JPH07285878A - Lilposome preparation - Google Patents

Abstract

PURPOSE:To obtain a lilposome preparation having improved retention in blood, capable of existing in high concentration for many hours, by avoiding intake of the lilposome preparation in reticuloendothelial system. CONSTITUTION:This lilposome preparation is a medicine-containing lilposome comprising a phospholipid as a membrane constituent component. The membrane of the lilposome is modified with an N-alkyl-N-acylamino acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liposome preparation, and more particularly to a liposome preparation which has a low uptake into the reticuloendothelial system and can be present in blood at a high concentration for a long time.

[0002]

2. Description of the Related Art In the field of medicine, the development and research of liposome preparations have been actively conducted as one of the main carriers of targeting preparations. When liposomes are administered into blood, they have the property of being taken up by the reticuloendothelial system such as the liver and spleen. Therefore, liposomes are advantageous for drugs that use the reticuloendothelial system as a targeting site. On the contrary, a drug used as a targeting site is a drawback.
In particular, it is a big problem to maintain the blood concentration of peptide drugs and the like having a short elimination half-life in blood.

Until the first half of 1980, research on reticuloendothelial system evading liposomes was focused on optimization of formulation of liposome membrane composition. For example, a method using lecithin having a high phase transition temperature (Biochemical Pharma).
, 32, 3381 (1983)), a method using sphingomyelin instead of lecithin (Bio).
chemical Pharmacology, 32,
609 (1983)), a method of adding cholesterol (Biochemica et Biophysica).
Acta, 761, 142, (1983)) and the like have been reported. Separately from this, since the latter half of 1980, ganglioside GM1 (FEBS L
ett. , 223, 42 (1987)), phosphatidylinositol (Proc. Natl. Acad. Sc).
i. U. S. A. , 85, 6949, (1988)),
Glucuronsan Induction (Chem. Pharm. Bul
l. , 38, 1663, (1990)) and the like to modify the liposome membrane surface.

Further, a method using glycophorin of a sialoglycoprotein and a ganglioside of a sialoglycolipid (JP-A-63-221837), a phospholipid whose acyl group is a saturated acyl group and a sugar having a sulfate group or a sialic acid group A method using a lipid as a membrane constituent (Japanese Patent Laid-Open No. 63-112512).
JP-A-64-25716), a method of using a phospholipid having an acyl group as a saturated acyl group and an anionic surfactant having a Kraft point of 37 ° C. or higher such as N-acylaminosulfonate as a membrane constituent component. (JP-A-64-14), a method using a glycolipid in which a sugar and a lipid are O-glycosidic bonds or S-glycosidic bonds (Japanese Patent Publication No. 1-808499).
No.), a method of using a nonionic surfactant such as fatty acid ester or polyoxyethylene (JP-A-4-2440).
17), polyethylene glycol, polylactic acid, a method using a hydrophilic polymer such as polyglycolic acid (JP-A-5-505173), and the like. Further, JP-A-5-501264 discloses a technique of introducing a PEG derivative onto the surface of a liposome to maintain the plasma concentration.

On the other hand, as an example of using an amino acid derivative in a liposome preparation, an amino acid N-acylated with a higher acyl group having 12 to 18 carbon atoms is used to prepare a liposome preparation into a reticulum such as liver and spleen. A method of selectively accumulating in the endothelium system (Japanese Patent Laid-Open No. 63-2921), charging with a charged substance such as a basic amino acid surfactant such as N-acyl arginine to retain the drug in the liposome at a high rate. For treating the formed liposomes (JP-A-4-1035)
No. 27) and the like are known.

However, the ganglioside used as a surface modifier is present in a small amount in nature,
In addition, commercialization is impossible because it is extremely difficult to synthesize. On the other hand, surfactants such as polyethylene glycol derivatives are said to have no toxicity, but since they are not biodegradable, they have not yet been put to practical use as surface modifiers.

[0007]

The object of the present invention is to prevent the uptake into the reticuloendothelium by adding a biodegradable substance, improve the blood retention, and maintain a high concentration over a long period of time. To provide a ribosome preparation that can be present in blood.

[0008]

[Means for Solving the Problems] The inventors of the present invention have proposed various liposome membrane compositions in order to stably retain intravenously administered liposome preparations in blood without being taken up by the reticuloendothelial system. The stagnation property was examined by using the modifier. As a result, surprisingly N-alkyl-N-
By modifying the liposome membrane with an acylamino acid,
It was found that the retention in blood can be remarkably improved by avoiding the uptake into the reticuloendothelial system, and the present invention has been completed.

That is, the present invention relates to a drug-containing liposome having a phospholipid as a membrane constituent, wherein the liposome membrane is modified with N-alkyl-N-acyl amino acid. More preferably, it relates to a liposome preparation characterized in that the molar ratio of N-alkyl-N-acylamino acid to phospholipid is in the range of 1 to 40%.

Any N-alkyl-N-acylamino acid used in the production of the liposome preparation of the present invention may be used as long as the nitrogen atom of the amino acid is substituted with an alkyl group and an acyl group. You can For example, the N-alkyl group is one having 1 or more carbon atoms, preferably one having 1 to 10 carbon atoms, more preferably one having 1 to 5 carbon atoms, and specifically, a methyl group, an ethyl group, n-propyl group, i-propyl group, n-butyl group,
Examples thereof include i-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, 2-methyl-butyl group, and the like, with methyl group and ethyl group being preferred.

The N-acyl group may be saturated or unsaturated as long as it is derived from an organic carboxylic acid, and is a straight chain or branched chain or cyclic group. However, it is preferable that the hydrophobic membrane has the same degree of hydrophobicity as the hydrophobic group of the phospholipid which is a constituent of the liposome membrane. As such an acyl group, those having 10 or more carbon atoms, and more preferably those having 10 to 20 carbon atoms can be mentioned. Specifically, decanoyl group, undecanoyl group, lauroyl group, tridecanoyl group, myristoyl group, pentadecanoyl group, palmitoyl group,
Heptadecanoyl group, stearoyl group, oleoyl group,
Elideyl group, linoleoyl group, linolenoyl group, 9
Examples thereof include a tetradecenoyl group and a 9-hexadecenoyl group, with a lauroyl group, a myristoyl group, an oleoyl group, and a palmitoyl group being preferable. Further, a fatty acid derived from a natural fat or oil can be used as the acyl group, and specifically, an acyl group derived from a fatty acid constituting the fat or oil such as coconut oil, sunflower oil or castor oil can be used. You can also Of these, cocoyl groups derived from coconut oil are preferred.

As the amino acid, a compound having an amino group and a carboxyl group in the molecule can be used, and naturally-occurring and non-natural amino acids such as α-amino acids, β-amino acids, acidic amino acids, basic amino acids, neutral amino acids and aromatic amino acids can be used. Various natural amino acids can be used. The amino acid used may be D-form or L-form,
It may be a mixture thereof. Specifically, glycine, alanine, β-alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, etc. Examples thereof include neutral and acidic amino acids such as glycine and alanine.

Specific examples of the N-alkyl-N-acylamino acid of the present invention are shown in the following Tables 1 to 22,

[0014]

[Table 1]

[0015]

[Table 2]

[0016]

[Table 3]

[0017]

[Table 4]

[0018]

[Table 5]

[0019]

[Table 6]

[0020]

[Table 7]

[0021]

[Table 8]

[0022]

[Table 9]

[0023]

[Table 10]

[0024]

[Table 11]

[0025]

[Table 12]

[0026]

[Table 13]

[0027]

[Table 14]

[0028]

[Table 15]

[0029]

[Table 16]

[0030]

[Table 17]

[0031]

[Table 18]

[0032]

[Table 19]

[0033]

[Table 20]

[0034]

[Table 21]

[0035]

[Table 22] Examples thereof include N-lauroyl-N-methylglycine (lauroylsarcosine), N-myristoyl-N-methylglycine (myristoylsarcosine), N-palmitoyl-N-methylglycine (palmitoylsarcosine), N-ole. Oil-N-methylglycine (oleoyl sarcosine), coconut oil fatty acid sarcosine (cocoyl sarcosine), N-lauroyl-N-methylalanine and the like are preferable.

The amount of N-alkyl-N-acylamino acid added is 1 mol% or more, preferably 1 mo1% to 40 mol%, and more preferably 10 mo1% or more and 40 mo1% or less with respect to the phospholipid that is a membrane constituent. Used in the range.

As the phospholipids constituting the liposome membrane, phosphatidylcholine, phosphatidylethanolamine, phosphatidyllucerin, phosphatidic acid,
Natural or synthetic phospholipids such as phosphatidylglycerol, sphingomyelin, egg yolk lecithin and soybean lecithin, hydrogenated phospholipids thereof, acylated phospholipids and the like can be used alone or in combination of two or more.

If necessary, other components such as a membrane stabilizer, an antioxidant and a charged substance may be added to the liposome of the present invention. Cholesterol or the like can be used as the membrane stabilizer, tocopherol, ascorbic acid, glutathione or the like can be used as the antioxidant, and stearylamine, dicetyl phosphate or the like can be used as the charged substance. Further, the aqueous solution on the inner and outer shells of the liposome contains substances that constitute a buffer solution such as phosphoric acid, acetic acid and citric acid, isotonic agents such as sodium chloride, glucose and glycerin, and preservatives such as chlorbutanol and paraben. You may use the added thing.

The drug encapsulated in the liposome may be a drug having any physical property such as water-soluble, fat-soluble and amphipathic properties. For example, cisplatin and its derivatives (eg, carboplatin, spiroplatin), adriamycin, daunomycin, epirubicin, daunorubicin, vincristine, vinblastine, mitomycin, mitomycin C, actinomycin, actinomycin D, bleomycin, 5-fluorouracil, dacarbazine,
Methotrexate, cytosine arabinoside, aclacinomycin, ansamitomycin, leucovorin, neocarzinostatin, prednisolone, L-asparaginase, cyclophosphamide, and other anticancer agents and concomitant agents with anticancer agents, gentamicin, streptomycin, kanamycin, sulfazecin, micro Nomycin, astromycin, sisomycin, amikacin, tobramycin, bekanamycin, cefaclor, cefmetazole,
Antibacterial agents such as cefpyramide sodium, cefotiam, cefazoline sodium, latamoxef sodium, cefmenoxime, sulbactam sodium, cefoperazone sodium, cefpyramide sodium, amoxicillin, bacampicillin, and tarampicillin.

Lymphokines such as natural type or gene recombinant interferon (α, β, γ) and natural type or gene recombinant interleukin (1, 2, 3, 4, 5, 6, 7) , Tumor necrosis factor (α, β), colony stimulating factor, G-CSF, GM-CSF, prourokinase, urokinase, erythropoietin, Tissue plasminogen activator, insulin, calcitonin, uricase, manganese superoxide desmutase ( SOD) or its derivative, gonadotropin-releasing factor, desmopressin, adrenocorticotropic hormone, cyclosporine, motilin, gramicidin S, enkephalin, glutathione, factor VII, alkaline phosphatase, bioactive peptides such as vaccines, proteins and enzymes, B Tamine A, Vitamin D, Vitamin E (tocopherol), Vitamin K, Thiamine, Riboflavin, Biotin, Choline, Vitamin B12, Vitamin C, Ubiquinone, Carnitine, Cyanocobalamin, and other vitamins and vitamin-acting factors, and antiprotozoal drug such as meglumine antimonate. , An antifungal agent such as amphotericin B,
Anticoagulants such as heparin, antiallergic agents such as oxatomide, amoxanox, muramyl peptide, muramyl dipeptide, muramyl tripeptide, levamisole, TM
Immunostimulants such as D-66, cardiovascular drugs such as propranolol, glutathione, antisense RNA against HIV and ras genes, actinoblanc, lidocaine, chlorpromazine, dibucaine, timolol, quinidine, pilocarpine, dopamine, serotonin, imipramine, diphen. Examples thereof include hydantoin, glutathione, quinine, chloroquinine, etc., but rather, peptide thread drugs having a short blood half-life are preferable.

The particle size of the liposome preparation of the present invention is not particularly limited, but if it is 300 nm or more, it is easily taken up by the spleen, and if it is less than 50 nm, it is difficult to embed the drug.
Those of 0 to 300 nm are preferable, and especially 100 to 2
It is preferably 00 nm. The liposome preparation of the present invention contains phospholipids, N-alkyl-N-acylamino acids, and if necessary, a membrane stabilizer, an antioxidant, etc., and halogenated hydrocarbons such as chloroform, methylchloroform and methylene chloride, Alcohols such as ethyl alcohol, hydrocarbons such as hexane and heptane, benzene, toluene,
It is dissolved in an organic solvent such as aromatic hydrocarbons such as xylene, diethyl ether, diisopropyl ether, ethylene glycol dimethyl ether, ethers such as tetrahydrofuran, etc., and the organic solvent is removed under reduced pressure, and then an isotonic solution containing a water-soluble drug is prepared. After the addition, the mixture can be stirred to obtain multilamellar vesicle-like liposomes, and if necessary, can be manufactured according to a known method such as sizing with an extruder. The temperature at which this sizing is performed is preferably equal to or higher than the phase transition temperature of the liposome composition.

The obtained liposome dispersion liquid was subjected to gel filtration,
By purifying according to a known method such as centrifugation, the liposome and the drug not encapsulated in the liposome,
It can be separated from the marker and the like. A suspension of the liposome of the present invention containing the above specific glycolipid by adding a buffer aqueous solution (Tris, phosphoric acid, HEPES, MOPS, etc.) prepared to be isotonic with the encapsulated liquid of the separated liposome. Is obtained. The liposome obtained by the above operation can be present in blood at high concentration for a long time.

The shape of the liposome preparation of the present invention is
Multi-lamellar vesicles (MLV), multi-unilamellar vesicles (MUV), small-lamellar vesicles (S
UV), large unilamellar vesicles (LUV), reverse phase evaporated vesicles (REV), and the like.

The amount of phospholipid used is not particularly limited and may be appropriately selected depending on the type of the encapsulated material used and the like. The temperature conditions for distilling off the solvent to form a lipid film are not particularly limited. Further, the pH and salt concentration at the time of liposome preparation are not particularly limited as long as the denaturation of the lipid and the liposome does not occur, but usually pH 7 is set and osmolality is set to about 0.3.

[0045]

EXAMPLES The present invention will be described in more detail with reference to the following examples using typical N-alkyl-N-acyl amino acids, but the present invention is not limited thereto.

Example 1 77 mg of hydrogenated soybean lecithin (HSPC), 19 mg of cholesterol (Chol) and 3.5 mg of oleoyl sarcosine were dissolved in 10 ml of chloroform in a 100 ml eggplant flask and subjected to rotary evaporation, followed by distillation under reduced pressure. Then, the lipid thin film was obtained. 1 in this lipid film
-Hydroxypyrene-3,6,8-trisulfonic acid trisodium salt (1-Hydroxypyrene-3,
6,8-trisulfonic acid, tris
Oxal salt (HPTS) in 2 ml of physiological saline, etc., and then mixed by vortexing to form multilamellar vesicle-like liposomes (MLV)
Got This MLV is 200
After sizing to nm, gel filtration was performed to obtain liposomes encapsulating HPTS.

Example 2 Example 1 was replaced with 2.7 mg lauroyl sarcosine instead of the oleoyl sarcosine used in Example 1.
A liposome encapsulating HPTS was obtained by the same method as described above.

Example 3 Using 2.8 mg of cocoyl sarcosine instead of oleoyl sarcosine used in Example 1, liposomes encapsulating HPTS were obtained in the same manner as in Example 1.

Example 4 Liposomes encapsulating HPTS were obtained in the same manner as in Example 1 except that 3.0 mg of myristoyl sarcosine was used instead of oleoyl sarcosine used in Example 1.

Example 5 Using the same method as in Example 1 except that 3.2 mg of palmitoyl sarcosine was used in place of the oleoyl sarcosine used in Example 1, liposomes encapsulating HPTS were obtained.

Example 6 A liposome encapsulating HPTS was obtained in the same manner as in Example 1 except that 2.8 mg of lauroylmethylalanine was used in place of the oleoyl sarcosine used in Example 1.

Reference Example 1 HPTS-encapsulated liposomes were obtained in the same manner as in Example 1 except that 80 mg HSPC and 20 mg Chol were used and oleoyl sarcosine was not used. Next, the sustaining effect in blood of the liposome preparation of the present invention obtained in the above Examples will be shown in Test Examples.

Experimental Example 1 Under pentobarbital anesthesia, the liposomes obtained in Examples 1 to 6 and Reference Example 1 as a control or an aqueous solution of HPTS was administered through the femoral vein of male Wistar rats. Two hours after administration, blood was collected from the jugular vein, treated with heparin, and then centrifuged to obtain plasma. HPTS in the obtained plasma was quantified by a high performance liquid chromatography method, and the result is shown in FIG. According to FIG. 1, all N-
Liposomes modified with alkyl-N-acyl amino acids showed significantly higher plasma concentrations compared to controls.

[0054]

INDUSTRIAL APPLICABILITY The liposome preparation of the present invention has little uptake into the reticuloendothelial system and can be present in blood at high concentration for a long time.

[0055]

[Brief description of drawings]

[0056]

FIG. 1 shows the results of Experimental Example 1. "% Dose"
Indicates the ratio (%) in the detected plasma to the dose.

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Claims (3)

[Claims] 1. A drug-containing liposome comprising a phospholipid as a membrane constituent, wherein the liposome membrane is N-alkyl-N.
A liposome preparation characterized in that it is modified with an acylamino acid. 2. The molar ratio of N-alkyl-N-acyl amino acid is 1 to 40 mol% with respect to the phospholipid.
The liposome preparation according to. 3. The liposome preparation according to claim 1, wherein the drug is a peptide drug.