WO2023024513A1 - Novel cationic lipid compound - Google Patents

Novel cationic lipid compound Download PDF

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Publication number
WO2023024513A1
WO2023024513A1 PCT/CN2022/084284 CN2022084284W WO2023024513A1 WO 2023024513 A1 WO2023024513 A1 WO 2023024513A1 CN 2022084284 W CN2022084284 W CN 2022084284W WO 2023024513 A1 WO2023024513 A1 WO 2023024513A1
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composition
lipid
lipids
compound
glycero
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PCT/CN2022/084284
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French (fr)
Chinese (zh)
Inventor
黄才古
黄铁强
谭俊荣
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广州谷森制药有限公司
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Publication of WO2023024513A1 publication Critical patent/WO2023024513A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules 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
    • A61K9/51Nanocapsules; Nanoparticles
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C213/00Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02Compounds 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
    • C07C219/04Compounds 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
    • C07C219/06Compounds 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention provides novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, steroids, and polymer-conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition combining a A method of delivering one or more therapeutic and/or prophylactic agents to a mammalian cell or organ and/or producing a polypeptide in a mammalian cell or organ.
  • lipid components such as neutral lipids, steroids, and polymer-conjugated lipids
  • the lipid nanoparticle compositions of the invention may also include, in specified proportions, one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, Lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents.
  • nucleic acids Efficient targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids presents a persistent medical problem. Specifically, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.
  • compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structured lipids (such as steroids) and/or polyethylene glycol-containing Lipids of alcohols (polymer-conjugated lipids).
  • Cationic lipids include, for example, amine-containing lipids that can be readily protonated.
  • RNA degradation in plasma has been used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides.
  • lipid nanoparticles for the delivery of oligonucleotides.
  • Improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids.
  • these preferred lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of the patient at effective doses of the nucleic acid does not create unacceptable toxicity and/or risk to the patient.
  • the present invention provides these and related advantages.
  • the present invention provides the following novel compounds and methods involving these compounds:
  • the present invention relates to compounds of the following structural formula (I):
  • R 1 and R 2 are independent hydrogen or hydroxyl, at least one of which is hydroxyl
  • the compound has one of the structures shown in Table 1 below
  • composition comprising any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.
  • compositions comprising any one or more of the compounds of structure (I) and a therapeutic and/or prophylactic agent.
  • the composition comprises any of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more compounds selected from the group consisting of neutral lipids, steroids, and polymer conjugates. Lipid excipients. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.
  • the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkaline (DPPC), 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) and mixtures thereof.
  • the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
  • the steroid is selected from the group consisting of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof .
  • the preferred steroid is cholesterol.
  • the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)
  • the composition ratio ranges from about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipids, about 10 to 55 mol% of steroids, and about 0 to 10 mol% of polymer-conjugated lipids. quality.
  • the therapeutic and/or prophylactic agent comprises a nucleic acid.
  • nucleic acid is RNA, and it is selected from following composition: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and its mixture.
  • the RNA is selected from mRNA.
  • the present invention relates to methods of administering therapeutic and/or prophylactic agents to a subject in need thereof, the method comprising preparing or providing any of the compositions described above and administering the composition to the subject combination.
  • the compounds of the invention can be administered as a drug substance, or can be formulated as a pharmaceutical composition.
  • the pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. Appropriate concentrations and dosages can be readily determined by those skilled in the art.
  • compositions of the present invention may be by any of the acceptable modes of administration for agents of similar utility.
  • the pharmaceutical composition of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques.
  • the pharmaceutical compositions of the invention are formulated so as to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a subject.
  • Compositions to be administered to a subject or patient are in the form of one or more dosage units, where a tablet may be a single dosage unit and a container of the compound in aerosol form of the invention may hold multiple dosage units. Current methods for the preparation of such dosage forms are known, or will be apparent, to those skilled in the art.
  • the composition to be administered will contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the associated disease or condition in accordance with the teachings of the invention.
  • compositions of the present invention may be in solid or liquid form.
  • the carrier is particulate, such that the composition is in tablet or powder form.
  • the carrier can be a liquid in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for administration by inhalation.
  • the pharmaceutical composition When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where forms considered solid or liquid herein include semi-solid, semi-liquid, suspension and gel forms.
  • the pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, flakes and the like.
  • Such solid compositions will generally contain one or more inert diluents or edible carriers.
  • binders such as gelatin, cellulose, etc.
  • excipients such as lactose, etc.
  • disintegrants such as alginic acid, etc.
  • lubricants such as magnesium stearate, etc.
  • Glidants such as silica gel, etc.
  • sweeteners such as sucrose or saccharin
  • flavoring agents such as mint
  • coloring agents such as sucrose or saccharin
  • liquid carriers other than materials of the above type, such as polyethylene glycol or oil.
  • compositions may be in liquid form, such as syrups, solutions, emulsions or suspensions.
  • Liquids can be used for oral administration or for injectable delivery, as two examples.
  • the compositions of Urim contain, in addition to the compounds of the present invention, one or more of sweetening agents, preservatives, dyeing/coloring agents and darkening agents.
  • one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.
  • the liquid pharmaceutical composition of the present invention may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that may be used as a solvent or suspending medium; antibacterial agents , such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenedipic acid; buffering agents, such as acetate, clematine, or phosphate; Reagents for tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose via
  • compositions of the invention may consist of dosage units which can be administered as an aerosol.
  • aerosol is used to denote systems ranging from those of a colloidal nature to those consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system which dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single-phase, bi-phasic or triphasic systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Preferred aerosols can be determined by those skilled in the art without undue experimentation.
  • compositions of the present invention can be prepared by methods well known in the field of pharmacy.
  • Pharmaceutical compositions intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers into a solution.
  • Surfactants can be added to promote the formation of a uniform solution or suspension.
  • Surfactants are compounds that interact non-covalently with the compounds of the invention in order to facilitate dissolution or uniform suspension of the compounds in aqueous delivery systems.
  • composition of the present invention is administered in a therapeutically effective amount, which will vary depending on a number of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; The patient's age, weight, general health, sex, and diet; mode and timing of administration; rate of excretion; drug combination; severity of the specific case, etc.
  • compositions of the invention may also be administered concurrently with, before or after administration of one or more other therapeutic agents.
  • combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of a composition of the invention and each active agent in its own separate pharmaceutical dosage formulation.
  • compositions of the invention and other active agents may be administered to a subject together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations.
  • the compound of the invention and the one or more additional active agents may be administered at substantially the same time, or sequentially at times staggered from each other; it is understood that combination therapy includes all such dosing regimens.
  • novel cationic lipid compounds have achieved more advantageous physical and chemical properties, including more suitable pKa and better chemical stability, and are used in mRNA nanoliposome compositions, which can realize anti-ionic nucleic acid
  • the drug is more effectively combined and delivered, and its chemical structure is more stable, which is convenient for synthesis and beneficial to be developed as a pharmaceutical excipient.
  • functional groups of intermediate compounds may need to be protected by suitable protecting groups.
  • suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, and the like.
  • Suitable protecting groups for amino include t-butoxycarbonyl, benzyloxycarbonyl and the like.
  • Suitable protecting groups for carboxylic acids include hydrocarbyl, aryl or arylhydrocarbyl esters. Protecting groups can be added or removed according to standard techniques, known to those skilled in the art and described herein.
  • all compounds of the present invention that exist in free base or free acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of compounds of the present invention may be converted to their free base or acid forms by standard techniques.
  • Compound B can be synthesized according to the representative route described in Example 1.
  • the particle size of the lipid nanoparticles is approximately 65-105 nm in diameter, and in some cases, approximately 75-100 nm in diameter.
  • liver tissue approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1/4" ceramic balls (MP Biomedicals) were added to each tube, and 500 ⁇ L of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue.
  • the FastPrep24 instrument MP Biomedicals
  • the Liver tissue was homogenized at 2x6.0m/s for 15 s. The homogenate was incubated at room temperature for 5 min, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega).
  • FLuc mRNA (L-6107) from Trilink Biotechnologies will express the luciferase protein, which was originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.
  • the pKa of the formulated cationic lipid correlates with the effectiveness of the LNP for delivery of nucleic acids.
  • the preferred pKa range is 5-7.
  • the pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-phthanesulfonic acid (TNS).
  • TNS 2-(p-toluidino)-6-phthanesulfonic acid
  • the vesicles were diluted to contain 24uM lipid in 2mL buffer solution containing 10mM HEPES, 10mM MES, 10mM sodium acetate, 130mM NaCl, wherein the pH value was 2.5-11. Aliquots of TNS solution were added to give a final concentration of 1 uM, and after vortex mixing, fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence Spectrophotometer at room temperature using excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH that yielded half the maximum fluorescence intensity.
  • lipid nanoparticles containing FLuc mRNA were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in Example 3.
  • Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., ( 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, the average PEG molecular weight is 2000).
  • PEG-DMG i.e., ( 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, the average PEG molecular weight is 2000.
  • Relative activity was determined by measuring luciferase expression in the liver. The activity was compared at doses of 0.3 and 1.0 mg mRNA/kg and expressed as ng

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Abstract

The present invention relates to a lipid compound, which can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids, and/or analogs thereof, and/or polymer conjugated lipids, so as to form lipid nanoparticles for delivering a therapeutic agent and/or preventive agent. In some embodiments, the lipid nanoparticles are used for delivering nucleic acids, for example, messenger RNA and/or antisense RNA. A method for treating and/or preventing various diseases by using the described lipid nanoparticles is also provided. In one embodiment, a compound having the following formula (I) structure is provided: or a salt or an isomer thereof or an N-oxide thereof, wherein R1 and R2 as defined herein also provide a pharmaceutical composition comprising one or more compounds represented by the foregoing structural formula (I) and a therapeutic agent and/or preventive agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from among neutral lipids, charged lipids, steroids, and polymer conjugated lipids. The described compositions are useful for forming lipid nanoparticles for delivering a therapeutic agent and/or preventive agent. In other embodiments, the present invention provides a method for administering a therapeutic agent and/or preventive agent to a subject in need thereof. The method comprises preparing a pharmaceutical composition comprising lipid nanoparticles containing a compound represented by structural formula (I) and a therapeutic agent and/or preventive agent, and delivering the composition to a subject.

Description

新型阳离子脂质化合物Novel Cationic Lipid Compounds 技术领域technical field
本发明提供新型阳离子脂质,其可用于与其他脂质组分(如中性脂质、类固醇和聚合物缀合的脂质)结合,以便形成一种核酸mRNA脂质纳米粒子组合物将一种或多种治疗剂和/或预防剂递送至哺乳动物细胞或器官和/或在哺乳动物细胞或器官中产生多肽的方法。除新型脂质外,本发明的脂质纳米粒子组合物还可以包括呈特定比例的一种或多种阳离子性和/或可离子化氨基脂质、包括多不饱和脂质在内的中性脂质、聚合物缀合的脂质、类固醇、和/或治疗剂和/或预防剂。The present invention provides novel cationic lipids that can be used in combination with other lipid components (such as neutral lipids, steroids, and polymer-conjugated lipids) to form a nucleic acid mRNA lipid nanoparticle composition combining a A method of delivering one or more therapeutic and/or prophylactic agents to a mammalian cell or organ and/or producing a polypeptide in a mammalian cell or organ. In addition to the novel lipids, the lipid nanoparticle compositions of the invention may also include, in specified proportions, one or more cationic and/or ionizable amino lipids, neutral lipids including polyunsaturated lipids, Lipids, polymer-conjugated lipids, steroids, and/or therapeutic and/or prophylactic agents.
背景技术Background technique
生物活性物质如小分子药物、蛋白质和核酸的有效靶向递送提出了一个持久的医学难题。确切地说,将核酸递送至细胞因这些物种的相对不稳定性和低细胞渗透性而变得困难。因此,需要开发有助于将治疗剂和/或预防剂如核酸递送至细胞的方法和组合物。Efficient targeted delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids presents a persistent medical problem. Specifically, delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of these species. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.
经研究证明,利用含脂质的纳米颗粒组合物、脂质体和脂质体复合物作运输媒介,可有效地将生物活性物质如小分子药物、蛋白质和核酸运送至细胞和/或细胞内隔室中。这些组合物一般包含一种或多种"阳离子性"脂质,包括多不饱和脂质在内的中性脂质(如磷脂)、结构性脂质(如类固醇)和/或含聚乙二醇的脂质(聚合物缀合的脂质)。阳离子脂质包括如可容易地质子化的含胺脂质。Studies have proved that using lipid-containing nanoparticle compositions, liposomes and liposome complexes as transport media can effectively deliver biologically active substances such as small molecule drugs, proteins and nucleic acids to cells and/or intracellularly in the compartment. These compositions generally comprise one or more "cationic" lipids, neutral lipids including polyunsaturated lipids (such as phospholipids), structured lipids (such as steroids) and/or polyethylene glycol-containing Lipids of alcohols (polymer-conjugated lipids). Cationic lipids include, for example, amine-containing lipids that can be readily protonated.
然而,在治疗环境中使用寡核苷酸目前面临着两个问题。第一,游离的RNA易于在血浆中核酸酶消化。第二,游离RNA进入存在相关翻译机制的细胞内隔室的能力受限。由阳离子脂质与其他脂质组分(如中性脂质、胆固醇、PEG、PEG化的脂质和寡核苷酸)形成的脂质纳米颗粒已用于阻止RNA在血浆中的降解并促进寡核苷酸的细胞摄取。However, the use of oligonucleotides in a therapeutic setting currently faces two problems. First, free RNA is readily digested by nucleases in plasma. Second, cell-free RNA has limited access to intracellular compartments where associated translation machinery exists. Lipid nanoparticles formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides have been used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides.
仍然有必要改进的用于递送寡核苷酸的阳离子脂质和脂质纳米颗粒。改进的脂质纳米颗粒会提供优化的药物递送,保护核酸不在血清中被降解和清除,其适于全身或局部递送,并且提供核酸的细胞内递送。另外,这些优选的脂质- 核酸颗粒应当是耐受良好的,并且提供足够的治疗指数,使得在有效剂量的核酸下的患者治疗不会对患者产生不可接受的毒性和/或风险。本发明提供这些优点和相关的优点。There remains a need for improved cationic lipids and lipid nanoparticles for the delivery of oligonucleotides. Improved lipid nanoparticles would provide optimized drug delivery, protect nucleic acids from degradation and clearance in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids. In addition, these preferred lipid-nucleic acid particles should be well tolerated and provide a sufficient therapeutic index such that treatment of the patient at effective doses of the nucleic acid does not create unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.
公开内容public content
本发明提供以下新型化合物和涉及这些化合物的方法:The present invention provides the following novel compounds and methods involving these compounds:
第一方面,本发明涉及以下结构式(I)的化合物:In a first aspect, the present invention relates to compounds of the following structural formula (I):
Figure PCTCN2022084284-appb-000001
Figure PCTCN2022084284-appb-000001
或其盐或其异构体或其N-氧化物,其中:or its salt or its isomer or its N-oxide, wherein:
R 1、R 2为独立的氢或羟基,其中至少含有一个为羟基 R 1 and R 2 are independent hydrogen or hydroxyl, at least one of which is hydroxyl
在各个不同的实施方案中,所述化合物具有以下表1中所示的结构之一In various embodiments, the compound has one of the structures shown in Table 1 below
表1代表性化合物Table 1 Representative Compounds
Figure PCTCN2022084284-appb-000002
Figure PCTCN2022084284-appb-000002
Figure PCTCN2022084284-appb-000003
Figure PCTCN2022084284-appb-000003
在一些实施方案中,提供了包含结构式(I)的化合物中的任一种或多种和治疗剂和/或预防剂的组合物。In some embodiments, there is provided a composition comprising any one or more of the compounds of structural formula (I) and a therapeutic and/or prophylactic agent.
在一些实施方案中,提供了包含结构(I)的化合物中的任一种或多种和治疗剂和/或预防剂的组合物。在一些实施方案中,所述组合物包含结构(I)的化合物中的任一种和治疗剂和/或预防剂以及一种或多种选自中性脂质、类固醇和聚合物缀合的脂质的赋形剂。其他药物可接受的赋形剂和/或载体也包括在组合物的各种实施方案内。In some embodiments, there is provided a composition comprising any one or more of the compounds of structure (I) and a therapeutic and/or prophylactic agent. In some embodiments, the composition comprises any of the compounds of structure (I) and a therapeutic and/or prophylactic agent and one or more compounds selected from the group consisting of neutral lipids, steroids, and polymer conjugates. Lipid excipients. Other pharmaceutically acceptable excipients and/or carriers are also included in various embodiments of the compositions.
在一些实施方案中,中性脂质选自1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆寇基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、鞘磷脂(SM)和其混合。在一些实施方案中,优选中性脂质为1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)。In some embodiments, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine Alkaline (DPPC), 1,2-Dimyristyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palm Acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM) and mixtures thereof. In some embodiments, the preferred neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
在一些实施方案中,类固醇选自胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚和其混合。在一些实施方案中,优选类固醇为胆固醇。In some embodiments, the steroid is selected from the group consisting of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof . In some embodiments, the preferred steroid is cholesterol.
在一些实施方案中,聚乙二醇化脂质为1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)In some embodiments, the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)
在一些实施方案中,所述组合物比例以下范围:约10~60mol%所述化合物、约0~30mol%中性脂质、约10~55mol%类固醇和约0~10mol%聚合物缀合的脂质。In some embodiments, the composition ratio ranges from about 10 to 60 mol% of the compound, about 0 to 30 mol% of neutral lipids, about 10 to 55 mol% of steroids, and about 0 to 10 mol% of polymer-conjugated lipids. quality.
在一些前述组合物的实施方案中,治疗剂和/或预防剂包括核酸。其中核酸 为RNA,其选自以下组成:siRNA、aiRNA、miRNA、dsRNA、shRNA、mRNA以及其混合物。在一些实施方案中,所述RNA选自mRNA。In embodiments of some of the foregoing compositions, the therapeutic and/or prophylactic agent comprises a nucleic acid. Wherein nucleic acid is RNA, and it is selected from following composition: siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and its mixture. In some embodiments, the RNA is selected from mRNA.
在其他不同的实施方案中,本发明涉及向有需要的受试者施用治疗剂和/或预防剂的方法,该方法包括制备或提供上述组合物中的任一种并向受试者施用该组合物。In other various embodiments, the present invention relates to methods of administering therapeutic and/or prophylactic agents to a subject in need thereof, the method comprising preparing or providing any of the compositions described above and administering the composition to the subject combination.
出于施用的目的,本发明的化合物(通常是脂质纳米颗粒与治疗剂和/或预防剂结合的形式)可以以原料药施用,或者可以配制为药物组合物。本发明的药物组合物包含结构(I)的化合物和一种或多种药物可接受的载体、稀释剂或赋形剂。结构(I)的化合物以有效形成脂质纳米颗粒并递送治疗剂和/或预防剂。本领域技术人员可以容易地确定适当的浓度和剂量。For purposes of administration, the compounds of the invention, typically in the form of lipid nanoparticles in combination with therapeutic and/or prophylactic agents, can be administered as a drug substance, or can be formulated as a pharmaceutical composition. The pharmaceutical composition of the present invention comprises a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents or excipients. Compounds of structure (I) are effective to form lipid nanoparticles and deliver therapeutic and/or prophylactic agents. Appropriate concentrations and dosages can be readily determined by those skilled in the art.
本发明组合物的施用可以通过任何用于类似效用的试剂的可接受施用方式来进行。本发明的药物组合物可以配制成固体、半固体、液体或气体形式的制剂,例如片剂、胶囊、粉末、颗粒、软膏、溶液、悬浮液、栓剂、注射剂、吸入剂、凝胶、微球和气溶胶。施用这类药物组合物的典型途径包括,但不限于,口服、局部、经皮、吸入、胃肠外、舌下、口含、直肠、阴道和鼻内途径。本文使用的术语胃肠外包括皮下注射,静脉内、肌内、皮内、胸骨内注射或输注技术。配制本发明的药物组合物以便允许经过对受试者施用该组合物后其中含有的活性成分是生物可利用的。待向对象或患者施用的组合物是一个或多个剂量单位的形式,其中,片剂可以是单剂量单位,而本发明气溶胶形式的化合物的容器可以容纳多个剂量单位。制备这些剂型的现行的方法是己知的,或者对于本领域技术人员是显而易见的。在任何情况下,待施用的组合物将会含有治疗有效量的本发明化合物或其药物可接受的盐,以便根据本发明的教导治疗相关的疾病或病况。Administration of the compositions of the present invention may be by any of the acceptable modes of administration for agents of similar utility. The pharmaceutical composition of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal routes. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions of the invention are formulated so as to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a subject. Compositions to be administered to a subject or patient are in the form of one or more dosage units, where a tablet may be a single dosage unit and a container of the compound in aerosol form of the invention may hold multiple dosage units. Current methods for the preparation of such dosage forms are known, or will be apparent, to those skilled in the art. In any event, the composition to be administered will contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treating the associated disease or condition in accordance with the teachings of the invention.
本发明的药物组合物可以是固体或液体的形式。一方面,载体是微粒,使得组合物是片剂或粉末形式。载体可以是液体,此时组合物是口服糖浆或可注射液体或气溶胶,所述气溶胶适用于吸入施用。The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is particulate, such that the composition is in tablet or powder form. The carrier can be a liquid in which case the composition is an oral syrup or an injectable liquid or an aerosol suitable for administration by inhalation.
当意图用于口服施用时,药物组合物优选为固体或液体形式,其中本文认为是固体或液体的形式包括半固体、半液体、悬浮液和凝胶形式。When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where forms considered solid or liquid herein include semi-solid, semi-liquid, suspension and gel forms.
作为用于口服施用的固体组合物,药物组合物可以配制成粉末、颗粒、压缩的片剂、丸剂、胶囊、咀嚼胶、薄片等形式。这类固体组合物通常将含有一种或多种惰性稀释剂或可食用载体。另外,可以存在以下的一种或多种:粘合剂,如明胶、纤维素等;赋形剂,如乳糖等;崩解剂,如海藻酸等;润滑剂,如硬脂酸镁等;助流剂,如硅胶等;甜味剂,如蔗糖或糖精;调昧剂,如薄荷等;以及着色剂。As solid compositions for oral administration, the pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewing gums, flakes and the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, there may be one or more of the following: binders, such as gelatin, cellulose, etc.; excipients, such as lactose, etc.; disintegrants, such as alginic acid, etc.; lubricants, such as magnesium stearate, etc.; Glidants, such as silica gel, etc.; sweeteners, such as sucrose or saccharin; flavoring agents, such as mint; and coloring agents.
当药物组合物是胶囊形式时,其可以含有上述类型材料之外的液体载体,如聚乙二醇或油。When the pharmaceutical composition is in capsule form, it may contain liquid carriers other than materials of the above type, such as polyethylene glycol or oil.
药物组合物可以是液体的形式,如糖浆、溶液、乳液或悬浮液。作为两种实例,液体可以用于口服施用或用于注射递送。当意图用于口服施用时,优边的组合物含有,除本发明化合物之外的,甜味剂、防腐剂、染色/着色剂和增昧剂中的一种或多种。在通过注射施用的组合物中,可以包括表面活性剂、防腐剂、润湿剂、分散剂、悬浮剂、缓冲剂、稳定剂和等渗剂中的一种或多种。Pharmaceutical compositions may be in liquid form, such as syrups, solutions, emulsions or suspensions. Liquids can be used for oral administration or for injectable delivery, as two examples. When intended for oral administration, the compositions of Urim contain, in addition to the compounds of the present invention, one or more of sweetening agents, preservatives, dyeing/coloring agents and darkening agents. In compositions administered by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.
本发明的液体药物组合物,不论其为溶液、悬浮液还是其他类似的形式,可以包括以下佐剂中的一种或多种:无菌稀释剂,如注射用水、盐水溶液、优选生理盐水、林格氏溶液、等渗氯化钠;不挥发性油类,如可用作溶剂或悬浮介质的合成的单甘酯或双甘酯,聚乙二醇、甘油、丙二醇或其他溶剂;抗菌剂,如尼泊金甲醋等;抗氧化剂,如抗坏血酸或亚硫酸氢钠;螯合剂,如乙二肢四乙酸;缓冲剂,如乙酸盐、拧棱酸盐或磷酸盐;以及用于调节张力的试剂,如氯化钠或葡萄糖;用作冷冻保护剂的试剂,如蔗糖或海藻糖。胃肠外制剂可以封装在玻璃或塑料制作的安瓿、一次性注射器或多剂量瓶中。生理盐水是优选的佐剂。可注射的药物组合物优选为无菌的。The liquid pharmaceutical composition of the present invention, whether it is a solution, a suspension or other similar forms, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol, or other solvents that may be used as a solvent or suspending medium; antibacterial agents , such as methylparaben, etc.; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenedipic acid; buffering agents, such as acetate, clematine, or phosphate; Reagents for tonicity, such as sodium chloride or glucose; reagents used as cryoprotectants, such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.
本发明的药物组合物可以由可作为气溶胶施用的剂量单位组成。术语气溶胶用于表示从胶体性质的系统到由加压包装组成的系统的各种系统。可以通过液化气或压缩气来递送,或者通过分散活性成分的适合的泵系统来递送。本发明化合物的气溶胶可以以单相、双相系统或三相系统来递送,以便递送活性成分。气溶胶的递送包括必要的容器、活化剂、阀、子容器等,其在一起可以形成试剂盒。本领域技术人员不需过度实验即可确定优选的气溶胶。The pharmaceutical compositions of the invention may consist of dosage units which can be administered as an aerosol. The term aerosol is used to denote systems ranging from those of a colloidal nature to those consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system which dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single-phase, bi-phasic or triphasic systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Preferred aerosols can be determined by those skilled in the art without undue experimentation.
本发明的药物组合物可以通过制药领域熟知的方法来制备。意图通过注射施用的药物组合物可以通过将本发明的脂质纳米颗粒与无菌的蒸馏水或其他载体结合成溶液来制备。可以加入表面活性剂以促进形成均匀的溶液或悬浮液。表面活性剂是与本发明化合物非共价地相互作用,以便促进所述化合物在水性递送系统中溶解或均匀悬浮的化合物。The pharmaceutical compositions of the present invention can be prepared by methods well known in the field of pharmacy. Pharmaceutical compositions intended to be administered by injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers into a solution. Surfactants can be added to promote the formation of a uniform solution or suspension. Surfactants are compounds that interact non-covalently with the compounds of the invention in order to facilitate dissolution or uniform suspension of the compounds in aqueous delivery systems.
本发明的组合物或其药物可接受的盐以治疗有效量施用,所述量将会根据多种因素变化,包括使用的具体治疗剂的活性;治疗剂的代谢稳定性和作用时长;受试者的年龄、体重、一般健康状况、性别和饮食;施用的方式和时间;排泄速率;药物组合;具体病例的严重性等。The composition of the present invention, or a pharmaceutically acceptable salt thereof, is administered in a therapeutically effective amount, which will vary depending on a number of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; The patient's age, weight, general health, sex, and diet; mode and timing of administration; rate of excretion; drug combination; severity of the specific case, etc.
本发明的组合物也可以在施用一种或多种其他治疗剂的同时、之前或之后施用。这类组合治疗包括施用本发明组合物和一种或多种另外的活性剂的单一药物剂量制剂,以及施用本发明组合物和各个在其自身单独药物剂量制剂中的活性剂。例如,本发明组合物和其他活性剂可以以单一口服剂量组合物(如片剂或胶囊)一起向受试者施用,或者各个试剂以不同的口服剂量制剂施用。当使用不同的剂量制剂时,本发明化合物和一种或多种另外的活性剂可以在基本同一时间施用,或者在相互交错的时间依次施用;应理解组合治疗包括所有的这些给药方案。Compositions of the invention may also be administered concurrently with, before or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation of a composition of the invention and one or more additional active agents, as well as administration of a composition of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, compositions of the invention and other active agents may be administered to a subject together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate oral dosage formulations. When different dosage formulations are used, the compound of the invention and the one or more additional active agents may be administered at substantially the same time, or sequentially at times staggered from each other; it is understood that combination therapy includes all such dosing regimens.
上述新型阳离子脂质化合物的结构修饰和设计,实现了更有优势的理化性质,包括更合适的pKa和更好的化学稳定性,用于mRNA纳米脂质体组合物,可实现对离子类核酸药物更有效结合并递送,同时其化学结构更稳定,便于合成和有利开发为药用辅料。The structural modification and design of the above-mentioned novel cationic lipid compounds have achieved more advantageous physical and chemical properties, including more suitable pKa and better chemical stability, and are used in mRNA nanoliposome compositions, which can realize anti-ionic nucleic acid The drug is more effectively combined and delivered, and its chemical structure is more stable, which is convenient for synthesis and beneficial to be developed as a pharmaceutical excipient.
上述化合物和组合物的制备方法在下文描述,和/或在本领域已知。Methods for the preparation of the above compounds and compositions are described below, and/or are known in the art.
本领域的技术人员将会认识到,在本文描述的方法中,中间化合物的官能团可能需要通过适合的保护基保护。这类官能团包括羟基、氨基和羧酸。用于羟基的适合的保护基包括三烷基甲硅烷基或二芳基烷基甲硅烷基、四氢呋喃基、苄基等。用于氨基的适合的保护基包括叔丁氧基羰基、苄氧基羰基等。用于羧酸的适合的保护基包括烃基、芳基或芳烃基酯。保护基可以根据标准技术添加或去除,所述标准技术是本领域技术人员已知的合本文中描述的。Those skilled in the art will recognize that in the methods described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl, tetrahydrofuranyl, benzyl, and the like. Suitable protecting groups for amino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for carboxylic acids include hydrocarbyl, aryl or arylhydrocarbyl esters. Protecting groups can be added or removed according to standard techniques, known to those skilled in the art and described herein.
本领域技术人员还将认识到,虽然本发明化合物的这类经保护的衍生物可以不由此具有药物活性,但其可以对哺乳动物施用并且之后在体内代谢形成具有药理活性的本发明化合物。这类衍生物因此可以被描述为“前药”。所以本发明化合物的前药包括在本发明的范围内。Those skilled in the art will also recognize that while such protected derivatives of compounds of the invention may not thereby be pharmaceutically active, they may be administered to a mammal and then metabolized in vivo to form pharmacologically active compounds of the invention. Such derivatives can thus be described as "prodrugs". Prodrugs of the compounds of the present invention are therefore included within the scope of the present invention.
此外,所有以游离碱或游离酸形式存在的本发明化合物可以根据本领域技术人员已知的方法用适当的无机或有机的碱或酸处理来转化为其药物可接受的盐。本发明化合物的盐可以通过标准技术转化为其游离碱或酸形成。In addition, all compounds of the present invention that exist in free base or free acid form can be converted to their pharmaceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of compounds of the present invention may be converted to their free base or acid forms by standard techniques.
提供了以下实施例,其目的在于举例展示,并非限定。The following examples are offered for purposes of illustration and not limitation.
以下实施例,除非另外指出,否则使用的所有溶剂和试剂都是商购得到并且以原样使用。In the following examples, unless otherwise indicated, all solvents and reagents used were obtained commercially and used as received.
以下描述的程序可用于合成化合物A与B。The procedure described below can be used to synthesize compounds A and B.
本文采用了以下缩写:This article uses the following abbreviations:
EDC.HCl:1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
DCM:二氯甲烷DCM: dichloromethane
DMAP:4-二甲氨基吡啶DMAP: 4-Dimethylaminopyridine
DIEA:N,N-二异丙基乙胺DIEA: N, N-Diisopropylethylamine
具体实施方式Detailed ways
实施例1:Example 1:
代表性路线representative route
化合物1的合成Synthesis of compound 1
Figure PCTCN2022084284-appb-000004
Figure PCTCN2022084284-appb-000004
1)化合物A的合成1) Synthesis of Compound A
Figure PCTCN2022084284-appb-000005
Figure PCTCN2022084284-appb-000005
化学式:C 22H 42O 3 Chemical formula: C 22 H 42 O 3
分子量:354.58Molecular weight: 354.58
依次向2-已基癸酸(2.5g,9.8mmol)和4-环氧乙基-1-丁醇(1.4g,12.1mmol)的DCM混合液中加入EDC.HCl(2.3g,12.0mmol),DIEA(5.0g,38.7mmol),DMAP(0.2g,1.6mmol)。在室温下反应24h,后用DCM稀释体系,依次用饱和碳酸氢钠水溶液、稀盐酸水溶液洗涤,硫酸钠干燥后,过滤浓缩,所得残余物通过柱层析(乙酸乙酯/正己烷)纯化。得化合物A(2.0g,5.6mmol,57%)。Add EDC.HCl (2.3g, 12.0mmol) to the DCM mixture of 2-hexyldecanoic acid (2.5g, 9.8mmol) and 4-oxiranyl-1-butanol (1.4g, 12.1mmol) successively , DIEA (5.0 g, 38.7 mmol), DMAP (0.2 g, 1.6 mmol). After reacting at room temperature for 24 h, the system was diluted with DCM, washed successively with saturated aqueous sodium bicarbonate and dilute aqueous hydrochloric acid, dried over sodium sulfate, concentrated by filtration, and the resulting residue was purified by column chromatography (ethyl acetate/n-hexane). Compound A (2.0 g, 5.6 mmol, 57%) was obtained.
2)化合物B的合成2) Synthesis of Compound B
Figure PCTCN2022084284-appb-000006
Figure PCTCN2022084284-appb-000006
化学式:C 28H 53NO 4 Chemical formula: C 28 H 53 NO 4
分子量:443.71Molecular weight: 443.71
将化合物A(2.0g,5.6mmol)与4-氨基-1-丁醇(2.0g,22.4mmol)混合,密闭升温至90℃反应3天。降温冷却,加入乙酸乙酯和水,分出有机相后使用无水硫酸钠进行干燥,真空浓缩,残余物通过硅胶柱(氨水/甲醇/DCM)纯化。得化合物B(0.8g,1.8mmol,32%)。Compound A (2.0 g, 5.6 mmol) and 4-amino-1-butanol (2.0 g, 22.4 mmol) were mixed, sealed and heated to 90° C. for 3 days. After cooling down, ethyl acetate and water were added, the organic phase was separated and dried over anhydrous sodium sulfate, concentrated in vacuo, and the residue was purified by silica gel column (ammonia/methanol/DCM). Compound B (0.8 g, 1.8 mmol, 32%) was obtained.
3)化合物C的合成3) Synthesis of compound C
Figure PCTCN2022084284-appb-000007
Figure PCTCN2022084284-appb-000007
化学式:C 21H 41BrO 2 Chemical formula: C 21 H 41 BrO 2
分子量:405.46Molecular weight: 405.46
依次向2-已基癸酸(2.5g,9.8mmol)和6-溴己醇(2.0g,12.0mmol)的 DCM混合液中加入EDC.HCl(2.3g,12.0mmol),DIEA(5.0g,38.7mmol),DMAP(0.2g,1.6mmol)。在室温下反应24h,后用DCM稀释体系,依次用饱和碳酸氢钠水溶液、稀盐酸水溶液洗涤,硫酸钠干燥后,过滤浓缩,所得残余物通过柱层析(乙酸乙酯/正己烷)纯化。得化合物C(3.2g,7.9mmol,81%)。Add EDC.HCl (2.3g, 12.0mmol), DIEA (5.0g, 38.7 mmol), DMAP (0.2 g, 1.6 mmol). After reacting at room temperature for 24 h, the system was diluted with DCM, washed successively with saturated aqueous sodium bicarbonate and dilute aqueous hydrochloric acid, dried over sodium sulfate, concentrated by filtration, and the resulting residue was purified by column chromatography (ethyl acetate/n-hexane). Compound C (3.2 g, 7.9 mmol, 81%) was obtained.
4)化合物1的合成4) Synthesis of compound 1
Figure PCTCN2022084284-appb-000008
Figure PCTCN2022084284-appb-000008
化学式:C 48H 95NO 6 Chemical formula: C 48 H 95 NO 6
分子量:782.29Molecular weight: 782.29
依次向化合物B(0.8g,1.8mmol)和化合物C(1.0g,2.5mmol)的乙醇混合物中加入DIEA(0.4g,3.2mmol)升温至65℃,搅拌24h,降温减压浓缩溶剂,残余物溶于乙酸乙酯,使用碳酸氢钠水溶液洗涤有机相。经过无水硫酸钠干燥后,真空浓缩。残余物通过硅胶柱(氨水/甲醇/DCM)纯化,得化合物1(0.56g,0.7mmol,39%)。Add DIEA (0.4g, 3.2mmol) to the ethanol mixture of Compound B (0.8g, 1.8mmol) and Compound C (1.0g, 2.5mmol) sequentially. Warm up to 65°C, stir for 24h, cool down and concentrate the solvent under reduced pressure, and the residue Dissolve in ethyl acetate and wash the organic phase with aqueous sodium bicarbonate. After drying over anhydrous sodium sulfate, it was concentrated in vacuo. The residue was purified by silica gel column (ammonia/methanol/DCM) to obtain compound 1 (0.56 g, 0.7 mmol, 39%).
C 48H 95NO 6,Ms m/z:[M+H +]783; 1H-NMR(300MHz,CDCl 3)δ:ppm 4.08(4H,t),3.48~3.42(3H,m),2.75~2.32(6H,m),2.30~2.00(2H,m),1.80~1.50(14H,m),1.35~1.10(52H,m),0.92~0.78(12H,m)。 C 48 H 95 NO 6 , Ms m/z: [M+H + ]783; 1 H-NMR (300MHz, CDCl 3 ) δ: ppm 4.08(4H,t), 3.48~3.42(3H,m), 2.75 ~2.32(6H,m), 2.30~2.00(2H,m), 1.80~1.50(14H,m), 1.35~1.10(52H,m), 0.92~0.78(12H,m).
实施例2:Example 2:
化合物2:Compound 2:
Figure PCTCN2022084284-appb-000009
Figure PCTCN2022084284-appb-000009
化学式:C 48H 95NO 7 Chemical formula: C 48 H 95 NO 7
分子量:798.29Molecular weight: 798.29
化合物B可根据实施例1描述的代表性路线合成。Compound B can be synthesized according to the representative route described in Example 1.
C 48H 95NO 7,Ms m/z:[M+H +]799; 1H-NMR(300MHz,CDCl 3)δ:ppm 4.12(4H,t),3.50~3.40(4H,m),2.62~2.32(6H,m),2.30~2.00(2H,m),1.70~1.35(20H,m),1.35~1.10(44H,m),0.95~0.80(12H,m)。 C 48 H 95 NO 7 , Ms m/z: [M+H + ]799; 1 H-NMR (300MHz, CDCl 3 ) δ: ppm 4.12(4H,t), 3.50~3.40(4H,m), 2.62 ~2.32(6H,m), 2.30~2.00(2H,m), 1.70~1.35(20H,m), 1.35~1.10(44H,m), 0.95~0.80(12H,m).
实施例3Example 3
利用脂质纳米颗粒组合物的荧光素酶mRNA体内评价In vivo evaluation of luciferase mRNA using a lipid nanoparticle composition
将阳离子脂质、DSPC、胆固醇和PEG-脂质以50:10:38:2或48:10:40:2的摩尔比溶解在乙醇中。以约10:1至30:1的总脂质与mRNA的重量比制备脂质纳米颗粒(LNP)。简而言之,将mRNA在10ml至50ml柠檬酸盐缓冲液(pH=4)稀释至0.15mg/m L。使用注射器泵,将脂质的乙醇溶液与mRNA水溶液以约1:5至1:3(体积/体积)的比例混合,总流速为10ml/min以上。然后去除乙醇,并通过透析用PBS替代外部的缓冲液。最后,将脂质纳米颗粒通过0.2um孔径的无菌过滤器过滤。使用Malvern Zetasizer Nano ZS通过准弹性光散射测定的脂质纳米颗粒的粒径为直径大约65-105nm,并且在一些情况下,直径大约75-100nm。Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol at a molar ratio of 50:10:38:2 or 48:10:40:2. Lipid nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10:1 to 30:1. Briefly, mRNA was diluted to 0.15 mg/mL in 10 ml to 50 ml citrate buffer (pH=4). Using a syringe pump, mix the ethanolic lipid solution with the aqueous mRNA solution at a ratio of about 1:5 to 1:3 (vol/vol) at a total flow rate of 10 ml/min or more. Ethanol was then removed and the external buffer was replaced by PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles, as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS, is approximately 65-105 nm in diameter, and in some cases, approximately 75-100 nm in diameter.
根据国家科学技术委员会制定的指南,在6-8周龄的雌性C57BL/6小鼠,8-10周龄CD-1小鼠上进行研究。通过尾静脉注射全身性给予不同剂量的mRNA脂质纳米颗粒,并在给药后的特定时间点(例如5小时)使动物安乐死。将肝脏和脾脏收集在预先称重的管中,确定重量,立即在液氮中快速冷冻,并且在-80℃下储存,直至用于分析。The study was performed on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice according to the guidelines established by the National Science and Technology Commission. Different doses of mRNA lipid nanoparticles were administered systemically via tail vein injection, and animals were euthanized at specific time points (eg, 5 hours) after administration. Livers and spleens were collected in pre-weighed tubes, weight determined, immediately snap frozen in liquid nitrogen, and stored at -80°C until analysis.
对于肝脏,切割约50mg以便在2mL FastPrep管(MP Biomedicals,Solon OH)中进行分析。向各个管中加入1/4"陶瓷球(MP Biomedicals),并将平衡至室温的500μL的Glo裂解缓冲液-GLB(Promega,Madison WI)加入到肝脏组织中。使用FastPrep24仪器(MP Biomedicals)将肝脏组织在2x6.0m/s下均匀化15秒。将匀浆在室温下孵育5分钟,然后在GLB中进行1:4稀释,并使用SteadyGlo荧光素酶测定系统(Promega)进行评估。具体地,将50uL的稀释的组织匀浆与 50μL的SteadyGlo底物反应,摇振10秒,接着孵育5分钟,然后使用SpectraMAX_L化学发光型酶标仪(美谷分子仪器(上海)有限公司)定量。通过使用BCA蛋白质定量试剂盒(上海易色医疗科技有限公司)来确定测定的蛋白质的量。然后将相对发光度单位(RLU)归一化成所测定蛋白质的总ug。为了将RLU转化成μg荧光素酶,用QuantiLum重组荧光素酶(Promega)生成了标准曲线。For liver, approximately 50 mg was cut for analysis in 2 mL FastPrep tubes (MP Biomedicals, Solon OH). 1/4" ceramic balls (MP Biomedicals) were added to each tube, and 500 μL of Glo Lysis Buffer-GLB (Promega, Madison WI) equilibrated to room temperature was added to the liver tissue. Using the FastPrep24 instrument (MP Biomedicals), the Liver tissue was homogenized at 2x6.0m/s for 15 s. The homogenate was incubated at room temperature for 5 min, then diluted 1:4 in GLB and evaluated using the SteadyGlo luciferase assay system (Promega). Specifically , 50uL of diluted tissue homogenate was reacted with 50μL of SteadyGlo substrate, shaken for 10 seconds, then incubated for 5 minutes, and then quantified using a SpectraMAX_L chemiluminescent microplate reader (Megu Molecular Instruments (Shanghai) Co., Ltd.). Use the BCA Protein Quantification Kit (Shanghai Yise Medical Technology Co., Ltd.) to determine the amount of assayed protein. The relative luminescence units (RLU) are then normalized to the total ug of assayed protein. In order to convert RLU into μg fluorescein Enzymes, standard curves were generated using QuantiLum recombinant luciferase (Promega).
来自Trilink Biotechnologies的FLuc mRNA(L-6107)将表达荧光素酶蛋白,其最初从萤火虫(Photinus pyralis)中分离出来。Fluc通常用于哺乳动物细胞培养物中以测量基因表达和细胞活力。其在底物萤光素存在下发射出生物性光。这种加帽并且聚腺昔酸化的mRNA被5-甲基胞苷和假尿苷完全取代。FLuc mRNA (L-6107) from Trilink Biotechnologies will express the luciferase protein, which was originally isolated from the firefly (Photinus pyralis). Fluc is commonly used in mammalian cell culture to measure gene expression and cell viability. It emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA is completely replaced by 5-methylcytidine and pseudouridine.
实施例4Example 4
所配制得脂质的pKa的测定Determination of the pKa of the prepared lipid
所配制的阳离子脂质的pKa与用于递送核酸的LNP的效果相关。优选的pKa范围是5~7。使用基于2-(对甲苯胺基)-6-荼磺酸(TNS)的荧光的分析,在脂质纳米颗粒中测定各阳离子脂质的pKa。如实施例3中所述,使用有序的方法来制备在PBS中的浓度为0.4mM总脂质的包含阳离子脂质/DSPC/胆固醇/PEG脂质(50/10/38/2mol%)的脂质纳米颗粒。将TNS在蒸馏水中制备成100uM储备溶液。将囊泡稀释成在2mL缓冲溶液中含24uM脂质,所述缓冲溶液含有10mM HEPES、10mM MES、10mM乙酸按、130mM NaCl,其中pH值为2.5~11。加入等份的TNS溶液以产生l uM的终浓度,并且在涡旋混合之后,在室温下使用321nm和445nm的激发波长和发射波长在SLM Aminco Series 2发光分光光度计中测量荧光强度。对荧光数据应用S形最佳拟合分析,并将pKa测量为产生半数最大荧光强度的pH。The pKa of the formulated cationic lipid correlates with the effectiveness of the LNP for delivery of nucleic acids. The preferred pKa range is 5-7. The pKa of each cationic lipid was determined in lipid nanoparticles using an assay based on the fluorescence of 2-(p-toluidino)-6-phthanesulfonic acid (TNS). As described in Example 3, a sequenced method was used to prepare cationic lipid/DSPC/cholesterol/PEG lipid (50/10/38/2 mol%) at a concentration of 0.4 mM total lipid in PBS. Lipid nanoparticles. TNS was prepared as a 100 uM stock solution in distilled water. The vesicles were diluted to contain 24uM lipid in 2mL buffer solution containing 10mM HEPES, 10mM MES, 10mM sodium acetate, 130mM NaCl, wherein the pH value was 2.5-11. Aliquots of TNS solution were added to give a final concentration of 1 uM, and after vortex mixing, fluorescence intensity was measured in a SLM Aminco Series 2 Luminescence Spectrophotometer at room temperature using excitation and emission wavelengths of 321 nm and 445 nm. Sigmoid best-fit analysis was applied to the fluorescence data, and pKa was measured as the pH that yielded half the maximum fluorescence intensity.
实施例5Example 5
使用体内荧光素酶mRNA表达的啮齿动物模型测定含有各种阳离子脂质的脂质纳米颗粒制剂的效能Determination of the Potency of Lipid Nanoparticle Formulations Containing Various Cationic Lipids Using a Rodent Model of In Vivo Luciferase mRNA Expression
为了比较的目的,如实施例3所述,使用有序混合方法,将这些脂质也用于配制含有FLuc mRNA(L-6107)的脂质纳米颗粒。使用以下摩尔比来配制脂质 纳米颗粒:50%阳离子脂质/10%二硬脂酰磷脂酰胆碱(DSPC)/38%胆固醇/2%PEG脂质("PEG-DMG",即,(1-(单甲氧基一聚乙二醇)-2,3一二肉豆蔻酰基甘油,平均PEG分子量为2000)。如实施例3所述,在经由尾静脉注射施用之后的5小时,通过测量肝脏中的荧光素酶表达来确定相对活性。在0.3和1.0mg mRNA/kg的剂量下比较所述活性,并表达成在如实施例3所述的施用之后5小时测量的ng荧光素酶/g肝脏。实施例3及4结果如表2所示。For comparison purposes, these lipids were also used to formulate lipid nanoparticles containing FLuc mRNA (L-6107) using the ordered mixing method as described in Example 3. Lipid nanoparticles were formulated using the following molar ratios: 50% cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38% cholesterol/2% PEG lipid ("PEG-DMG", i.e., ( 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, the average PEG molecular weight is 2000). As described in Example 3, 5 hours after administration via tail vein injection, by Relative activity was determined by measuring luciferase expression in the liver. The activity was compared at doses of 0.3 and 1.0 mg mRNA/kg and expressed as ng luciferase measured 5 hours after administration as described in Example 3 /g liver. Embodiment 3 and 4 results are shown in table 2.
表2与mRNA表现出活性的比较脂质Table 2. Comparative lipids exhibiting activity with mRNA
Figure PCTCN2022084284-appb-000010
Figure PCTCN2022084284-appb-000010
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对公开专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the scope of the disclosed patents. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (14)

  1. 具有以下结构(I)化合物:Have following structure (I) compound:
    Figure PCTCN2022084284-appb-100001
    Figure PCTCN2022084284-appb-100001
    或其盐或其异构体或其N-氧化物,其中:or its salt or its isomer or its N-oxide, wherein:
    R 1、R 2为独立的氢或羟基,其中至少含有一个为羟基。 R 1 and R 2 are independently hydrogen or hydroxyl, at least one of which is hydroxyl.
  2. 包含权利要求1中任一项所述的化合物和治疗剂和/或预防剂的组合物。A composition comprising a compound according to any one of claim 1 and a therapeutic and/or prophylactic agent.
  3. 如权利要求2所述的组合物,其还包含选自中性脂质、类固醇以及聚合物缀合的脂质中的一种或多种赋形剂。The composition of claim 2, further comprising one or more excipients selected from neutral lipids, steroids, and polymer-conjugated lipids.
  4. 如前述权利要求3所述的组合物,其中所述组分中的中性脂质选自以下的一种或多种物质混合:1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆寇基-sn-甘油-磷酸胆碱(DMPC)、1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和鞘磷脂(SM)。The composition as claimed in claim 3, wherein the neutral lipid in the component is selected from one or more of the following mixtures: 1,2-distearoyl-sn-glycero-3-phosphate Choline (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristyl-sn-glycerol-phosphocholine (DMPC), 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM).
  5. 如前述权利要求4所述的组合物,其中所述中性脂质为DSPC。The composition of claim 4, wherein the neutral lipid is DSPC.
  6. 如前述权利要求3~5所述的组合物,其中所述组分中的类固醇选自以下一种或多种物质混合:胆固醇、粪固醇、谷固醇、麦角固醇、菜油固醇、豆固醇、菜籽固醇、番茄碱、熊果酸、α-生育酚。The composition according to claims 3 to 5, wherein the steroid in the component is selected from one or more of the following mixtures: cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, Stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol.
  7. 如前述权利要求6所述的组合物,其中所述类固醇为胆固醇。A composition as claimed in claim 6, wherein said steroid is cholesterol.
  8. 如前述权利要求3~7所述的组合物,其中所述组分中的聚合物缀合的脂质为聚乙二醇化脂质。A composition as claimed in claims 3 to 7, wherein the polymer-conjugated lipid in said component is a pegylated lipid.
  9. 如前述权利要求中8所述的组合物,其中所述聚乙二醇化脂质为1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)The composition of claim 8, wherein the pegylated lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG)
  10. 如前述权利要求中任一项所述的组合物,其中所述治疗剂和/或预防剂 是能够引起免疫响应的疫苗或化合物,其中包括核酸。The composition of any one of the preceding claims, wherein the therapeutic and/or prophylactic agent is a vaccine or compound capable of eliciting an immune response, including a nucleic acid.
  11. 如前述权利要求10的组合物,其中所述核酸为RNA,其选自以下组成:siRNA、aiRNA、miRNA、dsRNA、shRNA、mRNA以及其混合物。The composition of claim 10, wherein the nucleic acid is RNA selected from the group consisting of siRNA, aiRNA, miRNA, dsRNA, shRNA, mRNA and mixtures thereof.
  12. 如前述权利要求11的组合物,其中所述RNA为mRNA。The composition of claim 11, wherein said RNA is mRNA.
  13. 向有需要的受试者施用治疗剂和/或预防剂的方法,所述方法包括制备或提供上述权利要求中任一项所述的组合物,并向所述对象施用所述组合物。A method of administering a therapeutic and/or prophylactic agent to a subject in need thereof, the method comprising preparing or providing a composition according to any one of the preceding claims, and administering the composition to the subject.
  14. 如前述权利要求中任一项所述的受试者为哺乳动物或人。The subject of any one of the preceding claims is a mammal or a human.
PCT/CN2022/084284 2021-08-23 2022-03-31 Novel cationic lipid compound WO2023024513A1 (en)

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