DE3309076C2 - Liposomes and methods of making them - Google Patents

Abstract

Steryl glucosides and steryl glucoside monopalmitates, which are known to have excellent hemostatic, vascular stabilizing and anti-shock effects, have a strong affinity for lipids, so that they can be enclosed by these in the form of liposomes and, as such, are excellent medicaments which can be administered parenterally, in particular are injectable. These injection solutions and the process for their preparation are the subject of the invention.

Description

The invention relates to injection solutions containing one or more steryl glucosides and / Sj;

or sterylglucoside monopalmitates as active ingredients and a method for their production according to the above g

pending claims. |

Steryl glucosides and steryl glucoside monopalmitates are contained in plants and can be extracted from them? '

and separated, mostly in the form of a mixture of /? - Sitosteryl-y ^ -D-glucoside, Stigmasteryl-yi-D-glu- %

koside and campesteryl glucoside and their fatty acid esters. The natural starting materials used are, for example, i

Soybeans, cottonseed, horse beans, mung beans, chickpeas, grapefruit residues, etc., where f

for example that of T. Kiribuchi et al. in Agricultural Biological Chemistry, Volume 30, No. 8, pages 770-778

(1966) is used. To obtain steryl glucoside from plant material, a 'u

3d mixture obtained by the process described above hydrolyzed with alkali. To sterylglucoside! Ίι

To win monopalmitate, the steryl glucosides obtained by the above hydrolysis are chemically ε see subjected to synthesis reaction.

The proportions and components of steryl glucosides extracted and separated from plants are in the &

Table 1 below. Ϊ;

I.

Table 1 f

/ f-sitosteryl- stigmasteryl- catnpesteryl-

^ D-glucoside /? - D-glucoside / 7-D-glucoside V

^: ·

Soybeans 56% 23% 21% '(,

Cottonseed 96% 4% 0% ί

Beans or chickpeas 87% 0% 3% f

Grapefruit residues 84% 9% 7% -;

Steryl glucosides can be synthesized from plant sterols, which are in the form of /? - Sitosterol, Stigmasterin, If

Campesterol or a mixture thereof can be obtained by known processes, for example by a #

such as is described in Chemical Reports, Volume 105, Pages 1097-1121. Based on the echo hi

The monopalmitates can be produced from a steryl glucoside. $ ■

Steryl glucosides are soluble in pyridine, somewhat soluble in dioxane, hardly soluble in alcohols and ketones and |?

almost insoluble in common organic solvents such as hydrocarbons and halogenated %

Solvents, as well as in water. V

The steryl glucosides show no significant differences in their physical and chemical properties | '

shafts, even if the starring groups are different or even in their extracts from plants;

Mix. On the other hand, steryl glucoside monopalmitates are soluble in non-polar solvents, somewhat soluble in i;

Alcohols and almost insoluble in water. The steryl glucoside monopalmitates show no differences in theirs

physical and chemical properties, even if different sterling groups are present or themselves; ■

in their mixtures extracted from plants. :

Steryl glucosides and steryl glucoside monopalmitates have hemostatic, vascular stabilizing and anti-shock effects

Effect and the like as described in Japanese Patent Applications Sho-54-11369 and Sho-53 109954; and represent useful drugs. In view of their pharmacological efficacy, it would be desirable of these compounds to be able to prepare injection solutions, but because they are insoluble in water it has hitherto been impossible to prepare aqueous solutions of them in order to make them injectable. Around therefore, to convert these compounds into injectable solutions, attempts have been made to convert them into non-aqueous solutions

b5 to dissolve solvents or to convert them into suspensions. The solubilities of these compounds in Propylene glycol, macrogol, and vegetable oils, often used as solvents for solutions for injection However, the first method is low, and it is not possible to obtain the desired concentrations to obtain. According to the latter method, the preparation of injection solutions was possible, but

when injected in vivo, the compound is released from the injected part to the body so slowly that the desired pharmaceutical effect cannot be expected. Deliver in any case both methods do not provide the desired injection solutions.

Thus, it was more difficult to dissolve using conventional injection solution preparation techniques Connections not possible, injectable solutions of steryl glucosides and of steryiglucoside monopalmitates and therefore a special technique is necessary. Attempts have already been made to this Compounds soluble in water through the use of hydrophilic solvents and solubilizing agents to do, for example, in Japanese Patent Applications Sho-53-31210 and Sho-53-20567 According to these methods, however, there are still some points that need improvement, namely the following:

1) Since the affinity of steryl glucosides and steryl glucoside monopalmitates for water is low for solubilizing given amounts of these compounds in water, comparatively large amounts of Solvent or surfactant required.

2) When sterilizing by heating, the surfactants separate and stick to the Ampoule wall.

3) When injecting intravenously, the pharmacological effect of these compounds is influenced by the solubilizing agent used. In fact, the desired effect is not achieved by using solubilizing ^{agents other than a few, such as HCO-6 (X R} ) (60 moles of polyoxyethylene and hydrogenated castor oil).

From DE-OS 27 59 171 is still the medicinal use of steryl glucosides as inhibitors of the Prostaglandin synthesis known. However, it is only known from this publication, solutions for the oral For administration, but not for injectable use.

Extensive studies were carried out within the scope of the invention to find a solution for injection which can be easily sterilized by heating, which contains high concentrations of the active ingredient Vascular stabilizing and anti-shock agent where a high dose is required and which is safe pharmaceutical effect even with intravenous injection. According to the invention it was found that this Object can be fully achieved by including these compounds in liposomes. The invention thus relates to liposomes with a novel, steryl glucoside or steryiglucoside monopalmitate containing composition and a process for their preparation.

Examples of the use of liposomes as carriers for pharmaceutical agents have been reported in recent times known from the literature, and its structure, composition and manufacturing process is in various ways To read reports, for example by D. H. Tyrell et al, Biochimica et Biophysica Acta MR 457, Pp. 259-302 (1976); from]. H. Render et al, Life Science, 20 (7), pp. 1109-1020 (1977).

Liposomes are generally obtained in the following forms: Thus, the solution of a lipid in Put the chloroform in a pear-shaped flask, evaporate the chloroform so that it adheres to the Flask wall forms a thin lipid membrane, then buffer and an aqueous solution of the pharmaceutical By means of added, and the lipid membrane is removed from the wall by stirring, whereby the aqueous solution of the pharmaceutical agent included in the resulting small spheres or droplets will. Therefore, it was necessary to have pharmaceutical agent not entrapped in the liposome to be removed by gel filtration or ultracentrifugation. In addition, if the liposome is in the above form is present, the aqueous solution of the pharmaceutical agent diffuses into the external within a short time aqueous layer even if the liposome has been stripped of free pharmaceutical agent, and it there is the disadvantage that the liposome can hardly be used as a practically applicable pharmaceutical preparation can be used.

After thorough studies, however, it was surprisingly found according to the invention that steryl glucosides or Sleryl glucoside monopalmitates have a strong affinity for a lipid that forms the liposome, and found that, by taking advantage of this property, these compounds are incorporated into the liposome lipid can be. Accordingly, the present invention relates to a liposome mixture in which steryl glucosides or steryl glucoside monopalmitates are entrapped in lipid by dissolving lipid in Chloroform, adding steryl glucosides or steryl glucoside monopalmitates to it, dissolving by adding a membrane stabilizer (such as cholesterol) or, if necessary, a compound containing charge carriers, Evaporation of the chloroform and stirring with physiological saline solution or buffer solution or is obtained by applying ultrasonic waves. The invention also extends to a method for Preparation of such a liposome mixture.

Examples of lipids which can be used according to the invention are natural fats, such as lecithin, sphingolipid phosphoglycides, Gangliosides, etc., or synthetic lipids such as dimyrstoyl, dipalmitoyl, distearyl and dioleyl phosphatidyl choline etc. Of these, natural or synthetic lecithins are preferable. Examples of stabilizers for the liposomal branch are cholesterol, ^ -sitosterol, stigmasterol, campesterol or from plant material extracted noise mixtures. Examples of compounds containing charge carriers are stearylamine, the Has positive charge carriers, and phosphuidic acid and diketylphosphoric acid, which have negative charge carriers exhibit.

Examples of steryl glucosides which can be used according to the invention are ^ -Sitosteryl - ^ - D-glucoside, stigmasieryl- _/ 3 ^> D-glucoside, campesteryl - ^ - D-glucoside, cholesteryl - ^ - D-glucoside and steryl glucoside mixtures, which borrowed the main b5 consist of the above steryl glucosides extracted from plant material. Examples of steryl glucoside monopalmitates, as they can be used according to the invention, are the 6-monopalmkates of the steryl glucosides mentioned above.

The ratio of the main constituent and lipid constituent, based on one part by weight of the main constituent, is 1 to 10 parts (preferably 3 to 5 parts) lipid, 0.1 to 5 parts (preferably 0.3 to 2 parts) sterol and 0.05 to 0.5 parts of the charge carrier compound. Depending on the extent and time of stirring and Ultrasonic irradiation results in multilamellar or unilamellar liposomes.

The liposome according to the invention containing steryl glucoside or steryl glucoside monopalmitate becomes parenteral administered and has the following benefits:

1) Because of the use of amounts as small as 3 to 5 parts by weight of lipid to 1 part by weight Main ingredient, it is possible to obtain an intravenous injectable solution for injection.

ίο 2) It is possible to prepare a solution for injection even if these main ingredients are on the order of 5 percent contains, and therefore it is possible to use injection solutions not only for hemostatic Purposes, but also for blood vessel stabilization and as an anti-shock agent, where high concentrations are used and administration doses are required.

3) The preparation according to the invention allows a certain pharmacological effect even with intravenous Injection.

4) It can be easily sterilized by heating.

In addition to the above-mentioned advantages, the liposome of the present invention has remarkable properties on: If it is filled into an ampoule along with nitrogen gas and kept in the dark, it is Stable for at least two years at room temperature and does not change in appearance or concentration.

Comparative experiments

In order to make the surprising effectiveness of the liposomes according to the invention clear, in the the following table shows the concentrations of glucosides, the concentrations of the added substances, the Ratios of the substances added to the glucosides and comments on the products obtained compiled by

30th a) Example 1 of Japanese Patent Publication 53-31210, Concentration concentrations of the Ethanol 10% (5000) 11). Use for the b) Example 1 of Japanese Patent Publication 53-20567, of the solubilizing agents obtained *) HCO 60 Ig (500) intravenous injection c) Example 4a of DE-OS 27 59 171 and solution Ethanol 5% (25) d) Example 1 of the subject of the application. Lecithin 4% (20) 35 Table 1 0.02% HCO 60 4% (20) possible Water soluble too Ethanol 60% (30,000) making connection 0.2 PVP 1% (500) not recommended Lecithin 0.4% (4) 40 Cholesterol 0.05% (0.5) a) Cholesteryl - ^ - D-glucoside 0.02% - b) /? - SitosteryI - /? - D-glucoside 0.1% possible 45 a) Japanese Patent Publication 53-31210 (Example c) soy asteryl glucoside d) / i-sitosteryl-p-D-glucoside 50

b) Japanese Patent Publication 53-20567 (Example 1).

c) DE-OS27 59 171 (Example 4a).

d) Invention (Example 1).

*) Ratio of solubilizing agent or additive to 1 part of the glucoside compound.
HCO 60 = polyoxyethylene derivative of hardened or hydrogenated castor oil.

The table above shows that in the solubilization of the glucosides according to the invention only proportionally small amounts of additives are required (see d)), while the other processes (see a), b) and c)) amounts of solubilizing agents from a few tens to several thousand times as large as the amount of glucoside are required. Solutions with a high glucoside content are therefore only according to the invention Process, but not producible in a known manner. Injection solutions with high concentrations, such as from 1%, as can be produced according to the invention, was previously, as the literature shows, only on the way of Salt formation possible. In contrast, the invention makes it possible to use steryl glucosides in the form of liposomes to be administered parenterally in high doses.

The invention is explained in detail below on the basis of examples.

example 1

20 mg Kigelblecithin are placed in a 50 ml pear-shaped flask. The contents are dissolved in 2 ml of chloroform, then 5 mg ^ -Sitosteryl-zi-D-glucoside and 2 mg cholesterol dissolved therein, and the chloroform was evaporated using a drum evaporator at 30 ⁰ C on a steam bath. Nitrogen gas is blown into the residue for 10 minutes. It is then dried in a vacuum desiccator for 6 hours, 5 ml of physiological saline solution is added, and then it is homogenized for 3 minutes under a stream of nitrogen using a probe-type ultrasonic wave homogenizer (Choompa Kogyo Co., 25 kHz, 150 W), whereby an almost transparent, pale yellow liquid is obtained. This is sterile filtered using a millipore filter Type GS, wherein a filtrate is obtained in which 99.6 ⁰ Xi of the identified in -SitosteryN ^ / iD-glucoside, which was present prior to filtration. The filtrate is filled into a 5 ml ampoule together with nitrogen gas and sterilized in an autoclave at 120 ° C. for twenty minutes. Neither the main component nor the lipid are separated out during sterilization. If the injection solution is stored in the dark, no deposition of foreign substances or change in concentration are observed for a period of more than 2 years at room temperature. Both this injection solution and a blank sample, which has been prepared with the omission of the main component such as the injection solution, are injected intravenously into mice (10 animals per group). Their tails are docked 1 cm from the end with a surgical knife, immersed in water, and the time until the bleeding ceases is measured (cf. Motohashi et al .. Tokyo (ikeikai Medical Journal, 75 (5), 1008,! 959 ) to determine the pharmacological effectiveness of the solutions according to the invention when injected intravenously The results are summarized in Table 2, from which it can be seen that while the average hemostatic time for the blank sample is 14.7 ± 0.82 minutes, the group , at which 0.2 mg / kg injection solution were administered, shows a haemostatic time of 12.3 + 0.27 minutes, so there is a considerable difference at a level of P <0.01, and the effectiveness of the injection solution according to the invention safe by intravenous administration.

Example 2

20 mg dipalmitoyl lecithin, 10 mg / f-sitosteryl-yi'-D-glucoside monopalmitate and 5 mg cholesterol are in 3 ml of chloroform are dissolved, and the mixture is applied in vacuo on a water bath at 30 ° C evaporated in a drum evaporator. Nitrogen gas is added to the obtained residue for 10 minutes blown in and dried in a vacuum desiccator for an additional 6 hours. There are 5 ml of phosphate buffer of pH 6.2 added (prepared by dissolving 1.8 g of trisodium phosphate, 6.4 g of sodium dihydrogen phosphate and 5.1 g of sodium chloride in distilled water for injections to 1 1), and the mixture is taken under Application of a probe-type ultrasonic wave homogenizer for 3 minutes homogenized, with a slightly cloudy liposome solution is obtained. This is filtered through a millipore filter of the HA type, whereby a filtrate is obtained which contains 99.2% of the / f-sitosteryl- ^ - D-glucoside monopalmitate. The filtrate will collect filled with nitrogen gas in an ampoule and with high pressure steam at 120 ° C for 20 minutes in sterilized in an autoclave. The pharmacological effectiveness of the solution for injection obtained by intravenous Dosage is illustrated in Table 2, No. 2.

Example 3

A pear-shaped flask with a capacity of 50 ml contains 4 mg of dioleylphosphatidylcholine and 4 mg of dipalmitoylphosphatidylcholine are added, the mixture is dissolved in 2 ml of chloroform, then 3 mg of cholesteryl - ^ - D-glucoside and 1 mg of cholesterol are dissolved in it, and the chloroform is on a 30 ° C water bath evaporated using a drum evaporator. Then refer to the example 1 described manner further treated, with 5 ml of liposome solution containing 3 mg of cholesteryl / f-D-glucoside can be obtained. The pharmacological effectiveness of the solution is illustrated in Table 2, No. 3.

Example 4

In a pear-shaped flask with a capacity of 50 ml, 15 mg of dipalmitoyl lecithin, dissolved in 2 ml of chloroform, then 5 mg of stigmasteryI - /? - D-glucoside, 5 mg of stigmasterine and 1 mg of stearylamine and the chloroform is dissolved on a 30 ° C water bath using a drum evaporator evaporated. Treatment is then continued in the manner described in Example 2, with 5 ml of liposome solution with a positive charge and a content of 5 mg stigmasteryl - /? - D-glycoside can be obtained. The pharmacological The effectiveness of the injection solution obtained is given in Table 2, No. 4.

B e i s ρ i e 1 5

Instead of 1 mg of stearylamine from Example 4, 1 mg of diketylphosphoric acid is added and then treated in the manner described in Example 4, 5 ml of liposome solution having a negative charge and a content of 5 mg of stigmasteryl - /? - D-glucoside being obtained. The pharmacological effectiveness of the injection solution is given in Table 2, No. 5. μ

Example 6

200 mg of egg yolk lecithin are placed in a pear-shaped flask with a capacity of 100 ml, dissolved in 5 ml of chloroform, then 50 ml of steryl glucoside and 25 ml of cholesterol extracted from soybeans are dissolved therein, and the chloroform is added to a water bath at 30 ^° C removed using a drum evaporator in vacuo. Nitrogen gas is blown into the residue for 10 minutes and dried in a vacuum desiccator for 6 hours. Then 5 ml of phosphate buffer are added as in Example 2 and homogenized for 5 minutes under a stream of nitrogen by means of a probe-shaped ultrasonic wave homogenizer, a slightly cloudy liposome solution being obtained. This is filtered with a millipore filter of the HA type, a filtrate being obtained which contains 98.2% of the sterylgucoside. This is then treated in the manner described in Example 1, 5 ml of liposome solution containing 50 mg of steryl glucoside being obtained. The pharmacological effectiveness of this injection solution is given in Table 2, No. 6.

Example 7

200 mg of egg yolk lecithin are placed in a pear-shaped flask with a capacity of 100 ml given, dissolved in 5 ml of chloroform, 50 mg of cholesterol and 50 mg of sterylglucoside monopalmitate, extracted from Cottonseed, are dissolved in it, and then treated further in the manner described in Example 6, whereby 5 ml liposome solution with a content of 50 mg steryl glucoside monopalmitate, extracted from cottonseed, can be obtained. The pharmacological effectiveness of this injection solution is given in Table 2, No. 7.

From Table 2 it is clear that the preparations according to the invention with intravenous injection a have good pharmacological effectiveness.

Table 2

Pharmacological activities of liposome injection solutions

No. Preparation Administer Time required for bleeding to stop (min)

according to the type of treatment 0.4 mg / kg a) 0.2 mg / kg a) 0.1 mg / kg a) blank sample

1 example 1 = Dose administered. intravenous 11.1 ± 1.55 **) 11.7 ± 0.87 **) 14.0 ± l, 13th 14.7 ± 0.82 2 Example 2 ρ <0.05. intravenous 9.5 ± 0.91 *) 11.1 ± 1.12 **) 13.8 ± 1.02 14.7 + 0.82 3 Example 3 ρ <0.01. intravenous 11.3 ± 1.20 *) 11.4 ± 0.78 *) 12.0 ± 0.96 14.7 ± 0.82 4th Example 4 intravenous 10.8 ± 0.91 ··) 11.6 ± 0.80 *) 13.3 ± 1.12 14.7 ± 0.82 5 Example 5 intravenous ll, 2 ± 0.81 *) 12.2 ± 0.75) 12.8 ± 1.00 14.7 ± 0.82 6th Example 6 intravenous 9.7 ± 0.88 **) 10.9 + 1.15 **) 12.1 ± 0.80 14.2 + 0.95 7th Example 7 intravenous 11.6 ± 0.73 *) 11.2 + 0.82 **) 12.5 ± 0.96 14.2 ± 0.95 Remarks: a) **)

Claims (4)

Patent claims: 1. Injection solution containing one or more steryl glucosides and / or steryl glucoside monopalmitates as active ingredients, characterized in that the active ingredients are enclosed in lipids are. 2. Injection solution; iach claim 1, characterized in that the lipid is natural or synthetic Is phosphatidylcholine 3. Injection solution according to claim 1 to 2, characterized in that the steryl glucoside y? -Silosteryl- / · - D-glucoside, stigmasteryl - /? - D-glucoside, campesteryl - ^ - D-glucoside or cholesteryl - ^ - D-glucoside and that ι ο Sterylglakosidmonopalmital is a monopalmitate of the named sterylglucosides 4. A method for the preparation of injection solutions according to claim 1 to 3, characterized in that one or more steryl glucosides or steryl glucoside monopalmitates are dissolved in a lipid solution optionally sterol and a compound containing a charge carrier in chloroform adds that one then evaporates the chloroform to form a lipid membrane on the container wall and that one after adding a physiological saline solution or buffer solution or the ultrasonic wave irradiation suspends