JPH04360832A - Liposome pharmaceutical - Google Patents

Abstract

PURPOSE:To obtain a liposome pharmaceutical, useful as a preventive and therapeutic agent for pneumocystis carinii pneumonia, having high therapeutic effects with hardly any side effects, capable of administering the active ingredient in a high content and having high safety. CONSTITUTION:A stable liposome pharmaceutical is obtained by preparing a medicine containing aculeacins, expressed by the formula (R1CO is long-chain saturated or unsaturated fatty acid residue or organic acid residue which may have benzene ring, pyridine, O, S or N in the molecule; R2 is H, lower alkyl or benzyl that may have branch or amino-lower alkyl in which NH2 may be substituted with mono- or dilower alkyl; R3 is H or CONH2; R4 is H or OH) and sparingly soluble in water as an active ingredient into a liposome pharmaceutical using glycyrrhizins, acyltaurines, cholic acids, sodium higher alkyl sulfates and/or polyoxyethylene sorbitan.monohigher alkylates as a liposome- forming adjuvant. The aforementioned pharmaceutical has the above-mentioned effects.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel liposome preparation of acreacins effective for the prevention and treatment of, for example, Pneumocystis carinii pneumonia.

0002

[Prior art] Pneumocystis carinii (Pneum)
ocystis carinii) is considered to be a type of protozoan, although its taxonomic position is controversial, and one genus and one species is known to date.

[0003] This substance is known to be a pathogen of pneumonia, and is commonly used in infants with low immunity due to congenital immunodeficiency or malnutrition, childhood diseases such as acute lymphocytic or myeloid leukemia, and autoimmunity in the elderly. disease, malignant tumors such as lung cancer, especially when large amounts of antitumor drugs, steroids, and immunosuppressants are used, or complications with infectious diseases such as AIDS, toxoplasma, cytomegalovirus, actinomycetes, and fungi. This can lead to Pneumocystis carinii pneumonia, which often leads to death from respiratory failure.

[0004]Currently, drugs that have been reported to be effective against Pneumocystis carinii pneumonia include a combination drug (ST combination drug) of sulfamethoxazole and trimethoprim, which are antibacterial agents, and pentamidine, which is an antiprotozoal drug. However, sulfa drugs are highly toxic to AIDS patients, and pentamidine itself is highly toxic, which limits their use and, accordingly, limits their therapeutic effects.

Under these circumstances, there are few side effects,
As a drug with more effective therapeutic effect, the following general formula (1) such as antibiotic acreasin Aα, Aγ, Dα, Dγ, etc.

[
0006

[Case 2]

(In the formula, R1 -CO- represents a long-chain saturated or unsaturated fatty acid residue or an organic acid residue which may contain a benzene ring, a pyridine ring, an oxygen atom, a sulfur atom or a nitrogen atom in the molecule) R2 represents a hydrogen atom, a lower alkyl group that may have a branched chain, a benzyl group, or an amino-lower alkyl group in which the amino group may be substituted with a mono-lower alkyl group or a di-lower alkyl group, and R3 is A drug containing an acreacin represented by the following formula (representing a hydrogen atom or a -CONH2 group, and R4 representing a hydrogen atom or a hydroxyl group) as an active ingredient has been proposed.

[0008] However, the above acreacins are extremely poorly soluble in water, and are also not soluble in chloroform or ether. It is also soluble in hydrophilic organic solvents, such as alcohols such as methanol, ethanol, isopropanol, and n-butanol, but these are unsuitable solvents for injectable preparations and cannot be used as solubilizers. Only. Therefore, it has been impossible to formulate the above-mentioned substance by conventional means.

[0009] On the other hand, ultra-microencapsulation of drugs by forming liposomes is generally carried out as a means of formulation, but when liposomes are produced only from phospholipids such as lecithin, it is not possible to form liposomes.
Otherwise, it is extremely difficult to form, and synthetic surfactants are usually used. However, liposomes formed using synthetic surfactants have many problems such as safety, skin irritation, and biodegradability, and are problematic for use in applications related to living organisms.

0010

SUMMARY OF THE INVENTION The present invention has been made in response to the above-mentioned problems, and provides a method for preventing and treating Pneumocystis carinii pneumonia containing acreacins represented by the general formula (1) as an active ingredient. The object of the present invention is to provide a liposome preparation that is highly safe and easy to use, and can be administered in a high content of the acreacins.

[0011]

[Means and effects for solving the problem] That is, the present invention solves the problem by the general formula (1)

0012

[Chemical formula 3]

(In the formula, R1 -CO- represents a long-chain saturated or unsaturated fatty acid residue or an organic acid residue which may contain a benzene ring, a pyridine ring, an oxygen atom, a sulfur atom or a nitrogen atom in the molecule) R2 represents a hydrogen atom, a lower alkyl group that may have a branched chain, a benzyl group, or an amino-lower alkyl group in which the amino group may be substituted with a mono-lower alkyl group or a di-lower alkyl group, and R3 is A preparation containing as an active ingredient at least one selected from the group consisting of acreacins represented by a hydrogen atom or a -CONH2 group, and R4 represents a hydrogen atom or a hydroxyl group, glycyrrhizins, glycyrrhizins, and the like as liposome formation aids. The present invention relates to a liposome formulation characterized in that it is formulated using one or more of acyl taurines, cholic acids, sodium higher alkyl sulfate, and polyoxyethylene sorbitan monohigher alkylate.

In the present invention, by forming the above-mentioned active ingredient, which is extremely poorly soluble in water, into liposomes together with a specific liposome-forming aid, the amount of acreasin, which is an active ingredient, per 1 part of the phospholipid used, such as lecithin, is reduced to 0.0%.
001 to 2 parts, and has been completed to contain the active ingredient at a particularly high concentration, furthermore, it becomes possible to effectively administer the above-mentioned active ingredient into the living body.

[0015] In the present invention, liposomes are microparticles that form a lipid bilayer membrane in water, mainly composed of phospholipids such as lecithin, and usually have a diameter of 10 μm or less. The phospholipid that is the main component forming the liposome in the present invention may be either a naturally occurring phospholipid or a synthetic phospholipid, such as egg yolk lecithin, soybean lecithin and their hydrogenated products, and dimyristoylphos. Fatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilinoleylphosphatidylcholine (DRPC), dioleylphosphatidylcholine (DOPC), etc. or a mixture of these phospholipids. Also,
Other phospholipids or additives include sphingomyelin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylinositol, cardiolipin, various fatty acids, fatty alcohols, etc. , antioxidants such as α-tocopherol and its esters,
Various cationic, anionic, and nonionic surfactants, parabens, phenol, preservatives such as benzalkonium chloride and benzethonium chloride, salts such as sodium chloride and potassium chloride, and sugars such as glucose as necessary. Alternatively, an isotonizing agent such as polyhydric alcohol such as polyethylene glycol or glycerin may be added as long as it does not pose a safety problem. Further, it is extremely preferable to further add cholesterol to the above-mentioned phospholipid in terms of stability of the liposome.

[0016] The above liposomes can be formed by using the following formation aids, and can be made into safe pharmaceutical preparations. That is, as the liposome formation aid, one or more of glycyrrhizins, acyltaurines, cholic acids, sodium higher alkyl sulfate, and polyoxyethylene sorbitan monohigher alkylate is used.

Among these, glycyrrhizin has the formula:

[
0018

[C4]

[0019] It is a general term for compounds that share a common basic skeleton represented by (wherein R11 to R15 are hydrogen atoms or appropriate substituents), and even if it is a single compound or a mixture of these compounds. Often, specifically 18α-glycyrrhizic acid (R11 in the above formula is glucuronyl-
Glucuronic acid residue, R12 is COOH group, R13 is CH
3 group, R14 is =O group, R15 is CH3 group), 18β-glycyrrhizinic acid (same as above), 18α
-Glycyrrhetinic acid (R11; OH group, R12; COO
H group, R13; CH3 group, R14; =O group, R15;
CH3 group), 18β-glycyrrhetinic acid (same as above), 3
β-glucuronyl-18β-glycyrrhetinic acid (R11
; Glucuronic acid residue, R12; COOH group, R13; C
H3 group, R14;=O group, R15; CH3 group), carbenoxolone (R11; COOCH2 CH2 CO
OH group, R12; COOH group, R13; CH3 group, R
14;=O group, R15; CH3 group), deoxoglycyrrhetinic acid (R11; OH group, R12; COOH group, R
13; CH3 group, R14; hydrogen atom, R15; CH3
group), 3α-dehydroxyglycyrrhetinic acid (R11
;=O group, R12; COOH group, R13; CH3 group,
R14;=O group, R15; CH3 group), hederagenin (R11; OH group, R12; CH3 group, R13; CO
OH group, R14; hydrogen atom, R15; CH3 group), 1
1-oxohederagenin (R11; OH group, R12; C
H3 group, R13; COOH group, R14;=O group, R1
5; CH2 OH group), oleanolic acid (R11; OH
group, R12; CH3 group, R13; COOH group, R14
; hydrogen atom, R15; CH3 group), 11-oxooleanolic acid (R11; OH group, R12; CH3 group, R
13; COOH group, R14; =O group, R15; CH3
Examples of the non-toxic salts include potassium salts, sodium salts, ammonium salts, hemisuccinates, and the like. Depending on the number of carboxylic acid groups of glycyrrhizin, it can be made into mono-, di-, tri-salts, etc., and usually 18β-glycyrrhizinic acid, dipotassium salt, monoammonium salt, diammonium salt, disodium salt, etc. Trisodium salt, 18β-glycyrrhetinic acid, carbenoxolone disodium, etc. are preferably used.

As the acyl taurine, acyl taurine salts or acyl methyl taurine salts are used, and their salts with alkali metals such as sodium and potassium may also be used, and those with an alkyl group of 10 to 25 carbon atoms, More preferably, the number is 12 to 20, and it may be saturated or unsaturated.

[0021] As cholic acids, deoxycholic acid,
Preferred are cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid and their alkali metal salts such as sodium and potassium.

Further, the active ingredient of the present invention has the above general formula (1
) These are compounds known as antibiotic acreacins (Japanese Patent Publication No. 59-2035).
Publications No. 0 to 3, Tetrahedron Lette
rs, 4147-4150 (1976), Helv.
Chim. Acta. , 62 (4) 1252-12
67 (1979), Japanese Patent Application No. 2-34470, Japanese Patent Application No. 2-216593).

In the above general formula showing the active ingredient, examples of the group R1 include, for example,

[0024]

[C5]

[0025]

[C6]

[0026]

[C7]

[0027]

[Chemical formula 8]

[0028]

[Chemical formula 9]

[0029]

[Chemical formula 10]

[0030]

[Chemical formula 11]

[0031] etc.

Examples of the group R2 include hydrogen atom, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, hexyl, 3-methylbutyl, 2-ethylbutyl,
Straight chain or branched carbon number 1-6 such as 1-ethylbutyl
lower alkyl group, benzyl group, 2-aminoethyl,
Amino-lower alkyl groups such as 3-aminopropyl, 4-aminobutyl, 2-aminopropyl, and 2-aminobutyl; mono-lower alkyl groups in which the amino group is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, etc. Alternatively, amino-lower alkyl groups such as 2-aminoethyl and 3-aminopropyl substituted with a di-lower alkyl group can be mentioned.

The group R3 is a hydrogen atom or -CO
Examples of the group R4 include a hydrogen atom or a hydroxyl group.

In the acreacins represented by the above general formula (1), R1 -CO- is a long chain saturated or unsaturated fatty acid residue, for example, a carbon number of 14 to 18 (C14 to C18).
), R2 is a hydrogen atom, R3 is a hydrogen atom,
Preferred are acreasin derivatives in which R4 is a hydrogen atom or a hydroxyl group;
1 -CO- is a myristic acid residue (C14), R4 is a hydroxyl group, acreacin Aα, R1 -CO- is a palmitic acid residue (C16), R4 is a hydroxyl group, acreacin Aγ, R1 -CO- is myristic acreasin Dα, R in which the acid residue, R4, is a hydrogen atom;
A good example is acreasin Dγ in which 1 -CO- is a palmitic acid residue and R4 is a hydrogen atom, and R1 -CO- is a stearic acid residue (C18), R4
Echinocandin C, R1 in which is represented by a hydrogen atom
-CO- is a linoleic acid residue (C18, 2 double bonds),
Examples include echinocandin B in which R4 is a hydroxyl group.

[0035] Furthermore, the composition of the liposome preparation of the present invention is such that cholesterol is 0.01 part for 1 part of phospholipid.
~1 part, forming aid 0.002-2 parts, preferably 0
．． 0.01 to 1 part of the active ingredient, preferably 0.001 to 2 parts, preferably 0.002 to 2 parts, particularly preferably 0.01 to 1 part.
Liposomes may be formed based on this composition.

[0036] Further, the production of liposomes will be exemplified. By specifically using the above-mentioned glycyrrhizin when forming liposomes, there is an advantage that liposomes can be formed efficiently. That is, the liposome formation aid of the present invention may be present in the above-mentioned phospholipids and other additives added as necessary, and liposomes may be produced by a conventional method. When producing liposome preparations, if glycyrrhizin, which is a liposome formation aid, is added or present in the presence of acreacins at the time of liposome formation, or at least before that, liposome preparations can be efficiently formed, and acreacins is efficiently encapsulated in liposomes.

[0037] As a method for producing liposome preparations, for example, in the preparation of reverse phase evaporation vesicle (REV) liposomes, phospholipids and cholesterol added as necessary are first mixed with an organic solvent such as diethyl ether, isopropyl ether, etc. After dissolving in chloroform or a mixed solvent of two or more of these, the liposome formation aids such as the aforementioned glycyrrhizins and the like are added to a solution in which, for example, various buffer solutions or various salts and isotonic agents such as glucose are appropriately added. An alcoholic or aqueous solution containing a drug-liposome formation aid to which acreacins has been added is added to prepare a W/O emulsion by a conventional method. By adding a liposome formation aid, emulsification is facilitated and a uniform and fine emulsion can be obtained. The preparation of liposomes from this W/O emulsion is described in the Proceedings of the National Academy of Sciences.
of the United States of America (
Proc. Natl. Acad. Sci. US
A) 75,4194 (1978) or Unexamined Japanese Patent Publication No. 1987-
It can be carried out according to the method described in No. 118415. The organic solvent used in emulsion preparation should be
Generally, about 0.1 to 10 times the amount is used. The amount of phospholipid used is about 5 to 200 μmol per 1 ml of the filling liquid, and it is generally preferable to dissolve it in an organic solvent in advance. The concentration of the alcoholic or aqueous solution containing acreacins and liposome formation aids can be selected as appropriate, but for example, the concentration of glycyrrhizins is 5 to 50
An example is about mM.

[0038] The emulsification method for obtaining a W/O type emulsion is as follows:
Conventional methods such as a stirring method, a pressure method, and an ultrasonic method can be employed, and the ultrasonic method is particularly preferred. In the case of the ultrasonic method, a uniform emulsion can be obtained by performing one or more treatments for several seconds to 20 minutes using a probe-type homogenizer at about 20 kHz.

The solvent is removed from the W/O emulsion thus obtained by a conventional method. This can be done by distilling off the solvent under reduced pressure using, for example, a rotary evaporator. The temperature at this time can be selected as appropriate, but if stability is not preferred, it is preferably 40 to 50°C or less. When the solvent is further removed by distillation, R
An EV liposome formulation is obtained. The present liposome preparation has a unilamellar or oligolamellar form (usually composed of about 10 or less lipid bilayer membranes), and the drug is encapsulated therein.

On the other hand, the phospholipid prepared by the same method as above and an organic solvent such as cholesterol added as necessary are distilled off under reduced pressure to form a dry film of phospholipid, and then acreasin - Adding an alcoholic or aqueous liquid containing a liposome-forming aid;
If dispersed at an appropriate temperature, a liposome preparation of multilamellar vesicles can be obtained, and during this dispersion, ultrasonication is performed using a probe-type homogenizer, or the previously obtained liposome preparation of multilamellar vesicles is treated with a probe-type homogenizer. By sonication, a liposome formulation of small unilamellar vesicles is obtained.

[0041] Furthermore, in the method for forming a dry film from phospholipids described above, a liposome formation aid may be added in advance to form a dry film.
A liposome preparation is obtained by dispersing an alcoholic or aqueous liquid containing acreacins into the thus obtained dry film containing the liposome-forming aid and subjecting it to ultrasonication.

Furthermore, the production of liposomes in the present invention is not particularly limited, and may be produced by any conventional method, for example, the following methods may be used. Multilamellar vesicles (1) Dry film method: Multilamellar vesicles (
MLV), a single or mixed solvent containing phospholipids is evaporated to dryness using an evaporator to form a dry film, and an aqueous drug solution is added to swell the film for 8 to 24 hours. (2) Freeze-saw method: This is a method in which MLV is subjected to freeze-thaw cycles to increase the rate of drug uptake into liposomes. (3) Freeze-drying method: If some peptides or proteins are denatured, decomposed, or associated with repeated freeze-saw methods, freeze-dry them only once or add an anti-ice agent (cryoprotectant). It is preferable to do so. (4) Reverse-phase evaporation method: Adding water to an ether solution of phospholipid and treating it with ultrasound produces a w/o emulsion. After distilling off the ether, shaking with a vortex mixer reverses the phase, and further under reduced pressure. Reverse phase Evaporation Vesicle (REV) when you remove Aether
Can be done. (5) Ca fusion method: When Ca ions are added to acidic phospholipid small lamellar vesicles (SUVs), membrane fusion occurs. When EDTA is added to this to remove excess Ca, large unilamellar vesicles are generated. (6) Cholic acid method: When MLV or SLV is mixed with a surfactant such as cholic acid or deoxycholic acid and then removed, LUV is produced. Small unilamellar vesicles (7) Ultrasonication method: When a suspension of MLVs is irradiated with ultrasonic waves of higher power, it gradually becomes SLVs. (8) Ethanol injection method: SUVs are generated when lipids are dissolved in ethanol and injected with a micro-microsyringe into a buffer solution above the phase transition temperature. (9) French press method: When MLV is placed in a French press cell and the extrusion operation is repeated at 20,000 psi, S
UV can be used.

The obtained liposomes can be made uniform in particle size by dialysis or filtration using a polycarbonate membrane. Mechanically, an extruder (NOF Liposome) may be used. In addition, centrifugation, dialysis, gel filtration, and the like are used to separate drugs trapped in liposomes from drugs that are not trapped.

The average particle size of the liposomes obtained in the present invention is 0.01 μm to 10 μm as measured by laser scattered light.
m, preferably 0.02 μm to 2 μm, more preferably 0.05 μm to 0.5 μm, and containing acreasin at a high concentration of up to about 2 parts per 1 part of phospholipid. can be obtained,
This provided an effective drug. Further, the preferable salt concentration of the liposome suspension obtained in the present invention is 0.01 to
50mM, more preferably 0.1-25mM. Further, the tonicity of the solution is preferably about 1/2 isotonic to 2 times hypertonic.

The liposome preparation of the present invention may be injected intravenously, intramuscularly, subcutaneously, intradermally, etc., or may be administered orally.
It may be applied to the mucous membranes of the oral cavity, nose, lungs, rectum, vagina, etc., or it may be administered transdermally. In addition, the preferred pH range of the liposome of the present invention is 3 to 10, more preferably 5 to 8, and furthermore, in formulation, glucose, sucrose, lactose, trehalose, mannitol, sorbitol, dextrin (e.g. T-40, T-70)
Freeze-dried additives such as and cyclodextrin, parabens, phenol, preservatives such as benzalkonium chloride and benzethonium chloride, salts such as sodium chloride and potassium chloride, sugars such as glucose and sucrose, propylene glycol and glycerin, etc. Isotonic agents such as polyhydric alcohols may also be used.

[0046]

【Example】

Example 1 Dipalmitoylphosphatidylcholine (DPPC) 0
．． 60g, cholesterol 0.06g, dipotassium glycyrrhizinate 0.06g and acreasin Aγ2
5 mg was dissolved in 60 ml of methanol, and the solvent was distilled off under reduced pressure using an aspirator. The solvent was further removed from this product using an oil rotary vacuum pump at a vacuum level of 0.1 Torr or less for several hours. Next, a phosphate buffer solution (
A liposome suspension was obtained by adding 15 ml of 5mM, pH 7.2) and stirring with shaking. Next, in a stainless steel container filled with ice, the mixture was heated at approximately 20 kHz using an ultrasonic homogenizer (manufactured by Nippon Seiki Seisakusho, model U-300).
z for 20 seconds and 10 seconds off for 5 to 10 times to produce acreasin Aγ-containing liposomes. Further, this solution was centrifuged at approximately 100,000 G or more, the supernatant liquid was discarded, and the resulting pellet was resuspended in 10 ml of chilled phosphate buffer containing 5% glucose (pH 7.2) and passed through a polycarbonate membrane. After sterilizing, add 2ml to a 5ml vial that has been sterilized in an autoclave.
A new liposome preparation was obtained by dispensing the solution and freeze-drying it. This product was stable even after 3 months at 4°C.

Example 2 Dimyristoylphosphatidylcholine (DMPC) 1
．． 20g and 50ml of cholesterol 0.12g
The lipid layer was dissolved in methanol, the solvent was distilled off under reduced pressure using an aspirator, and the mixture was further vacuumed at 0.1 Torr or less using an oil rotary vacuum pump for several hours to remove the solvent, thereby preparing a lipid layer film. Next, 25 mg of acreasin Dγ was added to 25 ml of an aqueous solution containing 0.24 g of glycyrrhizin ammonium, and the mixture was added to the lipid layer and dispersed by shaking. While cooling this liquid in a stainless steel container containing ice water, it was dispersed using an ultrasonic homogenizer to prepare liposomes containing acreasin. Furthermore, this liquid was heated at 100,000 G or more for 1
Centrifuge at below 0°C, remove the supernatant, redisperse the resulting pellet in chilled phosphate buffer containing 5% glucose, apply it to an extruder at room temperature, and incubate with nitrogen gas at a pressure of approximately 10 kg/cm2. After pressurizing and sizing with
The mixture was dispensed into vials in 2 ml portions and lyophilized to obtain a stable liposome preparation.

Example 3 Table 1 shows the effect of the encapsulation rate in the liposome preparation of the present invention. That is, DPPC 200mg,
Cholesterol 20mg, main drug acreasin Aγ
5 mg was dissolved in 20 ml of methanol together with the liposome formation aid shown in the table, and the solvent was dried under reduced pressure using an aspirator to prepare a dry film. As formation aids, ammonium glycyrrhizinate (GlyA), dipotassium glycyrrhizinate (GlyK2), stearylamine (
SA), stearoylmethyltaurine (SMT) was used (Formulations 1 to 5). In addition, liposomes were prepared by dissolving lipids and cholesterol in 20 ml of chloroform, distilling off the solvent under reduced pressure, adding a solution in which acreasin had been solubilized with a formation aid, and performing ultrasonic treatment (prescription). 6,
7). As shown in Table 1, liposomes could not be formed without the formation aid, but uniform liposomes were formed using the formation aid of the present invention. The encapsulation rate is better if the main drug is placed in a dry film together with the lipid in advance (the resulting liposome preparation is ultracentrifuged at 100,000 G at 4°C for 30 minutes, the precipitate is collected, and 2% Triton X-100 is added. The amount of acreacins was determined by the Lowry-Forin method (OD 750 nm) to determine the encapsulation rate.) was high. Moreover, the average particle size was 0.5 μm or less in all cases.

[0049]

[Table 1]

Example 4 Table 2 shows the effect of the encapsulation rate on the liposome preparations of the present invention. That is, DPPC 200mg,
Cholesterol 20mg, main drug acreasin Aγ
5 mg was dissolved in 20 ml of methanol together with the liposome formation aid shown in the table, and the solvent was dried under reduced pressure using an aspirator to prepare a dry film. Sodium deoxycholate and sodium cholate were used as formation aids (Formulations 1 to 3). In addition, fat and cholesterol (
Chol) was dissolved in 20 ml of chloroform, the solvent was distilled off under reduced pressure, and a solution in which acreasin was solubilized with a formation aid was added and subjected to ultrasonication to produce liposomes (Formulation 4). Furthermore, in Formulation 5, cholic acid was added after forming a dry film, and in Formulation 6, the main drug and a forming aid were added after forming a dry film to prepare liposomes. As shown in Table 2, liposomes could not be formed without the formation aid, but uniform liposomes were formed using the formation aid of the present invention. The encapsulation rate was higher when the main drug was placed in the dry film together with the lipids in advance. Moreover, the average particle size was 0.5 μm or less in all cases.

[0051]

[Table 2]

Example 5 Table 3 shows the particle size and encapsulation rate of liposomes when other phospholipids or additives are used in combination in addition to the combination of DPPC and cholesterol in Formulation 3 of Example 3. Indicated. Glycyrrhizin K2 (use ratio: 1) was used as a formation aid. In the table, DOPC is dioleyl phosphatidylcholine, DPPG is dipalmitoylphosphatidylglycerol, DMPG is dimyristoylphosphatidylglycerol, and DMPA is dimyristoylphosphatidic acid.

[0053]

[Table 3]

Example 6 Instead of acreasin Aγ in Example 1, acreasin A
Liposomes were prepared using α. The encapsulation rate of acreasin Aα into liposomes was 63.1%.

Example 7 Instead of acreasin Dγ in Example 2, acreasin D
Liposomes were prepared using α. The encapsulation rate of acreasin Dα into liposomes was 37.2%.

Example 8 According to Example 1, Tween 20, S was added as a forming aid.
The ability to form liposomes was examined using a surfactant of DS (sodium dodecyl sulfate). The results are shown in Table 4.

[0057]

[Table 4]

[0058]

[Effects of the Invention] As explained above, the present invention makes it possible to form a stable liposome formulation of poorly water-soluble acreacins, and as a result, provides a drug effective for the prevention and treatment of Pneumocystis carinii pneumonia. It became possible to do so.

Claims (13)

[Claims] Claim 1: General formula (1) [Formula 1] (wherein, R1 -CO- represents a long-chain saturated or unsaturated fatty acid residue, a benzene ring, a pyridine ring, an oxygen atom, a sulfur atom or a nitrogen atom in the molecule) R2 represents a hydrogen atom, a lower alkyl group which may have a branched chain, a benzyl group or an amino group, which may be substituted with a mono-lower alkyl group or a di-lower alkyl group. Indicates a good amino-lower alkyl group, R3 is a hydrogen atom or -
CONH2 group, R4 is a hydrogen atom or a hydroxyl group) A preparation containing as an active ingredient at least one selected from the group consisting of acreacins represented by a hydrogen atom or a hydroxyl group, glycyrrhizins, acyltaurines, A liposome formulation characterized in that it is formulated using one or more of cholic acids, sodium higher alkyl sulfate, and polyoxyethylene sorbitan monohigher alkylate. Claim 2: In the liposome preparation, phospholipid 1
0.01 to 1 part of cholesterol, 0.002 to 2 parts of formation aid, and 0.001 to 2 parts of active ingredient.
The liposome preparation according to claim 1, which is 3. In the acreacin represented by the general formula (1), R1 -CO- is a long-chain saturated or unsaturated fatty acid residue, R2 is a hydrogen atom, R3 is a hydrogen atom, R
The liposome preparation according to claim 1, which is an acreasin derivative in which 4 is a hydrogen atom or a hydroxyl group. 4. The liposome preparation according to claim 1, wherein R1 -CO- is a myristic acid residue (C14) and R4 is a hydroxyl group. 5. The liposome preparation according to claim 1, wherein R1 -CO- is a palmitic acid residue (C16) and R4 is a hydroxyl group. 6. The liposome preparation according to claim 1, wherein R1 -CO- is a myristic acid residue (C14) and R4 is a hydrogen atom. 7. The liposome preparation according to claim 1, wherein R1 -CO- is a palmitic acid residue (C16) and R4 is a hydrogen atom. 8. Glycyrrhizins include 18β glycyrrhetinic acid, 18α glycyrrhetinic acid, 18β glycyrrhetinic acid, 18α glycyrrhetinic acid, 3β-glucuronyl-18β glycyrrhetinic acid, carbenoxolone, deoxoglycyrrhetinic acid, 3α-hydroxyglycyrrhetinic acid, 3-epiglycyrrhetinic acid. acid, 3α-dehydroxyglycyrrhetinic acid, deoxoglycyrrhetinic acid, hederagenin, 11-oxohederagenin, oleanolic acid, 1
The liposome preparation according to claim 1, which is 1-oxooleanolic acid or a nontoxic salt or hemisuccinate salt thereof. Claim 9: The acyl taurine has 10 to 2 carbon atoms.
The liposome preparation according to claim 1, which is an acyl taurine or acyl methyl taurine having 5 saturated or unsaturated fatty acid residues or a non-toxic salt thereof. [Claim 10] The cholic acids are deoxycholic acid,
The liposome preparation according to claim 1, which is cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, or a nontoxic salt thereof. 11. The liposome preparation according to claim 1, wherein the sodium higher alkyl sulfate is sodium dodecyl sulfate. [Claim 12] The polyoxyethylene sorbitan monohigher alkylate is polyoxyethylene (20).
The liposome formulation according to claim 1, which is a monolaurate. Claim 13: Claim 1, wherein the liposome particle size (average) in the liposome preparation is 0.01 to 10 μm.
The liposome formulation according to any one of items 1 to 12.