Note: Descriptions are shown in the official language in which they were submitted.
- 2 - ~ 0 ~7~ 0~
The present in~ention relates to an alcoholic,
aqueous gel-like phospholipid composition and its use.
The present invention furth~rmore relates to topical
preparations contA i n ing it.
SGels are shape-ret~i~ing, easily deformable,
liquid-rich disperse systems composed o~ at least two
components. The best known and most widely distributed
gels are the aqueous gels, in which water is used as the
main component which is always present. Also known are
10the so-called organogels, in which the liquid main
component is an organic sol~ent and an added apolar
polymer which causes gelling and influences its strength.
Thus, wo 86/02264 describes a system of reversed
micelles which can be converted into corresponding gels
15by addition of suitable solvents, such as, for example,
squalene, Miglyol or vegetable oils.
Systems were also investigated in which gels are
formed from lecithin or general phospholipids, a solvent,
water or other required auxiliaries. However, such gels
20are often only used as interme~i~tes in order to form
liposome dispersions from them as the desired final
product.
Liposomes are spherical vesicles having a cover-
ing of one or more double layers (bilayer). They are
25preferably produced from lipids of natural origin. In the
ph~r~ceutical industry and in the cosmetics sector,
especially those liposomes composed of phospholipids play
a crucial role. The most important phospholipid sources
are soyabeans and other phospholipid-rich plants, for
30ex2mple rape or peanut, and to a lesser extent the
phospholipids are also obt~ine~ from eggs or from
animals.
Under certain conditions, phospholipids are able
to form liposomes in aqueous solution (Bangham, A.D.,
35Horne, R.W., J. Mol. Biol. 8 (1964), p. 660 et seq.).
Since then, numerous attempts have been made to prepare
stable liposomes which offer wide application possibili-
ties. The method proposed by RAnghAm (RAngh~m, A.D., et
_ 3 _ 2û~g(~7
.,,
al., Meth. in Membrane Biol. 1 (1976), pp. 1-68) of
dissolving phospholipids in an organic solvent, removing
the latter in a rotary evaporator to obtain, on the wall
of the flaslc, a lipid film which then forms liposomes on
dispersion in water or aqueous solution, cannot be
carried out on the industrial scale. The liposomes
prepared in this way can only be used for a small range
of applications.
Liposomes can be converted into smaller, uni-
lamellar vesicles by the sonication of multilamellar
liposomes, as can be obtained, for example! by the above
method, by means of ultrasound tC. Huang, Biochemistry 8
(1969), pp. 346-352).
Unilamellar liposomes can likewise be prepared at
relatively low pressures by means of the 'French press",
which consists in forcing multilamellar liposome
preparations prepared in a customary n~-nner through a
narrow opening (Hamilton, R.L., et al., J. Lipid Res. 21
(1980), pp. 981-992).
Another possibility for generating liposomal
solutions is the ethanol injection method of Batzri and
~orn (Batzri, S., Korn, E.D., Biochim. Biophys. Acta 298
(1976), pp. 1015-1019). In this method, the lipid dis-
solved in ethanol is injected into an aqueous buffer
solution so that liposomes form. This process, exactly
like the film method of Bangham, cannot be carried out on
the industrial scale. In both methods, the organic
possibly even toxic - solvent additionally has to be
le~oved in a complicated manner to obtain pharmaceutical
or cosmetic preparations.
Large unilamellar liposomes are also formed when
loaded lipids are susp~n~r~ in a buffer in the presence
of calcium cations (Papahad~opoulos, D., Ann. N.Y. Acad.
Sci. 308 (197~) p. 751). After removal of the calcium
cations, large unilamellar liposomes are then formed.
A recent method for the formation of liposomes
consists in ~ i ng phospholipids in a cationic detergent
to an organic solYent and transferring the lipid mixture
to a finely divided or finely structured surface such as
2~78~7
-- 4 --
....,~
molecular sieve, quartz or zeolites (D.D. Lasic, J. of
Coll. and Interface Sci. 124 82), (1988), pp. 428-435)
and then removing the solvent in vacuo. In this way,
phospholipid vesicles are formed directly.
The articles by Szoka, F. et al., in Ann. Rev.
Biophys. Bioeng. 9 (1980), pp. 467-508 and Lasic, D.D. in
Biochem. J. 256 (1988), pp. 1-11 give a general outline
of the most commonly used methods for liposome
preparation.
In EP-A-0 160 266 a liposome composition is
claimed which consists of a three-dimensional network of
liposomes and a networ~ material. For the networ~
material polysaccharides are preferably used in which the
liposomes are embedded.
According to WO 85/03640, loaded liposomes in a
gel matrix composed of starch or modified starch are
claimed.
In EP-A-0 069 307 a method for the preparation of
a liposome gel is described according to which an aqueous
or solvent-cont~ining lecithin solution is treated with
ultrasound. Depending on the sonication period and
sonication intensity, a more or less viscous gel is
formed. By prolonging the sonication time or by means of
mechanical stirring action, a liposome-contAi~i~g aqueous
solution is obtained as the final product.
A so-called preliposome gel is obtained from a
mixture of phospholipids, fatty acids and a hydrating
agent according to EP-A-0 211 647. Liposomes are formed
after addition of water or buffer solution.
A phospholipid-cont~ining, highly fluid gel is
claimed in WO 89/00077 for use as aerosol liposomes. The
system consists of lecithin, an organic solvent and a
little water. A broad span of liposome diameters in the
range from 100 to 2500 nm occurs here; a high solvent
content must be selected for a worthwhile application
range.
US-A-2,0gO,537 relates to a process for the
preparation of "water-cont~ining" lecithin (lecithin
hydrate), consisting of a homogeneous mixture of
2 ~
preferably 15-25% vegetable lecithin, preferably 8-25%
alcohol, in particular ethanol or isopropanol, and 58-78%
water as the rP~A i nder . The water-con~Aining lecithin is
obtained by heating water and alcohol preferably to about
71~C (160~F), adding the lecithin and stirring. After
cooling to room temperature, a phase separation occurs in
which the lowermost phase of the three phases contains
the lecithin hydrate. This lecithin phase, saturated with
alcohol, water and oil, is already adequately stable as
such and can be further purified by removal of the
alcohol or a part of the water in vacuo, the water-
cont~i ni ng lecithin being obtained. Alternatively, this
phase can be obtained as a gel by acidifying to pH 4 to
pH 6.
EP-A-0 158 441 relates to a li~uid composition
contAining a homogeneous mixture of at least one membrane
lipid, at least one water-miscible organic solvent, for
example ethanol or propylene glycol which serves as a
solvent fo~ the lipid, and optionally an amount of water,
which is characterized in that this composition spon-
taneously forms vesicles or liposomes on addition of more
water, the weight ratio of lipid:solvent being 40:1 to
1:20.
EP-A-0 240 346 describes a preparation process
for liposomes having an enlarged reservoir for active
substances using the following process steps:
1. preparation of a liposome with and without an
active-substance reservoir from a phospholipid;
2. dispersion of the liposome in an active substance-
contAini ng liquid;
3. addition of a slightly volatile organic solvent to
the dispersion with gel formation; and then
4. removal of this organic solvent by evaporation and
reconstitution of the liposomes.
The liposomes according to process step 1 are
either obtained as multilamellar liposomes in a ~nner
known per se or as unilamellar liposomes by ultrasonic
treatment. The liposomes according to process step 4 no
longer obtain (sic) substantial amounts of organic
~ ~ 7 8 ~ 7 ~
solvents after their working-up
The present application is directed towards the provision of an alcoholic, aqueous
gel-like phospholipid composition which is self-preserving, storable and transparent.
In the present invention, the gel-like phospholipid composition being a liposomal
5 gel, i.e. a system built up exclusively from liposomes, which consists of a phospholipid
concentrate of specific composition, alcohol and water in specific concentrations and
whose aqueous phase is virtually exclusively the internal phase.
Accordingly, in one aspect of the present invention, there is provided a liposomal
gel composition comprising an aqueous phospholipid composition which comprises: (a)
15 to 30 parts by weight of a phospholipid concentrate, consisting of: (i) 70 to 80 parts by
weight of phosphatidylcholine, (ii) 5 to 15 parts by weight of at least one acidic
phospholipid selected from the group consisting of phosphatidylethanolamine,
phosphatidic acid, N-acylphosphatidylethanolamine and ~ lu~es thereof, (iii) 5 to 25
parts by weight of at least one other phospholipid selected from the group consisting of
15 lysophosphatidylcholine, phosphatidylinositol and llli~UleS thereof, and (iv) 1 to 15 parts
by weight of at least one phosphorous-free lipid per 100 parts by weight of (i), (ii) and
(iii); (b) 20 to 14 parts by weight of at least one alcohol, and (c) 50 to 71 parts by weight
of an aqueous solution.
The gel-like phospholipid composition according to the invention has a
20 transparent structure which is homogeneous and substantially free of agglomerates and
other clouding agents and has a mean particle size of 200 nm + 20%. (Electron
microscopy, freeze-fracture). The liposomal solution obtained from the gel-like
phospholipid composition by dilution with aqueous solution preferably has an average
liposome size of 200 nm + 20% (det~rrnined by the laser light-sc~tt~rin~ method) and is
25 thus preferably employed in topical preparations, such as cosmetic or pharmaceutical
preparations, which require a liposome particle diameter of 100-400 nm, preferably 100-
200 nm. A particular advantage is that these liposomes remain transparent, in dependence
of active substance, not only in the unloaded state, but also in the loaded state.
Additionally, both the gel and the liposomal solution can be prepared in sterile and
30 pryogen-free form, according to German Pharmacopeia 9, so that they can be forrnnl~3ted
to give cosmetic and pharmaceutical preparations without additional, possibly allergenic,
preservatives. Furthermore, it has been surprising for the person skilled in the art that
B
' ,_
alcohol in concentrations of 14 to 20% by weight does not lead to destruction of the
liposome solution.
Finally, a liposomal solution can be obtained in an in(lllstri~lly simple mannerfrom the phospholipid composition (liposome gel) according to the invention without
having to carry out process steps which are industrially and energetically complex, that is
to say in particular without increasing the temperature or employing ultrasound.In accordance with another aspect of the present invention, there is provided a
method for preparing a liposomal solution comprising: (a) dissolving in at least one
alcohol a phospholipid concentrate comprising (i) 70 to 80 parts by weight of
phosphatidylcholine, (ii) 15 to 5 parts by weight of at least one phospholipid selected
from the group con.cisting of phosphatidylethanolamine, phosphatidic acid, N-
acylphosphatidylethanolamine, and mixlu~es thereof, (iii) 5 to 25 parts by weight of at
least one other phospholipid selected from the group consisting of
lysophosphatidylcholine, phosphatidylinositol and lllixLules thereof, and (iv) 1 to lS parts
by weight of at least one phosphorous-free lipid per 100 parts by weight of (i), (ii) and
(iii), (b) adding an aqueous solution to produce a liposomal gel comprising (i) 15 to 30
parts by weight of said phospholiid concentrate; (ii) 20 to 14 parts by weight of said one
alcohol, and (iii) S0 to 71 parts by weight of said aqueous solution, and (c) adding a
further amount of water to convert said liposomal gel to said liposomal solution.
The phospholipid concentrates, as one of the constituents of the phospholipid
composition according to the invention, are obtained from natural phospholipid nlixlul~s,
for example from oil seeds, such as soybean, rape, sunflower etc.
An enrichment process for the preparation of phospholipid concentrates of this
type is described in EP-A-0 069 770. Phospholipid concentrates of this type consist of
phospholipids (phosphatidylcholine, acidic phospholipids and other phospholipids) and
phosphorous-free associated lipids. The acidic phospholipids include
phosphatidylethanolamine, phosphatidic acid and also N-acylphosphatidylethanolamine.
The other phospholipids include lysophosphatidylcholine and phosphatidylinositol. The
phosphorous-free associated lipids include, inter alia, glycolipids and phytolipids. The
phosphorous-free associated lipids are present in the phospholipid
~,
2 ~ 7
-- 8 --
concentrates in 1-15 parts by weight, preferably
1-9 parts by weight, particularly preferably 1-5 parts by
weight, relative to 100 parts by weight of phospholipids.
The phospholipid concentrate described above thus
S has the following composition:
60.87-79.21% by weight of phosphatidylcholine
14.85- 4.35% by weight of acidic phospholipids
4.95-21.74% by weight of other phospholipids and
0.99-13.04% by weight of phosphorus-free associated
lipids.
A further preferred embodLment of the phospho-
lipid concentrate as a constituent of the phospholipid
composition according to the invention is a mixture of
80 parts by weight of phosphatidylcholine, 5-15 parts by
weight of acidic phospholipids, and 15-5 parts by weight
of other phospholipids, this concentrate furthermore
contA; n; ng 1-9 parts by weight of phosphorus-free
associated lipids per 100 parts by weight of the above
phospholipids. A preferred phospholipid concentrate of
this type thus has the following composition:
73.39-79.21% by weight of phosphatidylcholine,
4.~5-13.76% by weight of acidic phospholipids,
14.85- 4.59% by weight of other phospholipids and
0.99- 8.26% by weight of phosphorus-free associated
lipids.
The phospholipid concentrate is employed in
amounts from 15.00 to 30.00 parts by weight, preferably
20.20 to 30.00 parts by weight/100 parts by weight of the
phospholipid composition according to the invention.
The alcohol is employed in amounts of 14 to
2~ parts by weight, preferably about 16 parts by weight,
per 100 parts by weight of the phospholipid composition
according to the invention.
The aqueous solution is employed in amounts of 50
to 71.00 parts by weight, preferably 54.00 to 63.80 parts
by weight, per 100 parts by weight of the phospholipid
composition. Aqueous solution in the sense of the present
invention is understood as meaning once-distilled water,
tap water, purified water, German Pharmacopeia g,
20~7~7
g
d~mineralized water, and also buffer solutions, such as,
for example, phosphate buffer or a physiological saline
solution.
In the liposomal solution obtained by dilution of
S the phospholipid composition with stirring, the con-
stituents are present in the following concentrations:
The phospholipid concentrate is present in the
liposomal solution in amounts from 10.10 to 20.20 parts
by weight, preferably 10.10 parts by weight, relative to
100 parts by weight of the total limposomal (sic) solu-
tion. The alcohol is present in the liposomal solution in
amounts from about 16 parts by weight, relative to
100 parts by weight of the total liposomal solution. The
aqueous solution is present in the liposomal solution in
amounts from 63.80 to 73.90 parts by weight, relative to
100 parts by weight of the total liposomal solution.
According to a preferred embodiment of the present
invention, at least one biologically active substance can
be ~m; xed to the liposomal gel. Examples of active
substances of this type are anti-inflammatories such as
ketoprofen, bisabolol etc., anticoagulants such as
heparin, hirudin etc., antimycotics, and also spasmoly-
tics or circulation-promoting agents such as papaverine.
The present invention furthprmore relates to
topical preparations which contains (sic) at least one of
the phospholipid compositions described above in combina-
tion with at least one biologically active substance and
customary auxiliaries and additives. Biologically active
substances which are intended to be administered in
combination with gels are, for example, the active
substances described above.
The present invention furthP~ore relates to
phArmAceutical preparations which contain at least one
phospholipid composition of the type described above in
combination with at least one biologically active sub-
stance, preferably for the treatment of the indications
described above.
The present invention finally relates to cosmetic
preparations which contain at least one phospholipid
~7~07
-- 10 --
composition described above in combination with at least
one cosmetic active substance for care of the skin and
hair, it preferably being a caring agent penetrating into
the horny skin, such as, for example, urea, elastin etc.
A particularly preferred embodiment of the
present invention is a gel which consists of 20~ phospho-
lipid (having a content of 80% phosphatidylcholine) and
16% ethanol and which is specified by the following
parameters:
- Appearance golden brown, slightly
cloudy gel
- Transmission at least 50% (German
(c = 0.5~ in water, 660 nm) Pharmacopeia 9, vol. 6.19)
- Viscosity 5000 + 2000 mPa.s
(measured at 20~C)
Electron microscopical investigation by the
method of Muller, T. et al., Seifen Ole Fette Wachse 3,
88-89 (1989) shows, after use of the freeze-fracture
technique, the liposomal structure of the gel (Fig. 1).
The liposomes were detected by the method des-
cribed by ZellmAnn et al., ZEISS Application EM 902 Cryo,
1989 (Fig. 2).
The present invention is illustrated in greater
detail below by means of 2 figures which show preferred
embodiments of the invention.
The figures show:
Fig. 1: The liposomes of the sample, prepared by the
freeze-fracture technique, of the gel prepared
according to the invention are shown in the form
of an electron micrograph. The liposomes form a
vesicular gel. They are in close contact with one
another and additional water cannot be seen.
(1 measuring unit corresponds to 350 nm).
Fig. 2: The cryoelectron micrograph of a 3% strength
dispersion of the gel prepared according to the
invention in water shows that such a preparation
exclusively contains multilamellar liposomes.
(1 measuring unit corresponds to 350 nm).
The gel-like phospholipid composition is prepared
11- 2~7~-7
. ~,....
in an industrially particularly simple ~-~er by stirring
the phospholipid concentrate of dete~ine~ composition
with a detPrmine~ amount of alcohol for a short tLme and
inducing gel formation by addition of water and further
stirring. The stirring can be carried out using any
commercially available stirrer.
However, this stirrer must have a sufficiently
high speed so that thorough mixing is achieved in a short
time. In this process, the starting material is a phos-
pholipid composition which in general has a pH in therange from 5 to 8, preferably 6.5 to 7.5.
The invention will now be illustrated in greater
detail by exemplary embodiments, the following phospho-
lipid concentrate composition being used:
The phospholipid contents of this composition
consist of:
phosphatidylcholine 80~;
acidic phospholipids 15%;
other phospholipids 5%.
The phosphorus-free associated lipids lie at (sic) 5
parts by weight, relative to 100 parts by weight of
phospholipids, i.e. 4.76% by weight of phosphorus-free
associated lipids are present.
ExamPle 1
10.48 g of the phospholipid concentrate (con-
t~ i n; ng 10 g of phospholipids) are dissolved in 8 g of
ethanol with stirring. The solution has a viscosity of
806 mPa.s (at 25~C) and is homogeneous. The solution is
homogenized for 3 min. with 31.52 g of demineralized
water, a commercially availa~le high-speed laboratory
stirrer being used. A transparent gel cont~ini~g 20.96~
by weight of phospholipid concentrate (20% by weight of
phospholipids) i~ obt~inP~.
Exam~le 2
15.72 g of the phospholipid concentrate (con-
t~ i n i ng 15 g of phospholipids) are dissolved in 8 g of
ethanol analogously to Example 1. The solution is stirred
for 3 min. with 26.28 g of demineralized water until a
homogeneous, transparent gel is formed. The gel contains
- 12 - 2~ 7
, ,.
31.4% by weight of phospholipid concentrate (30% by
weight of phospholipids).
Example 3
15.72 g of the phospholipid concentrate (con-
taining 15 g of phospholipids) are dissolved in 8 g of 2-
propanol analogously to Example 1. 26.28 g of
demineralized water are added and the mixture is stirred
for a further 3 min. A transparent gel is formed which
contains 31.40~ by weight of phospholipid concentrate
(30.00 parts by weight of phospholipids).
ExamPle 4
10.48 g of the phospholipid concentrate (con-
t~in;ng 10 g of phospholipids) are dissolved in 8 g of
2-propanol as in Example 1. After addition of 36.52 g of
demineralized water, the mixture is stirred for 3 min.
and a transparent gel is obtained contAin;ng 19.05% by
weight of phospholipid concentrate (18.18~ by weight of
phospholipids).
The following examples show how liposomal solu-
tions are obtained from the phospholipid-cont~ining
liposomal gel by means of simple process steps.
Example 5
The entire amount of the phospholipid gel (50 g)
obtained in Example 1 is mixed with 42 g of 0.2 molar
phosphate buffer solution of pH 7.4 and stirred for
4 min. The resulting highly fluid dispersion is mixed
with 8 g of ethanol and additionally stirred for a
further minute to give the ready-to-use final product.
The proportions of phospholipid concentrate:ethanol:
aqueous solution are 20.96:16:73.04 (phospholipid:
ethanol:water are 10:16:74). The mean particle size,
measured by the laser light-scattering method, is 204 nm
(~ 20%).
Example 6
3S The entire amount of the phospholipid gel (S0 g)
obtained in Example 2 is mixed with 84 g of tap water,
stirred for 4 min. and 16 g of ethanol are then added.
After a further stirring time of 1 min., a liposomal
solution having an ethanol content of 16% by weight and
- 13 _ 20~7go7
,. .
10.48% by weight of phospholipid concentrate (corres-
ponding to 10% by weight of phospholipids) and a mean
particle size of lg4 nm (~ 20%) is obtained as the final
product. In spite of the use of tap water, which is
usually contAmi~Ated with microorganisms and salts, the
product obtains (sic) less than 100 microorganisms per
gram.
Example 7
111 g of physiological saline solution (0.~% by
weight sodium chloride) are added to the entire amount of
gel from Example 4 analogously to Example 6. After
stirring for 4 min., a further 24 g of 2-propanol are
added and the mixture is stirred for a further minute.
The mean particle size of the vesicles in the liposomal
solution is 200 nm (~ 20%).
ExamPle 8
The phospholipid gel obtained in Example 4 is
mixed with stirring with 37 g of 0.2 molar phosphate
buffer solution, stirred for 4 min., 8 g of 2-propanol
are added and the mixture is additionally stirred for a
further 1 min. The mean particle size of the vesicles in
the liposomal solution is 187 nm (+ 20%).
The number of microorganisms in the gel-like
phospholipid compositions according to the invention
according to Examples 1 to 4 and the liposomal solutions
obtained from these according to the invention according
to Examples S to 8 was determined in accordance with the
requirements of German Pharmacopeia 9 for Medicaments of
category 2, Preparations for topical or other types of
local application. In all cases, the number of
microorganisms was below 100 microorganisms/g of the
preparation and thus corresponds to the reauirements of
C~rmA~ Pharmacopeia 9.
PreParations contA i n ina bioloaicall~ active substances
The liposomes which are present in the gel
prepared according to the invention (Fig. 1) can be
loaded with various active substances. Surprisingly,
loading can be carried out both with lipophilic (for
example bisabolol) and with hydrophilic (for example
- 14 _ 2~7~6
papaverine x HCl) su~stances.
Pre~aration 1:
97.0 g of gel prepared according to the invention
as in Example 1 are stirred with 3.0 g of bisabolol at
50~C for 10 min by means of a propeller stirrer. 15.0 g
of this mixture are diluted with 85.00 g of demineralized
water. The mean particle size of a solution diluted to
O.01% phospholipid with ~Pmi neralized water was 215 nm
(laser light-scattering).
Pre~aration 2:
4.0 g of papaverine HCl are dissolved in 36.0 g
of ethanol and homogenized with 360.0 g of gel prepared
according to the invention as in Example 1 in a rapidly
stirring mixer. The mean particle size was 175 nm (laser
light-scattering).
Preparation 3:
1.43 g of hirudin (100,000 ATU/100 g) are stirred
with 98.57 g of the gel prepared according to the inven-
tion as in Example 1 in a Fanta bowl and homogenized in
a rapidly stirring mixer. The mean particle size was
151 nm (laser light-scattering) and the pH was 6.9.
PreParation 4:
1.98 g of heparin Na are dissolved in 40.0 g of
ethanol, 204.33 g of ~mi neralized water and 3.7 g of
NaCl by means of a magnetic stirrer. This solution is
homogenized with 250.0 g of gel prepared according to the
invention as in Example 1 using a rapidly stirring mixer.
The mean particle size of a solution diluted to 0.01%
phospholipid with ~p~inqralized water was 231 nm and the
pH was 6.5.
Preparation 5:
1,0 g of ketoprofen are stirred in a Fanta bowl
with 1.60 g of ethanol, 90.0 g of gel prepared according
to the invention as in Example 1, 8.4 g of demineralized
water and 0.60 g of 10% strength aqueous sodium hydroxide
solution. The mean particle size of a solution diluted to
0.01% phospholipid with demineralized water was 216 nm.
. - 15 ~ 07
Preparation 6:
5 g of urea are stirred with 20 g of the gel
prepared as in ExampLe 1 and then diluted to 3%
phospholipid with demineralized water. Liposomes having
a mean particle size of 175 nm are formed.
Pre~aration 7:
5 g of elastin are stirred with 20 g of the gel
prepared as in Example 1 and then diluted to 3% phospho-
lipid with ~mi neralized water. Liposomes having a mean
particle size of 171 nm are formed.
