WO2022190967A1 - リン脂質 - Google Patents

リン脂質 Download PDF

Info

Publication number
WO2022190967A1
WO2022190967A1 PCT/JP2022/008670 JP2022008670W WO2022190967A1 WO 2022190967 A1 WO2022190967 A1 WO 2022190967A1 JP 2022008670 W JP2022008670 W JP 2022008670W WO 2022190967 A1 WO2022190967 A1 WO 2022190967A1
Authority
WO
WIPO (PCT)
Prior art keywords
phospholipid
acid
lipid
lipid particles
present
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.)
Ceased
Application number
PCT/JP2022/008670
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
康治 冨田
雄希 井上
綾乃 横内
尚文 深田
典之 前田
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.)
Nippon Fine Chemical Co Ltd
Original Assignee
Nippon Fine Chemical 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 Nippon Fine Chemical Co Ltd filed Critical Nippon Fine Chemical Co Ltd
Priority to CN202280006255.1A priority Critical patent/CN116234538A/zh
Priority to JP2023505328A priority patent/JP7848179B2/ja
Priority to EP22766926.4A priority patent/EP4306531A4/en
Priority to US18/017,751 priority patent/US20230295199A1/en
Publication of WO2022190967A1 publication Critical patent/WO2022190967A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/650952—Six-membered rings having the nitrogen atoms in the positions 1 and 4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings

Definitions

  • the present invention relates to phospholipids and the like.
  • RNA-containing drugs and vaccines have been developed.
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • An extremely sophisticated delivery system is required for exogenously administered RNA to exhibit its original activity in vivo. This is because RNA is rapidly enzymatically degraded and hardly passes through the cell membrane. Therefore, the development of delivery systems is inevitably associated with the practical use of RNA-containing drugs and vaccines.
  • RNA As a delivery system for drugs such as RNA, it is known to administer drugs in the form of encapsulation in lipid particles. However, when administering negatively charged nucleic acids, there is a concern of cytotoxicity, since positively charged lipids are usually used to cause electrostatic interactions.
  • Patent Document 1 reports that charge-reversible phospholipids have siRNA-encapsulating properties and safety at physiological pH.
  • a phospholipid alcohol solution is usually used when producing lipid particles.
  • Patent Document 1 it was necessary to use t-butanol to dissolve the phospholipid.
  • the melting point of t-butanol is around room temperature, it will solidify depending on the temperature of use.
  • the present inventor thought that ethanol would be desirable because it does not solidify at around room temperature and is recognized as a pharmaceutical additive.
  • An object of the present invention is to provide a phospholipid suitable for preparing lipid particles, which has charge reversibility and ethanol solubility.
  • the inventors of the present invention found that the above problems can be solved with the phospholipid represented by the general formula (1).
  • the present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
  • R 1 and R 2 are the same or different and represent a chain hydrocarbon group.
  • R3 represents a hydrogen atom or a hydrocarbon group.
  • m represents an integer from 1 to 3.
  • Section 4 The phospholipid according to any one of Items 1 to 3, wherein said m is 2.
  • Item 5 The phospholipid according to any one of items 1 to 4, wherein said R 3 is a hydrogen atom or an alkyl group.
  • Item 6 A lipid particle containing the phospholipid (phospholipid A) according to any one of Items 1 to 5.
  • Item 7. The lipid particles encapsulating a drug.
  • Item 8 Item 7, wherein the drug is a polynucleotide.
  • Item 9 The lipid particle according to any one of Items 6 to 8, which contains a sterol.
  • Item 10 The phospholipid A according to any one of Items 6 to 9, wherein the phospholipid A is a phospholipid having an unsaturated chain hydrocarbon group, and further contains a phospholipid (phospholipid B) other than the phospholipid A. lipid particles.
  • Item 11 An alcohol solution containing the phospholipid according to any one of Items 1 to 5.
  • Item 12. The alcohol solution according to Item 11, wherein the alcohol is ethanol.
  • Item 13. A method for producing lipid particles, comprising the step of mixing the alcohol solution according to Item 11 or 12 and an acidic aqueous solution.
  • Item 14 A pharmaceutical containing the lipid particles according to any one of Items 6 to 10.
  • a phospholipid suitable for preparing lipid particles which has charge reversibility and ethanol solubility.
  • Example 1 shows an NMR chart of DOP-PPZ synthesized in Synthesis Example 1-1.
  • 1 shows an NMR chart of DOP-MPPZ synthesized in Synthesis Example 1-2.
  • ⁇ -Potential measurement results of Test Example 2 are shown.
  • the legend indicates the phospholipid used as lipid 1.
  • the horizontal axis indicates the pH at the time of measurement.
  • 3 shows the results of LDH assay in Test Example 3.
  • the vertical axis shows the relative value of damaged cells when the negative control is corrected to 1, and the siRNA concentration in the evaluation system and the phospholipid used as lipid 1 are shown below the column.
  • Negative control is a sample without lipid particles
  • Positive control is a sample with lysis buffer
  • Other cationic lipid is a lipid complex using Lipofectamine (registered trademark) 2000 instead of lipid particles.
  • a sample. The results of WST-8assay of Test Example 3 are shown. The vertical axis shows the relative value of living cells when the negative control was corrected to 1, and the siRNA concentration in the evaluation system and the phospholipid used as lipid 1 are shown below the column.
  • Negative control is a sample without lipid particles
  • Positive control is a sample with lysis buffer
  • Other cationic lipid is a lipid complex using Lipofectamine (registered trademark) 2000 instead of lipid particles.
  • a sample. 4 shows the results of a gene suppression test in Test Example 4.
  • FIG. The ordinate indicates the relative value of the PLK1 mRNA expression level when the control is corrected to 1, and the siRNA species and their concentrations in the evaluation system are shown below the columns.
  • Control is a sample added with R
  • R 1 and R 2 are the same or different and represent a chain hydrocarbon group.
  • R3 represents a hydrogen atom or a hydrocarbon group.
  • m represents an integer from 1 to 3.
  • the phospholipid represented by herein sometimes referred to as "the phospholipid of the present invention”). This will be explained below.
  • the chain hydrocarbon group represented by R 1 or R 2 is not particularly limited as long as it is a monovalent chain hydrocarbon group, and may be either linear or branched (preferably linear). contain.
  • the number of carbon atoms in the chain hydrocarbon group is not particularly limited as long as it is capable of forming lipid particles. Even more preferably 14-18.
  • the chain hydrocarbon group includes both a saturated chain hydrocarbon group and an unsaturated hydrocarbon group, preferably an unsaturated chain hydrocarbon group, more preferably an unsaturated chain hydrocarbon group containing a double bond. It is a hydrocarbon group, more preferably an unsaturated chain hydrocarbon group having only one double bond.
  • Chain hydrocarbon groups include, for example, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, tridecyl, tetradecyl, pentadecyl, 9-pentadecenyl, hexadecyl, heptadecyl, cis-9-heptadecenyl, 11- heptadecenyl, cis,cis-9,12-heptadecantrienyl, 9,12,15-heptadecantrienyl, 6,9,12-heptadecantrienyl, 9,11,13-heptadecantrienyl, nonadecyl, 8,11-nonadecadienyl, 5,8,11-nonadecatrienyl, 5,8,11,14-nonadecatetraenyl, heneicosyl, tri
  • At least one of R 1 and R 2 is preferably an unsaturated chain hydrocarbon group, more preferably both are unsaturated chain hydrocarbon groups.
  • the hydrocarbon group represented by R3 is not particularly limited as long as it is a monovalent hydrocarbon group.
  • the monovalent hydrogen group is preferably a chain hydrocarbon group, more preferably an alkyl group.
  • the number of carbon atoms in the hydrocarbon group is not particularly limited, but is, for example, 1-8, preferably 1-6, more preferably 1-4, still more preferably 1-2, and particularly preferably 1.
  • R 3 is preferably a hydrogen atom or an alkyl group, more preferably a hydrogen atom.
  • m is preferably 2.
  • the phospholipids of general formula (1) also include salt forms. Both acid salts and basic salts can be employed as the salts.
  • acid salts include inorganic salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, malic acid organic salts such as salts, citrates, methanesulfonates, and paratoluenesulfonates;
  • examples of basic salts include alkali metal salts such as sodium salts and potassium salts; Alkaline earth metal salts such as salts; Salts with ammonia; Morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl) Examples thereof include salts with organic amines such as amines.
  • the phospholipids of the present invention can be synthesized by various methods.
  • the compounds of the present invention are represented by, for example, the following reaction schemes:
  • R 1 , R 2 , R 3 and m are the same as above. ] or analogously.
  • the amount of the compound represented by the general formula (B) used is preferably 3 to 25 mol, and 8 to 18 mol, per 1 mol of the compound represented by the general formula (A), from the viewpoint of yield and the like. more preferred.
  • the amount of phospholipase D used is preferably 50 to 1000 U, more preferably 200 to 500 U, per 1 millimole of the compound represented by general formula (A), from the viewpoint of yield and the like.
  • 1 U is the amount of enzyme that can change 1 micromole ( ⁇ mol) of substrate per minute under optimal conditions (at a temperature of 30°C and acidity at which the chemical reaction proceeds the most) (1 micromole per minute ).
  • This reaction is performed in the presence of a solvent.
  • the solvent is not particularly limited as long as it can exhibit phospholipase D activity.
  • Various buffer solutions are preferably used as the solvent.
  • the buffer preferably includes an acetate buffer.
  • the pH of the solvent is preferably 4-7, more preferably 5-6.
  • the reaction system may contain various organic solvents (eg, ethyl acetate, etc.) for dissolving the compound represented by formula (A).
  • additives can be used as appropriate within a range that does not significantly impair the progress of the reaction.
  • the reaction temperature is not particularly limited as long as it can exhibit the activity of phospholipase D, and is usually 20-50°C, preferably 35-45°C.
  • the reaction time is not particularly limited as long as the activity of phospholipase D can be exhibited, and is usually 6 to 72 hours, preferably 12 to 24 hours.
  • the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. Also, the structure of the product can be identified by elemental analysis, MS (FD-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR and the like.
  • Ionizable lipids are acidic and positively charged, with a change in net charge from 0 to +1.
  • the change in net charge of the phospholipids (charge-reversible lipids) of the present invention can range from -1 to +2, which is a different focus.
  • the phospholipids of the present invention are ionized even under neutral conditions and may have different physicochemical properties from ionizable lipids.
  • the lipids of the present invention are also capable of behaving as amphipathic lipids even under neutral conditions and are therefore expected to be more stable and safer.
  • lipid particles that do not have a positive charge at the pH of body fluids (usually in the neutral range) and that enable more efficient expression of the effect of the drug contained therein. can.
  • Lipid particles contains the phospholipids of the present invention (herein, sometimes referred to as “phospholipid A”), containing lipid particles (herein, "the lipid particles of the present invention Also referred to as “lipid particles”). This will be explained below.
  • the lipid particles of the present invention are not particularly limited as long as they contain the phospholipid of the present invention as a particle-constituting lipid.
  • the phospholipids of the present invention contained in the lipid particles may be used singly or in combination of two or more.
  • the lipid particles of the present invention include, for example, particles in which an amphipathic lipid containing the phospholipid of the present invention constitutes an outer layer, and the lipids are arranged with the hydrophilic portion facing outward.
  • the particles include particles whose outer layer is composed of a single lipid membrane, and particles whose outer layer is composed of a lipid bilayer, preferably particles whose outer layer is composed of a single lipid membrane, and more preferably particles whose outer layer is composed of a single lipid membrane. in which the amphiphilic lipids are aligned with the hydrophilic portion facing outward.
  • the inner layer of the particles may consist of a homogeneous phase, either an aqueous phase or an oil phase, but preferably contains one or more reverse micelles.
  • the particle size of the lipid particles of the present invention is not particularly limited.
  • the particle size is preferably nano-sized, specifically for example 10 to 700 nm, preferably 20 to 500 nm, more preferably 40 to 200 nm, still more preferably 60 to 150 nm.
  • the lipid particles of the present invention do not have a positive charge at the pH of body fluid (usually in the neutral range). More specifically, the lipid particles of the present invention have a zeta potential of -80 to -1 mV, -60 to -10 mV, -60 to -20 mV in a pH 7.0 buffer.
  • the lipid particles of the present invention may contain other lipids as particle-constituting lipids in addition to the phospholipids of the present invention.
  • Specific examples of lipids include phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids, and the like.
  • phospholipids include phosphatidylcholines such as dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, myristoylpalmitoylphosphatidylcholine, myristoylstearoylphosphatidylcholine, palmitoylstearoylphosphatidylcholine; Phosphatidylglycerols such as lauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilinoleoylphosphati
  • glycolipids include glyceroglycolipids such as diglycosyldiglycerides, digalactosyldiglycerides, galactosyldiglycerides and glycosyldiglycerides; glycosphingolipids such as galactosylcerebrosides and gangliosides; stearyl glucosides and esterified stearyl glycosides.
  • sterols include cholesterol, cholesteryl hemisuccinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, phytosterol, stigmasterol, timosterol, ergosterol, sitosterol, campesterol, and brassicasterol.
  • the sterol has the effect of stabilizing the liposome membrane and adjusting the fluidity of the liposome membrane, so it is desirable that the sterol is contained as a constituent lipid of the liposome membrane.
  • saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 10 to 22 carbon atoms such as decanoic acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid and docosanoic acid. .
  • the above lipids may be used singly or in combination of two or more.
  • the lipid particles of the present invention preferably contain a phospholipid other than the phospholipid of the present invention (herein sometimes referred to as "phospholipid B") and/or a sterol, more preferably phospholipid X and a sterol including.
  • phospholipid B preferably has a saturated chain hydrocarbon group.
  • Phospholipid B is preferably phosphatidylcholine, particularly preferably dipalmitoylphosphatidylcholine.
  • Phospholipids B also include, for example, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleylphosphatidylcholine, palmitoyloleylphosphatidylcholine, and the like.
  • Preferred sterols include cholesterol.
  • the content thereof is, for example, 15 to 100 mol, preferably 30 to 70 mol, more preferably 40 to 60 mol, per 100 mol of the phospholipid of the present invention. , more preferably 45 to 55 mol.
  • the content is, for example, 5 to 70 mol, preferably 10 to 40 mol, more preferably 15 to 30 mol, still more preferably 17 to 27 mol, per 100 mol of the phospholipid of the present invention.
  • the content is, for example, 30 to 200 mol, preferably 60 to 140 mol, more preferably 80 to 120 mol, per 100 mol of the phospholipid of the present invention. It is preferably 90 to 110 mol, more preferably 95 to 105 mol.
  • the content of phospholipid B is, for example, 15 to 100 mol, preferably 30 to 70 mol, more preferably 40 to 60 mol, per 100 mol of sterol. mol, more preferably 45 to 55 mol.
  • the content is, for example, 5 to 70 mol, preferably 10 to 40 mol, more preferably 15 to 30 mol, still more preferably 17 to 27 mol, per 100 mol of sterol.
  • the total content of the phospholipid of the present invention and other lipids that are optionally blended is relative to 100 mol% of the lipid particles constituting the lipid particles of the present invention, for example It is 50 mol % or more, preferably 70 mol % or more, more preferably 90 mol % or more, still more preferably 95 mol % or more, and even more preferably 99 mol % or more.
  • part of the phospholipid can be modified with a water-soluble polymer such as PEG.
  • the content of the PEG-modified phospholipid is, for example, 0 to 50 mol%, preferably 0 to 30 mol%, more preferably 0 to 20 mol%, relative to 100 mol% of the lipid particles constituting the lipid particles of the present invention. It is preferably 0 to 15 mol %.
  • the lipid particles of the present invention preferably encapsulate a drug.
  • Drugs are not particularly limited, and examples include polynucleotides, peptides, proteins, sugars, low-molecular-weight compounds, and the like.
  • the drug is preferably negatively charged and water-soluble.
  • Polynucleotides can be preferably employed as such drugs.
  • Target diseases of drugs include, but are not particularly limited to, cancer (in particular, solid cancer).
  • the polynucleotide is not particularly limited as long as it can exhibit a function as a drug. , Cas protein expression vector, TALEN expression vector, etc.), nucleic acid vaccines, and the like.
  • Polynucleotides may be subjected to known chemical modifications as exemplified below. Substitution of the phosphate residue of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. to prevent degradation by hydrolases such as nucleases can be done.
  • a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc.
  • the hydroxyl group at the 2nd position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, CH 3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH2CH2NHC ( NH ) NH2 , CH2CONHCH3 , CH2CH2CN , etc.).
  • R is, for example, CH 3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH2CH2NHC ( NH ) NH2 , CH2CONHCH3 , CH2CH2CN , etc.
  • the base moiety pyrimidine, purine
  • phosphate moiety or hydroxyl moiety has been modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like.
  • BNA LNA
  • the conformation of the sugar portion is fixed to the N-type by bridging the 2' oxygen and 4' carbon of the sugar portion of a nucleotide, can also be preferably used.
  • the drug is preferably contained in the inner layer of the lipid particles of the present invention.
  • the drug is preferably contained within reverse micelles in the inner layer.
  • the molar ratio of the lipid particle-constituting lipid of the present invention to the drug is, for example, 500 or more, preferably 1000 or more when the drug is a polynucleotide such as siRNA. , more preferably 1,500 or more, still more preferably 1,900 or more, even more preferably 2,500 or more, and particularly preferably 3,200 or more.
  • the upper limit of the molar ratio is not particularly limited, and is 10,000, 7,000, 5,000, for example.
  • the lipid particles of the present invention may contain other components in addition to the above.
  • Other components include, for example, membrane stabilizers, charged substances, antioxidants, membrane proteins, polyethylene glycol (PEG), antibodies, peptides, sugar chains and the like.
  • Antioxidants can be contained to prevent oxidation of the film, and are used as necessary as a component of the film.
  • Antioxidants used as membrane constituents include, for example, butylated hydroxytoluene, propyl gallate, tocopherols, tocopherol acetate, concentrated mixed tocopherols, vitamin E, ascorbic acid, L-ascorbic stearate, palmitic acid. Examples include ascorbic acid, sodium hydrogen sulfite, sodium sulfite, sodium edetate, erythorbic acid, citric acid and the like.
  • Membrane proteins can be contained for the purpose of adding functions to membranes or stabilizing membrane structures, and are used as necessary as membrane constituents.
  • Membrane proteins include, for example, surface membrane proteins, integral membrane proteins, albumin, recombinant albumin and the like.
  • the content of other components is, for example, 10% or less, preferably 5% or less, more preferably 2% or less, still more preferably 1% or less, relative to 100% by mass of the lipid particles of the present invention.
  • the lipid particles of the present invention can be produced according to or according to known methods for producing lipid particles.
  • the lipid particles of the present invention can be preferably produced by a method including a step of mixing an alcoholic solution containing the phospholipid of the present invention and an acidic aqueous solution (Step 1).
  • the alcohol that is the solvent for the alcohol solution is not particularly limited as long as it can dissolve phospholipids.
  • preferred alcohols include ethanol, 2-propanol, t-butanol, and the like. Among these, ethanol is particularly preferred from the viewpoint of ease of handling, safety, and the like.
  • the acidic aqueous solution usually contains an acid in addition to the solvent water.
  • acids include organic acids and inorganic acids, preferably organic acids.
  • organic acids include maleic acid, formic acid, acetic acid, propionic acid, folic acid, isobutyric acid, valeric acid, isovaleric acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, ketoglutaric acid, adipic acid, Lactic Acid, Tartaric Acid, Fumaric Acid, Oxaloacetic Acid, Malic Acid, Isocitric Acid, Citric Acid, Benzoic Acid, Phthalic Acid, Isophthalic Acid, Terephthalic Acid, Hemimellitic Acid, Trimellitic Acid, Trimesic Acid, Mellophanic Acid, Prenic Acid, Pyromellitic Acid, Merit acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulf
  • Inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, boric acid, boronic acid, hydrofluoric acid, hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromous acid, bromous acid, bromine acid, perbromic acid, hypoiodous acid, iodous acid, iodic acid, periodic acid, phosphorous acid, phosphoric acid, polyphosphoric acid, chromic acid, permanganic acid, amberlyst.
  • One type of acid may be used alone, or two or more types may be used in combination.
  • the pH of the acidic aqueous solution is preferably 3-5.
  • the acidic aqueous solution preferably contains a water-soluble drug.
  • the mixing ratio of the acidic aqueous solution and the alcoholic solution is, for example, 1.5-10, preferably 2-8, more preferably 3-6.
  • Mixing is not particularly limited as long as the lipid and the drug can be mixed, but for example, vigorous stirring with a vortex or the like can be adopted.
  • the mixing time varies depending on the mixing mode, but is, for example, 10 seconds to 2 minutes, preferably 15 seconds to 1 minute.
  • step 1 can be performed at room temperature or under heating.
  • the temperature in step 1 is, for example, 5°C to 50°C, preferably 15°C to 45°C. If t-butanol is not used or the amount used is small, lipid particles can be prepared even if the temperature in step 1 is relatively low.
  • the temperature is, for example, less than 30°C and 25°C or less.
  • Step 1 can also be carried out using a reaction system using microchannels. In that case, various conditions can be appropriately adjusted according to the reaction system.
  • step 1 it is preferable to remove alcohol by dialysis.
  • the dialysis solvent water can usually be used.
  • the dialysis time is, for example, 4-48 hours, preferably 6-24 hours, more preferably 6-12 hours. During dialysis, it is preferable to replace the dialysis solvent as appropriate.
  • the lipid particles of the present invention can be frozen products, freeze-dried products, and the like.
  • Lipid Particles One aspect of the present invention relates to a medicine containing the lipid particles of the present invention (herein sometimes referred to as "the medicine of the present invention"). Moreover, the lipid particles of the present invention can also be used as a reagent.
  • the lipid particles of the present invention can exert the effects of drugs (for example, polynucleotides such as siRNA) more efficiently while reducing cytotoxicity. Therefore, the lipid particles of the present invention can be suitably used as drug carriers.
  • drugs for example, polynucleotides such as siRNA
  • the content of the active ingredient in the medicament of the present invention can be about 0.0001 to 100 parts by weight based on 100 parts by weight of the entire medicament of the present invention.
  • the dosage form of the medicament of the present invention is not particularly limited as long as the desired effect is obtained. It can be administered to mammals including humans by any route of administration.
  • a preferred mode of administration is parenteral administration, more preferably intravenous injection.
  • Dosage forms for oral administration and parenteral administration and production methods thereof are well known to those skilled in the art, and can be produced according to conventional methods by mixing the active ingredient with a pharmaceutically acceptable carrier or the like. can.
  • Dosage forms for parenteral administration include preparations for injection (e.g., drip injection, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection), external preparations (e.g., ointments, poultices, lotions). medicine), suppository inhalant, ophthalmic agent, ophthalmic ointment, nose drops, ear drops and the like.
  • an injectable preparation is prepared by dissolving the lipid particles of the present invention in distilled water for injection, and adding a solubilizer, a buffer, a pH adjuster, a tonicity agent, an analgesic, and a preservative, if necessary. , and stabilizers and the like can be added.
  • the medicament can also be a lyophilized formulation for extemporaneous preparation.
  • the medicament of the present invention may further contain other drugs effective in treating or preventing diseases.
  • the medicament of the present invention can also contain ingredients such as bactericides, antiphlogistic agents, cell activators, vitamins, and amino acids, if necessary.
  • the carrier used for formulating the medicament of the present invention includes excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and if necessary, stabilizers, emulsifiers, emulsifiers, Absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, etc. be able to.
  • the dosage of the medicament of the present invention is determined, for example, by route of administration, type of disease, degree of symptoms, patient age, sex, body weight, severity of disease, pharmacological findings such as pharmacokinetics and toxicological characteristics, It can be determined by the clinician based on a variety of factors, such as whether a drug delivery system is used and whether it is administered as part of a combination of other drugs.
  • the dosage of the drug of the present invention can be, for example, about 1 ⁇ g/kg (body weight) to 10 g/kg (body weight) per day.
  • the administration schedule of the medicament of the present invention can also be determined in consideration of factors similar to the dosage. For example, the above daily doses can be administered once a day to once a month.
  • Synthesis example 1 Synthesis of DOP-PPZ and DOP-MPPZ 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethylpiperazine) (DOP-PPZ) and 1,2-di-(9Z-octadecenoyl)-sn- Glycero-3-phosphomethylpiperazine (DOP-MPPZ) was synthesized according to the following scheme.
  • Synthesis Example 1-1 Synthesis of DOP-PPZ 3.01 g (3.82 mmol) of DOPC was dissolved in ethyl acetate, and 6.90 g of 1-(2-Hydroxyethyl) piperazine (53 mmol) was dissolved in 0.5 M acetic acid, pH 5.5. Buffer was added and warmed to 40°C. After heating, PLDP (Phospholipase D manufactured by Asahi Kasei Pharma Corp.) (1440 U) was added and stirred at 40°C. After 19 hours, disappearance of DOPC was confirmed by TLC. One unit is the amount of enzyme that can change 1 micromole ( ⁇ mol) of substrate per minute under optimal conditions (at a temperature of 30°C and acidity at which chemical reactions proceed the most) (1 micromole per minute ).
  • the resulting crude reaction product was dissolved in 36 ml of dioxane and filtered through a 0.2 ⁇ m membrane, then 18 ml of dioxane/4M HCl was added dropwise to the filtrate while cooling with ice, and the mixture was stirred for 30 minutes. After stirring, precipitated white crystals were filtered, and the crystals were suspended and washed with acetone three times. The obtained crystals were vacuum-dried overnight to obtain 2.01 g of white crystals.
  • Synthesis Example 1-2 Synthesis of DOP-MPPZ 1.00 g (1.27 mmol) of DOPC was dissolved in ethyl acetate, and 2.55 g of 4-Methylpiperazine-1-ethanol (17.7 mmol) was dissolved in the solution in 0.5 M acetate buffer, pH 5.5. The liquid was added and warmed to 40°C. After heating, PLDP (Phospholipase D manufactured by Asahi Kasei Pharma Corp.) (480 U) was added and stirred at 40°C. After 19 hours, disappearance of DOPC was confirmed by TLC. One unit is the amount of enzyme that can change 1 micromole ( ⁇ mol) of substrate per minute under optimal conditions (at a temperature of 30°C and acidity at which chemical reactions proceed the most) (1 micromole per minute ).
  • the resulting crude reaction product was dissolved in 12.5 ml of dioxane and filtered through a 0.2 ⁇ m membrane, then 6.8 ml of dioxane/4M HCl was added dropwise to the filtrate while cooling with ice, and the mixture was stirred for 30 minutes. After stirring, precipitated white crystals were filtered, and the crystals were suspended and washed with acetone three times. The obtained crystals were vacuum-dried overnight to obtain 0.51 g of white crystals.
  • Synthesis example 2 Synthesis of dioleoylphosphate-diethylenediamine conjugate (DOP-DD) DOP-DD (DOP-DEDA) was synthesized according to the following scheme. Specifically, it was synthesized according to the method described in Patent Document 1.
  • Test example 1 Ethanol solubility test Ethanol solubility test was carried out based on the general rules of the Japanese Pharmacopoeia.
  • Test example 2 Production of lipid particles and measurement of various physical properties ⁇ Test Example 2-1. Production of lipid particles> siRNA was added to 1 mM citrate buffer (pH 4.0) to prepare an acidic siRNA aqueous solution (25°C, siRNA concentration: 71.4 nM). On the other hand, lipids ((lipid 1) DOP-DEDA, DOP-PPZ, or DOP-MPPZ, (lipid 2) dipalmitoylphosphatidylcholine (DPPC), and (lipid 3) cholesterol (Chol)) were mixed at a ratio of 45:10:45.
  • RNA quantification reagent (RiboGreen reagent, manufactured by Thermo Fisher Scientific) was used. Specifically, it was carried out as follows. 2% Triton-X 100 or RNase free water was added to the lipid particle solution. The resulting solution, RNase free water, and RiboGreen reagent were mixed in wells of a 96-well black plate. After shaking the plate for 5 minutes, the fluorescence intensity of each well was measured.
  • encapsulation rate (%) (fluorescence intensity of total siRNA - fluorescence intensity of free siRNA) / (fluorescence intensity of total siRNA), encapsulation of siRNA in lipid particles rate was calculated.
  • Test example 3 Cytotoxicity evaluation test ⁇ Test Example 3-1. Production of lipid particles> Lipid particles were produced in the same manner as in Example 2-1.
  • ⁇ Test Example 3-2 Toxicity Evaluation Test> MDA-MB-231 human breast cancer cells were seeded in a 96-well plate (7 ⁇ 10 3 cells/well) and cultured at 37° C. for 24 hours. Lipid particle solution (containing 0.6/2/6 pmol of siRNA) or lipid complex solution (complex prepared using Lipofectamine® 2000 (manufactured by Thermo Fisher Scientific), containing 0.6/2/6 pmol of siRNA) was added dropwise to the wells and cultured at 37°C for 96 hours. Cytotoxicity of lipid particles and lipid complexes was evaluated using a Viability/Cytotoxicity Multiplex Assay Kit (Dojindo Laboratories).
  • Test example 4 Gene Suppression Test ⁇ Test Example 4-1. Production of lipid particles> Lipid particles were produced in the same manner as in Test Example 2-1 except that DOP-PPZ was used as lipid 1 and siRNA targeting the PLK1 gene or non-specific siRNA (si Control) was used as siRNA.
  • cDNA was synthesized from total RNA using First-Strand cDNA Synthesis Kit (manufactured by GE Healthcare Life Sciences). The amount of PLK1 mRNA was quantified by real-time PCR using cDNA as a template and TB Green (registered trademark) Premix Ex Taq (trademark) II (manufactured by Takara Bio Inc.).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
PCT/JP2022/008670 2021-03-09 2022-03-01 リン脂質 Ceased WO2022190967A1 (ja)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202280006255.1A CN116234538A (zh) 2021-03-09 2022-03-01 磷脂
JP2023505328A JP7848179B2 (ja) 2021-03-09 2022-03-01 リン脂質
EP22766926.4A EP4306531A4 (en) 2021-03-09 2022-03-01 Phospholipid
US18/017,751 US20230295199A1 (en) 2021-03-09 2022-03-01 Phospholipid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021037573 2021-03-09
JP2021-037573 2021-03-09

Publications (1)

Publication Number Publication Date
WO2022190967A1 true WO2022190967A1 (ja) 2022-09-15

Family

ID=83227160

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/008670 Ceased WO2022190967A1 (ja) 2021-03-09 2022-03-01 リン脂質

Country Status (5)

Country Link
US (1) US20230295199A1 (https=)
EP (1) EP4306531A4 (https=)
JP (1) JP7848179B2 (https=)
CN (1) CN116234538A (https=)
WO (1) WO2022190967A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024171859A1 (ja) * 2023-02-14 2024-08-22 静岡県公立大学法人 ｐＨ応答性リン脂質

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6041494A (ja) * 1983-04-11 1985-03-05 Meito Sangyo Kk 酵素法リン脂質一級アルコ−ル誘導体の製法
WO2018190017A1 (ja) * 2017-04-11 2018-10-18 日本精化株式会社 核酸導入用脂質誘導体

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE787457A (fr) * 1971-08-14 1973-02-12 Basf Ag Derives de l'acide chloro-2-ethanephosphonique
DE2752553C2 (de) * 1977-11-24 1985-07-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen Neue phospholipidartige Verbindungen, ihre Verwendung zur Herstellung von Pflanzenhybriden und Verfahren zu ihrer Herstellung
DE10207177A1 (de) * 2002-02-19 2003-09-04 Novosom Ag Fakultativ kationische Lipide
JP4442864B2 (ja) 2004-03-04 2010-03-31 竹本油脂株式会社 リン脂質誘導体及び遺伝子導入キャリア
WO2019005830A1 (en) * 2017-06-26 2019-01-03 Fordoz Pharma Corp. NANOSOME FORMULATIONS OF APREPITANT AND METHODS AND APPLICATIONS THEREOF

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6041494A (ja) * 1983-04-11 1985-03-05 Meito Sangyo Kk 酵素法リン脂質一級アルコ−ル誘導体の製法
WO2018190017A1 (ja) * 2017-04-11 2018-10-18 日本精化株式会社 核酸導入用脂質誘導体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP4306531A4 *
TOMITA, KOHJI ET AL.: "P-70 Development of Novel Functional Lipid with Piperazine Skeleton", PROCEEDINGS OF THE 36TH ANNUAL MEETING OF THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM (DDS); AUGUST 28-29, 2020, JAPAN SOCIETY OF DRUG DELIVERY SYSTEM (DDS), vol. 36, 10 August 2020 (2020-08-10), pages 197, XP009539658 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024171859A1 (ja) * 2023-02-14 2024-08-22 静岡県公立大学法人 ｐＨ応答性リン脂質

Also Published As

Publication number Publication date
US20230295199A1 (en) 2023-09-21
EP4306531A1 (en) 2024-01-17
JPWO2022190967A1 (https=) 2022-09-15
JP7848179B2 (ja) 2026-04-20
CN116234538A (zh) 2023-06-06
EP4306531A4 (en) 2025-02-19

Similar Documents

Publication Publication Date Title
EP3239132B1 (en) Cationic lipid
EP2608785B1 (en) Lipomacrocycles and uses thereof
AU2004257373A1 (en) Lipid encapsulated interfering RNA
KR102758330B1 (ko) 황화물을 포함하는 이온화 가능한 지질 및 이를 포함하는 지질나노입자
JP6230538B2 (ja) 安定なオキサリプラチン封入リポソーム水分散液及びその安定化方法
KR20240082390A (ko) 생분해성 에스터 결합을 포함하는 이온화 가능한 지질 및 이를 포함하는 지질나노입자
JP7016084B2 (ja) 核酸導入用脂質誘導体
KR20250111222A (ko) 신규 이온화 가능한 지질 및 이를 포함하는 지질나노입자
WO2022190967A1 (ja) リン脂質
JP7748394B2 (ja) リン脂質
KR20240082389A (ko) 생분해성 이황화 결합을 포함하는 이온화 가능한 지질 및 이를 포함하는 지질나노입자
JP2024084059A (ja) リン脂質
CN101597312A (zh) 维甲酰阿糖胞苷缀合物及其药质体、其制备方法和应用
EP4667475A1 (en) Ph-responsive phospholipid
CN101176721B (zh) 六甲蜜胺脂质体及其制备方法
EP4691476A1 (en) Method for producing nucleic acid-encapsulated lipid nanoparticles
WO2025242219A1 (zh) 含氯喹结构的化合物、含其的组合物和其应用
CN116284006A (zh) 可电离脂质化合物、包含其的脂质载体及应用
CN120965753A (zh) 一种可离子化脂质以及由其制备的脂质载体及用途
CN120230060A (zh) 一种含有哌嗪的阳离子脂质化合物及其纳米组合物和应用
CA3194742A1 (en) Formulated and/or co-formulated liposome compositions containing pd-1 antagonist prodrugs useful in the treatment of cancer and methods thereof

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2023505328

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22766926

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2022766926

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022766926

Country of ref document: EP

Effective date: 20231009