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EP4626858A1 - Nouveaux lipides ionisables et nanoparticules lipidiques les contenant - Google Patents

Nouveaux lipides ionisables et nanoparticules lipidiques les contenant

Info

Publication number
EP4626858A1
EP4626858A1 EP23898975.0A EP23898975A EP4626858A1 EP 4626858 A1 EP4626858 A1 EP 4626858A1 EP 23898975 A EP23898975 A EP 23898975A EP 4626858 A1 EP4626858 A1 EP 4626858A1
Authority
EP
European Patent Office
Prior art keywords
lipid
alkyl group
formula
group
amino
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
EP23898975.0A
Other languages
German (de)
English (en)
Inventor
Joo-Youp LEE
Vishnu SRIRAM
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.)
University of Cincinnati
Original Assignee
University of Cincinnati
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University of Cincinnati filed Critical University of Cincinnati
Publication of EP4626858A1 publication Critical patent/EP4626858A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/18—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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

Definitions

  • an ionizable lipid a lipid nanoparticle (LNP) formed therefrom, a composition including an mRNA formulated in the lipid nanoparticle, and a method of delivering an mRNA to a subject or cell comprising administering said composition.
  • LNP lipid nanoparticle
  • lipid-based nanoparticle compositions such as lipoplexes and liposomes have been used as packaging vehicles for biologically active substances to allow transport into cells and/or intracellular compartments.
  • These lipid-based nanoparticle compositions typically comprise a mixture of different lipids such as ionizable lipids, helper lipids or phospholipids, structural lipids (such as sterols or cholesterol), and lipid conjugates.
  • Ri is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R2;
  • R2 is a C2-C18 alkyl group or C2-C18 alkenyl group;
  • R3 is a C2-C18 alkyl group;
  • R4 is a C2-C6 alkyl group;
  • R5 is a C2-C18 alkyl group;
  • Re is a C2-C18 alky l group or C2-C18 alkenyl group, optionally substituted with R7;
  • R7 is a C2-C18 alkyl group or C2-C18 alkenyl group;
  • Rs and R9 are independently hydrogen or a C1-C2 alkyl group.
  • Ri is a C9-C11 alkyd group, optionally substituted with R2;
  • R2 is a Ce- Cs alkyd group;
  • R3 is a C4-C7 alkyd group;
  • R4 is a C2-C6 alkyl group;
  • R5 is a C4-C7 alkyd group;
  • Re is a C9-C11 alkyd group, optionally substituted with R7;
  • R7 is a Ce-Cs alkyd group; and
  • Rs and R9 are independently hydrogen or a C1-C2 alkyl group.
  • the compound having the structure of Formula (I) may be selected from the group consisting of the following compounds:
  • the sterol may be cholesterol or a derivative thereof.
  • composition comprising mRNA formulated in the lipid nanoparticle is provided.
  • FIG. 1 is a graph showing the transfection efficiency of LNPs comprising different ionizable lipids in Jurkat T cells after 20-hour incubation using 100 ng eGFP mRNA.
  • FIG. 2 is a graph showing the transfection efficiency of LNPs comprising different ionizable lipids in Jurkat T cells after 20-hour incubation using 100 ng FLuc mRNA.
  • FIG. 3 is a graph showing (a) particle size, and (b) surface charge of LNPs comprising different ionizable lipids.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 25 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3. 4, 5, 6. 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • “nested sub-ranges” that extend from either end point of the range are specifically contemplated.
  • a nested sub-range of an exemplary range of 1 to 25 may comprise 1 to 5, 1 to 10, 1 to 15, and 1 to 20 in one direction, or 25 to 20, 25 to 15, 25 to 10, and 25 to 5 in the other direction.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single subject) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • a gene product may be a transcript.
  • a gene product may be a polypeptide.
  • expression of a nucleic acid sequence may involve one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post- translational modification of a polypeptide or protein.
  • subject refers to any living organism to which a pharmaceutical can be administered.
  • subject includes, but is not limited to. humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult, child, and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term "pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • the tenn "pharmaceutically acceptable excipient, carrier, or diluent” or the like refer to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • salts refers to pharmaceutically acceptable organic or inorganic salts of an ionizable lipid of the present disclosure.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1 ’- m
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion.
  • the counter ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the phannaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
  • lipid encapsulated is meant to refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g, an anti-sense oligonucleotide (ASO), mRNA, siRNA, close ended DNA (ceDNA), viral vector, etc.), with full encapsulation, partial encapsulation, or both.
  • a nucleic acid e.g, an anti-sense oligonucleotide (ASO), mRNA, siRNA, close ended DNA (ceDNA), viral vector, etc.
  • ASO anti-sense oligonucleotide
  • mRNA e.g., mRNA, siRNA, close ended DNA (ceDNA), viral vector, etc.
  • ceDNA DNA
  • viral vector etc.
  • the nucleic acid may be fully encapsulated in the lipid particle (e.g., to form a nucleic acid containing lipid particle).
  • the structures depicted and described herein include all isomeric (e.g.. enantiomeric, diastereomeric, and geometric) forms of the structure; for example, tautomers, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Additionally, unless otherwise stated, the structures depicted and described herein include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or as therapeutic agents.
  • the present disclosure provides a novel ionizable lipid compound.
  • the present disclosure provides a compound having the following structure of Formula (I) or a pharmaceutically acceptable salt thereof: wherein.
  • Ri is a C2-C18 alkyl group or C2-C18 alkenyl group, optionally substituted with R2;
  • R2 is a C2-C18 alkyl group or C2-C18 alkenyl group;
  • R3 is a C2-C18 alkyl group;
  • R4 is a C2-C6 alkyl group;
  • R5 is a C2-C18 alkyl group;
  • Re is a C2-C18 alky l group or C2-C18 alkenyl group, optionally substituted with R7;
  • R7 is a C2-C18 alkyl group or C2-C18 alkenyl group;
  • Rs and R9 are independently hydrogen or a C1-C2 alkyl group.
  • Ri is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R2;
  • R2 is a C2-C12 alkyl group or C2-C12 alkenyl group;
  • R3 is a C2-C13 alkyd group;
  • R4 is a C2-C6 alky l group;
  • R5 is a C2-C13 alkyd group;
  • Re is a C5-C15 alkyl group or C5-C15 alkenyl group, optionally substituted with R7;
  • R7 is a C2-C12 alkyl group or C2-C12 alkenyl group;
  • Rs and R9 are independently hydrogen or a C1-C2 alkyd group.
  • Ri is a C9-C11 alkyd group, optionally substituted with R2;
  • R2 is a Ce- Cs alkyd group;
  • R3 is a C4-C7 alkyd group;
  • R4 is a C2-C6 alkyl group;
  • R5 is a C4-C7 alkyd group;
  • Re is a C9-C11 alkyd group, optionally substituted with R7;
  • R7 is a Ce-Cs alkyl group; and
  • Rs and R9 are independently hydrogen or a C1-C2 alkyl group.
  • the compound having structure of Formula (I) may be selected from the group consisting of the following compounds: [Table 2] 2. Lipid nanoparticle
  • the present disclosure also provides a lipid nanoparticle comprising the compound having any one of the structures selected from the group consisting of Formula (T), Formula (I- 1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Fomiula (1-13), Formula (1-14) and Formula (1-15).
  • the lipid nanoparticle may further comprise a phospholipid, a sterol, and a PEGylated lipid conjugate.
  • the lipid component of a lipid nanoparticle may include one or more neutral lipids such as phospholipids including one or more (poly) unsaturated lipids, as a helper lipid.
  • neutral lipids such as phospholipids including one or more (poly) unsaturated lipids, as a helper lipid.
  • phospholipids may assemble into one or more lipid bilayers structures.
  • helper lipids or phospholipids that can form part of the present lipid nanoparticle may include but are not limited to 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1.2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), l,2-d
  • the phospholipid may be phosphatidylcholine (PC), phosphatidylethanolamine (PE) phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PA phosphatidic acid
  • PG phosphatidylglycerol
  • a lipid nanoparticle may include at least one phospholipid selected from l,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-Dipalmitoyl-sn- glycero-3-Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and 1, 2-dioleoyl-sn- glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG).
  • DOPG 1, 2-dioleoyl-sn- glycero-3-phospho-rac-(l -glycerol) sodium salt
  • DOPG 1, 2-
  • the lipid nanoparticle may include from about 5% to about 15% on a molar basis of the phospholipids e.g., from about 5 to about 12%, from about 7 to about 12%, from about 7 to about 15%, or about 5%, about 10%, or about 15% on a molar basis.
  • the lipid component of a lipid nanoparticle may include one or more polymer conjugated lipids, such as PEGylated lipid (PEG-lipids) conjugates.
  • PEG-lipids PEGylated lipid conjugates
  • Exemplary polymer conjugated lipids may include but are not limited to PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, PEGylated dialkylglycerols, and mixtures thereof.
  • a PEG-lipid may be l,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG also referred herein as DMG-PEG).
  • the lipid nanoparticle may include the PEG-lipids at a molar ratio of from about 0.5% to about 5% e.g., from about 0.5 to about 3%, from about 1 to about 5%, from about 1 to about 3%, or about 0.5%, about 1%. about 1.5%, about 2%, about 2.5%, or about 3% of the total lipids.
  • the lipid component of a lipid nanoparticle may include one or more structural lipids.
  • structural lipids can stabilize the amphiphilic structure of a nanoparticle, such as but not limited to the lipid bilayer structure of a nanoparticle.
  • Exemplary structural lipids that can be used in connection with the present disclosure may include but are not limited to sterol, for example, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, and mixtures thereof.
  • the structural lipid may be cholesterol.
  • the structural lipid may include cholesterol or a corticosteroid (such as prednisolone, dexamethasone, prednisone, or hydrocortisone), or a combination thereof.
  • the lipid nanoparticles provided herein may comprise a steroid or steroid analogue.
  • the steroid or steroid analogue may be cholesterol.
  • the lipid nanoparticle may include the structural lipids at a molar ratio of from about 30% to about 55% e.g., from about 30 to about 50%, from about 35 to about 55%, from about 35 to about 50%, or about 38%, about 38.5%, about 39%, about 40%, about 45%, about 47%, or about 48% of the total lipids.
  • the lipid component of a lipid nanoparticle may include one or more ionizable lipids.
  • the lipid nanoparticle may comprise at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Fonnula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15).
  • the lipid nanoparticle may include one or more other ionizable lipids which are known in the art, in addition to the ionizable lipids described above.
  • Exemplary ionizable lipids that can be used in connection with the present disclosure may include but are not limited to 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethyl aminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • DLin-KC2-DMA 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4- dimethyl aminobutyrate
  • L319 di((Z)-non-2-en-l-yl) 9-((4- (dimethyl
  • the lipid nanoparticle may include the ionizable lipids at a molar ratio of from about 30% to about 60% e.g.. from about 35 to about 55%, from about 38 to about 52%, from about 35 to about 50%, from about 40 to about 55%, from about 40 to about 50%, or about 35%, about 40%, about 45%, about 50%, or about 55% of the total lipids.
  • the lipid nanoparticle may comprise (i) at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2). Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15), (ii) at least one phospholipid, e.g., 1,2- distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphochohne (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dipal
  • the lipid nanoparticle may comprise (i) at least one ionizable lipid compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Fonnula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15), (ii) at least one phospholipid, e.g., 1,2- distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3- Phosphatidylcholine (DPPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC), dipalmitoyl phosphatidyl
  • the lipid nanoparticle may be a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticle has a mean diameter of about 50-300 nm, or about 50-200 nm.
  • composition comprising mRNA formulated in the lipid nanoparticle
  • compositions comprising an mRNA formulated in the lipid nanoparticle which comprises the compound having any one of the structures selected from the group consisting of Formula (I), Formula (1-1), Formula (1-2), Formula (1-3), Formula (1-4), Formula (1-5), Formula (1-6), Formula (1-7), Formula (1-8), Formula (1-9), Formula (1-10), Formula (1-11), Formula (1-12), Formula (1-13), Formula (1-14) and Formula (1-15).
  • mRNA in the above composition, 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 mRNA from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.
  • the LNPs may be prepared at a molar ratio between the amine group of the ionizable lipid (N) and the phosphate group of the mRNA (P) from about 4: 1 to about 20: 1, e g., from about 5: 1 to about 10: 1, or from about 6: 1 to about 8: 1.
  • the LNPs may be prepared at an N/P ratio about 4-20, e.g., about 5-10, or 6-8.
  • the composition may additionally include a pharmaceutically acceptable carrier which is suitable for delivering an mRNA to a suitable in vivo or ex vivo site.
  • a pharmaceutically acceptable carrier can include, but is not limited to, an adjuvant, an excipient, etc.
  • composition of the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 5 mg/kg, from about 0.
  • 1 mg/kg to about 10 mg/kg from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 0.0001 mg/kg to about 1 mg/kg, from about 0.001 mg/kg to about 1 mg/kg, from about 0.005 mg/kg to about 1 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, or from about 0. 1 mg/kg to about 1 mg/kg in a given dose, where a dose of 1 mg/kg provides 1 mg of mRNA or nanoparticle per 1 kg of subj ect body weight.
  • a dose of about 0.005 mg/kg to about 5 mg/kg of mRNA or nanoparticle of the disclosure may be administrated.
  • a dose may be administered one or more rimes per day, in the same or a different amount, to obtain a desired level of mRNA expression and/or effect (e.g., a therapeutic effect).
  • the desired dosage may be delivered, for example, three times a day, two times a day. once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.
  • the specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level for any particular patient will depend upon a variety of factors including the severity and identify of a disorder being treated, if any; the one or more mRNAs employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed; and like factors well known in the medical arts.
  • the effective amount may be a total dose of 10 pg-300 pg. In some embodiments, the effective amount may be a total dose of 30 pg-100 pg or 50 pg-200 pg.
  • the composition of the present disclosure may be administered twice (e g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later.
  • twice e g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later.
  • composition comprising mRNA formulated in the lipid nanoparticle may be administered three or four times.
  • Lipids 1 to 15 were synthesized according to the following general scheme:
  • LNPs lipid nanoparticles
  • the LNP containing different ionizable lipids such as Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5.
  • Lipid 6 or Lipid 8 showed an eGFP mRNA transfection efficiency of > ⁇ 70% (Fig. 1). and the LNP containing different ionizable lipids such as Lipid 1.
  • Lipid 2, Lipid 3, Lipid 4, Lipid 5 or Lipid 6 showed Flue mRNA transfection efficiency measured by luciferase assay ranged between 10 3 -10 5 relative light units (RLU) (Fig. 2).
  • the LNPs containing different ionizable lipids such as Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10 or Lipid 11 w ere characterized for particle size and surface charge using dynamic light scattering and zeta potential measurements.
  • the LNPs showed a range of particle sizes of -100-150 nm (Fig. 3(a)).
  • the surface charge of the LNPs ranged from -3 mV to -9 mV (Fig. 3(b)). Since these LNPs contained ionizable lipids, their surface charge was close to neutral when they were measured at physiological pH of 7.4.
  • the polydispersity index (PDI) of the LNPs was measured to be about - 0.2.
  • the encapsulation efficiency of mRNA inside the LNP was detennined using QUANT-ITTM Ribogreen® RNA assay (Invitrogen). In particular, the encapsulation efficiency of the LNP containing Lipid 2 was measured to be about 80 %.
  • Experimental Example 3 In-vivo Whole-Body and Organ-Specific Flux Animals
  • mice Female Balb/c mice aged 7-9 weeks (Strain #: 000651, 18-22 grams in body weight) were purchased from The Jackson Laboratory. All animals were housed in pathogen-free conditions and were provided with w aler and food ad libitum. All procedures involving animal were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academys, USA) and were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.
  • mice were injected with D-Luciferin, Sodium Salt (GoldBio, catalog #: LUCNA) intraperitoneally (i.p.) 10 min prior to the imaging time point. Luciferin was diluted using PBS to a concentration of 15 mg/ml and was injected at a dosage of 150 mg/kg. Three minutes prior to imaging, mice were placed in a chamber filled with 3% isoflurane and 97% oxygen. Once the mice were completely anesthetized, they were moved into isoflurane- delivering nosecones in the imaging chamber positioned with ventral side up and maintained on 3% isoflurane and 97% oxygen. Images were acquired using IVIS Spectrum (PerkinElmer).
  • mice were imaged 6 hours after the administration of different LNPs at a dosage of 0.5 mg/kg.
  • the total flux was measured using Living image software. After euthanizing the mice and isolating the organs, the liver, spleen, and lymph nodes were measured for luciferase expression (Fig. 4).
  • LNP containing Lipid 1 or Lipid 2 showed atotal flux of ⁇ 10 9 p/sec/cm 2 /sr.
  • LNP containing Lipid 1 or Lipid 2 showed a liver radiance of ⁇ 10 8 p/sec/cm 2 /sr, a spleen radiance in the range of 10 7 - 10 8 p/sec/cm 2 /sr, and the lymph node radiance in the range of 10 6 - 10 7 p/sec/cm 2 /sr.

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Abstract

La présente invention concerne un composé lipidique ionisable, une nanoparticule lipidique contenant le composé lipidique ionisable, une composition contenant un ARNm formulé dans la nanoparticule lipidique, et une méthode d'administration d'un ARNm à un sujet ou à une cellule par administration au sujet ou à la cellule de la composition contenant un ARNm formulé dans la nanoparticule lipidique.
EP23898975.0A 2022-12-02 2023-12-01 Nouveaux lipides ionisables et nanoparticules lipidiques les contenant Pending EP4626858A1 (fr)

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