contradicted by context, the terms “a”, “an” and “the” are used interchangeably with “one or more” and “at least one”. Thus, for example, a composition comprising “a” conjugate of the invention can be interpreted as referring to a composition comprising “one or more” conjugates of the invention.
As used herein, the term "about" preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated. If the term “about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint ±10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint ±5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. If the term “about” is used in connection with the endpoint of an open-ended range, it preferably refers to the corresponding range starting from the lower endpoint -10% or from the upper endpoint +10%, more preferably to the range starting from the lower endpoint -5% or from the upper endpoint +5%, and even more preferably to the open-ended range defined by the exact numerical value of the corresponding endpoint. If the term “about” is used in connection with a parameter that is quantified in integers, such as the number of amino acid residues in a protein, the numbers corresponding to ±10% or ±5% of the indicated numerical value are to be rounded to the nearest integer (using the tie-breaking rule “round half up”).
As used herein, the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, ...” In addition thereto, this term also includes the narrower meanings of “consisting essentially of’ and “consisting of’. For example, the term “A comprising B and C” has the meaning of “A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., “A containing B, C and D” would also be encompassed), but this term also includes the meaning of “A consisting essentially of B and C” and the meaning of “A consisting of B and C” (i.e., no other components than B and C are comprised in A).
As used herein the term “production” (or “producing”, “produces”, “produce”), unless explicitly indicated otherwise may be used interchangeably with the term “preparation” (or “preparing”, “prepares”, “prepare”, respectively).
The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures, by practitioners of the chemical, biological and biophysical arts.
The “treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The “treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the “treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
The term “prevention” of a disorder or disease, as used herein, is also well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators or biomarkers. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term “prevention” comprises the use of a conjugate of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
In the context of the present invention the groups (A, B, C, and D) of the compound of formula (I) may herein above and below be referred to as “A”, “B”, “C”, and “D”, also, they may herein above and below further be referred to as “group A”, “group B”, “group C”, and “group D”. Further the terms “group” and “groups” may be used interchangeably with the terms “moiety” and “moieties” in the context of the compound of formula (I). Accordingly, the groups (A, B, C, and D) of the compound of formula (I) may herein above and below also be referred to “moiety A”, “moiety B”, “moiety C”, and “moiety D”. Similarly, the compounds A-B-C-D, A-C-D, L-B-C-D, and L-C-D may either be referred to as “A-B-C-D”, “A-C-D”, “L-B-C-D,” and “C-D” or as “compound A-B-C-D”, “compound A-C-D”, “compound L-B-C-D”, and “compound L-C-D”, respectively. Further, compound A-B-C-D and compound A-C-D may separately or collectively be referred to as “a compound of formula (I)”.
It is to be understood that the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments. In particular, the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in the P/A peptides and the conjugates according to the invention.
In this specification, a number of documents including patents, patent applications and scientific literature are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates.
In the following table (Table 1), the herein disclosed, non-limiting amino acid sequences are also reproduced. However, these sequences are also comprised in the appended sequence listing which is a specific part of this disclosure and the description of the present invention. In the following table, all amino acid sequences are provided in the one letter code. Further, the letter “X” at position 1 indicates an N- terminal pyroglutamate (“Pga” or “Pyrrolidone carboxylic acid”; used herein synonymously) protecting group and the letter “X” at position 22 or position 42 indicates a C-terminal s-aminohcxanoic acid (“Ahx” or “acp”; used herein synonymously) residue (linker).
Table 1: Herein disclosed, non-limiting amino acid sequences
Figure imgf000071_0001
Figure imgf000072_0001
The present invention is further described by reference to the following non-limiting figures. The figures show:
Figure 1: Reaction scheme for the coupling of PAS-polypeptides to di(tetradecyl)amine. In the presence of the non-nucleophilic base JV,JV-diisopropylethylamine (DIPEA, Hiinig’s base) and with a mixture of dichloromethane (DCM) and methanol as solvent the N-terminally protected PAS-polypeptide (e.g. Pga-PAS40) is activated via its C-terminus with 2-( I H-bcnzotriazol- l -yl)- ' ' " "- tetramethyluronium tetrafluoroborate (TBTU). The resulting hydroxybenzotriazole active ester of the peptide subsequently reacts selectively with the single amino group of di(tetradecyl)amine while free 1- Hydroxybenzotriazole (HOBt) is released.
Figure 2: Exemplary chromatogram from the RP-HPLC purification of Pga-PAS40- di(tetradecyl)amine. The flowthrough contains unreacted educts and side products, while Pga-PAS40- di(tetradecyl)amine eluted in a broad single peak during the DCM gradient.
Figure 3: Characterization of Pga-PAS20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 4: Characterization of Pga-PAS40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 5: Characterization of Pga-PAS100-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 6: Characterization of Pga-P/A20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 7: Characterization of Pga-P/A40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 8: Characterization of Pga-PAS100-K-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 9: Characterization of RNA encapsulation efficiency of PEG2k-, P/A-, and PAS-LNPs by agarose gel electrophoresis analysis. LNPs were prepared as described in Example 3 and applied to the wells of a 1% agarose gel and run on the E-Gel power snap electrophoresis system by Thermo Fisher Scientific, as described in Example 6. Lane M shows RiboRuler High Range RNAladder, lane 1 shows 20 pl of FLux mRNA as positive control and lanes 3 to 7 show Entries g to 1 from Example 3, respectively. Only in lane 1 a single clear band, corresponding to FLux mRNA, can be observed. Consequently, all tested LNPs (Entries g to 1 from Example 3) effectively encapsuled the enclosed mRNA. Figure 10: Characterization of transfection efficiency of PEG2k-, P/A-, and PAS-LNPs by luciferase assay. LNPs were prepared as described in Example 3 and transfected into HeLa, Jurkat, A549, HepG2, C2C12 cell lines according to the methods described in Example 8. jetMessenger and jetPEI were used as positive controls and untreated cells were used as negative control. One day after transfection, transfection efficiency was determined using the Luciferase Assay System (Promega GmbH, Walldorf, Germany). The luminescence signal was quantified with the Infinite 200 PRO multiplate reader. While transformation efficiencies varied for different cell lines, all tested LNPs showed high transformation efficiencies, in parts higher than the positive controls (jetMessenger and jetPEI).
Figure 11: Characterization of Pga-PAS40-di(decyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 12: Characterization of Pga-PAS40-di(dodecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 13: Characterization of Pga-PAS40-di(hexadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 14: Characterization of Pga-PAS40-di(octadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom).
Figure 15: Reaction scheme for the coupling of PAS-polypeptides with C-terminal cysteine to di(acyl)amines. Using a mixture of dichloromethane (DCM) and methanol as solvent the N-terminally protected PAS-Cys polypeptide (e.g., Pga-PAS20C) is activated via its thiol group using the bispecific crosslinker succinimidyl-tra«5-4-(N-maleimidylmethyl)cyclohexane-l-carboxylate (SMCC). In the presence of the non-nucleophilic base ' '-diisopropylcthylaminc (DIPEA, Hiinig’s base) the resulting SMCC active ester of the peptide reacts selectively with the single secondary amino group of di(tetradecyl)amine while free N-hydroxysuccinimide (NHS) is released (left reaction scheme). Alternatively, said bispecific crosslinker may first react with the single secondary amino group of di(tetradecyl)amine and only then the resulting intermediate reacts with the thiol group of the PAS-Cys polypeptide (right reaction scheme). The resulting compound comprises a PAS-polypeptide linked to a di(acyl)amine via a linker (i.e., /raw.s-4-(N-malcimidylmcthyl)cyclohcxanc- 1 -carboxylate (MCC).
Figure 16: Characterization of Pga-PAS20C-MCC-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (center) and deconvoluted ESI-MS spectrum (bottom). Examples
Certain embodiments of the invention will now be described with reference to the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.
Example 1: Coupling of di(tetradecyl)amine with P/A- and PAS-peptides via their single carboxy group
A 30 mM solution of the N-terminally blocked peptides Pga-P/A20-Ahx (P/A20; SEQ ID No.: 1, Almac group, London, UK), Pga-P/A40-Ahx (P/A40; SEQ ID No.: 2, Almac), Pga-PAS20 (PAS20; SEQ ID No.: 3, XL-protein, Freising, Germany), Pga-PAS40 (PAS40; SEQ ID No.: 4, XL-protein) or Pga-PASIOO (PAS 100; SEQ ID No.: 5, XL-protein) was prepared in a dichloromethane (DCM)Zmethanol mixture (5: 1). 1.5 molar equivalents of di(tetradecyl)amine (Ambeed, Arlington Hts, IL, USA) were dissolved as a solid in the P/A- or PAS-polypeptide solution, followed by addition of 2 molar equivalents of DIPEA base (N,N- Diisopropylethylamine; Merck) as a liquid and 1.5 molar equivalents of TBTU (2-(lH-Benzotriazole-l- yl)-l,l,3,3-tetramethylaminium tetrafluoroborate; 400 mM stock solution in dimethylformamide; DMF; Carl Roth, Karlsruhe, Germany) were added and the mixture was incubated under an argon cloud for 1 h at 25 °C (Figure 1). Alternatively, the P/A- or PAS-polypeptide was dissolved in methanol alone and a solution of di(tetradecyl)amine in DCM was added. To precipitate the unreacted di(tetradecyl)amine, 8 volumes of a 10: 1 methanol/water mixture were added and the mixture was incubated on ice for 20 min. The precipitate was removed by fdtration (Acrodisc 1 pm glass fiber syringe filter, PALL, Port Washingtom, NY, USA) and the coupling product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a VP 250/10 Nucleodur C18 Gravity column (Macherey-Nagel, Duren, Germany). For the 20-mer and 40-mer P/A- or PAS-polypeptide-coupled lipids, the mobile phase was 85% (v/v) MeOH, 10% (v/v) H2O, 5% (v/v) DCM, 0.1% (v/v) formic acid and the coupling products were eluted in a 0-100% gradient with MeOH/DCM (1: 1) (Figure 2). For Pga-PAS100-di(tetradecyl)amine 70% MeOH, 30%H20, 0.1% formic acid was used as mobile phase and a gradient with 0-100% with 90% MeOH (v/v), 10% (v/v) DCM and 0. 1% (v/v) formic acid was used for the elution.
The elution fractions were pooled, concentrated using a SpeedVac concentrator and diluted 1: 10 in H2O before freezing and lyophilization. Analytical reverse phase-HPLC runs were conducted on aResourceRPC polystyrene/divinylbenzene column (1 ml column volume, Cytiva) using the mobile phases described above for the preparative RP-HPLC runs of the respective P/A- or PAS-polypeptide-di(tetradecyl)amines. Furthermore, 10-50 pg of the P/A- or PAS— di(tetradecyl)amines was dissolved in 50% acetonitrile in H2O with 0.1% formic acid and electrospray ionization-mass spectrometry (ESI-MS) was performed using a maXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany) in the positive ion mode. Based on the RP-HPLC and ESI-MS data the P/A- and PAS-compounds are clearly uniform, as is evident from the presence of single peaks in all analytical RP-HPLC and ESI-MS analyses, shown in Figures 3 to 7. Example 2: Coupling of di(tetradecyl)amine with a Pga-PASIOO-K peptide via the s-amino group of its C-terminal lysine
The N-terminally protected Pga-PASIOO-K peptide (PAS100-K; SEQ ID No.: 6) was dissolved in 100 mM sodium bicarbonate buffer (pH 8.4) and iodoacetylated at the s-amino group of its C-terminal lysine side chain by incubation with iodoacetic acid N-hydroxy succinimide ester (CAS 39028-27-8, Apollo Scientific, Bredbury, UK) at 25 °C for 1 h. After dialysis against ultra-pure water and lyophilization, the activated PAS100-K peptide was dissolved in MeOH/DCM (1:7). To achieve alkylation, the 10-fold molar amount of di(tetradecyl)amine and the 2-fold molar amount of DIPEA were added and the coupling reaction was allowed to proceed for 24 h at 25 °C. The addition of 8 vol. eq. of H2O/MeOH (1: 10) and incubation on ice for 20 min resulted in the precipitation of unreacted di(alkyl)amine, which was removed by filtration (Acrodisc glass fiber 1 pm syringe filter, PALL). The coupling product was purified by RP-HPLC on a VP 250/10 Nucleodur C18 Gravity column (Macherey-Nagel, Duren, Germany). A mixture of 70% (v/v) MeOH, 30% (v/v) H2O and 0.1% (v/v) formic acid was used as mobile phase. The alkylated PAS- polypeptide was eluted from the Cl 8 column using a gradient of 0-100% of 90% MeOH (v/v), 10% (v/v) DCM and 0.1% formic acid. After evaporation of DCM under vacuum, the eluate was diluted in H2O and lyophilized. Based on RP-HPLC and ESI-MS analytics the Pga-PASlOO-K-compounds are clearly uniform, as is evident from the presence of a single peak in the analytical RP-HPLC and ESI-MS analysis, shown in Figure 8.
Example 3: Preparation of LNPs
CleanCap® Flue mRNA was obtained from TriLink BioTechnologies (San Diego, CA, USA). D-Lin-MC3-DMA was obtained from MedCHemExpress (Monmouth Junction, NJ, USA), cholesterol was purchased from Merck KGaA, DSPC was obtained from NOF (White Plains, NY, USA). All cell lines were supplied by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany).
An aqueous solution (or an aqueous phase) containing 0. 133 g/1 FLuc mRNA and 11 mM acetic acid was mixed with an ethanolic solution (or an ethanolic phase) containing 9.43 mM total lipid (50 mol% DLin- MC3-DMA, 38.5 mol% cholesterol, 10 mol% DSPC, 1.5 mol% PEG2k-compound, P/A-compound or PAS-compound) at a 3: 1 volume ratio by pipetting. The crude colloidal LNP dispersions were dialyzed against phosphate-buffered saline (PBS) for 3 h (3x buffer exchange). Purified colloidal LNP dispersions (i.e., LNPs or purified colloidal dispersions) were stored at 4 °C until further use.
The purified LNPs remained stable at 4 °C for at least 4 days. No signs of turbidity or precipitation were observed for any of the tested LNPs. Example 4: Determination of particle diameter (Z-average), polydispersity index (PDI), and Zetapotential of PEG2k-, P/A-, and PAS-LNPs
Results are shown in Table 2. Measurements were conducted using a Zetasizer NanoZS from Malvern Instruments GmbH (Herrenberg, Germany). A DTS 1070 clear disposable folded capillary cell from Malvern Panalytical GmbH (Kassel, Germany) was used. For particle diameter measurement, samples were diluted 1: 10 in RNAse free water corresponding to an RNA concentration of 5 ng/pl. For Zeta-potential measurement, purified (after dialysis) and crude (before dialysis) colloidal LNP dispersions from Example 3 were diluted 1:30 in RNAse free water corresponding to an RNA concentration of 1.67 ng/pl. Z-average (i.e., mean particle diameter), polydispersity index (PDI; i.e., the width of the fitted Gaussian distribution), as well as average Zeta-potential values were measured before and after dialysis and calculated from the data of at least 10 runs.
Z-average measurements confirm that LNP particle diameter is influenced by the molecular mass of the respective P/A-, and PAS-compounds. P/A20- and PAS20-compounds share a similar molecular mass as the PEG2k-compounds. Surprisingly, however, the corresponding LNPs showed an approx. 10% smaller particle diameter. Similarly, the molecular weight of P/A40- and PAS40-compounds is approx. 55% higher as compared to the PEG2k-compounds, however, the corresponding LNPs share a comparable particle diameter. Collectively, this demonstrates that P/A- and PAS-compounds, surprisingly, result in more compact LNPs.
Poly dispersity indices of PEG2k-, P/A-, and PAS-LNPs was comparable, thus, it is evident that P/A- and PAS-LNPs are similarly uniform as compared to PEG2k-LNPs.
While the average Zeta-potentials were comparable for all tested LNPs before dialysis, after dialysis the PEG2k-LNPs showed a higher Zeta-potential as compared to all tested P/A- and PAS-LNPs. This demonstrates that P/A- and PAS-LNPs have a higher capacity for shielding the charged membrane of LNPs, as compared to PEG2k-LNPs. This is particularly surprising, given the smaller particle diameters of P/A- and PAS-LNPs.
Table 2: Particle diameter, PDI, and Zeta-potential of PEG2k-, P/A-. and PAS-LNPs
Figure imgf000078_0001
Example 5: MTS cell viability assay using PEG2k-, P/A-, and PAS-compounds and -LNPs 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was conducted with He La cells. One day before transfection 10,000 cells were seeded into each well of a
96-well plate in a volume of 100 pl of the respective medium (containing 10% fetal bovine serum (FBS) and 30 pg/ml Gentamicin) and cultured for 24 h at 37 °C and 5% CO2. On day 2, the old medium was removed and 90 pl of fresh medium were added to the cells. For P/A- and PAS-compounds testing, compounds from Examples 1 and 2 were dissolved in sterile water to meet the final concentrations depicted in T able 3, samples were adjusted to a volume of 10 pl. For LNP testing, purified colloidal LNP dispersions from Example 3 were adjusted to an mRNA concentration of 5 to 20 ng/pl using RNAse free water for dilution. 10 pl of the respective diluted samples were added to the cells equaling an amount of 50 to 200 ng mRNA per well in a total volume of 100 pl, results are shown in Table 4. The cells were further incubated for 24 h at 37 °C and 5% CO2. On day 3, cell viability was determined using CellTiter 96 AQueous Non- Radioactive Cell Proliferation Assay (MTS) according to manufacturer’s protocol (Promega GmbH). The absorbance signal (at 490 nm) was quantified with a multiplate reader (Infinite 200 PRO, Tecan, Mannedorf, Switzerland). When 0.01, 0.1, or 1 pg/ml of any PEG2k-, P/A-, or PAS-compound were applied in the MTS assay, only minor reductions in cell viability were observed. However, when 10, or 100 pg/ml of PEG2k-compounds were applied, cell viability dropped to (or below) 0%. In contrast, cell viability remained drastically higher for P/A- and PAS-compounds as compared to PEG2k-compounds at 10 pg/ml, where all tested P/A- and PAS-compounds resulted in cell viability above 69%. Surprisingly, it is thus evident, that P/A- and PAS- compounds are less toxic towards human cells than PEG2k-compounds, when 10 pg/ml of the respective compounds are applied to HeLa cells in the MTS assay. It is, therefore, clear that the inventors surprisingly found that P/A- and PAS-compounds are superior for use as pharmaceutical component as compared to PEG2k-compounds . Viability of HeLa cells was barely affected by any tested dosages of any of the tested PEG2k-, P/A-, or PAS-LNPs. Thus, P/A- and PAS-LNPs are clearly non-toxic to human cells.
Table 3: MTS cell viability assay results for PEG2k-, P/A-, and PAS-compounds
Figure imgf000079_0001
Table 4: MTS cell viability assay results for PEG2k-, P/A-. and PAS-LNPs
Figure imgf000080_0001
Example 6: Characterization of PEG2k-, P/A-, and PAS-LNPs by agarose gel electrophoresis (AGE) RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3 was characterized by agarose gel electrophoresis. Assay was carried out using the E-Gel power snap electrophoresis system by Thermo Fisher Scientific. For the evaluation of the samples a 1% agarose gel with a volume capacity of 20 pL per well was used. Results are summarized in Figure 9. Here, lane “M” contains RiboRuler High Range RNA Ladder, Thermo Fisher Scientific, lane 1 contains mFluc mRNA (negative control), lane 2 contains mRFLuc-MC3 LNP-PEG2k-N,N- di(tetradecyl)acetamide (mRFluc-MC3 LNP l,2-Dimyristoyl-rac-glycero-3 -methoxypoly ethylengly col-2000), lane 3 contains mRFLuc-MC3 LNP-PEG2k-N,N-di(tetradecyl)acetamide, lane 4 contains mRFLuc-MC3 LNP-Pga-P/A20- Ahx-di(tetradecyl)amine, lane 5 contains mRFLuc-MC3 LNP-Pga-P/A40-Ahx-di(tetradecyl)amine, lane 6 contains mRFLuc-MC3 LNP-Pga-PAS20-di(tetradecyl)amine, lane 7 contains mRFLuc-MC3 LNP-Pga-PAS40- di(tetradecyl)amine and lane 8 contains mRFLuc-MC3 LNP-Pga-PAS100-K-di(tetradecyl)amine.
Agarose gel electrophoresis demonstrates full encapsulation of mRNA for all tested LNP compositions (within the detection range of SYBR Safe staining). Thus, based on this assay, LNPs prepared with P/A- or PAS-compounds exhibit similar mRNA encapsulation efficiency as the reference prepared with conventional PEG2k-compounds.
Example 7: Characterization of PEG2k-, P/A-, and PAS-LNPs by RiboGreen Assay
RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3 was further characterized by RiboGreen Assay. The Thermo Fischer Quant-iT RiboGreen RNA Assay Kit was used. The procedure was performed according to manufacturer’s protocol with slight adjustments. Samples were diluted to a theoretical RNA concentration of 0.4 pg/ml using either Tris-EDTA (TE)-buffer or Triton- buffer and added to a 96-well plate at a volume of 100 pl. For dissolution of LNPs in the presence of Tritonbuffer the plate was placed into an incubator for 10 minutes at 37 °C and 5% CO2. 100 pl dye solution were added to each well followed by thorough pipetting. Fluorescence signals were measured with the Infinite 200 PRO microplate reader at excitation and emission wavelengths of 480 nm and 520 nm, respectively. All samples and standards were measured in duplicates.
RNA encapsulation efficiency of all test P/A- and PAS-LNPs was higher as compared to PEG2k-LNPs, as is evident from Table 5. Surprisingly, P/A- and PAS-compounds are, thus, more effective at shielding LNP membranes as compared to PEG2k-LNPs.
Table 5: RiboGreen Assay results for PEG2k-, P/A-, and PAS-LNPs
Figure imgf000081_0001
Example 8: Determination of transfection efficiency of PEG2k-, P/A-, and PAS-LNPs by luciferase assay
Luciferase assay was conducted with several immortal cell lines (HeLa, Jurkat, C2C12, HepG2, A549). Cell lines were grown according to standard cell culture conditions.
Transfection and luciferase assay with adherent cells
HeLa, C2C12, HepG2, and A549 cells were used as adherent cells.
On day one, 10,000 cells per well were seeded into a 96-well plate in a volume of 100 pl of the respective medium (containing 10% FBS and 30 pg/ml Gentamicin) and cultured for 24 h at 37 °C and 5% CO2. Either Dulbecco's Modified Eagle Medium (DMEM), DMEM/F12, or RPMI1640 medium was used. On day two, old medium was removed and 90 pl of fresh medium (without FBS and antibiotics) were added to the cells. The purified colloidal LNP dispersions prepared according to Example 3 were adjusted to an mRNA concentration of 10 ng/pl using RNAse free water for dilution. 10 pl of the respective diluted samples were added to the cells equaling an amount of 100 ng mRNA per well in a total volume of 100 pl. After 4 h, old medium containing residual samples was removed and replaced with 100 pl of fresh medium (containing 10% FBS and 30 pg/ml Gentamicin). Cells were further incubated for 20 h at 37 °C and 5% CO2. On day three, transfection efficiency was determined using the Luciferase Assay System (Promega GmbH, Walldorf, Germany). The luminescence signal was quantified with the Infinite 200 PRO multiplate reader. Transfection and luciferase assay with suspension cells
Jurkat cells were used as suspension cells.
On day one, 50,000 cells per well were seeded into a 96-well plate in a volume of 90 pl of RPMI1640 medium (containing 10% FBS and 30 pg/ml Gentamicin). Samples were adjusted to an mRNA concentration of 10 ng/pl using RNAse free water for dilution. 10 pl of the respective diluted samples were added to the cells equaling an amount of 100 ng mRNA per well in a total volume of 100 pl. Cells were further incubated for 24 h at 37 °C and 5% CO2. On day two, transfection efficiency was determined using the Luciferase Assay System (Promega GmbH). The luminescence signal was quantified with the Infinite 200 PRO multiplate reader.
For all transfection experiments, jetMessenger and jetPEI were used as positive controls. Reagents were prepared according to manufacturer’s protocols and applied at an equal RNA dose per well as the test samples. Results are summarized in Figure 10.
While transformation efficiencies varied for different cell lines, all tested P/A- and PAS-LNPs showed high transformation efficiencies, partly higher than the positive controls (i.e., jetMessenger and jetPEI). Remarkably, P/A20- and PAS20-LNPs showed highest transformation efficiencies among the P/A- and PAS-LNPs and even higher transformation efficiencies for A549, Jurkat, HepG2, and C2C12 cells as compared to PEG2k-LNPs. Collectively, P/A- and PAS-LNPs can efficiently transfect various human cell lines.
Example 9: Coupling of di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine and di(octadecyl)amine with a Pga-PAS40 peptide via its single carboxylate group
A 30 mM solution of the N-terminally blocked Pga-PAS40 peptide (PAS40; SEQ ID No.: 4, XL-protein) was prepared in a dichloromethane (DCM)/methanol mixture (5: 1). 1.5 molar equivalents of di(decyl)amine (TCI Deutschland, Eschborn, Germany) or di(dodecyl)amine (TCI Deutschland) or di(hexadecyl)amine (Ambeed, Arlington Hts, IL, USA) or di(octadecyl)amine (Ambeed, Arlington Hts, IL, USA) was dissolved in the PAS40 solution, followed by addition of 2 molar equivalents of DIPEA base (N,N- diisopropylethylamine; Merck) and 1.5 molar equivalents of TBTU (2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium tetrafluoroborate; 400 mM stock solution in dimethylformamide; DMF; Carl Roth, Karlsruhe, Germany) and the mixture was incubated under an argon cloud for 1 h at 25 °C. To precipitate the unreacted di(alkyl)amine, 8 volumes of a 10: 1 methanol/water mixture were added, and the mixture was incubated on ice for 20 min. The precipitate was removed by filtration (Acrodisc 1 pm glass fiber syringe filter, PALL) and the coupling product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a VP 250/10 Nucleodur C18 Gravity column (Macherey-Nagel) with 85% (v/v) MeOH, 10 % (v/v) H2O, 5 % (v/v) DCM, 0.1 % (v/v) formic acid as mobile phase. The coupling product was eluted in a 0-100 % gradient with MeOH/DCM (1: 1).
The elution fractions were pooled, concentrated using a SpeedVac concentrator and diluted 1: 10 in H2O prior to freezing and lyophilization. Analytical reverse phase-HPLC was conducted on a Gemini 3 pm Cl 8 110 A column (150 x 4.6 mm, Phenomenex, Torrance, CA, USA) using the same mobile phases as described above for the preparative RP-HPLC of the respective PAS-di(alkyl)amines. Furthermore, 10-50 pg of the PAS-di(alkyl)amines was dissolved in 50 % (v/v) acetonitrile in H2O with 0.1 % (v/v) formic acid and electrospray ionization-mass spectrometry (ESI-MS) was performed using a maXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany) in the positive ion mode. All PAS-di(alkyl)amines were obtained in a uniform composition, as was evident from the presence of single peaks in all analytical RP-HPLC and ESI-MS analyses (shown in Figures 11 to 14).
Example 10: Coupling of di(tetradecyl)amine with a Pga-PAS20-C peptide via the thiol group of its C-terminal cysteine residue using a bispecific crosslinker
A 20 mM solution of the N-terminally blocked Pga-PAS20-C peptide (PAS20C; SEQ ID NO: 23, XL- protein) was prepared in a dichloromethane (DCM)/methanol mixture (5: 1). 1 molar equivalent of di(tetradecyl)amine (Ambeed) and 1 molar equivalent of the bispecific crosslinker siiccinimidyl-/raw.s-4- (N-maleimidylmethyl)cyclohexane-l -carboxylate (SMCC, TCI Deutschland) were dissolved in the PAS- polypeptide solution, followed by addition of 1 molar equivalent of DIPEA base (N,N- Diisopropylethylamine; Merck) (Figure 15). The mixture was incubated under an argon cloud for 2 h at 25 °C. To precipitate the unreacted di(alkyl)amine, 8 volumes of a 10: 1 methanol/water mixture were added and the mixture was incubated on ice for 20 min. The precipitate was removed by filtration (Acrodisc 1 pm glass fiber syringe filter, PALL) and the coupling product was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a VP 250/10 Nucleodur Cl 8 Gravity column (Macherey-Nagel) with 85 % (v/v) MeOH, 10 % (v/v) H2O, 5 % (v/v) DCM, 0.1 % (v/v) formic acid as mobile phase. The coupling products were eluted in a 0-100 % gradient with MeOH/DCM (1: 1).
The elution fractions were pooled, concentrated using a SpeedVac concentrator and diluted 1: 10 in H2O prior to freezing and lyophilization. Analytical reverse phase-HPLC was conducted on a Gemini 3 pm Cl 8 110 A column (150 x 4.6 mm, Phenomenex) using the same mobile phases as described above for the preparative RP-HPLC. Furthermore, 10-50 pg of the PAS-di(alkyl)amines was dissolved in 50 % (v/v) acetonitrile in H2O with 0.1 % (v/v) formic acid and electrospray ionization-mass spectrometry (ESI-MS) was performed using a maXis Q-TOF instrument (Bruker Daltonics) in the positive ion mode. The PAS- di(alkyl)amine was obtained in a uniform composition, as was evident from the presence of a single peak in the analytical RP-HPLC and ESI-MS analyses (shown in Figure 16).

Claims

1. A method for the preparation of a compound of the formula (I):
A-[B-]bC-D (I) wherein
A is a di(alkyl)amino group,
B is a linker, b is 1 or 0, so that B can be present or absent,
C is a polypeptide capable of forming a random coil conformation and comprises an amino acid sequence consisting of alanine, proline, and serine, or comprises an amino acid sequence consisting of alanine and proline,
D is an N-terminal protecting group, and wherein the method comprises the steps of a) the coupling of a di(alkyl)amine to a compound L-B-C-D or a compound L-C-D to yield a compound of formula (I), wherein L is a leaving group; and b) the purification of a compound of formula (I).
2. The method of claim 1, wherein A comprises two alkyl chains, and wherein said alkyl chains are independently a linear or branched alkyl chain.
3. The method of claim 1 or 2, wherein said two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.
4. The method of any one of claims 1 to 3, wherein A is a di(tetradecyl)amino group.
5. The method of any one of claims 1 to 4, wherein C comprises an amino acid sequence consisting of alanine and proline, and wherein C comprises at least one alanine and one proline residue.
6. The method of claim 5, wherein said proline residues constitute more than about 10 mol% and less than about 70 mol% of C.
7. The method of claim 5 or 6, wherein C comprises at least 95 mol% proline and alanine residues.
8. The method of any one of claim 1 to 4, wherein C comprises an amino acid sequence consisting of alanine, proline, and serine, and wherein C comprises at least one alanine, one proline residue, and one serine residue.
9. The method of claim 8, wherein said proline residues constitute more than 4 mol% and less than 40 mol% of C.
10. The method of claim 8 or 9, wherein C comprises at least 95 mol% proline, alanine, and serine residues.
11. The method of any one of claims 5 to 10, wherein C comprises an amino acid sequence consisting of about 10 to about 200 amino acids, preferably about 20, about 40, or about 100 amino acids.
12. The method of any one of claims 5 to 11, wherein C comprises no more than 6 identical consecutive amino acid residues.
13. The method of any one of claims 5 to 12, wherein C comprises an amino acid sequence selected from any one of SEQ ID NO: 9 to SEQ ID NO: 22, or multiples thereof.
14. The method of any one of claims 1 to 13, wherein D is selected from pyroglutamoyl, formyl, CO(Ci-4 alkyl), and homopyroglutamoyl, wherein the alkyl moiety comprised in said -CO(Ci-4 alkyl) is optionally substituted with one or two groups independently selected from -OH, -O(Ci-4 alkyl), -NH(CI.4 alkyl), -N(CI.4 alkyl)( Ci.4 alkyl) and -COOH.
15. The method of claim 14, wherein D is selected from pyroglutamoyl, formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl, propionyl,
2-hydroxypropionyl, 3 -hydroxypropionyl, 2-methoxypropionyl, 3 -methoxypropionyl.
2-ethoxypropionyl, 3 -ethoxypropionyl, succinyl, butyryl, 2-hydroxybutyryl
3 -hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3 -methoxybutyryl
4-methoxybutyryl, glycine betainyl, glutaryl, and homopyroglutamoyl, preferably pyroglutamoyl.
16. The method of any one of claims 1 to 15, wherein B comprises a natural or unnatural amino acid.
17. The method of claim 16, wherein B is -HN-(C2-i2 hydrocarbyl)-C(O)-, wherein it is preferred that
B is selected from -HN-(CH2)3 io-C(0)-, -HN-phenyl-C(O)-, and -HN-cyclohexyl-C(O)-, and wherein it is more preferred that B is selected from -HN-(CH2)4-C(O)-, -HN-(CH2)5-C(O)-, -HN-
Figure imgf000086_0001
18. The method of any one of claims 1 to 17, wherein L is replaced by A during the coupling of the di(alkyl)amine and the compound L-B-C-D or during the coupling of the di(alkyl)amine and the compound L-C-D.
19. The method of claim 18, wherein the compound L-B-C-D or the compound L-C-D comprises a group -C(O)-L, wherein L represents the leaving group, and is preferably selected from -OH and -OR3, wherein R3 is a C1-C6 alkyl.
20. The method of any one of claims 1 to 15, wherein b is 0.
21. The method of any one of claims 1 to 20, wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled in a single reaction.
22. The method of any one of claims 1 to 21, wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D each comprise only a single reactive group and wherein the di(alkyl)amine and compound L-B-C-D or compound L-C-D are coupled via said reactive group.
23. The method of any one of claim 1 to 22, wherein during the coupling, the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are comprised in (an) organic solvent(s), preferably said organic solvent(s) comprises dichloromethane and methanol.
24. The method of claim 23, wherein said organic solvent comprises about 3 volume parts, about 3.5 volume parts, about 4 volume parts, about 4.5 volume parts, about 5 volume parts, about 5.5 volume parts, about 6 volume parts, about 6.5 volume parts, about 7 volume parts, about 7.5 volume parts, or about 8 volume parts dichloromethane and about 1 volume part methanol, preferably 5 volume parts dichloromethane and about 1 volume part methanol.
25. The method of any one of claims 1 to 24, wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are coupled using a coupling reagent, and wherein said coupling reagent is 2-( IH-Benzotriazole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethylaminium tetrafluoroborate (TBTU) .
26. The method of any one of claims 1 to 25, wherein the di(alkyl)amine, and compound L-B-C-D or compound L-C-D are further coupled using a non-nucleophilic base, and wherein said non- nucleophilic base is selected from N,N -Diisopropylethylamine (DIPEA), 1,8-Diazabicycloundec- 7-ene (DBU), l,5-Diazabicyclo[4.3.0]non-5-ene (DBN), triethylamine (TEA), preferably DIPEA.
27. The method of any one of claims 1 to 26, wherein the purification of the compound comprises liquid chromatography of said compound, preferably high-pressure liquid chromatography.
28. A compound as defined by any one of claims 1 to 27, or a compound of formula (I) obtained/obtainable by the method of any one of claims 1 to 27.
29. Use of the compound of claim 28 for the preparation of a lipid nanoparticle.
30. A method for the preparation of a lipid nanoparticle comprising the compound of claim 28, wherein the method comprises the steps of a) the provision of the compound of claim 28 in an ethanolic solution; b) the mixing of said ethanolic solution with an aqueous solution, thereby preparing a lipid nanoparticle; and c) optionally the dialysis of said lipid nanoparticle.
31. The method of claim 30, wherein said ethanolic solution further comprises a cationic lipid, a noncationic lipid, and a sterol.
32. The method of claim 31, wherein said ethanolic solution comprises between about 2 mM and about 60 mM total lipid, preferably between about 7.5 mM and about 30 mM, more preferably 9.43 mM total lipid.
33. The method of any one of claims 30 to 32, wherein said ethanolic solution comprises between about 1 mol% and about 5 mol%, preferably about 1.5 mol% of the compound of claim 28.
34. The method of any one of claims 31 to 33, wherein said ethanolic solution comprises between about 45 mol% and about 55 mol%, preferably about 50 mol% of the cationic lipid.
35. The method of any one of claims 31 to 34, wherein said ethanolic solution comprises between about 7 mol% and about 13 mol%, preferably about 10 mol% of the non-cationic lipid.
36. The method of any one of claims 31 to 35, wherein said ethanolic solution comprises between about 35 mol% and about 42 mol%, preferably about 38.5 mol% of the sterol.
37. The method of any one of claims 31 to 36 wherein said sterol is cholesterol.
38. The method of any one of claims 31 to 37, wherein said cationic lipid is DLin-MC3-DMA ([(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl] 4-(dimethylamino)butanoate).
39. The method of any one of claims 31 to 38, wherein said non-cationic lipid is an anionic lipid, preferably wherein said anionic lipid is a phospholipid, more preferably distearoylphosphatidylcholine (DSPC).
40. The method of any one of claims 30 to 39, wherein said aqueous solution comprises one or more active ingredients.
41. The method of claim 40, wherein said one or more active ingredients are one or more nucleic acids, one or more polypeptides, one or more proteins, or combinations thereof, preferably one or more nucleic acids.
42. The method of claim 41, wherein said one or more nucleic acids are selected from the group consisting of mRNA, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA for gene editing systems, DNA, plasmids, antisense oligonucleotides, and combinations thereof, preferably mRNA.
43. The method of any one of claims 40 to 42, wherein said aqueous solution further comprises one or more acids.
44. The method of claim 43, wherein said one or more acids are selected from acetic acid, HEPES (4- (2 -hydroxyethyl)- 1 -piperazineethanesulfonic acid), and citric acid, preferably acetic acid.
45. The method of claim 44, wherein said aqueous solution comprises a concentration of between about 5 mM and about 50 mM, preferably between about 10 mM and about 30 mM, more preferably about 11 mM of said one or more acids.
46. The method of any one of claim 30 to 45, wherein step c) comprises the dialysis of said lipid nanoparticle using a buffer, preferably wherein said buffer is selected from the group of phosphate buffer, phosphate buffer saline, and phosphate buffer saline comprising sucrose, more preferably phosphate buffered saline.
47. A lipid nanoparticle as defined by the method of any one of claims 30 to 46, or obtained/obtainable by the method of any one of claims 30 to 46.
48. A lipid nanoparticle suspension comprising the lipid nanoparticle of claim 47.
49. A method for the preparation of a pharmaceutical composition, wherein the method comprises the formulation of the compound of claim 28, the lipid nanoparticle of claim 47, and/or the lipid nanoparticle suspension of claim 48 into a pharmaceutical composition.
50. The method of claim 49, wherein the method comprises the formulation of the compound of claim 28, the lipid nanoparticle of claim 47, and/or the lipid nanoparticle suspension of claim 48 using a pharmaceutically acceptable carrier.
51. The method of claim 49 or 50, wherein said pharmaceutical composition is formulated for intramuscular or intravenous administration.
52. The method of any one of claims 49 to 51, wherein said pharmaceutical composition is formulated for use as a medicament or for use as a vaccine.
53. A pharmaceutical composition as defined by the method of any one of claims 49 to 52, or obtained/obtainable by the method of any one of claims 49 to 52.
54. Use of the compound of claim 28, the lipid nanoparticle of claim 47, and/or the lipid nanoparticle suspension of claim 48 for the preparation of a pharmaceutical composition.
55. Use of the compound of claim 28, the lipid nanoparticle of claim 47, and/or the lipid nanoparticle suspension of claim 48 for the preparation of a non-pharmaceutical composition.
56. A method for the preparation of a non-pharmaceutical composition, wherein the method comprises formulating the compound of claim 28, the lipid nanoparticle of claim 47, and/or the lipid nanoparticle suspension of claim 48 into a non-pharmaceutical composition.
57. A non-pharmaceutical composition as defined by claim 56, or obtained/obtainable by the method of claim 56.
PCT/EP2024/080338 2023-10-27 2024-10-25 Methods for the coupling of di(alkyl)amines to polypeptides Pending WO2025088192A1 (en)

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