JP3383704B2 - Stable aqueous liposome dispersion - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an aqueous dispersion of liposomes. More specifically, the present invention relates to a liposome aqueous dispersion containing a hydroxy acid and an amino acid as a stabilizer.

[0002]

2. Description of the Related Art Liposomes are closed vesicles composed of lipid bilayers, and in recent years, drug delivery systems, diagnostic agents, artificial enzyme carriers, sensors,
Applied development is being carried out in various fields such as cosmetics. Particularly, liposomes encapsulating a drug have been actively studied for the purpose of stabilizing an unstable drug, gradually releasing the drug in a living body, and further specifically directing the drug to a lesion site.
However, the liposome itself is often unstable to heat, and even if it is prepared as a suspension, the liposome particles usually aggregate and fuse with each other, and further, the formation and coloring of a precipitate occur relatively quickly. It is known to end up. This is one of the major problems in commercializing the liposome preparation.

Several attempts have been made so far to improve the storage stability of liposomes in an aqueous dispersion state. However, most of them are mainly for inhibiting aggregation of liposomes, preventing precipitation, or preventing leakage of inclusions, and there have been few studies on appearance color change that can be most easily confirmed in practical use. . It is known that this coloring phenomenon is caused by a decomposition product generated by peroxidative decomposition of phospholipids generally used as a film-forming substance for liposomes. Then, as a means for solving this problem, a preparation in which vitamin E is added to a liposome film-forming substance has been proposed (JP-A-2-29).
5917), Vitamin E itself is very unstable to heat, light, and oxygen, and its decomposition causes a color change.

On the other hand, Japanese Patent Laid-Open No. 62-42733 discloses that a liposome prepared by using a specific hydrating agent and using dipalmitoylphosphatidylcholine, which is one of the synthetic phospholipids, as a liposome-forming substance is stored at room temperature. It is described that even after 1 year, it was stable with no color change. However, there is no description or suggestion of the stabilizing effect of the wettable powder on liposomes prepared using egg yolk lecithin or soybean lecithin. Also, liposomes using synthetic phospholipids are more stable than liposomes composed of natural phospholipids, but they are economically expensive and therefore have a difficulty in economics.

[0005]

Means for Solving the Problems The inventors of the present invention have earnestly studied for the purpose of obtaining an aqueous dispersion of liposomes having good storage stability by using natural phospholipid, which is inexpensive and highly safe, as a liposome film-forming substance. did. As a result, they have found that by adding a hydroxy acid and an amino acid to a drug-free liposome composed of natural phospholipids, it is possible to prevent the color change of the liposome when stored in an aqueous dispersion state. Furthermore, they have found that the same effect is observed in liposomes encapsulating drugs, and completed the present invention. It was also found that according to the present invention, leakage of the drug encapsulated in the liposome can be suppressed. Further, conventionally, an aqueous dispersion of liposomes was known to be most stable in the vicinity of neutral pH 6 to 7, but still it was not sufficiently stable in practical use. However, according to the present invention, it is possible to provide a liposome aqueous dispersion having practical stability not only in the vicinity of neutrality but also in a wide pH range of pH 4 to 12.

The present invention will be described in detail below. In the present invention, examples of the “liposome film-forming substance” include those containing egg yolk lecithin and / or soybean lecithin. Therefore, there is no particular limitation as long as it is a composition containing the above lecithin, hydrogenated products of these lecithin, or dimyristoylphosphatidylcholine which is a synthetic phospholipid,
The effect of the present invention can be expected even when dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dicetylphosphate, phosphatidylglycerol and the like are added to egg yolk lecithin and / or soybean lecithin. In preparing the liposome aqueous dispersion of the present invention, the amount of these liposome film-forming substances used is usually 0.0001 to 0.1 part by weight, preferably 0.001 to 0. 05 parts by weight. If desired, a film-forming auxiliary may be added to these liposome film-forming substances. Examples of the film formation auxiliary agent include sterols such as cholesterol, and aliphatic amines and fatty acids such as stearylamine and oleic acid. The amount of these film-forming aids used is not particularly limited, but usually 0.05 to 0.5 part by weight of sterols and 0 to 0 parts by weight of aliphatic amines and fatty acids are used with respect to 1 part by weight of the liposome film-forming substance. .05 to 0.2 parts by weight.

The hydroxy acid relating to the present invention is not particularly limited, but preferably at least one kind of lactic acid, tartaric acid, malic acid, citric acid or a pharmaceutically acceptable salt thereof is used. . Among them, citric acid or a pharmaceutically acceptable salt thereof is particularly preferable. The amino acid is preferably, but not limited to, glycine, alanine, leucine, serine, histidine, proline, hydroxyproline, cysteine, methionine, lysine, arginine, glutamic acid, aspartic acid or a pharmaceutically acceptable salt thereof. At least one of the possible salts is used. Among them, methionine, histidine, arginine, glutamic acid, aspartic acid or a pharmaceutically acceptable salt thereof is particularly preferable.

The requirement of the present invention resides in the combined use of at least one of the above hydroxy acids and amino acids. Therefore, several kinds of the above-mentioned hydroxy acids and several kinds of the amino acids may be mixed and used.
No limitation on the use of more than one type of mixture is intended. Furthermore, the concentration range in which the effect is obtained varies slightly depending on the types of hydroxy acid and amino acid to be combined.

For example, when sodium citrate and methionine are used, 0.05 to 5 parts by weight of sodium citrate (converted to citric acid) and methionine of 0. 008 to 0.8 parts by weight. When sodium citrate and histidine hydrochloride are used, 0.05 to 5 parts by weight of sodium citrate (converted to citric acid) and 0 of histidine hydrochloride are used with respect to 1 part by weight of the liposome membrane-forming substance. .0006 ~
0.3 parts by weight (converted to histidine). Also,
When sodium citrate and arginine are used, 0.05 to 5 parts by weight of sodium citrate (converted to citric acid) and 0.02 to 4 parts by weight of arginine are used with respect to 1 part by weight of the liposome membrane-forming substance. Is. Further, when sodium citrate and sodium glutamate or sodium aspartate are used, the liposome membrane-forming substance 1
0.05 to 5 parts by weight of sodium citrate (converted to citric acid) and 0.015 to 3 parts by weight of sodium glutamate or sodium aspartate (converted to glutamic acid or aspartic acid) relative to parts by weight.
Is.

The effect of the present invention can be obtained by using the hydroxy acid and the amino acid in combination in the above ratio with respect to the liposome membrane-forming substance. However, the amount of these stabilizers in the liposome aqueous dispersion is not less than a certain amount. If so, the operability is lowered due to the increase in the viscosity of the solution and the phase separation due to the floating of the liposome is not preferable. Furthermore, in the case of amino acids, the stabilization effect is also reduced. Therefore, the liposome aqueous dispersion 1 is usually used.
0.13 parts by weight or less of hydroxy acid to parts by weight,
The addition amount of the amino acid is limited depending on the kind, but for example, methionine is 0.02 parts by weight or less, histidine is 0.015 parts by weight or less, arginine is 0.05 parts by weight or less, and glutamic acid or aspartic acid is 0. 08
It is preferable to use less than or equal to parts by weight.

The drug held in the liposome of the present invention may be a hydrophilic drug or a lipophilic drug and is not particularly limited. For example, anticancer agents, antibiotics, chemotherapeutic agents, physiologically active substances, immunomodulators, anti-inflammatory agents, vitamins, analgesics and sedatives, diagnostic agents, prostaglandins, antiallergic agents, central nervous system agents, Examples include peripheral nervous system drugs, hormone drugs, metabolic drugs, drugs for blood and body fluids, drugs for digestive organs, ophthalmic agents, drugs for circulatory organs, drugs for respiratory organs, and the like. Examples of these are shown below, but the invention is not limited thereto. (1) Anticancer agents such as mitomycin, adriamycin, methotrexate, cisplatin, tegafur, vincristine, doxorubicin, etc. (2) Antibiotics such as amphotericin B, gentamicin, erythromycin, tetracycline, chloramphenicol, colistin methanesulfonate (3) Chemistry Therapeutic agents such as ofloxacin, norfloxacin, miconazole and the like (4) Physiologically active substances such as erythropoietin and the like (5) Immunomodulators such as interferon, interleukin, muramyl dipeptide, muramyl tripeptide, penicillamine, cyclosporine and the like (6) anti-inflammatory Agents such as fluorometholone, glycyrrhizinic acid, diclofenac, etc. (7) vitamin agents such as vitamin A, vitamin C, etc. (8) analgesic and sedative agents, eg For example, morphine, codeine, etc. (9) Diagnostic agents, such as fluorescein, etc. (10) Prostaglandins, such as prostaglandin E1, prostaglandin A1, etc. (11) Antiallergic agents, such as a) Antihistamines, such as chlorpheni Lamin, diphenhydramine, etc. b) Antiallergic agents, such as cromoglycic acid, tranilast, ketotifen, etc. (12) Central nervous system drugs, such as a) Hypnotics and anxiolytics, such as alprazolam, diazepam, etc. b) Antiepileptic drugs For example, phenobarbital c) antipyretic analgesic and anti-inflammatory agent, for example, pranoprofen, ketoprofen, etc. d) Neuropsychiatric agent, for example, amitriptyline, chlorpromazine, etc. , Lidocaine, benzocaine,
Tetracaine, dibucaine, etc. b) Anticonvulsants, such as atropine, afloqualone, etc. (14) Hormonal agents, such as a) Adrenal hormone agents, such as betamethasone, dexamethasone, hydrocortisone, triamcinolone, prednisolone, etc. b) Hormonal agents, such as insulin Etc. (15) Metabolic drugs, such as a) Liver disease agents, such as glycyrrhizic acid / DL-
Methionine etc. b) Enzymatic agents such as superoxide dismutase,
Urokinase, t-PA, etc. c) Drugs for diabetic complications, such as epalrestat, etc. (16) Drugs for blood and body fluids, such as hemostatics, such as carbazochrome, tranexamic acid, etc. (17) Drugs for digestive organs, such as anti Ulcer agents such as farnesol, geraniol, 2-N- {3- [3- (1-
Piperidinomethyl) phenoxy] propyl} amino-5
-Amino-1,3,4-thiadiazole (hereinafter abbreviated as TAS), etc. (18) Ophthalmic agent, for example, a) Glaucoma treatment agent, for example, epinephrine, timolol, carteolol, etc. b) Cataract treatment agent, for example, glutathione, Pyrenoxin, etc. c) Mydriatics, such as tropicamide, etc. d) Miotics, such as pilocarpine, carbachol, etc. (19) Cardiovascular drugs, such as a) Antihypertensives, such as reserpine, hydralazine, etc. b) Vasoconstrictors For example, phenylephrine, naphazoline, methoxamine, etc. c) Vasodilators, such as diltiazem, nifedipine, etc. d) Hyperlipidemic agents, such as clofibrate, pravastatin, etc. (20) Respiratory agents, for example a) Antitussives , For example, methylephedrine, ephedrine, etc. b) Bronchodilators, such as theophylline, or
Mention may be made of their pharmaceutically acceptable salts.

In the present invention, the "pharmaceutically acceptable salt" of hydroxy acid, amino acid or drug is, for example, a salt of sodium, potassium or the like for hydroxy acid, glutamic acid, diclofenac, betamethasone phosphate and the like. Can be illustrated. Further, for arginine, histidine, gentamicin, morphine, lidocaine, pilocarpine and the like, salts such as hydrochloric acid and sulfuric acid can be exemplified. For chlorpheniramine and timolol,
Mention may be made of the maleate salt.

Next, the method for producing the liposome aqueous dispersion of the present invention will be described. Although various known methods are available,
For example, after dissolving egg yolk lecithin and optionally a liposome film-forming substance such as cholesterol and a film-forming auxiliary in a suitable organic solvent such as chloroform, the solvent is distilled off under reduced pressure,
Prepare a thin film of lipid. The liposome aqueous dispersion of the present invention can be obtained by suspending this in water and then adding a hydroxy acid and an amino acid. The liposome aqueous dispersion of the present invention can also be obtained by suspending the lipid thin film produced as described above in an aqueous solution of hydroxy acid and amino acid. Further, in order to prepare the liposome aqueous dispersion of the present invention which holds the drug, the following may be carried out. That is, a hydrophilic drug may be dissolved in water in which a lipid thin film is suspended or an aqueous solution of a hydroxy acid and an amino acid and used in the same manner as described below. For a lipophilic drug, this may be used to form a liposome membrane. It may be used by mixing it in the organic solvent together with the substance and optionally the film-forming auxiliary. The aqueous dispersion of liposomes thus obtained, if necessary, can be repeatedly frozen and thawed to improve the encapsulation rate of the drug, or can be subjected to the extrusion method (MJ Hope, MBBally, G. et al.
Webb and P.M. R. Cullis ,: Bioc
him. Biophys. Acta 812, 55 (1
985)) and the like to control the particle size distribution.
Furthermore, unencapsulated drug can be removed by gel filtration, centrifugation, affinity chromatography and the like.

There are various possible uses of the thus obtained liposome aqueous dispersion of the present invention.
For example, moisturizers and pharmaceuticals for skin care can be processed and used as injections, eye drops, nasal drops, transdermal agents, inhalants, and oral agents. In processing the liposome aqueous dispersion of the present invention into these preparations, generally used isotonicity agents, pH adjusters, preservatives, stabilizers and the like are added within a range that does not impair the effects of the present invention. Good. Furthermore, the aqueous liposome dispersion of the present invention can be sterilized by an intermittent sterilization method. This process does not impair stability. Further, generally, as an antioxidant method for injections and the like, when the ampoule and the like are filled with the prepared liquid, ventilation replacement of nitrogen gas is performed. The liposome dispersion of the present invention is stable without nitrogen substitution, but nitrogen substitution may be used together, and in that case, higher stability can be expected.

[0015]

EXAMPLES Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.
The stability was evaluated by obtaining the degree of coloring, the relative encapsulation rate, and the drug residual rate. For the degree of appearance coloring,
Color difference meter (ND-504DE type: Nippon Denshoku Industries Co., Ltd.)
Was used to obtain and evaluate the color difference (ΔE) between the sample before storage and the sample after storage. That is, ΔE was calculated from the following formula by measuring L, a, and b of the samples before and after storage. ΔE = {(L ₁ −L ₂ ) ² + (a ₁ −a ₂ ) ² + (b ₁
-B _{²⁾ 2}} ^1/2 _{where, L 1, a 1, b} 1 before _{saving, L 2, a 2, b} 2 indicates the measurements of the sample after storage. In the display of appearance evaluation in the following examples, the above ΔE values are classified into five levels and shown by the following terms or symbols. No change: ΔE <2.5 * (slightly yellow): 2.5 ≦ ΔE <3.5 ** (light yellow): 3.5 ≦ ΔE <5.0 *** (yellow): 5.0 ≦ ΔE <8.0 *** (yellowish brown): ΔE ≧ 8.0 The relative encapsulation rate (%) and the drug residual rate (%) were calculated from the following formulas, respectively. Relative encapsulation rate (%) = (encapsulation rate after storage (%)) / (encapsulation rate before storage (%)) x 100 drug residual rate (%) = (total drug concentration after storage) / (before storage) (Total drug concentration) × 100 However, as for the total drug concentration, Triton X-100 was added to the liposome aqueous dispersion to break the liposomes, and the drug concentration of the liposome aqueous dispersion was determined. The unencapsulated drug concentration was determined by ultrafiltration of the liposome aqueous dispersion to remove the liposomes and determining the drug concentration in the filtrate. Encapsulation rate (%) = (total drug concentration-non-encapsulated drug concentration) / (total drug concentration) × 100

Example 1 Egg yolk phosphatidylcholine (PC purity: 95% or more, NOF Corporation) 0.25 g, cholesterol 0.125
20 g of a mixed solution of chloroform / methanol (10: 1)
After dissolving in 1 l, the solvent was removed by an evaporator to form a lipid thin film. After adding 5 ml of pH 7.4 phosphate buffer solution (isotonic with sodium chloride) to this lipid film and thoroughly shaking and stirring, this solution was made of a polycarbonate membrane filter (Nuclepore: Nomura Micro Science Co., Ltd.) having a pore size of 0.2 μm. (Manufactured by the company) was extruded and filtered to obtain a milky white liposome stock solution containing egg yolk phosphatidylcholine. Furthermore, soybean lecithin (PC purity: 97.3%, Nippon Seika Co., Ltd.) was used in place of egg yolk phosphatidylcholine, and a milky white liposome stock solution containing soybean lecithin was obtained in the same manner. To 1 volume of the obtained liposome stock solution, 1 volume of a solution containing various hydroxy acids and amino acids was added to obtain a liposome aqueous dispersion of the present invention. For comparison, 1 volume of phosphate buffer solution containing either hydroxy acid or amino acid, or simply phosphate buffer solution, was added to 1 volume of the above liposome stock solution.
The volume was added to obtain a comparative liposome aqueous dispersion. These were filled in a glass ampoule of 1 ml and stored at 60 ° C. to examine the appearance change. Table 1 shows the change in appearance after 1 week of storage. Coloring was observed when no stabilizer was added or when a hydroxy acid or amino acid was added alone. On the other hand, the liposome aqueous dispersion of the present invention showed almost no change in appearance as compared with the initials, indicating that the liposome aqueous dispersion of the present invention is stable.

[0017]

[Table 1]

Example 2 One volume of a solution containing various concentrations of sodium citrate and methionine, histidine hydrochloride, arginine or sodium glutamate to one volume of a liposome stock solution containing egg yolk phosphatidylcholine obtained in Example 1. Was added to obtain liposome aqueous dispersions to which various concentrations of stabilizer shown in Table 2 were added. These were stored at 60 ° C. in the same manner as in Example 1 and examined for appearance changes. Table 2 shows the change in appearance after 2 weeks of storage. It can be seen that the effect of the present invention is achieved by using 0.05 to 5 parts by weight of citric acid and the following amount of amino acid in combination with 1 part by weight of the liposome membrane-forming substance. The amount of amino acid blended is 0.008 to 0.8 parts by weight as methionine, 0.0006 to 0.3 parts by weight as histidine, 0.02 to 2 parts by weight as arginine, and 0.0 as glutamic acid.
15 to 3 parts by weight.

[0019]

[Table 2]

Example 3 Various liposome membrane-forming substances and membrane-forming auxiliary agents shown in Table 3 were dissolved in a mixture of chloroform / methanol (10: 1), and the solvent was removed by an evaporator to prepare lipids having various compositions. A thin film was formed. P on these lipid thin films
After adding 5 ml of H7.0 Britton Robinson buffer solution and thoroughly shaking and stirring, this solution was extruded and filtered through a polycarbonate membrane filter (Nuclepore: Nomura Micro Science Co., Ltd.) having a pore size of 0.2 μm to obtain various lipid compositions. A liposome stock solution was obtained. To 1 volume of the obtained liposome stock solution, 1 volume of a solution containing 4% sodium citrate and 0.4% sodium glutamate was added to obtain a liposome aqueous dispersion of the present invention. As a comparison, for 1 volume of the above-mentioned liposome stock solution,
Briton Robinson buffer was added in an amount of 1 volume each to obtain a liposome aqueous dispersion for comparison. These were stored at 60 ° C. in the same manner as in Example 1. Table 3 shows the change in appearance after 1 week of storage. Coloring was observed when the stabilizer was not included. On the other hand, the liposome aqueous dispersion of the present invention showed almost no change in appearance as compared with the initial dispersion. Therefore, it can be seen that the present invention shows a stabilizing effect in an aqueous dispersion of liposomes having various lipid compositions containing egg yolk lecithin or soybean lecithin.

[0021]

[Table 3]

Example 4 To a lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1 was added 5 ml of Briton-Robinson buffer having a pH of 3 to 12, and the mixture was shaken and stirred sufficiently. Extrusion and filtration were carried out using a polycarbonate membrane filter (Nuclepore: Nomura Micro Science Co., Ltd.) to obtain liposome stock solutions of various pHs. Solution 1 of the same pH containing 0.4% or 4% sodium citrate and 0.4% methionine per 1 volume of the obtained liposome stock solution of various pH
The volume was added to obtain a liposome aqueous dispersion of the present invention. For comparison, 1 volume of the Briton Robinson buffer solution having the same pH was added to 1 volume of the above-mentioned stock solution of liposomes having various pHs to obtain a liposome aqueous dispersion for comparison. These were stored at 60 ° C. in the same manner as in Example 1 and examined for appearance changes.
Table 4 shows the change in appearance after 1 week of storage. Although remarkable coloring was observed in the comparative example, the aqueous dispersion of liposomes of the present invention to which sodium citrate and methionine were added was stable at pH 4 to 12 as compared with the initials. Therefore, it can be seen that the present invention exhibits a stabilizing effect in the pH range of 4 to 12.

[0023]

[Table 4]

Example 5 5 ml of a pH 8.3 phosphate buffer solution (isotonic with sodium chloride) was added to the lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1, and the mixture was thoroughly shaken and stirred.
This solution was extruded through a polycarbonate membrane filter having a pore size of 0.2 μm (Nuclepore: manufactured by Nomura Micro Science Co., Ltd.) and filtered to obtain a milky white liposome stock solution. To 1 volume of the obtained liposome stock solution, 1 volume of a solution containing 0.4% sodium citrate and 0.4% methionine was added, and the liposome aqueous dispersion of the present invention containing a stabilizer in the outer aqueous phase of the liposome. A liquid was obtained. Furthermore, 10 ml of a pH 8.3 phosphate buffer solution (isotonic with sodium chloride) containing 0.2% sodium citrate and 0.2% methionine was added to the lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1. After sufficient shaking and stirring, the solution is extruded and filtered through a polycarbonate membrane filter having a pore size of 0.2 μm (Nuclepore: manufactured by Nomura Micro Science Co., Ltd.) to obtain a solution containing a stabilizer in the internal and external aqueous phases of the liposome. An aqueous liposome dispersion of the invention was obtained. As a comparison, the above-mentioned liposome stock solution 1
A 1-volume phosphate buffer solution was simply added to the volume to obtain a liposome aqueous dispersion for comparison. Further, the liposome aqueous dispersion of the present invention containing a stabilizer in the internal and external aqueous phases of the liposome is centrifuged at 100,000 × g, and then the precipitated liposomes are redispersed with a phosphate buffer to obtain an internal aqueous phase of the liposome. An aqueous dispersion of comparative liposomes containing only a stabilizer was obtained. These were stored at 60 ° C. in the same manner as in Example 1 and examined for appearance changes. Table 5 shows the change in appearance after 2 weeks of storage.
Coloring was observed when the stabilizer was not included and when the stabilizer was added only to the aqueous phase in the liposome. On the other hand, when the stabilizer is added to the internal / external aqueous phase of the liposome or only to the external aqueous phase, there is no change compared to the initial, and thus the effect of the present invention is stable in the internal / external or external aqueous phase of the liposome. It can be seen that this is achieved by adding an agent.

[0025]

[Table 5]

Example 6 5 ml of a pH 8.3 phosphate buffer solution (isotonic with sodium chloride) containing 0.4% betamethasone sodium phosphate was added to the lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1. The mixture was shaken and stirred sufficiently. This liposome aqueous dispersion was frozen using dry ice-acetone and then thawed in a water bath at 20 ° C. After repeating this freeze-thaw operation eight times, a polycarbonate membrane filter having a pore size of 0.2 μm (Nuclepore:
Extrusion filtration was carried out with Nomura Micro Science Co., Ltd.). Furthermore, this liposome aqueous dispersion is 100,000 ×
The unencapsulated drug was removed by centrifugation at g, and the precipitated liposome was redispersed with a phosphate buffer to obtain a liposome stock solution containing sodium betamethasone phosphate. A phosphate buffer solution containing 1.6% sodium citrate and 0.8% sodium aspartate or 0.4% sodium citrate and 0.2% sodium aspartate per 1 volume of the obtained liposome stock solution, respectively. 0.25 volume and 1 volume were added to obtain a liposome aqueous dispersion of the present invention having a liposome membrane-forming substance concentration of 0.04 and 0.025 parts by weight, respectively. Furthermore, after diluting the above-mentioned liposome stock solution with phosphate buffer 5 times and 50 times, 0.04% sodium citrate and 0.0
1 volume each of a phosphate buffer containing 2% sodium aspartate, 0.004% sodium citrate and 0.002% sodium aspartate was added, and the liposome membrane forming substance concentrations were 0.0025 and 0.00025 parts by weight, respectively. An aqueous dispersion of liposomes of the present invention was obtained. For comparison, samples without addition of sodium citrate and sodium aspartate were prepared by the same procedure. These were stored at 60 ° C. in the same manner as in Example 1. Table 6 shows
The change in appearance after 2 weeks of storage and the relative encapsulation rate are shown. When the stabilizer is not included, the concentration of the liposome film-forming substance is 0.0
At 025 parts by weight or more, a change in appearance was observed, and the relative encapsulation rate was also significantly reduced. In addition, the relative encapsulation rate was also markedly reduced when the liposome membrane-forming substance concentration was 0.00025 parts by weight where no change in appearance was observed. On the other hand, the liposome aqueous dispersion of the present invention showed almost no change in appearance and the relative encapsulation rate was almost 90% or more at all concentrations of the liposome film-forming substance. Therefore, it can be seen that the liposome aqueous dispersion of the present invention is stable regardless of the concentration of the liposome film-forming substance.

[0027]

[Table 6]

Example 7 To 1 volume of a stock solution of liposomes containing betamethasone sodium phosphate prepared in Example 1, 1 volume of a solution containing sodium citrate and various amino acids was added to prepare a liposome aqueous dispersion of the present invention. Obtained. For comparison, 1 volume of phosphate buffer solution containing either sodium citrate or amino acid, or simply 1 volume of phosphate buffer solution was added to 1 volume of the above liposome stock solution to obtain a liposome aqueous dispersion for comparison. . These were stored at 60 ° C. in the same manner as in Example 1. Table 7 shows the change in appearance after 1 week of storage.
Coloring was observed when no stabilizer was added or when a hydroxy acid or amino acid was added alone. In contrast, the aqueous liposome dispersion of the present invention showed almost no change in appearance as compared with the initial dispersion. Therefore, it can be seen that the aqueous liposome dispersion of the present invention is stable even when it contains a drug.

[0029]

[Table 7]

Example 8 (betamethasone sodium phosphate-encapsulated liposome) Solution 1 containing 4% sodium citrate and 0.4% sodium glutamate per 1 volume of the liposome stock solution containing betamethasone sodium phosphate prepared in Example 6 The volume was added to obtain a liposome aqueous dispersion of the present invention. For comparison, a sample in which sodium glutamate and sodium citrate were not added and nitrogen substitution was performed at the time of sealing the ampoule was prepared by the same operation. These were stored at 60 ° C. in the same manner as in Example 1 and examined for appearance changes. Table 8 shows appearance changes, relative encapsulation rates, and drug residual rates after 1 and 2 weeks of storage. The aqueous dispersion of liposomes for comparison showed a change in color over time despite the substitution with nitrogen, and a decrease in the encapsulation rate was observed accordingly. On the other hand, the aqueous dispersion of liposomes of the present invention showed no change in appearance even after 2 weeks at 60 ° C., and maintained the relative encapsulation rate and the drug residual rate at about 90%.

[0031]

[Table 8]

Example 9 (Liposome Encapsulating Morphine Hydrochloride) In Example 6, 0.5% was used instead of 5 ml of pH 8.3 phosphate buffer containing 0.4% betamethasone sodium phosphate.
Using 5 ml of a pH 6.0 phosphate buffer solution (isotonic with sodium chloride) containing morphine hydrochloride, a liposome stock solution containing morphine hydrochloride was obtained in the same manner as in Example 6 below. To 1 volume of the obtained liposome stock solution, 1 volume of a phosphate buffer containing 0.4% sodium citrate and 0.4% sodium glutamate was added to obtain a liposome aqueous dispersion of the present invention. For comparison, 1 volume of phosphate buffer containing either 0.8% sodium citrate or 0.8% sodium glutamate, or simply phosphate buffer, was added to 1 volume of the stock solution of morphine-encapsulated liposomes. In addition, a liposome aqueous dispersion for comparison was obtained. These were stored at 60 ° C. in the same manner as in Example 1. Table 9 shows changes in appearance, relative encapsulation rate, and drug residual rate after 2 weeks of storage. When the stabilizer was not contained, a change in appearance and a marked decrease in encapsulation rate and drug residual rate were observed. In addition, when sodium citrate or sodium glutamate was added alone, the appearance change and the decrease in the drug residual rate were slight, but the encapsulation rate was remarkable. On the other hand, the aqueous dispersion of liposomes of the present invention showed no change in appearance and no decrease in the drug residual rate, and the relative encapsulation rate was maintained at about 90%. From the results of Examples 8 and 9, it is understood that the aqueous dispersion of liposomes of the present invention can prevent the appearance change and further suppress the leakage of the drug.

[0033]

[Table 9]

Example 10 (Dexamethasone sodium phosphate-encapsulated liposome) In Example 6, 0.4% of the beta 8.3 phosphate buffer containing 0.4% betamethasone sodium phosphate was replaced with 0.4% of phosphate buffer.
Using 5 ml of pH 8.3 phosphate buffer containing dexamethasone sodium phosphate, a liposome stock solution containing sodium dexamethasone phosphate was obtained in the same manner as in Example 6 below.

Example 11 (Timolol Maleate Encapsulated Liposome) In Example 6, instead of 5 ml of pH 8.3 phosphate buffer containing 0.4% betamethasone sodium phosphate, pH 8.0 containing 5% timolol maleate. Phosphate buffer 5 ml
In the same manner as in Example 6 below, a stock solution of liposomes containing timolol maleate was obtained.

Example 12 (Liclofenac sodium-encapsulated liposome) A lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1 was added to a pH 8.0 phosphate buffer containing 0.2% diclofenac sodium (isotonic with sodium chloride). ) 5 ml was added and thoroughly shaken and stirred. This liposome aqueous dispersion was extruded and filtered through a polycarbonate membrane filter (Nuclepore: Nomura Micro Science Co., Ltd.) having a pore size of 0.2 μm to obtain a liposome stock solution containing diclofenac sodium.

Example 13 (Liposome encapsulating fluorometholone) 0.25 g of egg yolk phosphatidylcholine, 0.125 g of cholesterol and 1 mg of fluorometholone were added to hexane.
After dissolving in 40 ml of a mixed solution of methanol (10: 1), the solvent was removed by an evaporator to form a lipid thin film containing fluorometholone. After adding 5 ml of pH 8.0 phosphate buffer (isotonic with sodium chloride) to this lipid film and thoroughly shaking and stirring, this liposome aqueous dispersion was 0.2 μm.
Polycarbonate membrane filter with a pore size of Nucleopore (Nuclepore: Nomura Micro Science Co., Ltd.)
Extrusion filtration was carried out to obtain a liposome stock solution containing fluorometholone.

Example 14 (Sodium pranoprofen-encapsulated liposome) In Example 12, instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium, pH 7. Using 5 ml of 0 phosphate buffer, a liposome stock solution containing pranoprofen sodium was obtained in the same manner as in Example 12 below.

Example 15 (Tegafur-encapsulated liposome) In Example 12, 5 ml of pH 9.0 Britton Robinson buffer containing 4% tegafur was replaced with 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium.
In the same manner as in Example 12 below, a stock solution of liposome containing tegafur was obtained.

Example 16 (Gentamicin sulfate-encapsulated liposome) In Example 12, instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium, pH 6.0 briton robinson containing 0.3% gentamicin sulfate. Using 5 ml of the buffer solution, a liposome stock solution containing gentamicin sulfate was obtained in the same manner as in Example 12 below.

Example 17 (Chloropheniramine Maleate-Encapsulated Liposome) In Example 12, 0.5% chlorpheniramine maleate was replaced with 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium. A liposome stock solution containing chlorpheniramine maleate was obtained in the same manner as in Example 12 using 5 ml of pH 5.0 Briton Robinson buffer containing pH 5.0.

Example 18 (Liposome encapsulating sodium cromoglycate) In Example 12, pH 5.5 briton robinson containing 2% sodium cromoglycate was used instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% sodium diclofenac. Using 5 ml of the buffer solution, a liposome stock solution containing sodium cromoglycate was obtained in the same manner as in Example 12 below.

Example 19 (Urokinase-encapsulated liposome) In Example 12, 12,000 was used instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% sodium diclofenac.
Using 5 ml of pH 7.0 Britton Robinson buffer containing 0 unit / ml urokinase, a liposome stock solution containing urokinase was obtained in the same manner as in Example 12.

Example 20 (Lidocaine hydrochloride-encapsulated liposome) In Example 12, pH 6.0 Briton Robinson buffer containing 1% lidocaine hydrochloride was used in place of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium. 5
Using ml, a liposome stock solution containing lidocaine hydrochloride was obtained in the same manner as in Example 12 below.

Example 21 (Liposome encapsulating phenylephrine hydrochloride) In Example 12, pH 5.0 briton robinson containing 0.5% phenylephrine hydrochloride instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium. Using 5 ml of the buffer solution, a liposome stock solution containing phenylephrine hydrochloride was obtained in the same manner as in Example 12 below.

Example 22 (Liposome encapsulating pilocarpine hydrochloride) In Example 12, instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium, pH 5.0 briton robinson buffer containing 1% pilocarpine hydrochloride. Using 5 ml, a liposome stock solution containing pilocarpine hydrochloride was obtained in the same manner as in Example 12 below.

Example 23 (Carbochol-encapsulated liposome) In Example 12, 0.75% was used instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium.
Using 5 ml of pH 7.0 Briton-Robinson buffer containing carbachol, a stock solution of carbachol was prepared in the same manner as in Example 12.

Example 24 (TAS-encapsulated liposome) In Example 12, instead of 5 ml of pH 0.8 phosphate buffer containing 0.2% diclofenac sodium, 0.2% T was added.
PH 5.0 Briton Robinson buffer containing AS 5 ml
Using the same procedure as in Example 12, a stock liposome solution containing TAS was obtained.

Example 25 (Prostaglandin E ₁ Encapsulating Liposomes) In Example 13, 25 mg was used instead of 1 mg of fluorometholone.
A liposome solution containing prostaglandin E ₁ was obtained in the same manner as in Example 13 except that μg of prostaglandin E ₁ was replaced with pH 8.0 phosphate buffer solution and pH 5.0 briton-Robinson buffer solution. .

Example 26 (Tropicamide-encapsulated liposome) In Example 12, pH 5.0 Briton Robinson buffer containing 0.5% tropicamide was used in place of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium. Using 5 ml, a liposome stock solution containing tropicamide was obtained in the same manner as in Example 12 below.

Example 27 (Glutathione-encapsulated liposome) In Example 12, instead of 5 ml of pH 8.0 phosphate buffer containing 0.2% diclofenac sodium, pH 6.0 Briton Robinson buffer containing 2% glutathione 5 m
Using l, a liposome stock solution containing glutathione was obtained in the same manner as in Example 12 below.

Example 28 (Penicillamine-encapsulated liposome) The lipid thin film containing egg yolk phosphatidylcholine prepared in Example 1 contained 1% penicillamine at pH 5.0.
Britton Robinson buffer (5 ml) was added, and the mixture was thoroughly shaken and stirred. The aqueous dispersion of liposomes was extruded and filtered through a polycarbonate membrane filter (Nuclepore: Nomura Micro Science Co., Ltd.) having a pore size of 0.2 μm and filtered, and the unencapsulated drug was removed by centrifugation at 100,000 × g. The precipitated liposomes were redispersed in a Briton-Robinson buffer solution to obtain a liposome stock solution containing penicillamine.

1 volume of a stock solution of liposomes containing various drugs obtained in Examples 10 to 28, and 1 volume of a Briton-Robinson buffer solution containing sodium citrate and sodium glutamate, sodium aspartate or histidine hydrochloride at predetermined concentrations. Were added to obtain liposome aqueous dispersions of the present invention shown in Tables 10 to 13. For comparison, to 1 volume of the above-mentioned liposome stock solution, 1 volume of each of Briton-Robinson buffer solution containing either sodium citrate or the above amino acid or simply Briton-Robinson buffer solution was added to obtain 1 volume of the liposome dispersion for comparison. It was These were stored at 60 ° C. in the same manner as in Example 1. Table 10
13 shows the change in appearance after 1 week or 2 weeks of storage. Coloring was observed when no stabilizer was added or when sodium citrate or amino acid was added alone. On the other hand, the liposome aqueous dispersion of the present invention showed almost no change in appearance as compared with the initial dispersion. From the above results, it is understood that the liposome aqueous dispersion of the present invention is stable even if the drug contained therein is hydrophilic or lipophilic.

[0054]

[Table 10]

[0055]

[Table 11]

[0056]

[Table 12]

[0057]

[Table 13]

Example 29 (Injection) 2% sodium citrate obtained in Example 8, 0.2%
A betamethasone phosphate-encapsulated liposome containing sodium glutamate was diluted with a phosphate buffer (pH 8.3) containing 2% sodium citrate and 0.2% sodium glutamate, and a liposome aqueous dispersion containing 1 mg of betamethasone in 1 ml. Got Fill 2 ml glass ampoule with this dispersion liquid and heat sterilize at 70 ° C for 1 hour.
Injections were prepared by repeating 3 times every hour.

Example 30 (nasal drop) 2% sodium citrate obtained in Example 8 0.2%
The betamethasone phosphate-encapsulated liposome containing sodium glutamate is diluted with a phosphate buffer (pH 8.3) containing 2% sodium citrate and 0.2% sodium glutamate, and benzalkonium chloride is adjusted to 0.005%. To add 1m as betamethasone in 1ml
A liposome aqueous dispersion containing g was obtained. The dispersion is filtered using a 0.22 μm membrane filter,
A spray type nasal container was filled to obtain a nasal drop.

Example 31 (Coating agent) 2% sodium citrate obtained in Example 8, 0.2%
The betamethasone phosphate-encapsulated liposome containing sodium glutamate is diluted with a phosphate buffer (pH 8.3) containing 2% sodium citrate and 0.2% sodium glutamate, and benzalkonium chloride is adjusted to 0.005%. To add 1m as betamethasone in 1ml
A liposome aqueous dispersion containing g was obtained. The dispersion is filtered using a 0.22 μm membrane filter,
A plastic container with a sponge was filled to obtain a coating agent.

Example 32 (inhalant) 2% sodium citrate obtained in Example 8, 0.2%
The betamethasone phosphate-encapsulated liposome containing sodium glutamate is diluted with a phosphate buffer (pH 8.3) containing 2% sodium citrate and 0.2% sodium glutamate, and benzalkonium chloride is adjusted to 0.005%. To add 1m as betamethasone in 1ml
A liposome aqueous dispersion containing g was obtained. The dispersion is filtered using a 0.22 μm membrane filter,
It was filled in a nebulizer container to give an inhalant.

Example 33 (oral preparation) 2% sodium citrate obtained in Example 8 0.2%
The betamethasone phosphate-encapsulated liposome containing sodium glutamate was diluted with a phosphate buffer (pH 8.3) containing 2% sodium citrate and 0.2% sodium glutamate, and D-sorbitol was added to 10% and potassium sorbate was added. A liposome aqueous dispersion containing 0.1 mg of betamethasone in 1 ml was obtained by adding 0.1% of the mixture. This dispersion was filtered using a 0.45 μm membrane filter and filled in a glass container to give an oral preparation.

Example 34 (Injection) 0.2% sodium citrate obtained in Example 9, 0.
Phosphate buffer solution (pH 6.0) containing 0.2% sodium citrate and 0.2% sodium glutamate containing liposomes containing morphine hydrochloride containing 2% sodium glutamate.
To obtain a liposome aqueous dispersion containing 0.5 mg of morphine hydrochloride in 1 ml. 0.22μ of this dispersion
m membrane filter and then filled in a 1 ml glass ampoule to give an injection.

Example 35 (eye drops) 0.5% sodium citrate obtained in Example 11
Timolol maleate-encapsulating liposomes containing 0.5% sodium glutamate were added to 0.5% sodium citrate,
It is diluted with a phosphate buffer (pH 8.0) containing 0.5% sodium glutamate, and benzalkonium chloride is added to 0.005% to contain 2.5 mg of timolol in 1 ml. An aqueous dispersion of liposomes was obtained. This dispersion was filtered using a 0.22 μm membrane filter and filled in a 5 ml eye drop bottle to give an eye drop.

Example 36 (eye drops) 0.5% sodium citrate obtained in Example 12,
Diclofenac sodium-encapsulated liposomes containing 0.2% sodium glutamate were diluted with a phosphate buffer (pH 8.0) containing 0.5% sodium citrate and 0.2% sodium glutamate, and benzalkonium chloride was added to 0%. An aqueous dispersion of liposomes containing 1 mg as diclofenac sodium in 1 ml was obtained by adding so as to be 0.005%. This dispersion was filtered using a 0.22 μm membrane filter and filled in a 5 ml eye drop bottle to give an eye drop.

[0066]

INDUSTRIAL APPLICABILITY According to the present invention, liposomal water which is inexpensive and highly safe as a liposome membrane-forming substance, has no color change due to the use of natural phospholipids, and has excellent storage stability with little leakage of encapsulated drug. A dispersion is provided. Furthermore,
Since the effect of the present invention is achieved in a wide pH range, it is possible to select a pH suitable for the stability of the drug to be encapsulated, and therefore the liposome water having excellent storage stability even for the encapsulated drug. A dispersion is provided.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI A61K 31/27 A61K 31/27 31/35 31/35 31/41 31/41 31/415 31/415 31/435 31/435 31/44 31/44 31/485 31/485 31/505 31/505 31/557 31/557 31/57 31/57 31/70 31/70 38/46 47/16 47/16 A61P 7/02 A61P 7/02 9/02 9/02 25/04 25/04 27/02 27/02 29/00 29/00 31/04 31/04 35/00 35/00 37/08 37/08 39/02 39 / 02 43/00 113 43/00 113 A61K 37/54 (72) Inventor Takeshi Maeda 1-5-3 Nihombashi Muromachi, Chuo-ku, Tokyo Inside Wakamoto Pharmaceutical Co., Ltd. (56) Reference JP-A-62-201815 ( JP, A) JP 62-215513 (JP, A) JP 61-293910 (JP, A) JP 2-273539 (JP, A) JP 62-42733 (JP, A) JP 57-46921 (JP, A) Special Table, 4-504564 (JP, A) (58) Field (Int.Cl. ^7, DB name) A61K 9/127 A61K 47/12 A61K 47/16

Claims (5)

(57) [Claims] 1. A liposome aqueous dispersion containing egg yolk lecithin and / or soybean lecithin as a liposome membrane-forming substance, wherein the following parts by weight of citric acid or a pharmaceutically acceptable salt thereof and methionine, arginine, histidine, glutamic acid, A liposome aqueous dispersion liquid containing one or more kinds of the following amino acids in an amount of 1 part by weight or more selected from the group consisting of aspartic acid and pharmaceutically acceptable salts thereof. The amount of citric acid or a pharmaceutically acceptable salt thereof is 0.05 to about 1 part by weight of lipids forming the liposome membrane, converted to citric acid.
It is 5 parts by weight, and is 0.13 parts by weight or less in terms of citric acid with respect to 1 part by weight of the liposome aqueous dispersion. Methionine is 0 for 1 part by weight of liposome membrane-forming lipid.
008 to 0.8 parts by weight, and 0.02 parts by weight or less with respect to 1 part by weight of the liposome aqueous dispersion. Histidine or a pharmaceutically acceptable salt thereof is 0.1 in terms of histidine based on 1 part by weight of liposome membrane-forming lipid.
0006 to 0.3 parts by weight, and 0.015 parts by weight or less in terms of histidine based on 1 part by weight of the liposome aqueous dispersion. Arginine or a pharmaceutically acceptable salt thereof is 0.
02 to 4 parts by weight, and 0.05 parts by weight or less in terms of arginine per 1 part by weight of the liposome aqueous dispersion. Glutamic acid or aspartic acid or a pharmaceutically acceptable salt thereof is 0.01 in terms of glutamic acid or aspartic acid based on 1 part by weight of the lipid forming the liposome membrane.
5 to 3 parts by weight, and 0.08 in terms of glutamic acid or aspartic acid per 1 part by weight of the liposome aqueous dispersion.
It is less than or equal to parts by weight. 2. The liposome aqueous dispersion according to claim 1, wherein the liposome contains a drug. 3. The drug is an anticancer agent, an antibiotic, a chemotherapeutic agent,
Physiologically active substances, immunomodulators, anti-inflammatory agents, vitamins, analgesics and sedatives, diagnostic agents, prostaglandins, antiallergic agents, central nervous system agents, peripheral nervous system agents, hormone agents, metabolic agents Medicine, blood and body fluid medicine, digestive organ medicine,
An ophthalmic agent, a cardiovascular agent, a respiratory agent.
The liposome aqueous dispersion described. 4. The drug is tegafur, gentamicin, penicillamine, chlorpheniramine, cromoglicic acid, betamethasone phosphate, dexamethasone phosphate, diclofenac, fluorometholone, planoprofen, morphine, urokinase, lidocaine, prostaglandin E.
1,2-N- {3- [3- (1-piperidinomethyl) phenoxy] propyl} amino-5-amino-1,3,4
-The liposome aqueous dispersion according to claim 2, which is thiadiazole, timolol, glutathione, phenylephrine, tropicamide, pilocarpine, carbachol or a pharmaceutically acceptable salt thereof. 5. An injection, an eye drop, a nasal drop containing the liposome aqueous dispersion according to any one of claims 1 to 4.
Transdermal, inhalation, oral agents.