Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
_.
An aqueous liposome system and a method for the preparation of such a liposome
system
The present invention relates to an aqueous liposome system with the
characteristic
features of the preamble of claim 1 and a method for the prepararion of such a
liposome system.
to
Different use are known for aqueous liposome systems. Such systems may for
instance
be used in cosmetics or for the preparation of pharmaceutical products. They
are
characterised by the fact that they contain hollow vesicles, also known as
liposomes,
which have an inner sphere; containing an aqueous phase. Depending on the
respective
is use the aqueous phase in tree inner part of the hollow vesicles may contain
a
pharmaceutically or cosmetically active ingredient in a dissolved, a dispersed
or a
suspended form. The systems described by EP-A 0 309 519 and EP-A- 0 315 467
are
examples of such liposome systems whereby these liposome systems contain the
drug
pentamidine in the inner part of the: vesicles.
Furthermore aqueous liposome systems are known, which contain in the inner
part of
the vesicles only the aqueous phase., such liposome systems being called an
unloaded
liposome system which car,, if needed, be loaded subsequently with a
pharmaceutically
2s or cosmetically active product.
Both types of the above-de;~cribed aqueous liposome systems have as a barner
to the
outer phase a lipid double membrane.
2~.19~~°~
The: liposome systems known from the prior art have a frequent disadvantage,
that they
show the tendency to form unwanted sediments after a short time.
The; object of the subject invention is to provide an aqueous liposome system
which
comprises besides a non-toxic organic solvent at least one phospholipid, which
possesses a particularly high stability and does not lead to unwanted
sedimentation.
This is achieved by a liposome system with the characteristic features of
claim 1.
to The inventive aqueous liposome system which comprises optionally besides a
non-
toxic organic solvent at least one phospholipid, comprises besides said
phospholipid
constituting the liposome system a further substance, which is not a
phospholipid..
This further non-phospholipidi.c substance is a bile acid, a bile acid salt, a
bile acid
derivative and/or a salt of a bile acid derivative, the mass ratio between
said
1s phospholipid and said further non-phospholipidic substance varying between
1:0.001
and 1:0.1.
The inventive liposome system has a number of advantages. So, it could be
observed,
that the inventive liposome system even at a storage period of several months
or years
2o did not show any tendency to form sediments or deposits on the inner walls
of the
vessels. Furthermore, the inventive liposome system has a high transparency
and does
not show any opalescence as <io the liposome systems known in the art. Due to
the lack
of sedimentarion and deposit i:ormarion and the high transparency the
inventive
liposome system can be inspe~;,ted quil:e easily on the presence of foreign
particles ,
2s since such an inspection may be simply performed by looking through the
same.
The above advantageous properties of the inventive liposome system are thought
to
depend on a synergistic effect brought about by the presence of the small
amounts of
the further non-phospholipidi~;, substa~ace ( a bile acid, a bile acid salt, a
bile acid
3
21~.9Q~"~
derivative andlor a salt of a bile acid derivative), which is not present in
natural
phospholipid systems.
'Particularly good properties, in respect to the before-mentioned advantages
are shown
lby a first embodiment of the inventiive aqueous liposome system, in which the
mass
ratio of the phospholipid to the furdber non-phospholipidic substance ( a bile
acid, a
bile acid salt, a bile acid derivative and/or a salt of a bile acid
derivative) is between
1:0.03 and 1:0.1.
to As mentioned before, the irmentive phospholipid preparation comprises at
least one
further non-phospholipidic substance. This concerns in particular a salt, in
particular a
sodium and/or an ammonium salt, of cholic acid, deoxycholic acid,
ursodeoxycholic
acid and/or chenodeoxycholic acid but may also be the salt, in particular a
sodium
and/or an ammonium salt, of a bile; acid derivative, preferably glycocholic
acid and/or
t s taurocholic acid.
A further particularly suitalble embodiment of the inventive liposome system
provides,
that the inventive liposome system contains as the further non-phospholipidic
substance glycocholic acid, a salt o~f glycocholic acid and/or a derivative of
glycocholic
2o acid within the previously :mentioned range of mass ratios.
The concentration of the at: least one phospholipid varies between 5 % by
weight and
25 % by weight, in particular between 8 % by weight and 18 % by weight, these
concentration values relati~lg to the; final liposome system or to a liposome
system as is
2s offered commercially. It is obviou<.>, that depending on the respective
intended type of
treatment or the intended case to be treated, higher as well as lower
phospholipid
concentrations as the above values are possible.
A particularly high stability and a particularly homogeneous distribution of
the
liposomes with respect to their diameter is found at another embodiment of the
inventive liposome system, when it contains phosphatidylcholine, in particular
such a
phosphatidylcholine (3-sn-phosphatidlylcholine, Soya) which is gained from
soybean.
s In particular in cases where the phospholipid in the inventive liposome
system
constitutes to at least 90 % by weight and preferably to at least 95 % by
weight of
phosphatidylcholine, then such a liposome system possesses the advantages
which
were mentioned previously in~ even more pronounced form. Additionally, such a
special liposome system can ~,vith essentially less effort and for that reason
in about
~o haf of the usual time be reduced in a homogeneous way by extrusion, high
pressure
split homogenisation or ultrasound treatment to a wanted mean particle
diameter of
beaween 30 and 100 nm, in p~irticular of between 35 and 90 nm. Also such a
special
liposome system which contains mostly unilamellar to bilamellar liposomes of
the
said dimension, to be filtratedl easily , whereby preferably 0.2 p.m filters
are being
~s used.
A further advantage of the inventive liposome system is the fact, that it
possesses a pH
value which varies around ne utrality and lies preferably between 6.0 and 8.0
and more
preferably between 6.2 and 7.4.
A.~ stated previously, the inventive liposome system comprises mostly
unilamellar or
bilamellar liposomes with a particle diameter between 30 nm and 100 nm,
preferably
beaween 35 nm and 90 nm.
2s Tlae inventive liposome system can bc: used for pharmaceutical as well as
cosmetic
proposes, this use covering the unloaded liposomes as well as such liposome
systems
loaded with a respective pharmaceutically or cosmetically active ingredient.
2~.~.~05"~
If the inventive liposome s~~stem was used in the form of an unloaded liposome
system
without any encapsulated active ingredient, it could be observed that said
unloaded
system is perfectly suited for the treatment of atherosclerosis, increased
blood lipid
values as well as the treatment of liiver diseases of any type. Said unloaded
liposome
s system comprises preferably besidEa water and eventually an alcohol between
5 % by
weight and 20 % by weighvt of a rni.xture of phosphatidylcholine of the before-
mentioned purity and the fi.irther non-phospholipidic substance (bile acid, a
bile acid
salt, a bile acid derivative and/or a salt of a bile acid derivative of the
afore-mentioned
type) in the mass ratio as mentioned previously. Such a pharmaceutical product
is
~o particularly suitable for injection.
As has been mentioned before, the inventive liposome system may contain an
encapsulated cosmetic or pharmaceutical active ingredient. It has been
observed, that
such an encapsulated actin. ingredient has a greater therapeutic efficacy in
particular
is with respect to its duration., in comparison with conventional application
form, like e.g.
a tablet, a sugar-coated tablet or the like. This greater efficacy enables as
a rule the
reduction of the amount of the active ingredient, without this having a
negative effect
on the therapeutic efficacy. This depot effect may be explained by a regular
and long-
lasting release of the encapsulated active ingredient from the liposome
system. As a
2o result any unwanted side effects do not appear, or at least in a less
severe form. The
selection of the active ingredient depends on the type of treatment. It is
possible to
encapsulate in the inventive liposome system pentamidine, pentamidine salts,
in
particular pentamidine isetlhionate .and/or pentamidine derivatives dissolved
and/or
encapsulated, such a pharmaceutical product then preferably being used for the
2s parenteral and in particular for the pulmonary treatment of Pneumocystis
carinii
Pneumonia, the African sleeping diisease or of Kala-Azar.
It is particularly suitable, not to usc: the pharmaceutical or cosmetic active
ingredient
from the beginning in the preparation of the liposome system, but to add said
2~.~.9~~rr
pharmaceutically or cosmetically active ingredient only after the preparation
of the
liposome system and in particular immediately before use. For example, this
can be
oibtained, by mixing an aqueous unloaded liposome system with the active
ingredient,
whereby this active ingredient is present in dry form, or as a solution,
dispersion,
emulsion or suspension in a non-toxic organic solvent, or by dispersing a
dried
li~posome system in water in which th.e active ingredient has been dissolved.
Pharmaceutical or cosmetic products prepared in this way show a high
transparency,
which makes it an easy task to inspect them on the presence of unwanted
foreign
p~u-ticles.
io
If the inventive liposome system comprises as active ingredient doxorubicin
hydrochloride, then such a product m,ay be used for the treatment of cancer
disease.
If on the other hand, the inventive liposome system is to be used for the
treatment of
~s vv~al diseases, in particular of viral skin diseases, then the active
ingredient to be
encapsulated in the vesicles will be an virucidal agent, like e.g. rosmarinic
acid or
dextrane sulphate.
Furthermore the known active ingredients for the treatment of cancer, Aids,
liver
2o di<.;ease or virus disease can also be encapsulated.
The invention further relates to a metHod for the production of the above-
described
liposome system.
2s According to the inventive method for producing the liposome system
initially the at
least one phospholipid is dissolved or dispersed in an organic solvent
together with the
at least one further non-phospholipidic substance, which may be a bile acid, a
bile acid
salt, a bile acid derivative and/or a salt of a bile acid derivative, the mass
ratio being
1:x.001 to 0.01, preferably 1:0.3 to 1:0.1.
Subsequently this solvent is partially or completely evaporated from the
solution or
dispersion and the liposome system is then formed by the addition of water.
'The inventive method provides a number of advantages. Surprisingly it was
observed,
that the inventive method makes is possible to produce in a particularly
simple, rapid
and cheap manner aqueous liposome systems which possess a pronounced storage
stability, which do not lead to sedimentation, which do not lead to formation
of
deposits on the inner vessel walls and which show in addition a large
transparency,
allowing a particularly simple and rapid inspection on foreign particles.
Furthermore
to the liposome system produced according to the inventive method shows highly
reproducible particle sizes, in particular such unilamellar or bilamellar
liposomes being
produced, with particle diameters varying between 30 and 100 nm, preferably
between
35 and 90 nm. It is also possible, to~ perform sterile filtration of the
liposome system
produced according to the inventive method, where the use of a 0.2 pn filter
is being
~s preferred.
The organic solvent used for dissolving the at least one phospholipid and the
further
non-phospholipidic substance is a non-toxic solvent in which both the
phospholipid
and the non-phospholipidic; substance (bile acid, a bile acid salt, a bile
acid derivative
2o and/or a salt of a bile acid derivative) can be dissolved. It is preferred
to use as organic
solvent ethanol, 1-propanol, 2-propanol or benzyl alcohol.
Depending on the type of non-toxic organic solvent used and its mixing
properties with
water , the solvent is partiaaly or completely evaporatede from the solution
or
2s dispersion initially prepared according to the inventive method to
different final
amounts of the organic solvent. These final amounts may vary between 0 volume
and 20 volume %, preferably between 0 volume % and 10 volume %. When the
organic solvent used according to the inventive method is not water-miscible,
then the
organic solvent is removed to dryness.
I:f in the inventive method the reproducibility of the particle size of the
liposome
system should be further improved, so that in the liposome system which is
produced
by the inventive method the range of particle sizes is small, it is
recommended to
subject the liposome system, comprising the at least one phospholipid and the
further
non-phospholipidic substance (a bile acid, a bile acid salt, a bile acid
derivative and/or
a salt of a bile acid derivative) to an extrusion, a high pressure
homogenisation and/or
an ultrasound treatment. Preferably tlhis treatment is performed at a
temperature below
40 °C, in particular at a temperature lbetween 20 °C and 30
°C.
to
The duration of the extrusion, the high pressure split homogenisation or the
ultrasound
treatment is chosen to be sufJ:iciently long for the liposomes to show the
wanted mean
diameter. Said extrusion, high pressure split homogenisation or ultrasound
treatment is
performed until the liposomes formed possess a mean diameter of between 30 nm
and
is 100 nm, preferably between .35 nm and 90 nm.
A further embodiment of the inventive method is the sterile filtration of the
liposome
system produced according to the inventive method, preferably with a 0.2 p,m
filter.
2o The liposome systems produced according to the inventive method can be
filled
directly in corresponding ampoules in a condition ready for use. According to
a
particularly suitable embodiment of t1e inventive method the liposome system
which
is formed after the addition of water is dries carefully after the addition of
a suitable
additive in particular a carbohydrate, whereby lyophilisation constitutes the
best
2s method for this careful drying. According to this embodiment of the
inventive method
a powder-like liposome system is obtaiined, which can be reverted into the
vesicles by
the: addition of a suitable amount of water. It is not necessary to subject
the liposome
system formed after the addition of water to extensive agitation or for
instance to high
pressure split homogenisation or ultra~~ound treatment.
9
21~~05~
As has been explained before, it is preferred to use in the inventive method a
soybean
phospholipid and in particular such a soybean phospholipid which contains a
large
concentration of phosphatidylcholv:~e, preferably at least 90 % by weight
phosphatidylcholine.
Two possibilities exist to produce the liposome system according to the
inventive
method comprising the above-mentioned pharmaceutically or cosmetically active
ingredient.
According to the first possibility, tt~e active ingredient is added together
with the
phospholipid and the furthex non-phospholipidic substance ( a bile acid, a
bile acid
salt, a bile acid derivative and/or a salt of a bile acid derivative) in the
above-
mentioned mass ratio to the; organic; solvent used.
is According to a variant of tHus method, a phospholipid is used which is
loaded with the
active ingredient. For this purpose the selected phospholipid is soaked with
the non-
aqueous solution, dispersion or suspension containing the active ingredient
and
subsequently performing a careful drying of the phospholipid loaded with the
active
ingredient in this manner. Thereafter the phospholipid loaded with the active
2o ingredient is dissolved together with the further substance which is not a
phospholipid
in an organic solvent, whiclh may optionally be a different one from solvent
used for
the loading step. Hereafter the org<~nic solvent is removed completely or
partially as
was described above at the description of the inventive method, and when in
the final
step the water is added a liposome system with an encapsulated active
ingredient is
2s formed. This variant of the inventive method is especially recommendable in
cases
where the active ingredient shows a good storage stability.
According to the second possibility which is to be preferred when the active
ingredient
is better soluble in water than in thc: organic solvent, the at least one
phospholipid is
__ l0 2~~.°~57
dissolved or dispersed initially together with the at least one non-
phospholipidic
substance in the selected organic solvent. After the removal of the solvent
from the
solution or the dispersion the; active ingredient is added together with the
water,
whereby it is preferred to dissolve the active ingredient in the water.
A, modification of the above-described variant of the method, which is used
especially
when the active ingredient has a limited stability, is based on a powder-like
dried
liposome system. During the redispersion step the active ingredient is added
together
with the amount of water, so that the active ingredient is encapsulated into
contact with
liposome system only immediately prior to the use of such a product and is
therefore
pootected against ageing.
To prevent unwanted side effects, the; inventive method is performed under
protection
gas (inert gas).
is
As has been mentioned before at the description of the inventive aqueous
liposome
system, the preferred phospholipid concentration lies between 5 % by weight
and 25
b~~ weight, in particular between 8 % by weight and 18 % by weight, whereby,
depending on the intended use and the type of treatment, these concentration
values
2o may be higher and lower, as long as tlhe mass ratio of phospholipid to the
further non-
phospholipidic substance is v~~ithin thE: range given before. Accordingly a
required
amount of phospholipid is selected anal the mass ratio of phospholipid to the
at least
one non-phospholipidic substance is chosen according to the defined range (
1:0.001 to
1:0.1).
2s Preferred embodiments of the inventive liposome system and the inventive
method are
indicated in the subclaims.
Tine following examples are fi.u-ther illustrations of the inventive method.
11
2~.~.9Q5'~
For the preparation of sevexal aqueous liposome systems, the following
compositions I
to VIII are dissolved in 500 ml ethamol. Subsequently the solvent is removed
under
vacuum to dryness. The re~;idue wa.s dispersed in 1000 ml water and brought
thereafter
by high pressure split homogenisatiion to a mean particle diameter between 30
nm and i
s 100 nm. Hereafter the obtained liposome system was filtrated under sterile
conditions
through a 0.2 pm filter and filled under sterile conditions into ampoules.
To prevent unwanted oxidation reactions the preparative method was performed
under
inert gas (nitrogen).
The following compositions I to VfII were used according to the previously
mentioned ,
procedure, the indicated phosphatidylcholine being a highly pure soybean
phosphatidylclioline (3-sn-phosphatidylcholine, soya), which contained at
least 90
by weight phosphatidylcholine.
i
Composition I
100 g phosphatidylc;holine
2o Composition II
99,5 g phosphatidylcholine
0,5 deoxycholic acid sodium salt
Composition III
2s 99,5 g phosphatidylcholine
0,5 g chenodeoxycholic acid sodium salt
Composition IV
99,5 g phosphatidy~lcholine
12 21I9a5'~
0,5 g cholic acid sodium salt
~:omposition V
95 g phosphatidylcholine
s 5 g glycocholic acid sodium salt
~ om~osition VI
99 g phosphatidylcholine
1 g glycocholic acid sodium salt
to
Composition VII
99,5 g phosphatidylch~~line
0,5 g glycocholic acid sodium salt
i s composition VIII
99,7 g phosphatidylcholine
0,3 g glycocholic acid sodium salt
The aqueous liposome system of composition I had strongly differing properties
from
2o th.e compositions II to VIII as can be 'taken from the following table 1
13 2~ ~90.~'~
to 1e 1:
composition appearance mean particle transparency pH
size (nm) in % (660 nm)
I opalescent 123 ca 1 6,0
emulsion
II yellowish 48 89 6,1
transparent
fluid
III " 42 90 7,4
IV " 44 91 7,2
V " ~44 96 7,2
VI " 40 94 6,5
~I " 45 92 6,2
VIII " 45 89 6,2
The aqueous liposome systems II to VIII could easily and with a large yield be
to filtrates in a sterile manner through a 0.2 ~.m filter whereas filtration
of the aqueous
liposome system from composition I was not possible.
