[go: up one dir, main page]

WO2025194409A1 - Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations - Google Patents

Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations

Info

Publication number
WO2025194409A1
WO2025194409A1 PCT/CN2024/082878 CN2024082878W WO2025194409A1 WO 2025194409 A1 WO2025194409 A1 WO 2025194409A1 CN 2024082878 W CN2024082878 W CN 2024082878W WO 2025194409 A1 WO2025194409 A1 WO 2025194409A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmol
lipid
bis
propyl
residue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/082878
Other languages
English (en)
Inventor
Xi Zhu
Hong Chen
Chenxi HOU
Shin-Shay Tian
Xiaoping Zhao
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.)
Shanghai Vitalgen Biopharma Co Ltd
Original Assignee
Shanghai Vitalgen Biopharma Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Vitalgen Biopharma Co Ltd filed Critical Shanghai Vitalgen Biopharma Co Ltd
Priority to PCT/CN2024/082878 priority Critical patent/WO2025194409A1/fr
Publication of WO2025194409A1 publication Critical patent/WO2025194409A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Definitions

  • the present disclosure belongs to the field of drug delivery. Specifically, the present application relates to novel lipid compounds and nanoparticles comprising said lipid compounds for delivering one or more biologically active agents to a subject. Also provided are methods of preparing and using said lipid compounds and nanoparticles, as well as use of said lipid compounds and nanoparticles.
  • the present disclosure includes a sequence listing as a part of the disclosure.
  • Nucleic acid-based therapeutics have enormous potential therapeutical application. These therapeutic nucleic acids include messenger RNA (mRNA) , antisense oligonucleotides, siRNA, miRNA, ribozymes, guide RNA, DNAzymes, plasmids, plasmids-based interfering nucleic acids, closed-end DNA, and aptamers. Different types of nucleic acids are currently being developed as therapeutics for the treatment of a number of diseases.
  • mRNA messenger RNA
  • antisense oligonucleotides siRNA
  • miRNA miRNA
  • ribozymes guide RNA
  • DNAzymes DNAzymes
  • plasmids plasmids-based interfering nucleic acids
  • closed-end DNA and aptamers.
  • nucleic acids such as mRNA, plasmids, closed-end DNA
  • nucleic acids can be used to express specific protein or enzyme, as would be useful in the treatment of, for example, single gene diseases related to deficiency or misfunction of such protein or enzyme.
  • Nucleic acids encoding an antigen of a pathogen, including mRNA and DNA could be used as vaccine for the prophylaxis of infectious diseases, such as COVID-19.
  • Nucleic acids encoding one or more neoantigens could be used as tumor vaccines, which can effectively activate the body’s immune system to kill tumor cells, making individualized immunotherapy based on tumor neoantigens a new direction for the development of personalized immunotherapy for treating tumors.
  • nucleic acids can down-regulate or up-regulate intracellular levels of specific mRNA, which subsequently affect expression of specific cellular proteins. These nucleic acids, such as siRNAs and miRNAs, have extremely broad therapeutic applications. Targets may include mRNAs associated with disease-states, such as tumor suppressor, and mRNAs of infectious agents. Antisense oligonucleotide constructs have shown the ability to specifically down-regulate target proteins through degradation of the cognate mRNA. Nucleic acids such as miRNA inhibitors would be useful in the treatment of diseases related to deficiency of protein or enzyme.
  • nucleic acid-based therapeutics have enormous potential, effective delivery of these molecules to appropriate sites within a cell or organism are prerequisite to realize this potential.
  • RNA molecules are susceptible to nuclease digestion in plasma and have extremely short half-life.
  • nucleic acids are impermeable to cell membrane due to its unique molecular characteristics. Thus, these molecules can hardly gain access to the intracellular compartment where the relevant translation machinery resides. Therefore, nucleic acids are usually considered have no druggability in its naked form. Improvement of delivery vehicle for nucleic acid is crucial for these molecules to fulfill their therapeutic potentials.
  • Nanoscale delivery vehicles including lipid and polymeric nanoparticles have been used to facilitate the cellular uptake of the nucleotides and prevent their degradation in biological environment.
  • Lipid nanoparticles formed from ionizable cationic lipids with other lipid components, such as neutral lipids, cholesterol, PEGylated lipids, and oligonucleotides have been proved to be one of the most potent delivery systems.
  • Cationic lipids are critical for protecting the nucleic acid cargo from degradation and facilitating intracellular delivery of the nucleic acid.
  • cationic lipids with diverse structures have been developed to enhance mRNA delivery.
  • Several groups have reported that lipids with branched tails are beneficial for the delivery of mRNA when compared to linear lipids.
  • the cationic lipids should be biodegradable, well-tolerated and easily synthesized.
  • Xu group used simple Michael addition chemistry to synthesize a library of cationic lipidoids for mRNA delivery in vitro and in vivo. These lipidoids contain degradable bond such as ester and disulfide bond, and have shown great biocompatibility both in vitro and in vivo.
  • degradable bond such as ester and disulfide bond
  • the present inventors have developed novel cationic lipids with branched tails with sulfi ether bond, which can form nanoparticles for the intracellular delivery of biologically active agents (e.g., mRNA, ceDNA, gRNA) both in vitro and in vivo.
  • biologically active agents e.g., mRNA, ceDNA, gRNA
  • the present disclosure provides a lipid having formula (I) , or a salt, tautomer, or stereoisomer thereof,
  • m and n are independently selected from any integer ranging from 3 to 12; p is selected from any integer of 1, 2 or 3, and the sum of m and p is selected from any integer ranging from 6 to 9; o is selected from any integer ranging from 1 to 6;
  • X 1 is a bond, -C (O) O-, -OC (O) -, -OC (O) O-, or a biodegradable group;
  • R 1 is a hydrogen bond donor-containing group or hydrogen bond acceptor-containing group; both of R 2 are same and selected from C 1 -C 16 alkyl, substituted C 1 -C 16 alkyl, C 2 -C 16 alkenyl, substituted C 2 -C 16 alkenyl, C 3 -C 12 cycloalkyl, substituted C 3 -C 12 cycloalkyl and combinations of thereof;
  • R 3 is selected from C 4 -C 22 alkyl, substituted C 4 -C 22 alkyl, C 4 -C
  • the lipid of the first aspect has formula (II) , or a salt, tautomer, or stereoisomer thereof,
  • n and p are selected from any integer ranging from 6 to 9.
  • R 1 is selected from hydroxyl, carboxyl, C 1 -C 3 alkylamine, C 5 -C 12 heteroaryl, C 5 -C 12 heteroaryl ketone, C 3 -C 8 heterocycloalkane, C 3 -C 8 heterocycloalkenone, and combinations thereof with a carbonyl group, an amide group, an imide group, a sulfinyl group, a sulfonamido group, or a sulfonamide group.
  • R 1 is selected from one of the following formulas:
  • each R 2 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains; and/or R 3 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
  • each R 2 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains; and/or each R 4 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
  • said carbon atom with hydrogen atom (s) substituted by one or two side chains is the second or more distant carbon atom counting from the junction.
  • said one or two side chains are C 1 -C 4 alkyls.
  • each R 2 , R 3 and/or each R 4 is independently selected from one of the following formulas:
  • the lipid is selected from one of the following formulas:
  • the present disclosure provides a lipid nanoparticle (LNP) comprising the lipid of the present disclosure, a neutral lipid, a PEG lipid and a steroid.
  • LNP lipid nanoparticle
  • said neutral lipid is disteroylphosphatidylcholine (DSPC) or dioleoyl-phosphatidylethanolamine (DOPE) .
  • DSPC disteroylphosphatidylcholine
  • DOPE dioleoyl-phosphatidylethanolamine
  • said PEG lipid is PEG-DMG.
  • said steroid is cholesterol, sitosterol or stigmasterol.
  • said lipid is comprised with a molar ratio of about 40%to about 60%
  • said neutral lipid is comprised with a molar ratio of about 5%to about 20%
  • said PEG lipid is comprised with a molar ratio of about 0.5%to about 5%
  • said steroid is comprised with a molar ratio of about 25%to about 50%.
  • said lipid is comprised with a molar ratio of about 50%
  • said neutral lipid is comprised with a molar ratio of about 10%
  • said PEG lipid is comprised with a molar ratio of about 1.5%
  • said steroid is comprised with a molar ratio of about 38.5%.
  • said LNP further comprises a nucleic acid molecule.
  • said nucleic acid molecule is DNA or RNA.
  • said nucleic acid molecule is plasmid DNA or closed-ended DNA (ceDNA) .
  • said nucleic acid molecule is selected from a group consisting of messenger RNA (mRNA) , guide RNA (gRNA) , a short interfering RNA (siRNA) , an RNA interference (RNAi) molecule, a microRNA (miRNA) , an antagomir, an antisense RNA, a ribozyme, a small hairpin RNA (shRNA) , or a mixture thereof.
  • mRNA messenger RNA
  • gRNA guide RNA
  • siRNA short interfering RNA
  • RNAi RNA interference
  • miRNA microRNA
  • antagomir an antisense RNA
  • shRNA small hairpin RNA
  • the present disclosure provides a pharmaceutical composition comprising the LNP of the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of delivering a nucleic acid molecule to a cell in vivo or in vitro, comprising contacting said cell with the LNP of the present disclosure.
  • the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the LNP of the present disclosure, wherein said LNP comprising a nucleic acid molecule having therapeutic activity.
  • said LNP is administered by injection.
  • FIG. 1 illustrates the expression levels in HEK293 cells of GFP mRNA delivered by lipid nanoparticles with different lipids of the present disclosure.
  • FIG. 2 illustrates the expression levels in HepG2 cells of GFP mRNA delivered by lipid nanoparticles with different lipids of the present disclosure.
  • FIG. 3 illustrates the hEPO protein expression level following administration of lipid nanoparticles with different lipids of the present disclosure to mice, tested at 6 hours and 24 hours after injection.
  • lipid nanoparticle or its abbreviation “LNP” used herein refers to a drug carrier on the order of nanometers and composed of one layer of lipids.
  • lipid nanoparticle also contemplates “lipid-polymer hybrid nanoparticle” which include both lipid portions and hydrophobic polymer portions.
  • lipid refers to a group of organic compounds that are poorly soluble in water, while soluble in nonpolar solvents. Lipids include, but are not limited to, esters of fatty acids.
  • cationic lipid refers to positively charged amphiphiles consisting of a hydrophilic head group connected to a hydrophobic tail via a linker. Cationic lipid is positively charged so that it can bind to negatively charged molecules or entities such as membrane.
  • polymer conjugated lipid refers to a molecule comprising a lipid portion and a polymer portion.
  • PEGylated lipid refers to a molecule comprising a lipid portion and a polyethylene glycol portion.
  • neutral lipid or “helper lipid” in the context of LNP is interchangeable and refers to lipids that are uncharged or stay in a neutral zwitterionic form at a selected pH.
  • charged lipid refers to lipids that are positively charged or negatively charged.
  • alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated.
  • alkenyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more double bonds.
  • stereoisomers refer to compounds consisting of the same atoms bonded by the same bonds but having different three-dimensional structures.
  • tautomers refer to structural isomers in which a proton shifts from one atom of a molecule to another atom of the same molecule.
  • nucleotide e.g., a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • peptide , "polypeptide” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by one or more peptide bonds.
  • subject refers to an individual, preferably a vertebrate, more preferably a non-human mammal or human.
  • the non-human mammal can be rodents such as murines, or non-human primates such as simians.
  • subject may also encompass cells, tissues and progenies of a biological entity obtained in vivo or culture in vitro.
  • phrases “effective amount” or “therapeutically effective amount” refers to the quantity of a composition, e.g., a composition comprising the LNP that is sufficient to result in a desired activity when being delivered to a subject in need thereof.
  • Said desired activity may encompass delaying the manifestation of a disorder, arresting or delaying the progression of a disorder, or alleviating at least one symptom of a disorder.
  • the present application provides a group of new ionizable cationic lipids, which are suitable for preparing lipid nanoparticles.
  • the ionizable cationic lipids of the present application is a compound having formula (I) , or a salt, tautomer, or stereoisomer thereof,
  • m and n are independently selected from any integer ranging from 3 to 12; p is selected from any integer of 1, 2 or 3; o is selected from any integer ranging from 1 to 6;
  • X 1 is a bond, -C (O) O-, -OC (O) -, -OC (O) O-, or a biodegradable group;
  • R 1 is a hydrogen bond donor-containing group or hydrogen bond acceptor-containing group; both of R 2 are same and selected from C 1 -C 16 alkyl, substituted C 1 -C 16 alkyl, C 2 -C 16 alkenyl, substituted C 2 -C 16 alkenyl, C 3 -C 12 cycloalkyl, substituted C 3 -C 12 cycloalkyl and combinations of thereof;
  • R 3 is selected from C 4 -C 22 alkyl, substituted C 4 -C 22 alkyl, C 4 -C 22 alkenyl, substituted C 4 -C 22 alkenyl, C
  • the lipid of the first aspect is a compound having formula (II) , or a salt, tautomer, or stereoisomer thereof,
  • R 1 - (CH 2 ) O -and the central nitrogen atom (N) constitutes the head group of the lipid compound of the present application.
  • Head group of a cationic lipid is positively charged at acidic environment and is essential for the delivery of nucleic acid payloads, as it interacts with the negatively charged phosphate groups of nucleic acids.
  • mRNA has a more complex secondary structure, comprising single-and double-stranded regions with some nucleosides exposed to the surrounding environment, such as the solvent.
  • R 1 serves to provide a set of hydrogen bond donors and/or acceptors with varying hydrogen-bonding ability in the head group.
  • H-bond donor-or acceptor-containing groups can be those comprising nitrogen (N) , oxygen (O) , carbon (C) or fluorine (F) atoms serving as H-bond donor or H-bond acceptor.
  • Exemplary H-bond donor-or acceptor-containing groups may include but not limited to hydroxyl-containing group, carboxyl-containing group, carbonyl-containing group, amide, imide, sulfoxide, and sulfonamide.
  • R 1 is selected from hydroxyl, carboxyl, C 1 -C 3 alkylamine, C 5 -C 12 heteroaryl, C 5 -C 12 heteroaryl ketone, C 3 -C 8 heterocycloalkane, C 3 -C 8 heterocycloalkenone, and combinations thereof with a carbonyl group, an amide group, an imide group, a sulfinyl group, a sulfonamido group, or a sulfonamide group.
  • R 1 is selected from a group consisting of following formulae:
  • the sum of m and p is selected from any integer ranging from 6 to 9.
  • the sum of n and p is selected from any integer ranging from 6 to 9. In specific embodiments, the sum of m and p, and the sum of n and p have same value selected from the integer of 6, 7, 8 or 9.
  • p is 1, and X 1 is -C (O) O-.
  • each R 2 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains; and/or R 3 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
  • each R 2 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains; and/or each R 4 has at least one carbon atom with hydrogen atom (s) substituted by one or two side chains.
  • said carbon atom with hydrogen atom (s) substituted by one or two side chains is the second or more distant carbon atom counting from the junction.
  • said one or two side chains are C 1 -C 4 alkyls.
  • R 2 is branched alkyl or branched alkenyl.
  • a hyperbranched structure may augment the cone-shaped morphology, and therefore increase intracellular delivery efficiency, it is preferred in the present application.
  • both R 2 , R 3 and R 4 are preferably branched.
  • R 2 and/or R 3 may have one or more side chains.
  • R 2 and R 3 are independently selected from 2-methylhexyl or 2, 6-dimethyloctyl.
  • each R 2 , R 3 and/or each R 4 is independently selected from one of the following formulas:
  • the present disclosure provides a compound of Formula (I-I) or Formula (II-I) :
  • each L is independently absent, C 1-10 alkylenyl, or C 3-7 cycloalkylenyl; each R 5 and R 6 are independently hydrogen, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, and C 3 -C 12 cycloalkyl.
  • each -L-C (R 5 ) R 6 is independently selected from the group consisting of:
  • the branched tail containing bis-alkylthio group could easily been synthesized by conjugate R 2 -SH or R 3 -SH to the alkynyl group.
  • the intermediate structure containing bis-alkylthio group were synthesized using the following reactions:
  • the lipid compound is selected from any one of following Compounds as shown below, including Compounds 1-27, 41 and 42 which have a chemical structure of formula (I) , and Compounds 28-40 and 43 which have a chemical structure of formula (II) .
  • the cationic lipid can be a protonated salt of the amine cationic lipid.
  • the term “free form” refers to the amine cationic lipids in non-salt form.
  • the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • the compounds of the disclosure, or their pharmaceutically acceptable salts may contain one or more stereocenters and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R) -or (S) -or, as (D) -or (L) -for amino acids.
  • the present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (+) and (-) , (R) -and (D) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • the pharmaceutically acceptable salts of the instant cationic lipids can be synthesized from the cationic lipids of this invention which contain a basic or acidic moiety by conventional chemical methods.
  • the salts of the basic cationic lipids are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
  • the lipid components comprised in the LNPs of the present application comprises a neutral lipid, a polymer conjugated lipid, and a steroid. In another embodiment, the lipid components further comprise a charged lipid.
  • the neutral lipid such as a phospholipid helps the LNP to bind to and cross the cell membrane.
  • Exemplary neutral lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC) , dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC) , dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) , dioleoyl-phosphatidylethanolamine (DOPE) , palmitoyloleoylphosphatidylcholine (POPC) , palmitoyloleoylphosphatidylethanolamine (POPE) , dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexan
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • the molar ratio of the ionizable cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1, preferably 5: 1.
  • the polymer conjugated lipid can be a PEGylated lipid or PEG lipid.
  • the PEG lipid is a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy- polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2′, 3′-di (tetradecanoyloxy) propyl-1-O- ( ⁇ -methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ⁇ -methoxy (polyethoxy)
  • the steroid or steroid analogue is cholesterol, sitosterol or stigmasterol. In certain embodiments, the steroid or steroid analogue is cholesterol. In certain embodiments, the steroid or steroid analogue is beta-Sitosterol. In certain embodiments, the steroid or steroid analogue is Stigmastanol. In some of these embodiments, the molar ratio of the ionizable cationic lipid to cholesterol ranges from about 5: 1 to 1: 1, preferably 3: 1 to 1: 1.
  • the nanoparticle of the present disclosure comprises the following in a mole ratio of 50: 10: 38.5: 1.5 or 40: 10: 48.5: 1.5: (1) the cationic lipid of the present disclosure, preferable selected from Compound 15-22 and 31-35; (2) a neutral lipid selected from DSPC or DOPE; (3) a steroid which is cholesterol; and (4) polymer conjugated lipid which is PEG-DMG.
  • the LNPs of the present application are particularly suitable for delivering nucleic acid molecules into cells.
  • the nucleic acid molecule can be either DNA or RNA or a mixture thereof, such as chimeric oligonucleotides.
  • the nucleic acid molecule can comprise naturally occurring or modified polynucleotides.
  • the nucleic acid molecule can be a coding sequence or non-coding sequence.
  • the nucleic acid molecule can be DNA molecule such as plasmid DNA, closed-ended DNA or a mixture thereof.
  • the nucleic acid molecule can be RNA molecule such as messenger RNA (mRNA) , guide RNA (gRNA) , a short interfering RNA (siRNA) , an RNA interference (RNAi) molecule, a microRNA (miRNA) , an antagomir, an antisense RNA, a ribozyme, a small hairpin RNA (shRNA) , or a mixture thereof.
  • mRNA messenger RNA
  • gRNA guide RNA
  • siRNA short interfering RNA
  • RNAi RNA interference
  • miRNA RNA interference
  • miRNA microRNA
  • antagomir an antisense RNA
  • shRNA small hairpin RNA
  • Plasmid DNA or closed-ended DNA may have a lengths at a range of 500 to 500,000 base pairs.
  • mRNA may have a length at a range of 200 to 100,000 base pairs.
  • gRNA may have a length at a range of 30 to 1,000 base pairs.
  • siRNAi, miRNA, or shRNA may have a length at a range of 15 to 1,000 base pairs.
  • the nucleic acid molecules can comprise modifications, such as one or more modifications to the backbone, one or more modifications to the base and/or one or more modifications to the sugar moiety.
  • the following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present disclosure.
  • the compounds of this disclosure having a structure of Formula (I) may be prepared via step 1 ⁇ 4 according to the procedures illustrated in Scheme 1.
  • the compounds of this disclosure having a structure of Formula (II) may be prepared via step 1 ⁇ 3 according to the procedures illustrated in Scheme 1.
  • One of ordinary skill in the art will note that, during the reaction sequences and synthetic schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups.
  • X is a reactive moiety selected to facilitate the desired reaction (e.g., halo) .
  • Condensation reaction of A-1 and A-2 under appropriate conditions e.g., using EDCI and DMAP
  • A-3 reacts with mercapto A-4 under suitable conditions (e.g., AIBN, dioxane and heat) to afford A-5.
  • Halide A-5 is then reacted with primary amine of A-6 under suitable conditions (e.g. heat) to yield a compound of Formula (II) or A-7.
  • A-7 is then reacted with another halide A-5 using appropriate conditions (e.g., DIEA and heat) to yield a compound of Formula (I) .
  • Lipid compositions are prepared by combining a cationic lipid, such as lipid according to formula (I) , helper lipids (such as DSPC) , a steroid (such as cholesterol) , and a PEG lipid (such as 1, 2- dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as DMG-PEG2000) at concentrations of about 5 mg/mL and 25 mg/mL in ethanol. Solutions should be refrigerated for storage at, for example, -80 °C.
  • helper lipids such as DSPC
  • a steroid such as cholesterol
  • PEG lipid such as 1, 2- dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as DMG-PEG2000
  • Nanoparticles compositions including a therapeutic and/or prophylactic and a lipid component are prepared by combining the lipid solution with a solution including the therapeutic and/or prophylactic at lipid component to therapeutic and/or prophylactic wt: wt ratios between about 10:1about 20: 1.
  • the lipid solution is injected using a microfluidic based system at flow rates between 0.25 mL/min and 2.0 mL/min into the therapeutic and/or prophylactic solution to produce a suspension with a water to ethanol ratio between about 3: 1.
  • solutions of the RNA or pDNA are diluted with 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution at concentrations of 0.1-0.5 mg/mL.
  • Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange.
  • Formulations were dialyzed twice against Tris-HCl Solution (20 mM, pH 7.4) at volumes about 2500 times that of the primary product using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill. ) with a molecular weight cutoff of 10 KD at 0-4 °C for 2 h. Then 40%sucrose solution was added, the final nanoparticle composition solution of 0.03 mg/mL to and 0.1 mg/mL (mRNA or pDNA) are generally obtained and stored at -80 °C.
  • the physical and chemical properties of the lipid nanoparticle of the present application depend on the formulation of the LNPs.
  • the choice of the ionizable cationic lipid dramatically influences the size of the formulated LNPs.
  • the lipid nanoparticles have a mean diameter of from about 100 nm to about 350 nm, preferably 150 nm to 250 nm.
  • the lipid nanoparticles have a mean diameter of about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm or 300 nm.
  • Polydispersity index is a parameter describing the distribution of molecular weight of the particles in an LNP formulation.
  • a lager PDI describes a broader distribution of molecular weight.
  • the PDI of the formulation can be 0.10 to 0.50, preferably 0.20 to 0.45, more preferably 0.05 to 0.30.
  • the lipid nanoparticles are substantially non-toxic or having acceptable toxicity to the subject.
  • the lipid nanoparticles of the present application have a reduced liver toxicity as compared to nanoparticles comprising a different cationic lipid.
  • the liver toxicity can be measured based known method in the art.
  • the lipid nanoparticle of the present disclosure can provide desirable transduction efficiency, which can be measured by e.g. the amount or expression level of the payload.
  • the lipid nanoparticles of the present application provide a comparable or increased amount of payload or expression of payload as compared to a nanoparticle comprising a different cationic lipid.
  • the LNP formed by the ionizable cationic lipid and loaded with nucleic acid molecules may have diverse uses, depending on the type and function of the nucleic acid molecules.
  • the LNPs can be used for treating a disease or disorder.
  • the disease or disorder is caused by insufficiency of a protein
  • the LNP comprises nucleic acid molecule encoding for the protein.
  • the LNPs can be used for preventing a disease or disorder.
  • the LNPs can be formed as a vaccine, in which the nucleic acid molecule encodes for an antigen, such as an antigen derived from a pathogen, e.g. a bacterium, a fungi, or a virus, or an antigen derived from tumor cell, e.g. tumor associated antigen.
  • an antigen such as an antigen derived from a pathogen, e.g. a bacterium, a fungi, or a virus
  • tumor cell e.g. tumor associated antigen.
  • the LNPs can be used for treating a disease or disorder by affecting expression of specific cellular proteins.
  • the LNPs comprise nucleic acids, such as siRNAs and miRNAs, which down-regulate or up-regulate intracellular levels of target mRNA associated with a disease state.
  • the target mRNA can be the mRNA of a tumor suppressor or mRNAs of infectious agents.
  • the LNPs can be used for treating a disease or disorder by affecting expression of specific cellular proteins via genome editing.
  • the LNPs may comprise nucleic acid molecule encoding for genome editing tool.
  • the LNPs can be used to deliver mRNA of a nuclease, e.g. Cas nuclease, and/or a guide RNA, so as to conduct nuclease-mediated gene editing.
  • Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; and the like.
  • LAH Lithium Aluminum Hydride
  • PPTS Pyridinium p-toluenesulfonate
  • Compound 19 was synthesized with similar procedure as Compound 16 but using 2, 3-bis ( (3-butylheptyl) thio) propyl 7-bromoheptanoate and 3-propylhexyl 8- ( (2-hydroxyethyl) amino) octanoate.
  • Compound 065 was obtained as yellow oil.
  • the reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine 20 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • the reaction mixture was poured into water (50 mL) and extracted with DCM (80 mL, 50 mL, 30 mL) , The combined organic layers were washed with brine 30 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • reaction mixture was poured into water (5 mL) and extracted with DCM (5 mL, 3 mL, 2 mL) , The combined organic layers were washed with brine (3 mL) , dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give bis (2, 3-bis (heptylthio) propyl) 6, 6'- ( (3-chloropropyl) azanediyl) dihexanoate (240 mg, crude) as a colorless oil.
  • reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL, 30 mL, 20 mL) , The combined organic layers were washed with brine (20 mL) , dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • Compound 32 bis (2, 3-bis ( ( (2E, 6Z) -nona-2, 6-dien-1-yl) thio) propyl) 6, 6'- ( (2-hydroxyethyl) azanediyl) dihexanoate
  • Compound 37 was prepared using similar procedure as Compound 36 but using 3-butylheptane-1-thiol.
  • reaction mixture was poured into water (50 mL) and extracted with EtOAc (300 mL, 200 mL, 100 mL) , The combined organic layers were washed with brine 30 mL, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue.
  • Nanoparticles comprising the cationic lipid as synthesized in Example 1.
  • Nanoparticles were prepared by mixing aqueous phase which contains RNA with ethanol phase which contains the lipid components. The two phases were mixed by using a microfluidic mixing process with a standard staggered herringbone mixer.
  • the ethanol phase was prepared by adding (1) an ionizable cationic lipid, specifically a cationic lipid as prepared in Example 1, (2) a neutral lipid, specifically DSPC, (3) a steroid, specifically cholesterol, and (4) a PEG lipid, specifically DMG-PEG2000 into ethanol. Unless otherwise stated, the ratio of the lipid components was 50: 10: 38.5: 1.5 mol%of ionizable cationic lipid: DSPC: cholesterol: PEG-DMG.
  • the weight ratio of the RNA or DNA molecules to the ionizable cationic lipid was kept at 1: 10.
  • the lipid solution (ethanol phase) was injected at 1 mL/min, and aqueous phase was injected at 3 mL/min into the mixer.
  • the volume ratio of the aqueous phase to the ethanol phase was 3: 1.
  • the solution with LNP were obtained and subsequently processed by dialysis to remove ethanol and achieve buffer exchange.
  • Dialysis was conducted twice against Tris-HCl Solution (20 mM, pH 7.4) at volumes about 2500 times that of the mixed solution using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, Ill. ) with a molecular weight cutoff of 10 KD at 4 °C for 2 h. Then 40%sucrose solution was added and the final nanoparticle solution with 8%sucrose were stored at -80 °C.
  • a Zetasizer Nano ZS (BeNano, Bettersize) were used to determine the particle size and the polydispersity index (PDI) of the nanoparticle compositions in PBS or Tris-HCl.
  • Ultraviolet-visible spectroscopy was used to determine the concentration of a therapeutic and/or prophylactic (e.g., RNA) in the nanoparticle compositions.
  • concentration of a therapeutic and/or prophylactic e.g., RNA
  • concentration of the therapeutic and/or prophylactic agent e.g., RNA
  • concentration of the therapeutic and/or prophylactic agent e.g., RNA
  • concentration of the therapeutic and/or prophylactic agent e.g., RNA
  • a QUANT-IT TM RIBOGREEN RNA assay (Shanghai ShineGene Molecular Biotechnology Co., LTD. ) was used to evaluate the encapsulation of RNA by the nanoparticle composition.
  • the samples were diluted in TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) , and then100 ⁇ L of the diluted samples were transferred to a polystyrene 96 well plate. The plate was incubated at a temperature of 40 °C for 10 minutes.
  • the RIBOGREEN or PICOGREEN reagent is diluted 1: 200 in TE buffer, and 100 ⁇ L of this solution is added to each well.
  • the fluorescence intensity was measured using a microplate reader (Molecular Devices i3max) at an excitation wavelength of, for example, about 488 nm and an emission wavelength of, for example, about 525 nm.
  • the fluorescence values of the reagent blank were subtracted from that of each of the samples and the percentage of free RNA or DNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100) .
  • mGFP mRNA coding EGFP

Landscapes

  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne de nouveaux lipides pour administrer une ou plusieurs molécules biologiquement actives à un sujet. La présente invention concerne également une composition ou une nanoparticule comprenant ledit lipide, des procédés de préparation dudit lipide, l'utilisation dudit lipide et un procédé d'utilisation dudit lipide.
PCT/CN2024/082878 2024-03-21 2024-03-21 Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations Pending WO2025194409A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/082878 WO2025194409A1 (fr) 2024-03-21 2024-03-21 Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/082878 WO2025194409A1 (fr) 2024-03-21 2024-03-21 Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations

Publications (1)

Publication Number Publication Date
WO2025194409A1 true WO2025194409A1 (fr) 2025-09-25

Family

ID=97138140

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/082878 Pending WO2025194409A1 (fr) 2024-03-21 2024-03-21 Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2025194409A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596922A (zh) * 2011-06-07 2014-02-19 因塞拉有限公司 氨基脂质，及其合成和用途
US20160089436A1 (en) * 2014-09-26 2016-03-31 Snu R&Db Foundation Self-assembled pharmaceutical composition for photodynamic therapy
US20160158363A1 (en) * 2013-06-26 2016-06-09 Massachusetts Institute Of Technology Multi-tailed lipids and uses thereof
CN108478794A (zh) * 2018-03-29 2018-09-04 沈阳药科大学 光敏剂-化疗药“光化一体”小分子前药及其自组装纳米粒的构建
CN111087332A (zh) * 2019-12-11 2020-05-01 东南大学 一种阳离子氨基脂质及其合成方法与应用
CN111617246A (zh) * 2020-06-01 2020-09-04 沈阳药科大学 一种纯光敏剂自组装纳米粒及其制备和应用
CN115304495A (zh) * 2022-07-20 2022-11-08 北京八亿时空液晶科技股份有限公司 一种菲衍生物及其应用
CN118108613A (zh) * 2022-12-26 2024-05-31 北京新合睿恩生物医疗科技有限公司 一种阳离子脂质化合物及制备方法和应用、mRNA递送系统

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103596922A (zh) * 2011-06-07 2014-02-19 因塞拉有限公司 氨基脂质，及其合成和用途
US20160158363A1 (en) * 2013-06-26 2016-06-09 Massachusetts Institute Of Technology Multi-tailed lipids and uses thereof
US20160089436A1 (en) * 2014-09-26 2016-03-31 Snu R&Db Foundation Self-assembled pharmaceutical composition for photodynamic therapy
CN108478794A (zh) * 2018-03-29 2018-09-04 沈阳药科大学 光敏剂-化疗药“光化一体”小分子前药及其自组装纳米粒的构建
CN111087332A (zh) * 2019-12-11 2020-05-01 东南大学 一种阳离子氨基脂质及其合成方法与应用
CN111617246A (zh) * 2020-06-01 2020-09-04 沈阳药科大学 一种纯光敏剂自组装纳米粒及其制备和应用
CN115304495A (zh) * 2022-07-20 2022-11-08 北京八亿时空液晶科技股份有限公司 一种菲衍生物及其应用
CN118108613A (zh) * 2022-12-26 2024-05-31 北京新合睿恩生物医疗科技有限公司 一种阳离子脂质化合物及制备方法和应用、mRNA递送系统

Similar Documents

Publication Publication Date Title
JP7547335B2 (ja) 修飾されたアミン脂質
JP7114465B2 (ja) 薬剤の細胞内送達のための化合物および組成物
KR20210093871A (ko) 이온화 가능한 아민 지질
EP4352038A2 (fr) Lipides cationiques ionisables d'administration d'arn
WO2023086514A1 (fr) Lipides cationiques ionisables pour l'acheminement d'arn
CN116675624B (zh) 一种脂质化合物及脂质纳米颗粒
US20240391870A1 (en) Ionizable cationic lipids for rna delivery
WO2025153097A1 (fr) Utilisation de nanoparticules lipidiques vierges dans la préparation d'un produit d'administration in-vivo
WO2025153095A1 (fr) Composition lipidique et son procédé de préparation ainsi que son utilisation
WO2024230844A1 (fr) Lipides cationiques et leur procédé de préparation
WO2025194409A1 (fr) Composés lipidiques cationiques ionisables pour système d'administration et leurs utilisations
WO2000012454A1 (fr) Nouveaux lipides cationiques
WO2025002399A1 (fr) Composés lipidiques cationiques ionisables pour l'administration d'agents biologiquement actifs
US20250268826A1 (en) Ionizable lipidoid compositions and therapeutic uses thereof
WO2024156291A1 (fr) Lipides cationiques et nanoparticules lipidiques
TW202525781A (zh) 可離子化脂質及其用途
BR122024007324A2 (pt) Composto de lipídeos de amina modificados, composição de nanopartículas lipídicas (lnp) compreendendo o dito composto, usos da mesma e métodos para editar genes
WO2024211865A2 (fr) Lipides et composés de type lipide pour distribution de nanoparticules lipidiques thérapeutiques (lnp)
WO2025212932A1 (fr) Procédés de synthèse de lipides aminés
KR20250087641A (ko) 생물 활성 성분을 전달하기 위한 아미노 지질 화합물 및 지질 나노입자
WO2025106670A1 (fr) Compositions lipidoïdes ionisables et leurs utilisations thérapeutiques
CN120531703A (zh) 预制载体在制备用于免疫细胞和干细胞的体外基因递送的产品中的应用
CN120531702A (zh) 预制载体在制备用于免疫细胞和干细胞的体外基因递送的产品中的应用
CN120682115A (zh) 一种氨基酸骨架可电离脂质及其制备方法与应用
EA046244B1 (ru) Модифицированные аминовые липиды