U.S. patent application number 12/863251 was filed with the patent office on 2010-12-23 for composition for topical use.
Invention is credited to Jean Hilaire Saurat.
Application Number | 20100324109 12/863251 |
Document ID | / |
Family ID | 39494330 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100324109 |
Kind Code |
A1 |
Saurat; Jean Hilaire |
December 23, 2010 |
Composition for Topical Use
Abstract
The use, as a dermatological or cosmetic medicament, of
compounds capable of transiently interacting with the AhR receptor
(aryl hydrocarbon receptor) as agents for modulating skin functions
such as sebaceous function, skin healing, skin atrophy termed
"dermatoporosis", estrogen deprivation and defense against
infection, without inducing other toxic effects of the TCDD type.
The compounds that interact with the AhR receptor are chosen in
that they have a metabolism favorable to the dissociation of these
effects, in particular by virtue of in situ production from a
precursor and/or metabolization modulated in situ.
Inventors: |
Saurat; Jean Hilaire;
(Geneva, CH) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
39494330 |
Appl. No.: |
12/863251 |
Filed: |
January 23, 2009 |
PCT Filed: |
January 23, 2009 |
PCT NO: |
PCT/IB2009/050271 |
371 Date: |
July 16, 2010 |
Current U.S.
Class: |
514/419 |
Current CPC
Class: |
A61P 17/10 20180101;
A61P 31/04 20180101; A61P 17/00 20180101; A61P 17/02 20180101; A61K
31/00 20130101; A61K 31/352 20130101; A61K 9/0014 20130101; A61K
31/407 20130101 |
Class at
Publication: |
514/419 |
International
Class: |
A61K 31/405 20060101
A61K031/405; A61P 17/02 20060101 A61P017/02; A61P 17/10 20060101
A61P017/10; A61P 31/04 20060101 A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2008 |
EP |
08150554.7 |
Claims
1-13. (canceled)
14. A composition for topical use, transiently inducing activation
of AhR receptors of skin tissues, said composition comprising an
active substance selected from the group consisting of AhR agonist
ligands with a short half-life and in situ precursors of said
ligands.
15. The composition as claimed in claim 14, wherein said active
substance is selected from the group consisting of endogenous AhR
ligands.
16. The composition as claimed in claim 14, wherein said active
substance is a precursor selected from the group consisting of the
metabolic proligands of said AhR ligands.
17. The composition as claimed in claim 14, wherein said active
substance is a said precursor that is activable so as to give an
AhR ligand under the effect of a physical agent.
18. The composition as claimed in claim 14, wherein said active
substance is a said precursor that is activable so as to give an
AhR ligand under the effect of UV radiation.
19. The composition as claimed in claim 14, wherein said active
substance is a said precursor that is activable so as to give an
AhR ligand under the effect of an endogenous agent present in the
pathological condition treated.
20. The composition as claimed in claim 14, wherein said active
substance is a said precursor that is activable so as to give an
AhR ligand under the effect of an exogenous agent present in the
pathological condition treated.
21. The composition as claimed in claim 14, wherein said ligands
have half-lifetimes in the human organism of between 2 h and 96
h.
22. The composition as claimed in claim 14, wherein said ligands
have half-lifetimes in the human organism of between 6 h and 24
h.
23. The composition as claimed in claim 14, wherein said active
substance has: an ability to activate the AhR receptor, an ability
to modulate a gene regulated by AhR, a short half-life in the human
organism of between 2 hours and 96 h, a measurable positive effect
on a recognized criterion of sebaceous hyperactivity, of wound
healing and of dermatoporosis, of bacterial colonization of the
skin, and/or of estrogen deprivation.
24. The composition as claimed in claim 14, for treating and/or
preventing diseases comprising sebaceous gland hypertrophy.
25. The composition as claimed in claim 14, for treating and/or
preventing bacterial skin infections.
26. The composition as claimed in claim 14, for treating and/or
preventing diseases comprising skin colonization by bacterial
species.
27. The composition as claimed in claim 14, for treating and/or
preventing diseases comprising Propionibacterium acnes and
Staphylococcus aureus.
28. The composition as claimed in claim 14, for treating and/or
preventing skin function problems associated with estrogen
deprivation.
29. The composition as claimed in claim 14, for treating and
healing skin wounds.
30. The composition as claimed in claim 14, for treating and/or
preventing skin atrophy.
31. The composition as claimed in claim 14, for treating and/or
preventing skin atrophy in the context of dermatoporosis.
32. The composition as claimed in claim 14, wherein said active
substance is selected from the group consisting of: Catechin,
Epigallocatechin 3-gallate, Quercetin, Apigenin, Silibinin,
Resveratrol, Yangonin, Tryptophan, Indole-3-carbinol, Tryptamine,
Kynurenine, 6-Formylindolo[3,2-b]carbazole (FICZ), alkyl and
alkenyl derivatives of the formyl group, 3,3'-Diindolylmethane,
Tryptanthrine, Malassezin, Indirubin, Indigo, ITE [Methyl
2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylate], YH439
{(Isopropyl-2-(1,3-dithioethan-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carb-
amoyl] acetate}, Hispidin, alpha-Naphthoflavone,
beta-Naphthoflavone, Ellipticine, Carvedilol, Triclabendazole,
Rutaecarpine, Ethyl .beta.-carboline-3-carboxylate and
Esomeprazole.
Description
[0001] The present invention relates to a composition for topical
use, intended for the treatment and/or prevention of skin function
dysfunctions. The present invention relates more specifically to a
pharmaceutical composition for topical use, intended for the
treatment and/or prevention of dysfunctions of sebaceous function,
of healing, of atrophy termed "dermatoporosis (Ref Dermatology
2007.215. 284-294)", of estrogen deprivation of the skin and of
defense against infection.
[0002] Treatments already exist for the dysfunctions mentioned
above, but they are not, however, without faults. Thus, by way of
examples: [0003] it is known that a vitamin A derivative,
isotretinoin (or 13-cis-retinoic acid), is active in the treatment
of diseases such as hyperseborrhoea and acne by inducing atrophy of
the sebaceous glands. However, this substance must be administered
systemically and not topically, since, when administered topically,
it does not atrophy the sebaceous gland. [0004] In order to
facilitate the healing of skin wounds, it is known practice to
protect the tissues undergoing reconstruction, in particular by
means of hydrocolloid dressings, but this technique provides
superficial mechanical protection without specifically stimulating
the intrinsic processes of tissue repair per se, when a deficiency
exists in the production of growth factors, in particular vascular
growth factors. [0005] It has been proposed to treat dermatoporosis
by means of retinoids and of hyaluronic acid fragments, but the
first can be irritant and the second can be difficult to prepare in
their optimal size and viscosity. [0006] In the case of estrogen
deprivation of the skin, which contributes to the skin becoming
fragile and to dermatoporosis, it is known practice to administer
estrogens systemically or topically. However, this supply of
exogenous hormones has the drawback of modifying, as a whole, the
patient's hormone balance and, according to several publications,
of increasing the risk of appearance of certain cancers. [0007] In
order to reinforce the capacities for defense against infection in
the event of exposure or of susceptibility to bacterial skin
infections, long-term antibiotic use has already been proposed, but
this method has the fault of selecting resistant strains and of not
increasing the intrinsic defenses of the individual.
[0008] There remains, therefore, an unmet need to develop new
medicaments against the dysfunctions mentioned above, and in
particular pharmaceutical compositions for topical use which make
it possible to avoid the drawbacks associated with systemic
administration.
[0009] Compounds which interact with the AhR receptor (aryl
hydrocarbon receptor), the prototype of which is
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mostly xenotoxic
compounds, inducing various types of tissue lesions and other
manifestations of poisoning, which are dose- and species-dependent.
The skin holds a major place among these manifestations of dioxin
poisoning, partly due to the acknowledged expression of the AhR
receptor by the epithelial and mesenchymal cells of the skin. For
this reason, the therapeutic and/or preventive use of such
compounds as active agents that modulate skin functions appears, in
principle, to be excluded.
[0010] During human poisoning with TCDD, a complex syndrome is
observed at the level of the skin. The detailed study of this
syndrome in humans and in experimental animals has led the
applicant to observe the cellular and molecular mechanisms that
form these toxic lesions. Surprisingly, the applicant has
discovered certain favorable effects of exposure to these toxic
compounds, described below, and has found means for obtaining them,
in the absence of the toxic effects mentioned above, for modulating
skin functions, in particular sebaceous function, defense against
infection, healing, skin atrophy termed "dermatoporosis (Ref.
Dermatology 2007.215. 284-294)", and estrogen deprivation.
[0011] Consequently, the invention relates to compounds which are
agonists of the AhR receptor and/or the proteins involved in its
cellular activation pathways, these compounds acting in the context
of the invention as agents for modulating skin functions, in
particular sebaceous function, defense against infection, healing,
atrophy termed "dermatoporosis", and estrogen deprivation of the
skin.
[0012] According to a first aspect of the invention, the subject of
said invention is a pharmaceutical composition for topical use,
intended for transiently inducing the activation of AhR receptors
of skin tissues, said composition comprising an active substance
chosen from metabolizable agonist ligands of AhR, and from the in
situ precursors of said ligands.
[0013] The toxicity of xenotoxic AhR ligands, such as TCDD, appears
to be essentially due to the fact that these substances are stored
by the human organism, in the tissues of which they exhibit
sustained half-lives, sometimes of the order of 4-12 years. As a
result, the TCDD molecules continually released from the storage
sites such as the fat tissue, to the target tissues expressing the
AhR receptor, such as the skin, continuously activate the AhR
signaling pathway, thereby leading to pathological effects. Thus,
unlike with TCDD, through the use of defined metabolizable AhR
ligands, the half-life of which in the organism can be evaluated in
hours or in days, the topical activation of skin AhRs results only
in a beneficial activation of certain skin functions, TCDD
poisoning having made it possible to identify these functions for
the first time in humans.
[0014] In this perspective, the active substance can be chosen from
endogenous AhR ligands: the applicant considers that there are very
probably endogenous ligands, or exogenous, in particular
environmental, ligands, which give the AhR signaling pathway a
physiological function. These ligands essentially have a short
lifetime.
[0015] Thus, according to a 2.sup.nd aspect of the invention, the
subject of said invention is a composition for topical use,
intended for transiently inducing the activation of AhR receptors
of skin tissues, said composition comprising an active substance
chosen from AhR agonist ligands with a short half-life and the in
situ precursors of said ligands.
[0016] The ligands used in the context of the present invention
preferably have half-life times in the human organism of between 2
and 96 hours, and more specifically, and according to the
application, between 6 and 24 hours.
[0017] The present invention is therefore completely distinct from,
and even antinomic to, certain prior art inventions which have
proposed the use of AhR receptor antagonist ligands, with a view to
preventing gene expression of the type of those that can be induced
by exposure to TCDD and other xenotoxic AhR ligands. These
inventions, described in particular in documents WO2004/041758,
WO2007/060256 and WO2007/128725, were based on extrapolations drawn
from in vitro experiments and on the premise that activation of the
AhR pathway would by definition be harmful. On the contrary, the
applicant has noted that the activation, in humans and experimental
animals, of the AhR pathway can have favorable effects for the
treatment of diseases such as abnormalities of sebaceous function,
of healing, of atrophy termed "dermatoporosis", of defense against
infection and of estrogen deprivation of the skin.
[0018] The ligands according to the invention are chosen such that
they preferably meet four criteria: [0019] 1. An ability to
activate the AhR receptor. [0020] 2. An ability to modulate a
specific gene regulated by AhR. [0021] 3. A short half-life in the
human organism, preferably of between 2 hours and 96 h, and more
specifically, and according to the application, between 6 and 24
hours. [0022] 4. Preferably, a measurable positive effect on a
recognized criterion of sebaceous hyperactivity, of wound healing,
of dermatoporosis, of bacterial colonization of the skin, and/or of
estrogen deprivation.
[0023] The modulated gene may in particular be the CYP1A1 gene.
This modulated gene may also be one of the numerous genes described
in the present application as being modulated by exposure to TCDD
and the promoter of which expresses an AhR-binding site. The
applicant considers that the modulation of the CYP1A1 gene is
proposed as a paradigm based on current observations and knowledge;
thus, it does not exclude the desired effects being obtained by
means of AhR ligands which do not have activation of CYP1A1
expression as their predominant effect. The applicant considers,
furthermore, that the lifetime should be a determining element in
the beneficial effects/toxic effects dissociation by inducing a
transient activation of AhR. However, the applicant does not
exclude the possibility that certain ligands, the half-life of
which departs from the numerical values indicated above, having
this property of beneficial effects/toxic effects dissociation.
This is because the duration of activation of the AhR receptor in
the skin might not depend exclusively on the half-life of the
ligand in the organism.
[0024] In general, the dissociation between beneficial effect
according to the invention and toxic effects is obtained by
stimulating the AhR receptor in a modulated and transient manner,
i.e. in a manner close to the physiological action of the nontoxic,
natural endogenous/exogenous ligands. This dissociation can also be
obtained by the formation of a ligand, in situ, from a topically
administered precursor. Said active substance can be a precursor
chosen from the metabolic proligands of AhR ligands. Said active
substance may also be a precursor that can be activated so as to
give an AhR ligand under the effect either of a physical agent, in
particular under the effect of UV radiation, or of any other
biological process, such as metabolic activation within a skin
tissue by the saprophytic or pathogenic flora.
[0025] The invention will be understood more clearly by those
skilled in the art through the description hereinafter of several
specific applications, with reference to the corresponding examples
and to the accompanying figures, in which:
[0026] FIG. 1 shows the disappearance of sebaceous glands owing to
exposure to orally administered toxic doses of TCDD in humans;
[0027] FIG. 2 illustrates the reproduction of this phenomenon of
sebaceous gland disappearance by topical application of TCDD in
mice: FIG. 2 shows the disappearance of the sebaceous glands of the
ear skin of C57BL/6 mice treated once a day for 45 d with 2 mcg/ml
TCDD (bottom photograph) compared with the control (top
photograph).
[0028] FIG. 3 illustrates the reduction in sebaceous glands through
topical application of 4 ligands according to the invention: FIG. 3
shows the reduction in the surface area of the sebaceous glands in
the ears of C57GL/6 mice treated once a day for 45 d with various
ligands according to the invention.
[0029] FIG. 4 shows the major inhibition of the gene expression of
key enzymes of sebaceous lipogenesis in humans exposed to TCDD, by
analysis of the whole transcriptome (Affymetrix) in human skin
exposed to TCDD: lipo/sebogenesis enzyme genes are greatly
underexpressed. The appendix to FIG. 4 is a table which gives the
symbols of the genes of which the expression is strongly repressed
compared with the controls in human skin exposed to TCDD.
[0030] FIGS. 5 and 6 show the healing times of superficial wounds
in the absence or in the case of exposure to TCDD in humans. FIG. 5
shows the percentage surface area healed as a function of time of 4
dermabrasions in the case of the presence of TCDD in the skin, in
comparison with 4 controls (C1-C4).
[0031] FIG. 6 shows the mean values calculated on the basis of the
data of FIG. 5. Thus, the time to complete healing is brought back
from 9.3 (gray bars) to 5 days (black bars).
[0032] FIG. 7 shows the healing times of experimental wounds in a
culture of human keratinocytes in vitro, treated with TCDD and with
ligands according to the invention.
[0033] FIG. 8 shows the expression of the messenger RNAs of growth
factors and of their receptors in human skin exposed to TCDD in
vivo.
[0034] FIG. 9 shows the healing of a chronic human wound to which a
solution of Trp generating AhR activation according to the
invention has been applied for 10 days. Contraction of the chronic
wound is noted; the arrows show the development of epithelial
healing with strips of keratinocytes migrating from the edges.
[0035] FIG. 10a shows the induction, in the skin, of messenger RNAs
of estrogen receptors and of aromatase by exposure to TCDD in a
man.
[0036] FIG. 10b shows the strong ectopic and focal expression of
the ER-alpha receptor protein in the pilosebaceous structures on a
specimen of the skin of an individual exposed to TCDD.
[0037] FIG. 11 shows the concentration of estradiol (E2) in human
skin exposed to TCDD, determined by ELISA on fragments of human
skin.
[0038] FIG. 12 shows the decrease in staphylococcus sp.
colonization of human skin exposed to TCDD (data over four
months).
[0039] FIG. 13 shows the decrease in the colonization of human
facial skin by Propionibacterium acnes, in skin exposed to TCDD
(data over four months).
[0040] FIGS. 14 and 15 show the AhR receptor activation values in
two cell types using various products with the aim of identifying
active substances which activate AhR but the half-life of which is
short. FIG. 14 shows the AhR activation of HEP G2 cells by various
ligands according to the invention. The values represent the
percentage activation compared with that obtained with 0.01 .mu.M
TCDD, using the concentration, in .mu.M, most active in the range
tested for each substance. FIG. 15 shows the AhR activation of
keratinocytes by various ligands according to the invention. The
values represent the percentage activation compared with that
obtained with 0.01 .mu.M TCDD, using the concentration, in .mu.M,
most active in the range tested for each substance.
[0041] FIG. 16 shows the activation of AhR by various extracts of
common human skin bacteria in the presence of tryptophan.
[0042] FIG. 17 shows the induction of a specific biological
activity, CYP1A1, by AhR-activating ligands according to the
invention. The tests are carried out using the "P450-GLO assay" kit
from Promega.
[0043] As regards the sebaceous glands, the useful effect of TCDD,
for the purpose of the present invention, is the gradual decrease
in the size of the glands, and also the decrease in the expression
of the genes of key enzymes in sebaceous lipogenesis.
[0044] This decrease is observed in humans, from a morphological
(histology) point of view, after a period of 2 to 4 weeks after
exposure and becomes complete around week 8 (FIG. 1). The
comparative data concerning normal human skin, presented in FIG. 1,
were published by Montagna (1963).
[0045] Strong and focal expression of the CYP1A1 enzyme (mRNA and
immunohistochemistry) is observed at the level of the sebaceous
glands, and then in the hamartomatous metaplasia thereof in MADISH.
This indicates a strong AhR-dependent activation at the level of
the sebaceous glands and demonstrates, for the first time, that
this organ is an AhR-dependent target, which can therefore be
modulated by agonists of this receptor. This effect is not observed
on the other skin structures (hairs, sweat glands, differentiation
of the epidermis), which has led the applicant to consider that the
effect on the sebaceous gland results from an inhibition of the
differentiation of the sebaceous stem cell via the TCDD/AhR
pathway. Thus, our analysis of the transcriptome of human skin
exposed to TCDD indicates that the molecular target downstream of
the TCDD/AhR pathway might be the human analog of blimt (a murine
transcription factor which ensures the promotion of the sebaceous
stem cell (Cell 126, 597-609, 2006)).
[0046] This thus results in a very considerable decrease in the
enzymes of sebaceous lipogenesis, as attested to by the analysis of
the transcriptome of human skin exposed to TCDD (FIG. 4).
[0047] It should also be emphasized that the TCDD-exposed human
skin immunohistochemistry reveals strong and focal expression of
estrogen receptors at the level of the sebaceous glands (FIG.
10b).
[0048] These data have led the applicant to design a new approach
for modulating sebaceous hypertrophy. This approach comprises the
activation of AhR-dependent pathways in the sebaceous glands by
means of agonists of this receptor. It involves the inhibition of
sebaceous stem cell differentiation factors and of sebocyte
proliferation, the inhibition of sebaceous lipogenesis enzymes, and
also the local stimulation of estrogen production pathways and the
local overexpression of estrogen receptors.
[0049] This effect of gradual decrease in the size of the sebaceous
glands can be reproduced in animals by topical application of TCDD.
This effect is specific since, in humans, there is no atrophy of
the other secondary structures such as hairs and sweat glands.
Thus, the present invention uses the effect of specific inhibition
of the sebaceous gland by means of an AhR ligand for the prevention
and treatment of diseases comprising a hypertrophy of these glands,
such as hyperseborrhoea and acne, by inducing sebaceous gland
atrophy. For this application, this invention offers a spectrum of
activity that is potentially identical to that of oral
isotretinoin, but via topical administration. The invention also
identifies the key enzymes of sebaceous lipogenesis and also the
factors which ensure promotion of the sebaceous stem cell as a
target for AhR activators, but also potentially other chemical
modulators that may have a therapeutic application. A therapeutic
application against sebaceous gland tumors and analogs thereof,
such as carcinomas with sebaceous differentiation in Muir-Torre
syndrome, is also envisioned.
[0050] The AhR ligands used to reproduce this effect are chosen on
the basis of a reduction of at least 40% in the size and number of
sebaceous glands, without the induction of toxic signs, and also a
reduction in the expression of the key enzymes of sebaceous
lipogenesis. The means for obtaining the effect on sebaceous glands
without inducing other toxic effects of the TCDD type, particularly
the formation of hamartomatous structures (Metabolizing Acquired
Dioxin Induced Skin Hamartomas, MADISH), comprise the use of
compounds that transiently interact with the AhR receptor, such
that the kinetics are favorable to this temporal dissociation
between favorable effect and toxic effects.
[0051] As regards healing and atrophy of the skin termed
"dermatoporosis (Ref. Dermatology 207.215. 284-294)", the useful
effect of TCDD noted by the applicant manifests itself through
impressively quick wound healing. Thus, the applicant has observed
that epidermal-dermal wounds, the average healing time of which is
ordinarily 10 days, are covered in 3 days. This effect is in line
with the induction by TCDD of several growth factors (Vascular
Endothelial Growth Factor VEGF, amphiregulin, TGFb, PDGF, IGF,
hepatocyte growth factor hepapoietin A; scatter factor) and of
receptors thereof, and also of thrombospondin 1. These data have
been obtained by analysis of the whole transcriptome (Affymetrix)
in several samples of skin from a man exposed to TCDD, in
comparison with human controls. The effect of TCDD is further
characterized by a very substantial accumulation of hyaluronate in
the dermis, in parallel with hyperexpression of hyaluronate
synthases.
[0052] A subject of the invention is therefore a pharmaceutical
composition that can be administered topically, intended for
treating and preventing skin healing problems and delayed skin
healing, in particular associated with dermatoporosis, this
composition comprising at least one compound that interacts as an
agonist with the AhR receptor. The means for obtaining the desired
effect without inducing other toxic effects of the TCDD type
comprise the use of compounds of which the kinetics of interaction
with the AhR receptor are favorable to this dissociation: the
invention in particular proposes in situ production from a
precursor such as tryptophan (Trp) or a Trp derivative,
administered in a pharmaceutical carrier, enabling transcutaneous
bioavailability, followed by exposure to UV radiation, or else the
application, in a chronic wound, of Trp in a suitable carrier,
enabling the generation, in situ, by the medium, of AhR ligand
according to the example shown in FIGS. 7, 9, 14-17, the
interaction with the AhR receptor being limited by the
metabolization in situ by phase 1 enzymes.
[0053] Another subject of the invention is therefore a
pharmaceutical composition that can be administered topically,
intended for treating and preventing skin atrophy termed
dermatoporosis (Ref. Dermatology 2007.215. 284-294), which is
characterized by a depletion of hyaluronate resulting in a loss of
the viscoelasticity of the skin, this composition comprising at
least one compound that interacts as an agonist with the AhR
receptor, or a precursor of said compound, of the type described
above.
[0054] As regards estrogen deprivation, the useful effect of TCDD
manifests itself through an activation of the estrogen production
pathways and also through an overexpression of estrogen receptors.
Estrogens are endogenous hormones that are very important in many
skin functions: estrogen deprivation leads to problems with each of
these functions. It is in particular a factor involved in
dermatoporosis. It is moreover known that the local administration
of estrogens is a conventional means, which is nevertheless not
without risk, for combating hyperseborrhoeic conditions and
androgenetic alopecia. Exposure to TCDD leads, in the skin, to a
strong expression of the genes for estrogen receptors and for the
aromatase enzyme (FIG. 10), and also an increase in cutaneous
estradiol (FIG. 11). The immunohistochemistry of these specimens
reveals a strong and very focal expression of the receptors at the
level of the pilosebaceous structures. Thus, the applicant has
identified for the first time the local induction, by TCDD, of a
state of hyperestrogenism focused on a skin structure which is
known to be hormone-dependent. It is highly probable that this
contributes to the decrease in the size of the sebaceous
glands.
[0055] A subject of the invention is therefore a pharmaceutical
composition that can be administered topically, intended for
treating and preventing skin function problems associated with
estrogen deprivation, this composition comprising at least one
compound that interacts as an agonist with the AhR receptor. The
means for obtaining the desired effect on estrogen expression
without inducing other toxic effects of the TCDD type comprise the
use of compounds of which the kinetics of interaction with the AhR
receptor are favorable to this dissociation: the invention in
particular proposes in situ production from a precursor such as
tryptophan or a Trp derivative, administered in a pharmaceutical
carrier enabling transcutaneous penetration, followed by exposure
to UV radiation, the interaction with the AhR receptor being
limited by the metabolization in situ by phase 1 enzymes.
[0056] As regards the defense against infection, the useful effect
of TCDD noted by the applicant manifests itself through a marked
resistance to skin infections. This resistance coincides with a
continual stabilization of the surface flora to the benefit of
nonpathogenic saprophytic species. This resistance results in an
absence of superinfection by pathogenic microorganisms. It is in
parallel with and probably partly associated with an overexpression
of defensins, including beta-defensin. It is also in parallel with
and no doubt at least partly associated with an overexpression of
filaggrin. The two observations that have a therapeutic implication
are, on the one hand, the reduction in staphylococcal colonization
and, on the other hand, the reduction in colonization by P.
acnes.
[0057] A subject of the invention is therefore a pharmaceutical
composition that can be administered topically, intended for
treating and preventing the effects of bacterial skin infections,
in particular in individuals with a high susceptibility, and also
other situations involving chronic carriers, this composition
comprising at least one compound that interacts as an agonist with
the AhR receptor. The means for obtaining the desired effect on
skin infections without inducing other toxic effects of the TCDD
type comprise the use of compounds in which the kinetics of
interaction with the AhR receptor are favorable to this
dissociation: the invention in particular proposes in situ
production from a precursor such as tryptophan or a Trp derivative,
administered in a pharmaceutical carrier enabling transcutaneous
penetration, followed by exposure to UV radiation, or else the
generation, in situ, by the medium, of AhR ligand according to the
example shown in FIGS. 7, 9, 14-17. It will in particular be noted
that certain saprophytic and pathogenic strains of the skin surface
generate AhR activation which is potentiated by the presence of
tryptophan (FIG. 16).
[0058] Among the compounds which bind to AhR and are capable of
producing the effects according to the invention, mention may be
made, by way of nonlimiting examples, of: [0059] Flavonoids [0060]
Flavanols with alkyl, alkenyl, alkoxyl or carboxyl substituents in
positions 4 to 6 and 3' to 5'. [0061] Flavanones with alkyl,
alkenyl, alkoxyl or carboxyl substituents in positions 5, 6 and 3'
to 5'. [0062] Flavones with alkyl, alkenyl, alkoxyl or carboxyl
substituents in positions 3, 5, 6 and 3' to 5'. [0063] Flavonols
with alkyl, alkenyl, alkoxyl or carboxyl substituents in positions
5, 6 and 3' to 5'. [0064] Isoflavones with alkyl, alkenyl, alkoxyl
or carboxyl substituents in positions 5, 6 and 3' to 5'. [0065]
Anthocyanidins with alkyl, alkenyl, alkoxyl or carboxyl
substituents in positions 4 to 6 and 3' to 5'. [0066]
Epigallocatechin gallate; esterification in one or other of
positions 5, 7, 3' to 5', 3'' to 5'' with fatty acids or retinoic
acid. [0067] Stilbenes [0068] cis-Stilbenes with hydroxyl, alkyl,
alkenyl, alkoxyl or carboxyl substituents in positions 1 to 5 and
1' to 5'. [0069] trans-Stilbenes with hydroxyl, alkyl, alkenyl,
alkoxyl or carboxyl substituents in positions 1 to 5 and 1' to 5'.
[0070] Tryptophan derivatives [0071] Indole-3-carbinol; alkyl,
alkenyl, alkoxyl or carboxyl derivatives on the hydroxyl group.
[0072] Tryptamine. [0073] Kynurenine and ester of the carboxylic
group with retinol. [0074] Indoleacetic acid and ester of the
carboxylic group with retinol. [0075] Indolepyruvic acid and ester
of the carboxylic group with retinol. [0076]
6-Formylindolo[3,2-b]carbazole and alkyl or alkenyl derivatives of
the formyl group. [0077] 3,3'-Diindolylmethane. [0078]
Trythanthrine. [0079] Malassezin. [0080] Indirubin. [0081] Indigo.
[0082] Other ligands [0083] ITE [methyl
2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylate] (endogenous, see
U.S. 20020183524). [0084] YH439
{(isopropyl-2-(1,3-dithioethan-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carb-
amoyl] acetate}. [0085] a-Naphthoflavone. [0086] b-Naphthoflavone.
[0087] Hispidin. [0088] Carvedilol. [0089] Rutaecarpine. [0090]
Ethyl .beta.-carboline-3-carboxylate. [0091] Esomeprazole. [0092]
Fatty acid metabolites, in particular arachidonate metabolites.
[0093] The ligands are chosen in that they preferably meet four
criteria: [0094] 1. An ability to activate the AhR receptor, as
shown in FIGS. 14, 15 and 16 on two cell systems. [0095] 2. An
ability to modulate a gene regulated by AhR, for example CYP1A1,
which is shown in FIG. 17. [0096] 3. A short half-life in the human
organism, preferably of between 2 hours and 96 h, and more
specifically, and according to the application, between 6 and 24
hours. [0097] 4. A measurable positive effect on a recognized
criterion of sebaceous hyperactivity, of wound healing and of
dermatoporosis, of bacterial colonization of the skin, and/or of
estrogen deprivation according to, but non exclusively, the
examples given here for each of these effects.
[0098] Among the preferred AhR ligands, mention may be made of:
[0099] Flavonoids [0100] a. Catechin [0101] b. Apigenin [0102] c.
Epigallocatechin 3-gallate [0103] d. Quercetin [0104] e. Silibinin
[0105] Stilbenes and derivatives [0106] f. Resveratrol [0107] g.
Yangonin [0108] Tryptophan and tryptophan derivatives [0109] h.
Indole-3-carbinol [0110] i. Tryptamine [0111] j. Kynurenine [0112]
k. 6-Formylindolo[3,2-b]carbazole (FICZ) illustrated in FIGS. 3, 14
and 15 and alkyl or alkenyl derivatives of the formyl group [0113]
l. 3,3'-Diindolylmethane [0114] m. Trythanthrine [0115] n.
Malassezin [0116] o. Indirubin [0117] p. Indigo [0118] Other
ligands [0119] q. ITE [Methyl
2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylate] (endogenous, see
U.S. 20020183524) [0120] r. YH439
{(Isopropyl-2-(1,3-dithioethan-2-yl-idene)-2-[N-(4-methylthiazol-2-yl)car-
bamoyl] acetate} [0121] s. Hispidin, illustrated in FIGS. 3, 14, 15
[0122] t. alpha-Naphthoflavone [0123] u. beta-Naphthoflavone [0124]
v. Ellipticine [0125] w. Carvedilol illustrated in FIGS. 14, 15, 17
[0126] x. Triclabendazole [0127] y. Rutaecarpine illustrated in
FIGS. 3, 14, 15, 16, 17 [0128] z. Ethyl
.beta.-carboline-3-carboxylate (B-CCE) illustrated in FIGS. 3, 14,
15, 16, 17 [0129] aa. Esomeprazole illustrated in FIGS. 14, 15.
[0130] The methods for delivering the AhR receptor ligand under the
optimum conditions for the desired effect comprise: [0131] a.
Direct application of the active ligand in a suitable galenic
carrier such as a lotion, gel, cream, ointment, foam, impregnated
dressing. [0132] b. Topical application, in a suitable galenic
carrier, of a proligand, which will be metabolized to a ligand by
the epidermal enzymes. [0133] c. Application of a proligand to the
surface of the skin to be treated, which proligand will be
activated to give a ligand by means of the effect of a physical
agent such as UV radiation; for example, tryptophan and UVB or UVA,
optionally in combination with riboflavin; or else, depending on
the indication, by means of the endogenous or exogenous agents
present in the disease treated: by way of example, activation of
AhR ligand in the fluid of a chronic wound treated with tryptophan,
or by Propionibacterium acnes on a seborrheic/acneic face treated
with tryptophan (FIGS. 7, 9, 14, 15 and 16).
EXAMPLE 1
Sebaceous Glands
[0134] The sebaceous glands, which are present in large numbers in
normal human skin, disappear during exposure to toxic doses of
TCDD. FIG. 1 shows the number of sebaceous glands per square
centimeter on various areas of the human body, which is distributed
from 50 to 900 depending on areas and individuals. Thus, on the
face of an adult, the number is between 400 and 900/cm.sup.2.
[0135] FIG. 1 shows that, when there is exposure to TCDD, the
sebaceous glands disappear in all the areas studied. For each site,
3 to 10 skin biopsies were analyzed after fixing with formol and
staining with hematin-eosin. At least 10 sections per block were
analyzed. Out of a total of 62 biopsies, totaling 620 sections, no
sebaceous gland could be observed, whereas the number and structure
of the sweat glands and the hairs were conserved. These data were
obtained on specimens of human skin containing from 930 to 2900 ppt
of TCDD measured by gas chromatography/mass spectrometry.
[0136] The effect on the sebaceous glands can be reproduced in
animals by topical application of TCDD. A 95% reduction in the
number of sebaceous glands can be observed in C57BL/6 mice after 45
days of application of 2 mcg/ml TCDD (FIG. 2).
[0137] To reproduce the useful effect of TCDD, in a therapeutic
reduction approach the reduction in the surface area of the
sebaceous glands in the ears of C57BL/6 mice treated once a day for
45 d with various ligands according to the invention can be
studied. The surface area of the sebaceous glands is measured on
three serial sections per animal. A reduction of greater than 40%
is sought, and obtained here with a concentration, a treatment time
and an application frequency considered to be minimal. Isotretinoin
is shown by way of example of a product used for the topical and
systemic treatment of acne.
[0138] The topical application of an AhR ligand such as
6-formylindolo[3,2-b]carbazole (FICZ), at a concentration of 100
mcg/ml, makes it possible to obtain a significant decrease in the
sebaceous glands, of the order of 40%. Other ligands according to
the invention (see FIGS. 14, 15 and 17) also lead to a decrease in
the surface area of the sebaceous glands. The data indicate that
the optimum doses for a reduction of greater than 40% may be beyond
the concentrations, the time and the application frequency shown in
FIG. 3 and illustrate the concept of the invention.
[0139] FIG. 16 shows the activation of AhR of HEP G2 cells by
various extracts of common human skin bacteria in the presence of
tryptophan (trp) F1: G+ flora; F2: G+ flora; PA: P. acnes; SE: S.
epidermidis. The activation value of 25.00 on the Y-axis
corresponds to 1/6 of the AhR stimulation by 10 nM TCDD in this
same system (157.00). It can be seen that, in particular with P.
acnes, an increase in the activation is observed with the addition
of tryptophan (trp). These observations illustrate the method of
activation of AhR by tryptophan partly by interaction with the
surface flora.
[0140] The effect shown in FIG. 16, where it is seen that
Propionibacterium acnes (PA) generates an AhR activation, which is
highly accentuated by the addition of tryptophan, can be taken
advantage of in the context of this invention. This is because PA
is one of the key players in sebaceous gland diseases, and it is
present in large amounts in facial skin. It could therefore
contribute to producing therapeutic amounts of AhR ligands in the
presence of tryptophan, or also other aromatic amino acids such as
tyrosine and phenylalanine. In order to explore the relevance of
this approach, the applicant carried out a test by applying a gel
containing 1% tryptophan to the face of two hyperseborrheic men for
3 weeks, and observed a decrease in the seborrhea which could be
evaluated at 40% of the starting value using sebumetric measurement
of the casual sebum levels.
[0141] FIG. 4 shows the considerable reduction of messenger RNAs of
several sebaceous lipogenesis enzymes in human skin exposed to
TCDD. This observation identifies a new target for medicaments
aimed at modulating sebaceous function, a concept exemplified here
by AhR activation.
EXAMPLE 2
Healing and Skin Atrophy
[0142] The healing time of superficial epidermal-dermal wounds
(dermabrasions) is considerably decreased by TCDD. FIG. 5 shows the
percentage of surface area healed as a function of time of 4
dermabrasions in the case of the presence of TCDD in the skin
(TCDD1-4, 930 to 2900 ppt TCDD measured by gas chromatography/mass
spectrometry) in comparison with 4 controls (C1-C4). FIG. 6 shows
the mean values calculated from the data of FIG. 5. Thus, the time
to complete healing is reduced from 9.3 to 5 days.
[0143] FIG. 7 shows, in a model of wound healing in vitro on a
culture of human keratinocytes, that the gap in the culture is
repaired with TCDD and with active substances according to the
invention in proportions similar to what is observed after
application of growth factors. The wound is induced by means of an
incision with a yellow micropipette tip (10-200 .mu.l). Migration
index=value obtained with the ligand/that of the control without
ligand. These results indicate a strong stimulation of healing with
TCDD at nM concentrations, and with FICZ and tryptophan at .mu.M
concentrations. This indicates an activity of the AhR activation
pathway on the migration of keratinocytes which is found in vivo in
humans (FIGS. 5, 6, 10).
[0144] At the highest concentrations, a decrease in the effect, or
even an inhibition of healing, is observed with TCDD and FICZ.
Tryptophan (Trp) generates, under the conditions of this culture,
an AhR binding and activation activity (see also FIGS. 14 and 15
and 16).
[0145] The expression of the messenger RNAs of vascular endothelial
growth factors (VEGFs) and of their receptors is increased in skin
exposed to TCDD. FIG. 8 shows the ratio (RT PCR) on fragments of
human skin containing 1800 ppt of TCDD, measured by gas
chromatography/mass spectrometry, in comparison with the
controls.
[0146] Contraction of chronic wounds and the development of
epithelial healing with strips of keratinocytes was observed after
application of a tryptophan gel generating, in the wound, factors
capable of activating AhR (FIGS. 15-16). Also noted is thickening
of the dermatoporotic skin around the wound which, in the opinion
of the specialists, has a less atrophic and younger appearance.
FIG. 9 is representative of a series of 10 cases of chronic wounds
on legs exhibiting dermatoporosis.
EXAMPLE 3
Estrogenic Effect
[0147] The expression of the messenger RNAs of the various estrogen
receptors ESRR and ER, of the estrogen responsive finger protein
(EFP) ligase and of the estrogen-producing aromatase enzyme is
increased in skin exposed to TCDD. FIG. 10a shows the RT PCR ratios
on fragments of human skin containing 1800 ppt of TCDD, measured by
gas chromatography/mass spectrometry, in comparison with the
controls: exposing a man to high doses of TCDD significantly
increases, in the skin, the expression of messenger RNAs of
estrogen receptors and of associated proteins, and also of
aromatase, thus corresponding to a state of hyperestrogenism. FIG.
10b shows that, in these skin fragments, there is a strong ectopic
and focal expression of the ER-alpha receptor protein in the
pilosebaceous structures, which indicates that the local induction
of a state of hyperestrogenism by TCDD is focused on a skin
structure which is known to be hormone-dependent. The
immunohistochemistry reveals a strong and very focal expression of
the estrogen receptor alpha, ER.alpha., at the level of the
pilosebaceous structures.
[0148] FIG. 11 shows the increase in the concentration of estradiol
(E2) in human skin exposed to TCDD, determined by ELISA on
fragments of human skin containing 1800 ppt of TCDD (measured by
gas chromatography/mass spectrometry) in comparison with the
controls (f<40: women under the age of 40; f>40: women over
the age of 40; males 50 years old). These results demonstrate for
the first time in humans the local induction by TCDD of a state of
hyperestrogenism focused on a skin structure which is known to be
hormone-dependent.
EXAMPLE 4
Defense Against Infection
[0149] As shown in FIG. 12, the colonization of human skin by
staphylococcus sp (number of CFU, colony forming units, per
cm.sup.2) is very greatly decreased in the event of exposure to
TCDD. This figure shows the values obtained on 4 samples taken 1
month apart from a human individual expressing more than 900 ppt of
TCDD in the skin, measured by gas chromatography/mass spectrometry.
The same effect is observed on P. acnes colonization, and
illustrated in FIG. 13.
EXAMPLE 5
In Vitro Methods for Identifying Ligands According to the
Invention
AhR Activation Tests
[0150] FIGS. 14, 15 and 16 show AhR activation in HEP G2 cells and
normal human keratinocytes (NHKs) stably transfected with the
lentivector plox-XRE TATA-Luc.
[0151] The Hep G2 cells are cultured in DMEM (Gibco)+10% fetal
bovine serum+penicillin+streptomycin, the NHKs in keratinocyte SFM
medium (Gibco)+penicillin+streptomycin. At D0, the Hep G2 cells are
seeded into 12-well plates in a proportion of approximately 30,000
cells/cm.sup.2. The NHKs are seeded into 6-well plates in a
proportion of approximately 15,000 cells/cm.sup.2. After 24 h, the
medium is replaced with fresh medium and the cells are transduced
with the lentivector plox-XRE TATA-Luc. After 48 h, the cells are
subcultured and maintained in culture, and tested for their
reactivity to TCDD.
[0152] The tests are carried out using the luciferase reporter
assay system kit from Promega. At D0, the cells are seeded at a
density of approximately 60% confluence, and then treated, at D1,
with the test substance diluted to various concentrations in the
appropriate culture medium. At D2, the cells are lysed in CLB
buffer, and the lysate is clarified by centrifugation for 5 min at
10 000 g. The luciferase activity is measured in 20 microliters of
lysate as recommended by the supplier, using the luminoskan
luminometer (Thermo). After treatment for 24 h with 10 nM of TCDD,
a 100-fold induction of the luciferase activity is observed in the
Hep G2-plox-XRE TATA-Luc cells and a 10-fold induction is observed
in the NHK-plox-XRE TATA-Luc cells.
[0153] FIG. 14 shows the AhR activation in HEP G2 cells with
various ligands according to the invention. The values represent
the percentage activation compared to that obtained with 0.01 .mu.M
TCDD, using the concentration, in .mu.M, most active in the range
tested for each substance. It is noted that ethyl
.beta.-carboline-3-carboxylate (B-CCE), tryptophan, rutaecarpine,
hispidin and FICZ exhibit the greatest activities.
[0154] FIG. 15 shows, with the same representation, the activity of
the same ligands on normal human keratinocytes. Similar results are
noted.
Test for Potentiation by Bacterial Flora
[0155] FIG. 16 shows the AhR activation in HEP G2 cells with
various extracts of common human skin bacteria in the presence of
tryptophan (trp); F1: G+ flora; F2: G+ flora; PA: P. acnes; SE: S.
epidermidis. The activation value 25.00 on the Y-axis corresponds
to 1/6 of the AhR stimulation by 10 nM TCDD in this same system
(157.00). It is seen that, in particular with P. acnes, an increase
in the activation is observed with the addition of tryptophan.
These observations illustrate the concept of AhR activation by
tryptophan, partly by interaction with the surface flora.
CYP1A1 Activity Test
[0156] The tests are carried out using the P450-GLO assay kit from
Promega. At D0, the cells are seeded at a density of approximately
60% confluence, and then treated at D1 with the test substance
diluted to various concentrations in the appropriate culture
medium. At D2, the CYP1A1 activity is measured as recommended by
the supplier, using the nonlytic protocol on a cell monolayer.
After treatment for 24 h with 10 nM of TCDD, a 250-fold induction
of the CYP1A1 activity was observed in the Hep G2 cells and a
20-fold induction was observed in the NHK cells, as shown in FIG.
17. FIG. 17 shows, likewise, the induction of this AhR-dependent
biological activity with ligands according to the invention, said
ligands being carvedilol, B-CCE and rutaecarpine.
[0157] As will be noted by those skilled in the art, the results of
tests disclosed above illustrate a methodology for selection of
molecules that are candidates for use in the production of a
composition according to the invention.
* * * * *