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WO2010058840A1 - Moyens pour libérer un mélange depuis un liposome et procédé pour évaluer des caractéristiques de libération - Google Patents

Moyens pour libérer un mélange depuis un liposome et procédé pour évaluer des caractéristiques de libération Download PDF

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Publication number
WO2010058840A1
WO2010058840A1 PCT/JP2009/069710 JP2009069710W WO2010058840A1 WO 2010058840 A1 WO2010058840 A1 WO 2010058840A1 JP 2009069710 W JP2009069710 W JP 2009069710W WO 2010058840 A1 WO2010058840 A1 WO 2010058840A1
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Prior art keywords
liposome
solution
drug
release
liposomes
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Japanese (ja)
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宏司 中村
敬亮 吉野
真代 近藤
恵子 山下
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Terumo Corp
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Terumo Corp
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Priority to US13/130,513 priority Critical patent/US20110223675A1/en
Priority to JP2010539260A priority patent/JPWO2010058840A1/ja
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    • 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

Definitions

  • the present invention relates to a drug release means from a liposome and a method for evaluating drug release of a liposome preparation using the release means.
  • Liposomes are closed vesicles formed by phospholipids discovered by Bangham in the 1960's, and research was initially progressing as a biological membrane model. Since then, the application of DDS has been studied taking advantage of the characteristics of this liposome, and now it is well known as one of the DDS media.
  • a hydration method (Bangham method) is generally well known. Although it is also referred to as a sonication method or an extrusion method due to some difference in operation, the basic operation is the same and it is the simplest liposome production method.
  • a liposome can be formed by preparing a phospholipid in a thin film state, adding an aqueous solvent thereto to hydrate and swell, and performing ultrasonic treatment or extrusion.
  • the drug When encapsulating a fat-soluble drug, the drug is dissolved together at the stage of preparing the phospholipid film, and the drug is incorporated into the phospholipid film.
  • a water-soluble drug when encapsulated, the drug is dissolved in an aqueous solvent that hydrates and swells, and the drug is encapsulated in an aqueous phase inside the liposome (hereinafter referred to as “inner aqueous phase”) by sonication or extrusion. To do.
  • these preparation methods are the simplest methods, but have a problem of low drug encapsulation efficiency.
  • a fat-soluble drug it is not possible to encapsulate the drug beyond the lipid mole because it is not taken into the inner aqueous phase but is incorporated into the lipid membrane.
  • a water-soluble drug it is taken into the inner aqueous phase, but can be encapsulated only in the ratio of the inner aqueous phase to the outer aqueous phase (referred to as the aqueous phase outside the liposome, the same applies hereinafter), and several tens of percent of the total drug It is the limit to enclose in the inner water phase.
  • This method is referred to as a passive loading method.
  • Patent Documents 1 and 2 As a method for solving the problem of drug encapsulation efficiency, there is a remote loading method (Patent Documents 1 and 2). According to the remote loading method, a drug can be stably introduced with high encapsulation efficiency.
  • a buffer adjusted to an appropriate pH is used for the outer aqueous phase of the liposome.
  • This outer aqueous phase uses a medium lacking ammonium ions (for example, NaCl or saccharide), and the liposome has an inner aqueous phase and an outer aqueous phase in which the liposome is not destroyed by the difference in osmotic pressure between the two. Adjusted to pressure.
  • the ammonium ion inside the liposome is in equilibrium with ammonia and protons.
  • Non-protonated ammonia can freely pass through the lipid bilayer of the liposome and migrate outside the liposome. For this reason, a phenomenon occurs in which the equilibrium is continuously shifted inside the liposome.
  • This remote loading method is a drug-limited encapsulation method that can be used for conventional drugs that can exist in a charged state when dissolved in a suitable aqueous medium.
  • the drug can permeate the liposome membrane according to the formed gradient and be encapsulated within the liposome.
  • a drug to which the introduction method can be applied can be encapsulated with an encapsulation efficiency close to 100% (Patent Documents 3 to 5, Non-Patent Document 1).
  • Liposomes encapsulating a drug by the remote loading method are stored as a formulation in a container such as a vial, the ionic gradient at the time of encapsulation is maintained, the encapsulated drug is retained in the aqueous phase in the liposome, and no release occurs.
  • release may be caused by some factor, and the release profile is known to vary depending on the drug to be encapsulated.
  • the method for evaluating the drug release characteristics of the liposome preparation is a method capable of simultaneously evaluating the drug release characteristics by the external environment and the physicochemical characteristics of the liposome membrane.
  • the drug release characteristics from liposomes can be evaluated in vivo at the laboratory level, or in vitro using biological or biological components such as serum and plasma (hereinafter referred to as “conventional method”). 3 ”).
  • a simple liposome preparation is used in which the drug retention ability of the liposome is judged by imitating the state in the liposome in a test tube and observing the state before forming the liposome.
  • a method for evaluating drug release characteristics is disclosed (Patent Document 8, hereinafter referred to as “Conventional Method 4”).
  • the liposome preparation is mainly administered parenterally, and the drug is stably held in the liposome (particularly, in the liposome preparation prepared by the remote loading method), and simply buffered. Since the drug release does not occur only by dispersing in the liquid, and the drug release is not caused by the collapse of the liposome, it is not appropriate to divert it as a drug release test method for the liposome preparation.
  • Conventional method 3 has problems such as accuracy and reproducibility due to differences in individual animals, differences in lots of biological components, storage stability of biological components, and the like when implemented at an industrial level. Not useful as a sex test method.
  • Conventional method 4 is a method that can relatively compare many drugs and is useful from the viewpoint of screening for drugs that can be encapsulated in liposomes.
  • the actual drug release characteristics of liposomal formulations cannot be measured directly. Therefore, it is not appropriate to use this method as a drug release test method for liposome preparations.
  • the present invention is intended to solve the problems of conventional drug release test methods, and uses a living body or a living body-derived substance such as a human body, a laboratory animal, cultured cells, serum or plasma.
  • a drug from a liposome that measures the drug release of a liposome preparation encapsulating the drug in an in vitro system is easy, accurate and excellent in reproducibility, and can achieve the In Vivo / In Vitro correlation (IVIVC). It is an object of the present invention to provide a release means and a method for evaluating drug release properties of a liposome preparation.
  • the present invention causes liposomes encapsulating a drug in an inner aqueous phase to be present in a solution to which a shift reagent is added, and the concentration of the drug in the solution. It was found that the drug release properties of the liposome preparation can be evaluated by measuring the above, and the present invention has been completed.
  • a deprotonation reagent or a protonation reagent is used as the shift reagent.
  • the “shift reagent” is used to form an ionic gradient opposite to the ionic gradient formed at the time of drug encapsulation between the inner aqueous phase and the outer aqueous phase of the liposome encapsulating the drug. And weaken the retention of the drug dissolved and retained in the inner aqueous phase by weakening the ionic gradient formed to retain the drug and causing a shift (shift) in the chemical equilibrium of the inner aqueous phase. It is a reagent that creates an environment in which drugs are easily released from the inside of liposomes. In short, it is thought that the action of the shift reagent is to weaken the ionic gradient formed between the inner aqueous phase and the outer aqueous phase of the liposome, which is formed to hold the drug.
  • deprotonation reagent and “protonation reagent” which are one embodiment of “shift reagent” have the following characteristics: (1) It can permeate the phospholipid membrane of the liposome without being ionized; and (2) In the aqueous phase in the liposome, the deprotonating agent acts as a Bronsted base to deprotonate the drug, and the protonating agent is Bronsted. Acts as an acid to protonate the drug.
  • shift reagent may be used as a term meaning a substance that produces the above-mentioned “deprotonation reagent” or “protonation reagent” in a solution.
  • deprotonation reagent ammonium acetate
  • protonation reagent ammonia is produced in solution as a deprotonation reagent.
  • the deprotonating reagent or protonating reagent permeates the phospholipid membrane of the liposome and moves from the outer aqueous phase to the inner aqueous phase, and acts as a Bronsted base or Bronsted acid in the inner aqueous phase,
  • the chemical equilibrium shift (shift) associated with the drug dissolved and retained is caused according to Le Chatelier's principle, and the drug retention in the inner aqueous phase is reduced.
  • the drug moves from the inner aqueous phase to the outer aqueous phase. It is considered to move to.
  • the liposome encapsulating the drug is present in a solution containing a deprotonating reagent or a protonating reagent, the encapsulated drug is released into the solution, and the release is stopped if desired. The concentration of the released drug is measured, and the drug release property of the liposome preparation is evaluated.
  • a deprotonation reagent or a protonation reagent may be included in the solution by adding a shift reagent into the outer aqueous phase solution of the liposome.
  • the drug release from the liposome internal aqueous phase to the external aqueous phase may be started by heating for a predetermined time.
  • the release of the drug from the liposome aqueous phase to the outer aqueous phase may be stopped by natural cooling by stopping heating, or by forced cooling such as ice cooling after stopping heating. Good.
  • the liposome is preferably one in which a drug is encapsulated by a remote loading method.
  • the drug is preferably an amphiphilic compound, more preferably an amphiphilic amphoteric compound, an amphiphilic weak basic compound or an amphiphilic weak acidic compound, and an amphiphilic weak base. More preferably.
  • a solution to which a shift reagent such as an external aqueous phase solution is added preferably does not contain the shift reagent before adding the shift reagent.
  • a solution to which a shift reagent such as an external aqueous phase solution is added is a deprotonation reagent that is a Bronsted base and a protonation reagent that is a conjugate acid or a Bronsted acid as one embodiment of the shift reagent. And no conjugated base thereof.
  • the solution to which a shift reagent such as an external aqueous phase solution is added preferably does not contain a biological component such as serum or plasma.
  • water, physiological saline, Ringer's solution, or buffer solution can be used as the solution to which the shift reagent is added, and a buffer solution is preferably used.
  • the pH of the buffer solution is preferably determined in consideration of the stability of the components constituting the liposome membrane, such as drugs and phospholipids encapsulated in the liposome, and is preferably about pH 5-9.
  • Liposomes encapsulating a drug (denoted as “A”) are allowed to exist in a solution containing a deprotonating reagent (denoted as “B”), and the deprotonating reagent is in a non-ionized state in a liposomal lipid. It passes through the membrane and moves from the outer aqueous phase to the inner aqueous phase.
  • the deprotonation reagent (B) accepts a hydrogen ion (proton) from a drug (HA + ) existing in a cationized state and is protonated (HB + ), while the drug (HA + ) is deprotonated.
  • the protonation reagent is deprotonated by donating hydrogen ions (protons) (A).
  • A Drug (non-ionized state); HA + : drug (cationized state); B: Deprotonation reagent (non-ionized state); HB + : deprotonation reagent (cationized state); and H + : hydrogen ion (proton).
  • the deprotonation reagent may be present when the shift reagent is present in the aqueous solvent.
  • the deprotonation reagent is ammonia (NH 3 )
  • the shift reagent only needs to generate ammonia in an aqueous solvent, may be ammonia itself, or ammonium such as ammonium acetate (CH 3 COONH 4 ). It may be a salt.
  • the deprotonation reagent is preferably ammonia or a low molecular amine having a molecular weight of 500 or less, and ammonia is particularly preferred.
  • Liposomes encapsulating a drug are present in a solution containing a protonating reagent (denoted as “HD”), and the protonated reagent is allowed to pass through the liposome lipid membrane in a non-ionized state. Permeate and move from the outer water phase to the inner water phase.
  • the protonating reagent (HD) is deprotonated by donating a hydrogen ion (proton) to a drug (C ⁇ ) existing in an anionized state (D ⁇ ), while the drug (C ⁇ ) is protonated.
  • a hydrogen ion (proton) is received from the reagent and protonated (HC).
  • the protonation reagent may be present when the shift reagent is present in the aqueous solvent.
  • the shift reagent when the protonating reagent is citric acid, the shift reagent only needs to generate citric acid in an aqueous solvent, and may be citric acid itself or a citrate such as sodium citrate. .
  • the present invention is as follows.
  • [1] A method for evaluating drug releasability of a liposome preparation, wherein a liposome encapsulating a drug is present in a solution to which a shift reagent is added, and the concentration of the drug in the solution is measured.
  • [2] The method according to [1], wherein chemical equilibrium shift is caused in the inner aqueous phase of the liposome, and the drug is released into the outer aqueous phase of the liposome.
  • [3] The method according to [1] or [2], wherein the liposome encapsulating the drug is a liposome encapsulating the drug by a remote loading method.
  • [21] A method for releasing a drug from a liposome, wherein a liposome encapsulating a drug is present in a solution to which a shift reagent is added.
  • the method according to [21] or [22], wherein the liposome encapsulating the drug is a liposome encapsulating the drug by a remote loading method.
  • the deprotonation reagent or protonation reagent is a non-ionized agent that permeates the liposome lipid membrane, moves from the outer aqueous phase to the inner aqueous phase, and is ionized to be retained in the inner aqueous phase.
  • the drug is an amphiphilic weakly basic compound.
  • the amphiphilic weak basic compound is epirubicin, daunorubicin, idarubicin, mitoxantrone, carcinomycin, N-acetyladriamycin, ruvidazone, 5-imidodaunomycin, N-acetyldaunomycin, all anthracillin drugs, Daunolin, topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, TAS103, 7- (4-methyl-piperazinomethylene) -10,11-ethylenedioxy-20 (S)- Camptothecin, 7- (2-isopropylamino) ethyl-20 (S) -campto
  • an accurate and highly reproducible test result can be obtained by directly measuring the drug release characteristics of a liposome preparation without using a living body such as a laboratory animal or a living body such as serum. And IVIVC (In Vivo / In Vitro Correlation) is achieved. And the method of this invention can confirm the release property for every lot of the manufactured liposome formulation, and can be used suitably as a quality control method of a liposome formulation.
  • FIG. 6 is a graph showing drug release from liposomes composed of phospholipids having different phase transition temperatures.
  • FIG. It is a graph which shows the release
  • a liposome means a closed vesicle formed of a phospholipid bilayer, but may also mean a liposome preparation that is a suspension comprising such a liposome.
  • the membrane structure of the liposome of the present invention is not particularly limited, and may be any of unilamellar vesicles (Unilamella vesicles), multilamellar vesicles (MLV) made of a single lipid bilayer membrane, or other structures. Further, in the case of unilamellar vesicles, any of SUV (Small Unilamellar Vesicle), LUV (Large Uniellalar Vesicle) or other structures may be used.
  • SUV Mall Unilamellar Vesicle
  • LUV Large Uniellalar Vesicle
  • the particle size of the liposome of the present invention is preferably set within a range where the EPR effect can be used. More specifically, the particle size of the liposome is preferably 200 nm or less, and more preferably 50 to 200 nm. However, this is not the case when it is not required to use the EPR effect.
  • EPR Enhanced Permeability and Retention
  • phospholipid that is the main membrane material constituting the phospholipid membrane of the liposome of the present invention phospholipids known to those skilled in the art can be used singly or in combination, but the phase transition point is the in vivo temperature.
  • the temperature is preferably higher than (35 to 37 ° C), more preferably 40 ° C or higher.
  • phosphatidylcholine lecithin
  • an amphiphilic substance having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group in the molecule an amphiphilic substance having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group in the molecule.
  • Phosphatidylglycerol phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, glycerophosphate such as phosphatidylinositol; sphingophospholipids such as sphingomyelin (SM); natural or synthetic diphosphatidylphospholipids such as cardiolipin and derivatives thereof;
  • Examples of the hydrogenated product include hydrogenated soybean phosphatidylcholine (HSPC).
  • the phospholipid is a hydrogenated phospholipid such as HSPC, SM, or the like, in which the drug encapsulated in the liposome has a phase transition temperature that does not easily leak in a living body such as blood.
  • the phospholipid membrane of the liposome of the present invention can also contain membrane components other than phospholipid as long as the liposome can be stably formed.
  • membrane components other than phospholipids for example, lipids not containing phosphoric acid (other membrane lipids), membrane stabilizers, antioxidants and the like can be contained as desired or necessary.
  • lipids include, for example, fatty acids.
  • membrane stabilizer examples include sterols such as cholesterol and saccharides such as glycerol and sucrose that reduce membrane fluidity.
  • antioxidants examples include ascorbic acid, uric acid or a tocopherol homologue, that is, vitamin E.
  • Tocopherol has four isomers, ⁇ , ⁇ , ⁇ , and ⁇ , and any of them may be used in the present invention.
  • the lipid of the liposome membrane component is the phospholipid of the main membrane material, other membrane lipids, lipids such as sterol as the membrane stabilizer, and lipids contained in the membrane modifier described later It is used in the meaning including all lipids other than drugs.
  • the phospholipid is preferably 20 to 100 mol%, more preferably 40 to 100 mol%, and the other lipids are preferably 0 to 80 mol%, more preferably Is 0 to 60 mol%.
  • membrane modifying components that can be included in the liposome preparation can be included within a range that does not impair the object of the present invention.
  • Liposome-surface modification / hydrophilic polymer The surface of the liposome of the present invention may be modified.
  • film modifying components include hydrophilic polymers and other surface modifiers.
  • the hydrophilic polymer chain can be stably distributed on the outer surface by retaining the lipid portion, which is a hydrophobic portion, in the membrane.
  • the hydrophilic polymer is not particularly limited.
  • water-soluble polysaccharides such as glucuronic acid, sialic acid, dextran, pullulan, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and derivatives thereof such as glycolipids are exemplified.
  • a particularly preferred hydrophilic polymer is polyethylene glycol (PEG). This is because it has the effect of improving the blood retention, but is not limited to this reason.
  • the molecular weight of PEG is not particularly limited, but is preferably 500 to 10,000 Da, more preferably 1,000 to 7,000 Da, and still more preferably 2,000 to 5,000 Da.
  • the lipid (hydrophobic part) of the hydrophilic polymer lipid derivative examples include phospholipid, long-chain aliphatic alcohol, sterol, polyoxypropylene alkyl, glycerin fatty acid ester, and the like. More specifically, when the hydrophilic polymer is PEG, a phospholipid derivative or cholesterol derivative of PEG is exemplified.
  • the phospholipid is preferably phosphatidylethanolamine, and the acyl chain is usually a saturated fatty acid of about C 14 -C 20 such as dipalmitoyl, distearoyl or palmitoyl stearoyl.
  • distearoyl phosphatidylethanolamine derivative (PEG-DSPE) of PEG is a readily available general-purpose compound.
  • the use timing of the membrane-modifying component is not particularly limited, but membrane modification with a hydrophilic polymer is difficult to be affected by the efficiency of distribution and the influence of the drug on the hydrophilic polymer in the inner aqueous phase. Therefore, it is preferable to selectively distribute the hydrophilic polymer from the outer surface of the liposome membrane, particularly from the outer membrane of the lipid bilayer membrane to the outer liquid side. For this reason, in this invention, after making a liposome, it is desirable to add especially after the sizing process.
  • the liposome modification rate with the hydrophilic polymer is preferably 0.1 to 10 mol%, more preferably 0.1 to 5 mol%, as a ratio of the hydrophilic polymer weight to the membrane (total lipid).
  • the drug-encapsulated liposome of the present invention is preferably produced using a remote loading method.
  • the method of the remote loading method is not particularly limited, but examples include a method using a citrate buffer or ammonium sulfate.
  • the term “remote loading method” is used in the usual sense that is known to those skilled in the art, and an empty liposome in which the drug is not encapsulated is produced, and the drug is introduced into the liposome by adding the drug to the external solution of the liposome. Means the method.
  • the drug added to the external liquid is actively transferred to the liposome and taken into the liposome.
  • a solubility gradient, an ion gradient, a pH gradient, or the like is used as the driving force.
  • a solubility gradient, an ion gradient, a pH gradient, or the like is used.
  • there is a method of introducing a drug into a liposome using an ion gradient formed across a liposome membrane for example, there is a technique in which a drug is added to liposomes formed in advance by a remote loading method with respect to a Na + / K + concentration gradient (Patent Document 3).
  • a proton concentration gradient is generally used.
  • the liposome membrane has an inner (inner aqueous phase) pH lower than the outer (outer aqueous phase) pH. It is done.
  • the pH gradient can be formed by an ammonium ion gradient and / or a concentration gradient of an organic compound having an amino group that can be protonated.
  • a method for performing remote loading by introducing ionophore into a liposome membrane has also been disclosed (US Patent Application Publication No. 2006/0193904).
  • the drug retained in the drug-encapsulated liposome of the present invention is not particularly limited as long as it is encapsulated in the liposome by the remote loading method and can be retained, but an amphiphilic compound is preferable, and an amphiphilic weakly basic compound is More preferred.
  • the acid dissociation constant pKa of the amphiphilic weak basic compound is preferably 5 to 8.
  • Amphiphilic weakly basic compounds include epirubicin, daunorubicin, idarubicin, mitoxantrone, carcinomycin, N-acetyladriamycin, ruvidazone, 5-imidodaunomycin, N-acetyldaunomycin, all anthracillin drugs, daunolin and topotecan , 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, TAS103, 7- (4-methyl-piperazinomethylene) -10,11-ethylenedioxy-20 (S) -camptothecin, 7 -(2-Isopropylamino) ethyl-20 (S) -camptothecin, CKD-602, UCN-01, propranolol, pentamidine, dibucaine, bupivacaine, tetracaine, procaine, chlorpromazi Vinblastine, vincristine
  • Amphiphilic acidic compounds are also preferred drugs, nonsteroidal anti-inflammatory drugs such as prednisolone, methylprednisolone, dexamethasone, aspirin, indomethacin, ibuprofen, felbinac, diclofenac, naproxen, mefenamic acid, phenylbutazone, etc.
  • Preferred examples include angiotensin converting enzyme (ACE) inhibitors such as anti-inflammatory drugs (NSAIDs), captopril, benazepril and enalapril.
  • ACE angiotensin converting enzyme
  • ⁇ Liposome-inner aqueous phase solution The selection of the counter ion is important for the internal aqueous phase of the liposome used to encapsulate the amphiphilic weakly basic compound in the liposome.
  • the counter ion encapsulated in the liposome together with the amphiphilic weakly basic drug is hydroxide, sulfate, phosphate, glucuronate, citrate, carbonate, bicarbonate, Includes nitrates, cyanates, acetates, benzoates, bromides, chlorides, and other inorganic or organic anions, or anionic polymers such as dextran sulfate, dextran phosphate, dextran borate, carboxymethyldextran, etc. You can choose from non-limiting examples.
  • the pH of the inner aqueous phase varies depending on the remote loading method. For example, when citric acid is used, it is necessary to previously form a pH gradient between the inner aqueous phase and the outer aqueous phase. In that case, it is preferable that ⁇ pH is 3 or more. In the case of other remote loading methods, a pH gradient is formed by chemical equilibrium, so that no special consideration is required.
  • the outer aqueous phase solution does not contain a shift reagent before mixing with the shift reagent.
  • the shift reagent does not include substances corresponding to shift reagents exemplified by deprotonating agents and their conjugate acids, protonating agents and their conjugate bases, and the like.
  • the outer aqueous phase solution before mixing with the shift reagent preferably does not contain ammonia and ammonium ions.
  • saccharides such as NaCl and / or glucose or sucrose are preferably used as solutes.
  • the pH and osmotic pressure of the outer aqueous phase solution are preferably adjusted with a buffer solution.
  • PH is preferably adjusted in the range of 5.5 to 7.5. This is due to consideration of lipid degradation and pH disparity during in vivo administration, but is not limited thereto. However, when a pH gradient inside and outside the liposome is formed using citric acid, it is desirable that the pH is around 7.0 as described above.
  • the osmotic pressure of the inner aqueous phase and the outer aqueous phase of the liposome is not particularly limited as long as it is adjusted to an osmotic pressure within a range where the liposome is not destroyed by the difference in osmotic pressure between the two, but physical stability of the liposome is not limited. Considering the characteristics, the smaller the osmotic pressure difference between the inner aqueous phase and the outer aqueous phase, the better.
  • the shift reagent as used in the present invention forms an ionic gradient opposite to the ionic gradient formed at the time of drug encapsulation between the inner aqueous phase and the outer aqueous phase of the liposome encapsulating the drug, By weakening the ionic gradient formed to hold the drug and causing shift (shift) of the chemical equilibrium of the inner aqueous phase, weakening the retention of the drug dissolved and held in the inner aqueous phase, liposomes It is a substance that is thought to create an environment in which drugs are easily released from the inside. For example, a deprotonation reagent or a protonation reagent is exemplified.
  • the deprotonation reagent or the protonation reagent permeates in a state where it does not ionize the phospholipid membrane of the liposome and passes through the outer aqueous phase.
  • the deprotonating agent acts as a Bronsted base to deprotonate the drug
  • the protonating agent acts as a Bronsted acid to protonate the drug
  • shift chemical equilibrium shift
  • the deprotonation reagent is preferably ammonia or an amino compound, particularly preferably ammonia.
  • the amino compound is particularly preferably a low molecular amino compound having a molecular weight of 500 or less. This is in consideration of the permeability of the lipid membrane, but is not necessarily limited thereto.
  • amino group structures include ammonia, primary amines, secondary amines, and tertiary amines.
  • ammonia As the deprotonation reagent, ammonia, methanolamine, ethanolamine, ethylenediamine, triethylamine and the like are preferably exemplified, but ammonia is particularly preferable.
  • the shift reagent that generates the deprotonation reagent in the buffer solution may be the deprotonation reagent itself or a salt composed of a conjugate acid and an anion of the deprotonation reagent.
  • Such anions include hydroxide ions, sulfate ions, phosphate ions, glucuronic acid ions, citrate ions, carbonate ions, bicarbonate ions, nitrate ions, cyanate ions, acetate ions, benzoate ions, bromide ions.
  • anionic polyelectrolytes exemplified by dextran sulfate, dextran phosphate, dextran borate, carboxymethyldextran, and the like, and chloride ions, and other inorganic or organic anions.
  • a salt composed of such a conjugate acid and conjugate base sulfate, phosphate, glucuronate, citrate, carbonate, bicarbonate, nitrate, cyanate, acetate
  • benzoate Preferred examples include salts of bromides, chlorides, and other inorganic or organic salts, and anionic polyelectrolytes.
  • the shift reagent that generates the protonation reagent in the buffer solution may be the protonation reagent itself or a salt composed of a conjugate base and a cation of the protonation reagent.
  • the concentration of deprotonating reagent or protonating reagent may affect the drug release rate.
  • the concentration is excessive, the release rate becomes too fast, it is difficult to control the release rate, and it is difficult to obtain reproducible data.
  • the concentration is too low, the chemical equilibrium shift is not sufficient, and the drug concentration cannot be measured.
  • the final concentration of the shift reagent in the mixed system composed of the liposome preparation and the solution added with the shift reagent is preferably 0.1 to 150 mM.
  • the specific values of the pH and osmotic pressure of the mixed system consisting of a solution containing a liposome preparation and a shift reagent are not particularly limited, but compared with the release in vivo if mimicking the biological environment conditions.
  • the pH in the mixed system is preferably in the range of 5.0 to 9.0, and the osmotic pressure is preferably in the range of 20 to 400 mOsm.
  • the pH and osmotic pressure are preferably adjusted with a buffer.
  • the mixed system of the liposome preparation and the solution to which the shift reagent is added can be heated at a predetermined temperature for a predetermined time to promote the release.
  • This temperature needs to take into account the phase transition temperature in the lipid of the liposome, and can be changed according to the lipid composition of the liposome.
  • a temperature in the vicinity of body temperature for example, in the range of 30 to 40 ° C., from the advantage that it can be compared with the release in vivo by imitating the ecological environment condition as described above.
  • the predetermined time is preferably set to a time after shifting from the initial release rate to a stable release rate, and is preferably set to a time with the least error as a test method.
  • a time that is too long is not preferable, and it is preferable to set it within a range of 1 to 180 minutes.
  • setting according to the drug, the shift reagent, and the lipid composition is required, it is not limited to this range.
  • the mixed system of the liposome preparation and the solution to which the shift reagent is added can be heated at a predetermined temperature for a predetermined time to promote the release.
  • the release can be stopped by cooling or adding stop liquid.
  • the stop solution is a solution that can stop the release from the liposome, and is characterized in that no shift reagent is added.
  • the addition of the stop solution means that the concentration of the added shift reagent is diluted, and there is a possibility that the released drug is encapsulated again in the liposome.
  • the role of the stop solution is to suppress the drug release caused by the added shift reagent. Due to such concerns, the pH of the stop solution is such that the pH gradient between the liposome inner and outer water phases is small, and the pKa of the added shift reagent is taken into account, and the permeability of the added shift reagent to the liposome membrane is significantly reduced. It is preferable that the setting is made to be able to be performed.
  • the pH is desirably in the range of 1.0 to 5.0.
  • the pH is preferably adjusted using a buffer.
  • Liposomes are known to accelerate the diffusion of drugs outside the liposome above the lipid phase transition temperature. Since the purpose of this stop solution is to stop drug diffusion, the temperature of the stop solution is preferably as low as possible. As a practical example that can be implemented, for example, ice cooling.
  • a method for separating the drug various methods known to those skilled in the art can be used, and the method is not particularly limited.
  • a method for separating a drug a dialysis membrane, gel permeation chromatography, a filtration method using a filter, a flow-through cell method which is one of an ultracentrifuge and a dissolution test apparatus and the like are preferably exemplified.
  • the drug release evaluation method of the present invention it is possible to calculate the release amount and release rate by separating the released drugs and then quantifying these drugs.
  • a known test method known to those skilled in the art may be used for the analysis and quantification method of the drug.
  • a quantification method using an ultraviolet absorptiometer and a fluorescence spectrophotometer, a high performance liquid chromatograph HPLC method, etc. are preferably exemplified. .
  • the container or instrument used in the drug release test of the present invention can use an existing dissolution tester from a microtube, and is not particularly limited.
  • the stop solution may not be added.
  • the stop solution preparation method, lipid concentration measurement method, particle size measurement method, drug quantification method, and release rate calculation method in this additional example are shown below.
  • ⁇ Stopping solution preparation method 5.84 g of sodium chloride and 15.60 g of sodium dihydrogen phosphate dihydrate were dissolved in 900 mL of water. Next, a phosphate buffer solution was added to the solution to adjust to pH 3.0, and water was further added to make the total volume 1000 mL. This was used as a stop solution (also referred to as “release stop solution” in this specification). The osmotic pressure of the stop solution prepared according to the above preparation method was 300 mOsm.
  • phospholipid concentration (unit: mg / mL) in the liposome dispersion was measured using a phospholipid quantification kit (Phospholipid C Test Wako, Wako Pure Chemical Industries, Ltd.).
  • VCR Quantification Method 100 ⁇ L of VCR-containing liposomes prepared according to the description of Preparation Examples described later were dispersed in 2 mL of methanol to obtain a sample solution. Separately, 100 ⁇ L of VCR aqueous solutions having different concentrations were taken and dispersed in 2 mL of methanol to prepare a standard curve standard solution. About these liquids, it measured by the HPLC method according to the following measurement conditions. The VCR concentration was calculated from a calibration curve formula.
  • Measurement condition column C8 column (250 ⁇ 4.6 mm, 5 ⁇ m) Measurement wavelength: 298 nm
  • Measurement condition column C8 column (250 ⁇ 4.0 mm, 5 ⁇ m) Measurement wavelength: 278 nm
  • Mobile phase 130 mL of acetonitrile was added to 870 mL of phosphate buffer at pH 3.0 and 0.02 mol / L.
  • Flow rate 1 mL / min
  • Injection volume 10 ⁇ L
  • Column temperature 40 ° C
  • Measurement condition guard column GL Kart Inertsil ODS-2 (manufactured by GL Sciences Inc.) Column: Inertsil ODS-2 (250 ⁇ 4.6 mm, 5 ⁇ m) (manufactured by GL Sciences Inc.) Measurement wavelength: 254 nm
  • HSPC hydrogenated soybean phosphatidylcholine (molecular weight: 790; manufactured by Lipoid)
  • DPPC Dipalmitoylphosphatidylcholine (Molecular weight: 734.15; manufactured by NOF Corporation)
  • DSPC Distearoyl phosphatidylcholine (molecular weight: 790.15; manufactured by NOF Corporation)
  • DMPC Dimyristoylphosphatidylcholine (molecular weight: 677.94; manufactured by NOF Corporation)
  • PEG5000-DSPE Polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (molecular weight 6081; manufactured by NOF Corporation) Chol.
  • lipid dispersion 0.71 g of HSPC and Chol. Each 0.29 g was weighed. These were mixed with 1 mL of absolute ethanol, heated and dissolved in a constant temperature layer at 70 ° C. to obtain a lipid ethanol solution. The osmotic pressure of this ethanol solution was set to 500 mOsm by adding a 250 mM citric acid aqueous solution and sucrose to this ethanol solution. Next, 9 mL of a liquid adjusted to pH 2 was added to this ethanol solution and further heated to obtain a lipid dispersion.
  • VCR-containing liposomes Table 1 shows the VCR concentration and lipid concentration of liposomes after removal of unencapsulated drug (hereinafter referred to as “LIP1”) produced according to Preparation Example 1, and the VCR concentration and particle diameter of liposomes after filtration. .
  • LIP1 unencapsulated drug
  • Example 1 Influence of shift reagent concentration on drug releasability
  • the influence of shift reagent concentration on the release of drug-loaded liposomes into the liposome external solution was examined.
  • ammonium acetate was used as a shift reagent.
  • shift reagent solution Ammonium acetate was dissolved so that the ammonium ion concentration was 5, 10, 25, 50, and 100 mM, and the pH of the solution was adjusted to 7.0 using a 0.1 M sodium hydroxide test solution. Furthermore, sodium chloride was added to this solution to adjust the solution osmotic pressure to 300 mOsm to obtain a shift reagent solution.
  • FIG. 4 shows the results of verifying the influence of the VCR release from the VCR-encapsulated liposomes on the shift reagent concentration in the shift reagent solution.
  • Example 2 Relationship between pH of Shift Reagent Solution and Release of Drug
  • the effect of pH of the shift reagent solution on the release from the liposome carrying the drug to the liposome external solution was examined.
  • ammonium acetate was used as a shift reagent.
  • shift reagent solution Ammonium acetate was dissolved so that the ammonium ion concentration was 50 mM, and the pH of the solution was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0. Were adjusted respectively. Furthermore, sodium chloride was added to this solution to adjust the solution osmotic pressure to 300 mOsm to obtain a shift reagent solution.
  • FIG. 6 shows changes in VCR release behavior when the pH of the shift reagent solution is set to 4.0, 5.0, 6.0, 7.0, or 8.0. It became clear that the release behavior of the VCR depends on the pH of the shift reagent solution, and in this example, it became clear that the release of the VCR was promoted as the pH increased.
  • Example 3 Relationship between Osmotic Pressure of Shift Reagent Solution and Drug Release Property
  • the influence of the osmotic pressure of the shift reagent solution on the release of the drug-loaded liposome into the external liposome solution was examined.
  • ammonium acetate was used as a shift reagent.
  • FIG. 7 is a graph showing the influence of the osmotic pressure of the shift reagent solution on the VCR release from the liposome.
  • the release rate of the VCR was greatly influenced by the osmotic pressure, and it became clear that the release of the VCR was promoted as the osmotic pressure of the shift reagent solution was lower.
  • lipid dispersion 0.71 g of HSPC and Chol. Each 0.29 g was weighed. These were mixed with 1 mL of absolute ethanol, heated and dissolved in a constant temperature bath at 70 ° C. to obtain a lipid ethanol solution. The osmotic pressure of the ethanol solution was adjusted to 500 mOsm by adding a 250 mM citric acid aqueous solution and sucrose to the ethanol solution. Next, 9 mL of a liquid adjusted to pH 2.5 was added to this ethanol solution and further heated to obtain a lipid dispersion.
  • Example 4 VCR release in a shift reagent solution containing an amino compound
  • a primary amine (2-aminoethanol, ethylenediamine) or secondary amine (diethylamine) is used as a shift reagent
  • ammonium acetate which is an ammonium salt
  • LIP2 was used as the liposome.
  • FIG. 8 shows the influence of the shift reagent on VCR release from liposomes. Although the release behavior differs depending on the amino compound used, it became clear that VCR can be released, and it was shown that amino compounds other than ammonium salts can be selected as shift reagents in this release test.
  • Examples 1 to 4 As shown in Examples 1 to 4, it was found that the release of the drug from the liposomes is affected by the solution properties of the shift reagent solution. That is, it is suggested that the release of the drug from the liposome varies greatly depending on the external environment. From this point of view, it was suggested that when evaluating drug release assuming in vivo, it is desirable to adjust to solution characteristics close to that of a living body.
  • aqueous PEG5000-DSPE solution (concentration: about 0.04 g / mL) was added to a total lipid amount (the sum of PC 1 and Chol.) Of 0. An amount corresponding to 75 mol% was prepared.
  • This aqueous PEG5000-DSPE solution was heated in a thermostat set at 65 ° C. in advance.
  • This aqueous PEG5000-DSPE solution was mixed with the suspension of liposomes after sizing. After mixing, the mixture was further heated for 30 minutes in a thermostatic bath set at 65 ° C. to obtain a suspension of PEG-modified liposomes.
  • Example 5 Relationship between liposome membrane composition and drug release properties
  • liposomes were examined using liposomes of LIP3 to LIP7.
  • FIG. 9 shows drug release properties from liposomes having different phospholipid and cholesterol composition ratios.
  • a clear difference was observed in the drug release between the ratio of phospholipid to cholesterol between 54:46 and 60:40. This result is presumed to be a change in drug release due to a change in membrane fluidity because it is known that the membrane fluidity changes depending on the cholesterol content. Therefore, the difference in release obtained in this example was greatly influenced by the fluidity of the liposome, and it became clear that the fluidity of the membrane can be evaluated by the method for evaluating drug release of the present invention.
  • FIG. 10 shows drug release properties from liposomes composed of phospholipids having different phase transition temperatures. It was revealed that VCR release increases in the order of DMPC>DPPC> DSPC. On the other hand, the membrane fluidity shown as the phase transition temperature of lipid is higher in the order of DMPC>DPPC> DSPC. It has been clarified that the drug release from the liposome exhibits a higher release property as the membrane fluidity is higher. Therefore, the relationship between the drug release from the liposomes obtained in this Example and the phase transition temperature was consistent with previous findings.
  • Liposome membrane Drug release from liposomes involves the structure of the liposome membrane, particularly fluidity, in pharmacokinetic studies using animals and in vitro release experiments using biological components. Increasing with increase. This is because the liposome membrane serves as a diffusion barrier when the drug is released from the liposome membrane. Therefore, in addition to the particle size of the liposome, the liposome membrane physical properties are very important in the preparation quality control. From these viewpoints, there is a need for a method for evaluating membrane physical properties in liposome preparations and a test method for drug release from liposome preparations. In this example, as a result of examining drug release properties using preparations having different phospholipid and cholesterol ratios and preparations having different phospholipid phase transition temperatures, these preparations may show different release behaviors.
  • Preparation Example 4 Production of DXR-containing liposome and VCR-containing liposome Production of DXR-containing liposomes and VCR-containing liposomes VCR-containing liposomes and DXR-containing liposomes were produced according to the following steps.
  • lipid dispersion 0.71 g of HSPC and Chol. Each 0.29 g was weighed. These were mixed with 1 mL of absolute ethanol, heated and dissolved in a constant temperature layer at 70 ° C. to obtain a lipid ethanol solution. The osmotic pressure of this ethanol solution was set to 500 mOsm by adding a 250 mM citric acid aqueous solution and sucrose to this ethanol solution. Next, 9 mL of a liquid adjusted to pH 2.5 was added to this ethanol solution and further heated to obtain a lipid dispersion.
  • DXR-containing liposome the mass ratio (DXR / HSPC) of DXR and HSPC is 0.14 (w / w) in the suspension of the liposome after the external liquid replacement. To this was added DXR aqueous solution. This was heated for 30 minutes in a constant temperature bath at 60 ° C., and DXR encapsulation was performed. After DXR encapsulation, a liposome suspension was obtained.
  • VCR-containing liposomes In the production of VCR-containing liposomes, an aqueous solution of VCR was added to the suspension of liposomes after replacement of the outer solution so that the mass ratio of VCR to HSPC (VCR / HSPC) was 0.22 (w / w). It was. This was heated for 30 minutes in a constant temperature bath at 60 ° C., and VCR encapsulation was carried out, and after suspension of VCR, a liposome suspension was obtained.
  • Example 6 Evaluation of DXR and VCR release from liposomes
  • LIP8 and LIP9 were used as liposomes.
  • FIG. 11 shows the behavior of DXR release or VCR release from liposomes.
  • DXR-containing liposomes (LIP8) released almost no drug, whereas VCR-containing liposomes (LIP9) increased the amount of drug released over time.
  • Example 7 Pharmacokinetics in blood of DXR-containing liposomes and VCR-containing liposomes
  • the pharmacokinetics in blood of DXR-containing liposomes and VCR-containing liposomes were evaluated.
  • LIP8 DXR-containing liposomes
  • LIP9 VCR-containing liposomes
  • Method A drug dose of 0.10 ⁇ mol / kg was injected from the tail vein of SD male rats. After the injection, blood was collected over time from the rat tail vein. The obtained blood was centrifuged (5000 rpm, 10 minutes) to obtain serum. After adding 200 ⁇ L of methanol to 40 ⁇ L of this serum, further centrifugation was performed (3500 rpm, 10 minutes), and the supernatant was collected to obtain a sample solution. For each sample solution, DXR quantification and VCR quantification were performed according to the methods described above.
  • the measurement wavelength was an excitation wavelength of 485 nm
  • the fluorescence wavelength was 590 nm
  • the injection amount was 100 ⁇ L.
  • the injection volume was 50 ⁇ L.
  • FIG. 12 is a diagram showing the blood pharmacokinetics of DXR and VCR.
  • Table 6 is a table showing the results of calculating pharmacokinetic parameters from the pharmacokinetics obtained in FIG. 12 according to a 1-compartment model. As shown in FIG. 12, the drug pharmacokinetics of the liposome differ depending on the drug, and as shown in Table 6, the retention of the drug is more in the VCR-containing liposome (LIP9) than in the DXR-containing liposome (LIP8). Was found to be low.
  • the elimination half-life is lower than that of the DXR-containing liposome, and the drug is released in the blood of the VCR. That is, in the DXR-containing liposome, the drug is retained in the liposome even in an in vivo environment, and for the VC-containing R liposome, the drug is released into the liposome external solution in the in vivo environment.
  • the method for evaluating drug release of the present invention it is possible not only to evaluate changes in the membrane structure of liposomes and environmental changes in the inner and outer aqueous phases, but also to release drugs from liposomes in blood in vivo. It is a system that can be evaluated without performing it, and is a method that can realize In Vivo / In Vitro Correlation that is performed in oral formulations based on this evaluation method.
  • lipid dispersion 7.06 g of HSPC and Chol. Each 2.94 g was weighed. These were mixed with 10 mL of absolute ethanol, heated and dissolved in a constant temperature bath at 70 ° C. to obtain a lipid ethanol solution. Separately from this, 90 mL of an aqueous ammonium sulfate solution having a concentration of 250 mM was prepared and preheated in a constant temperature bath at 70 ° C. This ammonium sulfate aqueous solution was added to the ethanol solution and further heated to obtain a lipid dispersion.
  • VCR / HSPC the mass ratio of VCR to HSPC
  • CFX aqueous solution was added so that it might become 04 (w / w). This was heated in a constant temperature bath at 55 ° C. for 30 minutes, and after suspension of CFX, a liposome suspension was obtained.
  • Liposome characteristics of VCR-containing liposomes and CFX-containing liposomes Table 7 shows the liposome characteristics of VCR-containing liposomes (hereinafter referred to as “LIP10”) and CFX-containing liposomes (hereinafter referred to as “LIP11”) produced according to Preparation Example 5.
  • Example 8 Release from liposomes loaded with different drugs into the external solution of liposome
  • liposomes used were LIP10 and LIP11.
  • shift reagent solution 1.9 g of ammonium acetate was weighed, 210 mL of 0.2 mol / L disodium hydrogen phosphate solution and 40 mL of 0.2 mol / L sodium dihydrogen phosphate solution were added, and water was further added to make 500 mL. A shift reagent solution at pH 6.5 was prepared.
  • FIG. 13 shows changes over time in the drug release rate of the VCR-containing liposome (LIP10) and the CFX-containing liposome (LIP11). It can be seen from FIG. 13 that even if the same membrane is used, the release properties are greatly different depending on the drugs to be enclosed. This is considered to be derived from the affinity for the membrane.
  • the drug release evaluation method using the shift reagent shown in this example is a method that can evaluate the affinity of the drug to the membrane.
  • Preparation Example 6 Production of DXR-containing liposomes having different phospholipids Production of DXR-containing liposomes DXR-containing liposomes having phospholipids of HSPC or DMPC were produced according to the following steps.
  • lipid dispersion 0.70 g of HSPC and Chol. Each 0.29 g was weighed. DMPC 0.67 g and Chol. Each 0.33 g was weighed. These were mixed with 1 mL of absolute ethanol, heated and dissolved in a constant temperature bath at 70 ° C. to obtain a lipid ethanol solution. Separately, 9 mL of an ammonium sulfate solution having a concentration of 250 mM was prepared and heated in advance in a constant temperature bath at 70 ° C. This ammonium sulfate aqueous solution was added to the ethanol solution and further heated to obtain a lipid dispersion.
  • Table 8 shows the ribosome properties of DXR-containing liposomes (LIP12 and LIP13) produced according to Preparation Example 6. DXR quantification, lipid concentration measurement, and particle size were performed by the methods described above for liposomes after filter filtration.
  • Example 9 DXR release behavior when ethylenediamine is used as a shift reagent
  • DXR release behavior when ethylenediamine is used as a shift reagent is shown.
  • LIP12 and LIP13 were used for the liposome.
  • DXR-containing liposomes (LIP12 and LIP13 prepared in Preparation Example 6) were diluted 10-fold with the shift reagent solution and heated at 37 ° C. Samples were taken out at 0, 2, 4 hours after the start of heating. Samples were stored under ice cooling until use. The released DXR was quantified by the above-mentioned “quantification method of released DXR”.
  • Example 6 It was already described in Example 6 that DXR was hardly released when the release property of DXR from DXR-containing liposomes was evaluated using the shift reagent solution shown in Example 8.
  • Example 9 250 mM ethylenediamine / phosphate buffer (pH 7.4) was used as the shift reagent solution, but it was revealed that DXR can be released as shown in FIG.
  • Example 9 the DXR release properties of liposomes prepared using different phospholipids as lipid membranes are compared. There was a clear difference in DXR release between LIP12 using HSPC as the phospholipid and LIP13 using DMPC. The inventors speculate that this difference may be based on changes in drug release properties due to differences in membrane fluidity.
  • the drug release property evaluation method using the shift reagent shown in Example 9 evaluates the release characteristics even in liposomes that are difficult to release the drug by selecting the shift reagent according to the type of drug or the liposome characteristics. Is possible.
  • the characteristic change obtained by the evaluation method shown in Example 9 is not only an evaluation of drug release from liposomes, but also an evaluation method that can capture minute physicochemical changes of liposomes that cannot be detected by existing measuring devices. It is assumed that there is.
  • the drug release test method of the present invention can be used in quality control for evaluating whether or not the drug release characteristics of a liposome preparation are within a predetermined range.

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Abstract

La présente invention concerne un moyen pour libérer un médicament depuis un liposome qui est utile pour le contrôle qualité d'une préparation de liposomes, a des simplicité, exactitude et reproductibilité satisfaisantes, et peut également atteindre une corrélation in vivo-in vitro (IVIVC), et un procédé pour évaluer des caractéristiques de libération de médicament d'une préparation de liposomes. Le moyen pour libérer un médicament depuis un liposome cause un déplacement d'équilibre chimique à l'intérieur du liposome et le procédé permet d'évaluer les caractéristiques de libération de médicament par détermination quantitative du médicament libéré à l'extérieur du liposome.
PCT/JP2009/069710 2008-11-20 2009-11-20 Moyens pour libérer un mélange depuis un liposome et procédé pour évaluer des caractéristiques de libération Ceased WO2010058840A1 (fr)

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JP2019527351A (ja) * 2016-07-06 2019-09-26 サムヤン バイオファーマシューティカルズ コーポレイションＳａｍｙａｎｇ Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｃｏｒｐｏｒａｔｉｏｎ 難水溶性薬物を含む高分子ミセル製剤のインビトロ放出試験方法及び評価方法

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CN116338071B (zh) * 2022-10-27 2025-11-25 江苏汉斯通药业有限公司 一种培化磷脂酰乙醇胺中杂质的检验方法

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