U.S. patent application number 11/752189 was filed with the patent office on 2007-09-20 for complete nutrient medium for use as a cosmetic and cosmetic use thereof.
This patent application is currently assigned to Jean-Noel THOREL. Invention is credited to Hugues Gatto, Jean-Noel Thorel.
Application Number | 20070219157 11/752189 |
Document ID | / |
Family ID | 26231681 |
Filed Date | 2007-09-20 |
United States Patent
Application |
20070219157 |
Kind Code |
A1 |
Thorel; Jean-Noel ; et
al. |
September 20, 2007 |
COMPLETE NUTRIENT MEDIUM FOR USE AS A COSMETIC AND COSMETIC USE
THEREOF
Abstract
A cosmetic composition contains an aqueous complex nutritive
base comprising a plurality of amino acids, at least one vitamin, a
plurality of assimilable organic components, and at least one
inorganic salt. The cosmetic composition does not contain a
biological extract of animal or cellular origin, or a living
nourishing substrate. A cosmetic method comprises contacting human
skin with the cosmetic composition.
Inventors: |
Thorel; Jean-Noel; (Paris,
FR) ; Gatto; Hugues; (Albertville, FR) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 19928
ALEXANDRIA
VA
22320
US
|
Assignee: |
Jean-Noel THOREL
Paris
FR
|
Family ID: |
26231681 |
Appl. No.: |
11/752189 |
Filed: |
May 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10892376 |
Aug 11, 2004 |
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11752189 |
May 22, 2007 |
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08860231 |
Jul 25, 1997 |
6821780 |
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PCT/FR96/00037 |
Jan 9, 1996 |
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10892376 |
Aug 11, 2004 |
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Current U.S.
Class: |
514/45 ; 435/371;
514/23; 514/440; 514/506; 514/579; 514/663; 514/738 |
Current CPC
Class: |
A61K 8/365 20130101;
A61K 8/27 20130101; C12N 2500/95 20130101; A61K 8/345 20130101;
A61K 8/494 20130101; A61K 8/55 20130101; A61K 8/19 20130101; A61K
8/67 20130101; A61K 8/41 20130101; A61K 8/64 20130101; A61K 8/25
20130101; A61K 8/447 20130101; C12N 5/0629 20130101; A61K 45/06
20130101; A61K 31/7052 20130101; A61K 8/673 20130101; A61K 8/492
20130101; A61K 8/49 20130101; A61K 8/20 20130101; A61K 8/23
20130101; A61K 8/34 20130101; A61K 8/60 20130101; A61K 8/606
20130101; A61K 8/4946 20130101; A61K 8/442 20130101; A61K 8/44
20130101; A61K 31/70 20130101; A61K 8/4913 20130101; A61K 8/675
20130101; A61K 8/24 20130101; A61K 8/4986 20130101; A61Q 19/00
20130101 |
Class at
Publication: |
514/045 ;
435/371; 514/023; 514/440; 514/506; 514/579; 514/663; 514/738 |
International
Class: |
A61K 31/70 20060101
A61K031/70; A61K 31/7052 20060101 A61K031/7052; C12N 5/08 20060101
C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 9, 1995 |
FR |
95-00327 |
Jan 9, 1995 |
FR |
95-00329 |
Claims
1. A method of cosmetic treatment, comprising contacting only an
area of human skin whose integrity has not been breached by injury
with a treatment composition comprising an aqueous complex
nutritive base comprising: a plurality of amino acids, at least one
vitamin, a plurality of assimilable organic components each
selected from the group consisting of i-Inositol, Putrescine 2HCl,
Sodium pyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and
D-Glucose, and at least one inorganic salt, wherein said treatment
composition does not comprise a biological extract of animal or
cellular origin, or a living nourishing substrate, or a cellular
growth stimulating compound or factor, or a hormone.
2. The method of claim 1, wherein the pH and osmolarity of said
complex nutritive base are per se close to physiological
conditions.
3. The method of claim 1, wherein said treatment composition
consists essentially of components that are biomimetic to skin.
4. The method of claim 1, wherein said amino acids include at least
one amino acid selected from the group consisting of L-Alanine,
L-Arginine HCl, L-Asparagine, L-Aspartic acid, L-Cysteine
HCl.H.sub.2O, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine
HCl.H.sub.2O, L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine,
L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,
L-Tyrosine 2 Na 2H.sub.2O, and L-Valine.
5. The method of claim 1, wherein said at least one vitamin
includes at least one vitamin selected from the group consisting of
d-Biotin, Folic acid, Nicotinamide, Ca D-Pantothenate, Pyridoxine
HCl, Riboflavin, Thiamine HCl, and Vitamin B.sub.12.
6. The method of claim 1, wherein said complex nutritive base
supports per se viable in vitro growth of human epidermal
keratinocytes, with no proliferation of any transformed human
epidermal keratinocyte.
7. The method of claim 1, wherein the complex nutritive base does
not have per se cytotoxic manifestations to skin.
8. The method of claim 1, wherein said method is for maintaining
the integrity and balance of the superficial cells of the skin.
9. A method of cosmetic treatment, comprising contacting only an
area of human skin whose integrity has not been breached by a wound
with a treatment composition comprising an aqueous complex
nutritive base comprising: a plurality of amino acids, at least one
vitamin, a plurality of assimilable organic components each
selected from the group consisting of i-Inositol, Putrescine 2HCl,
Sodium pyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and
D-Glucose, and at least one inorganic salt, wherein said treatment
composition does not comprise a biological extract of animal or
cellular origin, or a living nourishing substrate, or a cellular
growth stimulating compound or factor, or a hormone.
10. The method of claim 9, wherein the pH and osmolarity of said
complex nutritive base are per se close to physiological
conditions.
11. The method of claim 9, wherein said treatment composition
consists essentially of components that are biomimetic to skin.
12. The method of claim 9, wherein said amino acids include at
least one amino acid selected from the group consisting of
L-Alanine, L-Arginine HCI, L-Asparagine, L-Aspartic acid,
L-Cysteine HCl.H.sub.2O, L-Glutamic acid, L-Glutamine, Glycine,
L-Histidine HCl.H.sub.2O, L-Isoleucine, L-Leucine, L-Lysine HCl,
L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine 2 Na 2H.sub.2O, and L-Valine.
13. The method of claim 9, wherein said at least one vitamin
includes at least one vitamin selected from the group consisting of
d-Biotin, Folic acid, Nicotinamide, Ca D-Pantothenate, Pyridoxine
HCl, Riboflavin, Thiamine HCl, and Vitamin B.sub.12.
14. The method of claim 9, wherein said complex nutritive base
supports per se viable in vitro growth of human epidermal
keratinocytes, with no proliferation of any transformed human
epidermal keratinocyte.
15. The method of claim 9, wherein the complex nutritive base does
not have per se cytotoxic manifestations to skin.
16. The method of claim 9, wherein said method is for maintaining
the integrity and balance of the superficial cells of the skin.
17. A method of cosmetic treatment, comprising contacting human
skin with a treatment composition comprising an aqueous complex
nutritive base comprising: a plurality of amino acids, at least one
vitamin, a plurality of assimilable organic components each
selected from the group consisting of i-Inositol, Putrescine 2HCl,
Sodium pyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and
D-Glucose, and at least one inorganic salt, wherein said treatment
composition does not contain a biological extract of animal or
cellular origin, or a living nourishing substrate, and wherein said
treatment composition consists essentially of components that are
biomimetic to skin.
18. The method of claim 17, wherein said complex nutritive base
supports per se viable in vitro growth of human epidermal
keratinocytes.
19. The method of claim 17, wherein the pH and osmolarity of said
complex nutritive base are per se close to physiological
conditions.
20. The method of claim 17, wherein said amino acids include at
least one amino acid selected from the group consisting of
L-Alanine, L-Arginine HCI, L-Asparagine, L-Aspartic acid,
L-Cysteine HCl.H.sub.2O, L-Glutamic acid, L-Glutamine, Glycine,
L-Histidine HCl.H.sub.2O, L-Isoleucine, L-Leucine, L-Lysine HCl,
L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine 2 Na 2H.sub.2O, and L-Valine.
21. The method of claim 17, wherein said at least one vitamin
includes at least one vitamin selected from the group consisting of
d-Biotin, Folic acid, Nicotinamide, Ca D-Pantothenate, Pyridoxine
HCl, Riboflavin, Thiamine HCl, and Vitamin B.sub.12.
22. The method of claim 17, wherein the complex nutritive base does
not have per se cytotoxic manifestations to skin.
23. The method of claim 17, wherein said method is for maintaining
the integrity and balance of the superficial cells of the skin.
24. A cosmetic composition, comprising an aqueous complex nutritive
base comprising: a plurality of amino acids, at least one vitamin,
a plurality of assimilable organic components each selected from
the group consisting of i-Inositol, Putrescine 2HCl, Sodium
pyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and D-Glucose,
and at least one inorganic salt, wherein said cosmetic composition
does not comprise a biological extract of animal or cellular
origin, or a living nourishing substrate, or a cellular growth
stimulating compound or factor, or a hormone.
25. The cosmetic composition of claim 24, wherein the pH and
osmolarity of said complex nutritive base are per se close to
physiological conditions.
26. The cosmetic composition of claim 24, wherein said cosmetic
composition consists essentially of components that are biomimetic
to skin.
27. The cosmetic composition of claim 24, wherein said amino acids
include at least one amino acid selected from the group consisting
of L-Alanine, L-Arginine HCl, L-Asparagine, L-Aspartic acid,
L-Cysteine HCl.H.sub.2O, L-Glutamic acid, L-Glutamine, Glycine,
L-Histidine HCl.H.sub.2O, L-Isoleucine, L-Leucine, L-Lysine HCl,
L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine 2 Na 2H.sub.2O, and L-Valine.
28. The cosmetic composition of claim 24, wherein said at least one
vitamin includes at least one vitamin selected from the group
consisting of d-Biotin, Folic acid, Nicotinamide, Ca
D-Pantothenate, Pyridoxine HCl, Riboflavin, Thiamine HCl, and
Vitamin B.sub.12.
29. The cosmetic composition of claim 24, wherein said complex
nutritive base supports per se viable in vitro growth of human
epidermal keratinocytes, with no proliferation of any transformed
human epidermal keratinocyte.
30. The cosmetic composition of claim 24, wherein the complex
nutritive base does not have per se cytotoxic manifestations to
skin.
31. A cosmetic composition, comprising an aqueous complex nutritive
base comprising: a plurality of amino acids, at least one vitamin,
a plurality of assimilable organic components each selected from
the group consisting of i-Inositol, Putrescine 2HCl, Sodium
pyruvate, Thymidine, Adenine (HCl), DL-Lipoic acid and D-Glucose,
and at least one inorganic salt, wherein said cosmetic composition
does not contain a biological extract of animal or cellular origin,
or a living nourishing substrate, and wherein said cosmetic
composition consists essentially of components that are biomimetic
to skin.
32. The cosmetic composition of claim 31, wherein said complex
nutritive base supports per se viable in vitro growth of human
epidermal keratinocytes.
33. The cosmetic composition of claim 31, wherein the pH and
osmolarity of said complex nutritive base are per se close to
physiological conditions.
34. The cosmetic composition of claim 31, wherein said amino acids
include at least one amino acid selected from the group consisting
of L-Alanine, L-Arginine HCl, L-Asparagine, L-Aspartic acid,
L-Cysteine HCl.H.sub.2O, L-Glutamic acid, L-Glutamine, Glycine,
L-Histidine HCl.H.sub.2O, L-Isoleucine, L-Leucine, L-Lysine HCl,
L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine 2 Na 2H.sub.2O, and L-Valine.
35. The cosmetic composition of claim 31, wherein said at least one
vitamin includes at least one vitamin selected from the group
consisting of d-Biotin, Folic acid, Nicotinamide, Ca
D-Pantothenate, Pyridoxine HCl, Riboflavin, Thiamine HCl, and
Vitamin B.sub.12.
36. The cosmetic composition of claim 31, wherein the complex
nutritive base does not have per se cytotoxic manifestations to
skin.
37. The cosmetic composition of claim 31, said aqueous complex
nutritive medium comprising at least four of said assimilable
organic components.
Description
[0001] The present invention relates to a complex nutrient medium,
to its applications and more especially to its use for
manufacturing a composition for topical use, and in particular for
topical cosmetic or medicinal use.
[0002] The composition obtained according to the invention enables
an extracellular environment which is entirely suited to the
epidermis to be obtained, by supplying in particular: [0003] an
optimized nutritional provision, both in respect of vitamins and
trace elements and in respect of essential amino acids, [0004] cell
growth factors directed towards replacing the morphogenic cellular
interactions, [0005] and pH and osmolarity characteristics close to
physiological conditions.
[0006] Generally speaking, according to the invention, the
nutritional agent consists of a complex nutrient medium comprising
compounds which are both biocompatible, biomimetic and bioavailable
in respect of the skin, excluding any biological extract of animal
origin, such as foetal calf serum, or of cellular origin.
[0007] The complex nutrient medium adopted according to the
invention has a composition suitable for permitting, on its own and
in an aqueous medium, viable in vitro culture of an inoculum of
human epidermal keratinocytes, with at least one clonal
proliferation of the latter at the first passage, without a living
nourishing substrate such as fibroblasts.
[0008] "Biocompatible" is understood to mean the property according
to which the compound is harmless to the skin.
[0009] "Biomimetic" is understood to mean the fact that the
compound is present in the natural state in the skin.
[0010] "Bioavailable" is understood to mean the property according
to which the compound is assimilable by human epidermal
keratinocytes, both in vitro and in vivo.
[0011] By routine tests, a person skilled in the art is in a
position to formulate a complex nutrient medium according to the
invention, in particular by carrying out with the said medium in
vitro culturing of keratinocytes, the growth of which can be
observed, for example under a microscope.
[0012] In this connection, the following documents have already
described media suited to in vitro culturing of keratinocytes, the
viability and growth of which can be determined by the tests
currently in use, and be directly assessed by observation under a
microscope: [0013] Boyce ST, Ham RG, Calcium-regulated
differentiation of normal human epidermal keratinocytes in defined
clonal culture and serum-free serial culture, J. Invest. Dermatol.
1983; 81: 33S-40S [0014] Boyce ST, Ham RG, Cultivation, frozen
storage, and clonal growth of normal human epidermal
keratino-cytesin [sic] serum-free media, J. Tissue Culture Methods.
1985; 9: 83-93.
[0015] Where necessary, the content of these publications is
incorporated in the present description.
[0016] The complex nutrient medium according to the invention
comprises amino acids, one or more vitamins, one or more cell
growth factors and one or more inorganic salts.
[0017] A composition of the invention for topical use comprises a
phase which is biocompatible with the superficial parts of the
human body, in which phase at least the said nutrient medium as
defined above is distributed homogeneously.
[0018] In a composition according to the present invention, the
biocompatible phase in which the nutritional agent is distributed
can constitute the excipient, or one of the components of the
excipient, of the said composition.
[0019] Since all of the compounds present in the nutrient medium
according to the invention are water-soluble, two methods of
formulation may be employed in order to obtain a composition for
topical use:
[0020] 1) Aqueous continuous phase, containing the nutrient medium
according to the invention: [0021] in the form of an aqueous gel,
with the aid of a nonionic water-soluble polymer of the
polysaccharide or cellulose ether type (polymers compatible with
the high ionic strength of the medium); [0022] in the form of an
emulsified system (oil-in-water emulsion employing surfactants that
withstand high ionic strengths); [0023] in the form of a cosmetic
serum.
[0024] 2) Oily continuous phase, the discontinuous phase containing
the nutrient medium according to the invention: [0025] in
emulsified form, on the understanding that the ionic strength of
the discontinuous phase entails instability of the emulsion; it is,
however, possible to formulate lamellar or cylindrical phases
having better stability, or alternatively a two-phase system
reemulsified immediately before use by simple shaking; [0026] by
encapsulation: [0027] in a rigid capsule of the polysaccharide
type, dispersed in the lipid phase, [0028] in a soft capsule of the
gelatin type, dispersed in the discontinuous phase.
[0029] The use of liposomes as an encapsulation delivery agent can
be envisaged in the form of a liposomal gel in an aqueous
continuous phase.
[0030] A composition according to the invention can serve as a
cosmetic base. Its nutritional provision is considerably
advantageous for improvement of the viability, maintenance of the
integrity and the balance of the superficial cells of the skin. In
particular, it enables the primary intrinsic qualities of the skin
to be preserved on a long-lasting basis, its resistance to damage
to be increased and, where appropriate, its return to a state of
balance to be promoted.
[0031] Another subject of the invention is a cosmetic preparation
comprising a base defined above, in which the complex nutrient
medium constitutes either an active principle, or an excipient in
the presence of other active principles which it is capable of
potentiating.
[0032] The complex nutrient medium of the invention can also be
used for the preparation or production of a medicament.
[0033] The use of such a medium on a weakened skin (irritated or
dehydrated skins, older skins, etc) enables the skin to return to a
satisfactory state, in terms both of trophicity and of hydration of
the superficial layers of the epidermis.
[0034] A medicinal composition comprising a complex nutrient medium
according to the invention can serve as a pharmaceutical
formulation base, in particular a nutrient pharmaceutical
formulation base.
[0035] It possesses, in addition, pharmacological properties which
will be demonstrated in the examples. According to an advantageous
application of a medicinal composition of the invention, it is
intended for the preservative treatment of grafts after they are
removed. It will preferably take the form of a sterile solution
which is especially suitable for the cleaning and maintenance of
grafts in third-degree burns victims.
[0036] In addition, a composition as defined above has efficacious
properties for preventing or treating disorders of cicatrization
such as bedsores, varicose ulcers, stretch marks and keloids,
and/or a delay of cicatrization.
[0037] More generally speaking, a composition according to the
invention can be incorporated in any preparation for use in a
pharmaceutical formulation, as an active principle optionally with
other active principles, but also as an excipient as a result of
its capacity to potentiate the action of specific active
principles.
[0038] The characteristics, applications and advantages of the
present invention are described in greater detail in Examples 1 to
4 and FIGS. 1 to 4 below.
[0039] Example 1 gives an example of a formulation of a composition
of the invention.
[0040] Example 2 demonstrates the properties of a composition of
the invention compared to known media, in support of the attached
drawing in which:
[0041] FIG. 1 is a sectional view of human epidermis after 36 hours
of culture in a standard commercial medium designated MCDB 153,
marketed, in particular, by IRVINE SCIENTIFIC and GIBCO-BRL,
[0042] FIG. 2 is a sectional view of human epidermis after 36 hours
of culture in a buffered saline solution (PBS), a balanced saline
solution commonly used in cell culture, and
[0043] FIG. 3 is a sectional view of human epidermis cultured in
the nutrient medium of the invention, described in Example 1, at
different culture times:
[0044] A: after 12 hours
[0045] B: after 24 hours
[0046] C: after 36 hours
[0047] Example 3 demonstrates the absence of stimulation of the
proliferation of transformed cells by a composition of the
invention compared to a standard composition, in support of FIG. 4
which depicts a diagram showing the multiplication of transformed
cells cultured on a medium of the invention and a standard
medium.
[0048] Example 4 illustrates the pharmacological properties of a
composition of the invention: a) on the treatment of grafts; b) on
cicatrization.
EXAMPLE 1
[0049] Formulation of a composition of the invention TABLE-US-00001
TABLE 1 Concentration COMPONENTS in mg/l. Amino acids L-Alanine 9.2
L-Arginine HCL [sic] 421.4 L-Asparagine (anhydrous) 14.2 L-Aspartic
acid 4.0 L-Cysteine HCl.cndot.H.sub.2O 42.0 L-Glutamic acid 14.8
L-Glutamine 1754.4 Glycine 7.6 L-Histidine HCl.cndot.H.sub.2O 50.0
L-Isoleucine 6.0 L-Leucine 131.2 L-Lysine HCl 54.0 L-Methionine
13.5 L-Phenylalanine 10.0 L-Proline 34.6 L-Serine 126.1 L-Threonine
24.0 L-Tryptophan 9.3 L-Tyrosine 2 Na 2H.sub.2O 11.7 L-Valine 70.3
Vitamins and cell growth factors d-Biotin 0.02 Folic acid 0.80
Nicotinamide 0.04 Ca D-Pantothenate 0.30 Pyridoxine HCl 0.06
Riboflavin 0.04 Thiamine HCl 0.30 Vitamin B.sub.12 0.41 i-Inositol
18.0 Putrescine 2 HCl 0.20 Sodium pyruvate 55.0 Thymidine 0.73
Adenine (HCl) 24.0 DL-Lipoic acid 0.20 Inorganic components Sodium
chloride 6800.0 KCl 112.0 Na.sub.2 HPO.sub.4 284.0
CuSO.sub.4.cndot.5H.sub.20 0.003 Sodium acetate 300.0 (anhydrous)
D-Glucose 1080.0 HEPES (piperazine) 6600.0 Phosphorylethanolamine
0.06768 Ethanolamine 0.04684 Sodium sulphate 3.4 Sodium bicarbonate
1160.0 FeSO.sub.4.cndot.7H.sub.2O 1.39 MgCl.sub.2.cndot.6H.sub.2O
120.0 CaCl.sub.2.cndot.2H.sub.2O from 13.0 to 22.05
ZnSO.sub.4.cndot.7H.sub.2O 0.144 (NH4).sub.6
MO.sub.7O.sub.24.cndot.4H.sub.2O 0.00120
Na.sub.2SiO.sub.3.cndot.5H.sub.2O 0.142 MnCl.sub.2.cndot.4H.sub.2O
0.00002 SnCl.sub.2.cndot.2H.sub.2O 0.00011 NH.sub.4 VO.sub.3
0.00057
EXAMPLE 2
[0050] The cytocompatibility and the performance features of the
complex nutrient medium described in Example 1 were tested on
cultures of human keratinocytes in a monolayer, and on human
epidermis reconstituted in vitro.
[0051] The nutrient medium according to Example 1 permits the
culture of keratinocytes in a monolayer under optimal conditions of
viability for at least 36 hours without the slightest cytotoxic
effect manifesting itself.
[0052] In contrast, a traditional survival solution such as PBS
(phosphate buffered saline, a balanced saline solution commonly
used in cell culture) proves cytotoxic from 12 hours of incubation
onwards.
[0053] In agreement with FIG. 3, the nutrient medium according to
the example permits culture of normal human epidermis reconstituted
under optimal conditions of viability, without cytotoxic
manifestations even after 36 hours (FIG. 3C) of contact. The
cultures displayed basal, prickle, mast and intact, orthokeratotic
cornified cell layers, of regular and normal stratification.
[0054] On comparing FIG. 3C with FIG. 1, the latter illustrating
the use of a standard commercial medium (MCDB 153, marketed, in
particular, by IRVINE SCIENTIFIC), it is seen that the performance
features of the medium of the invention are equally good.
[0055] In contrast, the use of PBS induces, in agreement with FIG.
2, the appearance of keratinocytes in a terminal phase of
differentiation at the level of the basal and prickle strata, with
more or less pronounced signs of necrosis. A total detachment of
the epidermis is also noted, with complete loss of structuring of
the different keratinocytic strata.
EXAMPLE 3
Effects of a Composition of the Invention on the Growth of
Transformed Epidermal Cells
[0056] The composition used for this study is the one described in
Example 1, comprising the medium termed medium 1.
[0057] The effect of the composition 1 on the growth of a
spontaneously transformed line of human keratinocytes was tested
over 4 days of culture by comparison with cells cultured on a
standard medium (DMEM, Dulbeco Modified Epidermal Medium +foetal
calf serum).
[0058] The cells are first inoculated into the standard medium and
grow until the 2nd day after inoculation into this medium. On the
2nd day, the batch of cells is divided into two, one batch
continuing to be cultured in standard medium, the other in medium
1.
[0059] The results are collated in FIG. 4, in which the curve
obtained with the points --.quadrature.-- corresponds to the
composition of the invention and that obtained with the points
--.quadrature.-- corresponds to the composition of standard medium.
The points were duplicated and the counts originate from
quadruplicates. The results are corrected for the standard error of
the mean, SEM. The arrow seen in the diagram corresponds to the
dividing of the batch on the second day of culture.
[0060] The morphology of the cells differs according to the medium
employed. That of the cells cultured in medium 1 resembles more
closely that obtained using a semi-defined medium for epithelial
cells, of the GIBCO-BRL KSFM type (cells with looser junctions,
less pavemental appearance, etc).
[0061] No significant difference is noted in the growth of this
line in accordance with the different media, up to confluence (days
6 to 7, not shown here).
[0062] It is concluded that the composition 1 has no stimulatory
effect on the proliferation of transformed keratinocytes.
EXAMPLE 4
Effects of a Composition of the Invention on the Taking of Human
Skin Grafts and the Prevention of Cicatrization Disorders
[0063] The composition tested is the one described in Example 1,
comprising the medium termed medium 1.
[0064] The effects of the composition 1 on the taking of human skin
grafts and the prevention of cicatrization disorders were studied
on a mouse model (athymic mouse lacking cell-mediated
immunity).
[0065] Two types of grafts were employed: cultured epidermis and
human skins originating from plastic surgery. The grafts were
irrigated for 30 days with 1 ml of composition 1 (one application
daily) for the group A mice and 1 ml of buffered saline solution
(PBS) for the group B mice (20 animals per group). Compresses of
tulle gras were applied after each irrigation in order to prevent
the grafts from drying out.
[0066] A clinical observation of the grafts was carried out on D-7,
D-15 and D-30.
[0067] Two parameters were evaluated: the necrosis of the cultured
epidermis and the cicatrization.
[0068] a) The Necrosis of the Cultured Epidermis ("Taking of
Grafts")
[0069] Scoring is performed from 0 to 3: 0=no sign of necrosis;
1=slight inflammation and superficial degradation of the graft;
2=partial necrosis; 3=total necrosis.
[0070] The results are collated in Table 2. TABLE-US-00002 TABLE 2
Score D-7 D-15 D-30 GROUP A MICE (20 grafts in total, treated with
the nutrient composition) 0 9/20 12/20 16/20 1 7/20 4/20 0/20 2
3/20 2/20 2/20 3 1/20 2/20 2/20 GROUP B MICE (20 grafts treated
with the buffered saline solution) 0 2/20 4/20 7/20 1 8/20 6/20
3/20 2 6/20 5/20 5/20 3 4/20 5/20 5/20
[0071] The composition 1 improves the taking of the grafts of
cultured human epidermis on athymic mice compared to a traditional
survival solution (PBS). Significant differences are noted from 7
days of treatment onwards, for a final improvement of more than
50%.
[0072] b) The Cicatrization (with the Grafted Whole Skins)
[0073] Scoring is performed from 0 to 3: 0=no cicatrization
disorder; 1=delay of cicatrization; 2=delay with abnormality of the
cicatrization (granulation of the cicatrix); 3=hypertrophic
cicatrix.
[0074] The results are collated in Table 3. TABLE-US-00003 TABLE 3
Score D-7 D-15 D-30 GROUP A MICE (20 grafted whole skins treated
with the nutrient composition) 0 20/20 16/20 15/20 1 0/20 3/20 2/20
2 0/20 1/20 2/20 3 0/20 0/20 1/20 GROUP B MICE (20 grafted whole
skins treated with the buffered saline solution) 0 16/20 10/20 5/20
1 4/20 7/20 8/20 2 0/20 3/20 3/20 3 0/20 0/20 4/20
[0075] The composition 1 significantly improves the cicatrization
processes; this effect is especially marked after 30 days of
treatment.
* * * * *