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WO2024177424A1 - Nouveau lipide ionisable et nanoparticule lipidique le contenant - Google Patents

Nouveau lipide ionisable et nanoparticule lipidique le contenant Download PDF

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Publication number
WO2024177424A1
WO2024177424A1 PCT/KR2024/002375 KR2024002375W WO2024177424A1 WO 2024177424 A1 WO2024177424 A1 WO 2024177424A1 KR 2024002375 W KR2024002375 W KR 2024002375W WO 2024177424 A1 WO2024177424 A1 WO 2024177424A1
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Prior art keywords
lipid
alkyl
glycero
peg
independently
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English (en)
Korean (ko)
Inventor
김경진
김욱일
김다영
이주영
이지은
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ST Pharm Co Ltd
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ST Pharm Co Ltd
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    • 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
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic 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
    • A61K48/0008—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
    • A61K48/0033—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 non-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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07C229/26—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 amino group bound to the carbon skeleton, e.g. lysine
    • 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
    • C07C237/08—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 singly-bound oxygen atoms
    • 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
    • C07C271/20—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 nitrogen atoms not being part of nitro or nitroso groups

Definitions

  • the present invention relates to novel ionizable lipids comprising a biodegradable bond. Specifically, the present invention relates to ionizable lipids comprising an ester and/or amide bond as a biodegradable bond, lipid nanoparticles prepared using the same, and uses thereof.
  • Drug Delivery System is a technology designed to efficiently deliver the required amount of drugs while reducing the side effects of drugs and maximizing their efficacy and effectiveness.
  • conventional viral vectors have been proven to be effective as drug delivery vehicles, but the use of viruses as gene delivery systems is limited due to several drawbacks such as immunogenicity, limitations in the size of injected DNA, and difficulties in mass production.
  • lipid-DNA conjugates lipoplex
  • polymer-DNA conjugates polyplex
  • lipid-DNA conjugates are widely used at the cellular level because they bind to nucleic acids and deliver nucleic acids well into cells, but in vivo, they often cause inflammation in the body when injected locally (Filonand and Phillips, Biochim. Biophys/Acta, 1329, 345-356, 1997), and when injected intravascularly, they accumulate mainly in tissues such as the lungs, liver, and spleen, which are the first passage organs (Ren et al., Gene Therapy. 7, 764-768, 2000).
  • the present inventors have made extensive efforts to develop a novel material having an excellent drug encapsulation rate and capable of efficiently delivering anionic drugs such as nucleic acids to a target organ or cell, and as a result, have confirmed the excellent drug delivery effect of a novel ionizable lipid including a biodegradable bond of the present invention, thereby completing the present invention.
  • One object of the present invention is to provide an ionizable lipid comprising a biodegradable bond of novel structure, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a lipid nanoparticle comprising the ionizable lipid, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a drug delivery composition comprising the lipid nanoparticle and an anionic drug.
  • the ionizable lipid including the biodegradable bond of the present invention stably delivers anionic drugs when manufacturing lipid nanoparticles, and particularly shows excellent effects in nucleic acid delivery, so that it can be usefully used in related technical fields such as lipid nanoparticle-mediated gene therapy.
  • an ionizable lipid including a biodegradable bond represented by the following chemical formula 1 stably and effectively delivers a drug and has fewer side effects such as liver toxicity when producing lipid nanoparticles, thereby completing the present invention.
  • One embodiment of the present invention for achieving the above object is an ionizable lipid represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • R 1 is each independently -C 1-4 alkyl
  • R 2 is independently -H, -C 1-4 alkyl, or -COO-R 2A ,
  • R 2A is -(CH 2 )-aryl, -(CH 2 )-heteroaryl, -aryl, or -heteroaryl,
  • R 3 is each independently -H, -C 2-16 alkyl, or -C 2-16 alkenyl
  • n is an integer from 0 to 2.
  • R 1 is each independently -C 1-3 alkyl
  • R 2 is independently -H, -C 1-3 alkyl, or -COO-R 2A ,
  • R 2A is -phenyl or -benzyl
  • X is -O-
  • R 3 can each independently be -H, -C 4-12 alkyl, or -C 4-12 alkenyl,
  • R 1 is all -CH 3 ,
  • R 2 is independently -H, -CH 3 , or -COO-R 2A ,
  • R 2A is -phenyl or -benzyl
  • X is -O-
  • R 3 is each independently -H, -C 6-10 alkyl, or -C 6-10 alkenyl
  • n 1
  • R 1 is each independently -C 1-3 alkyl
  • R 2 is independently -H, -C 1-3 alkyl, or -COO-R 2A ,
  • R 2A is -phenyl or -benzyl
  • R 3 is each independently -H, -C 4-12 alkyl, or -C 4-12 alkenyl
  • R 1 is all -CH 3 ,
  • R 2 is independently -H, -CH 3 , or -COO-R 2A ,
  • R 2A is -phenyl or -benzyl
  • R 3 is each independently -H, -C 6-10 alkyl, or -C 6-10 alkenyl
  • n 1
  • the compound represented by the chemical formula 1 may be selected from the compounds described in Table 1 below.
  • alkyl means a straight-chain or branched-chain acyclic saturated hydrocarbon, unless otherwise specified.
  • C 1-6 alkyl may mean alkyl having 1 to 6 carbon atoms.
  • the term "ionizable lipid” means an amine-containing lipid that can be easily protonated, and is also called a lipid analogue. Since the charge state of the ionizable lipid can change depending on the surrounding pH, it plays a role in allowing the drug to be encapsulated into lipid nanoparticles with high efficiency through electrostatic interaction with an anionic drug, and contributes to forming the structure of the lipid nanoparticle.
  • the ionizable lipid of the present invention is characterized by having a form in which an amine head including one tertiary amine is bonded to an alkyl chain including a biodegradable ester bond and/or an amide bond and optionally including a double bond, and has an advantage in that it has superior efficacy such as tissue specificity and gene expression rate in vivo when delivering nucleic acids, and is easily decomposed in vivo after drug delivery, so that side effects such as hepatotoxicity are not significant.
  • stereoisomer means a compound of the present invention having the same chemical formula or molecular formula but having different stereochemistry. Each of these stereoisomers and mixtures thereof are also included in the scope of the present invention.
  • a solid bond ( ) is a wedge-shaped solid line combination representing the absolute arrangement of the stereocenter ( ) or wedge-shaped dotted line join ( ) may be included.
  • the compound of formula 1 of the present invention may exist in the form of a "pharmaceutically acceptable salt".
  • a salt an acid addition salt formed by a pharmaceutically acceptable free acid may be useful, but is not limited thereto.
  • pharmaceutically acceptable salt means any organic or inorganic acid addition salt, or base addition salt of the compound, which has an effective effect that is relatively nontoxic and harmless to the patient, and the side effects due to the salt do not reduce the beneficial efficacy of the compound represented by formula 1.
  • Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. Equimolar amounts of the compound and the acid or alcohol in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction.
  • a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile.
  • organic acids and inorganic acids can be used as the free acid, and inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid can be used, and organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, or hydroiodic acid can be used, but are not limited thereto.
  • inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid
  • organic acids such as methanesulfonic acid, p-tol
  • a pharmaceutically acceptable metal salt can be prepared using a base.
  • An alkali metal salt or an alkaline earth metal salt can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate.
  • the metal salt can be prepared, for example, sodium, potassium, or calcium salts, but is not limited thereto.
  • a corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).
  • Lipid nanoparticles comprising ionizable lipids comprising biodegradable bonds
  • lipid nanoparticle comprising an ionizable lipid represented by the above-described chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • the lipid nanoparticle of the present invention may comprise one kind of ionizable lipid represented by the chemical formula 1, or two or more kinds.
  • an ionizable lipid other than that of the present invention may be additionally included.
  • the above lipid nanoparticles may further include, but are not limited to, one or more selected from helper lipids, structural lipids, and PEG-lipids.
  • auxiliary lipid plays a role in wrapping and protecting the core formed by the interaction of the ionizable lipid and the drug within the lipid nanoparticle, and combines with the auxiliary lipid bilayer of the target cell to facilitate passage through the cell membrane and endosomal escape during intracellular delivery of the drug.
  • auxiliary lipid may be any auxiliary lipid capable of promoting the fusion of lipid nanoparticles, and examples thereof include, without limitation, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine.
  • DOPE dioleoylphosphatidylethanolamine
  • DSPC distearoylphosphatidylcholine
  • POPC palmitoyloleoylphosphatidylcholine
  • EPC egg phosphatidylcholine
  • DSPE disearoylphosphatidylethanolamine
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • PE dipalmitoylphosphatidylethanolamine
  • 1-PA 1,2-dilinoleoyl-sn-glycero-3-phosphocholine
  • 1,2-diarachidonoyl-sn-glycero-3-phosphocholine 1,2-diarachidonoyl-sn-glycero-3-phosphocholine
  • 1,2-diarachidoyl-sn-glycero-3-phosphocholine 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine
  • 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine ME 16:0 PE
  • 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1,2-diphytanoyl-sn
  • the above structural lipids provide morphological rigidity to the lipid charging within the lipid nanoparticles and serve to enhance the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles.
  • the above structural lipids may be, but are not limited to, cholesterol, cholesterolol, spinasterol, fecosterol, sitosterol, ergosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or mixtures thereof.
  • PEG-lipid refers to a form in which lipid and PEG are conjugated, and means a lipid having a polyethylene glycol polymer, which is a hydrophilic polymer, bound to one end.
  • the PEG-lipid contributes to the stability of the nanoparticles in the serum within the lipid nanoparticles, and plays a role in preventing aggregation between nanoparticles.
  • the PEG-lipid protects the nucleic acid from a decomposing enzyme when the nucleic acid is delivered in the body, thereby enhancing the stability of the nucleic acid in the body, and can increase the half-life of a drug encapsulated in the nanoparticle.
  • the PEG-lipid may be, for example, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-c-DMA, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or a mixture thereof, but is not limited thereto.
  • the molar ratio of the ionizable lipid: the auxiliary lipid: the structural lipid: the PEG-lipid may be 20 to 60:5 to 30:20 to 60:1 to 5.
  • the molar ratio may be 30 to 60:5 to 20:30 to 50:1 to 2, and more specifically, 36.5 to 50:10 to 15:38.5 to 47:1.5, but is not limited thereto.
  • the lipid nanoparticle of the present invention exhibits a positive charge under acidic pH conditions, and thus can easily form a complex with a therapeutic agent such as a nucleic acid or anionic drug exhibiting a negative charge through electrostatic interaction, thereby enabling high-efficiency encapsulation of anionic drugs, and can be used as an intracellular or in vivo drug delivery composition. Therefore, the lipid nanoparticle of the present invention can be usefully used for the delivery of not only nucleic acids but also all forms of drugs having anions. That is, the lipid nanoparticle of the present invention can be ultimately manufactured in a form (encapsulated form) additionally including an anionic drug.
  • encapsulation refers to encapsulating a delivery substance to surround it and efficiently introduce it into a living body
  • the encapsulation efficiency refers to the content of a drug encapsulated in a lipid nanoparticle with respect to the total drug content used in manufacturing.
  • the above anionic drug may be a nucleic acid, a low molecular weight compound, a peptide, a protein, a protein-nucleic acid structure, or an anionic biopolymer-drug conjugate, but is not limited thereto, as long as it can be stably and efficiently delivered by forming a lipid nanoparticle together with the ionizable lipid of the present invention.
  • the nucleic acid may be, but is not limited to, siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme or a mixture thereof.
  • the weight ratio of ionizable lipid/nucleic acid in the above lipid nanoparticles may be 1 to 20.
  • the weight ratio of ionizable lipid/mRNA may be 5 to 15, specifically 10 to 15, and more specifically 12 to 12.53, but is not limited thereto.
  • the lipid nanoparticle may have a diameter size of, for example, 50 to 180 nm, specifically 70 to 160 nm, and more specifically 100 to 150 nm, but is not limited thereto.
  • Drug delivery and pharmaceutical compositions comprising lipid nanoparticles
  • Another aspect of the present invention is a drug delivery composition comprising an anionic drug-containing lipid nanoparticle according to the present invention.
  • Another aspect of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an anionic drug-containing lipid nanoparticle according to the present invention as an active ingredient.
  • the lipid nanoparticle of the present invention forms a stable complex with anionic drugs such as nucleic acids and exhibits low cytotoxicity and effective cellular uptake, and is therefore effective in delivering anionic drugs. Accordingly, the lipid nanoparticle has a preventive or therapeutic effect on related diseases depending on the type of anionic drug and the type of nucleic acid used, and has unlimited potential for use as a drug delivery composition.
  • the term "treatment" refers to an intervention to alter the natural course of an individual or cell having a disease, which may be performed during the progression of the pathological condition or to prevent it.
  • the desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease condition, and improving the prognosis.
  • the present invention includes all acts of improving the course of the disease by administering lipid nanoparticles comprising an ionizable lipid including a biodegradable bond, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and an anionic drug as active ingredients.
  • prevention refers to all acts of inhibiting or delaying the onset of the disease by administering the lipid nanoparticles.
  • the lipid nanoparticles of the present invention are used for treatment or prevention purposes, they are administered to a subject in a therapeutically effective amount.
  • therapeutically effective amount used in the present invention refers to an effective amount of anionic drug-containing lipid nanoparticles.
  • therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. And it can be administered singly or in multiple doses.
  • the dosage of the pharmaceutical composition of the present invention can be determined by an expert according to various factors such as the patient's condition, age, sex, and complications. Since the effective ingredient of the pharmaceutical composition of the present invention has excellent safety, it can be used in amounts exceeding the determined dosage.
  • composition containing the above lipid nanoparticles as an active ingredient can be administered orally, intramuscularly, intravenously, intraarterially, subcutaneously, intraperitoneally, pulmonaryly, and intranasally, but is not limited thereto.
  • composition of the present invention may further comprise one or more pharmaceutically acceptable carriers for administration.
  • Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components.
  • Other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets.
  • composition of the present invention may be a patch, a solution, a pill, a capsule, a granule, a tablet, a suppository, or the like.
  • These preparations can be prepared by conventional methods used in formulation in the art or by methods disclosed in a literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and can be formulated into various preparations depending on each disease or ingredient.
  • Step 2 The precursor 2 (877 mg, 1.84 mmol) obtained in Step 1 was dissolved in anhydrous DCM (9.2 mL, 0.2 M), and decanoic acid (380 mg, 2.20 mmol), EDC ⁇ HCl (422 mg, 1.2 eq), DIPEA (1.28 mL, 4 eq), and DMAP (44.9 mg, 0.2 eq) were sequentially added dropwise, and the mixture was stirred for 1 h in a similar manner to Step 1 to obtain precursor 3 (977.7 mg, 1.55 mmol, yield: 84.0%) in the form of a transparent oil.
  • LC-MS (ESI, m/z) 632.5 (M+H + ).
  • Step 4 Precursor 4 (650 mg, 1.31 mmol) obtained in Step 3 was dissolved in DCM (4.35 mL, 0.3 M), and then precursor 1 (604 mg, 1.96 mmol), EDC ⁇ HCl (501 mg, 2 eq), and DMAP (31.9 mg, 0.2 eq) were sequentially added dropwise, and the mixture was stirred for 1 h in a similar manner to Step 1 to obtain compound 4 (467 mg, 0.59 mmol, yield: 45.4%) as a transparent oil.
  • LC-MS (ESI, m/z) 788.6 (M+H + ).
  • Step 1 Benzyl (2-hydroxyethyl)carbamate (200 mg, 1.02 mmol) and 2-hexyldecanoic acid (315 mg, 1.22 mmol) were treated in a similar manner to Step 2 of Example 1-1 to obtain precursor 5 (336.9 mg, 0.77 mmol, yield: 76%) as a transparent oil.
  • LC-MS (ESI, m/z) 434.3 (M+H + ).
  • Step 2 Precursor 5 (100 mg, 0.231 mmol) obtained in Step 1 was treated in a similar manner to Example 1-2 to obtain precursor 6 (71.5 mg, 0.239 mmol, yield: quant.) in the form of a transparent oil.
  • LC-MS (ESI, m/z) 300.3 (M+H + ).
  • Step 3 Precursor 6 (400 mg, 1.33 mmol) and precursor 1 (577 mg, 1.87 mmol) obtained in Step 2 were treated in a similar manner to Step 2 of Example 1-1 to obtain compound 7 (170 mg, 0.288 mmol, yield: 21.6%) as a transparent oil.
  • LC-MS (ESI, m/z) 590.3 (M+H + ).
  • Precursor 8 (910.2 mg, 2.148 mmol) obtained in Step 1 was dissolved in anhydrous THF (2.75 mL, 0.78 M) under N 2 gas, and slowly added dropwise over 15 minutes while lowering the temperature of the reaction solution to 10 °C or lower. The reaction solution was warmed to room temperature and reacted for 5 hours. After completion of the reaction, the temperature of the reaction solution was lowered to 10 °C or lower, and 20% NH 4 Cl solution (30 mL) was slowly added dropwise. After filtering with Celite, it was washed three times with MTBE (5 mL). The organic layer was extracted with MTBE (12 mL) and washed with 15% NaCl solution (12 mL).
  • Step 4 The precursor 10 (147 mg, 0.320 mmol) obtained in Step 3 was treated in a similar manner to Step 2 of Example 1-1 to obtain compound 11 (104.2 mg, 0.139 mmol, yield: 64.2%) as a transparent oil.
  • LC-MS (ESI, m/z) 750.5 (M+H + ).
  • Lipid nanoparticles comprising an ionizable lipid containing a biodegradable bond of the present invention were prepared. Specifically, a method for preparing lipid nanoparticles (hereinafter, mRNA-encapsulated LMPs) comprising an ionizable lipid (compound 1, 2, 3, or 8) of the present invention and an anionic drug (SARS-CoV-2 Spike mRNA) is described in detail in Examples 2-1 to 2-2 according to Table 3 below.
  • mRNA-encapsulated LMPs comprising an ionizable lipid (compound 1, 2, 3, or 8) of the present invention and an anionic drug (SARS-CoV-2 Spike mRNA) is described in detail in Examples 2-1 to 2-2 according to Table 3 below.
  • Example 2-1 Preparation of mRNA-encapsulated lipid nanoparticles (mRNA-encapsulated LNP 1, mRNA-encapsulated LNP 2, or mRNA-encapsulated LNP 8)
  • the ionizable lipid (compound 1, compound 2, or compound 8) prepared in Example 1, a phospholipid (DOPE, Avanti Polar Lipids, CAS: 4004-05-1 or DSPC, Avanti Polar Lipids, CAS: 816-94-4) as an auxiliary lipid, a structural lipid (cholesterol, Sigma-aldrich, CAS: 57-88-5), and a PEG-lipid (PEG-2000-ceramide, Avanti Polar Lipids, CAS: 212116-78-4) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an oily phase.
  • the mRNA was dissolved in 100 mM sodium acetate buffer to prepare an aqueous phase.
  • the volume ratio of the lipid phase, aqueous phase, and buffer for dilution (PBS, pH 7.4) was set to 1:1:4, respectively, and mixed at a flow rate of 12 mL/min using a microfluidic mixing device (PNI Ignite, Precision Nanosystems, NIN0001) to produce mRNA-encapsulated lipid nanoparticles (mRNA-encapsulated LNP 1, mRNA-encapsulated LNP 2, or mRNA-encapsulated LNP 8).
  • Example 2-2 Preparation of mRNA-encapsulated lipid nanoparticles (mRNA-encapsulated LNP 3)
  • the ionizable lipid (compound 3) prepared in Example 1, a phospholipid (DOPE, Avanti Polar Lipids, CAS: 4004-05-1) as an auxiliary lipid, a structural lipid (cholesterol), and a PEG-lipid (PEG-2000-ceramide, Avanti Polar Lipids, CAS: 212116-78-4) were dissolved in ethanol at a molar ratio of 36.5:15:47:1.5 to prepare an oily phase.
  • the mRNA was dissolved in 100 mM sodium acetate buffer to prepare an aqueous phase.
  • the volume ratio of the lipid phase, aqueous phase, and buffer for dilution was set to 1:1:4, respectively, and mixed at a flow rate of 12 mL/min using a microfluidic mixing device (PNI Ignite, Precision Nanosystems, NIN0001) to produce mRNA-encapsulated lipid nanoparticles (mRNA-encapsulated LNP 3).
  • mRNA SARS-CoV-2 Spike mRNA
  • mRNA-encapsulated lipid nanoparticles manufactured by the method of Example 2 were examined. Specifically, 5 ⁇ L of mRNA-encapsulated lipid nanoparticles was taken and diluted 100-fold with 1 x PBS buffer. Approximately 500 ⁇ L of the diluted sample was placed in a disposable cuvette (Malvern, ZEN0040), and the size (Z-Average) and polydispersity index (PDI) values were measured using a Zetasizer (Malvern, Zetasizer Ultra).
  • the encapsulation efficiency (drug loading rate, %) of the mRNA-encapsulated lipid nanoparticles was measured using Ribogreen analysis (Quant-iTTM RiboGreen® RNA kit, Invitrogen).
  • the mRNA-encapsulated lipid nanoparticles were diluted with 1 x TE buffer to a final mRNA concentration of 4 to 7 ⁇ g/mL per 3 mL, to make 2 mL.
  • Drug loading rate (%) (Fluorescence of Triton LNP(+) - Fluorescence of Triton LNP(-))/(Fluorescence of Triton LNP(+)) X 100
  • hEPO Human Erythropoietin
  • the lysate sample of HEK-293T cells transfected with lipid nanoparticles (mRNA-encapsulated LNP 1, mRNA-encapsulated LNP 3) encapsulating hEPO mRNA by the method of Example 2 was diluted to fall within the appropriate concentration range. 100 ⁇ L of the diluted sample was dispensed into each well of a 96-well plate, and 200 ⁇ L of the hEPO Conjugate reagent was dispensed. After 2 hours, the solution in each well was removed, 200 ⁇ L of the substrate reagent was dispensed, and the expression of the hEPO protein was confirmed by measuring the absorbance at a specific wavelength (450 nm).
  • the lipid nanoparticles containing the ionizable lipid of the present invention effectively delivered mRNA to HEK-293T cells and showed high expression of hEPO protein, thereby delivering anionic drugs to cells with excellent efficiency (Table 5).
  • hEPO mRNA was delivered to mice, and then bioluminescence was confirmed to measure the expression amount of hEPO protein.
  • lipid nanoparticles encapsulating hEPO mRNA by the method of Example 2 were injected into the muscle of BALB/c mice at a concentration of 0.1 ⁇ g/ ⁇ L.
  • 50 ⁇ L of blood was collected, plasma was separated, and the concentration of hEPO protein was measured using an hEPO Quantikine ELISA kit (R&D Systems).
  • 100 ⁇ L of the diluted sample was dispensed into each well of a 96-well plate, and 200 ⁇ L of the hEPO Conjugate reagent was dispensed.
  • the solution in each well was removed, 200 ⁇ L of the substrate reagent was dispensed, and the absorbance was measured at a specific wavelength (450 nm).
  • the lipid nanoparticles containing the ionizable lipid of the present invention effectively delivered mRNA into a living body, and a significant level of hEPO protein was detected in the blood, and anionic drugs could be delivered into a living body with excellent efficiency (Table 6).
  • fLUC mRNA was delivered to mice, and then the expression level of fLUC (firefly Luciferase) protein was measured by confirming bioluminescence through IVIS spectrum (preclinical tomographic optical imaging).
  • lipid nanoparticles (mRNA-encapsulated LNP 1, mRNA-encapsulated LNP 3) encapsulating fLUC mRNA by the method of Example 2 were administered at a concentration of 0.1 g/L into the right quadriceps muscle of BALB/c mice.
  • 100 ⁇ L of luciferin reagent was injected at each time point (0.5 h, 6 h, 24 h, 48 h, 72 h, and 6 days), and then the in vivo luminescence of each mouse was confirmed using IVIS Spectrum equipment (IVIS Spectrum, PerkinElmer). The total luminescence value was calculated by measuring luminescence for 6 days after lipid nanoparticle administration (Table 7).
  • the lipid nanoparticles containing the ionizable lipid of the present invention expressed high levels of protein at the injection site for at least 48 hours.

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Abstract

La présente invention concerne un nouveau lipide ionisable contenant une liaison biodégradable. Spécifiquement, le lipide ionisable contenant une liaison ester et/ou amide biodégradable selon la présente invention délivre de manière stable un médicament anionique lorsqu'il est préparé en nanoparticules lipidiques, et présente un excellent effet, en particulier, dans l'administration d'acides nucléiques, et peut ainsi être efficacement utilisé dans des domaines techniques associés tels que la thérapie génique médiée par des nanoparticules lipidiques.
PCT/KR2024/002375 2023-02-24 2024-02-23 Nouveau lipide ionisable et nanoparticule lipidique le contenant Ceased WO2024177424A1 (fr)

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