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WO2025157953A1 - Lipides ionisables et nanoparticules lipidiques les contenant - Google Patents

Lipides ionisables et nanoparticules lipidiques les contenant

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Publication number
WO2025157953A1
WO2025157953A1 PCT/EP2025/051733 EP2025051733W WO2025157953A1 WO 2025157953 A1 WO2025157953 A1 WO 2025157953A1 EP 2025051733 W EP2025051733 W EP 2025051733W WO 2025157953 A1 WO2025157953 A1 WO 2025157953A1
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WIPO (PCT)
Prior art keywords
lipid
glycero
ethyl
dimethylamino
ionizable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/051733
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English (en)
Inventor
Javier GIMÉNEZ WARREN
Juan HEREDERO GARCÍA
Juan Enrique MARTÍNEZ OLIVÁN
Álvaro PEÑA MORENO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Certest Biotec SL
Original Assignee
Certest Biotec SL
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Filing date
Publication date
Application filed by Certest Biotec SL filed Critical Certest Biotec SL
Publication of WO2025157953A1 publication Critical patent/WO2025157953A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C327/00—Thiocarboxylic acids
    • C07C327/20—Esters of monothiocarboxylic acids
    • C07C327/32—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C07C327/34—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups with amino groups bound to the same hydrocarbon radicals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C327/00—Thiocarboxylic acids
    • C07C327/20—Esters of monothiocarboxylic acids
    • C07C327/32—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups

Definitions

  • the present disclosure relates to ionizable lipids comprising at least one thioester moiety and one amide moiety and to lipid nanoparticles (LNPs) comprising said ionizable lipids.
  • LNPs lipid nanoparticles
  • These LNPs can be used as non-viral vectors for the delivery of active ingredients, including polynucleotides, to cells.
  • nucleic acid therapies are excellent modalities for rapid vaccination designs. On top of that, they avoid pathogen culture.
  • Polynucleotides such as RNA for systemic delivery, can be successfully encapsulated in gene delivery systems containing them. Said delivery systems must fulfil a few requisites such as to be safe and non-toxic, be on the nanometer scale, provide protection to avoid degradation of the polynucleotides, remain intact in the system for the sufficient period of time in order to reach their target, and be easily degraded once they have released the load.
  • RNA does not involve the risk of being stably integrated into the genome of the transfected cell.
  • RNA degrades more easily in vivo, and therefore there is a lower risk of generating undesired anti-RNA antibodies that would reduce therapy efficacy and could produce very serious side effects.
  • RNA-based therapies the low transfection rates and the limited protein production efficiency. For these reasons, the dosage is increased in order to obtain the desirable therapeutic effects although, consequently, a higher dose also means elevated costs and, more importantly, some undesirable side effects observed.
  • LNPs have become one of the more potent intracellular delivery technologies for encapsulation and delivery of active principles, such as for example genetic material (e.g. mRNA) in vaccines.
  • active principles such as for example genetic material (e.g. mRNA) in vaccines.
  • LNPs used in commercial mRNA vaccines usually comprise four types of lipids in their composition, in particular, an ionizable or a cationic lipid, a structural lipid which is a sterol such as cholesterol, a PEG-modified lipid, and a non-cationic lipid such as a phospholipid.
  • ionizable lipids are crucial in LNPs.
  • Ionizable lipid properties have a great impact on the protection of the genetic material encapsulated inside the LNP, as they allow the structure and physicochemical properties of the genetic material to be maintained until the LNP reaches the target (e.g. a tissue, a cell) where the genetic material is to be released.
  • the target e.g. a tissue, a cell
  • Ionizable lipids usually present the following general structure:
  • the hydrophilic moiety comprises an ionizable tertiary amine, a functional group where a change in pH influences its formal charge.
  • ester groups easily hydrolysable by enzymes, facilitates the degradation of the ionizable lipid once the genetic material has been released into the tissue/cell of interest, what improves its biocompatibility and biodegradability.
  • the proximity of ester groups to the hydrophilic moiety has also been described to have a great impact on the potency of the lipid.
  • the lipid commercially known as SM-102 lipid and the lipid known as ALC-0315, depicted below, are respectively comprised in the commercial SARS-CoV-2 vaccine formulations Spikevax (Moderna) and Comirnaty (Pfizer).
  • the structures comprise a tertiary ionizable amine as well as two ester groups.
  • LNPs lipid nanoparticles
  • the present inventors have developed a new ionizable lipid that allows obtaining lipid nanoparticles (LNPs) that can be effectively used as non-viral vectors for delivery of active ingredients, including polynucleotides, to cells.
  • LNPs comprising the ionizable lipid of the present disclosure show enhanced/improved transfection rates and biodegradability without compromising their stability.
  • ionizable lipids of Formula (I) as defined herein below: Formula (I) comprising a polar head, at least one thioester moiety, at least one stereogenic center, at least one amide moiety, and a moiety X selected from amide, ester or thioester, wherein Q, X, R1, R2, m, p and t are as defined in the present disclosure, are particularly useful in the preparation of LNPs, which are capable of encapsulating an active agent.
  • transfection rates in vivo of LNPs prepared with the ionizable lipids of the present disclosure and comprising an active agent are unexpectedly high compared to other commercial or standard compositions known in the art.
  • the ionizable lipids of the present disclosure provide a new tool to overcome some of the limitations of known LNPs.
  • the higher transfection efficiency of the LNPs of the present disclosure may allow reducing the therapeutic dose of polynucleotide, such as RNA, required in gene therapy and vaccination.
  • polynucleotide such as RNA
  • the high degradability of the thioester moiety could be detrimental for the stability of the ionizable lipids of the present disclosure.
  • the present inventors have found that not only the biodegradability, and hence cytotoxicity are better than other known alternatives, but also the stability of the ionizable lipids of Formula (I) is unexpectedly good which may provide easier handling and storage conditions.
  • a first aspect of the present disclosure relates to an ionizable lipid of formula or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein
  • R’ is selected from the group consisting of H, methyl, and ethyl
  • X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m is selected from 0, 1 , 2, 3, 4, 5, and 6; p is selected from 0, 1 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;
  • Another aspect of the invention relates to a LNP comprising the ionizable lipid of formula (I) as defined herein.
  • the LNP of the present disclosure can further comprise a pharmaceutically active agent and, as such, be formulated in a pharmaceutical with excipients and carriers.
  • a pharmaceutical composition comprising the LNP comprising a pharmaceutically active agent as defined herein and a pharmaceutically acceptable excipient or carrier.
  • the LNP comprising a pharmaceutically active agent or the pharmaceutical composition of the present disclosure may be used in medicinal applications.
  • another aspect of the invention relates to the LNP comprising a pharmaceutically active agent or the pharmaceutical composition of the present disclosure for use in medicine, particularly for use in a method for treating a disease or disorder in a subject in need thereof; or for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy.
  • the method comprises administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition.
  • Another aspect of the invention relates to the use of the LNP as defined herein as an encapsulation agent for an active ingredient.
  • FIG. 1 Related to Example 4, depicts protein expression in mice administered with LNPs prepared with the following lipids of the invention: VC-LC-1272, VC-LC-1285 and VC-LC-1289 containing mRNA for luciferase as the active ingredient.
  • FIG. 2 depicts a comparative hydrolytic cleavage of thioester and ester bonds, of the ionizable lipids VC-LC-1282, SM-102, and VC-LC-0729.
  • moiety refers to a specific segment or functional group of a molecule or compound.
  • subject refers to any mammal, including both human and nonhuman mammals.
  • C1-C# alkyl refers to saturated linear or branched hydrocarbon which contains from 1 to # carbon atoms, and which is optionally substituted.
  • alkyl groups include, without being limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl, isodecyl, undecyl, dodecyl, tetradecyl, and hexadecyl.
  • C2-C# alkenyl refers to an unsaturated linear or branched hydrocarbon chain which comprises from 2 to # carbon atoms and at least one or more double bonds, and which is optionally substituted.
  • alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl, dodecenyl, tetradecenyl and hexadecenyl.
  • C2-C# alkynyl refers to an unsaturated linear or branched hydrocarbon chain which comprises from 2 to # carbon atoms and at least one or more triple bonds, and which is optionally substituted.
  • alkynyl groups include, without being limited to, ethynyl, prop-1 -ynyl, prop-2-ynyl, 1- methylprop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3- ynyl.
  • heterocycle comprising at least one N atom refers to an optionally monosubstituted or multi-substituted cyclic system including one or more rings, where at least one ring includes at least one nitrogen.
  • optionally substituted means that the number of substituents can be equal to or different from zero. Unless otherwise indicated, it is possible that optionally substituted groups are substituted with as many optional substituents as can be accommodated by replacing a hydrogen atom with a non-hydrogen substituent on any available carbon or nitrogen atom.
  • polynucleotide is interchangeably used with “nucleic acid” and refers to a polymer of nucleotides, either ribonucleotides or deoxyribonucleotides.
  • a polynucleotide formed by ribonucleotides may be referred to as “RNA polynucleotide”, “ribonucleic acid” or simply “RNA”; and a polynucleotide formed by deoxyribonucleotides may be referred to as "DNA polynucleotide", “deoxyribonucleic acid” or simply "DNA”.
  • the polynucleotide may be single- or double-stranded, optionally incorporating synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA.
  • "Artificial polynucleotide” refers to a polynucleotide with a sequence that does not occur in nature or that has been altered by human intervention.
  • mRNA messenger RNA
  • mRNA refers to any RNA polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, or ex vivo.
  • an mRNA is single stranded and comprises an ORF in its structure.
  • open reading frame or “ORF” refers to a sequence of several nucleotide triplets that encodes a polypeptide, that is, that can be translated into a polypeptide sequence.
  • DNA construct refers to an artificial polynucleotide including a sequence of interest operatively linked to an expression promoter, said promoter controlling expression of the sequence of interest.
  • expression vector refers to a vector used to introduce a specific nucleic acid, typically a DNA construct, into a target cell for expression of the nucleic acid by the cell.
  • suitable expression promoters and expression vectors include those conventionally used in molecular biology and known to the skilled person.
  • polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). "Polypeptide” refers to both short chains, commonly referred as peptides, oligopeptides, or oligomers, and to longer chains generally referred to as proteins.
  • therapeutically effective amount refers to the amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of the disease to which it is addressed.
  • the particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, the particular circumstances of the individual subject to be treated, and similar considerations.
  • pharmaceutical also encompasses the concept of “veterinary composition”. Thus, they relate to compositions that are therapeutically effective when administered by any desired or applicable route to any animal, including humans.
  • the term "antigen” is a compound that can be recognized by immunoglobulin receptors of B cells, or by the T-cell receptor when complexed with MHC.
  • an "antigen” is a polypeptide.
  • nanoparticle refers to a particle with at least two dimensions at the nanometer scale, particularly with all three dimensions at the nanoscale, where the nanoscale is in the range of about 1 nm to about 500 nm. Particularly, when the nanoparticle is substantially rod-shaped with a substantially circular cross-section, such as a nanowire or a nanotube, the “nanoparticle” refers to a particle with at least two dimensions at the nanoscale, these two dimensions being the cross-section of the nanoparticle.
  • size or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.
  • lipid nanoparticle refers to a nanoparticle whose external envelope is totally or partially made of lipids.
  • the "polydispersity index (PDI)" is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution.
  • zeta potential is the electrokinetic potential of a lipid, e.g., in a particle composition.
  • Z potential is also defined as the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. It is generally accepted as a quantification of the magnitude of the charge, and it is often the only available path for characterization of double-layer properties.
  • apparent pKa refers to an experimentally determined value resulting from the average ratio of all the ionized to deionized groups in a nanoparticle. Apparent pKa is different to the intrinsic pKa of any individual molecule, and it is an important parameter for the performance of nanoparticles encapsulating RNAs.
  • the apparent pKa of nanoparticles can be measured by different techniques known in the art. For example, acid-base titration and 2-(p-toluidino)-6-naphtalene sulfonic acid (TNS) fluorescent methods are widely used in the art.
  • Nanoparticles with an optimum pKa carry negligible charges at physiological pH, which prevent nonspecific binding and toxicity in the body.
  • the optimum pKa of nanoparticles plays an important role in the endosomal escape mechanism and in the release of RNAs in the cytosol to exert therapeutic effect.
  • a first aspect of the invention refers to an ionizable lipid of formula
  • pharmaceutically acceptable salts encompasses any salt formed from pharmaceutically acceptable non-toxic acids including inorganic or organic acids. There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable.
  • the preparation of pharmaceutically acceptable salts of the ionizable lipids of the present disclosure can be carried out by methods known in the art. For instance, they can be prepared from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of the ionizable lipids of the present disclosure with a stoichiometric amount of the appropriate pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture of them.
  • Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, or phosphoric acid; and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, malic, lactic, formic, propionic, glycolic, camphorsulfuric, mandelic, benzenesulfonic, p-toluenesulfonic, oxalic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, lysine, and procaine.
  • the ionizable lipids of the present disclosure and their salts may differ in some physical properties, but
  • stereoisomer refers to all isomers of individual ionizable lipids that differ only in the orientation of their atoms in space.
  • stereoisomer includes enantiomers, racemates, racemic mixtures, geometric isomers (cis/trans or syn/anti or E/Z), and diastereomers.
  • the present invention relates to each of these stereoisomers and also mixtures thereof.
  • the preparation processes described herein can be modified to give enantiopure compounds as well as mixtures of stereoisomers. It is possible to prepare specific stereoisomers or specific mixtures by various processes including the use of stereospecific reagents or by introducing chiral centers into the compounds during its preparation process. In addition, it is possible to separate stereoisomers once the compound has been prepared by standard resolution techniques known to the skilled person.
  • the pharmaceutically acceptable salts, or stereoisomer of the ionizable lipids or of their pharmaceutically acceptable salts are always contemplated even if they are not specifically mentioned.
  • the ionizable lipids of the present disclosure also include isotopes of the structure depicted.
  • isotopes refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes include, without being limited to, tritium, deuterium, 13 C or 14 C, or 15 N.
  • a compound or salt of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • the ionizable lipids of the present disclosure are characterized by their retention time, mass spectrometry, size distribution, polydispersity index, and Z-potential. These parameters can be measured by methods well-known in the art. Some of them are indicated in more detail in the examples below.
  • R’ is selected from the group consisting of H and methyl and m is selected from 0, 1 , 2, 3 and 4.
  • the ionizable lipid of Formula (I) is a compound of Formula (IA)
  • X is -NH-C(O)- or -C(O)-NH-; or X is -C(O)-O- or -O-C(O)-; or X is -C(O)-S- or -S-C(O)-.
  • Q is: wherein Ra and Rb are independently linear or branched C1-C6 alkyl, optionally substituted with a hydroxyl group.
  • Q is a 5-membered ring or a 6-membered ring comprising one N atom and, optionally, a second heteroatom selected preferably from N and O.
  • Q is selected from the following structures:
  • X is selected from -NH-C(O)- and -C(O)-NH-;
  • R1 is selected from C the group consisting of C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 branched alkyl;
  • R2 is selected from the group consisting of C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 or C24 branched alkyl and C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23 and C24 linear alkenyl or alkdieny
  • ionizable lipids of Formula (I) include the compounds depicted in Table 1 below. Table 1
  • the ionizable lipid of the present disclosure is a compound selected from the group consisting of: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC-LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC- 1311), (VC-LC-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC- LC-1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), (VC-LC-1545).
  • the ionizable lipid of Formula (IA) is a compound selected from the group consisting of: (VC-LC-1272), (VC-LC-1284), (VC-LC-1285), (VC-LC-1286), (VC- LC-1287), (VC-LC-1288), (VC-LC-1289), (VC-LC-1293), (VC-LC-1297), (VC-LC-1311), (VC-LC-1369), (VC-LC-1371), (VC-LC-1373), (VC-LC-1374), (VC-LC-1376), (VC-LC- 1377), (VC-LC-1378), (VC-LC-1539), (VC-LC-1540), (VC-LC-1541), (VC-LC-1543), (VC- LC-1545) described herein; or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them.
  • the ionizable lipid of Formula (I) can be prepared according to any of the following synthetic schemes. Any person skilled in the art will know which reactants are required to obtain any particular ionizable lipid according to the present disclosure following the synthetic methods depicted in the schemes below or an analogous method thereof.
  • Ionizable lipids of Formula (I) wherein X is an amide moiety can be prepared by the synthetic method, or an analogous one, depicted in scheme 1 below.
  • Ionizable lipids of Formula (I) wherein X is an ester moiety can be prepared by the synthetic method, or an analogous one, depicted in scheme 2 below.
  • the present invention also relates to a LNP comprising an ionizable lipid of formula (I) as defined herein.
  • LNPs typically have a core-shell structure comprising an inner core and an external shell.
  • LNP formulations with several lipidic components of different nature such as an ionizable lipid, a sterol, a PEGylated lipid, and a non-cationic lipid (also referred to as “helper lipid”) such as a phospholipid, comprise several phases that separate into a hydro- phobic core region formed by the ionizable lipid and cholesterol, and a surrounding shell formed by the helper lipid, cholesterol and PEGylated lipids covering the surface.
  • the ionizable lipid of formula (I) forms part of the internal shell of the LNP and, optionally, a pharmaceutically active agent is encapsulated or loaded in the inner core.
  • the LNP further comprises at least one lipid selected from the group consisting of a non-cationic lipid; a sterol, a steroid precursor or steroid derivative; and a PEG-modified lipid.
  • the LNP comprises the ionizable lipid of the present disclosure and a non-cationic lipid.
  • non-cationic lipids include, without being limited to, 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocoline (SOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), 1 ,2-diundecanoyl-sn- glycero-phospho
  • the non-cationic lipid is DSPC.
  • the non-cationic lipid is DOPE.
  • the lipid- containing particle comprises DSPC and DOPE.
  • the LNP optionally in combination with one or more features of the various embodiments described above, the LNP further comprises a sterol or a sterol precursor.
  • the LNP comprises the ionizable lipid of the present disclosure and a sterol, a steroid precursor, or a steroid derivative.
  • sterols include, without being limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol, and mixtures thereof.
  • the sterol is cholesterol.
  • sterol precursors include, without being limited to, a triterpene, a triterpenoid, or a steroid precursor of this kind.
  • Non-limiting examples of triterpenes, triterpenoids and other steroid precursors include squalene, achilleol, polypodatetrane, lanostane, cucurbitacin, hopane, oleanane, chamaecydin, lupine, and mixtures thereof.
  • the term “steroid derivative” refers to a derivative of the simplest steroid containing the nucleus gonane, also known as cyclopentanoperhydrophenantrene, which contains seventeen carbon atoms arranged in four fused rings.
  • a steroid derivative can include different modifications such as different functional groups attached to the four-ring core or the oxidation state of the rings. Examples of steroid derivatives include, without being limited to cholic acid, lanosterol and p-sitosterol.
  • the LNP comprises the ionizable lipid of the present disclosure, a non-cationic lipid as defined above, and a sterol, a steroid precursor, or a steroid derivative as defined above.
  • the LNP further comprises a PEG-modified lipid.
  • PEG-modified lipid refers to a lipid comprising a polyethylene moiety.
  • PEG- modified lipids include, without being limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified phosphatidylcholine, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, PEG-DPG, or a combination thereof.
  • the LNP further comprises a conjugated lipid.
  • conjugated lipid examples include, without being limited to, polysarcosine (pSar) lipids and derivatives such as, for example, N-tetradecyl-pSar25, N-hexadecyl-pSar25, N- octadecyl-pSar25, N-dodecyl-pSar25, DMG-pSar25, 18:1 PE (DOPE) pSar25, N- TETAMINE-pSar25, N-TETAMINE-pSar35, N-TETAMINE-pSar45, N-TETAMINE-pSar45- Maleimide.
  • pSar polysarcosine
  • the LNP comprises the ionizable lipid of the present disclosure, a non-cationic lipid as defined above, and a PEG-modified lipid or a conjugated lipid as defined above.
  • the LNP comprises a lipid component comprising or consisting of the ionizable lipid as disclosed herein, a non-cationic lipid, a sterol, and a PEG-modified lipid.
  • the LNP comprises a lipid component comprising the ionizable lipid as disclosed herein and at least one of distearolyphosphatidycholine (DSPC), cholesterol, and DMG-PEG2000.
  • the lipid component comprises or consists of the ionizable lipid as disclosed herein, DSPC, cholesterol, and DMG-PEG2000.
  • the LNP comprises from 25 to 60 mol% of a ionizable lipid; from 0.1 to 10 mol% of a PEG-modified lipid or, alternatively, of a conjugated lipid, particularly of a PEG-modified lipid; from 10 to 45 mol% non-cationic lipid; and from 10 to 40 mol% sterol.
  • the LNP comprises from 32 to 50 mol% ionizable lipid, from 1 to 8 mol% PEG-modified lipid or a conjugated lipid, particularly PEG-modified lipid; from 12.5 to 42 mol% non-cationic lipid; and from 15 to 38.5 mol% sterol. More particularly, the LNP comprises from 35 to 47 mol% ionizable lipid, from 1 to 4 mol% PEG-modified lipid, from 30 to 38 mol% non-cationic lipid, and from 15 to 25 mol% sterol.
  • the ionizable lipid is in an amount from 25 to 64 mol%
  • the PEG-modified lipid is in an amount from 0.1 to 1.5 mol%
  • the sterol is in an amount from 35 to 74 mol%.
  • mol% refers to a component's molar percentage relative to the total moles of all lipid components in the LNP (i.e., total mols of ionizable lipid, PEG-modified lipid; non-cationic lipid; and sterol).
  • the molar ratio of ionizable lipid to the non-cationic lipid ranges from 6:1 to 1:2, or from 2:1 to 1 :1.
  • the molar ratio of ionizable lipid to the sterol ranges from 5:1 to 1:2, or from 2:1 to 1 :1.
  • the molar ratio of ionizable lipid to the PEG-modified lipid ranges from 120:1 to 2:1 , or from 100:1 to 10:1.
  • the LNPs of the invention may comprise one or more pharmaceutically active agents.
  • the term “pharmaceutically active agent” refers to an agent that has pharmacological activity and is used for curing, mitigating, treating or preventing a disease in a subject, in particular a human.
  • pharmaceutically active agents include low molecular weight drugs, polynucleotides, peptides, antibodies, proteins, and combinations thereof.
  • polynucleotide refers to a natural or an artificial deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the polynucleotide may comprise at least one chemical modification selected from the group consisting of pseudouridine, N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine or ml ⁇ P), N6- methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'- thiouridine, 5-methylcytosine (also referred to 5mC), 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine
  • the polynucleotide is partially modified with at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, of N1-methylpseudouridine or fully modified with N1-methylpseudouridine, 5- methoxyuridine or a combination thereof.
  • the active agent is selected from the group consisting of a polynucleotide, a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, and an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide.
  • the polynucleotide is a ribonucleic acid (RNA).
  • the RNA is selected from the group consisting of a short interfering RNA (siRNA), a self-replicating RNA (srRNA), circular RNA (circRNA), a self-amplifying RNA (saRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small RNA (sRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.
  • siRNA short interfering RNA
  • srRNA self-replicating RNA
  • circRNA circular RNA
  • saRNA self-amplifying RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • siRNA small interfering RNA
  • siRNA small RNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin
  • the RNA is an mRNA.
  • the ionizable lipid to RNA ratio (N/P; where N represents the moles of amine present in the ionizable lipid and P represents the moles of phosphate present in the polynucleotide backbone) in the LNP ranges from 20:1 to 2:1 , particularly from 10:1 to 3:1.
  • the polynucleotide is an isolated artificial polynucleotide.
  • the LNP comprises an ionizable lipid as defined herein, a non-cationic lipid, a sterol, a PEG-modified lipid, and a polynucleotide.
  • the LNPs containing one or more polynucleotides may be prepared by standard methods, for instance, microfluidic mixing as disclosed in Hassett, K. J. et al., ("Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines", 2019, Mol. Ther. Nucleic Acid, vol. 15, pp. 1-11), or by manual/bulk mixing as disclosed in Wang X., Liu S., Sun Y., et al. (“Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038/s41596-022-00755-x). Both methods are known in the art and the skilled person would know how to proceed in each specific case.
  • SORT selective organ-targeting
  • the process for the preparation of LNPs comprises: i) preparing a first alcoholic mixture comprising the ionizable lipid of the present disclosure and, optionally, at least one lipid selected from the group consisting of a non-cationic lipid, a sterol, and a PEG- modified lipid in a suitable alcohol such as for example ethanol; ii) preparing a second aqueous composition comprising a polynucleotide and an acidification buffer; and iii) mixing i) with ii) in a microfluidic mixer.
  • the microfluidic mixer allows thorough and rapid mixing of the lipid phase and the polynucleotide phase in a microscale device. Depending on the process parameters, and in particular on the total flow rate, the skilled person will be able to modulate the size of the LNPs.
  • the polynucleotide encodes a polypeptide, particularly, wherein the polypeptide is an antigen. More particularly, the antigen is selected from the group consisting of a viral protein, a bacterial protein, and a tumor-associated antigen.
  • the polypeptide is an antibody or a fragment thereof.
  • the antibody or a fragment thereof is a therapeutic antibody or a fragment thereof.
  • the antigen is a SARS-CoV-2 antigen, particularly a SARS-CoV-2 spike antigen.
  • another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the LNP as defined herein and a pharmaceutically acceptable excipient or carrier.
  • pharmaceutically acceptable excipient or carrier refers to pharmaceutically acceptable materials, compositions, or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
  • compositions of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils.
  • Excipients such as colouring agents, coating agents, sweetening, and flavouring agents can be present in the composition, according to the judgment of the formulator.
  • the pharmaceutical composition disclosed herein is administered orally, intranasally, intravenously, intraperitoneally, intramuscularly, intradermally, subcutaneously, topically, or by intra-articular administration.
  • compositions of the present disclosure 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 invention with sterile, distilled water or other carrier so as to form a solution.
  • Some excipients or carriers can be added to ease the formation of a homogeneous solution or suspension.
  • the LNP comprising a pharmaceutically active agent, or the pharmaceutical composition of the present disclosure may be used in therapeutic applications. In particular, they may be used as non-viral vectors of general use for biomedical applications, such as vaccines or gene therapy, being effective for transfection of genetic material into eukaryotic cells.
  • an aspect of the invention relates to a LNP or a pharmaceutical composition as defined herein for use in a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition or of the pharmaceutical composition as defined herein.
  • Another aspect of the invention relates to a LNP or a pharmaceutical composition as defined herein for use in a method of inducing an immune response in a subject, for use in a method for the therapeutic immunization of a subject, for use as a vaccine, or for use in gene therapy.
  • the subject is a human.
  • the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and reno-vascular diseases, and metabolic diseases.
  • the LNP or the pharmaceutical composition described herein can be used in vaccine therapy, in the enhancement of the efficacy of a conventional vaccine and/or as a novel vaccine form for use against infectious pathogens, such as viruses, bacteria, fungi, protozoa, prions, and helminths (worms); or for use in treating diseases such as cancer and proliferative diseases.
  • infectious pathogens such as viruses, bacteria, fungi, protozoa, prions, and helminths (worms)
  • worms helminths
  • the pharmaceutical composition is a vaccine.
  • the pharmaceutical composition is a vaccine and further comprises an adjuvant.
  • the skilled person would know, based on its common general knowledge, which excipients, carriers, and adjuvants to include in the vaccine depending on the intended use.
  • Reagents were purchased from Sigma-Aldrich, TCI Chemicals, Fluorochem, or VWR. All possible wise combinations shown previously were prepared following a three-step reaction protocol.
  • DL-homocysteine thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Then triethylamine (252 pL, 1 .8 mmol) was added followed by N-(3-dimethylaminopropyl)-N z -ethylcarbodiimide hydrochloride (249.2 mg, 1.3 mmol), 4-(dimethylamino)pyridine (24.4 mg, 0.2 mmol) and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred overnight under an argon atmosphere at room temperature.
  • step 1 The product from step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and was added to a solution of N,N-Dimethylethylenediamine (780 pL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere. The solution was then stirred at room temperature for 30 minutes, after which the solvent was evaporated under reduced pressure.
  • Step 3 Final step in the synthesis of lipid VC-LC-1272
  • reaction crude was redissolved in 40 mL of dichloromethane and washed two times using distilled water (2 x 10 mL) and finally with saturated brine (10 mL).
  • the organic layer was dried with anhydrous MgSO4, filtered, and evaporated under reduced pressure.
  • the resulting residue was purified by flash chromatography (gradient of dichloromethane/eluent A: 100/0 to 0/100). After removing the solvent under reduced pressure, VC-LC-1272 is afforded as an oil (31 % yield).
  • Sample preparation for the reaction monitoring 5 pL of the reaction crude are dissolved in 100 pL of HPLC-MS-grade acetonitrile in a HPLC sample tube. The tube is vortexed manually for 5 seconds and placed inside the HPLC sample chamber.
  • the resulting product was purified by flash chromatography using a gradient of hexane /ethyl acetate (100/0 to 0/100). The fractions containing the product were combined and evaporated under reduced pressure yielding VC-LC-9099 as a yellowish oil (1.33 g, 84%).
  • steps 2 and 3 which are analogous to the corresponding steps followed for the synthesis of lipid VC-LC-1272 described in Example 1 , were carried out. This allowed for the obtention of a combined 21% yield (quantitative and 21% yield for steps 2 and 3, respectively).
  • VC-LC-1285 was characterised by HPLC-ELSD-MS. Retention time: 9.37 min, MS (ES): experimental m/z [M+H]+ 723.85 ⁇ theoretical m/z [M+H]+ 723.61.
  • VC-LC-1285 was characterised by HPLC-ELSD-MS following the general procedure detailed above and successful detection of the analyte was performed by an evaporative light dispersion detector and by a simple quadrupole mass detector in positive mode with an spectrophotometer Waters Acquity QDa.
  • VC-LC-9261 was characterised by HPLC-ELSD-MS. Retention time: 4.17 min, MS (ES): 473.65 experimental m/z [M+H]+ ⁇ theoretical m/z [M+H]+ 473.34 ⁇
  • Step 4 Final step in the synthesis of lipid VC-LC-1311 - -
  • VC-LC-1311 was characterised by HPLC-ELSD-MS. Retention time: 6.98 min, MS (ES): 601.56 experimental m/z [M+H]+ ⁇ theoretical m/z [M+H]+ 601.44 ⁇
  • VC-LC-9241 was characterised by HPLC-LSD-MS following the general procedure detailed above. Retention time: 6.98 min, MS (ES): 601.56 experimental m/z [M+H]+ ⁇ theoretical m/z [M+H]+ 601.44 ⁇
  • Encapsulation of mRNA comprising in the 5’ to 3’ direction a sequence encoding for luciferase of SEQ ID NO:1 into LNPs was performed in some cases by microfluidics or, alternatively, by manual means.
  • a mixture of the ionizable lipid of the invention DSPC (Merk 850365P):cholesterol (Sigma C3045):DMG-PEG2000 (Cayman 33945-1) were dissolved into ethanol at the respective molar percentages of 50:10:38.5:1.5, and with a lipid-nitrogen-to-phosphate ratio (N:P) ratio of 5,5:1.
  • LNPs were obtained by manual methods, e.g. by manual/bulk mixing as disclosed in Wang X., Liu S., Sun Y., et al. (“Preparation of selective organtargeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissuespecific mRNA delivery”, 2022, Nat. Protoc., doi:10.1038/s41596-022-00755-x), the mRNA aqueous solution was carefully added to the ethanol solution, and the resulted solution was homogenized by pipetting up and down for 4-5 times. The resulting LNPs were immediately diluted 1 :1 with Tris buffer and dialyzed overnight against Tris buffer containing 15% sucrose. The resulting LNP solution was then collected, and the encapsulated mRNA was assessed by Quant-IT® Ribogreen (Invitrogen R11490) following the manufacturer’s instructions.
  • SORT selective organtargeting
  • RNA encapsulation was assessed by Quant-IT® Ribogreen following the manufacturer’s instructions.
  • the LNP solution was passed through a 0.22 mm filter and the LNPs were stored at -80 °C until required.
  • Table 6 provides the results of several parameters for LNPs comprising the specific ionizable lipids shown therein in the following standard formulation: ionizable lipidmon-cationic lipid:sterol:PEG-modified lipid 50:10:38.5:1.5.
  • each of the compounds tested had an acceptable particle size for the purpose sought.
  • the encapsulation efficiency measured as the percentage of RNA entrapped in the LNP, ranged from 100% to approximately 15%. Nevertheless, this is only an indicative of how easily the components of the nanoparticle interact between them to form a LNP able to entrap RNA. It does not indicate the intracellular transfection efficiency.
  • the intracellular transfection efficiency of the mRNA (encoding for the expression of luciferase) is measured as luminescence given as total flux (p s -1 ). The total flux is a bioluminescence measurement in photons per second or average radiance in each pixel integrated over the region of interest.
  • the encapsulation efficiency means the approximate percentage of the RNA that was encapsulated when contacted with the components of the LNP.
  • the value of the total flux observed indicated that the LNPs were not immediately degraded in vivo after injection, and that transfection of the cells was indeed very successful, hence the contents encapsulated inside the LNPs effectively reached the cytosol after the endosomal escape.
  • Example 4 Protein expression in mice muscle administered with selected examples of LNPs prepared with the lipids of the invention and containing mRNA as active ingredient.
  • mice Female BALB/c mice (Charles River Laboratories), 8-10-week-old, weighting 18-23 g, were acclimatized to new conditions upon arrival to experimental facilities for 3-7 days. Housing conditions were room temperature 20-24 °C, humidity 50-70 %, and light intensity 60 lux with a light-dark cycle of 12 hours.
  • LNPs produced as indicated above ionizable lipid:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5
  • containing 1 pg of the indicated mRNA in 30 l final volume were injected intramuscularly.
  • RNA-LNP inoculation mice were anaesthetized by inhalation with 4% of isoflurane using a vaporizer. The maintenance of the anaesthesia was performed at 1.5% of isoflurane. Then, D-luciferin (Quimigen, 12507) was injected intraperitoneally at 150 mg/kg, normally -200 pL of the stock at 15 mg/mL in PBS for a 20 g mouse.
  • Luciferase images were acquired 10 minutes after luciferin inoculation using the I VIS Lumina XRMS Imaging System following manufacturer's instructions.
  • Table 7 shows selected examples of LNPs comprising ionizable lipids of Formula (I) with different combinations of R1 and R2 in order to prove that the general structure of Formula (I) produces the desired effect, independently of the selected substituents.
  • Table 7 demonstrates that LNPs comprising ionizable lipids of Formula I effectively encapsulate polynucleotides and produce high levels of cellular transfection in vivo after administration.
  • Figure 1 depicts images of the total flux measured in mice which had been administered LNPs formulated with ionizable lipids VC-LC-1272, VC-LC-1285 and VC-LC-1289.
  • Example 5 Comparative study of the hydrolytic cleavage of selected functional groups.
  • DL-homocysteine thiolactone hydrochloride (270.4 mg, 1.76 mmol) was dissolved in 5 mL of anhydrous dichloromethane at room temperature. Then triethylamine (1620 pL, 1.8 mmol) was added followed by EDC hydrochloride (249.2 mg, 1.3 mmol), 4- (dimethylamino)pyridine (24.4 mg, 0.2 mmol) and 2-hexyldecanoic acid (256.42 mg, 1 mmol). The reaction mixture was stirred at room temperature overnight under an argon atmosphere. Then, the reaction crude was washed two times using distilled water (2 x 10 mL) and finally with saturated brine (10 mL).
  • step 1 The product from step 1 (VC-LC-9018, 249 mg, 0.7 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL) and was added to a solution of N,N-Dimethylethylenediamine (756 pL, 7.03 mmol) in anhydrous tetrahydrofuran (1.0 mL) under argon atmosphere. The solution was then stirred at room temperature for 30 minutes, after which the solvent was evaporated under reduced pressure.
  • Step 4 Final step of the synthesis of lipid VC-LC-0729 Scheme 18. Synthetic route for Step 4 in the preparation of lipid VC-LC-0729.
  • SM-102 is commercially available and was purchased from BOCSI (95% purity, Catalogue Number B2699-358154).
  • Sample preparation for the reaction monitoring In a HPLC sample tube, 5 pL of the reaction crude are dissolved in 100 pL of HPLC-MS-grade tetra hydrofuran. Then, the sample tube is vortexed manually for 5 seconds and placed inside the HPLC sample chamber.
  • Detection Evaporative light scattering detector plus simple quadrupole mass detector in positive mode (Waters Acquity QDa).
  • ELSD Evaporative light dispersion detector (Waters ELSD 2424).
  • MS Simple quadrupole mass detector (Waters Acquity QDa).
  • Tables 8 and 9 summarize the data obtained for the appearance of the expected fragments produced during the hydrolysis of ionizable lipids using selected ion recording (SIR) HPLC-MS
  • ionizable lipids of the present disclosure containing at least one amide moiety and one thioester moiety, have a superior degradability compared to SM-102 and VC-LC-0729, which do not contain amide and thioester (SM-102), or which contain an amide moiety but do not contain a thioester moiety (VC-LC-0729);
  • VC-LC-1284 demonstrates that the ionizable lipids of the present disclosure do not degrade until they reach their target and release their cargo in the system, but when they do their degradability occurs faster which is advantageous in order to avoid several problems such as a strong immunity response.
  • Example 6 Study of protein expression in mice muscle administered with selected examples of LNPs prepared with ionizable lipids comprising shorter R1, excluded from Formula (I).
  • Encapsulation of mRNA comprising in the 5’ to 3’ direction a sequence encoding for luciferase of SEQ ID NO:1 into LNPs was performed in by microfluidics in the same manner as previously described in Example 3.
  • Table 11 provides the results of several parameters for LNPs comprising the specific ionizable lipids shown therein in the following standard formulation: ionizable lipidmon-cationic lipid:sterol:PEG-modified lipid 50:10:38.5:1.5.
  • the compounds tested had acceptable particle size, P.D.I., Zeta potential, apparent pKa and encapsulation efficiency over 90% both for VC-LC-1544 and VC-LC-1546.
  • Table 12 shows that LNPs comprising ionizable lipids VC-LC-1544 and VC-LC-1546, comprising linear butyl or linear nonyl as R1 substituents, effectively encapsulate polynucleotides, but produce very low levels of cellular transfection in vivo after administration.
  • the total flux observed indicates that the cellular transfection was 3 orders or magnitude lower compared to that observed for LNPs comprising ionizable lipids of Formula (I) and Formula (IA).
  • Example 7 Biodegradability tests in vivo of LNPs comprising an ionizable lipid of Formula (I) encapsulating mRNA.
  • LNPs comprising the ionizable lipids of the present disclosure and a commercially available one for comparison
  • liver tissue was diluted in 300 pL of isopropanol/ethanol, adding an internal control. Liver tissue was dissociated using a GentleMACS Dissociator (Miltenyi). The sample was sonicated for 15 minutes and centrifuged at 10000g for 15 minutes. Supernatats were collected and concentrated using a SpeedVac system and subsequently diluted with a 1 :1 mixture of isopropanol and ethanol and analyzed against calibration standards. Chromatographic separation and quantification were accomplished with a liquid chromatography (LC)-MS system.
  • LC liquid chromatography
  • Example 8 Stability of LNPs over time.
  • Quant-IT® Ribogreen Invitrogen R11490
  • Example 9 Toxicity of LNPs comprising ionizable lipids of Formula (I)
  • liver enzymes alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate aminotransferase (AST), as indicators of liver malfunction, and albumin and urea, as markers of liver synthetic function and renal function, were measured in vivo in mice to determine toxicity of ionizable lipids of Formula (I) of the present disclosure.
  • ALP alkaline phosphatase
  • ALT alanine transaminase
  • AST aspartate aminotransferase
  • LNPs comprising an ionizable of Formula (I) of the present disclosure (ionizable lipid VC- LC-1272:helper lipid: sterol: PEG-modified lipid 50:10:38.5:1.5) encapsulating mRNA for Firefly Luciferase were prepared as previously described.
  • the LNPs were diluted in Tris buffer containing 15% sucrose and administered through the tail vein in a final volume of 250 pL using a 27 G syringe.
  • R’ is selected from the group consisting of H, methyl, and ethyl
  • X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m and p are independently selected from 0, 1 , 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;
  • a lipid nanoparticle comprising an ionizable lipid of formula (I): or a pharmaceutically acceptable salt thereof, or a stereoisomer of any one of them, wherein
  • R’ is selected from the group consisting of H, methyl, and ethyl
  • X is selected from the group consisting of -NH-C(O)-, -C(O)-NH-, -C(O)-O-, -O-C(O)-, -S- C(O)-, -C(O)-S-; m and p are independently selected from 0, 1 , 2, 3, 4, 5, and 6; t is selected from 1 , 2 and 3;
  • lipid nanoparticle according to claim 22 wherein from the ionizable lipid of formula (I) the compounds S-oleoyl-N-acetyl-L-cysteine-3-(dimethylaminopropylamine)-amide, and S-oleoyl-N-acetyl-L-cysteine-3(dimethylaminopropylamine)-amide hydrochloride are excluded.
  • lipid nanoparticle according to clauses 22 or 23, further comprising: i) a non-cationic lipid; optionally, wherein the non-cationic lipid is selected from the group consisting of: 1 ,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero- phosphocholine (DM PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-stearoyl-2- oleoyl-sn-glycero-3-phosphocoline (SOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-diundecanoyl-sn- glycero-phosphocholine (DLIPC), 1-palmitoyl
  • lipid nanoparticle according to clause 26 wherein the pharmaceutically active agent is selected from the group consisting of a polynucleotide, a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide, and an expression vector comprising a DNA construct comprising a promoter operatively linked to a sequence encoding a polynucleotide.
  • the polynucleotide is a natural or an artificial deoxyribonucleic acid (DNA) or a natural or artificial ribonucleic acid (RNA); further optionally, wherein the polynucleotide comprises at least one chemical modification selected from the group consisting of pseudouridine, N1 -methylpseudouridine (also referred to as 1 -methylpseudouridine or ml ⁇ P), N6-methyladenosine (also referred to as m6A), 2-thiouridine (also referred to as s2U), 4'-thiouridine, 5-methylcytosine (also referred to 5mC), 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- pseudouridine,
  • the chemical modification is N1 -methylpseudouridine, 5-methoxyuridine or a combination thereof; particularly the chemical modification is N1- methylpseudouridine further optionally, wherein the polynucleotide is a ribonucleic acid (RNA); further optionally, wherein the RNA is selected from the group consisting of a short interfering RNA (siRNA), a self-replicating RNA (srRNA), circular RNA (circRNA), a selfamplifying RNA (saRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a small RNA (sRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof; particularly the RNA is an mRNA.
  • siRNA short interfering RNA
  • srRNA self-re
  • a pharmaceutical composition comprising the lipid nanoparticle as defined in any one of clauses 22 to 28 and a pharmaceutically acceptable excipient or carrier.

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Abstract

L'invention concerne un lipide ionisable de formule (I) ou un sel pharmaceutiquement acceptable de celui-ci, ou un stéréoisomère de l'un quelconque d'entre eux ; une nanoparticule lipidique qui comprend le lipide ionisable, en particulier, en tant qu'agent d'encapsulation, qui comprend éventuellement un agent pharmaceutiquement actif ; et une composition pharmaceutique qui contient la nanoparticule lipidique. L'invention concerne une nanoparticule lipidique ou une composition pharmaceutique la contenant destinées à être utilisées en médecine, et l'utilisation des nanoparticules lipidiques en tant qu'agent d'encapsulation.
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