JPH04244017A - Liposome preparation - Google Patents

Abstract

PURPOSE:To obtain a liposome preparation increasing durability in blood while avoiding complement to the reticuloendothelial (RES) system such as liver, spleen and lung without adding a substance to cause physiological action to become side effects to human body. CONSTITUTION:A liposome preparation containing phospholipid and preferably <=3wt.% nonionic surfactant, preferably polyoxyethylene hardened castor oil, polyoxyethylene cetyl alcohol or polyoxyethylene sorbitan monooleate as an essential component, having slight complement to RES and long durability in blood.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to liposome preparations that have increased persistence in blood by containing a nonionic surfactant in phospholipids.

0002

BACKGROUND OF THE INVENTION In the pharmaceutical field, liposome preparations encapsulating various drugs have been actively developed as target-directed preparations. Liposomes have the property that when administered into the blood, they are trapped in the reticuloendothelial system (RES) of the liver, spleen, lungs, etc. This is an advantageous property when the target organ of the drug is the liver or lungs, but for drugs that aim for systemic effects, drugs whose target site is other than the RES, or drugs that are highly toxic to the liver, spleen, etc. This is a big drawback.

[0003] So far, ganglioside GM1, which is an in vivo information transmitter, has been used as a means to avoid the adhesion of liposomes to RES.
Lett. ), vol. 223, p. 42, 1987" and phosphatidylinositol "Proceedings of the National Academy of Sciences U.S.A. (Proc. Natl.A.)
cad. Sci. U. S. A. ), vol. 85, 6949
Page, 1988, and glucuronic acid derivatives that are involved in the metabolic effects of drugs in vivo, Chem.Phar.
m. Bull. ) Vol. 38, p. 1663, 1990" has been reported. Furthermore, JP-A No. 63-313724 discloses a technique in which by introducing a sialic acid derivative into liposomes, the presence rate in blood 2 hours after administration is increased by about 1.5 times compared to the original liposomes. There is.

On the other hand, JP-A-62-95134 claims a method for producing liposomes containing phospholipids and 1 to 15% by weight of a hydrophilic nonionic surfactant based on the phospholipids. Although a specific example of a method for producing liposomes in which about 10 and 15% of nonionic surfactants are added to the phospholipids is shown, the sustained effect of the drug contained when using such liposomes is etc. are not listed.

[0005]

[Problems to be Solved by the Invention] The purpose of the present invention is to avoid supplementation to the RES without adding substances that cause physiological effects that cause side effects to the human body, and to increase persistence in the blood. An object of the present invention is to provide a liposome formulation.

[0006]

SUMMARY OF THE INVENTION The present invention relates to a liposome formulation containing a phospholipid and 3% or less by weight of a nonionic surfactant.

[0007] As the nonionic surfactant in the present invention, general surfactants such as sorbitan fatty acid ester and fatty acid monoglyceride can be used, but preferably one containing a polyoxyethylene group in its molecular structure. Use a nonionic surfactant. Specifically, polyoxyethylene hydrogenated castor oil, polyoxyethylene alcohols such as polyoxyethylene cetyl alcohol, polyoxyethylene isoalcohols such as Emalfogen BC, and polyoxyethylene p-tert-octylphenols such as polyoxyethylene octylphenyl ether. , polyoxyethylene nonylphenols such as polyethylene glycol mono(nonylphenyl) ether, polyoxyethylene fatty acid esters such as sorbitan monooleate, and polyoxyethylene sorbitol esters such as polyoxyethylene sorbitan monooleate.

[0008] The amount of nonionic surfactant added is less than 10% by weight based on the phospholipid, which is a membrane component.

[0009] Phospholipids constituting liposomes include:
Natural or synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated phospholipids, glycerophosphoglyceroglycolipids, etc., alone or Use a mixture of two or more types.

[0010] If necessary, liposomes may contain sterols such as cholesterol as a membrane stabilizer, tocopherol as an antioxidant, or stearylamine, dicetyl phosphate, ganglioside, etc. as a charged substance. good.

The inner and outer aqueous solutions of the liposome contain substances constituting the buffer such as phosphoric acid, acetic acid, and citric acid, tonicity agents such as sodium chloride, glucose, and glycerin, and antioxidants such as ascorbic acid and glutathione. Preservatives such as chlorobutanol, paraben, etc. may be added.

[0012] The liposome may be in any shape such as a multilamellar vesicle, a large unilamellar vesicle, or a small unilamellar vesicle.

The drug to be encapsulated in the liposome may be of any physical properties such as water-soluble, fat-soluble, amphiphilic, etc., and includes cisplatin, carboplatin, adriamycin, daunomycin, epirubicin, daunorubicin, vincristine, vinblastine, mitomycin C, and actinomycin. D, anticancer drugs and combination drugs with anticancer drugs such as bleomycin, 5-fluorouracil, dacarbazine, methotrexate, cytosine arabinoside, leucovorin, neocarzinostatin, prednisolone, L-asparaginase, cyclophosphamide, interferon, colony stimulation various growth factors such as interleukin, tumor necrosis factor, tissue plasminogen activator, insulin, calcitonin, uricase,
Superoxide dismutase, gonadotropin-releasing factor, desmopressin, adrenocorticotropic hormone, cyclosporin, motilin, gramicidin S, enkephalin, glutathione, factor VIII, urokinase, alkaline phosphatase, vaccines and other bioactive peptides, proteins and enzymes, Gentamicin, streptomycin, kanamycin, micronomycin (sulfate), astromycin (sulfate), sisomicin (sulfate), amikacin, tobramycin, bekanamycin, cefaclor, cefmetazole, cefpiramide sodium,
Antibacterial agents such as cefotiam, cefazolin sodium, latamoxef sodium, cefmenoxime, sulbactam sodium, cefoperazone sodium, cefpiramide sodium, amoxicillin, bacampicillin, talampicillin, antifungal agents such as amphotericin B, vitamin A, vitamin D, vitamin E ( tocopherol), vitamins and vitamin-like agents such as vitamin K, thiamine, riboflavin, biotin, choline, vitamin B12, vitamin C, ubiquinone, and carnitine, antiprotozoal drugs such as meglumine antimonate, anticoagulants such as heparin, oxatomide , antiallergic agents such as amoxanox, immunostimulants such as muramyl dipeptide, muramyl peptide, levamisole, lidocaine, chlorpromazine, dibucaine, propranolol, timolol, quinidine,
pilocarpine, dopamine, serotonin, imipramine,
Diphenhydantoin, quinine, chloroquinine, etc. are used.

[0014] The method for preparing the nonionic surfactant-containing liposome of the present invention will be explained below. However, the method for preparing the liposome of the present invention is not particularly limited, and a general method for preparing liposomes can be used.

Preparation method Phospholipid and nonionic surfactant are dissolved in an organic solvent such as alcohol or chloroform, adding an antioxidant if necessary, and after removing the organic solvent under reduced pressure, the salt containing the water-soluble drug is dissolved. By adding water and shaking, multilamellar vesicle-like liposomes can be obtained.

[0016] If necessary, drugs that are not encapsulated within the liposomes may be removed by means such as centrifugation. In addition, using a polycarbonate membrane with a pore size of 100 nm, an extruder [Lipex Biomembrane (Li
The sizing may be carried out using the following method (manufactured by PEX Biomembranes). The obtained liposomes can be used as they are, but if necessary, excipients such as mannitol, lactose, glycine, etc. can be added and lyophilized.

Liposomes are generally used as injections, but can also be used as oral preparations, nasal drops, eye drops, transdermal preparations, suppositories,
It can also be processed and used as an inhalant.

Examples and reference examples are shown below.

[0019]

【Example】

Example 1 After dissolving 1 g of soybean phosphatidylcholine, 40 mg of α-tocopherol as a drug in liposome, and 10 mg of polyoxyethylene hydrogenated castor oil [HCO-60* (*trade name is shown below)] in 10 ml of methanol, Approximately 40
Methanol was removed under reduced pressure using a rotary evaporator while heating in a constant temperature bath at ℃. Distilled water for injection was added to the obtained lipid membrane so that the final liquid volume was 10 ml, and the mixture was vigorously shaken using a vortex mixer to obtain multilamellar vesicle-like liposomes (composition 1). . This solution was sized 10 times using an extruder equipped with a polycarbonate membrane with a pore size of 100 μm. The particle size of the obtained liposomes was determined by dynamic light scattering to be approximately 10
It was confirmed that it was 0 nm.

Example 2 Soybean phosphatidylcholine 1g, α-tocopherol 4
0mg, polyoxyethylene hydrogenated castor oil (HCO-6
0) Liposomes (composition 2) with a particle size of about 100 nm were prepared in the same manner as in Example 1 using a 30 mg dose.

Example 3 Soybean phosphatidylcholine 1g, α-tocopherol 4
0mg, polyoxyethylene sorbitan monooleate (
Tween 80*) (10 mg) was dissolved in 10 ml of isopropyl alcohol, and the procedure was then carried out in the same manner as in Example 1 to prepare liposomes (composition 3) with a particle size of about 100 nm.

Example 4 Soybean phosphatidylcholine 1g, α-tocopherol 4
0 mg, and Tween 80 (30 mg) were dissolved in 10 ml of isopropyl alcohol, and then operated in the same manner as in Example 1 to prepare liposomes (composition 4) with a particle size of about 100 nm.

Example 5 Soybean phosphatidylcholine 1g, α-tocopherol 4
0 mg of polyoxyethylene cetyl alcohol ether (Brij 58*) was dissolved in 10 ml of isopropyl alcohol, and the procedure was then carried out in the same manner as in Example 1 to prepare liposomes (composition 5) with a particle size of about 100 nm.

Reference Example 1 Soybean phosphatidylcholine 1g, α-tocopherol 4
Dissolve 0mg in 10ml of isopropyl alcohol,
Thereafter, the same procedure as in Example 1 was carried out to prepare liposomes (composition a) having a particle size of about 100 nm.

Reference Example 2 1 g of soybean phosphatidylcholine, 4 α-tocopherol
Dissolve 0mg in 10ml of isopropyl alcohol,
This was mixed with 5 ml of chloroform in which 55 mg of phosphatidylinositol was dissolved. The solvent was removed under reduced pressure using a rotary evaporator while heating in a constant temperature bath at about 40°C. A final liquid volume of 20 ml is applied to the obtained lipid membrane.
Add distilled water for injection so that
) was prepared.

Reference Example 3 Soybean phosphatidylcholine 1g, α-tocopherol 4
Dissolve 0mg in 10ml of isopropyl alcohol,
This was mixed with 5 ml of chloroform in which 26.8 mg of glucuronic acid palmitate was dissolved. Thereafter, the same procedure as in Example 1 was carried out to prepare liposomes (composition c) with a particle size of about 100 nm.

Reference Example 4 900 mg of soybean phosphatidylcholine, 40 mg of α-tocopherol, polyoxyethylene hydrogenated castor oil (HC
Liposomes (composition d) with a particle size of about 100 nm were prepared in the same manner as in Example 1 using 100 mg of O-60).

Next, the sustained effect of the liposome of the present invention in blood will be shown in a test example.

Test Example 1 Cannulae were inserted into the femoral vein and carotid artery of a male Wistar rat under urethane anesthesia. Compositions 1 to 5 and, as a control, composition a, which did not contain a nonionic surfactant, were administered at 1 ml/kg through the femoral vein. Blood was collected from the carotid artery over time until 2 hours after administration, and after heparin treatment,
Centrifugation was performed to obtain plasma. The α-tocopherol concentration in the obtained plasma was determined by high performance liquid chromatography, and the results are shown in FIG.

According to FIG. 1, compositions 1 to 5 showed higher plasma α-tocopherol concentrations than control composition a, and an increase in the persistence of the liposomes in the blood was observed.

Test Example 2 Composition 2, composition a, and compositions b and c as comparative examples were administered in the same manner as in test example 1, and the concentration of α-tocopherol in plasma was measured after administration. The results are shown in Figure 2.

According to FIG. 2, composition 2 showed a higher concentration of α-tocopherol in plasma than compositions b and c, and an increase in the persistence of the liposomes in the blood was observed.

Test Example 3 Cannulae were inserted into the femoral vein and carotid artery of a male Wistar rat under urethane anesthesia. 1 m of Compositions 1 to 5, Composition a as a control, and Composition d containing 10% nonionic surfactant as a comparative example were administered from the femoral vein.
1/kg was administered. Rats were decapitated 2 hours after administration.
The liver, spleen, and lungs were removed, and the amount of α-tocopherol in each organ was determined by high performance liquid chromatography. The organ distribution (%) when each organ, liver, spleen, and lungs were taken as RES for the dose of α-tocopherol was calculated, and the results are shown in Table 1.

According to Table 1, compositions 1 to 5 were found to have lower overall distribution rates to the liver, spleen, and lungs that constitute RES than control composition a and composition d.

[0036]

[Table 1]

[0037]

Effects of the Invention The present invention provides liposomes containing a nonionic surfactant that are not captured by RES and persist for a long time in blood.

[Brief explanation of the drawing]

[Figure 1] Long-lasting effect of nonionic surfactant-added liposomes in blood

[Explanation of symbols]

---X--- Composition a ───○─── Composition 1 ───●─── Composition 2 ───□─── Composition 3 ───■─── Composition 4 ───◎─── Composition 5

[Figure 2] Comparison of sustained effects in blood between nonionic surfactant-added liposomes and known liposomes

[Explanation of symbols]

---X--- Composition a ───●─── Composition 2 ---★--- Composition b ---☆--- Composition c

Claims (2)

[Claims] 1. A liposome preparation containing a phospholipid and a nonionic surfactant in an amount of 3% by weight or less. 2. The liposome preparation according to claim 1, wherein the nonionic surfactant is a nonionic surfactant containing a polyoxyethylene group.