GB2135878A

GB2135878A - Liposome containing steryl glycoside

Info

Publication number: GB2135878A
Application number: GB08306237A
Authority: GB
Inventors: Masanobu Kawamata; Koichi Ushimaru; Shuji Yamane
Original assignee: Nippon Shinyaku Co Ltd
Current assignee: Nippon Shinyaku Co Ltd
Priority date: 1981-10-29
Filing date: 1983-03-07
Publication date: 1984-09-12
Also published as: FR2542999A1; CH658001A5; DE3309076C2; GB8306237D0; GB2135878B; JPS5874619A; DE3309076A1; KR840001278A; FR2542999B1; BE896217A

Abstract

Liposomes contain one or more of steryl glycoside and steryl glucoside monopalmitate as effective ingredient(s). In a method of manufacturing the liposomes, one or more steryl glucosides and steryl glucoside monopalmitate is/are dissolved in a solution of lipid and, if necessary, sterol and a charging agent in chloroform, then chloroform is evaporated therefrom to form lipid membrane on the wall of the container, and then stirred or subjected to ultrasonic wave irradiation after addition of a medium. The steryl glucosides and steryl glucoside palmitates are derived e.g. from cholesterol sitosterol, campesterol and stigmasterol.

Description

SPECIFICATION Liposome and manufacture method therefor The present invention relates to a liposome composition of steryl glucoside or steryl g lucoside monopalmitate as well as a method of manufacturing the same.

Steryl glucosides and steryl glucoside monopalmitates are contained in plants and they can be extracted and separated, mostly in a form of a mixture of beta-sitosteryl-beta-D-glucoside, stigmasteryl-beta-Dglucoside and campesteryl glucoside as well as fatty acid esters thereof, from various natural materials such as, for example, soybean, cotton seed, gram, chickpea, grape fruit grounds, and the like by a method of, for example, T. Kiribuchi, et al disclosed in Agricultural Biological Chemistry, volume 30, number 8, pages 770 to 778 (1966). In order to afford steryl glucoside from plant materials, a mixture obtained by the above-given method is hydrolyzed with an alkali. In order to manufacture steryl glucoside monopalmitates, the steryl glucosides obtained by the above hydrolysis method are subjected to chemical synthetic reaction.

Rates of constituents of steryl glucosides extracted and separated from plants are given in Table 1.

TABLE 1 beta-Sitosteryl- Stigmasteryl- Campesteryl beta-D-glucoside beta-D-glucoside beta-D-glucoside Soybean 56% 23% 21% Cotton Seed 96% 4% 0% Gram or Chickpea 87% 0% 3% Grape Fruit Grounds 84% 9% 7% Steryl glucosides can be synthesized from plant sterols obtained in a form of beta-sitosterol, stigmasterol, campesterol or a mixture thereof by a known method such as, for example, that reported in Chemische Berichte, volume 105, pages 1097 to 1121. Starting from the resulting steryl glucoside can be manufactured their monopalmitates.

Steryl glucosides are soluble in pyridine, somewhat soluble in dioxane, sparingly soluble in alcohols and ketones, and nearly insoluble in usual organic solvents such as hydrocarbons and halogen-containing solvents as well as in water.

There is no substantial difference in physical and chemical properties in steryl glucosides even when the sterol moieties differ or even in mixtures thereof extracted from plants. On the other hand, steryl glucoside monopalmitates are soluble in non-polar solvents, somewhat soluble in alcohols and nearly insoluble in water. There is no difference in physical and chemical properties in steryl glucoside monopalmitates even when the sterol moieties differ or even in mixtures thereof extracted from plants.

Steryl glucosides and steryl glucoside monopalmiates exhibit hemostatic action, vascular strengthening action, anti-shock action and the like as disclosed in Japanese Examined Patent Publication Sho-54-11369 and Unexamined Patent Publication Sho-53-109954 and are useful compounds are pharmaceuticals. It is desired that those compounds are used as injection solutions in view of their pharmacological actions but, because of their insolubility in water, it has been impossible to make them dissolved in aqueous solvents to prepare injectable solutions. Thus, in order to prepare those compounds into injectable solutions, dissolving them into non-aqueous solvents or making them into suspensions have been attempted.However, in the former attempt, solubilities of the compounds into propylene glycol, Macrogol and vegetable oils which are frequently used as solvents for injection solutions are low and it is not possible to obtain desired concentrations. In the latter attempt, preparation of the injection solution is possible but, when it is injected in vivo, dissolution of the compound from the injected part into body is so slow that desired pharmaceutical effect cannot be expected. At any rate, both attempts cannot afford desired injection solutions.

Thus, by way of the conventional technique of preparing injection solution of hardly soluble compounds, it has not been possible to offer injectable solutions of steryl glucosides and of steryl glucoside monopalmitates and, therefore, special technique is required. Against such a requirements, the present inventors have already succeeded in water solubilization of those compounds by the use of hydrophilic solvents and solubilizing agents as disclosed, for example, in Japanese examined patent publications Sho-53-31210 and Sho-53-20567. According to such methods, however, there are still several points which are to be improved.

They are, for example, as follows.

1) Since the affinity of steryl glucosides and steryl glucoside monopalmitates to water is very low, comparatively large amount of solvents or surface active agents are necessary for solubilizing definite amounts of such compounds in water.

2) When sterilized by heating, surface active agents separate out and adhere on the wall of an ampoule.

3) When injected intravenously, pharmacological effect of the said compound is affected by the solubilizing agent used. In fact, desired effect is not achieved by the use of solubilizing agents except a few such as HCO-60 (Trademark) (polyoxyethylene 60 moles and hardened castor oil).

The present inventors have conducted extensive studies in order to find injection solution which can be subjected to steriiization by heating easily, contains high concentrations of active ingredient suitable for the field of blood vessel strengthening and anti-shock where high dose is required, and affords sure pharmaceutical effect even by intravenous injection and, as a result, the present inventors have found that such objects can be all attained by making the said compounds included in liposome and accomplished the present invention. Thus the present invention is a liposome which is a novel composition of steryl glucosides and steryl glucoside monopalmiates and a manufacture method thereof.

Examples of use of liposome as a carrier for pharmaceuticals have been recently found in literatures and its structure, composition and manufacture methods are found in various reviews. For instance, Tyrell, D. H., et al: Biochmica et Biophysica Acta MR 457, pages 259 to 302 (1976); Flender, J. H., et al: Life Science, 20(7), pages 1 109-1020(1977).

Liposomes are generally obtained in the following forms. Thus, a solution of lipid in chloroform is placed in an egg plant type flask, chloroform is evaporated therefrom so that thin membrane of the lipid is formed on the wall of the flask, then a buffer and an aqueous solution of the pharmaceutical are added thereto, and the lipid membrane is taken off from the wall by stirring whereupon an aqueous solution of the pharmaceutical is included in the resulting small globe or vehicle. Therefore it has been necessary to remove pharmaceuticals not incorporated in the liposome by gel filtration or by ultracentrifugation.Furthermore, so far as the liposome is in the above form, aqueous solution of the pharmaceutical in liposome diffuses out to outer aqueous layer within short time even when the liposome is separated from free pharmaceuticals and there is a disadvantage that liposome can hardly be used as practical pharmaceutical preparations.

However, after extensive studies by the present inventors, they have unexpectedly found that steryl glucosides or steryl glucoside monopalmitates have strong affinity with lipid which constitutes liposome, noted that, by utilizing such a property, those compounds can be included in liposome lipid and accomplished the present invention.Thus, the present invention relates to a liposome composition in which steryl glucosides or steryl glucoside monopalmitates are included in lipid obtained by dissolving lipid in chloroform, adding steryl glucosides or steryl glucoside monopalmitates thereto, making them dissolved by addition of membrane stabilizer (such as cholesterol) or charging agent if necessary, evaporating chloroform therefrom, and by stirring with physiological saline solution or buffer solution or by subjecting to ultrasonic wave as well as a manufacture method of the said liposome composition.

Examples of lipids used in the present invention are natural lipids such as lecithine, sphingolipid phosphoglycerides, gangliosides, etc. or synthetic lipids such as dimyristoyl-, dipalmitoyl-, distearyl-, and dioleylphosphatidyl choline, etc. and, among them, natural or synthetic lecithins are preferred. Examples for stabilizers for liposome membrane are cholesterol, beta-sitosterol, stigmasterol, campesterol or a mixed sterols extracted from plant materials. Examples of charging agents are stearylamine which charges positive electric charge and phosphatidic acid and dicetylphosphoric acid which charge negative electric charges.

Examples of steryl glucosides in the present invention are beta-sitosteryl-beta-D-glucoside, stigmasterylbeta-D-glucoside, campesteryl-beta-D-glucoside, cholesteryl-beta-D-glucoside and steryl glucosides mixture mainly composed of the above steryl glucosides extracted from plant materials. Examples of steryl glucoside monopalmitates used in this invention are 6-monopalmitates of the above-given steryl glucosides.

The ratio of main ingredient and lipid constituent is, to one part by weight of the main ingredient, one to ten parts (preferably three to five parts) of lipids, 0.1 to 5 parts (preferably 0.3 to 2 parts) of sterols, and 0.05 to 0.5 part of charging agent. Depending upon the strength and time for stirring and ultrasonic wave irradiation, multilamella or unilamella liposome is obtained.

The present invention liposome containing steryl glucosides or steryl glucoside monopalmitate is administered by parenteral route and exhibits the following advantages.

1) Due to the use of so small amount as three to five parts by weight of lipid to one part by weight of the main ingredient, it is possible to afford injection solution suitable for intravenous injection.

2) It is possible to prepare injection solution containing even five or around percent of main ingredient and, therefore, it is possible to offer injection solutions not only for hemostatic purpose but also for blood vessel strengthening and antishockfields requiring high administration doses.

3) The present invention composition afford definite pharmacological effect even by intravenous injection.

4) Sterilization by heating can be done easily.

Besides the above given advantages, the present invention liposome exhibits marked properties that, when filled in an ampoule together with nitrogen gas and kept in dark place, it is stable at least for two years at room temperatures giving no changes both in appearance and the concentration.

Representative examples of the present invention are as follows.

Example 1 Yolk lecithin (20 mg) is placed in a 50 ml egg plant form flask, the content is dissolved in 2 ml of chloroform, then 5 mg of beta-sitosteryl-beta-D-glucoside and 2 mg of cholesterol are dissolved therein, and chloroform is evaporated therefrom on a steam bath of 30"C using a rotary evaporator. Nitrogen gas is blown into the residue for ten minutes, dried for six hours in a vacuum desiccator, 5 ml of physiological saline solution is added thereto, and homogenized for three minutes under a nitrogen stream using a proble type ultrasonic wave homogenizer (Choompa Kogyo Co.; 25KHz, 150W) to give nearly transparent pale yellow liquid. This is subjected to a sterile filtration using millipore filter GS type to give a filtrate in which solution is given in Table 2, No.7.

Out of Table 2, it is quite apparent that the present invention compositions exhibit pharmacological activity by intravenous injection.

TABLE 2 Pharmacological activities of liposome injection solutions Compo- Route Time required for Bleeding Stopping (minute) sition of No. according Adminis to tration 0.4 mg/kgaJ 0.2 mglkga' 0,1 1mglkga) Blank 1 Example 1 intravenous 11.1+1.55** 11.7+0.87** 14.0t1.13 14.7t0.82 2 Example2 intravenous 9.5+0.91** 11.1+1.12** 13.8+1.02 14.7+0.82 3 Example3 intraneous 11.3+1.20** 11.4+0.78** 12.0-'0.96 14.7It0.82 4 Example4 intraneous 10.8+0.91** 11.6+0.80** 13.3+1.12 14.7+0.82 5 Example 5 intravenous 11.2+0.81** 12.2+0.75* 12.8+1.00 14.7+0.82 6 Example6 intraveous 9.7+0.88** 10.9+1.15** 12.or0.80 14.2i0.95 7 Example7 intraveous 11.6+0.73** 11.2+0.82** 12.5+0.96 14.2+0.95 a'Dose administered p p < 0.05 ** p < 0.01

Claims

1. Liposome containing one or more of steryl glucoside and steryl glucoside monopalmitate as effective ingredient(s).

2. Liposome according to Claim 1 in which it is in a form of simple lamella.

3. Liposome according to Claim 1 in which it is in a form of multi-layered lamella.

4. Liposome according to any of Claims 1 to 3 in which the effective ingredient(s) is (are) contained in lipid.

5. Liposome according to any of Claims 1 to 4 in which the lipid is natural or synthetic phosphatidyl choline.

6. Liposome according to Claim 5 in which the natural phosphatidyl choline is yolk lecithin.

7. Liposome according to Claim 5 in which the synthetic phosphatidyl choline is at least one of dimyristoil phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearyl phosphatidyl choline and dioieyl phosphatidyl choline.

8. Liposome according to any of Claims 1 to 7 in which sterol is contained as a stabilizer for the liposome.

9. Liposome according to Claim 8 in which the sterol is at least one of cholesterol, beta-sitosterol, stigmasterol and campesterol.

10. Liposome according to any of Claims 1 to 9 in which the liposome contains a substance which donates positive charge.

11. Liposome according to Claim 10 in which the substance donating positive charge is stearylamine.

12. Liposome according to any of Claims 1 to 9 in which the liposome contains a substance which donates negative charge.

13. Liposome according to Claim 12 in which the substance donating negative charge is phosphatidic acid or dicetylphosphoric acid.

14. Liposome according to any of Claims 1 to 13 in which the steryl glucoside is beta-sitosteryl-beta-Dglucoside, stigmasteryl-beta-D-glucoside, campesteryl-beta-D-glucoside or cholesteryl-beta-D-glucoside and the sterylglucoside monopalmitate is a monopalminate of the above-given steryl glucosides.

15. A method of manufacturing liposome, characterized in that, one or more of steryl glucoside and steryl glucoside monopalmitate is/are dissolved in a solution of lipid and, if necessary, sterol and a charging agent in chloroform, then chloroform is evaporated therefrom to form lipid membrane on the wall of the container, and then stirred or subjected to ultrasonic wave irradiation after addition of a medium.

solution is given in Table 2, No.7.

TABLE 2 Pharmacological activities of liposome injection solutions Compo- Route Time required for Bleeding Stopping (minute) sition of No. according Adminis to tration 0.4 mg/kgaJ 0.2 mg/kpaJ 0.1 mglkga' Blank 1 Example 1 intravenous 11.1+1.55** 11.7+0.87** 14.0+1.13 14.7+0.82 2 Example2 intravenous 9.5+0.91** 11.1i1.12** 13.81.02 14.70.82 3 Example 3 intraneous 11.3+1.20** 11 Ai0.78** 1 2.0t0.96 1 4.7t0.82 4 Example4 intraneous 10.8+0.91** 11.6+0.80** 13.3+1.12 14.7+0.82 5 Example5 intravenous 11.2+0.81** 12.2+0.75* 12.8+1.00 14.70.82 6 Example6 intraveous 9.7+0.88** 10.9+1.15** 12.1+0.80 14.2+0.95 7 Example7 intraveous 11.6+0.73** 11.2+0.82** 12.5+0.96 14.2+0.95 a)Dose administered * p < 0.05 ** p < 0.01 CLAIMS

2. Liposome according to Claim 1 in which it is in a form of simple lamella.

7. Liposome according to Claim 5 in which the synthetic phosphatidyl choline is at least one of dimyristoil phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearyl phosphatidyl choline and dioleyl phosphatidyl choline.