CA2048471C

CA2048471C - Water-containing liposome system

Info

Publication number: CA2048471C
Application number: CA002048471A
Authority: CA
Inventors: Jorg Hager; Manfred Durr; Ernst Lunebach
Original assignee: A Natterman und Cie GmbH
Current assignee: A Natterman und Cie GmbH
Priority date: 1990-08-06
Filing date: 1991-08-06
Publication date: 2002-02-26
Anticipated expiration: 2011-08-06
Also published as: JPH04265151A; DE59106973D1; DK0470437T3; GR3018395T3; EP0470437A1; EP0470437B1; CA2048471A1; DE4122744C2; ES2082055T3; ATE130756T1; JP2758297B2; DE4122744A1

Abstract

An aqueous liposome system which contains at least one phospholipid and which may contain a non-toxic organic solvent. In addition to the at least one phospholipid the liposome system contains at least one phospholipidic charge carrier.

Description

The present invention is directed to a water-containing liposome system containing at least one phospholipid and which may contain a non-toxic organic solvent, and to a method of producing such a liposome system.
Phospholipidic liposome systems are known for different kinds of applications. These systems are for example used in the cosmetic field or for the production of pharmaceutical products. The respective active ingredients are encapsulated in spheres (vesicles) designated liposomes. These liposomes preferably contain an aqueous phase in their interior in which the respective active ingredient is dissolved, dispersed or emulsified. The liposomes are confined towards the outside by a lipid double. membrane.
EP A 03 09 519 and EP A 03 15 467 describe liposome systems which encapsulate the active ingredient pentamidine and which are used as pharmaceutical products.
The known liposome systems often have the disadvantage that they have the tendency to form undesired sediments even after a short time.
The present invention is based on the problem of providing a water-containing phospholipidic liposome system which has an especially high stability and thus does not tend to form sediments.
According to the present invention a liposame system comprises at least one phosgholipid and at least one phospholipidic charge carrier in addition to the phoispholipid. The system may contain a non-toxic organic solvent.

The inventive liposome system has a number of advantages. The inventive liposome system, even at an extremely long storage time of several months up to years, did not show any tendency to form sediments or deposits on the walls of the vessels. Furthermore, the inventive liposome system has a high transparency and is not dull or opaque which is the case with the known liposome systems. This has the result that an inspection with regard to the presence of foreign particles can be carried out without any difficulty with the inventive liposome system since it is only necessary to inspect the corresponding liposome dispersions by looking through the same. Moreover, it is adapted to be filtered in a sterile manner so that the inventive liposome systems are especially suited for pharmaceutical, cosmetic or diabetic applications.
The above-described advantageous effects of the inventive liposome system are attributed to the fact that the presence of the negative phospholipidic charge carrier yields a synergistic effect.
One preferred embodiment of the inventive liposome system, containing as phospholipidic charge carrier at least one salt, preferably a sodium salt and/or ammonium salt, of phosphatidylglycerol and/or of the derivatives thereof has especially good results with respect to the above-cited advantages. Preferably, the salt is the corresponding salt of dimyristoylphosphatidylglycerol and/or dipalmitoylphosphatidylglycerol.
In principle, the phosphatidylglycerol, which is present as a corresponding salt and thus forms the preferred negative phospholipidic charge carrier according to the above-described embodiments, can be isolated from any natural substance, as for example from oil seeds, rape, sun flowers etc., and can be used 2~~8~~1 correspondingly, possibly after a purification. However, it is especially advantageous if the above-cited salts of the phosphatidylglycerol or the corresponding derivatives are isolated from soya beans so that a soya phosphatidylglycerol alkaline salt, especially sodium salt or potassium salt, or a soya phosphatidylglycerol derivative alkaline salt, preferably sodium salt or potassium salt, is used as the negative charge carrier in the inventive liposome system.
Preferably the mass ratio of the at least one phospholipid to the at least one negative phospholipidic charge carrier varies between 50:1 to 400:1, more preferably between 100:1 to 200:1. The above-mentioned small amounts of the negative charge carrier are sufficient to give the above-cited stability during storage and to give a high transparency to the phospholipidic liposome system produced herefrom. An embodiment of the inventive liposome system which contains phosphatidylcholine as phospholipid has an especially long durability as well as an especially high distribution of the liposomes. Especially in the case if the phosphatidylcholine is ultra-pure phosphatidylcholine, i.e. phosphatidylcholine containing less than about 10% by weight impurities, a liposome system produced herefrom preferably containing the above-described soya phosphatidylglycerol sodium salt as negative phospholipidic charge carrier has the above-described advantageous characteristics. Furthermore, such a specific liposome system can be homogeneously eomminuted to a desired mean particle diameter of between 50 nm and 180 nm, preferably between 70 nm and 130 nm, by high-pressure split homogenisation or ultrasonic treatment with essentially less effort and thus in about half of the time. Such a specific liposome system can also be filtered in a sterile manner without any problems. For this, preferably 0.2 ~m filters are used.

2~ ~~~"~~.

As regards the phospholipid concentration of the inventive liposome system, the same preferably varies between 0.5% by weight and 20% by weight.
As already described above, the inventive liposome system can be used not only fox pharmaceutical but also for cosmetic purposes.
When the inventive liposome system is used for pharmaceutical purposes, two preferred possibilities exist:
According to the first possibility the inventive liposome system is used as a blank liposome system, i.e.
the liposome system as such is already pharmaceutically active. With regard to such a system it could be observed that the same can be used in an excellent manner for the treatment of atherosclerosis (arteriosclerosis), increased blood fat values as well as hepatopathies of any genesis. Such a system preferably contains water, possibly alcohol and between 5% by weight and 15% by weight of a mixture of phosphatidylcholine and negative charge carrier in the above-mentioned mass ratio. Such a pharmaceutical product is especially advantageously injected in its therapeutic application.
According to the second possibility an active ingredient ~s encapsulated in the inventive liposome system. such an encapsulated active ingredient has an improved therapeutic effect compared with the known products without negatively influencing the aim'of the medical treatment. This effect is attributed to the fact that the active ingredients encapsulated in the liposome system are delivered especially uniformly during a longer period of time in the therapeutical treatment so that undesired secondary effects do not occur or will be at least substantially reduced.

L

The selection of the active ingredient depends on the field of application. Thus, for example, pentamidine, pentamidine salts, especially pentamidine isethionate, and/or pentamidine derivatives can be solved and/or encapsulated in the inventive liposome system so that such a pharmaceutical product is preferably used for the parenteral and especially pulmonary treatment of pneumocystis-carinii-pneumonia (PcP), of the African sleeping sickness or of kala-azar.
However, it is especially advantageous if the above-cited active ingredient is not used from the beginning of the production of the liposome system but is added only at a point of time immediately prior to the application.
This can be done by mixing an aqueous liposome system 1.5 with the active ingredient as a dry substance or by first dispersing a dried liposome system in water and subsequently mixing the same with the active ingredient.
A pharmaceutical product produced in such a manner has a high transparency. In certain cases similar effects are attained by combining blank liposome preparations with the active ingredient without encapsulating the active ingredient into the liposome.
When the inventive liposome system contains Doxorubicin x H~1 as active ingredient, it can be used as corresponding pharmaceutical product for the treatment of cancer diseases.
When the inventive liposome system is to be used for they treatment of virus diseases, especially virus diseases of the skin; it is preferred to encapsulate a corresponding virucidal active ingredient, preferably rosemary acid or dextrane sulfate.
Furthermore, known active ingredients for the treatment of cancer, ATDS, liver diseases and virus diseases can be encapsulated or agglomerated in the inventive liposome system.
Moreover, the invention is directed to a method of producing the above-described liposome system.
In the method of producing the inventive liposome system the phospholipid, preferably the above-described phosphatidylchaline or ultra-pure phosphatidylcholine, together with the phospholipidic charge carrier, preferably Soya phosphatidylglycerol sodium salt, is dissolved or dispersed in an organic solvent.
Thereafter, the solution or dispersion is concentrated, and a corresponding amount of water is added in order to form the corresponding liposome system.
Preferably, ethanol, 1-propanol and/or 2-propanol are used as the solvent in the above-described inventive method.
The solution or dispersion produced in the beginning is concentrated to different residual volumes dependent on the non-toxic organic solvent that is used and its ability to be mixed with or its; compatibility with water.
If, for example, the above-mentioned alcohols axe used as non-toxic organic solvents, it is preferred to concentrate the corresponding solution of the phospholipid with the negative phospholipidic charge carrier to a residual volume of between 3 vol. (volume) %
and 30 vol. %, preferably of 5 vol. % to 10 vol. %. With such organic solv~nta which cannot be mixed with water it is recommended to concentrate until dryness.
vln order-to produce a liposome system with the inventive method which is characterized by especially uniform and adjusted mean liposome diameters, it is preferred to subject the resulting liposome system to a 7 _.
high-pressure split homogenisation or to an ultrasonic treatment after the addition of water. Preferably, these treatments are carried out until the liposomes formed thereby have a mean diameter between 50 nm and 180 nm.
In addition, hereafter the liposome system treated in such a manner can be filtered in a sterile manner by means of a 0.2 ~m filter.
Then, the resulting liposome systems can be either directly filled into corresponding ampoules in a condition ready for application or can be carefully dried, especially freeze dried, preferably after the addition of suitable stabilising substances, especially carbohydrates, so that a powder-like liposome system develops which, by means of the addition of a suitable amount of water, again forms the desired vesicles which are ready for application without taking extensive measures for agitation or mixing.
Again, two preferred possibilities exist in order to produce the embodiment of the inventive liposome system having one of the above-cited active ingredients.
According to the first possibility the active ingredient together with the phospholipid and the phospholipidic charge carrier is added to the organic solvent directly at the beginning of the method.
According to a variant of this method the used phospholipid is loaded with the active ingredient dissolved, dispersed or emulsified in a non-aqueous solvent, and after carefully drying the phospholipid loaded in such a manner together with the phospholipidic charge carrier is dissolved in an organic solvent which may be different from the first solvent. Thereafter, the organic solvent, as described above, is concentrated, and water is added so that the active ingredient-liposome-_ 2~r~~ ~7~.
system is formed wherein the active ingredient can be encapsulated. This variant of the method is especially preferred for such cases in which the active ingredient is stable with regard to its storage.
According to the second possibility, which is preferred especially if the active ingredient cannot be dissolved in the organic solvent used at first but can be better dissolved in water, at first the aqueous liposome system is produced in the above-described manner wherein thereafter the active ingredient is added together with the water.
A modification of the above-described method, which is especially used if the active ingredient has only a limited durability, is based on a powder-like dried liposome system: According to this modification the active ingredient is added during the redispersing step together with the used water so that the active ingredient comes into contact with the liposome system only immediately prior to the use of such a product.
In order to exclude undesired secondary effects the inventive method is preferably carried out under a protective inert gas.
In the following the inventive method is discussed in'detail by means of examples.
Example 1 Production of a blank liposome system 99,5 g of ul ra-pure phosphatidylcholine, i.e. less than 10% by weight impurities, and 0.5 g soya phosphatidylglycerol sodium salt (EG) were solved in 500 ml ethanol DAB (German pharmacopeia 9) and subsequently _ g _ dried under vacuum. The obtained phospholipid mixture was dispersed in water for injection purposes to 1000 ml under agitation and inert gas and thereafter was brought to a mean particle diameter of <100 nm by means of a high-pressure split homogeniser in five cycles. The resulting liposome system was thereafter filtered through a 0.2 ~Cm filter under sterile conditions and Was filled into 10.0 ml ampoules under a gas atmosphere. The phosphatidylcholine/soya-PG sodium system liposome system produced according to example 1 had the following characteristics:
phospholipid content: 10% (m/V) appearance: transparent, slightly opalescent liquid pH: 6.1 viscosity: 2.6 mPa.s osmotic pressure: 0.49 (% NaC1) transmission (660 nm) 75%

mean particle diameter (laser light dispersion): 75 nm sterility: corresponds to examination for sterility, DAB 9 (German pharmacopeia) endotoxin content (Limulustest):corresponds to requirements of DAB

electron microscopic characterization (cryofixation): 40-100 nm unilamellar liposomes, seldom bilamellar liposomes On account of is composition this product can be used in the following fields application:
of atherosclerosis, increased fat values, blood hepatopathies of any genesis.

~~~8~'~1 Example 2 500 g of phospholipid mixture consisting of 497.5 g ultra-pure phosphatidylcholine, i.e. less than loo by weight impurities, and of 2.5 g Soya-PG-sodium salt produced according to example 1 were dispersed in 6.5 1 water for injection purposes under agitation and inert gas. Thereafter, it was filled up with water for injection purposes to 8.0 1. In a separate vessel 2 kg of maltose were dissolved in 1.5 1 water for injection purposes under heating to 70°C. The phospholipid system was brought to a mean particle diameter of 56 nm by several cycles in a nigh-pressure split homogeniser (APV
Gaulin, type LAB 60), mixed with the maltose solution under agitation and inert gas, filled up with water for injection purposes to 10.0 1, sterile filtered, filled under aseptic conditions, and freeze dried. The lyophilisate formed after the freeze drying had the following characteristics:
appearance: crystalline, slight yellow dry powder content of residual moisture according to Karl Fischer: <0.7~
content of phospholipids: 500 mg/Vial sterility: corresponds to examination for sterility according to DAB 9 endotoxin content (Limulustest): corresponds to requirements of DAB 9 3.0 After redispersion of the lyophilisate with 8.3 ml water For injection purposes a liposome system with the following characteristics was obtained:

- 11 - ~~~~~~~1 appearance: transparent, slightly opalescent liquid pg: 6.5 viscosity: 2.7 mPa.s transmission (660 nm): 720 mean particle diameter (laser light dispersion method): 60 nm The phospholipid liposome system produced according to example 1 and the lyophilisate produced according to example 2 can be used for the following purposes of application: atherosclerosis, increased blood fat values, hepatopathies of any genesis. The lyophilisate produced according to example 2 has the advantage of an increased stability compared with the aqueous liposome system produced according to example 1.
The phospholipid mixture produced according to example 1 and consisting of phosphatidylcholine and soya-PG-sodium salt can be used not only for the production of unloaded, sterile filtratable phosphatidylcholine liposome systems (examples 1 + 2) but also for the production of loaded sterile liposome systems (examples 3-5).
Example 3 i0 g of the inventive phospholipid mixture were dissolved together with 0.1 g propidiumiodide (DNA-~arker) in ethanol according to example 1 and dispersed in 100 ml water for injection purposes after drying under vacuum; inert gas and cooling; Thereafter, an ultrasonic treatment was caxried out, also under inert gas and cooling, until a mean particle diameter of the liposomes of 80 nm (laser, light dispersion) was attained, Then, the liposome system was sterile filtered through a 0.2 um filter, and a half thereof was filled into brown 1.0 ml ampoules under inert gas. The proportion of liposomal-bound propidiumiodide was determined in the sterile liposome system loaded with propidiumiodide by means of a dialysis method (DianormR apparatus, cellulosetriacetate membrane NMGT 20000). According to this, the liposomal-bound propidiumiodide proportion was 29%. In the second half which was sterile filtered the proportion of non-liposomal-bound propidiumiodide was separated by means of ultrafiltration through a cellulosetriaeetate membrane NMGT 20000. The liposome dispersion was once again sterile filtered through a 0.2 ~m filter and filled into brown 1.0 ml ampoules under inert gas. The liposome dispersion obtained had the following characteristics:
phospholipid content: 100 mg/ml propidiumiodide content: 0.285 mg/ml pH; 7.2 viscosity: 1.7 mPa.s mean particle diameter (laser light dispersion): 129 nm Example 4 18.4 g of the phospholipid mixture described in example 1 together with 0.2 g quinoline yellow were dissolved in ethanol, dried under vacuum, dispersed with water for injection purposes ad 200 ml and thereafter subjected to an ultrasonic treatment under cooling. The liposome system obtained was thereafter sterile filtered and filled into 5.0 ml injection bottles under aseptic conditions. The sterile filtered liposome dispersion had the following characteristics:
appearance: transparent, opalescent yellow liquid pg: 6.4 _ 13 _ mean particle diameter (laser light dispersion method): 75 nm transmission (660 nm): 330 sterility: corresponds to examination for sterility, DAB 9 quinoline yellow, liposomal-bound: 1.38 mg/ml quinoline yellow, non-liposomal-bound: 3.2 mg/ml The non-liposomal-bound quinoline yellow proportion was separated far determining the proportion of liposomal-bound quinoline yellow by means of ultrafiltration through a cellulosetriacetate membrane NMGT 20000, and the proportion of quinoline yellow was photometrically determined in the liposome dispersion and in the filtrate.
The inventive phospholipid mixture formed according to example 2 into a sterile dry powder is also suited for the extemporated (ready to use) production of liposomes loaded with active water-soluble substances:
Example 5 A sterile dry powder corresponding to 500 mg phospholipid mixture described in example l and 2000 mg carrier substance were dispersed with 5.0 ml Doxorubicin HC1-solution (10:0 mg Doxorubicin HC1). The liposome redispersate obtained (68 mi) loaded with Doxorubicin HC1 had a content of phospholipids of 73.5 mg/ml and a total content of Doxorubicin HC1 of 0.735 mg/ml. The proportion of liposomal-bound Doxorubicin HC1 was determined to be 0.58 mg/ml and corresponds to an inclusion xate of about ?80.

The determination of the liposomal-bound Doxorubicin HC1 proportion was carried out with the dialysis method by means of liposomates, amount 5.0 ml, duration 5 h.
Example 6 Production of a blank liposome system 100 g of ultra-pure phosphatidylcholine, i.e. less than 10% by weight impurities, and 0.502 g Soya phosphatidylglycerol sodium salt (PG) were dissolved in 500 ml ethanol DAB 9 and subsequently adjusted to a dry substance content of 92% by weight under vacuum. The obtained phospholipid mixture consisting of 91.54% by weight phosphatidylcholine, 0.46% by weight soya phosphatidylglycerol sodium salt, 6% by weight ethanol and 2% by weight water was dispersed in water for injection purposes to 1000 ml under agitation and inert gas and thereafter brought to a mean particle diameter of <100 nm by means of high-pressure split homogenises in five cycles. The obtained liposome system was thereafter filtered through a 0.2 ~m filter under sterile conditions and was filled into 10:0 ml ampoules under inert gas:
The phosphatidylcholine/Soya-PG-sodium-system liposome system produced according to example 6 had the following characteristics:
phosphatidylcholi,ne content 10% (m/V) appearance: transparent, slightly opalescent liquid pH, fi.1 viscosity: 2.6 mPa.s osmoticpressure: 0.49 (% NaC1) transmission-(660 nm): 75%
mean garti.cle diameter (laser light dispersion): 75 nm sterility: corresponds to examination for sterility, DAB 9 endotoxin content (Limulustest): corresponds to requirements of DAB 9 On account of its composition this product can be used in the following fields of application:
atherosclerosis, increased blood fat values, hepatopathies of any genesis.
Example 7 Production of a blank liposome system 100 g of ultra-pure phosphatidylcholine, i.e. less than 10% by weight impurities, and 0.502 g soya phosphatidylglycerol sodium salt (PG) were dissolved in 500 ml ethanol DAB 9 and thereafter adjusted to a dry substance content of 92% by weight under vacuum. The obtained phospholipid mixture consisting of 91:54% by weight phosphatidylcholine, 0.46% by weight Soya phosphatidylglycerol sodium salt, 6% by weight ethanol and 2% by weight water was-disperse in water for injection purposes to 8333 ml under agitation and inert gas and thereafter brought to a mean particle diameter of <100 nm by means of a high-pressure split homogeniser at 500 bar in five Cycles. The obtained liposome system was thereafter filtered through a 0.2 ~m filter under sterile conditions and filled into 10.0 ml ampoules under inert gas: The phosphatidylaholine/soya-FG-sodium-system liposome system groduced according to example 2 had the 'following characteristics:
phosphatidy~.choline content: 1.2% (m/V) 2~s~8!~71 appearance: transparent, slightly opalescent liquid pH: 6.19 viscosity: 1.4 mPa.s transmission (660 nm): 82%

mean particle diameter (laser light dispersion): 58 nm sterility: corresponds to examination for sterility, DAB 9 endotoxin content (Limulustest): corresponds to requirements of DAB

Claims

1. A liposome composition comprising at least one uncharged phospholipid, said uncharged phospholipid comprising at least 90% by weight of phosphatidylcholine, at least one negatively charged phospholipid, and water forming an aqueous phase for said liposome composition, wherein said negatively charged phospholipid is a salt of phosphatidylglycerol, wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 50:1 to 400:1, and the total phospholipid content of the liposome composition is between 0.50 and 20% by weight.

2. The liposome composition of claim 1 wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 100:1 to 200:1.

3. The liposome composition of claim 1 or 2 wherein said negatively charged phospholipid is a sodium or ammonium salt of a phosphatidylglycerol.

4. The liposome composition of any one of claims 1 to 3 wherein said negatively charged phospholipid is a salt of dimyristoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol.

5. The liposome composition of any one of claims 1 to 4 wherein said negatively charged phospholipid is a salt of a soya phosphatidylglycerol.

6. The liposome composition of any one of claims 1 to wherein said uncharged phospholipid has less than 10% by weight impurities.

7. The liposome composition of any one of claims 1 to 6 further comprising at least one pharmaceutically active substance.

8. The liposome composition of claim 7 wherein said pharmaceutically active substance is selected from the group consisting of doxorubicin.HCl, pentamidine, a pentamidine salt, rosemarinic acid, a salt of rosemarinic acid, and dextran sulfate.

9. The liposome composition of any one of claims 1 to 8 further comprising an organic solvent present in said aqueous phase.

10. The liposome composition of claim 9 wherein said organic solvent is selected from the group consisting of ethanol, propanol-1, propanol-2, and mixtures thereof.

11. A composition suitable for forming liposomes, comprising at least one uncharged phospholipid, said uncharged phospholipid comprising at least 90% by weight of phosphatidylcholine, and at least one negatively charged phospholipid, wherein said negatively charged phospholipid is a salt of phosphatidylglycerol, wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 50:1 to 400:1, and the total phospholipid content of the composition is between 0.5 and 20% by weight, said liposomes being formed when water is added to said composition.

12. The composition of claim 11 wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 100:1 to 200:1.

13. The composition of claim 11 or 12 wherein said negatively charged phospholipid is a sodium or ammonium salt of a phosphatidylglycerol.

14. The composition of any one of claims 11 to 13 wherein said negatively charged phospholipid is a sodium or ammonium salt of dimyristoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol.

15. The composition of any one of claims 11 to 14 wherein said negatively charged phospholipid is a salt of a soya phosphatidylglycerol.

16. The composition of any one of claims 11 to 15 further comprising at least one pharmaceutically active substance.

17. The composition of claim 16 wherein said pharmaceutically active substance is selected from the group consisting of doxorubicin.HCl, pentamidine, a pentamidine salt, rosemarinic acid, a salt of rosemarinic acid, and dextran sulfate.

18. A composition suitable for forming liposomes, comprising at least one uncharged phospholipid, said uncharged phospholipid comprising at least 90% by weight of phosphatidylcholine, at least one negatively charged phospholipid, wherein said negatively charged phospholipid is a salt of phosphatidylglycerol, wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 50:1 to 400:1, and at least one organic solvent which is selected from the group consisting of ethanol, propanol-1, propanol-2, and mixtures thereof, and the total phospholipid content of the composition is between 0.5 and 20% by weight, said liposomes being formed when water is added to said composition.

19. The composition of claim 18 wherein the mass ratio of the uncharged phospholipid to the negatively charged phospholipid is in the range of 100:1 to 200:1.

20. The composition of claim 18 or 19 wherein said negatively charged phospholipid is a sodium or ammonium salt of a phosphatidylglycerol.

21. The composition of any one of claims 18 to 20 wherein said negatively charged phospholipid is a sodium or ammonium salt of a dimyristoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol.

22. The composition of any one of claims 18 to 21 wherein said negatively charged phospholipid is a salt of a soya phosphatidylglycerol.

23. The composition of any one of claims 18 to 22 further comprising at least one pharmaceutically active substance.

24. The composition of claim 23 wherein said pharmaceutically active substance is selected form the group consisting of doxorubicin.HCl, pentamidine, a pentamidine salt, rosemarinic acid, a salt of rosemarinic acid, and dextran sulfate.

25. The composition of any one of claims 18 to 24 wherein said composition contains said solvent in a concentration between 3% by volume and 30% by volume.

26. The composition of claim 25 wherein said composition contains said solvent in a concentration between 5% by volume and 10% by volume.

27. A method for the preparation of a liposome composition according to one of the claims 1 to 10, wherein initially the phosphatidylcholine, comprising less than 10%
by weight of impurities, is dissolved or dispersed with the negatively charged phospholipid in a mass ratio of 50 : 1 to 400 : 1, in an organic solvent, subsequently the solution or the dispersion is concentrated and thereafter water is added, leading to the formation of the liposome composition.

28. The method according to claim 27 wherein the mass ratio is of 100 : 1 to 200 : 1.

29. The method according to claim 27 or 28 wherein ethanol, 1-propanol and/or 2-propanol is used as organic solvent.

30. The method according to any one of claims 27 to 29 wherein the solution or dispersion is concentrated to a residual volume of the solvent of 0 % by volume to 30 % by volume.

31. The method according to claim 30 wherein the residual volume is 5 % by volume to 10 % by volume.

32. The method according to one of claims 27 to 31 wherein the liposome composition resulting after the addition of water is subjected to a high pressure split homogenisation or an ultrasonic treatment.

33. The method according to claim 32 wherein the high pressure split homogenisation or the ultrasonic treatment is performed for such a time, until the resulting liposomes have a mean diameter between 50 nm and 180 nm.

34. The method according to one of claims 27 to 33 wherein the liposome composition is filtered through a 0,2 µm filter.

35. The method according to one of the claims 27 to 34 wherein the liposome composition formed after the addition of water is gently dried after the addition of a suitable excipient.

36. The method according to claim 35 wherein said gentle drying comprises lyophilising.

37. The method according to claim 35 or 36 wherein said excipient comprises a carbohydrate.

38. The method according to any one of claims 27 to 37 wherein a pharmaceutically active substance is dissolved, emulsified or dispersed with the phosphatidylcholine and the negatively charged phospholipid in the organic solvent.

39. The method according to any one of claims 27 to 38 wherein the liposome composition is dried, and the dried liposome composition is taken up in water, to which the at least one pharmaceutically active substance has been added.