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WO2025100737A1 - Nouveau lipide ionisable et composition de nanoparticules lipidiques le comprenant - Google Patents

Nouveau lipide ionisable et composition de nanoparticules lipidiques le comprenant Download PDF

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Publication number
WO2025100737A1
WO2025100737A1 PCT/KR2024/014825 KR2024014825W WO2025100737A1 WO 2025100737 A1 WO2025100737 A1 WO 2025100737A1 KR 2024014825 W KR2024014825 W KR 2024014825W WO 2025100737 A1 WO2025100737 A1 WO 2025100737A1
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bis
pyridin
lipid
compound
ribonucleic acid
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Korean (ko)
Inventor
기민효
최미화
박지영
박소현
백문정
이국화
노진석
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Medicibio Co Ltd
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Medicibio Co Ltd
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Priority claimed from KR1020240062988A external-priority patent/KR20250066398A/ko
Application filed by Medicibio Co Ltd filed Critical Medicibio Co Ltd
Publication of WO2025100737A1 publication Critical patent/WO2025100737A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals

Definitions

  • the present invention relates to a novel ionized lipid and a lipid nanoparticle composition comprising the same.
  • the present invention was studied as a project (S3368433) of the Regional Specialized Industry Promotion+(R&D) project hosted by the Small and Medium Business Technology Information Promotion Agency of the Ministry of SMEs and Startups, “Development of a novel biodegradable ionized lipid material as a core carrier of lipid nanoparticle technology for gene delivery.”
  • Nucleic acid-based medicines are used not only as therapeutic agents, but also as preventive agents that defend against diseases by injecting genes that can express antigens for specific diseases.
  • gene-based vaccines are divided into DNA vaccines, RNA vaccines, and viral vector vaccines.
  • RNA vaccines inject mRNA encoding antigens into the human body to express the antigens in the human body and induce antibody formation against them. They do not have the potential risks of infection that viral vector-based vaccines have or genetic mutations that DNA vaccines have, and they have the advantage of being able to be developed quickly, so they have been in the spotlight as an effective response to COVID-19 that broke out in 2019.
  • lipid nanoparticles as lipid delivery vehicles were applied to the development of patisiran, the first siRNA new drug approved by the FDA in 2018, as well as the mRNA vaccine for COVID-19, which was approved for emergency use in 2020. Since then, lipid nanoparticle technology has been recognized as a clinically useful delivery system for nucleic acid drugs such as siRNA and mRNA.
  • lipid nanoparticles are generally used in a form in which four components, ionized lipid, phospholipid (helper lipid), cholesterol (structural maintenance lipid), and PEG-lipid, are mixed in a certain ratio.
  • phospholipid helper lipid
  • cholesterol structural maintenance lipid
  • PEG-lipid lipid-based carriers
  • the purpose of the present invention is to provide a novel ionizable lipid compound.
  • Another object of the present invention is to provide a lipid nanoparticle composition comprising the compound.
  • Another object of the present invention is to provide medical uses of the lipid nanoparticle composition.
  • the present invention provides a compound selected from the compound represented by the following chemical formula I, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:
  • the present invention provides a lipid nanoparticle composition comprising the compound described above.
  • the present invention provides a drug delivery composition comprising the lipid nanoparticle composition described above; and a preventive or therapeutic agent.
  • the present invention relates to a novel ionized lipid compound and a lipid nanoparticle composition using the same.
  • the lipid nanoparticle exhibits excellent gene encapsulation rate and in vivo gene transfer rate, and also exhibits excellent gene transfer ability in a liver damage model, and thus can be usefully utilized in the development of gene therapeutic agents for various indications.
  • Figure 1 illustrates a manufacturing process of compound 1 according to one embodiment of the present invention.
  • Figure 2 shows the manufacturing process of compound 2.
  • Figure 3 shows the manufacturing process of compound 3.
  • Figure 4 shows the manufacturing process of compound 4.
  • Figure 5 shows the manufacturing process of compound 5.
  • Figure 6 shows the manufacturing process of compound 6.
  • the present inventors synthesized a novel ionizable lipid compound and confirmed that a lipid nanoparticle composition manufactured using the same can deliver genes into a living body with excellent efficiency, thereby completing the present invention.
  • the present invention provides a compound selected from the group consisting of a compound represented by the following chemical formula I, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:
  • n 0 or 1
  • R 1 is one of hydrogen, (C1-C20) alkyl, (C2-C20) alkenyl and (C2-C20) alkynyl,
  • R 1 is hydrogen or (C1-C10) alkyl
  • the compound may be selected from the following group of compounds:
  • compound 1 (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (hereinafter, compound 1) has a structure as shown in the following chemical formula 1-1, and the molecular formula is C 49 H 90 N 2 O 4 and the molecular weight is 771.25:
  • compound 2 bis(2-butyloctyl)10,10'-(pyridin-4-ylazanethyl)bis(decanoate) (hereinafter, compound 2) has a structure as shown in the following chemical formula 1-2, a molecular formula of C 49 H 90 N 2 O 4 , and a molecular weight of 771.25:
  • compound 3 bis(2-butyloctyl)8,8'-(pyridin-4-ylazanethyl)dioctanoate (hereinafter, compound 3) has a structure as shown in the following chemical formula 1-3, a molecular formula of C 45 H 82 N 2 O 4 , and a molecular weight of 715.14:
  • compound 5 bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanethyl)dioctanoate (hereinafter, compound 5) has a structure as shown in the following chemical formula 1-5, a molecular formula of C 53 H 98 N 2 O 4 , and a molecular weight of 827.36:
  • compound 6 (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 6) has a structure as shown in the following chemical formula 1-6, and the molecular formula is C 52 H 95 N 3 O 6 and the molecular weight is 858.33:
  • compound 7 bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanethyl)dihexanoate (hereinafter, compound 7) has a structure as shown in the following chemical formula 1-7, a molecular formula of C 49 H 90 N 2 O 4 , and a molecular weight of 771.25:
  • compound 8 bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanethyl)dihexanoate (hereinafter, compound 8) has a structure as chemical formula 1-8, a molecular formula of C 45 H 82 N 2 O 4 , and a molecular weight of 715.14:
  • compound 9 ((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 9) has a structure as shown in the following chemical formula 1-9, and the molecular formula is C 55 H 101 N 3 O 6 and the molecular weight is 900.41:
  • compound 10 ((3-((3-methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 10) has a structure as shown in the following chemical formula 1-10, a molecular formula of C 56 H 103 N 3 O 6 , and a molecular weight of 914.43:
  • compound 11 has a structure as shown in the following chemical formula 1-11, a molecular formula of C 55 H 101 N 3 O 6 , and a molecular weight of 900.41:
  • compound 12 has a structure as shown in the following chemical formula 1-12, and the molecular formula is C 49 H 90 N 2 O 4 S 4 and the molecular weight is 899.51:
  • compound 13 bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridin-4-ylazanethyl)dihexanoate (hereinafter, compound 13) has a structure as shown in the following chemical formula 1-13, a molecular formula of C 49 H 90 N 2 O 4 S 4 , and a molecular weight of 899.51:
  • the present invention provides a lipid nanoparticle composition
  • a lipid nanoparticle composition comprising a compound selected from the compound represented by the above chemical formula I, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.
  • the above compound can act as an ionizing lipid.
  • the above lipid nanoparticle composition may further include at least one selected from the group consisting of neutral lipids, steroids, and polymerized lipids.
  • the above neutral lipid may be selected from phospholipids or glycolipids.
  • the phospholipids are dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may be at least one selected from the group consisting of phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine, and the glycolipid may be at least one selected from the group consisting of glucosylceramide, galactosylceramide, glu
  • the above steroid may be at least one selected from the group consisting of cholesterol, bile acid derivatives and cholic acid derivatives, but is not limited thereto.
  • the above polymer polymerized lipid may be a pegylated lipid having a structure in which a water-soluble polymer and a lipid are combined, and the pegylated lipid may be at least one selected from the group consisting of PEG coupled to dialkyloxypropyl (PEG-DAA); PEG coupled to diacylglycerol (PEG-DAG) such as PEG-c-DOMG and PEG-DMG; PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-DLPE, PEG-DMPE and PEG-DSPE); PEG coupled to ceramide (PEG-CER); PEG coupled to cholesterol or a derivative thereof; PEG-modified phosphatidic acid; PEG-modified dialkylamine; and PEG-modified dialkylglycerol, but is not limited thereto.
  • PEG-DAA PEG coupled to dialkyloxypropyl
  • PEG-DAG diacylglycerol
  • the pegylated lipid may be a functional group coupled to a side that is not coupled to lipid (functionalized PEG).
  • the functional groups that can be used at this time may be at least one selected from the group consisting of succinyl, carboxylic acid, maleimide, n-hydroxysuccinimide, amine, biotin, cyanuric, and folate, but are not limited thereto.
  • the lipid nanoparticle composition can be manufactured by including ionized lipids, neutral lipids, steroids, polymer polymerized lipids, etc.
  • the lipid composition excluding effective ingredients such as genes such as RNA and DNA, may include 20 to 65 mol% of ionized lipids, 2.5 to 30 mol% of neutral lipids, 20 to 60 mol% of steroids, and 0.5 to 5 mol% of polymer polymerized lipids.
  • ionized lipid:neutral lipid:steroid:polymer polymerized lipid is 60:5:33.5:1.5, 50:10:38.5:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 25:25:48.5:1.5, 59.5:5:33.5:2.0, 49.5:10:38.5:2.0, 39.5:15:43.5:2.0, 29.5:20:48.5:2.0, 24.5:25:48.5:2.0, 59:5:33.5:2.5, 49:10:38.5:2.5, 39:15:43.5:2.5, It can be composed in one mol% ratio selected from the group consisting of 29:20:48.5:2.5, 24:25:48.5:2.5, 58.5:5:33.5:3.0, 48.5:10:38.5:3.0, 38.5:15:43.5:3.0, 28.5:20:48.5:3.0, 23.5:25:48.5:3.0, 58:5:33.5:3.5, 48:10:3
  • the lipid nanoparticle composition may further include a preventive or therapeutic agent, and the preventive or therapeutic agent may be a gene including RNA, DNA or a mixture thereof, and specifically, may be at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzy
  • the active ingredient of the composition may be an anionic biopolymer-drug conjugate such as various anionic peptides, protein drugs, protein-nucleic acid structures, hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc.
  • an anionic biopolymer-drug conjugate such as various anionic peptides, protein drugs, protein-nucleic acid structures, hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc.
  • the lipid nanoparticle composition including the ionized lipid, neutral lipid, steroid, polymerized lipid, etc. of the present invention can be prepared as a lipid solution by being dissolved in a solvent miscible with ethanol or water.
  • the active ingredient can be prepared as an active ingredient solution by being dissolved in a citric acid or acetic acid buffer having a pH of 4.0 ⁇ 1.0.
  • the lipid solution and the active ingredient solution can be prepared as lipid nanoparticles by being mixed at a volume ratio of 1:3 at a flow rate of about 10 to 15 mL/min using a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems).
  • the present invention provides a drug delivery composition comprising the lipid nanoparticle composition; and a preventive or therapeutic agent.
  • the above preventive or therapeutic agent may be a gene including RNA, DNA or a mixture thereof, and specifically, may be at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA)
  • the lipid nanoparticle according to the present invention has excellent safety and stability, and can function as a delivery vehicle so that a gene containing RNA, DNA or a mixture thereof as an active ingredient can exhibit its effect well in a cell. Accordingly, the novel lipid nanoparticle using the ionized lipid exhibits an excellent gene encapsulation rate and an in vivo gene delivery rate, and on the other hand, exhibits an excellent gene delivery function even in a liver damage model, and thus can be usefully utilized as a drug delivery composition.
  • the above drug delivery composition can be administered to mammals including humans by various routes including parenteral administration, and parenteral administration can be applied intravenously, subcutaneously, intraperitoneally or locally, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route and time of administration, but can be appropriately selected by those skilled in the art.
  • the drug delivery composition When formulating the drug delivery composition according to an example, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.
  • diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerol, and gelatin.
  • the drug delivery composition of the present invention can be administered while containing a pharmaceutically effective amount of a preventive or therapeutic agent.
  • the effective dosage level of the preventive or therapeutic agent can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concomitant drug, and other factors well known in the medical field.
  • the composition according to an example can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses.
  • the composition can be administered at 0.01 to 100 mg/kg, 0.1 to 50 mg/kg, or 1 to 10 mg/kg.
  • compound 1-b (9.74 g, 85.3 mmol) was added to xylene (100 mL), compound 1-a (10.0 g, 85.3 mmol) was added, and the mixture was stirred at 140°C for 12 hours under nitrogen gas.
  • PE petroleum ether
  • compound 2-a (11.0 g, 39.5 mmol) and compound 2-b (3.0 g, 17.9 mmol, 2.70 mL) were added to DMF (50 mL), followed by adding potassium carbonate (K 2 CO 3 ) (12.4 g, 89.7 mmol) and potassium iodide (KI) (7.45 g, 44.9 mmol), and stirred at 80 °C for 12 hours.
  • Purified water 500 mL was added to the mixture, and the extracted solution was washed with brine (300 mL ⁇ 3 times). After drying over Na 2 SO 4 , the mixture was concentrated under reduced pressure and purified on silica gel (100% DCM) to obtain compound 2-c as a yellow oil.
  • compound 3-a (25.1 g, 100 mmol) and compound 3-b (7.60 g, 45.5 mmol), K 2 CO 3 (31.4 g, 227 mmol), and sodium iodide (NaI) (17.0 g, 114 mmol) were added to DMF (150 mL) and stirred at 80°C for 12 h.
  • DCM/MeOH 100/0 to 97/23
  • compound 4-a (380 mg, 1.00 mmol) and compound 4-b (646 mg, 3.01 mmol) were added to DMF (10 mL), followed by adding EDCI (578 mg, 3.01 mmol) and DMAP (368 mg, 3.01 mmol), and stirred at 30°C for 12 h. Saturated sat. NaHCO 3 (50 mL) was added to the mixture, and the solution was extracted with EA (50 mL ⁇ 4 times), and washed with brine (50 mL ⁇ 2 times).
  • compound 5-a 350 mg, 0.925 mmol
  • compound 5-b (673 mg, 2.77 mmol) were added to DMF (10 mL), followed by addition of EDCI (532 mg, 2.77 mmol) and DMAP (339 mg, 2.77 mmol), and stirred at 30°C for 14 h. The mixture was washed with saturated sat.
  • the results of 1 H NMR analysis of compound 6 were as follows.
  • Lipid nanoparticles encapsulating Firefly Luciferase mRNA were prepared using the compound (ionized lipid) prepared in Example 1 above, dioleoylphosphatidylethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and PEG 2000-DMG (polymerized lipid).
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC distearoylphosphatidylcholine
  • PEG 2000-DMG polymerized lipid
  • Ionized lipids, DOPE/DSPC, cholesterol, and PEG 2000-DMG were mixed in a molar % ratio of 49:10:38.5:2.5 and dissolved in ethanol at a concentration of 4.8 mg/mL to prepare a lipid solution.
  • Firefly Luciferase mRNA was dissolved in a citric acid buffer solution (pH 4.0 ⁇ 1.0) or an acetic acid buffer solution (pH 5.0 ⁇ 1.0) at a weight ratio of 1:16 for mRNA: ionized lipid to prepare an active ingredient solution.
  • the lipid solution: active ingredient solution was passed through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of approximately 15 mL/min at a volume ratio of 1:3 to prepare lipid nanoparticles.
  • the manufactured lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube to ensure less than 1% ethanol content, and the final concentration was prepared to be 0.2 mg/mL based on mRNA.
  • lipid nanoparticles encapsulating Firefly Luciferase mRNA were prepared using MC3 ionized lipid, SM-102 ionized lipid, distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and DMG-PEG2000 (polymer polymerized lipid).
  • DSPC distearoylphosphatidylcholine
  • cholesterol steroid
  • DMG-PEG2000 polymer polymerized lipid
  • MC3, DSPC, cholesterol, and DMG-PEG2000 or SM-102, DSPC, cholesterol, and DMG-PEG2000 were prepared in a mol % ratio of 50:10:38.5:1.5 and used as lipid nanoparticles for comparison.
  • the size and surface charge of lipid nanoparticles according to the ionized lipid prepared in Example 1 were measured.
  • concentration of mRNA contained in each lipid nanoparticle was diluted with PBS to 1 ⁇ g/mL, and the diameter and polydispersity index (PDI) of LNPs were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).
  • DLS dynamic light scattering
  • Ribosomal RNA standard (100 ⁇ g/mL in TE buffer) was taken 5 ⁇ L and diluted with 245 ⁇ L TE buffer or 0.4% Triton-TE buffer, respectively, to prepare a stock solution for a calibration curve.
  • TE buffer 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in DEPC-treated water.
  • 50 ⁇ L of the diluted nanoparticle solution was added with 50 ⁇ L of TE buffer or 2% Triton-TE buffer, respectively.
  • the solution was incubated at 37°C for about 10 minutes. After that, 40 ⁇ L of each culture solution was placed in a microplate, 60 ⁇ L of TE buffer and 100 ⁇ L of Quant-iTTM RibogreenTM RNA Reagent were sequentially added, mixed, and the fluorescence intensity was measured.
  • the measured fluorescence intensity was substituted into the calibration curve to calculate the gene amount (TO) of the TE buffer and the gene amount (T2) of the 2% Triton-TE buffer, and then the gene inclusion rate was calculated using the following mathematical equation 1.
  • the gene inclusion rate was confirmed and is shown in Table 2. As a result, all showed good gene inclusion rates.
  • lipid nanoparticle-encapsulated luciferase gene was performed into 7-week-old C57BL/6 mice, and the mice were sacrificed after 4 hours and their livers were removed.
  • the weight of the liver tissue section was measured and transferred to a tube containing 3 mm metal beads.
  • 500 ⁇ l of Glo-Lysis Buffer (Promega, WI, USA) was added per 50 mg, and the tissue was homogenized using a bead homogenizer. Then, the supernatant of the centrifuged homogenate was taken and diluted with Glo-Lysis Buffer at an appropriate ratio.
  • the diluted tissue homogenate and Steady-Glo Luciferase Assay solution were transferred to a 96-well white plate at a 1:1 ratio (50 ⁇ l each) and reacted at room temperature.
  • the luminescence value was detected using a GloMax Discover microplate reader (Promega).
  • a calibration curve was generated using recombinant luciferase (Promega) as a standard, and the luciferase expression level of the sample (ng/g tissue) was derived.
  • the lipid nanoparticles using the ionized lipid of the present invention exhibited excellent gene delivery effects through systemic administration by showing a good level of luciferase expression in liver tissue when injected intravenously.
  • Luciferase expression level (Mean ⁇ SD, ng/g liver) 1 96,246 ⁇ 27,097 2 21,597 ⁇ 11,480 3 8,248 ⁇ 1,030 4 29,027 ⁇ 853 5 46,333 ⁇ 32,091 MC3 13,845 ⁇ 3,787
  • lipid nanoparticles loaded with EPO mRNA were intravenously administered to 7-week-old C57BL/6 mice at a dose of 0.5 mg/kg per gene.
  • EPO mRNA TriLink BioTechnologies, CA, USA
  • blood was collected 6 hours after administration, and the collected blood was treated with anticoagulation to separate plasma.
  • EPO enzyme-linked immunosorbent assay (ELISA) kit R&D Systems, MN, USA
  • the analysis was performed according to the method suggested by the manufacturer. More specifically, the plasma sample was diluted 1:3000 using the specimen diluent in the kit, and 100 ⁇ L of each (1:1 ratio) with the assay diluent was added to the microwell and reacted at room temperature for 2 hours. Then, after removing all wells, 200 ⁇ L of EPO conjugate was added and reacted for another 2 hours at room temperature, and after washing four times with 300 ⁇ L of washing buffer, 200 ⁇ L of substrate solution was added and reacted for 20 minutes.
  • ELISA enzyme-linked immunosorbent assay
  • the lipid nanoparticles using the ionized lipids of the present invention showed a good level of EPO expression in the blood compared to the lipid nanoparticles using the ionized lipids MC3 and SM-102 used for comparison when injected intravenously, confirming an excellent gene delivery effect through systemic administration.
  • EPO expression level (Mean ⁇ SD, ng/mL) 1 4,424 ⁇ 588 MC3 384 ⁇ 73 SM-102 1,154 ⁇ 364
  • liver damage was induced in 6-week-old C57BL/6 mice by intraperitoneal administration of CCl4 diluted to 20% in olive oil at 2 mL/kg three times a week for 2 weeks.
  • the ALT and AST levels in the plasma were checked the day after the final administration of CCl4.
  • liver damage was confirmed to have been induced.
  • a single intravenous injection of 1 mg/kg based on the loaded luciferase gene of each lipid nanoparticle was administered to mice with induced liver damage (liver damage model) and mice administered olive oil as a vehicle (normal model). The mice were sacrificed 6 hours after administration, and their livers were removed.
  • the weight of the liver tissue section was measured and transferred to a tube containing 3 mm metal beads.
  • 500 ⁇ L of Glo-Lysis Buffer (Promega, WI, USA) was added per 50 mg, and the tissue was homogenized using a bead homogenizer. Then, the supernatant of the centrifuged homogenate was taken and diluted with Glo-Lysis Buffer at an appropriate ratio.
  • the diluted tissue homogenate and Steady-Glo Luciferase Assay solution were transferred to a 96-well white plate at a 1:1 ratio (50 ⁇ L each) and reacted at room temperature.
  • the luminescence value was detected using a GloMax Discover microplate reader (Promega).
  • a calibration curve was generated using recombinant luciferase (Promega) as a standard, and the luciferase expression level of the sample (ng/g tissue) was derived.
  • the lipid nanoparticles using the ionized lipid of the present invention showed the characteristic of maintaining excellent gene transfer ability without difference from the normal animal model when intravenously injected into a liver injury animal model.
  • the lipid nanoparticles using SM-102 used as a comparison showed the characteristic of decreased gene transfer ability in a liver injury animal model.
  • Luciferase expression level (Mean ⁇ SD, ng/g liver) Normal model Liver damage model 1 65,704 ⁇ 12,003 86,676 ⁇ 8,499 SM-102 21,152 ⁇ 197 4,040 ⁇ 1,438

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Abstract

La présente invention concerne un nouveau lipide ionisable et une composition de nanoparticules lipidiques le comprenant. Les nanoparticules lipidiques formées à partir d'un nouveau composé lipidique ionisable, selon la présente invention, présentent un excellent taux d'encapsulation de gène et un excellent taux d'administration de gène in vivo, et de plus, présentent une excellente fonction d'administration de gène même dans un modèle d'endommagement du foie, et peuvent ainsi être utilisées utilement en tant que composition pour l'administration de médicament.
PCT/KR2024/014825 2023-11-06 2024-09-30 Nouveau lipide ionisable et composition de nanoparticules lipidiques le comprenant Pending WO2025100737A1 (fr)

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KR10-2023-0151385 2023-11-06
KR20230151385 2023-11-06
KR1020240062988A KR20250066398A (ko) 2023-11-06 2024-05-14 신규한 이온화 지질 및 이를 포함하는 지질 나노입자 조성물
KR10-2024-0062988 2024-05-14

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021204175A1 (fr) * 2020-04-09 2021-10-14 Suzhou Abogen Biosciences Co., Ltd. Compositions de nanoparticules lipidiques
US20220218816A1 (en) * 2020-04-09 2022-07-14 Suzhou Abogen Biosciences Co., Ltd. Nucleic acid vaccines for coronavirus
KR20220103923A (ko) * 2019-09-19 2022-07-25 모더나티엑스, 인크. 치료제의 세포내 전달을 위한 신규한 헤드기 지질 화합물 및 조성물
KR102560772B1 (ko) * 2022-03-21 2023-07-28 주식회사 메디치바이오 신규한 이온화지질 및 이를 이용한 지질나노입자 조성물
CN116514696A (zh) * 2023-06-29 2023-08-01 艾斯拓康医药科技(北京)有限公司 可离子化脂质及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220103923A (ko) * 2019-09-19 2022-07-25 모더나티엑스, 인크. 치료제의 세포내 전달을 위한 신규한 헤드기 지질 화합물 및 조성물
WO2021204175A1 (fr) * 2020-04-09 2021-10-14 Suzhou Abogen Biosciences Co., Ltd. Compositions de nanoparticules lipidiques
US20220218816A1 (en) * 2020-04-09 2022-07-14 Suzhou Abogen Biosciences Co., Ltd. Nucleic acid vaccines for coronavirus
KR102560772B1 (ko) * 2022-03-21 2023-07-28 주식회사 메디치바이오 신규한 이온화지질 및 이를 이용한 지질나노입자 조성물
CN116514696A (zh) * 2023-06-29 2023-08-01 艾斯拓康医药科技(北京)有限公司 可离子化脂质及其应用

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