Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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.
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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
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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~ ~~~"~~.
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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
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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
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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
9
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
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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:
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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
12 -
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.
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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
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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
- 16 -
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
