WO2025178465A1 - Nouveau lipide ionisable et composition de nanoparticules lipidiques l'utilisant - Google Patents

Nouveau lipide ionisable et composition de nanoparticules lipidiques l'utilisant

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Publication number
WO2025178465A1
WO2025178465A1 PCT/KR2025/099467 KR2025099467W WO2025178465A1 WO 2025178465 A1 WO2025178465 A1 WO 2025178465A1 KR 2025099467 W KR2025099467 W KR 2025099467W WO 2025178465 A1 WO2025178465 A1 WO 2025178465A1
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Prior art keywords
compound
oxy
heptanedioate
dec
oxopropyl
Prior art date
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Pending
Application number
PCT/KR2025/099467
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English (en)
Korean (ko)
Inventor
기민효
최미화
박지영
박소현
백문정
이국화
노진석
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.)
Medicibio Co Ltd
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Medicibio Co Ltd
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Publication date
Priority claimed from KR1020250020723A external-priority patent/KR102917928B1/ko
Application filed by Medicibio Co Ltd filed Critical Medicibio Co Ltd
Publication of WO2025178465A1 publication Critical patent/WO2025178465A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
    • 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
    • C07C229/24—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 more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Definitions

  • the present invention relates to a novel ionized lipid, a lipid nanoparticle composition using the same, and uses thereof.
  • Nucleic acid-based medicines are being used not only as therapeutic agents, but also as preventative agents that protect against diseases by injecting genes that can express antigens for specific diseases.
  • Gene-based vaccines are categorized into DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject mRNA encoding an antigen into the body, causing the antigen to be expressed in the body and inducing antibody formation. RNA vaccines do not have the potential risks of infection associated with viral vector-based vaccines or genetic mutation associated with DNA vaccines. Furthermore, they can be developed quickly, drawing attention as an effective response to the COVID-19 outbreak in 2019.
  • lipid nanoparticles as lipid delivery vehicles were applied to the development of patisiran (ONPATTRO ® ), the first siRNA new drug approved by the FDA in 2018, as well as the mRNA vaccine for COVID-19, which received emergency use approval 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 are mixed in a certain ratio: ionized lipid, phospholipid (helper lipid), cholesterol (structure-maintaining lipid), and PEG-lipid.
  • ionized lipid phospholipid (helper lipid), cholesterol (structure-maintaining lipid), and PEG-lipid.
  • phospholipid helper lipid
  • cholesterol structure-maintaining lipid
  • PEG-lipid PEG-lipid
  • lipid nanoparticles can be utilized in the development of gene therapy.
  • most lipid nanoparticles have problems with gene delivery capacity being reduced and not maintained when re-administered to maintain the therapeutic efficacy. Therefore, the development of novel ionizable lipid carriers to address this issue is needed.
  • Local administration of lipid nanoparticles can be utilized in the development of mRNA vaccines or topical therapeutics. However, in this case, unnecessary systemic exposure of lipid nanoparticles can lead to adverse effects. Therefore, the development of ionizable lipid carriers with high local protein expression rates is required.
  • the purpose of the present invention is to provide a novel ionizable lipid compound.
  • Another object of the present invention is to provide lipid nanoparticles using the above ionized lipid compound.
  • Another object of the present invention is to provide medical uses of the above lipid nanoparticles.
  • the present invention provides a compound selected from a compound in which R 1 , R 2 , R 3 and (R 4 )-X are coordinated around a central carbon as shown in the following chemical formula 1, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:
  • the present invention provides a lipid nanoparticle composition comprising the above compound.
  • the present invention provides a composition for drug delivery comprising the lipid nanoparticle composition described above.
  • Lipid nanoparticles containing novel ionized lipids according to the present invention exhibit excellent gene encapsulation rates and in vivo gene transfer rates, and have been confirmed to exhibit excellent gene transfer ability even upon re-administration, and thus can be usefully utilized in the development of gene therapeutic agents for various indications.
  • the present invention provides a compound selected from compounds in which R 1 , R 2 , R 3 and (R 4 )-X are coordinated around a central carbon as shown in the following chemical formula 1, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:
  • R 1 , R 2 and R 3 may each be independently represented by the following chemical formula 1-A:
  • L may be (C2-C5)alkylene
  • R may be selected from (C4-C25) alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl with or without
  • the above chemical formula 1-A corresponding to the lipid tail may be selected from the following structural formulas 1 to 3, or a mixed form thereof, but is not limited thereto:
  • X may be selected from amino substituted with one or more (C1-C4)alkyl; or a cyclic hetero group selected from the group consisting of piperidine, piperazine, and imidazole, and the cyclic hetero group may be substituted or unsubstituted with (C1-C4)alkyl, hydroxy, or hydroxy(C1-C4)alkyl, but is not limited thereto.
  • the present invention may include compounds represented by the following chemical formulas:
  • the compound is (1) di((z)-dec-4-en-1-yl) 4-(3-(((Z)-dec-4-en-1-yl)oxy)-3-oxopropyl)-4-(3-(piperidin-1-yl)propanamido)heptanedioate, (3) di((z)-dodec-5-en-yl) 4-(((4-(dimethylamino)butoxy)carbonyl)amino)-4-(3-oxo-3-(((Z)-tridec-6-en-1-yl)oxy)propyl)heptanedioate, (4) di((z)-dodec-5-en-1-yl) 4-(3-(((Z)-dodec-5-en-1-yl)oxy)-3-oxopropyl)-4-(3-(piperidin-1-yl)propanamido)heptanedioate, (5) di((z)
  • the above compound may include a pharmaceutically acceptable salt form exhibiting the same or similar activity.
  • “pharmaceutically acceptable” means that the compound or salt thereof is not toxic to cells or humans exposed to it, and thus has a safety and efficacy profile suitable for administration to humans.
  • the above salt may be used in the form of either a pharmaceutically acceptable basic salt or an acid salt.
  • the basic salt may be used in the form of either an organic basic salt or an inorganic basic salt, and may be selected from the group consisting of sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt.
  • the acid salt is useful as an acid addition salt formed by a free acid.
  • Inorganic acids and organic acids can be used as free acids, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid, nitric acid, etc.
  • organic acids such as citric acid, acetic acid, maleic acid, malic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, stearic acid, etc. can be used, but are not limited thereto, and all salts formed using various inorganic acids and organic acids commonly used in the art can be included.
  • the compound may include not only the above salts, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared by conventional methods.
  • Addition salts can be prepared by conventional methods, and can be prepared by dissolving in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of organic base, or adding an aqueous base solution of an inorganic base, and then precipitating or crystallizing.
  • the addition salt can be obtained by evaporating the solvent or excess base from the mixture and then drying, or by suction filtration of the precipitated salt.
  • the present invention provides a lipid nanoparticle composition comprising the above compound.
  • the above compound can act as an ionizable lipid.
  • the lipid nanoparticle composition comprising the above compound has excellent safety and stability, and can function as a delivery vehicle so that a gene including RNA, DNA, or a mixture thereof as an active ingredient can effectively exhibit its effect within a cell. Accordingly, the lipid nanoparticles comprising the novel ionizable lipid compound exhibit excellent gene encapsulation rates and in vivo gene delivery rates, and on the other hand, exhibit excellent gene delivery functions even upon re-administration, and thus can be usefully utilized as a drug delivery composition.
  • the lipid nanoparticle composition may further comprise one or more selected from the group consisting of neutral lipids, steroids, and polymerized lipids.
  • 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 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 bound to dialkyloxypropyl (PEG-DAA); PEG bound to diacylglycerol (PEG-DAG) such as PEG-c-DOMG and PEG-DMG; PEG bound to phospholipids such as phosphatidylethanolamine (PEG-DLPE, PEG-DMPE and PEG-DSPE); PEG bound to ceramide (PEG-CER); PEG bound 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 bound to dialkyloxypropyl
  • PEG-DAG PEG bound to diacylglycerol
  • the pegylated lipid may be a functionalized PEG in which a functional group is combined on a side that is not combined with lipid.
  • the functional group 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 is not limited thereto.
  • the above ionized lipids can be prepared into lipid nanoparticles, including neutral lipids, steroids, polymerized lipids, etc.
  • the lipid nanoparticle composition may contain 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 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 comprise a prophylactic or therapeutic agent.
  • the agent may be a gene comprising RNA, DNA or a mixture thereof composed of single strands or double strands, 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) and guide ribonucleic acid for
  • the active ingredient of the above composition may be an anionic biopolymer-drug conjugate such as various anionic peptides, protein drugs, protein-nucleic acid structures, or 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, or hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc.
  • the present invention provides a drug delivery composition comprising the lipid nanoparticle composition and a preventive or therapeutic agent.
  • the above drug delivery composition can be administered to mammals, including humans, via various routes, including parenteral administration.
  • Parenteral administration can be applied intravenously, subcutaneously, intraperitoneally, or locally.
  • the dosage varies depending on the patient's condition and weight, the extent of the 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 is manufactured using diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.
  • the drug delivery composition of the present invention can be administered containing a pharmaceutically effective amount of a preventive or therapeutic agent.
  • the effective dosage level of the preventive or therapeutic agent can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route and excretion rate, the duration of treatment, factors including concomitant medications, and other factors well known in the medical field.
  • the composition can be administered as an individual therapeutic agent or in combination with another therapeutic agent, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.
  • the composition can be administered at 0.001 to 10 mg/kg, 0.01 to 5 mg/kg, or 0.1 to 1 mg/kg.
  • compound 1-a (1.57 g, 10.0 mmol) was dissolved in dichloromethane (29 mL, hereinafter referred to as DCM) at room temperature, and then compound 1-b (4.16 g, 10.0 mmol) and 4-dimethylaminopyridine (122 mg, 1.00 mmol, hereinafter referred to as DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.88 g, 15.0 mmol, hereinafter referred to as EDCI) were added, followed by stirring at room temperature for 16 hours.
  • DCM dichloromethane
  • EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • 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 prepared in a molar % ratio of 50:10:38.5:1.5 (composition indicated as A) or 49:10:38.5:2.5 (composition indicated as B) 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 at pH 4.0 ⁇ 1.0 or an acetate buffer solution at pH 5.0 ⁇ 1.0 (composition indicated as *) at a weight ratio of 1:16 mRNA:ionized lipid to prepare an active ingredient solution.
  • lipid nanoparticles were prepared by passing the mixture through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of approximately 15 mL/min so that the lipid solution:active ingredient solution had a volume ratio of 1:3.
  • the manufactured lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube to ensure that the ethanol content was less than 1%, and the final concentration was manufactured to be 0.2 mg/mL based on mRNA.
  • lipid nanoparticles encapsulating Firefly Luciferase mRNA were prepared using SM-102 ionizable lipid, distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and DMG-PEG2000 (polymeric lipid).
  • DSPC distearoylphosphatidylcholine
  • cholesterol steroid
  • DMG-PEG2000 polymeric lipid
  • lipid nanoparticles containing the compound of 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 lipid nanoparticles (LNPs) were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).
  • DLS dynamic light scattering
  • lipid nanoparticles including the compound of Example 1
  • FLuc mRNA a reporter gene, FLuc mRNA
  • LDL receptor low-density lipoprotein receptor
  • HeLa cell line Kel Cell Line Bank, Seoul
  • human cervical cancer cell line with high expression of LDL receptor on the cell surface was used, and cell culture was performed at 37°C and 5% CO 2 using MEM (minimum essential medium) medium (Gibco) containing 10% fetal bovine serum (Gibco, NY, USA) and penicillin/streptomycin (Gibco).
  • MEM minimum essential medium
  • Gibco fetal bovine serum
  • lipid nanoparticle treatment Before lipid nanoparticle treatment, HeLa cell lines were cultured in 96-well plates at a density of 2 ⁇ 10 4 cells per well, and after stabilization for 16 to 24 hours, lipid nanoparticles at a concentration of 0.25 ⁇ g/mL based on the encapsulated mRNA were diluted in cell culture medium and treated to the cells.
  • the culture medium in the wells was removed, washed with PBS, and 100 ⁇ L of Glo-Lysis Buffer (Promega, WI, USA) was added to lyse the cells at room temperature for 5 minutes.
  • 50 ⁇ L of each cell lysate and Steady-GloTM Luciferase Assay solution were added at a 1:1 ratio to a 96-well white plate, and after incubation at room temperature for 5 minutes, the luminescence value was detected using a GloMax Discover microplate reader (Promega).
  • a calibration curve was created using recombinant luciferase (Promega) as a standard, and the luciferase protein concentration (pg/mL) of the cell lysate was derived from this.
  • lipid nanoparticles were intravenously injected once at a dose of 1 mg/kg based on the luciferase gene loaded into 7-week-old C57BL/6 mice, and after 4 hours, the mice were sacrificed and their livers were removed.
  • the excised liver tissue was transferred to a tube containing 3 mm metal beads, 500 ⁇ L of Glo-Lysis Buffer (Promega) per 50 mg was added, and the tissue was homogenized using a bead homogenizer. The homogenate was centrifuged at 1,000 g for 10 minutes, and the supernatant was diluted to an appropriate ratio using Glo-Lysis Buffer. The diluted tissue homogenate and Steady-Glo Luciferase Assay (Promega) solution were each transferred to a 96-well white plate at a 1:1 ratio, and reacted at room temperature for 5 minutes.
  • Glo-Lysis Buffer Promega
  • the luminescence value was detected using a GloMax Discover microplate reader (Promega), and a calibration curve was generated using recombinant luciferase (Promega) as a standard, through which the luciferase expression level of the sample (ng/g tissue) was derived.
  • composition B showed similar or higher delivery efficacy compared to composition A.
  • EPO enzyme-linked immunosorbent assay (ELISA) kit R&D Systems, MN, USA
  • plasma samples were diluted 1:5000 using the specimen diluent included in the kit, mixed with 100 ⁇ L of assay diluent in a 1:1 ratio in microwells, and incubated for 2 hours at room temperature. After removing all the solution in each well, 200 ⁇ L of EPO conjugate was added, and incubated for another 2 hours at room temperature. After washing four times with 300 ⁇ L of washing buffer, 200 ⁇ L of substrate solution was added, and incubated for 20 minutes.
  • ELISA enzyme-linked immunosorbent assay
  • the lipid nanoparticles including the compound of Example 1 showed a higher level of EPO expression in the blood compared to the lipid nanoparticles including the ionized lipid Lipid 5 used as a control during repeated intravenous injections, and when administered again, it was confirmed that, unlike the Lipid 5 lipid nanoparticles, the lipid nanoparticles including the compound of Example 1 maintained a high level of EPO expression well.
  • Lipid 5 is known to be a carrier that maintains a good gene delivery ability during repeated administration among carriers to date, it can be confirmed that the compound of Example 1 has characteristics that are advantageous for repeated administration.

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Abstract

La présente invention concerne un nouveau lipide ionisé et une composition de nanoparticules lipidiques l'utilisant. Les nanoparticules lipidiques comprenant le nouveau lipide ionisé selon la présente invention présentent un excellent taux d'encapsulation génique et un excellent taux d'administration de gène in vivo, ainsi qu'une excellente capacité d'administration de gène même lorsqu'elles sont réadministrées, et peuvent ainsi être avantageusement utilisées dans le développement d'agents thérapeutiques géniques pour diverses indications.
PCT/KR2025/099467 2024-02-19 2025-02-19 Nouveau lipide ionisable et composition de nanoparticules lipidiques l'utilisant Pending WO2025178465A1 (fr)

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KR10-2024-0023279 2024-02-19
KR20240023279 2024-02-19
KR10-2025-0020723 2025-02-18
KR1020250020723A KR102917928B1 (ko) 2024-02-19 2025-02-18 신규한 이온화 지질 및 이를 이용한 지질 나노입자 조성물

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635487A (en) * 1994-12-29 1997-06-03 Wolff; Jon A. Amphipathic, micellar delivery systems for biologically active polyions
US20170197903A1 (en) * 2014-08-07 2017-07-13 Takeda Pharmaceutical Company Limited Cationic lipid
KR20200104360A (ko) * 2017-12-27 2020-09-03 다케다 야쿠힌 고교 가부시키가이샤 핵산-함유 지질 나노-입자 및 그의 용도
US20200368173A1 (en) * 2017-08-04 2020-11-26 Kyowa Kirin Co., Ltd. Nucleic acid-containing lipid nanoparticle
KR20230052895A (ko) * 2020-07-17 2023-04-20 제너레이션 바이오 컴퍼니 폴리뉴클레오타이드를 감소된 크기의 지질 나노입자로 캡슐화하는 방법 및 신규한 지질 나노입자 제형

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635487A (en) * 1994-12-29 1997-06-03 Wolff; Jon A. Amphipathic, micellar delivery systems for biologically active polyions
US20170197903A1 (en) * 2014-08-07 2017-07-13 Takeda Pharmaceutical Company Limited Cationic lipid
US20200368173A1 (en) * 2017-08-04 2020-11-26 Kyowa Kirin Co., Ltd. Nucleic acid-containing lipid nanoparticle
KR20200104360A (ko) * 2017-12-27 2020-09-03 다케다 야쿠힌 고교 가부시키가이샤 핵산-함유 지질 나노-입자 및 그의 용도
KR20230052895A (ko) * 2020-07-17 2023-04-20 제너레이션 바이오 컴퍼니 폴리뉴클레오타이드를 감소된 크기의 지질 나노입자로 캡슐화하는 방법 및 신규한 지질 나노입자 제형

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