CA1195249A - Liposome and manufacture method therefor - Google Patents

Abstract

A B S T R A C T
Aqueous liposome compositions including steryl glucosides and/or steryl glucoside palmitates are described, together with a process for their preparation. These compositions find pharma-ceutical use due to their hemostatic action. These compositions also exhibit improved release and stability over known compo-sitions containing these steryl compounds.

Description

~S2~g The present invention re]ates to a liposome composition of steryl glucoside or steryl glucoside monopalmitate as well as a method of manufacturing the same.
Steryl glucosides and steryl glucoside monopalmi-tates 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-D-glucoside and campesteryl glucoside as well as fatty acid esters thereof, from various natural materials such as, for example, soybean, cotton seed, gram, chickpea, grape 10 fruit grounds, and the like by a method of, for example, T.
Kiribuchi, et al disclosed in Agricultural siological 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 ~ith an alkali. In order to manufacture steryl glucoside monopalmitates, the steryl glucosides obtained by the above hydrolysis method are subjected to chemical synthetic reaction.
Ratios of constituents of steryl glucosides extracted and separated from plants are given in Table 1.
Table beta-Sitosteryl- Stigmasteryl- Campesteryl beta-D-glucoside beta-D-gluco- beta-D-_ side. glucoside Soybean 56% 23% 21%
Cotton Seed 96% 4% o%
Gram or Chickpea 87% o% 3%
Grape Fruit Grounds 8~% 9% 7%

, . .. .

Steryl glucosides can be synthesized from plant sterolsobtained in a form of beta-sitosterol, stigmas-terol, campesterol or a mixture thereof by a known method such as, for example, that reported in Chemische Berichte, volume 105, pages 1097 to 1121. The corresponding monopalmitates can be prepared from the resulting steryl glycosides.
Steryl glycosides 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 dif~erence in physical and chemical properties in steryl glucosides even when the sterol moieties differ or even ln 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 stervl glucoside monopalmitates 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 as pharmaceuticals. It is desired that those compounds are used as injection solutions in view o~ their pharmacologi~al ~ctions, but beca~se or their ~5~

insolubility in water, it has been impossible to make them dissolve in aqueous solvents to prepare injectable solutions.
Thus, in order to prepare those compounds into injectable solu-tions, dissolving them into non-aqueous solvents or making them into suspensions have been attempted. However, solubilities of the compounds into propylene glycol, Macrogol (Trade Mark) and vegetable oils which are frequently used as non-aqueous solvents for injection solutions are low and it is not possible to obtain the desired concentrations. Whilst preparation of the injection solution as a suspension is possible but, when it is injected in vivo, dissolution of the compound from the injected part into body is so slow that the desired pharmaceutical effect cannot be expected. At any rate, both attempts do not afford the desired injection solutions.
Thus, the conventional techniques of preparing injection solution of hardly soluble compounds, do not ofEer injectable solutions of steryl glucosides and of steryl glucoside monopalmi-tates and, therefore, a special technique is required. Methods have been described whereby water solubilization of these com-pounds is achieved, by the use of hydrophilic solvents and solu-bilizing agents as is 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 S291~

glucoside monopalmitates for water is very low, comparatively large amount of solvents or surface active agents are necessary for solubilizing deEinite amounts of such compounds in water.

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

3) When injected intranvenously, the pharmacological effect of the compound is affec-ted by the solubilizing agent used.
In fact, the desired effect is only achieved by the use of particular solubilizing agents, such as HCO-60 (Trade Mark)(a polyoxyethylene- hardened castor oil adduct containing about 60 moles ethylene oxide).
This invention seeks to provide an injection solution which can be subjected to sterilization by heating easily, contains high concentrations of active ingredient suitable for the field of blood vessel strengthening and anti-shock treatment, where high dose is required, and affords sure pharmaceutical effects by intravenous injection. According to this invention, the steryl glucosides, or steryl glucoside monopalmitate compounds are included in a liposome. Thus, the present invention provides a novel liposome composition of steryl glucosides and steryl glucoside monopalmitates, and a manufacture method thereof.
Examples of use of a liposome as a carrier for pharmaceu-ticals 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;Biochemica et siophysica Acta MR 457, pages 259 to 302 (1976); Flender, J. H., et al:

~ D~ _ 5~

Life Science, 20t7), pages 1109-1020 (1977).
Liposomes are generally obtained in -the following forms.
Thus, a solution ofalipid in chloroform is placed in an egg-plant shaped flask, and the chloroform is evaporated therefrom so that thin membrane of the lipid is formed on the wall of the flaskO
Then a buffer and an aqueous solution of the pharmaceutical are added thereto, and the lipid membrane is removed from the wal]
by stirring, whereupon an aqueous solution of the pharmaceutical is included in the resulting small globes or vesicles. It has been necessary to remove pharmaceuticals not incorporated in the liposome by gel filtration or by ultra-centrifugation. Further-more, so far as the lipsome is in the above form, the aqueous solution of the pharmaceutical in the liposome diffuses out to the outer aqueous layer within a short time, even when the lipsome is separated from free pharmaceuticals and there is a disadvantage that liposome can hardly be used as practical pharmaceutical preparations.
However, it has now unexpectedly been found that steryl glucosides or steryl glucoside monopalmitates have a strong affinity with the lipid which constitutes the liposome. By utilizing such a property, those compounds can be included in liposome lipid. Thus, the present invention relates to a liposome composition in which steryl glucosides or steryl glucoside mono-palmitates are included in a lipid obtained by dissolving the lipid in chloroform, adding steryl glucosides or steryl glucoside monopalmitates thereto, ensuring dissolution by the addition of a membrane stabilizer (such as cholesterol) or a "charging agent"
if necessary, evaporating chloroform therefrom, and then stirring with physiological saline solution, or buffer solution, or by subjecting the mixture to ultrasonic waves, as well as to the method of manufacturing for the said liposome composition.
Examples of lipids used in the present invention are natural lipids such as lecithin, sphingolipid phosphoglycerides, gangliosides, etc. or synthetic lipids such as dimyristoyl-, dipalmitoyl-, distearyl-, and dioleylphosphatidyl choline, etc.
The natural or synthetic lecithins are preferred. Examples for stabilizers for the liposome membrane are cholesterol, beta-sito-sterol, stigmasterol, campesterol or a mixed sterols extracted from plant materials. Examples of "charging agents" are substan-ces which ~ill impart an ionic charge, such as stearyl~mine which charges with a positive electric charge being cationic and phos-phatidic acid and dicetylphosphoric acid which charges with a negative, electric charge, being anionic.
Examples of steryl glucosides in the present invention are beta-sitosteryl-beta-D-glucoside, stigmasteryl-beta-D-glucoside, campesteryl-beta-D-glucoside, cholesteryl-beta-D
glucoside and ster~l 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 ingredients in the lipid composition is as follows:

~3L95~ ~9 to one part by weight of the main ingredient, one to ten par-ts (preferably three to five parts) of lipids~ Ool to 5 parts (pre-ferably 0.3 to 2 parts) of membrane stabilizing sterols, and 0.05 to 0.5 part of charging agent. Depending upon the strength and time for stirring an ultrasonic wave irradiation, a multilamella or a unilamella liposome composition is obtained.
The liposome containing steryl glucosides or steryl glucoside monopalmitate of the present invention are administered by the parenteral route and exhibit 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 an injection solution suit-able for intravenous injection.
2) It is possible to prepare injection solutions con-taining about 5 percent of main ingredient and, therefore, it is possible to offer injection solutions not only for hemostatic purposes but also for blood vessel strengthening and antishock therapy requiring high administration doses.
3) The compositions afford predictable pharmacological effects even by intravenous injection.

4) Sterilization by heating can be done easily.
Besides the above given advantages, the liposome composition of the present invention 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 givin~ no changes both in appearance and the concen-tration.

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 water bath at 30C using a rotary evaporator. Nitrogen gas is blown into the residue for ten minutes. The residue is dried for six hours in a vaccum desiccator, 5 ml of physiological saline solution is added thereto, and the whole homogenized for three minutes under a nitrogen stream using a probe type ultrasonic wa~e homogenizer (Choompa Kogyo Co.; 25KH~, 150W) to give a nearly transparent pale yellow liquid. This is subjected to a sterile filtration using a millipore filter, GS type, to give a filtrate in which 99.6% of beta-sitO~teryl-beta-D-glucoside as compared with its amount before filtration is identified. The filtrate is filled in a 5 ml ampoule together with nitrogen gas and sub~ected to a sterilization in an autocalve at 120C for twenty minutes. Separation of neither main ingredient nor lipid is observed after the sterilization. When the injection solution is stored in the dark, neither separation of foreign matters nor lowering of concentration of the main ingredient is observed after more than two years at room temperatures. Both this injection solution and a blank solution prepared by omitting the main ingredient from the above injection solution are injecked intravenously to mice (ten mice per group). Their tails (1 cm 4~

from the end) are cut with a surgical knife, soa~ed in water, and the time necessary until the bleeding stops is measured (cf.
Motohashi, et al.: Tokyo Jikeikai Medical Journal, 75(5), 1008, 1959) to e~aluate the pharmacological activity of the pres-ent injection solutions by intravenous injection. The results are as given in Table 2 wherefrom it is apparent that, while the average hemostatic time in the blank group is 14.7 + 0.82 minutes, the group administered with 0.2 mg/kg of the injection solution shows the hemostatic time of 12.3 + 0.27 minutes. Thus, there is a significant difference within a level of P ~ 0.01 and the effectiveness of the present in~ention injection solution by the intravenous route is demonstrated.
Example 2 Dipalmitoyl lecithin (20 mg), lO mg of beta-sitosteryl-beta-D~glucoside monopalmitate and 5 mg of cholesterol are dissolved in 3 ml of chloroform and the mixture is evaporated in vacuo on a water bath at 30~C using a rotary evaporator.
Nitrogen gas is blown into the resulting residue for ten minutes which is then dried for six more hours in a vacuum desiccator.
To this is added 5 ml of phosphate buffer of pH 6.2 (prepared by dissolving 1.8 grams of trisodium phosphate, 6.4 grams of sodium dihydro~en phosphate and 5.1 grams of sodium chlsxide in a distilled water for injection to make onelitre). The mix-ture is homogenized for three minutes using a probe type ultra-sonic wave homogenizer to afford a slightly turbid liposome solution. This is filtered with a millipore filter HA type to give a filtrate in which 99.2% of beta-sitosteryl-beta-D-gluco-_ 9 ~

side monopalmitate is contained. The filtrate is Eilled in an ampoule together with nitrogen gas and sterilized with high pressure steam at 120C for twenty minutes in an autoclave. The pharmacological effect of the resulting injection solution by the intravenous route is given in Table 2, No.2.
Example 3 In a 50 ml egg plant type flask are placed 4 mg of dioleyl phosphatidyl choline and 4 mg of dipalmitoyl phosphatidyl choline, the mixture is dissolved in 2 ml of chloroform, then 3 mg of cholesteryl-beta-D-glucoside and 1 mg of cholesterol are dissolved therein. The chloroform is then evaporated on a water bath at 30C using a rotary evaporator. This is further treated as in Example 1 to afford 5 ml of liposome solution containing 3 mg oE cholesteryl-beta-D-glucoside. The pharmacological activity of the solution is given in Table 2, No. 3.
Example 4 In a 50 ml egg plant type flask is placed 15 mg of di-palmitoyl leclthin, which is dissolved in 2 ml of chloroform, 5 mg of sti.gmasteryl-beta-D-glucoside, Smg of stigmasterol and 1 mg of stearylamine are then dissolved. The chloroform is evaporated therefrom on a water bath at 30C using an evaporator. This product is treated in the same way as Example 2 to give 5 ml of liposome solution having a positive charge and containing 5 mg of stigmasteryl beta-D-glucoside. The pharmacological activity of the resultlng injection solution is given in Table 2, No.4.
Example 5 Instead of 1 mg of stearylamine in Example 4 is added 5~g 1 mg of dicetylphosphoric acid. The whole is then treated as in Example 4 -to ~ive 5 ml of liposome solution with a negative charge and containing 5 mg of stigmasteryl beta-D-glucoside. The pharmacological activity of the injection solution is given in Table 2, No.5.
Example 6 Yolk lecithin (200 mg) is placed in a 100 ml egg plant type flask, and dissolved in 5 ml of chloroform. Then 50 mg of steryl glucoside and 25 mg of cholesterol extrac-ted from soybean are dissolved therein. The chloroform is evaporated therefrom in vacuo on a water bath at 30C using a rotary evaporator.
Nitrogen gas is blown onto the residue for ten minutes, and -the residue dried for six hours in a vacuum desiccator. To the residue is added 5 ml of the phosphate buffer which is used in Example 2, and the mixture homogenized for five minutes under a nitrogen stream using a probe type ultrasonic wave homogenizer to give slightly trubid liposome solution. This is filtered using a millipore filter H~ type to give a filtrate containing 98 ! 2 percent of the initial steryl glucoside~ This product is treated in the same ~ay as Example 1 to give 5 ml of liposome solution containing 50 mg of steryl glucoside. The pharmacological actiVity of this injection solution is given in Table 2, No.6.
Example 7 In a 100 ml egg plant -type flask is placed 200 mg of yolk lecithin, dissolved in addition of 5 ml of chloroform, 50 mg of cholesterol and 50 mg of s-teryl glucoside monopalmitates extracted from cotton seed are then dissolved therein, and the whole treated as in Example 6 to give 5 ml of a liposome solution containing 50 mg of steryl glucoside monopalmitates extracted from cotton seed. The pharmacological activity of this injection solution is given in Table 2, No.7.
From Table 2, it is apparent that the compositions of this invention exhibit pharmacological activity by intravenous injection.

-Pharmacological Activities of L~osome Injection Solutions No. Compo- Route _ sition of Time required for Bleeding Stopping (minute) according Adminis- _ _ ~_ to tration 0.4 mg/kga) 0.2 mg/kga) 0.1 mg/kga) j Blank 1 Example 1 intravenous 11.1+1.55 11.7~0.87 14.0~1.13 14.7+0.82 2 Example 2 intravenous 9.5+0.91 11.1+1.12 13.8-~1.02 "
3 Example 3 intravenous 11.3+1.20 11.4+0.78 12.0+0.96 "
4 Example 4 intravenous 10.8+0.91 11.6+0.80 13.3+1.12 "
Example 5 intravenous 11.2+0.81 12.2~0.75 12.8+1.00 "

6 Example 6 intravenous 9.7+0.88 10.9+1.15 12.1-}0.80 14.2+0,95

7 Example 7 intravenous 11.6+0.73 11.2+0.82 12.5+0.96 "

a) Dose administered *) p ~ 0.05 **) p ~ 0.01

Claims (26)

THE EMBODIMENTS OF THE INVENTIQN IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of an aqueous liposome composition containing at least one steryl glucoside or steryl glucoside monopalmitate, which process comprises:
a) dissolving a lipid in chloroform;
b) dissolving, in the chloroform solution from (a), a steryl glucoside and/or a steryl glucoside monopalmitate, and a membrane stabilizer;
c) evaporating the chloroform from the solution; and d) dispersing the thus obtained liposome composition into a pharmaceutically acceptable aqueous solution.

2. A process according to claim 1, wherein, in step (b), an ionic charging agent additionally is dissolved in the chloroform.

3. An aqueous liposome composition containing, as effective ingredient, at least one steryl glucoside or steryl glucoside monopalmitate whenever prepared by the process of claim 1 or 2 or by obvious chemical equivalents thereof.

4. A process according to claim 1 or 2, wherein the lipid is chosen from natural or synthetic lipids.

5. A process according to claim 1 or 2, wherein the lipid is chosen from lecithin, sphingolipid phosphoglycerides, gangliosides, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearyl phosphatidyl choline or dioleyl phosphatidyl choline.

6. A process according to claim 1 or 2, in which the steryl glucoside or steryl glucoside monopalmitate is chosen from beta-sitosteryl-beta-D-glucoside, Stigmasteryl-beta-D-glucoside, campesteryl-beta-D-glucoslde or cholesteryl-beta-D-glucoside and the monopalmitates thereof.

7. A process according to claim 1 or 2, wherein the membrane stabilizer is a sterol.

8. A process according to claim 1 or 2, wherein the membrane stabilizer is a sterol chosen from at least one of cholesterol, beta-sitosterol, stigmasterol and campesterol.

9. A process according to claim 2, wherein the charging agent is one which provides a cationic charge.

10. A process according to claim 2, wherein the charging agent is one which provides an anionic charge.

11. A process according to claim 9, in which the charging agent is stearylamine.

12. A process according to claim 10, in which the charging agent is phosphatidic acid or dicetylphosphoric acid.

13. A process for preparing an aqueous liposome composition containing beta-sitosteryl-beta-D-glucoside as active ingredient which comprises dissolving beta-sitosteryl-beta-D-glucoside, cholesterol and yolk lecithin in chloroform, evaporating the chloroform, drying the residue, and homogenizing the dried residue in a physiological saline solution.

14. An aqueous liposome composition including beta-sitosteryl-beta-D-glucoside as an active ingredient whenever prepared by the process of Claim 13 or by an obvious chemical equivalent thereof.

15. A process for preparing an aqueous liposome composition containing beta-sitosteryl-beta-D-glucoside monopalmitate as active ingredient which comprises dissolving beta-sitosteryl-beta-D-glucoside monopalmitate, dipalmitoyl lecthin, and cholesterol in chlorofrm; evaporating the chloroform; drying the residue;
and homogenizing the dried residue in a phosphate buffer contain-ing Na3PO4, NaH2PO4 and NaC1 and having a pH of 6.2.

16. An aqueous liposome composition including beta-sitosteryl-beta-D-glucoside monopalmitate as an active ingredient whenever prepared by the process of Claim 15 or by an obvious chemical equivalent thereof.

17. A process for preparing an aqueous liposome composition containing cholesteryl-beta-D-glucoside as active ingredient which comprises dissolving cholesteryl-beta-D-glucoside, dioleyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, and cholesterol in chloroform; evaporating the chloroform; drying the residue; and homogenizing the dried residue in a physiological saline solution.

18. An aqueous liposome composition containing cholesteryl-beta-D-glucoside as an active ingredient whenever prepared by the process of Claim 17 or by an obvious chemical equivalent thereof.

19. A process for preparing an aqueous liposome composition containing stigmasteryl-beta-D-glucoside as active ingredient which comprises dissolving stigmasteryl-beta-D-glucoside, dipalmitoyl lecithin, stigmasterol and stearylamine in chloroform;
evaporating the chloroform; drying the residue; and homogenizing the dried residue in a buffer containing Na3PO4, NaH2PO4 and NaC1 and having a pH of 6.2.

20. An aqueous liposome composition containing stigmasteryl-beta-D-glucoside as an active ingredient whenever prepared by the process of Claim 19 or by an obvious chemical equivalent thereof.

21. A process for preparing an aqueous liposome composition containing stigmasteryl-beta-D-glucoside as active ingredient which comprises dissolving stigmasteryl-beta-D-glucoside, dipalmitoyl lecthin, stigmasterol and dicetylphosphoric acid in chloroform; evaporating the chloroform; drying the residue; and homogenizing the dried residue in a buffer containing Na3PO4, NaH2PO4 and NaC1 and having a pH of 6.2.

22. An aqueous liposome composition containing stigmasteryl-beta-D-glucoside as an active ingredient whenever prepared by the process of Claim 21 or by an obvious chemical equivalent thereof.

23. A process for preparing an aqueous liposome composition containing, as active ingredient soybean sterylglucoside which comprises dissolving soybean sterylglucoside, cholesterol and yolk lecithin in chloroform; evaporating the choroform; drying the residue; and homogenizing the residue in a buffer containing Na3PO4, NaH2PO4, and NaCl and having a pH of 6.2.

24. An aqueous liposome composition containing soybean sterylglucoside whenever prepared by the process of claim 23 or by an obvious chemical equivalent thereof.

25. A process for preparing an aqueous liposome composition containing, as active ingredient cotton seed sterylglucoside which comprises dissolving cotton seed sterylglucoside, cholesterol and yolk lecithin in chloroform; evaporating the chloroform; drying the residue; and homogenizing the residue in a buffer containing Na3PO4, NaH2PO4, and NaCl and having a pH of 6.2.

26. An aqueous liposome composition containing cotton seed sterylglucoside whenever prepared by the process of claim 25 or by an obvious chemical equivalent thereof.