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WO2025204688A1 - Composition liquide pour la protection d'une membrane phospholipidique - Google Patents

Composition liquide pour la protection d'une membrane phospholipidique

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Publication number
WO2025204688A1
WO2025204688A1 PCT/JP2025/008174 JP2025008174W WO2025204688A1 WO 2025204688 A1 WO2025204688 A1 WO 2025204688A1 JP 2025008174 W JP2025008174 W JP 2025008174W WO 2025204688 A1 WO2025204688 A1 WO 2025204688A1
Authority
WO
WIPO (PCT)
Prior art keywords
liquid composition
component
phospholipid
composition according
protecting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/008174
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English (en)
Japanese (ja)
Inventor
奈央樹 坂口
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.)
Daicel Corp
Original Assignee
Daicel Corp
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Filing date
Publication date
Application filed by Daicel Corp filed Critical Daicel Corp
Publication of WO2025204688A1 publication Critical patent/WO2025204688A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This disclosure relates to a liquid composition for protecting phospholipid membranes. More specifically, this disclosure relates to a liquid composition containing magnesium ions and a specific buffer solution, which is used to protect various structures having phospholipid membranes, such as cells, extracellular vesicles, and liposomes, from various types of damage, including damage caused by freezing, vibration, and contact with hydrophobic materials.
  • phospholipid membranes have a similar structure to biological membranes, and offer excellent biocompatibility, flexibility, and the potential for a wide range of applications, their similar structure makes it difficult to ensure their stability.
  • Patent Document 2 In the field of cells, highly membrane-permeable materials such as DMSO or ethylene glycol have been used as protective agents when cryopreserving cells. In recent years, the addition of dextran or glucose as protective agents has also been considered (Patent Document 2). Furthermore, the use of synthetic polymers such as trehalose, a type of sugar, and carboxypolylysine as protective agents has also been considered. Trehalose in particular has been reported to have high activity in protecting membranes during freezing, and its combination with other materials or the delivery of trehalose into cells has also been considered (Patent Document 3).
  • the present disclosure aims to provide a protective agent with excellent protective effects for phospholipid membranes.
  • Item 1 A liquid composition for protecting a phospholipid membrane, comprising (A) a magnesium salt and (B) an acetate buffer, a citrate buffer, a carbonate buffer, and/or a succinate buffer.
  • Item 2. The liquid composition according to Item 1, wherein the component (A) is selected from the group consisting of magnesium chloride, magnesium sulfate, and magnesium gluconate.
  • Item 3. The liquid composition according to Item 1 or 2, further comprising (C) a polyhydric alcohol.
  • Item 4 The liquid composition according to Item 3, wherein the component (C) is glycerin.
  • Item 6. The liquid composition according to Item 5, wherein the component (D) is mannitol.
  • Item 7. The liquid composition according to any one of Items 1 to 6, further comprising (E) polyethylene glycol.
  • Item 8. The liquid composition according to any one of Items 1 to 7, further comprising (C) glycerin, (D) mannitol, and (E) polyethylene glycol.
  • Item 11 The liquid composition according to any one of Items 7 to 10, wherein the component (B) is a mixed solution of an acetate buffer and a citrate buffer, and the content of the component (E) is 15 mg/mL or more.
  • Item 12 The liquid composition according to any one of Items 1 to 11, wherein the component (B) comprises an acetate buffer and a citrate buffer, or an acetate buffer, a citrate buffer, or a succinate buffer.
  • Item 13 The liquid composition according to any one of Items 1 to 11, wherein the component (B) comprises an acetate buffer and a citrate buffer, or an acetate buffer, a citrate buffer, or a succinate buffer.
  • Item 14 The liquid composition according to Item 13, wherein the component (F) is selected from the group consisting of arginine, arginine dipeptide, glutamic acid, proline, and salts thereof.
  • Item 15. The liquid composition according to any one of Items 1 to 14, wherein the content of the component (A) is 0.1 mg/mL or more.
  • Item 16 The liquid composition according to any one of Items 1 to 15, wherein the content of the component (A) is 0.1 mM or more in terms of magnesium ion concentration.
  • Item 18 The liquid composition according to any one of Items 3 to 17, wherein the content of the component (C) is 0.01 mg/mL or more.
  • Item 19 The liquid composition according to any one of Items 5 to 18, wherein the content of the component (D) is 0.01 mg/mL or more.
  • Item 20 The liquid composition according to any one of items 7 to 19, wherein the content of component (E) is 0.01 mg/mL or more.
  • Item 21 The liquid composition according to any one of Items 13 to 20, wherein the content of the component (F) is 1 mg/mL or more.
  • Item 22 The liquid composition according to any one of Items 13 to 20, wherein the content of the component (F) is 1 mg/mL or more.
  • Item 29. A pharmaceutical composition comprising cells, extracellular vesicles, and/or liposomes and the liquid composition according to any one of Items 1 to 28.
  • Item 30. The pharmaceutical composition according to Item 29, which is contained in a container made of a hydrophobic material.
  • Item 31. The pharmaceutical composition according to Item 29 or 30, which is in a frozen state.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 6.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 7.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 8.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 15.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 16.
  • 1 shows the results of evaluation of the protection of phospholipid membranes of fluorescent liposomes against freezing damage, obtained in Test Example 16.
  • the magnesium salt which is the component (A), is an active ingredient of the liquid composition for protecting a phospholipid membrane of the present disclosure.
  • the component (A) alone has the effect of protecting a phospholipid membrane.
  • the component (A) is not particularly limited as long as it is water-soluble and pharmaceutically acceptable, and examples thereof include magnesium chloride, magnesium sulfate, magnesium gluconate, etc. These magnesium salts may be used alone or in combination of two or more.
  • the upper limit of the content of component (A) in the liquid composition for protecting phospholipid membranes of the present disclosure is not particularly limited, and can be determined appropriately by a person skilled in the art taking into consideration factors such as osmotic pressure.
  • Examples of the content include 300 mg/mL or less, 250 mg/mL or less, 210 mg/mL or less, 150 mg/mL or less, 110 mg/mL or less, 70 mg/mL or less, 55 mg/mL or less, 35 mg/mL or less, 25 mg/mL or less, and 15 mg/mL or less.
  • preferred concentrations include 8 mg/mL or less, 6 mg/mL or less, 4 mg/mL or less, 3 mg/mL or less, 2 mg/mL or less, 1.5 mg/mL or less, 1 mg/mL or less, 0.8 mg/mL or less, 0.6 mg/mL or less, and 0.4 mg/mL.
  • Specific ranges for the content of component (A) in the liquid composition for protecting phospholipid membranes of the present disclosure can be any combination of the above upper and lower limits, and include, for example, 0.1 to 300 mg/mL, 0.1 to 250 mg/mL, 0.5 to 210 mg/mL, 0.5 to 150 mg/mL, 0.7 to 110 mg/mL, 0.7 to 70 mg/mL, 0.9 to 55 mg/mL, 0.9 to 35 mg/mL, 1 to 25 mg/mL, 1 to 15 mg/mL, 1 to 8 mg/mL, 1.5 to 6 mg/mL, 2 to 4 mg/mL, and 2.5 to 3 mg/mL.
  • the content of component (A) in the liquid composition for protecting a phospholipid membrane of the present disclosure may be, in terms of magnesium ion concentration, for example, 0.1 mM or more, 0.2 mM or more, 0.3 mM or more, or 0.4 mM or more.
  • the upper limit of the range of magnesium ion concentration in the liquid composition for protecting phospholipid membranes of the present disclosure can be determined appropriately by one of ordinary skill in the art, taking into consideration factors such as osmotic pressure, but examples include 1500 mM or less, 1300 mM or less, 1000 mM or less, 710 mM or less, 510 mM or less, 310 mM or less, 210 mM or less, 110 mM or less, 75 mM or less, 55 mM or less, or 35 mM or less.
  • preferred concentrations include 22 mM or less, 15 mM or less, 12 mM or less, 7 mM or less, 5 mM or less, 3.5 mM or less, 2.5 mM or less, 1.5 mM or less, or 0.8 mM or less.
  • component (B) examples include acetate buffer, citrate buffer, carbonate buffer, and/or succinate buffer (hereinafter also referred to as "predetermined buffer”). These buffers may be used alone or in combination of two or more.
  • component (B) includes acetate buffer, citrate buffer, and/or succinate buffer.
  • component (B) includes a combination of acetate buffer and citrate buffer.
  • the phospholipid membrane protective effect can be further enhanced by incorporating component (E) described below (for example, incorporating component (E) at 15 mg/mL or more).
  • component (B) includes a combination of acetate buffer, citrate buffer, and succinate buffer.
  • Acetate buffer, citrate buffer, carbonate buffer, and succinate buffer may be aqueous solutions of acetic acid, citric acid, carbonic acid, and succinic acid (hereinafter also referred to as “predetermined acids”) and their conjugate bases (hereinafter referred to as “conjugate bases of predetermined acids”), respectively.
  • the content of component (B) include a total concentration of the buffering agents constituting the buffer solution of 5 mM or more, and from the viewpoint of further enhancing the phospholipid membrane protective effect, the content is preferably 8 mM or more, 10 mM or more, or 13 mM or more, and more preferably 16 mM or more, 18 mM or more, 20 mM or more, 25 mM or more, 30 mM or more, 35 mM or more, 40 mM or more, 45 mM or more, or 48 mM or more.
  • the range of the content of component (B) in the liquid composition for protecting phospholipid membranes of the present disclosure is not particularly limited in terms of its upper limit, and can be determined appropriately by a person skilled in the art taking into consideration factors such as osmotic pressure.
  • Examples of the total concentration of the buffer include 80 mM or less, 70 mM or less, 65 mM or less, 60 mM or less, 55 mM or less, 50 mM or less, 47 mM or less, 42 mM or less, 37 mM or less, 32 mM or less, 27 mM or less, 22 mM or less, and 17 mM or less.
  • the specific range of the content of component (B) in the liquid composition for protecting phospholipid membranes of the present disclosure can be any combination of the upper and lower limits of the total concentration of the buffering agent described above, and examples include 8 to 80 mM, 10 to 70 mM, 13 to 65 mM, 16 to 65 mM, 18 to 60 mM, and 20 to 60 mM.
  • the polyhydric alcohol of component (C) is not particularly limited as long as it is pharmaceutically acceptable, but examples include dihydric alcohols such as propylene glycol, 1,3-butylene glycol, ethylene glycol, isoprene glycol, diethylene glycol, and dipropylene glycol; and trihydric alcohols such as glycerin. Note that the sugars and sugar alcohols of component (D) and polyethylene glycol of component (E), described below, are distinct from component (C). These polyhydric alcohols may be used alone or in combination of two or more.
  • component (C) in the liquid composition for protecting phospholipid membranes disclosed herein is not particularly limited and can be set appropriately depending on the desired level of phospholipid membrane protection effect.
  • the specific range of the content of component (C) in the liquid composition for protecting phospholipid membranes of the present disclosure can be any combination of the above upper and lower limits, and examples include 0.01 to 150 mg/mL, 0.1 to 100 mg/mL, 1 to 80 mg/mL, 1 to 70 mg/mL, 8 to 60 mg/mL, 8 to 55 mg/mL, 8 to 40 mg/mL, 10 to 30 mg/mL, and 10 to 20 mg/mL.
  • the liquid composition for protecting a phospholipid membrane of the present disclosure may contain a sugar and/or a sugar alcohol as component (D) for the purpose of enhancing the phospholipid membrane protecting effect.
  • Sugars are not particularly limited as long as they are pharmaceutically acceptable, and examples include monosaccharides and disaccharides.
  • Monosaccharides include glucose, fructose, galactose, mannose, etc.
  • Disaccharides include sucrose, trehalose, maltose, lactose, etc. These sugars may be used alone or in combination of two or more.
  • Sugar alcohols are not particularly limited as long as they are pharmaceutically acceptable, but examples include sorbitol, xylitol, isomaltose, erythritol, maltitol, mannitol, lactitol, palatinose, and reduced palatinose. These sugar alcohols may be used alone or in combination of two or more.
  • sugar alcohols are preferred, and mannitol is more preferred, from the viewpoint of further enhancing the phospholipid membrane protective effect.
  • the range of the content of component (D) in the liquid composition for protecting phospholipid membranes of the present disclosure is not particularly limited in terms of its upper limit, and can be determined appropriately by a person skilled in the art taking into consideration factors such as osmotic pressure. Examples include 100 mg/mL or less, 70 mg/mL or less, 60 mg/mL or less, 55 mg/mL or less, 50 mg/mL or less, 45 mg/mL or less, 40 mg/mL or less, 35 mg/mL or less, 30 mg/mL or less, or 20 mg/mL or less.
  • the liquid composition for protecting a phospholipid membrane of the present disclosure may contain polyethylene glycol as component (E) for the purpose of enhancing the phospholipid membrane protecting effect.
  • the average molecular weight of polyethylene glycol is not particularly limited, but examples include 270 or more. From the viewpoint of further enhancing the phospholipid membrane protective effect, it is preferably 350 or more, 400 or more, or 500 or more, more preferably 550 or more, 600 or more, 800 or more, 1000 or more, 1500 or more, 2000 or more, or 2500 or more, and even more preferably 3000 or more, or 3400 or more.
  • the range of the average molecular weight of polyethylene glycol is not particularly limited, even in terms of its upper limit, but examples include 30,000 or less, 25,000 or less, 20,000 or less, 15,000 or less, 10,000 or less, 8,000 or less, 6,000 or less, 4,000 or less, 2,000 or less, 1,000 or less, 900 or less, 700 or less, 500 or less, and 450 or less.
  • Specific ranges for the average molecular weight of polyethylene glycol can be any combination of the above upper and lower limits, and include, for example, 270 to 30,000, 270 to 25,000, 270 to 20,000, 270 to 15,000, 350 to 10,000, 350 to 8,000, 350 to 6,000, and 350 to 4,000.
  • the average molecular weight of polyethylene glycol was measured according to the test method for polyethylene glycol 200 (p. 1378) in the Quasi-drug Ingredients Standards 2006 (Yakuji Nipposha). Specifically, 42 g of phthalic anhydride was added to a 1 L light-tight glass-stoppered bottle containing 300 mL of freshly distilled pyridine, vigorously shaken to dissolve, and then allowed to stand for at least 16 hours. 25 mL of this solution was accurately measured and placed in a 200 mL pressure-resistant glass-stoppered bottle. Approximately 0.8 g of polyethylene glycol was added, tightly sealed, and placed in a water bath heated to 98°C ⁇ 2°C.
  • the bottle was removed from the water bath and allowed to cool to room temperature.
  • 50 mL of 0.5 N sodium hydroxide solution was added, and the resulting solution was titrated with 0.5 N sodium hydroxide solution.
  • Five drops of phenolphthalein-pyridine solution (1 ⁇ 100) were used as an indicator. The end point of the titration is when the solution turns pale red and lasts for 15 seconds. The obtained value is applied to the following formula to calculate the average molecular weight.
  • Average molecular weight ⁇ amount of sample (g) ⁇ 4000 ⁇ /(a-b) where a: Amount of 0.5N sodium hydroxide solution consumed in the blank test (mL) b: Amount of 0.5N sodium hydroxide solution consumed in the test of the sample (mL)
  • polyethylene glycol examples include PEG200 (average molecular weight 190-210), PEG300 (average molecular weight 285-315), PEG400 (average molecular weight 380-420), PEG1000 (average molecular weight 950-1050), PEG2000 (average molecular weight 1800-2200), PEG4000 (average molecular weight 2700-3400), PEG6000 (average molecular weight 7400-9000), and PEG20000 (average molecular weight 18000-25000).
  • PEG200 average molecular weight 190-210
  • PEG300 average molecular weight 285-315
  • PEG400 average molecular weight 380-420
  • PEG1000 average molecular weight 950-1050
  • PEG2000 average molecular weight 1800-2200
  • PEG4000 average molecular weight 2700-3400
  • PEG6000 average molecular weight 7400-9000
  • PEG20000 average molecular weight 18000-25000.
  • component (E) can be a combination of multiple polyethylene glycols with different average molecular weights.
  • the difference in average molecular weight of the multiple polyethylene glycols can be, for example, 2,000 or more, preferably 2,100 to 5,000 or 2,100 to 4,000, and more preferably 2,200 to 3,500.
  • component (E) in the liquid composition for protecting phospholipid membranes disclosed herein is not particularly limited and can be set appropriately depending on the desired level of phospholipid membrane protection effect.
  • the content of component (E) include 0.01 mg/mL or more, and from the viewpoint of further enhancing the phospholipid membrane protective effect, the content is preferably 0.1 mg/mL or more, more preferably 0.8 mg/mL or more, 1 mg/mL or more, or 1.3 mg/mL or more, and even more preferably 1.5 mg/mL or more, 3 mg/mL or more, 5 mg/mL or more, 7 mg/mL or more, 10 mg/mL or more, or 15 mg/mL or more.
  • the range of the content of component (E) in the liquid composition for protecting phospholipid membranes of the present disclosure is not particularly limited in terms of its upper limit, and can be determined appropriately by a person skilled in the art taking into consideration factors such as osmotic pressure. Examples include 200 mg/mL or less, 150 mg/mL or less, 100 mg/mL or less, 70 mg/mL or less, 50 mg/mL or less, 30 mg/mL or less, 20 mg/mL or less, 15 mg/mL or less, 10 mg/mL or less, 6 mg/mL or less, 4 mg/mL or less, or 2 mg/mL or less.
  • Amino acids include asparagine, aspartic acid, alanine, arginine, cysteine, glutamine, glutamic acid, glycine, proline, serine, histidine, lysine, threonine, etc.
  • Dipeptides are any compound in which two of the above amino acids are peptide-bonded. Salts are not particularly limited as long as they are pharmaceutically acceptable, but examples include alkali metal salts such as potassium salts and sodium salts, alkaline metal earth salts such as calcium salts, ammonium salts, triethanolamine salts, etc.
  • amino acids preferred are arginine, histidine, glycine, aspartic acid, glutamic acid, proline, and threonine; dipeptides comprising at least one of these (preferably two of these); and salts thereof. More preferred are arginine, glycine, glutamic acid, proline, dipeptides comprising at least one of these, and salts thereof. Even more preferred are arginine, arginine dipeptide (preferably arginine glutaminate consisting of arginine and glutamic acid), glutamic acid, proline, and salts thereof.
  • a more specific range of the osmotic pressure (mOsm/kg) of the liquid composition of the present disclosure is, for example, 200 to 1220 mOsm/kg, preferably 400 to 840 mOsm/kg, and more preferably 500 to 800.
  • the osmotic pressure is measured by the freezing point depression method. This osmotic pressure measurement can be performed, for example, using an OSMOMAT 3000 osmotic pressure analyzer.
  • the liquid composition of the present disclosure is used to protect phospholipid membranes.
  • the liquid composition of the present disclosure can be used to protect phospholipid membrane structures from damage that may occur to phospholipid membranes and improve their storage stability.
  • the types of damage are not particularly limited, but examples include freezing damage (particularly damage caused by repeated freezing and thawing once or 2 to 5 times, preferably 3 to 5 times, and more preferably 4 to 5 times), damage caused by contact with hydrophobic materials (polyolefins (polyethylene, polypropylene, etc.), polyvinyl chloride, polyethylene terephthalate, polycarbonate, polystyrene, etc.), vibration damage, and heat damage.
  • Phospholipid membrane structures include membrane particles containing phospholipids, such as cells (somatic cells, germ cells, stem cells, etc.), extracellular vesicles (exosomes, etc.), liposomes, and blood cells (red blood cells, white blood cells).
  • phospholipids such as cells (somatic cells, germ cells, stem cells, etc.), extracellular vesicles (exosomes, etc.), liposomes, and blood cells (red blood cells, white blood cells).
  • Preferred examples of these phospholipid membrane structures include those used as active ingredients or carriers thereof (specifically, carriers encapsulating active ingredients) in therapeutic or diagnostic pharmaceuticals.
  • the pharmaceutical composition of the present disclosure is excellent in protecting phospholipid membranes from damage caused by contact with hydrophobic materials, and therefore, in a preferred form, can be housed in a container made of a hydrophobic material.
  • Hydrophobic materials are as described above in "1-10. Uses.”
  • Specific examples of containers include common containers such as tubes, syringes (prefilled syringes), centrifuge tubes, cryovials, culture flasks, culture containers and other containers, bags (non-frozen bags, frozen bags), petri dishes, dishes, and well plates.
  • Specific examples of containers include, in addition to the common containers listed above, hollow structures such as catheter tubes.
  • the pharmaceutical composition of the present disclosure has excellent protection effects for phospholipid membranes from freezing damage, and therefore, in a preferred form, can be frozen. In this case, it may be frozen in a container made of the above-mentioned hydrophobic material.
  • Reagents used Sterile water (manufactured by Nacalai Tesque) D-PBS (Dulbecco's phosphate buffered saline: manufactured by Nacalai Tesque), hereinafter also referred to as PBS.
  • Physiological saline Otsuka saline injection: manufactured by Otsuka Pharmaceutical Co., Ltd.
  • ⁇ TE buffer pH 7.5 x 20 Promega) Sephadex G100 (Cytiva) 0.1 mol/L phosphate buffer solution, pH 7.4 (Nacalai Tesque) - Sodium carbonate, anhydrous (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) - Sodium acetate (for molecular biology) (Nacalai Tesque) 1 mol/L acetic acid (Nacalai Tesque) - Trisodium citrate dihydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Citric acid monohydrate (Nacalai Tesque) Disodium succinate hexahydrate (Nacalai Tesque)
  • Magnesium chloride hexahydrate (Nacalai Tesque) Magnesium sulfate heptahydrate (Nacalai Tesque) ⁇ Magnesium gluconate (Tokyo Chemical Industry Co., Ltd.)
  • Equipment used Electronic balance (Shimadzu Corporation, TX223N) - Electronic balance (Sartorius, secura125D) - pH meter (HORIBA, Ltd., LAQUA_D200_2) - Ultrasonic irradiation device (BRANSONIC, CPX2800H-J) ⁇ Medicinal refrigerator (Panasonic, MPR-414FRS-PJ) - Biomedical refrigerator (Panasonic, MDF-U538D-PJ) - Ultra-low temperature freezer (PHcbi, MDFDU300H-PJ) ⁇ CO2 incubator (Panasonic, MCO-5AC-PJ) - Clean bench (Panasonic, MHE-S1301 A2PJ) - Fully automated cell counter (Bio-Rad, TC-20) - Culture microscope (Olympus, CKX53) - Disposable cell counting chamber (Digital Bio, C-Chip) ⁇ Fluorescence microscope (Keyence, BZ-X
  • evaluation samples liquid compositions for protecting lipid membranes in Examples and liquid compositions in Comparative Examples
  • Evaluation samples with the compositions shown in each table were prepared.
  • buffer solutions of various concentrations were prepared, and then the pH was adjusted appropriately with a pH adjuster. After that, various other reagents weighed using an electronic balance were dissolved to prepare the evaluation samples.
  • water was used as the evaluation sample.
  • the medium for culturing or preparing cells was obtained as a 10% serum-containing RPMI medium by adding 50 mL of FBS and the recommended amount of penicillin-streptomycin mixed solution to 500 mL of RPMI medium.
  • pyranine solution 1.0 mL of pyranine solution (pyranine: 35 mM, DPX: 50 mM, MES: 25 mM, pH 7.4) was added to the thin film, which was then dispersed using an ultrasonic irradiation device. The outer aqueous layer was then replaced with PBS or physiological saline and a Sephadex G100 column to obtain a dispersion of fluorescent-encapsulated liposomes.
  • [G] Cell Preparation Cells were prepared from the spleens of Balb/c mice or C57BL/6 mice donated after euthanasia. Specifically, 5.0 mL of 10% serum-containing RPMI medium was added to the spleen removed from the specimen, and the spleen was then processed using a 100 ⁇ m Falcon cell strainer to obtain a cell suspension. The cells were then washed with PBS and hemolyzed using a hemolysis reagent. After multiple washes with PBS, the cells were cultured in 10% serum-containing RPMI medium for 2-8 hours to minimize the effects of the hemolysis treatment. The cells were then passed through a 100 ⁇ m Falcon cell strainer again, washed multiple times with PBS, and counted to obtain a spleen cell dispersion. Cell counts were performed using a Digital Bio disposable cell counting chamber (C-Chip).
  • C-Chip Digital Bio disposable cell counting chamber
  • Blood samples (blood cell samples) were prepared from Balb/c mice or C57BL/6 mice transferred after euthanasia. Specifically, Balb/c mice or C57BL/6 mice transferred after euthanasia were dissected, and the blood remaining in the heart was collected using a syringe. Immediately afterwards, the blood (whole blood) was mixed with 3.2 wt% citric acid solution at a ratio of 9:1 (vol/vol) for anticoagulation. This yielded blood samples (blood cell samples).
  • 2970 ⁇ L of PBS and 30 ⁇ L of the post-freeze-thaw sample were added to a disposable cell for fluorescence measurement, and the fluorescence intensity was measured to determine the destruction rate of fluorescent-encapsulated liposomes.
  • the fluorescent substance in the liposomes leaks out as the phospholipid membrane is destroyed, weakening the effect of the quencher and increasing the fluorescence intensity; therefore, a higher fluorescence intensity indicates a greater degree of destruction.
  • the destruction rate of fluorescent-encapsulated liposomes was calculated as a relative value (%), with 100 representing a completely destroyed state when 30 ⁇ L of 10-fold diluted Triton-X100 was added to a fluorescent-encapsulated liposome dispersion and stored.
  • Lx is the fluorescence intensity of the sample after freeze-thawing
  • L0 is the fluorescence intensity of a sample (without a history of freezing) prepared by mixing PBS or physiological saline with a fluorescent-encapsulated liposome dispersion
  • L100 is the fluorescence intensity of a sample (without a history of freezing) prepared by adding 30 ⁇ L of 10-fold diluted Triton-X100 to a fluorescent-encapsulated liposome dispersion.
  • the concentration of fluorescent-encapsulated liposomes was measured as a phospholipid concentration using a lab assay-phospholipid and adjusted to approximately 0.2 mg/mL with PBS or physiological saline.
  • the fluorescence intensity was measured using a fluorescence spectrophotometer (excitation: 416 nm, emission: 512 nm).
  • the freeze-thawing of the cell samples was performed in the same manner as for the fluorescent substance-encapsulated liposomes: the cell samples were placed in a deep freezer, thoroughly frozen, and then thawed in room-temperature water. When storing the cell samples for a specified period after freezing, they were left in the deep freezer for the specified period. Cell viability was measured for each of the cell samples subjected to each procedure.
  • Cell viability was measured by trypan blue exclusion. Specifically, for each cell sample, 0.4% trypan blue solution and the cell sample were mixed at a 1:1 (vol/vol) ratio, applied to a counting slide, and immediately counted using an automated cell counter. When DMSO was present, the counting slide was photographed under a fluorescence microscope, and cell viability was determined by visual counting. At least five photographs were used per sample.
  • the degree of liposome destruction (damage to the phospholipid membrane) can be evaluated by comparison with Reference Example 1a.
  • the surfactant Triton was added, the fluorescence intensity increased significantly from 25.5 to 899, and the liposomes were destroyed (Comparative Example 1).
  • Storage in water at 4°C for 24 hours did not result in substantial destruction (Reference Example 1b), but destruction was confirmed after 10 minutes at 90°C (Comparative Example 2).
  • liposome destruction occurred even after a single freezing cycle, and the degree of destruction increased with each repeated freeze-thaw cycle.
  • the acetate buffer, citrate buffer, or carbonate buffer (component (B)) alone was found to have a protective effect on phospholipid membranes (Reference Examples 8 to 10). Furthermore, when the magnesium salt (component (A)) was combined with the acetate buffer, citrate buffer, or carbonate buffer (component (B)), the combination was found to improve the protective effect on phospholipid membranes, particularly in the low concentration range of component (A) (Examples 1a, 1b, 2a, 2b, and 3a, 3b). This improvement was particularly significant when the component (B) was combined with acetate buffer or citrate buffer (Examples 1a, 1b, and 2a, 2b).
  • Test Example 6 the protective effect of phospholipid membranes against freezing damage was evaluated when component (A) in Test Example 6 was replaced with other components, according to "(J-1) Evaluation of Phospholipid Membrane Protection 1 - Evaluation of Protective Effect of Fluorescent Element-Encapsulating Liposomes against Freezing Damage.”
  • the evaluation samples used are shown in Table 6 below, and the membrane destruction rate is shown in Figure 6.
  • magnesium salts were found to have a protective effect on phospholipid membranes over a wide pH range from 5 to 10. In particular, they were found to have a stable and excellent protective effect on phospholipid membranes in the pH range from 5 to 8. Thus, magnesium salts are so effective at protecting phospholipid membranes that they exert a high protective effect even in basic pH ranges.
  • liquid compositions for protecting phospholipid membranes containing components (A) and (B) (Examples 1k and 1h)
  • liquid compositions for protecting phospholipid membranes (Examples 4 to 17) further containing (component (C), component (D), component (E), or component (F))
  • component (C) Example 4
  • component (D) Example 6
  • component (E) Examples 10 to 13
  • component (F) Examples 14 and 15
  • component (C) which is a polyhydric alcohol, component (D) which is a sugar or sugar alcohol, and component (E) which is polyethylene glycol showed improved protective effects on phospholipid membranes across the entire concentration range (Example 4 system, Example 6 system, Examples 11 to 13 system, Example 16 system). Furthermore, as shown in Table 9A, the level of improvement in the protective effects on phospholipid membranes was higher than when glycerin was used as component (C) (Example 4b, Example 4h, Example 4i) and when ethylene glycol was used as component (C) (Exa, Example 16b, Example 16c).
  • component (B) As shown in Table 10, the combination of component (B) and additional components (component (C), component (D), and component (E)) alone was effective in protecting phospholipid membranes (Reference Examples 17, 18, and 19), but by further combining it with component (A), an even more effective protection of phospholipid membranes was obtained (Examples 6, 11, and 12).
  • component (E) combined with components (A), (B), and (C) demonstrated improved protective effects on phospholipid membranes across the entire concentration range (Example 21 system, Example 22 system).
  • the (D) component combined with the (A), (B), (C), and (E) components all showed improved protective effects on phospholipid membranes (Example 23 system, Example 24 system, Example 25, Example 28 system). Furthermore, the level of improvement in the protective effects on phospholipid membranes was higher than when mannitol was used as the (D) component (Example 23 system) or when other sugars were used as the (D) component (Example 24 system, Example 25, Example 28 system c).
  • components (C) and (D) combined with components (A), (B), and (E) showed improved protective effects on phospholipid membranes across the entire concentration range.
  • Example 29 when components with different molecular weights were used in combination as component (E) (Example 29 system), an improved protective effect on phospholipid membranes was observed, similar to the results of Test Example 15. Furthermore, compared to when only acetate buffer was used as component (B) (Example 34 system), when acetate buffer and citrate buffer were combined as component (B) (Examples 36 and 38 systems), the level of improvement in the protective effect on phospholipid membranes due to the combination of (E) components with different molecular weights was greater.
  • liquid compositions for protecting phospholipid membranes could be prepared over a wide range of osmotic pressures.
  • exosomes in PBS were damaged over time after transfer (0 times), and the phospholipid concentration, i.e., the amount of remaining exosomes, decreased.
  • exosomes in the liquid composition for protecting phospholipid membranes received excellent protective effects even after being left to stand for 6 hours, and showed the same phospholipid concentration, i.e., the same amount of remaining exosomes, as at the initial stage (standing time 0 hours).
  • Example 40 stabilized the phospholipid membranes of exosomes and protected the exosomes from damage caused by contact with the hydrophobic material (the polypropylene that makes up the Eppendorf tube).
  • Example 48c when a citrate buffer was included (Example 48c, Example 49c), the amount of hemoglobin leakage was suppressed to a level approaching that of Reference Example 20, which did not receive thermal stimulation, demonstrating a significant effect in protecting the phospholipid membrane.

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Abstract

La présente invention a pour but de procurer un agent protecteur présentant une excellente efficacité protectrice pour une membrane phospholipidique. La composition liquide pour la protection d'une membrane phospholipidique contient (A) un sel de magnésium, (B) une solution tampon d'acétate, une solution tampon de citrate, une solution tampon de carbonate et/ou une solution tampon de succinate, et contient de préférence en outre au moins l'un des composés suivants : (C) un alcool polyhydrique, (D) un saccharide et/ou un alcool de sucre, (E) du polyéthylèneglycol, et (F) un acide aminé. La composition liquide pour la protection d'une membrane phospholipidique présente une excellente efficacité protectrice pour la membrane phospholipidique.
PCT/JP2025/008174 2024-03-26 2025-03-06 Composition liquide pour la protection d'une membrane phospholipidique Pending WO2025204688A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271825A1 (en) * 2013-03-14 2014-09-18 Zoneone Pharma, Inc. Pharmaceutical formulations of chelating agents as a metal removal treatment system
US20160220604A1 (en) * 2013-09-17 2016-08-04 Fresenius Medical Care Deutschland Gmbh Magnesium-liposome complexes
JP2018523682A (ja) * 2015-08-18 2018-08-23 アステラス インスティテュート フォー リジェネレイティブ メディシン 臨床製剤
JP2021528450A (ja) * 2018-06-27 2021-10-21 ブレス テラポイティクス ゲーエムベーハーＢｒｅａｔｈ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｇｍｂｈ 凍結乾燥形態の医薬組成物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271825A1 (en) * 2013-03-14 2014-09-18 Zoneone Pharma, Inc. Pharmaceutical formulations of chelating agents as a metal removal treatment system
US20160220604A1 (en) * 2013-09-17 2016-08-04 Fresenius Medical Care Deutschland Gmbh Magnesium-liposome complexes
JP2018523682A (ja) * 2015-08-18 2018-08-23 アステラス インスティテュート フォー リジェネレイティブ メディシン 臨床製剤
JP2021528450A (ja) * 2018-06-27 2021-10-21 ブレス テラポイティクス ゲーエムベーハーＢｒｅａｔｈ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｇｍｂｈ 凍結乾燥形態の医薬組成物

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