Note: Descriptions are shown in the official language in which they were submitted.
132~
The present invention relates essentially to a
composition based on hydrated lipidic lamellar phases
or on liposomes containing pregnenolone or a pregnenolone
ester.
The invention can be applied in particular in
the cosmetic or pharmaceutical sector, especially the
dermatological sector, for the preparation of compo-
sitions with regenerating or revitalizing activity, in
which it is incorporated.
Steroids ~ave already been used for the prepara-
tion of pharmaceutical or cosmetic compositions fortreating the skin. US patent 2 791 534 and British
patent 768 129, for example, disclose the use of
steroids as agents with antiwrinkle activity.
The effect of hormones on the skin has been
studied more particularly and it is indicated in the
article published in J. Soc. Cosmetic Chemists, volume
18, pages 549 to 562 (19 August 1967) that testosterone
produces a rejuvenating effect on the skin and that
progesterone and pregnenolone, which sre intermediates
in the biosynthesis of testosterone, produce a similar
effect, although to a much lesser degree, which is
particularly weak in the case of pregnenolone.
It is for this reason that pregnenolone, either
in the free form or in the form of an ester, has often
been used, for treating the skin, in combination with
other active agents.
Thus US patent 4 474 763 describes an anti-
wrinkle composition containing pregnenolone and
elastin, which is a skin proteln known for lts anti-
wrinkle activity.
,
:
~26~
-- 2
Likewise US patent 3 326 901 discloses a com-
position with an anti-irritating and antidehydrating
action which contains pregnenolone and allantoin.
The antidehydrating effect of pregnenolone on
the skin has also been mentioned more recently in
French patent application no. 2 405 069. This patent
application proposes a hormonal pharmaceu~ical com-
position containing a water-soluble estrogen (estrone),
a liposoluble estrogen (estradiol), an androgen (tes-
tosterone) and, if appropriate, pregnenolone to give~he composition an antidehydrating effect. According
to the author of this patent application, only the
sex hormones, i.e. male hormones and female hormones,
are capable of staving off the skin deterioration
phenomena due to the combined action of age and climatic
, aggression; on the other hand, it is indicated that
pregnenolone plays no part in the skin restoration
processes.
; Analysis of the prior art therefore shows that
the use of pregnenolone or a pregnenolone ester alone,
in skin treatment, has always been limited because of
~ the very weak activity of this substance.
; Furthermore, it is already known to use
hydrated lipidic lamellar phases or liposomes in
pharmaceutical compositions or cosmetic compositions
incorporating a variety of active principles (French
patent application A-2 521 565).
It has now been discovered, totally surprisingly
and unexpectedly, that pregnenolone or its esters have
an enhanced regenerating and revitalizing sctivity on
the skin and superficial body growths when this substance
is at least partially incorporated in a hydrated
lipidic lamellar phase or in liposomes. In particular,
a quite remarkable improvement has been observed in
the activity of pregnenolone on protein synthesis.
13264~9
-- 3
It is thus possible to deduce that the activity
of pregnenolone o~ pregne-~olone esters is subject to
some kind of potentiating effect in hydrated lipidic
lamellar phases or in liposomes.
Thus the effect of the present invention is to
solve the new technical problem of providing a novel
formulation of pregnenolone or a pregnenolone ester
which makes it possible to potentiate their efficacy so
as to enable them to be used in cosmetic or pharma-
ceutical compositions, especially dermatological
compositions, with regenerating or revitalizing
activity.
Thus, according to a first aspect, the present
invention provides a composition based on hydrated
lipidic lamellar phases or on liposomes, wherein
pregnenolone or a pregnenolone ester, preferably preg
nenolone acetate, sulfate or palmitate, is at least
partially incorporated in the said hydrated lipidic
lamellar phases or the said liposomes.
In another advantageous embodiment of the
invention, a sterol, preferably p-sitosterol, is also
at least partially incorporated in the above-mentioned
hydrated lipidic lamellar phases or in said liposomes of
the abo~e-mentioned composition.
More particularly, the sterol is incorporated
in the lipidic phase of the hydrated lipidic lamellar
phases or of the liposomes in a concentration by weight
of between about 1% and 20%, preferably of about 10%.
In one modified embodiment of this composition,
the pregnenolone or the pregnenolone ester of the said
composition, by itself or mixed with the sterol, is
introduced into the lipidic phase of the hydrated
lipidic lamellar phases or of the liposomes.
More psrticularly, the concentration of preg-
nenolone or pregnenolone ester is between about 0.05%
132~4~
- 4 -
and 20%, preferably between about 1~ and 15% and most
preferably between about 1% and 5% of the weight of the
said lipidic phase, so that the sum of the concentrations
in this phase of pregneno]one or pregnenolone ester, on
S the one hand, and any sterol present, on the other, does
not exceed about 25% and preferably about 20%.
In another modified embodiment of this com-
position, the pregnenolone ester of the said composition
is introduced into the aqueous phase of the hydrated
lipidic lamellar phases or of the liposomes. In this
case, of course, the solubility of the said ester in
water must be such that at least the desired concentra-
tion can be obtained in the said lamellar phases or the
said liposomes.
More particularly, the concentration of the
pregnenolone ester is between about 0.001% and 5% and
preferably between about 0.01% and 2% of the weight of the
said aqueous phase.
The incorporation of the pregnenolone or its
esters, and if appropriate the sterol, into the hydrated
lipidic lamellar phases or into the liposomes according
to the invention can be carried out by known processes.
These are chosen more particularly according to the
degree of lipophilicity or hydrophilicity of the com-
pound to be incorporated.
In a preferred embodiment of the compasitionbased on hydrated lipidic lamellar phases or on liposomes
according to the invention, a preparative technique
described in French patent F~ -A-2 521 565, in
combination, if appropriate, with a technique described
in French patent FR-A-2 534 587, is chosen.
According to a second aspect, the present
invention further relates to a cosmetic or pharmaceutical
composition, especially a dermatological composition,
with regenerating or revitalizing activity, which
132~
: 5
comprises a composition based on hydrated lipidic
, lamellar phases or on liposomes, as defined previously.
The proportion by weight of the pregnenolone
or the pregnenolone ester is preferably between about
;~ 5 0.0005% and 5%, more preferably between about 0.002~ and
1% and most preferably between about 0.01 and 0.5% by
weight, relative to the total weight of the composition.
- Embodiments of the invention will now be described by
way of example only with reference to the accompanying drawings
in which:
Figures 1 and 2 each represent a plot of the
multiplylng power of substance ver8u8 the content of pregnenolone
acetates contained in the sub8tances.
:
Other objects, characteristics and advantages
of the invention will become more clearly apparent from
the following explanatory description referring to
several illustrative Examples, which cannot in any way
limit the scope of the invention. In these Examples,
the percentages are given by weight, unless indicated
otherwise.
EXAMPLE 1
A - Preparation_of a composition in the form of a lipidic
Dowder containin~ pre~nenolone acetate
100 mg of pregnenolone acetate and 1 g of ~-
sitosterol are dissolved in 100 ml of methylene chloride
and 8.9 g of soya lecithin are added, if appropriate in
the presence of a lipophilic antioxidant, for example
0.2 g of ~-tocopherol.
~2~
- 5a -
The solution obtained is atomized at 65C in
the manner described in French patent ~ ~ A-2
521 565. This gives a lipidic powder composed of small,
substantially spherical particles with a diameter of
about 1 to 40 nm.
B - Preparation of a comDosition in the ~ osome
suspension which is advanta~eouslv homoQenized
5 g of the powder obtained in step A are dis-
persed in the appropriate amount of water to give a
final volume of 250 ml. This operation is carried out
at ordinary temperature for 1 h, with magnetic stirring.
`- 1326~9
-- 6
This gives a liposome suspension after homogeni-
zation either by means of ultrasound or by means of a
homogenizer under pressure, for example according to
the process described in French patent F~ -A-2
534 487.
If, for example, homogenization is effected by
treatment with ultrasound for 15 min, liposomes with a
size of between 135 and 186 nm are obtained.
250 ml of a homogenized liposome suspension in
which the proportion of lipidic phase is 2% are therefore
obtained after ~his step B.
EXAMPLES 2 to 7
Compositions in the form_of l~ome suspensions con-
tainin~ pre~nenolone acetate
Several compositions in the form of liposome
suspensions are prepared by the process described in
` Example 1, the proportions of the constituents of the
lipidic phase being varied.
Table I below indicates the compositions of the
different suspensions obtained.
EXAMPLES 8 to 10
; Compositions in the form of liposome suspensions con-
taininR pre~nenolone
Different liposome compositions are prepared by
~5 the process of Example 1, the pregnenolone acetate being
replaced with pregnenolone and the proportions of the
constituents of the lipidic phase being varied. The
compositions of the different liposome suspensions are
shown in Table I.
-- 132~9
. - 7
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~,
',
O CO O O O ~ O
~ __ _
., ~ O O O O O O _l
Ot) 00 O O _l ~I O O
~ ~ _ ._ _ .__ ... __ O
r- a~ o o o ~ c~ o
_ O
~0~ C~l O O ~I ~ O
_ . .__ o- - _
~ ~ U~ I~ 1-~ O O N O O
. _ _ _
'."' ~ a) ~ ~o o ~ o~ o
;. H _ _ _
~ ~ 0~ O U~ O ~ O O
,`, ~`I O O O O O C~l O
. _.
~`I
0, ~ O ~ O O
C _ __ _
~ ~1 ~ a~ ~n Il~
S b C C C O C
. ~1 -01 -01 ~ -O~ O
~ ~ C ~ C ~ C O~ C
_~ O OJ ~ ~J) ~r~ O~ 4
~J 4 ~ t~ C 0 C ta C
O ~ b U b .,~ ~I) Ql b
U~ ~ P.ta ~ ~ ~0 C~
. _ ~ .
~ ~q e
. C t~ ~ .,~ o .. .4
c o rl ~c ta r1 0 00
~ .,, ~, _ a) .. m a)
,~ o,l ~ e ~ c 3
e o ~ 30
I:Ll ~ v a.l~ o c
. . . j
1326~9
-- 8 --
EXAMPLE 11
StudY_of the influence of the sterol on the stabilitv of
the liposomes containin~ Pre~nenolone or a pre~nenolone
ester
. _
The aim is to determine the influence of ~-
sitosterol on the stability of the liposomes according
to the invention.
This is done by preparing a reference suspension
according to the process of Example 1, the composition
of the lipidic phase being 90% of soya lecithin and 10
of ~-sitosterol.
The stability of the liposomes is evaluated by
determination of their size using an appropriate
instrument such as a NANOSIZER . Measurements are taken
at time zero (when preparation is complete) at room
temperature and then on day 8 and day 15 at 4C, room
temperature and 40C. The polydispersity, which
quantifies the scatter of the liposome sizes, and the
mean size, are shown in Table II for each measurement.
The quality of the liposome suspension, one of the
criteria of which is the stability, is generally better
the smaller the values of these quantities and the more
uniform they are with time.
The stability of the liposomes can therefore be
evaluated by measuring their size since it is known
that, if they are unstabla, the liposomes tend to fuse
together, they increase in size and, beyond a certain
size, they form a sediment.
Table II clearly shows that the incorporation
of pregnenolone acetate is favored by the presence of
~-sitosterol. When the powder contains no ~-sitosterol
(Ex. no. 7), sedimentation is observed after 8 d at 40C
and the values of the polydispersity and size are large.
In the other cases, the size of the liposomes
remains substantially constant and at an acceptable
` 1326~
~ _ 9 _
'
value.
Moreover Table II shows that the incorporation
of ~-sitosterol in a proportion of more than about
15% by weight (Ex. no. 6) in the lipidic powder causes
sedimentation phenomena and detracts from the stability
~; of the liposomes containing pregnenolone acetate.
The advantage of the presence of ~-sitosterol
is clearly apparent, its concentration preferably being
10% of the weight of the lipidic phase (Ex. no. 4).
.
:
- 1326~
-- 10 --
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~J ,,~ c c ~ a~ ~ a) c
'D O O O a) a~ O O E c ,-~ ~ E c u~ E O
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E Q. :~ N ~ O ~ N 11
o ~ ~1 o ~ ~ E~
V 0 r~ ~ D U~ oq V Cl:
_ __ . _ _ --,.
:~ ~32g~
-- 1 1 --
F.XAMPLE 12
Evaluation of the multiplyin~ power o~_the compositions
accordin~ to the invention on human fibroblasts in
culture
The method of evaluating the multiplying power
of substances on cells in culture is derived from the
; method of Mrs. ADOL,PHE et al., J. Cosmetic Sci. (1983),
6, 55.
- Human fibroblasts are cultivated in a Petri
dish (35 mm) in MEM (Eagle's minimum essential medium)
containing 5% of calf serum.
After 24 h (37C, 5% C02), the medium is
replaced with a mixture consisting of MEM and the test
~ substance at different concentrations. This mixture
; 15 has a calf serum concentration of 2.5~. A reference
containing 2.5% of calf serum but no additional sub-
stances is prepared in parallel.
The cells are then incubated at 37C for 7 d
in a humid atmosphere containing 5% of C02. Three
experiments are carried out for each culture.
After washing with phosphate buffer and detach-
ment of the cells with trypsin, the cells are counted
using an appropriate instrument.
The multiplying power of the substances is
expressed by the following formula:
N.P. - N 2.5
A.P. , x 100
N 2.5
in which A.P. represents the multiplying power of the
cells, N.P. represents the number of cells cultivated
in the presence of the test substance and N 2.5
represents the number of cells cultivated in the
reference, i.e. in the presence of ~.5% of calf seruDI.
The results obtained for different concentrations
-` 1 3 2 ~
- 12 -
of pregnenolone acetate, abbreviated to PREAC, are
represented in Figure 1 in the form of a histogram.
` Figure 1 shows that, either free or incorporated
in liposomes, the pregnenolone acetate has no significant
. 5 effect on the increase in the number of human fibro- blasts in culture~
~, .
' EXAMPLE 13
c Evaluation of the stimulatinR activity on protein
.'~ synthesis
The activity on the protein content is evaluated
by a method derived from the one described by JOHNSON
and J.A. LOTT (1978), Clin. Chem. 24.
When the culture performed in Example 12 above
is complete, the dishes are washed twice with phosphate
buffer and then treated with 1 ml of sodium hydroxide
solution (0.5 N).
After shaking, 30 ~1 of cellular supernatant
are added to 2 ml of a solution of Coomassie blue.
The protein concentration is obtained by
measurement on a proti-analyzer (Marius Instruments),
which automatically evaluates the ratio of the optical
densities:
OD 590 nm
OD 465 nm
i
The activity on the "protein content" for each
substance at each concentration is expressed by the
formula:
TS ~ T2 5
T.P. = x 100
T2.5
132g~
- 13 -
in which T.P. represents the activity on the protein
content, TS represents the protein content obtained on
cultures in the presence of the test product and T2 5
represents the protein content obtained on cultures
in the presence of the reference, i.e. in the presence
of 2.5% of calf serum.
Figure 2 shows the histogram of the results
for the activity on the protein content at different
pregnenolone acetate concentrations.
Figure 2 very clearly demonstrates the stimula-
ting action on protein synthesis of pregnenolone
acetate incorporated in liposomes, whereas, in the free
state, this same product exhibits practically no action.
In conclusion, the tests performed on cultures
of human fibroblasts show the value of the compositions
according to the invention in maintaining or restoring
the normal biological processes of the skin and super-
ficial body growths through their stimulating action on
the synthesis of tissue proteins.
On the one hand, it is a known fact that this
synthesis is slowed down when the cells divide. The
absence of a particular action of the compositions
according to the invention on cell division does not
therefore constitute a handicap. On the other hand,
the existence of tissue proteins is known to be important,
in both quantity and quality, for the good physio-
logical condition of the skin. The stimulating action
of the compositions according to the invention on the
synthesis of such proteins is therefore of very great
interest, especially in cosmetics and in dermatology.
EXAMPLE 14
Gelled composition based on liposomes
soya lecithin 0.89 g
~-sitosterol 0.10 g
1326~ ~9
- 14 -
pregnenolone acetate 0.01 g
gelled excipient qs 100 g
First of all a lipidic powder is prepared
according to Example l-A. This powder is then dispersed
in water at a concentration of 4% according to Example
~` l-B and, if necessary, stabilized by means of a cos-
metically or pharmaceutically acceptable preservative
at the usual concentration.
The suspension obtained is then gelled by
mixing 1 part of suspension with 3 parts of an aqueous
gel of CarbopolR 940 separately prepared in conventional
manner at a concentration of 5%.
The gelled composition obtained can be used
either for incorporation in cosmetic or pharmaceutical
compositions, or as such, with added fragrances if
desired, to care for the skin on the body or face,
advantageously by daily application.
EXAMPLE lS
Cosmetic com~osition in the form of a cream
soya lecithin 4 g
~-sitosterol 0.5 g
pregnenolone acetate O.S g
excipient for oil-in-
water emulsion qs 100 g
A suspension of liposomes according to Example
2, on the one hand, and an oil-in-water emulsion, on
the other, are prepared separately. These two prepara-
tions are then mixed in proportions of 1 volume of
liposome suspension to 3 volumes of emulsion.
The cream obtained is applied to the face,
preferably in the evening.
:
1326~9
..
-- 15 --
s EXAMPLE 16
Cicatrizant dermatolo~ical composition
soya lecithin 8 g
~-sitosterol 1 g
pregnenolone acetate 1 g
gelled excipients qs lO0 g
` This composition is obtained from the powder
prepared according to Exa~.ple 4 and dispersed at a
concentration of 20% by means of a homogenizer under
pressure. The dispersion obtained is then mixed with
1 volume equivalent of a 5% gel of CarbopolR 940.
This composition has excellent cicatrizant
properties. It is indicated in particular for the
prevention and treatment of vergeture by application
twice daily.
The invention also covers processes of preparation of the
above-defined composition per se and of the above-defined
cosmetic or pharmaceutical composition. Generally, those processes
of preparation comprise incorporating at least partially
pregnenolone or a pregnenolone ester, such as, in particular,
pregnenolone acetate, sulfate or palmitate, in hydrated lipidic
lamellar phases or in liposomes. Particular embodiments are set
forth with the above-said composition particular embodiments.
