Water containing liposome system

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

The present invention is directed to a water-containing liposome system and 
to a method of producing such a liposome system. 
Phospholipidic liposome systems are known for different kinds of 
applications. So, 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 correspondingly 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 pentamidin and which are used as 
pharmaceutical products. 
The known liposome systems have often the disadvantage that they have the 
tendency to form undesired sediments even after a short time. 
The present invention is based on the problem to provide a water-containing 
phospholipidic liposome system which has an especially high stability and 
thus does not tend to the formation of sediments. 
The inventive water-containing liposome system has at least one 
phospholipidic charge carrier in addition to the at least one 
phospholipid. 
The inventive liposome system has a number of advantages. So, it could be 
observed that 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 difficulties with the inventive liposome 
system since it is only necessary for this to inspect the corresponding 
liposome dispersions by looking through the same. Moreover, it is adapted 
to be filtrated in a sterile manner so that the inventive liposome systems 
are especially suited for a pharmaceutical or cosmetic application. 
The above-described advantageous effects of the inventive liposome system 
are attributed to the fact that obviously the presence of the negative 
phospholipidic charge carrier has the result of a synergistic effect. 
An embodiment of the inventive liposome system containing as phospholipidic 
charge carrier at least one salt, preferably a sodium salt and/or an 
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. 
On principle, according to the inventive liposome system 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 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 preferably 
used as negative charge carrier in the inventive liposome system. 
With regard to the mass ratio of the at least one phospholipid and the at 
least one negative phospholipidic charge carrier it is to be stated that 
this mass ratio varies between 50:1 to 400:1, preferably between 100:1 to 
200:1. It could be observed that already the above-cited small amounts of 
the negative charge carrier are sufficient to give the above-cited 
stability during storage and 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 comminuted 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 filtrated in a sterile manner without 
any problems. For this, preferably 0.2 .mu.m filters are used. 
As regards the phospholipid concentration of the inventive liposome system, 
it is to be stated that the same varies between 0.5% by weight and 20% by 
weight. 
As already described above, the inventive liposome system can be used not 
only for pharmaceutical but also for cosmetic purposes. 
If the inventive liposome system is used for pharmaceutical purposes, two 
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 contains preferably water, 
possibly alcohol and between 5% by weight and 15% by weight of a mixture 
of phosphatidylcholine and negative charge carrier in the above-cited mass 
ratio. Such a pharmaceutical product is especially injected when applied. 
According to the second possibility an active ingredient is encapsulated 
into the inventive liposome system. Such an encapsulated active ingredient 
has an improved therapeutical effect compared with the known product 
without negatively influencing the aim of the medical treatment. This 
effect is attributed to the fact that the active ingredients encapsulated 
into the liposome system are delivered especially uniformly during a 
longer period of time during the therapeutical treatment so that undesired 
secondary effects do not occur or will be at least substantially reduced. 
The selection of the respective active ingredient depends on the respective 
field of application. So, for example pentamidin, pentamidin salts, 
especially pentamidin isethionate, and/or pentamidin 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 with the active 
ingredient as dry substance or at 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. 
If the inventive liposome system contains Doxorubicin.times.HCl as active 
ingredient, it can be used as corresponding pharmaceutical product for the 
treatment of cancer diseases. 
If the inventive liposome system is to be used for the treatment of virus 
diseases, especially virus diseases of the skin, it is preferred to 
encapsulate a corresponding virucide active ingredient, preferably 
rosemary acid or dextrane sulfate. 
Furthermore, also the known active ingredients for the treatment of cancer, 
AIDS, liver diseases or 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. 
The inventive method of producing the inventive liposome system is based on 
the fundamental cognition that at first the used phospholipid, especially 
the above-described phosphatidylcholine or ultra-pure phosphatidylcholine, 
together with the phospholipidic charge carrier, especially the 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, propanol 1 and/or propanol 2 are used as solvent in 
the above-described inventive method. 
The solution or dispersion produced in the beginning is concentrated to 
different residual volumes dependent on the used non-toxic organic solvent 
and its ability to be mixed or its compatibility with water. If, for 
example, the above-cited alcohols are 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 solvents which cannot be mixed with water 
it is recommended to concentrate until the dry condition. 
In 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 
high-pressure split homogenisation or 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 
filtrated in a sterile manner by means of a 0.2 .mu.m filter. 
Then, the produced liposome systems can be either directly filled into 
corresponding ampoules in a condition ready for application or can be 
carefully dried, especially freeze dried, after the addition of suitable 
assisting substances, especially carbon hydrates, 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 making necessary extensive measures for agitation or 
mixing. 
Again, two 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 inventive method. According to a 
variant of this method the used phospholipid is loaded with the active 
ingredient solved, 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 are solved in an organic solvent 
which is possibly different from the first solvent. Thereafter, the 
organic solvent, as described above, is concentrated, and hereafter the 
water is added so that the active ingredient-liposome-system is formed 
wherein the active ingredient can be encapsulated. This variant of the 
method is especially preferred for such cases according to 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 solved in the organic solvent used at first 
but can be better solved 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 protection gas (inert gas). 
Preferred embodiments of the inventive method are indicated in the 
subclaims. 
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 ultra-pure phosphatidylcholine, i.e. less than 10% by weight 
impurities, and 0.5 g soya phosphatidylglycerol sodium salt (PG) were 
dissolved in 500 ml ethanol DAB (German pharmacopeia 9) and subsequently 
dried under vacuum. The obtained phospholipid mixture was dispersed in 
water for injection purposes ad 1000 ml under agitation and inert gas and 
thereafter was brought to a mean particle diameter of &lt;100 nm by means of 
a high-pressure split homogenisator in five cycles. The resulting liposome 
system was thereafter filtrated through a 0.2 .mu.m 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 (% NaCl) 
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 9 
electron microscopic characterization (cryofixation): 40-100 nm unilamellar 
liposomes, seldom bilamellar liposomes 
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 2 
500 g of phospholipid mixture consisting of 497.5 g ultra-pure 
phosphatidylcholine, i.e. less than 10% by weight impurities, and of 2.5 g 
soya-PG-sodium salt produced according to example 1 were dispersed in 6.5 
l water for injection purposes under agitation and inert gas. Thereafter, 
it was filled up with water for injection purposes to 8.0 l. In a separate 
vessel 2 kg of maltose were dissolved in 1.5 l water for injection 
purposes under heating to 70.degree. C. The phospholipid system was 
brought to a mean particle diameter of 56 nm by several cycles in a 
high-pressure split homogenisator (APV Gaulin, type LAB 60), was mixed 
with the maltose solution under agitation and inert gas, was filled up 
with water for injection purposes to 10.0 l, was sterile filtrated, was 
filled under aseptic conditions and was 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: &lt;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 
After redispersion of the lyophilisate with 8.3 ml water for injection 
purposes a liposome system with the following characteristics was 
obtained: 
appearance: transparent, slightly opalescent liquid 
pH: 6.5 
viscosity: 2.7 mPa.s 
transmission (660 nm): 72% 
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 
10 g of the inventive phospholipid mixture were solved together with 0.1 g 
propidiumiodide ((DNA-marker) 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 
carried 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 filtrated through a 0.2 .mu.m 
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 (Dianorm.sup.R apparatus, cellulosetriacetate 
membrane NMGT 20000). According to this, the liposomal-bound 
propidiumiodide proportion was 29%. In the second half which was sterile 
filtrated the proportion of non-liposomal-bound propidiumiodide was 
separated by means of ultrafiltration through a cellulosetriacetate 
membrane NMGT 20000. The liposome dispersion was once again sterile 
filtrated through 0.2 .mu.m and for 1.0 ml filled into brown ampullas 
under inert gas. The obtained liposome dispersion 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 obtained liposome 
system was thereafter sterile filtrated and filled into injection bottles 
for 5.0 ml under aseptic conditions. The sterile filtrated liposome 
dispersion had the following characteristics: 
appearance: transparent, opalescent yellow liquid 
pH: 6.4 
mean particle diameter (laser light dispersion method): 75 nm 
transmission (660 nm): 33% 
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 for 
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 transferred 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 1 and 2000 mg carrier substance were dispersed with 5.0 ml 
Doxorubicin HCl-solution (10.0 mg Doxorubicin HCl). The obtained liposome 
redispergate (6.8 ml) loaded with Doxorubicin HCl had a content of 
phospholipids of 73.5 mg/ml and a total content of Doxorubicin HCl of 
0.735 mg/ml. The proportion of liposomal-bound Doxorubicin HCl was 
determined with 0.58 mg/ml and corresponds to an inclusion rate of about 
78%. 
The determination of the liposomal-bound Doxorubicin HCl 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 ad 1000 ml 
under agitation and inert gas and thereafter brought to a mean particle 
diameter of &lt;100 nm by means of high-pressure split homogenisator in five 
cycles. The obtained liposome system was thereafter filtrated through a 
0.2 .mu.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 has the following 
characteristics: 
phosphatidylcholine content appearance: 10% (m/V) transparent, slightly 
opalescent liquid 
pH: 6.1 
viscosity: 2.6 mPa.s 
osmotic pressure: 0.49 (% NaCl) 
transmission (660 nm): 75% 
mean particle 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: atherosklerosis, 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 phophatidylglycerol sodium salt, 6% by weight ethanol and 2% 
by weight water was dispersed in water for injection purposes ad 8333 ml 
under agitation and inert gas and thereafter brought to a mean particle 
diameter of &lt;100 nm by means of a high-pressure split homogenisator at 500 
bar in five cycles. The obtained liposome system was thereafter filtrated 
through a 0.2 .mu.m filter under sterile conditions and filled into 10.0 
ml ampoules under inert gas. The phosphatidylcholine/soya-PG-sodium-system 
liposome system produced according to example 7 had the following 
characteristics: 
phosphatidylcholine content: 1.2% (m/V) 
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 9