U.S. patent application number 15/128717 was filed with the patent office on 2018-05-31 for method for evaluating the harmful effects of urine on children's skin.
This patent application is currently assigned to Laboratoire Expanscience. The applicant listed for this patent is Caroline BAUDOUIN, Philippe MSIKA. Invention is credited to Caroline BAUDOUIN, Philippe MSIKA.
Application Number | 20180149640 15/128717 |
Document ID | / |
Family ID | 51014475 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180149640 |
Kind Code |
A2 |
BAUDOUIN; Caroline ; et
al. |
May 31, 2018 |
Method for Evaluating the Harmful Effects of Urine on Children's
Skin
Abstract
The invention relates to biomarkers of the skin of children and,
in particular, that of infants, having altered expression in the
presence of urine. Such markers are particularly advantageous as
they allow the skin's response to urine to be monitored. The
inventors have developed methods for evaluating the efficacy in
vitro of formulations in preventing the harmful effects of urine on
a child's skin, using a skin model specifically capable of
reproducing the characteristics of children's skin.
Inventors: |
BAUDOUIN; Caroline;
(Rambouillet, FR) ; MSIKA; Philippe; (Versailles,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAUDOUIN; Caroline
MSIKA; Philippe |
Rambouillet
Versailles |
|
FR
FR |
|
|
Assignee: |
Laboratoire Expanscience
Paris La Defense Cedex
FR
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170241989 A1 |
August 24, 2017 |
|
|
Family ID: |
51014475 |
Appl. No.: |
15/128717 |
Filed: |
March 31, 2015 |
PCT Filed: |
March 31, 2015 |
PCT NO: |
PCT/EP2015/057119 PCKC 00 |
371 Date: |
September 23, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5023 20130101;
G01N 33/5088 20130101; G01N 2500/10 20130101; C12Q 2600/158
20130101; C12Q 2600/136 20130101; C12Q 1/6883 20130101; G01N
2500/04 20130101; G01N 33/6881 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12Q 1/68 20060101 C12Q001/68; G01N 33/68 20060101
G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2014 |
FR |
1452803 |
Claims
1.-13. (canceled)
14. A method, comprising: a) contacting an active agent or a
formulation with a reconstructed skin model, said model being
obtained from a skin sample from a child; b) contacting the
reconstructed skin model after step a) with urine; and c) measuring
the expression level of at least one biological marker in the skin
model after step b), wherein said biological marker is: a skin
inflammation marker selected from the group consisting of
prostaglandin E2, PTGS2, IL-1.alpha., IL-8, TRPV1 and SPR; a
barrier function marker selected from the group consisting of
CNDSM, IVL, SMPD and CLDN1; or a stem cell marker selected from the
group consisting of KRT19, BIRC5, LRIG1, ITGA6, ITGB1 and
ITGB4.
15. The method of claim 14, further comprising the steps of: d)
measuring a reference expression level of said at least one
biological marker in a control reconstituted skin model, wherein
said control reconstituted skin model has not been contacted with
the active agent or the formulation, or wherein said control
reconstituted skin model has been contacted with a reference active
agent or formulation ; and e) preparing a cosmetic or
pharmaceutical composition comprising the candidate active agent or
candidate formulation when: (i) the expression level of said at
least one biological marker measured in step c) is less than or
equal to the reference expression level of said at least one
biological marker measured in step d) when said at least one
biological marker is a skin inflammation marker, or (ii) the
expression level of said at least one biological marker measured in
step c) is greater than or equal to the reference expression level
of said at least one biological marker measured in step d) when
said at least one biological marker is a barrier function marker or
stem cell marker.
16. The method of claim 14, further comprising the steps of: d)
measuring a reference expression level of said at least one
biological marker in a control reconstituted skin model, wherein
said control reconstituted skin model has not been contacted with
the active agent or the formulation, or wherein said control
reconstituted skin model has been contacted with a reference active
agent or formulation; e) measuring the cell viability in the skin
model and in the control skin model; and f) preparing a cosmetic or
pharmaceutical composition comprising the candidate active agent or
candidate formulation when: (i) the cell viability in the skin
model of step b) is greater than or equal to the cell viability in
control skin model of step d); and (ii) the expression level of
said at least one biological marker measured in step c) is less
than or equal to the reference expression level of said at least
one biological marker measured in step d) when said at least one
biological marker is a skin inflammation marker, or (iii) the
expression level of said at least one biological marker measured in
step c) is greater than or equal to the reference expression level
of said at least one biological marker measured in step d) when
said at least one biological marker is a barrier function marker or
a stem cell marker.
17. The method of claim 14, wherein step c) comprises the
measurement of a combination of biological markers, said
combination comprising: at least one skin inflammation marker and
at least one barrier marker; or at least one skin inflammation
marker and at least one stem cell marker; or at least one barrier
marker and at least one stem cell marker.
18. The method of claim 14, wherein step c) comprises the
measurement of a combination of biological markers, said
combination comprising at least one skin inflammation marker and at
least one barrier marker and at least one stem cell marker.
19. The method of claim 14, wherein the skin sample comes from a
donor selected from the group consisting of newborns, between 0 and
1 month of age, infants, between 1 month and 2 years of age, and
children, between 2 years and 16 years of age.
20. The method of claim 14, wherein the reconstructed skin model is
selected from suspended skin cell cultures, monolayer skin cell
cultures, bilayer skin cell cultures, reconstructed skin cultures
and reconstructed mucosal cultures.
21. The method of claim 14, wherein the cells of the reconstructed
skin model come from a skin tissue explant or from stem cells
differentiated into skin cells.
22. The method of claim 14, wherein the reconstructed skin model
comprises fibroblasts or keratinocytes.
23. The method of claim 15, further comprising administering an
effective amount of the cosmetic or pharmaceutical composition of
step e) to a human patient for preventing or reducing the harmful
effects of urine on patient's skin.
24. The method of claim 16, further comprising administering an
effective amount of the cosmetic or pharmaceutical composition of
step f) to a human patient for preventing or reducing the harmful
effects of urine on patient's skin.
25. A method, comprising: a) contacting a reconstructed skin model
with urine, said model being obtained from a skin sample from a
child; b) contacting an active agent or a formulation with the
reconstructed skin model after step a); and c) measuring the
expression level of at least one biological marker in the skin
model after step b), wherein said biological marker is: a skin
inflammation marker selected from the group consisting of
prostaglandin E2, PTGS2, IL-1.alpha., IL-8, TRPV1 and SPR; a
barrier function marker selected from the group consisting of
CNDSM, IVL, SMPD and CLDN1; or a stem cell marker selected from the
group consisting of KRT19, BIRC5, LRIG1, ITGA6, ITGB1 and
ITGB4.
26. The method of claim 25, further comprising the steps of: d)
measuring the reference expression level of said at least one
biological marker in a control reconstituted skin model, wherein
said control reconstituted skin model has not been contacted with
the active ingredient or the formulation of step a), or wherein
said control reconstituted skin model has been contacted with a
reference active agent or formulation ; and e) preparing a cosmetic
or pharmaceutical composition comprising the candidate active agent
or candidate formulation of step a) when: (i) the expression level
of said at least one biological marker measured in step c) is less
than or equal to the reference production level of said at least
one biological marker measured in step d) when said at least one
biological marker is a skin inflammation marker, or (ii) the
production level of said at least one biological marker measured in
step c) is greater than or equal to the reference production level
of said at least one biological marker measured in step d) when
said at least one biological marker is a barrier function marker or
stem cell marker.
27. The method of claim 25, further comprising the steps of : d)
measuring a reference expression level of said at least one
biological marker in a control reconstituted skin model, wherein
said control reconstituted skin model has not been contacted with
the active ingredient or the formulation, or wherein said control
reconstituted skin model has been contacted with a reference active
agent or formulation; e) measuring the cell viability in the skin
model of step b) and in the control skin model of step d); and f)
preparing a cosmetic or pharmaceutical composition comprising the
candidate active agent or candidate formulation of step a) when:
(i) the cell viability in the skin model of step b) is greater than
or equal to the cell viability in control skin model of step d) ;
and (ii) the expression level of said at least one biological
marker measured in step c) is less than or equal to the reference
expression level of said at least one biological marker measured in
step d) when said at least one biological marker is a skin
inflammation marker, or (iii) the expression level of said at least
one biological marker measured in step c) is greater than or equal
to the reference expression level of said at least one biological
marker measured in step d) when said at least one biological marker
is a barrier function marker or a stem cell marker.
28. The method of claim 25, wherein step c) comprises the
measurement of a combination of biological markers, said
combination comprising: at least one skin inflammation marker and
at least one barrier marker; or at least one skin inflammation
marker and at least one stem cell marker; or at least one barrier
marker and at least one stem cell marker.
29. The method of claim 25, wherein step c) comprises the
measurement of a combination of biological markers, said
combination comprising at least one skin inflammation marker and at
least one barrier marker and at least one stem cell marker.
30. The method of claim 25, wherein the skin sample comes from a
donor selected from the group consisting of newborns, between 0 and
1 month of age, infants, between 1 month and 2 years of age, and
children, between 2 years and 16 years of age.
31. The method of claim 25, wherein the reconstructed skin model is
selected from suspended skin cell cultures, monolayer skin cell
cultures, bilayer skin cell cultures, reconstructed skin cultures
and reconstructed mucosal cultures.
32. The method of claim 25, wherein the cells of the reconstructed
skin model come from a skin tissue explant or from stem cells
differentiated into skin cells.
33. The method of claim 25, wherein the reconstructed skin model
comprises fibroblasts or keratinocytes.
34. The method of claim 26, further comprising administering an
effective amount of the cosmetic or pharmaceutical composition of
step e) to a human patient for preventing or reducing the harmful
effects of urine on patient's skin.
35. The method of claim 27, further comprising administering an
effective amount of the cosmetic or pharmaceutical composition of
step f) to a human patient for preventing or reducing the harmful
effects of urine on patient's skin.
Description
[0001] The skin is a set of cells and macromolecules grouped
together in the form of a resistant and flexible tissue, covering
the entire body. It consists of two joined layers, the epidermis
and the dermis, with which subcutaneous tissues may be
associated.
[0002] The epidermis, the principal role of which is that of
protecting the body, forms the uppermost layer of the skin and
ensures the impermeability and resistance thereof. Four separate
cellular layers may be identified in the skin, a basal layer
(stratum basalis), a spinous layer (stratum spinosum), a granular
layer (stratum granulosum) and a corneal layer (stratum corneum).
While various cell types coexist in the epidermis, keratinocytes
represent the large majority (90%). The characteristic activity
thereof is the synthesis of keratins, fibrous water-insoluble
proteins which represent 95% of the total proteins of the
epidermis.
[0003] The principal function of the skin is to establish a
protective barrier against environmental attacks while allowing
some exchanges between the internal environment and the external
environment. The barrier function is provided first and foremost by
the corneal layer, which makes the skin impermeable and
hydrophobic, thus protecting the dermis from a massive influx of
water. It is also the corneal layer which resists chemical attacks.
It is composed of cells, called corneocytes, which are dead and
anucleate but filled with keratins and other products such as
lipids, fatty acids and ceramides. Corneocytes are joined together
by specific tight junctions, corneodesmosomes, forming a compact
layer the cohesion of which is further strengthened by a lipid
cement. Beneath the granular layer, tight junctions in the granular
layer also participate in the skin barrier function (see, for
example, Hogan et al., J Allergy, 2012: 901940, 2012).
[0004] Each day the skin must deal with various attacks. It is
exposed, for example, to chemical agents such as soap as well as to
physical stresses such as friction against clothing and exposure to
the sun. The epidermis and epidermal appendices must thus be
constantly renewed to keep the skin in good condition. It is the
stem cells which make these maintenance and repair processes
possible. More particularly, the regenerative capacity of the
epidermis is conferred by adult stem cells which enable the regular
replacement of differentiated cells eliminated during
keratinization. Epidermal stem cells thus give rise to
keratinocytes, which will ultimately differentiate into
corneocytes. This process is particularly crucial for barrier
function maturation and maintenance.
[0005] Adaptation to extrauterine life is a process that begins at
birth and continues throughout the first year of life. The first
months of postnatal life constitute a period of structural and
functional reorganization of the skin which allows physiological
adaptation to the extrauterine environment. For example, the
immaturity of the skin of newborns is underlined by the different
structure and molecular composition of the stratum corneum compared
to that of adults. These are incomplete and thus continue to
develop during the first 12 months at least after birth (Chiou et
al., Skin Pharmacol Physiol, 17: 57-66, 2004; Nikolovski et al., J
Invest Dermatol, 128: 1728-1736, 2008; Stamatas et al., Pediatr
Dermatol, 27: 125-131, 2010; Telofski et al., Dermatol Res Pract,
2012: 198789, 2012). Furthermore, the results of two recent
clinical studies (Fluhr et al., Br J Dermatol, 166(3): 483-90, 2012
and Fluhr et al., Br J Dermatol, 2014, doi: 10.1111/bjd.12880)
suggests that the skin of infants has a certain immaturity in its
capacity to capture water and to regulate the related mechanisms.
In addition, this work showed that the epidermal barrier is
organized structurally from birth to 2 years of age and is thus not
completely competent during this period. This helps explain the
fragility of infants' and young children's skin and the
susceptibility thereof to chemical, physical and microbial
attacks.
[0006] Moreover, incomplete skin maturation can have significant
clinical consequences. It is thus important to enable the skin to
be built and to develop correctly and harmoniously, without which
its functional and structural organization could be compromised. In
this respect, it is crucial to preserve the epidermis's barrier
function and capacity of renewal.
[0007] Diaper rash, or diaper dermatitis, is a dermatological
lesion common in babies wearing diapers (one child in three),
resulting in skin irritation, that is sometimes painful, appearing
where the diaper contacts the skin. It generally results from the
conjunction of several factors, the most important being extended
contact between the baby's skin and urine. Urine produces ammonia
by fermentation and thus will contribute toward modifying skin pH
which will then play an important and harmful role in the
physiopathology of the diaper rash. Moreover, the effects of urine
are added to those of perspiration trapped in skin folds and
beneath the diaper which soak the skin, creating a damp and
occlusive environment and considerably increasing pH and barrier
inflammation and degradation and thus amplifying the risks of
friction-generated lesions and thus irritation. In this context,
urine thus increases the risk of loss of skin barrier integrity,
which may lead to a more serious form of diaper rash, with open
wounds which may become infected and which must be treated by a
doctor.
[0008] The pharmaceutical or cosmetic products available on the
market generally contain zinc oxide, vitamins (A, D and D3), or
combinations thereof. These active agents are incorporated in a
cream or an aqueous paste or an ointment by mixing them with
semi-solid bases such as, for example, mineral oils, paraffin,
lanolin, etc. They may be used to prevent or reduce the harmful
effects of urine, such as diaper rash. However, while some of these
products provide relief during episodes of mild redness/irritation,
they show only a very relative efficacy against more significant
diaper rashes. And purely medicated products often have a
pharmacology and a concentration of ingredients which make them
difficult to use in the long term as they are potentially
irritating or allergenic. Moreover, the purpose of these products
is only to alleviate diaper dermatitis, not to preserve the barrier
function of the skin or the capacity of renewal of the epidermis.
There thus still remains a need, by integrating knowledge of
children's skin and the role of aggravating factors (like urine and
pH) to identify and characterize active agents and improved
formulations for dealing with diaper rash best.
FIGURE LEGENDS
[0009] FIG. 1: Production of PGE2, lipid inflammation mediator
[0010] FIG. 2: Expression of the PTGS2 gene, lipid inflammation
mediator
[0011] FIG. 3: Expression of the IL1alpha and IL8 genes, protein
inflammation mediators
[0012] FIG. 4: Expression of the TRPV1 and SPR genes, neurogenic
inflammation mediators
[0013] FIG. 5: Expression of the IVL, SMPD, CSN and CLDN1 genes,
barrier function markers
[0014] FIG. 6: Expression of the KRT19, ITGB1, ITGB4, ITGA6, BIRC5,
Lrig1 genes, stem cell markers
DESCRIPTION
[0015] The present Inventors showed that children's skin, and in
particular infants' skin, is particularly sensitive to the harmful
action of urine. In particular, they were able too identify
biological markers the expression of which is altered in the
presence of urine. Such markers are particularly advantageous
because they make it possible to follow the skin's response to
urine.
[0016] The Inventors developed methods for evaluating the in vitro
efficacy of active ingredients and formulations in preventing the
harmful effects of urine on children's skin, using a skin model
specifically able to reproduce the characteristics of children's
skin, and in particular that of very young children such as
infants. The studies of the prior art rested on the use of adult's
skin to analyze the skin's response to urine (Degouy et al.,
Toxicol In Vitro, 28(1): 3-7. 2014). However, the skin's properties
evolve during the first years of life (see, for example, Fluhr et
al., Exp Dermatol., 19(6): 483-492, 2010; Fluhr et al., Br J
Dermatol, 166(3): 483-90, 2012 and Fluhr et al., Br J Dermatol.,
2014, doi: 10.1111/bjd.12880) and it is unlikely that it is
possible to determine precisely the effects of urine on children's
skin from samples of adult's skin.
[0017] On the other hand, the Inventors developed reconstructed
skin models derived from samples from children and were able to
test the effect of urine on these models. They were thus able to
observe that the expression of certain biological markers was
altered when these models of reconstructed children's skin were
contacted with urine. Certain markers, such as markers of
inflammation, were thus more strongly expressed, whereas the
expression of others, such as stem cell markers or those of the
barrier function, was decreased. On the other hand, the variations
of expression of these markers were reduced, or became less marked,
when the models were treated with active agents or formulations
known to alleviate diaper rash. This result underlines the
physiological relevance of these markers. The importance of the use
of models of reconstructed skin from children and not from adults
in order to isolate such markers therefrom is further
reinforced.
[0018] The term "child", according to the invention, refers to an
individual under 16 years of age. Thus, the category of children
according to the invention includes newborns, between 0 and 1 month
of age, infants, between 1 month and 2 years of age, and children
per se, at least 2 years of age. A "newborn" as used herein may
equally well be born at full term or prematurely.
[0019] To remove any ambiguity, the term "child" used in the
present application without any further clarification should be
understood in the most general meaning thereof, i.e. as referring
to a person under 16 years of age. An "adult" according to the
present invention is a person who is not a child, in order words a
person over 16 years of age.
[0020] Preferably, the method according to the invention may be
used regardless of the ethnic or geographic origin of the skin, or
the phototype thereof. It may thus be of Caucasian, African, Asian,
South American, Melanesian or other origin; it may further have the
phototype I, II, III, IV, V or VI, without affecting the invention.
Indeed, the invention aims at identifying biological markers
characterizing any skin type and depending only on the donor's
age.
[0021] The methods of the invention thus rest on the use of a
suitable skin model, reproducing children's skin, as well as the
use of biological markers, the expression of which is affected by
urine in a particular manner in children's skin. The invention thus
makes it possible to determine precisely which active agents have
an advantageous effect on the prevention or treatment of the
harmful effects caused by contacting the skin with urine. The
methods of the invention are also suitable for evaluating the
activity of formulations. The Inventors were thus able to show that
certain formulations were more effective than others in preventing
and/or limiting the effects of urine, thus showing the utility of
the approach undertaken.
[0022] According to a first aspect, the invention has as an object
a method for evaluating the in vitro efficacy of an active agent or
a formulation in preventing the harmful effects of urine on
children's skin, characterized in that said method comprises the
following steps: [0023] a) contacting said active agent or said
formulation with a reconstructed skin model, said model being
obtained from a skin sample from a child; [0024] b) exposing the
reconstructed skin model of step a) to urine; [0025] c) measuring
the expression level of at least one biological marker in the skin
model of step b); and [0026] d) evaluating the efficacy of said
active agent or said formulation as a function of the level of step
c).
[0027] In a first preferred embodiment, the reconstructed skin
model is exposed to urine in step b) in the presence of the active
agent or the formulation. According to another preferred
embodiment, the active agent or the formulation is removed prior to
exposing said reconstructed skin model to urine during said step
b).
[0028] Furthermore, the invention also has as an object a method
for evaluating the in vitro efficacy of an active agent or a
formulation in reducing the harmful effects of urine on children's
skin, characterized in that said method comprises the following
steps: [0029] a) exposing a reconstructed skin model to urine, said
model being obtained from a skin sample from a child; [0030] b)
contacting said active agent or said formulation with the
reconstructed skin model of step a); [0031] c) measuring the
expression level of at least one biological marker in the skin
model of step b); and [0032] d) evaluating the efficacy of said
active agent or said formulation as a function of the level of step
c).
[0033] Persons skilled in the art will easily understand that steps
a) and b) may be carried out simultaneously or successively,
according to need. In other words, the reconstructed skin model may
be contacted with both urine and the active agent or formulation
tested. Alternatively, the skin model may first be exposed to
urine, then contacted with the active agent or the formulation.
[0034] The expression "the efficacy of a formulation or an active
agent in preventing or reducing the harmful effects of urine on
children's skin", in the meaning of the present application, refers
to the ability of the formulation or the active agent to cancel or
decrease said harmful effects on children's skin. Prevention refers
in this case to a treatment administered before the harmful effects
of urine develop, whereas reduction refers to a treatment
administered once the effects of urine appear.
[0035] According to a more preferred embodiment, the sample donor
is more particularly a donor between 0 and 1 month of age, between
1 month and 2 years of age or between 2 years and 16 years of age.
In other words, according to this embodiment, the sample donor is
selected from the group consisting of newborns, between 0 and 1
month of age, infants, between 1 month and 2 years of age, and
children, between 2 years and 16 years of age. More preferentially,
the sample donor is a newborn or an infant.
[0036] In a first embodiment, the expression level of said
biological marker of step a) is compared to a reference expression
level.
[0037] It is important to verify that the active agents and the
formulations of the invention are well tolerated. For example,
certain products currently marketed may cause irritation if used
regularly. Such an effect can only worsen a developing or existing
diaper rash.
[0038] According to another aspect, the invention thus has as an
object a method for evaluating the tolerance of an active agent or
a formulation when children's skin is exposed to urine, said method
comprising the following steps: [0039] a) contacting said active
agent or said formulation with a reconstructed skin model, said
model being obtained from a skin sample from a child; [0040] b)
exposing the reconstructed skin model of step a) to urine in the
presence of the active agent or the formulation; [0041] c)
measuring the expression level of at least one biological marker in
the skin model of step b); and [0042] d) determining whether said
active agent or said formulation is well tolerated by children's
skin as a function of the level of step c).
[0043] The method of the invention may further comprise a
comparison of the cell viability in the reconstructed skin model
treated with the active agent, the composition or the formulation
and in the control reconstructed skin model, i.e. treated with
urine alone. In this case, the active agent or the cosmetic
formulation is well tolerated by children's skin if the cell
viability of the reconstructed skin model is not affected by the
presence of the agent or the formulation.
[0044] According to another preferred embodiment, the method of the
invention thus comprises an additional step of determining the cell
viability in the reconstructed skin model exposed to urine and
treated with the active agent or the cosmetic formulation,
determining the cell viability of the control reconstructed skin
model and comparing the two.
[0045] Many tests for determining cell viability are available to
persons skilled in the art and are commonly used in cosmetics
science. In particular, mention may be made of the MTT test,
described for example in Mosman et al. (J Immunol Methods, 65(1-2):
55-63, 1983).
[0046] In another aspect, the invention makes it possible to
isolate formulations or active agents having an effect in
preventing the harmful effects of urine on children's skin. As the
experimental examples show, the invention makes it possible in
particular to distinguish active agents or formulations according
to their activity for preventing the harmful effect of urine on
children's skin. The invention thus is particularly suited to
identifying appropriate formulations or active agents for this very
specific skin.
[0047] The invention thus also has as an object a method for
identifying an active agent or a formulation for preventing the
harmful effects of urine on children's skin, characterized in that
said method comprises the following steps: [0048] a) contacting a
candidate active agent or formulation with a reconstructed skin
model, said model being obtained from a skin sample from a child;
[0049] b) exposing the skin model of step a) to urine; [0050] c)
measuring the expression level of at least one biological marker in
the skin model of step b); and [0051] d) determining whether said
candidate active agent or formulation is a formulation or an active
agent for preventing the harmful effects of urine on children's
skin as a function of the level of step c).
[0052] In a first preferred embodiment, the reconstructed skin
model is exposed to urine in step b) in the presence of the active
agent or the formulation. According to another preferred
embodiment, the active agent or the formulation is removed prior to
exposing said reconstructed skin model to urine during said step
b).
[0053] Likewise, the method of the invention makes it possible to
isolate active agents or formulations for reducing the harmful
effects of urine on children's skin. According to this embodiment,
this method comprises the following steps: [0054] a) exposing a
reconstructed skin model to urine, said model being obtained from a
skin sample from a child; [0055] b) contacting a candidate active
agent or formulation with the skin model of step a); [0056] c)
measuring the expression level of at least one biological marker in
the skin model of step b); and [0057] d) determining whether said
candidate active agent or formulation is a formulation or an active
agent for reducing the harmful effects of urine on children's skin
as a function of the level of step c).
[0058] Persons skilled in the art will easily understand that steps
a) and b) may be carried out simultaneously or successively,
according to need. In other words, the reconstructed skin model may
be contacted with both urine and the active agent or formulation
tested. Alternatively, the skin model may be exposed to urine
first, then contacted with the active agent or the formulation.
[0059] The candidate formulation is a formulation for preventing or
reducing the harmful effects of urine on children's skin if said
candidate formulation modulates the expression of at least one
biological marker of the invention. This modulation may correspond,
as the case may be, and in particular according to the nature of
the biological marker, to an increase or a reduction in the
expression of said marker. For example, it may be advantageous to
isolate formulations minimizing the harmful effects of urine on
markers preferentially expressed in stem cells, said formulations
making it possible to preserve the capacity of renewal of
children's fragile skin. Likewise, it would be advantageous to
identify formulations minimizing the harmful effects of urine on
barrier markers in children, in order to maintain the integrity of
the skin barrier. Finally, it could be desirable to isolate
formulations that would not induce markers of inflammation.
[0060] In the same way, the candidate active agent is an active
agent for preventing or reducing the harmful effects of urine on
children's skin if said candidate active agent modulates the
expression of at least one biological marker of the invention. This
modulation may correspond, as the case may be, and in particular
according to the nature of the biological marker, to an increase or
a reduction in the expression of said marker.
[0061] The term "urine" refers to a liquid consisting of about 95%
water and about 5% organic matter and mineral salts, mainly acid
chlorides, sulfates and phosphates of soda, potassium and
magnesium. The organic matter of urine comprises in particular
dyes, urea, uric acid and creatinine. Urine is typically produced
by the body of vertebrates, preferentially humans, from which it
evacuates waste. There exist also artificial or synthetic urines,
the composition of which is simplified compared to that of natural
urines, but which also come under the definition of the term
"urine" as used herein. Methods for manufacturing synthetic urine
are well-known to persons skilled in the art. Persons skilled in
the art can refer, for example, to patent EP 1 045 707 B1 or to the
articles by Shmaefsky, (Am. Biol. Teacher, 52: 170-173; 1990; Am.
Biol. Teacher, 57: 428-430, 1995) and Degouy et al. (Toxicol In
Vitro, 28(1): 3-7, 2014). Preferably, the synthetic or artificial
urine as defined in the present application comprises urea,
creatinine, ammonium acetate and citric acid. Thus, the synthetic
or artificial urine according to the invention advantageously
comprises urea at a concentration between 9 and 24.5 g/l, sodium
chloride between 3 and 14 g/l, creatinine between 0.6 to 2.3 g/l,
ammonium salts such as, for example, ammonium acetate, between
0.003 and 4.2 g/l and citric acid between 0.09 and 0.95 g/l.
[0062] Preferentially, the pH of the synthetic urine according to
the invention is between 6 and 10 and more preferentially between 7
and 9; more preferentially, the pH of the synthetic urine according
to the invention is equal to 8.
[0063] The expression "harmful effects of urine", in the meaning of
the present application, refers to pathological reactions ensuing
from exposure of the skin to urine. These pathological reactions
comprise in particular erythema, i.e. redness, accompanied by a
sensation of discomfort and pain, alteration of the barrier and the
skin, formation of lesions and desquamation.
[0064] pg,14 the meaning of the invention, the expression "the
efficacy of a formulation in preventing or reducing the harmful
effects of urine on children's skin" refers to the ability of the
formulation to decrease at least one of the above-mentioned harmful
effects.
[0065] First, the formulation or active agent of interest is
contacted with a reconstructed skin culture obtained from a sample
from a child. This contacting of the active agent of interest with
the skin model may be made directly. Alternatively, it could be
advantageous to formulate the active agent of interest, for example
so as to obtain a liquid composition, in order to facilitate its
contact with the skin model. Thus, according to an embodiment of
the invention, the method further comprises a step of formulating
the active agent, in particular in the form of a liquid solution,
in particular an aqueous solution, prior to the step of contacting
said active agent with a skin model.
[0066] The Inventors previously showed that the expression profiles
of specific categories of genes (for example, barrier,
inflammation, defense and stem cell genes) evolve as a function of
age (application PCT/EP2013/064926). Persons skilled in the art can
thus easily characterize skin at the molecular level from birth to
adulthood. More particularly, persons skilled in the art will note
that children's skin cells have a specific expression profile of
genes involved in specific physiological processes, in particular
cell metabolism, the stress response, inflammation, immunity,
apoptosis, growth/proliferation and the cell cycle, cell signaling,
migration and differentiation, the epidermal barrier, adhesion and
pluripotent stem cells of the skin.
[0067] In the meaning of the invention, the reconstructed skin
model obtained from a skin sample from a child may be any tissue
model comprising skin cells, in particular keratinocytes, and
wherein said skin cells were obtained from a sample from a
child.
[0068] The term "skin sample", in the meaning of the invention,
refers to any sample containing skin cells. The skin samples
according to the invention thus include fresh skin explants
obtained directly from the patient, suspended skin cell cultures,
monolayer skin cell cultures, bilayer skin cell cultures and tissue
models, including reconstructed skin cultures and reconstructed
mucosal cultures. As it is often difficult to work on fresh
explants, it is particularly advantageous, within the scope of the
present invention, to use skin cell cultures. Advantageously, the
skin cells according to the invention comprise normal, healthy or
diseased cells, or cells derived from lines. For example, the skin
cells placed in culture may be cells obtained from skin tissue
explant. The term "explant" or "skin explant" as used herein refers
to a sample of skin cells or tissue, which may be obtained for
surgical purposes or to perform analyses.
[0069] In particular, an explant may be obtained during surgical
excision. The term "excision" as used herein refers to a surgical
procedure consisting of cutting (excising) a portion of varying
width and depth of skin in order to treat a defect or growth
thereof. Excision is performed either to remove a cancerous or
suspected cancerous tumor, or to treat a benign skin defect which
is unwanted, for functional or cosmetic reasons. An excision
according to the invention includes for example skin samples
obtained after plastic surgery (mammoplasty, abdominoplasty,
face-lift, foreskin removal, otoplasty, i.e. ear pinback,
syndactyly or supernumerary fingers, etc.).
[0070] An explant also may be obtained by biopsy. The term "biopsy"
as used herein refers to a sample of skin cells or tissue taken for
the purposes of analysis. Several types of biopsy procedures are
known and performed in the field. The most common types include (1)
incisional biopsy, wherein only a tissue sample is taken; (2)
excisional biopsy (or surgical biopsy) consisting of total ablation
of a tumor growth, thus carrying out a therapeutic and diagnostic
procedure, and (3) needle biopsy, wherein a tissue sample is taken
with a needle, which may be wide or fine. Further types of biopsies
exist, such as for example smears or curettage, and are also
included in the present invention.
[0071] Alternatively, said skin cells may be obtained by stem cell
differentiation (Guenou et al., Lancet, 374(9703): 1745-1753, 2009;
Nissan et al., Proc. Natl. Acad. Sci., 108(36): 14861-14866, 2011;
Kraehenbuehl et al., Nature Methods, 8: 731-736, 2011).
[0072] The skin cells according to the invention, whether obtained
from a biopsy or obtained by stem cell differentiation, include at
least one type of cells habitually present in the hypodermis,
dermis and/or epidermis. These cells thus include, among others,
keratinocytes, melanocytes, fibroblasts, adipocytes, endothelial
cells, mast cells, Langerhans cells and/or Merkel cells.
Preferentially, the skin cells according to the invention include
at least keratinocytes and/or fibroblasts. More preferentially, the
skin cells according to the invention include keratinocytes and/or
fibroblasts.
[0073] Numerous skin cell culture methods are known to persons
skilled in the art. Any of these methods may be used to culture the
skin cells according to the invention. Advantageously, the skin
cells are cultured and/or stored under conditions maintaining, at
least partially, a cell metabolism and/or cell functions. The skin
cell culture according to the invention thus includes equally well
suspended skin cell cultures, monolayer skin cell cultures, bilayer
skin cell cultures and tissue models, including reconstructed skin
cultures and reconstructed mucosal cultures.
[0074] For example, suspended skin cell cultures are routinely
employed in a very large number of laboratories, for several
decades. Similarly, monolayer or bilayer skin cell cultures have
been known and used for a very long time.
[0075] Furthermore, numerous tissue models, including in particular
reconstructed skin models and reconstructed mucosal models (Rosdy
et al., In Vitro Toxicol., 10(1): 39-47, 1997; Ponec et al., J
Invest Dermatol., 109(3): 348-355, 1997; Ponec et al., Int J
Pharm., 203(1-2): 211-225, 2000; Schmalz et al., Eur J Oral Sci.,
108(5): 442-448, 2000; Black et al., Tissue Eng, 11(5-6): 723-733,
2005; Dongari-Batgtzoglou et Kashleva, Nat Protoc, 1(4): 2012-2018,
2006; Bechtoille et al., Tissue Eng, 13(11): 2667-2679, 2007; Vrana
et al., Invest Ophthalmol Vis Sci, 49(12): 5325-5331, 2008;
Kinicoglu et al., Biomaterials, 30(32): 6418-6425, 2009; Auxenfans
et al., Eur J Dermatol, 19(2): 107-113, 2009; Kinicoglu et al.,
Biomaterials, 32(25): 5756-5764, 2011; Costin et al., Altern Lab
Anim, 39(4): 317-337, 2011; Auxenfans et al., J Tissue Eng Regen
Med, 6(7): 512-518, 2012; Lequeux et al., Skin Pharmacol Physiol,
25(1): 47-55, 2012; EP 29 678; EP 285 471; EP 789 074; EP 1 451 302
B1; EP 1 878 790 B1; EP 1 974 718; US 2007/0148,771; US
2010/0,099,576; WO 02/070729; WO 2006/063864; WO 2006/063865; WO
2007/064305) are available to persons skilled in the art and are
included in the scope of the invention.
[0076] Advantageously, the tissue model comprises reconstructed
skin models and reconstructed mucosal models. Preferably, the
reconstructed skin model is selected from the group comprising
dermis models, principally containing stromal cells, and more
particularly fibroblasts, epidermis models essentially consisting
of keratinocytes, hypodermis models, skin models including a dermis
and an epidermis, and skin models comprising a dermis, an epidermis
and a hypodermis. The models comprising at least a dermis form
connective type tissues, whereas the models comprising at least an
epidermis form stratified epithelia comprising characteristic
layers of the tissue in question. For example, in the epidermis
models, it is possible to identify a basal layer (stratum basalis),
a spinous layer (stratum spinosum), a granular layer (stratum
granulosum) and a corneal layer (stratum corneum). Furthermore,
preferably, the reconstructed mucosal model according to the
invention is a mucosal model of the mouth, gum, vagina or
cornea.
[0077] Advantageously, said model is a connective type tissue model
of dermal matrix comprising a matrix substrate preferably selected
from: [0078] an inert substrate selected from the group consisting
of a semi-permeable synthetic membrane, in particular a
semi-permeable nitrocellulose membrane, a semi-permeable nylon
membrane, a Teflon membrane or sponge, a polycarbonate or
polyethylene, polypropylene, semi-permeable polyethylene
terephthalate (PET) membrane, an inorganic semi-permeable Anopore,
cellulose acetate or ester (HATF) membrane, a semi-permeable
Biopore-CM membrane, a semi-permeable polyester membrane, a
polyglycolic acid membrane or film.
[0079] This group includes for example the Skin.sup.2.TM. model
ZK1100 and Dermagraft.RTM. and Transcyte.RTM. dermal models
(Advanced Tissue Sciences); [0080] a cell culture-treated plastic
(formation of a dermal sheet: Michel et al., In Vitro Cell. Dev
Biol.-Animal, 35: 318-326, 1999); [0081] a gel or a membrane based
on hyaluronic acid (Hyalograft.RTM. 3D--Fidia Advanced Biopolymers)
and/or collagen (such as for example an equivalent dermis or
collagen lattices) and/or fibronectin and/or fibrin; this group
includes for example the Vitrix.RTM. dermal model (Organogenesis);
[0082] an optionally surfaced porous matrix (for example an
equivalent dermis), produced from collagen suitable for containing
one or more glycosaminoglycans and/or optionally chitosan
(EP0296078A1, WO 01/911821 and WO 01/92322).
[0083] This group also includes for example the Mimederm.RTM.
dermal model (Coletica).
[0084] These matrix substrates comprise stromal cells, particularly
fibroblasts.
[0085] Advantageously, said skin model is an epidermis model
comprising a matrix substrate preferably selected from: [0086] an
inert substrate selected from the group consisting of a
semi-permeable synthetic membrane, in particular a semi-permeable
nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon
membrane or sponge, a polycarbonate or polyethylene, polypropylene,
semi-permeable polyethylene terephthalate (PET) membrane, an
inorganic semi-permeable Anopore, cellulose acetate or ester (HATF)
membrane, a semi-permeable Biopore-CM membrane, a semi-permeable
polyester membrane;
[0087] this group includes the Reconstructed epidermis models
(Skinethic.RTM.) and the EpiDerm.RTM. model (Mattek Corporation);
[0088] a film or a membrane based on hyaluronic acid and/or
collagen and/or fibronectin and/or fibrin.
[0089] In this group, particular mention may be made of the models:
Laserskin.RTM. (Fidia Advanced Biopolymers), Episkin.RTM.
(L'Oreal).
[0090] These models may be inoculated with fibroblasts in the
dermal part.
[0091] These models, wherein fibroblasts may be optionally
integrated, act as a substrate for keratinocyte inoculation and
epidermis reconstruction. Advantageously, beside keratinocytes,
pigment cells, immunocompetent cells, nerve cells are introduced;
preferably, the immunocompetent cells are Langerhans cells.
[0092] Advantageously, said tissue model is a reconstructed skin or
mucosal tissue model comprising a dermal or chorion matrix
substrate, preferably selected from: [0093] an inert substrate
selected from the group consisting of a semi-permeable synthetic
membrane, in particular a semi-permeable nitrocellulose membrane, a
semi-permeable nylon membrane, a Teflon membrane or sponge, a
polycarbonate or polyethylene, polypropylene, semi-permeable
polyethylene terephthalate (PET) membrane, an inorganic
semi-permeable Anopore, cellulose acetate or ester (HATF) membrane,
a semi-permeable Biopore-CM membrane, a semi-permeable polyester
membrane, said inert substrate optionally containing stromal cells,
particularly fibroblasts, [0094] a gel based on collagen and/or
hyaluronic acid and/or fibronectin, and/or fibrin comprising
stromal cells, particularly fibroblasts, [0095] an optionally
surfaced porous matrix, produced from collagen suitable for
containing one or more glycosaminoglycans and/or optionally
chitosan, these porous matrixes incorporating stromal cells,
particularly fibroblasts, [0096] a de-epidermized dermis or dead
dermis, of human or animal origin.
[0097] In this group, particular mention may be made of the
following models: Mimeskin (Coletica), EpidermFT.TM.,
EpiAirway.TM., EpiOccular.TM. EpiOral.TM., EpiGingival.TM.,
EpiVaginal.TM. (MatTek corporation), Human Corneal Epithelium
(HCE), Human Oral Epithelium (HOE), Human Gingival Epithelium
(HGE), Human Vaginal Epithelium (HVE) (Skinethic.RTM.),
Phenion.RTM. Full Thickness Skin Model (Phenion), Apligraf.RTM.
(Organogenesis), ATS-2000 (CellSystems.RTM. Biotechnologie
Vertrieb) and Skin.sup.2.TM. (ZK1200-1300-2000 Advanced Tissue
Science).
[0098] Furthermore, models specifically intended for tissue therapy
are available which also may be used within the scope of the
present invention. Mention may be made of the Epidex (Modex
Therapeutiques), Epibase.RTM. (Laboratoire Genevrier), Epicell.TM.
(Genzyme), Autoderm.TM. and Transderm.TM. (Innogenetics)
models.
[0099] The matrix substrate is then inoculated with keratinocytes
to reconstruct the epidermis and eventually obtain a reconstructed
skin.
[0100] Advantageously, the skin model used comprises a model
wherein at least one complementary cell type has been incorporated,
such as endothelial cells (EC) and/or immune cells such as
lymphocytes, macrophages, mast cells, dendritic cells and/or
adipose cells and/or skin appendages, such as hair on the body and
head, sebaceous glands.
[0101] After the formulation of interest and the skin culture of
the invention have been contacted, the reconstructed skin model
thus treated may be exposed to urine.
[0102] The expression "expose the reconstructed skin model to
urine", in the meaning of the invention, refers to any exposure or
contacting of said reconstructed skin model with urine, whether
natural or synthetic.
[0103] Thus, according to a preferred embodiment, exposure of said
reconstructed skin model to urine according to the invention
comprises or consists of exposure to natural urine, i.e. urine
produced naturally by a human being. According to a more preferred
embodiment, exposure of the reconstructed skin model to urine
according to the invention comprises or consists of exposure to
artificial or synthetic urine, i.e. urine the composition of which
is determined and constant. According to an even more preferred
embodiment, exposure of the reconstructed skin model to urine
according to the invention comprises or consists of exposure to
synthetic urine comprising urea, creatinine, ammonium acetate and
citric acid. Preferentially, the pH of the synthetic urine to which
the skin culture is thus exposed is between 6 and 10 and more
preferentially between 7 and 9; still more preferentially, the pH
of the synthetic urine according to the invention is equal to
8.
[0104] After having exposed the reconstructed skin model of the
invention to urine, persons skilled in the art will be able to
proceed to measuring the expression level of the biological markers
of the invention.
[0105] The term "biological marker" in the meaning of the present
application refers to a characteristic which is objectively
measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacological responses to a
therapeutic intervention. A biological marker thus refers to a
whole range of various substances and parameters. For example, a
biological marker may be a substance the detection of which
indicates a particular diseased state (for example the presence of
activated protein C as a marker of infection), or on the contrary a
substance the detection of which indicates a specific physiological
state. The biological marker according to the invention is
preferentially a gene, gene products such as transcripts thereof
and peptides derived from transcripts thereof, a lipid, a sugar or
a metabolite.
[0106] According to an embodiment of the present invention, the
biological marker is a gene, gene products such as transcripts or
peptides, a lipid, a sugar or a metabolite the changes in
expression of which, in particular the expression level, correlate
with a physiological state of children's skin. According to a
particular embodiment, the biological marker is a peptide having an
enzymatic activity.
[0107] Persons skilled in the art seeking to determine the class to
which a genetic or protein marker belongs can easily consult the
relevant scientific literature or refer to public databases such
as, for example, those contained in the National Center for
Biotechnology Information website
(http://www.ncbi.nlm.nih.gov/guide/).
[0108] The Inventors particularly selected markers the variation of
the expression level of which varies after exposure to urine in a
surprising and unexpected manner in children. The selected markers
thus have a particular advantage within the scope of the method of
the invention, insofar as their expression level is measured on a
skin model reproducing the characteristics of children's skin.
[0109] The Inventors thus showed that markers of inflammation are
particularly expressed after children's skin was treated with
urine. Inflammation is a normal defense reaction of the body, but
it can contribute to reducing the skin's integrity. Moreover, the
Inventors showed that at same time, the expression of barrier
markers decreases.
[0110] Finally, stem cell markers, and consequently the skin's
capacity of renewal, are also affected. On the other hand,
contacting the skin with a formulation active against the effects
of urine makes it possible to prevent and correct the variations of
expression of said markers, which underlines the relevance
thereof.
[0111] The biological marker of the invention is thus
advantageously a marker selected from the group of skin
inflammation markers, barrier function markers and markers
preferentially expressed in stem cells.
[0112] The expression "skin inflammation markers", in the meaning
of the invention, refers to markers the variation of expression of
which correlates with skin inflammation.
[0113] The term "inflammation" according to the invention refers to
the set of defense reaction mechanisms by which the body
recognizes, destroys and eliminates all substances foreign to it.
"Skin inflammation" corresponds more particularly to an immune
system reaction to an attack on the skin, such as an environmental
attack, optionally causing a wound, even vascular damage as the
case may be. Skin inflammation may appear as an erythema,
characterized by redness associated with local vasodilatation, an
edema, characterized by swelling, and a sensation of heat.
Moreover, skin inflammation is accompanied by a variation of
expression level or concentration of genetic or protein markers
well-known to persons skilled in the art, who can refer, for
example, to Vahlquist (Acta Derm Venereol; 80: 161; 2000).
[0114] The triggering and the continuation of the inflammation, and
the diffusion thereof from the initial source, require factors
which are synthesized locally or which are in the inactive
precursor state in the circulation. Specific processes in the
inflammation reaction can be differentiated according to the
mediator type synthesized. Thus, the skin inflammation according to
the invention includes at least three distinct processes, protein
inflammation, lipid inflammation and neurogenic inflammation.
[0115] The term "protein inflammation" as used herein refers to the
production, in response to an external attack, of protein
inflammatory mediators, such as the cytokines IL-1, IL-2, IL-6, IL8
and TNF.alpha., the complement system, or proteins involved in
coagulation, as the case may be. The term "lipid inflammation" as
used herein refers to the production to said external attack of
lipid mediators, in particular prostaglandins and leukotrienes,
both synthesized from arachidonic acid, as well as activation of
the enzymes responsible for this production (Shimizu, Annu Rev
Pharmacol Toxicol., 49: 123-150, 2009). The protein and lipid
mediators thus produced will induce a reaction cascade within the
skin involving other inflammatory cells, in particular immune and
vascular cells. The clinical result is expressed particularly by
redness or edema.
[0116] In response to an external attack, the neurosensory system
had to be stimulated and associated with the inflammatory reaction
implementing then other cellular actors such as nerves (or nerve
endings) and cells such as mast cells. The term "neurogenic
inflammation" as used herein refers to the release by nerve
endings, in response to an external attack, of specific mediators,
in particular neuropeptides (in particular tachykinins including
substance P, and Calcitonin Gene-Related Peptide or CGRP); also
participating in the neurogenic inflammation according to the
invention is the activity of particular receptors such as the
substance P receptor or the receptor TRPV1. Neurogenic inflammation
most often results in a sensation of pain and/or discomfort and/or
of itching (pruritus).
[0117] The skin inflammation marker according to the invention is
preferably selected from protein inflammation markers, lipid
inflammation markers and neurogenic inflammation markers.
[0118] Preferentially, the protein inflammation marker is selected
from the group consisting of interleukins, preferably IL1.alpha.
and IL8. Human interleukin IL1.alpha. has a protein sequence
represented by the sequence of NCBI reference: NP_000566. This
protein is encoded by the human IL1A gene (NCBI reference: Gene ID:
3552), the sequence of which corresponds to NCBI reference:
NM_000575. The protein sequence of human interleukin IL-8
corresponds to the sequence of NCBI reference: NP_000575. This
protein is encoded by the human IL8 gene (NCBI reference: Gene ID:
3576). The sequence thereof is accessible under NCBI reference:
NM_000584.
[0119] The lipid inflammation marker is advantageously selected
from prostaglandins, including in particular prostaglandin E2, and
enzymes of the synthesis thereof from arachidonic acid, in
particular PTGS2.
[0120] Prostaglandin E2 (PGE2) is a well-known arachidonic acid
derivative obtained by the action of cyclooxygenase. Two isoforms
of cyclooxygenase (COX) exist: cyclooxygenase 1, which is
constitutive in tissues, and cyclooxygenase 2, which is induced by
inflammatory phenomena. Proinflammatory stimulation (trauma,
cytokines, etc.) thus lead to the synthesis of PGE 2, which is
responsible for vasodilatation (generating redness and edema),
sensitization of nociceptors to bradykinin and histamine
(responsible for pain) and fever (with cytokines IL1 and IL6).
[0121] The enzyme cyclooxygenase 2, also called
prostaglandin-endoperoxide synthase (PTGS), is encoded by the human
PTGS2 gene (NCBI reference: Gene ID: 5743). The sequence of this
gene is available under the NCBI reference: NM_000963 and the
protein sequence under NCBI reference: NP_000954.
[0122] Preferably, the marker of neurogenic inflammation is
selected from neuropeptides and neuropeptide receptors, in
particular the receptors TRPV1 and SPR. The receptor TRPV1
(Transient Receptor Potential Vanilloid 1) is a cation-channel type
membrane protein of the TRP family having the sequence of NCBI
reference: NP_061197. In the skin, TRPV1 is expressed by
keratinocytes, mast cells and nerve fibers. In response to an
aggressor, TRPV1 activation leads to the production of cytokines
and neuropeptides and is an actor in neurogenic inflammation. The
gene encoding receptor TRPV1 is the TRPV1 gene (NCBI reference:
Gene ID: 7442), the sequence of which has the NCBI reference: NM
_018727.
[0123] Substance P receptor (SPR; also known as neurokinin 1
receptor, NK1R, or tachykinin receptor 1, TACR1) is a G
protein-coupled receptor (GPCR) which transmits the signal of
substance P (SP) and other tachykinins. It is encoded by the human
TACR1 gene, the sequence of which has the NCBI reference:
NM_001058. Its peptide sequence is the sequence of NCBI reference:
NP_001049.
[0124] According to the invention, the skin inflammation marker is
thus preferably selected from the group consisting of IL1.alpha.,
IL8, PTGS2, PGE2, TRPV1 and SPR.
[0125] The present Inventors further showed that urine induces a
reduction in the expression of barrier function markers and markers
preferentially expressed in stem cells.
[0126] The "barrier markers" according to the invention comprise
markers which are expressed specifically in the outermost layers of
the epidermis and which participate in the barrier function.
[0127] As persons skilled in the art well know, the main function
of the skin is to establish a protective barrier against
environmental attacks while allowing certain exchanges between the
internal environment and the external environment. This barrier
function is chiefly provided by the stratum corneum of the
epidermis. Intercellular lipids and corneodesmosomes, and the
cornified envelope of corneocytes, are the key components.
[0128] However, beneath the stratum corneum, tight junctions
constitute a second line of the barrier function. These junctions
constitute in the stratum granulosum a selective paracellular
diffusion barrier preventing the penetration of harmful molecules.
Tight junctions are made up of various transmembrane proteins such
as in particular claudins, occludin and ZO1.
[0129] The barrier functions provided by the stratum corneum and
the tight junctions are closely linked. Indeed, the alteration of
one can influence the formation of the other.
[0130] Preferentially, the barrier function markers according to
the invention are markers expressed in the stratum corneum or
markers expressed in the tight junctions of the stratum granulosum.
In a more preferential embodiment, said epidermal barrier marker is
selected from the group consisting of CDSN (corneodesmosin), IVL
(involucrin), SMPD (sphingomyelinase or sphingomyelin diesterase),
and CLDN1 (claudin 1).
[0131] The corneodesmosome is the only junction structure of the
corneal layer, which underlines the importance of this structure
for maintaining corneal layer integrity. One of the principal
components of the corneodesmosome is corneodesmosin. It is the only
specific protein of the corneodesmosome: it thus plays an essential
role within this junction structure. Its sequence corresponds to
that under reference NP_001255. The CDSN gene (NCBI reference: Gene
ID: 1041) encodes corneodesmosin and has the sequence of reference
NM_001264.
[0132] Involucrin, having sequence NP_005538 and encoded by the IVL
gene (NCBI reference: Gene ID: 3713), which itself has sequence
NM_005547, participates in the formation of the cornified envelope
of corneocytes.
[0133] Sphingomyelinase (SMase) or sphingomyelin diesterase is a
hydrolase involved in sphingolipid metabolism. It cleaves
sphingomyelins into phosphocholine and ceramides 2 and 5, which are
part the intercellular lipid matrix which ensures the
watertightness of the corneal layer. Sphingomyelinase is a protein
the sequence of which is represented by NCBI reference: NP_000534.
The gene encoding this enzyme is the SMPD gene (NCBI reference:
Gene ID: 6609) having the sequence corresponding to reference
NM_000543.
[0134] Tight junctions represent one mode of cell adhesion, in
epithelial tissues. They block the circulation of fluids between
cells and thus ensure watertightness between two tissue
compartments. They are located at the apex of epithelial cells
where they form a continuous band around which watertightness is
ensured. The CLDN 1 gene (NCBI reference: Gene ID: 9076) encodes
the claudin 1 protein which is one of the most important components
of tight junctions. This protein has a sequence corresponding to
that of NCBI reference NP_066924. The CLDN1 gene sequence is
accessible under reference NM_021101.
[0135] The expression "markers preferentially expressed in stem
cells" according to the invention refers to the markers, and more
specifically to the genes and proteins, which are specifically
present in epidermal stem cells.
[0136] The expression "stem cell of the epidermis" or "epidermal
stem cell", in the meaning of the present invention, refers to an
epidermal cell capable of long-term renewal. The epidermal stem
cells of the invention comprise, among others, follicular stem
cells, sebaceous stem cells and basal stem cells, the latter also
being called interfollicular epidermal stem cells. The terms
"follicular stem cells", "sebaceous stem cells" and "basal stem
cells", in the meaning of the invention, refer to stem cells
located in the region of the hair follicle bulge, in sebaceous
glands and in the basal layer of the epidermis, respectively. In a
preferential embodiment of the invention, the epidermal stem cells
of the invention are basal stem cells.
[0137] More precisely, the term "epidermal stem cell", in the
meaning of the present invention, refers to a cell bestowed with a
high potential for long-term renewal. The term "potential for
renewal" as used herein refers to the capacity to undergo at least
one cell division cycle. A "high potential for long-term renewal"
thus represents a cell's capacity to enter several successive cell
division cycles. It is well-known that cells differentiated from
the skin are not capable of carrying out several successive
divisions (Fortunel and Martin, J Soc Biol, 202(1): 55-65, 2008).
It is understood herein that "successive" does not mean
"consecutive" and that there may be periods during which a stem
cell according to the invention remains quiescent without however
losing its high potential for long-term renewal.
[0138] Conservation of a high potential for long-term renewal is
expressed by asymmetrical division producing two different cells.
The first daughter cell is a stem cell identical to the mother stem
cell, while the second is a transit amplifying cell that divides in
a limited manner over a short period of time and then enters the
differentiation process. Advantageously, the epidermal stem cells
of the invention thus are further capable of generating at least
one type of epidermal cell by differentiation. In other words, the
transit amplifying cell is capable of giving rise to at least one
type of epidermal type by differentiation. Preferentially, said
epidermal cell is a keratinocyte. More preferentially, the transit
amplifying cell is capable of giving rise to all the types of
epidermal cells by differentiation.
[0139] Preferentially, the markers expressed in stem cells are
markers that participate in the functions and the protection of
stem cells. Mention may be made, for example, of the markers
.DELTA.Np63, BIRC5 (survivin), FN1 (fibronectin 1), MCSP
(melanoma-associated chondroitin sulfate proteoglycan), LRIG1
(leucine-rich repeats and immunoglobulin-like domains protein 1),
GJA1 (connexin 43), NID1 (nidogen 1), KRT15 (keratin 15), KRT19
(keratin 19), EGFR (epidermal growth factor receptor), CD71
(transferrin receptor), DSG3 (desmoglein 3), ITGB1BP1 (integrin
beta1 binding protein), ITGA6 (integrin alpha 6), ITGB1 (integrin
beta1) and ITGB4 (integrin beta 4) or markers involved in the
signaling and regulation of stem cell activity such as Wnt/beta
catenin, sonic hedgehog (SHH), NOTCH1 (notch homolog 1,
translocation-associated). .DELTA.Np63 and survivin are markers of
resistance to apoptosis, thus having a role in stem cell survival.
Cytokeratins 15 and 19 are positive stem cell markers, cytokeratin
15 being a marker of their survival. MCSP colocalizes with
integrins in cells that do not divide, whereas integrin beta1
(marker of basal membrane adhesion to the extracellular matrix) and
integrin alpha 6 (constituting hemidesmosomes, marker of
keratinocytes binding together) are surface proteins that take part
in intercellular communication, regulating the
differentiation/proliferation processes as well as interaction with
the niche. Transferrin receptor CD71 is a known surface marker for
stem cells which is used to isolate, in a population of integrin
alpha6-positive cells, cells with high clonogenic capacity.
Finally, Lrig1 is an epidermal growth factor receptor (EGFR)
antagonist, thus maintaining quiescent stem cells, whereas receptor
EGFR, which is a marker whose absence characterizes stem cells, in
contrast leads the cells down the proliferation pathway.
[0140] Preferentially, the marker preferentially expressed in the
stem cells of the invention is selected from the group consisting
of the markers KRT19 (keratin 19), BIRC5 (survivin), LRIG1
(leucine-rich repeats and immunoglobulin-like domains protein 1),
ITGA6 (integrin alpha 6), ITGB1 (integrin beta1) and ITGB4
(integrin beta 4). These markers are well-known to persons skilled
in the art. The KRT19 (NCBI reference: Gene ID: 3880), BIRC5 (NCBI
reference: Gene ID: 332), LRIG1 (NCBI reference: Gene ID: 26018),
ITGA6 (NCBI reference: Gene ID: 3655), ITGB1 (NCBI reference: Gene
ID: 3688) and ITGB4 (NCBI reference: Gene ID: 3691) genes thus
correspond to the sequences represented by the following Genbank
accession numbers: NM_002276, NM_001012270, NM_015541, NM_000210,
NM_002211 and NM_000213, respectively. The proteins keratin 19,
survivin, leucine-rich repeats and immunoglobulin-like domains
protein 1, integrin alpha 6, integrin beta1 and integrin beta 4
correspond, in turn, to the sequences represented by the following
Genbank accession numbers: NP_002267, NP_001012270, NP_056356,
NP_000201, NP_002202 and NP_000204, respectively.
[0141] It will be further evident to persons skilled in the art
that the method of the invention will allow an evaluation of the
efficacy of the formulation or the active agent which will be all
the more complete when a large number of markers of different types
are used.
[0142] According to a preferred embodiment, the method of the
invention comprises a step c) of measuring the expression level of
a combination of biological markers. Said combination according to
the invention comprises or consists of: [0143] at least one skin
inflammation marker and at least one barrier marker as defined
above; or [0144] at least one skin inflammation marker and at least
one marker preferentially expressed in stem cells, as defined
above; or [0145] at least one barrier marker and at least one
marker preferentially expressed in stem cells, as defined
above.
[0146] In a more preferential embodiment, said combination
comprises at least one skin inflammation marker and at least one
barrier marker and at least one marker preferentially expressed in
stem cells, as defined above.
[0147] The use of combinations of markers comprising at least one
marker of each of the various types indicated above is particularly
advantageous.
[0148] For each of these markers, the term "expression level"
refers to the cellular concentration of said marker. Thus the
expression level of prostaglandin E2 corresponds to the
concentration of said lipid in the cell. If the marker is a gene,
the "expression level" in the meaning of the invention corresponds
to the cellular concentration of at least one product of the gene
of said marker. More precisely, the expression level of said
biological marker corresponds to the quantity or to the cellular
concentration of the transcript of said gene or of the protein
derived from said transcript. According to a preferred embodiment,
the expression level of said biological marker corresponds to the
quantity or to the cellular concentration of the transcript of said
gene. According to another embodiment, the expression level of said
biological marker corresponds to the quantity or to the cellular
concentration of the protein derived from said transcript.
[0149] The expression "measuring the expression level of a
combination of biological markers", in the meaning of the present
application, refers to measuring the expression level of each
marker of the combination. The expression of a gene may be measured
for example at the nucleotide level, by measuring the quantity of
transcripts of said gene, and also may be measured for example at
the peptide level, by measuring for example the quantity of
proteins derived from said transcript. Thus, the expression
"measuring the expression level of said gene" in the meaning of the
invention refers to measuring the quantity of the gene product in
its peptide form or its nucleotide form.
[0150] Generally, the expression of the biological marker according
to the invention will be detected in vitro from the reconstructed
skin model.
[0151] In a particular embodiment, the method of the invention may
comprise one or more intermediate steps between obtaining the
reconstructed skin model and measuring the expression of the
biological marker, said steps corresponding to extracting from said
reconstructed skin model a lipid sample, an mRNA (or corresponding
cDNA) sample or a protein sample. Said sample may then be used
directly to measure the expression of the marker. The preparation
or extraction of mRNA (and the reverse transcription thereof into
cDNA), proteins or lipids from a cellular sample is a routine
procedure well-known to persons skilled in the art.
[0152] With regard to lipid mediators such as prostaglandin E, it
may not even be necessary to prepare a lipid sample. Indeed, this
mediator is secreted in the culture medium. It is then easy for
persons skilled in the art to assay prostaglandin E2 from said
culture medium. Several methods for assaying and quantifying
prostaglandin E2 have thus been described in the art, including in
particular ELISA methods. Such a method is thus detailed in the
experimental section of the present application and persons skilled
in the art can refer thereto. It should also be noted that kits are
commercially available for assaying prostaglandin E2 (for example,
from CissBio Assays or from Pierce).
[0153] Once a sample of mRNA (or corresponding cDNA) or protein is
obtained, the expression of the marker, in terms of either mRNA
(i.e. in all the mRNA or cDNA present in the sample) or proteins
(i.e. in all the proteins present in the sample), can be measured.
The method used to that end thus depends on the type of
transformation (mRNA, cDNA or protein) and on the type of sample
available.
[0154] When the expression of the marker is measured at the mRNA
(or the corresponding cDNA) level, any technology commonly used by
persons skilled in the art may be used. These technologies for
analyzing levels of gene expression, such as transcriptome
analysis, for example, include well-known methods such as PCR
(polymerase chain reaction, if starting with DNA), RT-PCR (reverse
transcription-PCR, if starting with RNA) and quantitative RT-PCR,
or nucleic acid chips (including DNA chips and oligonucleotide
chips) for a higher throughput.
[0155] The term "nucleic acid chips" as used herein refers to
several different nucleic acid probes attached to a substrate,
which may be a microchip, a glass slide or a microsphere-size bead.
The microchip may be composed of polymers, plastics, resins,
polysaccharides, silica or a material containing silica, carbon,
metals, inorganic glass or nitrocellulose.
[0156] The probes may be nucleic acids such as cDNA (cDNA chips),
mRNA (mRNA chips) or oligonucleotides (oligonucleotide chips), said
oligonucleotides typically having a length of between roughly 25
and 60 nucleotides.
[0157] To determine the expression profile of a particular gene, a
nucleic acid corresponding to all or part of said gene is marked
and then contacted with the chip under hybridization conditions,
leading to the formation of complexes between said marked target
nucleic acids and probes complementary to this nucleic acid
attached to the surface of the chip. The presence of the marked
hybrid complexes is then detected.
[0158] These technologies make it possible to follow the expression
level of one gene in particular or several genes, and even of all
the genes of the genome (full genome or full transcriptome) in a
biological sample (cells, tissues, etc.). These technologies are
used routinely by persons skilled in the art and thus it is not
necessary to detail them herein. Examples of implementations of the
invention based on analysis of gene expression (cDNA chips) and on
quantitative PCR are described in the experimental section.
[0159] Alternatively, it is possible to use any current or future
technology making it possible to determine the expression of genes
on the basis of the quantity of mRNA in the sample. For example,
persons skilled in the art can measure the expression of a gene by
hybridization with a marked nucleic acid probe, such as, for
example, with a Northern blot (for mRNA) or a Southern blot (for
cDNA), but also by techniques such as the serial analysis of gene
expression (SAGE) method and derivatives thereof, such as LongSAGE,
SuperSAGE, DeepSAGE, etc. It is also possible to use tissue chips
(also known as tissue microarrays, or TMAs). The tests commonly
employed with tissue chips include immunohistochemistry and
fluorescent in situ hybridization. For the analysis of mRNA levels,
tissue chips may be coupled with fluorescent in situ hybridization.
Finally, it is possible to use massively parallel sequencing to
determine the quantity of mRNA in the sample (RNA-Seq, or whole
transcriptome shotgun sequencing). For that purpose, several
methods of massively parallel sequencing are available. Such
methods are described in, for example, U.S. Pat. No. 4,882,127,
U.S. Pat. No. 4,849,077; U.S. Pat. No. 7,556,922; U.S. Pat. No.
6,723,513; WO 03/066896; WO 2007/111924; US 2008/0020392; WO
2006/084132; US 2009/0186349; US 2009/0181860; US 2009/0181385; US
2006/0275782; EP-B1-1141399; Shendure Et Ji, Nat Biotechnol.,
26(10): 1135-45. 2008; Pihlak et al., Nat Biotechnol., 26(6):
676-684, 2008; Fuller et al., Nature Biotechnol., 27(11):
1013-1023, 2009; Mardis, Genome Med., 1(4): 40, 2009; Metzker,
Nature Rev. Genet., 11(1): 31-46, 2010.
[0160] When the expression of the marker is measured at the protein
level, it is possible to employ specific antibodies, in particular
in well-known technologies such as immunoprecipitation,
immunohistology, Western blot, dot blot, ELISA or ELISPOT, protein
chips, antibody chips, or tissue chips coupled with
immunohistochemistry. Other techniques that may be used include
FRET or BRET techniques, methods of microscopy or histochemistry,
notably including methods of confocal microscopy and electron
microscopy, methods based on the use of one or more excitation
wavelengths and a suitable optical method, such as an
electrochemical method (voltammetry and amperometry techniques),
atomic force microscopy, and radio frequency methods, such as
multipolar resonance spectroscopy, confocal and non-confocal,
detection of fluorescence, luminescence, chemiluminescence,
absorbance, reflectance, transmittance, and birefringence or
refractive index (e.g., surface plasmon resonance, ellipsometry, a
resonant mirror method, etc.), flow cytometry, radioisotope or
magnetic resonance imaging, analysis by polyacrylamide gel
electrophoresis (SDS-PAGE), HPLC-mass spectrometry and liquid
chromatography-mass spectrophotometry/mass spectrometry (LC-MS/MS).
All of these techniques are well-known to the skilled person and it
is not necessary to detail them herein.
[0161] The expression "a reference expression level of a biological
marker", in the meaning of the present application, refers to any
expression level of said marker used as a reference. For example, a
reference expression level may be obtained by measuring the
expression level of the marker of interest in a children's skin
model, under particular conditions. Persons skilled in the art will
be able to choose these particular conditions as a function of
intended purpose when implementing the invention.
[0162] For example, in a preferred embodiment, the reference
expression level of a biological marker is the expression level of
said marker obtained in a children's skin model not treated with
the formulation or active agent of interest, and exposed to UV.
[0163] According to another embodiment, the reference expression
level of a biological marker is the expression level of said marker
obtained in a children's skin model, contacted with a reference
formulation or active agent, and exposed to urine.
[0164] When the reference expression level is an expression level
obtained in a skin model exposed to urine, persons skilled in the
art will easily understand that the conditions of urine exposure
for the skin model used in the method of the invention and for the
model used to obtain a reference expression level are
preferentially the same. Thus, preferentially, the composition of
the artificial urine used, and the exposure time used in the method
of the invention and for the model used to obtain a reference
expression level, are preferentially the same.
[0165] For example, persons skilled in the art can use as a
reference formulation any formulation known in the prior art for
its effect in preventing the harmful effects of urine on the
skin.
[0166] Preferentially, the reference formulation is selected from a
continuous oil phase emulsion diaper-change formula, a continuous
fatty phase ointment diaper-change formula, an aqueous paste
diaper-change formula, a continuous oil phase emulsion
diaper-change formula, a diaper-change liniment and diaper-change
wipes. More preferentially, the continuous oil phase emulsion
diaper-change formula corresponds to the formulation of Table 1,
the continuous fatty phase ointment diaper-change formula to that
of Table 2, the aqueous paste diaper-change formula to that of
Table 3, the continuous oil phase emulsion diaper-change formula to
that of Table 4, the diaper-change liniment to that of Table 5 and
the diaper-change wipes to that of Table 6.
TABLE-US-00001 TABLE 1 Diaper-change formula P1 - continuous oil
phase emulsion RAW MATERIAL % WATER q.s. ZINC OXIDE 5 to 20%
CAPRYLIC/CAPRIC TRIGLYCERIDE 5 to 20% COCO-CAPRYLATE/CAPRATE 1 to
10% POLYGLYCERYL-2-DIPOLYHYDROXYSTEARATE 1 to 10% GLYCERIN 1 to 10%
WAXES 1 to 5% PERSEA GRATISSIMA OIL 1 to 5% POLYGLYCERYL-3
DIISOSTEARATE 1 to 5% MAGNESIUM SULFATE 0 to 2% STEARALKONIUM
HECTORITE 0 to 2% PRESERVATIVES 0 to 2% PERSEA GRATISSIMA FRUIT
EXTRACT/AVOCADO 0 to 5% PERSEOSE HELIANTHUS ANNUUS SEED OIL 0 to 5%
UNSAPONIFIABLES UNDECYL DIMETHYL OXAZOLINE 0 to 5% ETHYL LINOLEATE
0 to 5% CAPRYLOYL GLYCINE 0 to 5%
TABLE-US-00002 TABLE 2 Diaper-change formula P2 - continuous fatty
phase ointment RAW MATERIAL % ZINC OXIDE 10 to 30% FISH LIVER
OIL/VITAMIN B5 10 to 30% LANOLIN 10 to 30% PETROLEUM JELLY q.s.
ANTIOXIDANT 0 to 2% FRAGRANCE/ESSENTIAL OIL 0 to 2%
TABLE-US-00003 TABLE 3 Diaper-change formula P3 - aqueous paste RAW
MATERIAL % VITAMIN B5 1 to 10% ZINC OXIDE 1 to 20% TITANIUM DIOXIDE
1 to 5% MINERAL FILLER (KAOLIN, TALC, SILICA) 1 to 20% ACTIVE AGENT
1 to 5% PRESERVATIVES 0 to 2% WATER q.s. GLYCERIN 1 to 10%
SEQUESTRANT 1 to 5%
TABLE-US-00004 TABLE 4 Diaper-change formula - continuous oil phase
emulsion RAW MATERIAL % VITAMIN B5 1 to 10% CETYL ALCOHOL 10 to 30%
STEARYL ALCOHOL 10 to 30% PETROLEUM JELLY/PARAFFIN 1 to 10%
ANTIOXIDANT 0 to 2% PLANT OIL 1 to 10% WAX 1 to 10% WATER q.s.
TABLE-US-00005 TABLE 5 Diaper-change liniment RAW MATERIAL % PLANT
OIL 20 to 50% WATER + CALCIUM HYDROXIDE q.s. STABILIZER 1 to 5%
PERSEA GRATISSIMA FRUIT EXTRACT/AVOCADO 0 to 5% PERSEOSE HELIANTHUS
ANNUUS SEED OIL 0 to 5% UNSAPONIFIABLES UNDECYL DIMETHYL OXAZOLINE
0 to 5% ETHYL LINOLEATE 0 to 5% CAPRYLOYL GLYCINE 0 to 5%
PRESERVATIVES 0 to 2% ANTIOXIDANT 0 to 2%
TABLE-US-00006 TABLE 6 Diaper-change wipes RAW MATERIAL % WATER
q.s. GLYCERIN 0 to 2% PEG-40 HYDROGENATED CASTOR OIL 0 to 2%
FRAGRANCE 0 to 2% PRESERVATIVES 0 to 2% PERSEA GRATISSIMA FRUIT
EXTRACT/AVOCADO 0 to 5% PERSEOSE HELIANTHUS ANNUUS SEED OIL 0 to 5%
UNSAPONIFIABLES UNDECYL DIMETHYL OXAZOLINE 0 to 5% ETHYL LINOLEATE
0 to 5% CAPRYLOYL GLYCINE 0 to 5% pH ADJUSTER 0 to 1%
[0167] According to another preferred embodiment, the reference
expression level of a biological marker is the expression level of
said marker obtained in a skin model obtained from a skin sample
from a child, said model not being treated with the formulation or
active agent of interest, and not exposed to urine.
[0168] According to another preferred embodiment, the reference
expression level of a biological marker is the expression level of
said marker obtained in a skin model obtained from a skin sample
from a child, said model not being treated with the formulation or
active agent of interest, but being exposed to urine.
[0169] According to another embodiment, the reference expression
level of a biological marker is the expression level of said marker
obtained in a skin model obtained from a skin sample from a child,
treated with the formulation or active agent of interest, and not
exposed to urine.
[0170] Persons skilled in the art will further easily understand
that the comparison of step d) is preferably carried out between
the measurements of expression levels obtained for skin models
obtained from skin samples from children, or from similar or even
identical histological structures. The expression "similar
histological structures", in the meaning of the present
application, means that the relative proportions of cell types
comprised in the compared skin models are similar. Thus, it is
preferable that the relative proportions of cell types comprised in
the skin model of step a) do not differ by more than 5% from the
relative proportions of cell types comprised in the skin model used
to obtain the reference expression level of step d). The expression
"relative proportion of a cell type", in the meaning of the present
application, refers to the ratio of the number of cells
corresponding to this cell type to the total number of cells
comprised in the skin model. Thus, for example, it is preferable
that the proportion of keratinocytes to the total number of cells
in the skin model of step a) does not differ by more than 5% from
the proportion of keratinocytes to the total number of cells in the
skin model used to obtain the reference expression level of step
d). The expression "identical histological structures", in the
meaning of the present application, means that the relative
proportions of cell types comprised in the compared skin models are
identical. In the meaning of the present invention, the relative
proportions of cell types comprised in the nipple skin model of
step a) are identical to the relative proportions of cell types
comprised in the skin model used to obtain the reference expression
level of step d) when they do not differ by more than 0.1%.
Advantageously, the proportion of keratinocytes to the total number
of cells in the skin model of step a) does not differ by more than
0.1% from the proportion of keratinocytes to the total number of
cells in the skin model used to obtain the reference expression
level of step d).
[0171] Persons skilled in the art will also easily understand that
the comparison of step d) is preferably carried out between the
measurements of expression levels obtained for skin models which
are of similar, or even identical, size, volume or weight. Thus, it
is preferable that the size, the volume or the weight of the skin
model of step a) does not differ by more than 5% from the size, the
volume or the weight of the skin model used to obtain the reference
expression level of step d). More preferentially, the size, the
volume and the weight of the skin model of step a) do not differ by
more than 5% from the size, the volume and the weight of the skin
model used to obtain the reference expression level of step c).
Even more preferentially, the size, the volume and the weight of
the skin model of step a) do not differ by more than 0.1% from the
size, the volume and the weight of the skin model used to obtain
the reference expression level of step d).
[0172] Alternatively, if the skin models differ by more than 5% in
terms of size, volume and weight, persons skilled in the art will
be able to normalize the level obtained in step c) and the
reference level of step d) using a normalization factor.
[0173] This normalization factor, for example, could be a directly
accessible physical marker such as the mass of the cells of the
sample, or the mass of a cellular component, such as the mass of
cellular DNA or the mass of cellular protein.
[0174] It also may be advantageous to use as the normalization
factor the expression level of a gene which is expressed at the
same level in all, or nearly all, of the body's cells. In other
words, according to a particular embodiment of the present
invention, the expression level of a housekeeping gene is used as
the normalization factor. According to another embodiment, the
level obtained in step c) and the reference level of step d) are
normalized using the expression level not of housekeeping genes but
of the proteins they encode. A housekeeping gene is a gene
expressed in all cell types which encodes a protein having a basic
function necessary for survival of all cell types. A list of human
housekeeping genes can be found in Eisenberg et al. (Trends in
Genet, 19: 362-365, 2003). The housekeeping genes according to the
invention include for example the following genes: B2M, TFRC,
YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1,
HPRT1, IPO8 and HMBS.
[0175] Persons skilled in the art will thus be able to easily
evaluate the efficacy of the formulation of interest as a function
of the comparison in step d).
[0176] According to another aspect, the invention has as an object
a kit for implementing a method according to the invention,
comprising the means necessary for measuring the expression level
of at least one marker selected from skin inflammation markers,
barrier function markers and markers preferentially expressed in
stem cells. Preferably, the skin inflammation marker is selected
from prostaglandin E2, PTGS2, IL-1.alpha., IL-8, TRPV1 and SPR, the
barrier function marker is selected from CNDSM, IVL, SMPD and
CLDN1, and the marker preferentially expressed in stem cells is
selected from KRT19, BIRC5, LRIG1, ITGA6, ITGB1 and ITGB4.
[0177] According to a particular embodiment, the kit according to
the invention further comprises the means necessary for measuring
the expression level of a combination of biological markers
selected from the group comprising or consisting of: [0178] at
least one skin inflammation marker and at least one barrier marker
as defined above; or [0179] at least one skin inflammation marker
and at least one marker preferentially expressed in stem cells, as
defined above; or [0180] at least one barrier marker and at least
one marker preferentially expressed in stem cells, as defined
above.
[0181] In a more preferential embodiment, said combination
comprises at least one skin inflammation marker and at least one
barrier marker and at least one marker preferentially expressed in
stem cells, as defined above.
[0182] The following examples are provided for illustration
purposes and unless otherwise indicated are not intended to be
limiting.
EXAMPLES
[0183] 1. Effect of Urine on Reconstructed Epidermises of
1-Year-Old Infants--Model Set-Up
[0184] 1.1. Introduction
[0185] Diaper rash or irritative diaper dermatitis affects more
than 35% of infants with a peak frequency around 10-12 months of
age. Our research program on children's skin, centered on the
epidermis, made it possible to show that:
[0186] Infants' skin is more fragile, and it less effectively
fulfills its barrier role against external attacks and infections.
The barrier being in the process of being built, it mobilizes its
stock of stem cells (abundantly present at birth), thus making it
more vulnerable. Finally, infants' skin has an inflammatory
potential which only needs to be developed.
[0187] In the buttocks area, beneath the diaper, the damp
atmosphere and occlusion quickly change these parameters toward
more fragility, more inflammation and higher pH, which creates an
imbalance. Moreover, in this context, extended contact with urine
and stools (exogenous factors) can cause redness and pain, clinical
signs of diaper rash.
[0188] Thus, associated with the known exogenous factors, these
discoveries make it possible to clarify the mechanisms and factors
which trigger diaper rash, namely an alteration of the barrier with
an in-demand and vulnerable stock of stem cells and a strong
inflammatory response.
[0189] To go further in understanding the action mechanisms and to
be able to have a model for evaluating cosmetic diaper-change
products, we developed a model which minimizes the harmful effects
of urine on epidermises of 1-year-old infants (peak appearance of
diaper rash) and studied the parameters found modulated thereby by
analyzing the markers of various inflammation pathways, barrier
function markers and stem cell markers.
[0190] To that end, epidermises were reconstructed with
keratinocytes from a 1-year-old infant.
[0191] The effect of an artificial urine preparation was evaluated
on these epidermises by analyzing the production of an inflammatory
mediator (ELISA) and the gene expression (RT-qPCR) of the
inflammation markers, barrier function markers and stem cell
markers.
[0192] 1.2. Material and Methods
[0193] The epidermises were produced with keratinocytes from a
1-year-old donor (foreskin removal) according to the model derived
from the method of Poumay et al. (Arch Dermatol Res; 296: 203-211,
2004). After two days of submerged culture, the reconstructed human
epidermises (RHEs) were cultured at the air/liquid interface for 10
days.
[0194] For each batch, at D10, the epidermises were incubated for
24 hours.
[0195] After incubation, the epidermises were treated (urine) or
not (control) topically with an artificial urine preparation
(composed of urea, creatinine, sodium chloride, ammonium acetate,
citric acid, pH 8) then incubated overnight (16 hours).
[0196] All the experimental conditions were carried out with
n=4.
[0197] Assay of the Inflammatory Mediator PGE2 in the Culture
Supernatants
[0198] After incubation, the quantities of PGE2 present in the
culture supernatants were measured using an ELISA kit according to
the supplier's specifications.
[0199] Analysis of the Differential Expression of Genes
[0200] The expression of markers was evaluated by RT-qPCR on the
messenger RNA extracted from the RHEs of each treatment.
[0201] The analysis of gene expression was carried out with n=2
using a PCR array containing genes of interest and two reference
(housekeeping) genes. Total RNA from each sample was extracted
using TriPure Isolation Reagent.RTM. according to the protocol
recommended by the supplier. The quantity and the quality of the
RNA were evaluated by capillary electrophoresis (Bioanalyzer,
Agilent). Complementary DNA (cDNA) was synthesized by reverse
transcription of the RNA in the presence of oligo(dT) and the
enzyme "Transcriptor Reverse Transcriptase". The cDNA obtained was
quantified by spectrophotometry, then the quantities of cDNA were
adjusted.
[0202] The polymerase chain reactions were carried out by
quantitative PCR with the "Light Cycler" system (Roche Molecular
Systems Inc.) according to the procedure recommended by the
supplier. The reaction mixture for each sample was: 10 ng/.mu.l
cDNA, primers for the various markers used, reaction mixture
containing the enzyme taq DNA polymerase, the marker SYBR Green I
(DNA intercalating agent) and MgCl.sub.2.
[0203] The incorporation of fluorescence into the amplified DNA is
measured continuously during the PCR cycles.
[0204] The quantitative analysis of the results is based on the
collection of threshold cycles (Ct). The threshold cycle is the
point at which the fluorescence emission signal is statistically
and significantly higher than the background. The threshold cycle
is directly correlated with the initial number of copies of target
DNA.
[0205] For each sample, the expression level of the gene of
interest is normalized by the expression level of the most stable
reference gene, which under these experimental conditions is the
GAPDH gene.
[0206] Table 7 lists the genes which were studied.
[0207] .DELTA.Ct is thus calculated:
.DELTA.Ct=Ct.sub.gene of interest-.DELTA.Ct.sub.reference gene
[0208] In a second step, the variation, as a function of the
treatment, of the number of copies of the gene of interest was
determined and .DELTA..DELTA.Ct is calculated:
.DELTA..DELTA.Ct=.DELTA.Ct.sub.control-.DELTA.Ct.sub.urine
[0209] Finally, relative quantity (RQ) is calculated:
RQ=(1+E).sup..DELTA..DELTA.Ct.
[0210] Considering that E (efficacy) is equal to 1, relative
quantity (RQ) is thus:
RQ=2.sup..DELTA..DELTA.Ct
TABLE-US-00007 TABLE 7 Classification and name of genes Cluster
name Abbreviation Gene name Housekeeping RPS28 Ribosomal protein
28S GAPDH Glyceraldehyde-3-phosphate dehydrogenase Inflammation
IL1A Interleukin 1, alpha IL8 Interleukin 8 TRPV1 Transient
receptor potential vanilloid, member 1 TACR1 or Tachykinin receptor
1 or Substance P SPR receptor PTGS2 Prostaglandin-endoperoxidase
synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
Epidermal IVL Involucrin differentiation, CDSN Corneodesmosin
Barrier function SMPD1 Sphingomyelin phosphodiesterase 1, acid
CLDN1 Claudin 1 Stem cells KRT19 Keratin 19 ITGB1 Integrin, beta 1
ITGB4 Integrin, beta 4 ITGA6 Integrin, alpha 6 LRIG1 Leucine-rich
repeats and immunoglobulin-like domains 1 BIRC5 Baculoviral IAP
repeat-containing 5 or survivin
[0211] 1.3. Results and Conclusions
[0212] 1.3.1. Inflammation
[0213] Infants' skin has a heightened inflammatory potential which
in contact with "aggressor" agents will transform into a genuine
inflammatory response causing redness and pain. In this context,
exposure of the buttocks area to urine (and stools) in an occlusive
environment will generate an inflammatory reaction involving three
distinct and interconnected pathways: protein inflammation, lipid
inflammation and neurogenic inflammation.
[0214] In the skin, the keratinocyte is one of the first cells
taking part in initiation of the inflammatory reaction (early
inflammation) in response to an environmental attack.
[0215] The "attacked" keratinocyte will then produce inflammatory
mediator proteins (type cytokines IL1 and IL8) and/or lipids (like
PGE2 and enzymes generating same from arachidonic acid such as
PTGS2) which will induce a reaction cascade within the skin then
involving other immune and vascular inflammatory cells. The
clinical result is expressed as redness or edema.
[0216] In reaction to an attack, the neurosensory system can be
stimulated and associated with the inflammatory reaction
implementing then other cellular actors such as nerves (or nerve
endings) and cells such as mast cells. This inflammatory reaction,
described as neurogenic, also involves the production of specific
mediators, which can be neuropeptides (such as substance P), and
also receptors such as the substance P receptor and the receptor
TRPV1*, in particular. The result is a sensation of pain and/or
discomfort and/or itching (pruritus).
[0217] *The receptor or nociceptor TRPV1 (Transient Receptor
Potential Vanilloid 1) is a cation-channel type membrane protein of
the TRP family. In the skin, TRPV1 is expressed by keratinocytes,
mast cells and nerve fibers. In response to an aggressor, TRPV1
activation leads to the production of cytokines and neuropeptides
and is an actor of neurogenic inflammation.
[0218] Treating the reconstructed epidermises with the artificial
urine preparation induced strong production of the lipid mediator
of inflammation, PGE2 (FIG. 1).
[0219] Moreover, urine induced the overexpression of genes, lipid
inflammation markers (PTGS2, enzyme of the prostaglandins synthesis
pathway), protein inflammation markers (IL1alpha and IL8) and
neurogenic inflammation markers (substance P receptor, SPR, and
TRPV1) (FIGS. 2, 3 and 4, respectively).
[0220] 1.3.2. Barrier Function
[0221] The outermost layer of the epidermis, the stratum corneum
(SC), constitutes, thanks to its particular structure, a protective
barrier against environmental attacks. The SC is the final product
of epidermal differentiation, where keratinocytes are transformed
into corneocytes; these corneocytes being coated with an
intercellular lipid matrix composed of ceramides, cholesterol and
essential fatty acids. After extrusion of the lamellar bodies, the
lipid precursors are metabolized to mature lipids. In the case of
the ceramide precursors, it is the enzymes acid sphingomyelinase
and beta glucocerebrosidase which transform them into ceramides 2
and 5 (for sphingomyelinase) and into ceramides 1, 3, 4, 6, 7, 8
and 9 (for glucocerebrosidase). Corneocytes are surrounded by a
protein cornified envelope composed of numerous proteins such as
involucrin, loricrin, small proline-rich proteins and sciellin in
particular, solidly joined together by the action of
transglutaminases.
[0222] Corneodesmosomes are modified, specialized desmosomes which
attach cells to each other in the SC and ensure their cohesion.
Corneodesmosomes are composed of transmembrane glycoproteins such
as desmoglein 1, desmocollin 1 and corneodesmosin.
[0223] The epidermal barrier function is principally provided by
the stratum corneum, and intercellular lipids and corneodesmosomes
and the cornified envelope of corneocytes are its key
components.
[0224] However, beneath the SC, tight junctions constitute a second
line of the barrier function. They form at the level of the stratum
granulosum anchor points between cells, forming a physical barrier
at the apical pole of the cells. These junctions thus constitute a
selective paracellular diffusion barrier preventing the penetration
of harmful molecules. The tight junctions are composed of various
transmembrane proteins such as, in particular, claudins, occludin
and ZO1.
[0225] The barrier functions provided by the SC and the tight
junctions are closely linked. Indeed, the alteration of one can
influence the formation of the other.
[0226] In our experimental conditions, urine induced a decrease in
the gene expression of key markers of the barrier function such as
involucrin (cornified envelope), acid sphingomyelinase (ceramide
maturation), corneodesmosin (corneodesmosome) and claudin 1 (tight
junction) (FIG. 5).
[0227] 1.3.3. Stem Cells
[0228] The stem cells of tissue in permanent renewal are
classically defined as being rare and relatively quiescent cells.
They have a unique capacity of self-renewal and of tissue
regeneration which enables them to ensure the homeostasis and
integrity of the tissue wherein they reside.
[0229] Among epidermal stem cells, interfollicular stem cells
located in the basal layer constitute the principal epidermal stem
cell reservoir. These cells reside in an anatomical and functional
microenvironment, the niche, which helps maintain their
characteristics, in particular when physiological conditions
change. Interfollicular stem cells and their niches are involved in
maintaining epidermal integrity and regeneration.
[0230] The stem cells are identifiable only by following several
markers: [0231] Keratin 19 is a marker of proliferation of stem
cells maintaining their characteristics. [0232] Integrins beta 1,
beta 4 and alpha 6 (cell surface receptors) are identified as
markers of stem cells, and more particularly of the interaction
with their niche. They also help regulate the processes of
differentiation and proliferation [0233] Survivin (BIRC5) is a
marker of stem cells regulating the cell cycle. Marker of
resistance to apoptosis, it has a role in the survival of stem
cells faced with attacks (like UV). [0234] Lrig1 is an epidermal
growth factor receptor antagonist and a marker maintaining the
quiescent stem cells.
[0235] Treatment with artificial urine strongly limited the
expression of several markers characteristic of stem cells and of
their niche (FIG. 6).
[0236] Treatment with an artificial urine preparation of
epidermises reconstructed with skin cells of 1-year-old infants
made it possible to show, in these experimental conditions, that
urine has a harmful effect on barrier function markers and on stem
cell markers. Moreover, urine induced significant inflammation
involving various pathways, on the one hand nonspecific
inflammation with various protein and lipid markers and, on the
other, neurogenic inflammation.
[0237] Once validated, this model was used to evaluate the efficacy
of cosmetic products formulated for application during a diaper
change on the buttocks area of infants.
[0238] 2. Evaluation of the Efficacy of Cosmetic Diaper-Change
Products on the Model of Reconstructed 1-Year-Old Infant
Epidermises "Attacked" with Artificial Urine
[0239] 2.1. Material and Methods
[0240] Epidermises were reconstructed in the same manner as
described above.
[0241] Reconstructed epidermises derived from keratinocytes of the
skin of 1-year-old infants were or were not treated topically with
the test products and were incubated for 24 hours. After
incubation, the epidermises again were or were not treated
topically with the test products and then were exposed to the
artificial urine (also applied topically). The RHEs were then
incubated overnight (16 hours). All the experimental conditions
were carried out with n=4.
[0242] Four cosmetic diaper-change products, of distinct
formulations, were tested and named P1, P2, P3 and P4.
[0243] These products are described in Tables 1 to 4,
respectively.
[0244] The assay of the inflammatory mediator PGE2 in the culture
supernatants and the analysis of the differential expression of
genes were carried out as described above.
[0245] For each sample, the expression level of the gene of
interest is normalized by the expression level of the most stable
reference gene, which in these experimental conditions is the GAPDH
gene. The calculation of relative quantities was carried out as
described above.
[0246] After calculation of the relative quantities of the genes of
interest in the various conditions, Control (without urine, without
test product), Urine (without test product) and Product P (test
product+urine), the percentage of modulation (stimulation or
inhibition) exerted by the test product on the harmful effect of
urine is calculated according to the following formulas:
Stimulation(%)=(RQ.sub.test product-RQ.sub.urine).times.100
Inhibition(%)=(RQ.sub.urine-RQ.sub.test product).times.100
[0247] Statistical analysis to calculate the significance of the
results: between-group comparisons were carried out using the
unpaired bilateral Student's t-test; *p<0.05; **p<0.01;
***p<0.001.
[0248] 2.2. Results and Conclusion
[0249] 2.2.1. Inflammation
TABLE-US-00008 TABLE 8 % inhibition of the expression of the IL1A
and IL8 genes by the test products (compared to urine) Mean
Inhibition Protein Infl. IL1 and Test product IL1A IL8 IL8
Significance P1 43 29 36 * P2 24 15 20 P3 32 30 31 * P4 39 0 20
[0250] Only products P1 and P3 inhibit IL1A and IL8.
TABLE-US-00009 TABLE 9 % inhibition of the expression of the PTGS2
gene and the production of PGE2 by the test products (compared to
urine) Test Mean Lipid Infl. PGE2 product PGE2 PTGS2 and PTGS2
Significance P1 75 88 82 ** P2 103 74 89 *** P3 28 76 52 * P4 58 75
67 *
[0251] All the products inhibit the lipid markers. P2 and P1
perform best.
TABLE-US-00010 TABLE 10 % inhibition of the expression of the TRPV1
and SPR genes by the test products (compared to urine) Test product
TRPV1 SPR Mean Neurogenic Infl. Significance P1 89 51 70 *** P2 65
23 44 ** P3 72 0 36 P4 93 30 62 **
[0252] All the products, except P3, effectively inhibit the markers
of neurogenic inflammation, P1 being most effective.
[0253] This analysis of the inflammation markers makes it possible
to show on the one hand the relative efficacy of the test products
to modulate inflammation induced by urine and, on the other, to
rank the products according to their efficacy.
[0254] P1>P2>P4>P3. Product P1 is the only product able to
inhibit all the inflammation markers, of whatever type, to a
significant degree.
[0255] 2.2.2. Barrier
TABLE-US-00011 TABLE 12 % stimulation of barrier markers by the
test products (compared to urine) Test IVL CDSN SMPD1 CLDN1 product
Significance Significance Significance Significance P1 123 ** 64
*** 106 *** 41 ** P2 226 *** 32 * 51 ** 23 * P3 74 * -14 115 ***
-21 P4 80 * 31 90 *** 2
[0256] Products P1 and P2 perform well to stimulate the expression
of the various barrier markers, and P1 stimulates all the markers
to a greater degree, except for the greater stimulation of
involucrin by P2. Products P3 and P4 have no effect on the markers
corneodesmosin and claudin 1.
[0257] This analysis of the barrier markers makes it possible to
show on the one hand the relative efficacy of the test products to
strengthen and protect the barrier altered by urine and, on the
other, to classify the products according to their efficacy.
[0258] P1>P2>P4>P3. Product P1 is the most effective
product for strengthening and protecting the epidermal barrier.
[0259] 2.2.3. Stem Cells
TABLE-US-00012 TABLE 14 % stimulation of stem cell markers by the
test products (compared to urine) Test product KRT 19 ITGB1 ITGA6
ITGB4 BIRC5 Lrig1 P1 93*** 62** 45* 90*** 48** 91** P2 51* 83***
77* 66** 14 27* P3 116*** 33 20 -6 48** 32* P4 80*** 78* 36* -43
42** 73*
[0260] To identify interfollicular stem cells in the epidermis it
is important to follow in parallel several markers specific to stem
cells and to their niche. The test products stimulate to varying
degrees the expression of the markers KRT19, ITGB1, A6 and B4,
BIRC5 and Lrig1 altered by urine. P1 stimulates in a significant
manner all the stem cell markers. P2 and P4 stimulate five of the
six stem cell markers. P3 acts only on three of the markers.
[0261] This analysis of the stem cell markers makes it possible to
show on the one hand the relative efficacy of the test products in
protecting stem cells altered by urine and, on the other, to
classify the products according to their efficacy.
[0262] P1>P2>P4.gtoreq.P3. Product P1 is the most effective
product in protecting stem cells of the basal layer of the
epidermis.
* * * * *
References