WO2009127249A1

WO2009127249A1 - Method for identifying compounds suitable for stimulation of stem cell production in skin

Info

Publication number: WO2009127249A1
Application number: PCT/EP2008/054575
Authority: WO
Inventors: Igor Arturovic Petropavlov; Igor Anatolievich Pomytkin; Tamara Alexsandrovna Vitkalova
Original assignee: United Technologies UT AG
Current assignee: United Technologies UT AG
Priority date: 2008-04-16
Filing date: 2008-04-16
Publication date: 2009-10-22
Anticipated expiration: 2010-10-16
Also published as: RU2011119212A; RU2485969C2

Abstract

The present invention relates to compounds suitable for stimulation of stem cell production in skin and method for identifying thereof, said method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the candidate compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin. Further, the present invention relates to compositions comprising compounds suitable for stimulation of stem cell production in skin.

Description

United Cosmeceuticals GmbH Wagistrasse 13 CH-8952 Schlieren/ZH Switzerland

METHOD FOR IDENTIFYING COMPOUNDS SUITABLE FOR STIMULATION OF STEM CELL PRODUCTION IN SKIN

Field of the Invention [0001] The present invention relates to compounds suitable for stimulation of stem cell production in skin and method for identifying thereof.

Background of the invention [0002] Stem cell is a cell that possesses two properties: (1 ) retains the ability to self-renewal through mitotic cell division while maintaining the undifferentiated state and (2) has the potency to differentiate into any type of a specialized cell. Stem cells could be recognized by specific stem cell markers. Such markers are well-known from the art. Rao R et al., Biology of Reproduction, 71 :1772-1778, 2004. Usually, stem cell markers represent gene products that are functionally associated with maintenance of undifferentiated stem cell state, maintenance of pluripotency, multipotency, or sometimes unipotency. The example of the reliable stem cell marker is OCT4, which also named OCT3/4 or Pou5f1. It is a transcription factor of the POU family that is critically involved in maintenance of the undifferentiated stem cell state and pluripotency. Watson CM et al.. Cell Struct. Func, 26: 123-129, 2001. Pesce M et al.. Stem Cells, 19: 271 -278, 2001.

[0003] At least three populations of endogenous stem cells are identified in adult skin. The stem cell population is found in the bulge region of the hair follicle. Potten CS et al.. Experientia 39,1125-1129, 1983. Watt FM. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353,831 -837, 1998. Lavker RM et al.. Proc. Natl. Acad. Sci. USA. 97,13473-13475, 2000. Another stem cell population is known to reside in the interfollicular basal layer of the epidermis. Tumbar T et a!.. Science 303,359-363, 2004. Taylor G et a!.. Cell 102,451 -461 , 2000. And still another stem cell population is found in the mesenchymal compartment. This population is capable to form a variety of cell types in vitro and even is able to generate an epidermal layer consisting of different cell types in a 3-dimensional model of skin. Crigler L et a!.. FASEB Journal. 2007:21 :2050-2063. Because of the capacity of endogenous stem cells to regenerate skin, there is a need in agents that are able to stimulate production of stem cells in skin to promote skin regeneration and renovation process under unfavorable circumstances such as trauma, disease, or aging. However, reliable identification of such agents is limited by the absence of suitable methods.

[0004] It is an object of the present invention to provide compounds suitable for stimulating stem cell production in skin and methods for identifying thereof.

Detailed Description of the Invention [0005] The present invention provides a method for identifying a candidate compound suitable for stimulating stem cell production in skin, said method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the candidate compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin.

[0006] Further, the present invention provides a compound suitable for stimulating stem cell production in skin, said compound identifiable by a method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin.

[0007] Further, the present invention provides a composition for stimulating stem cell production in skin comprising a compound suitable for stimulating stem cell production in skin, said compound identifiable by a method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin; and a cosmetically acceptable diluent or carrier.

[0008] Preferably, the animal of the present invention is a mammal. More preferably, the mammal is a mouse.

[0009] The term "stem cell" refers to a cell possessing two properties: (1 ) self-renewal and (2) potency, wherein the term "self-renewal" means the ability of the cell to go through numerous cycles of cell division while maintaining the undifferentiated state; and wherein the term "potency" means the capacity of the cell to differentiate into specialized cell types. Preferably, the stem cell of the present invention is pluripotent, multipotent, or unipotent stem cell. More preferably, the stem cell of this invention is pluripotent stem cell. The term "pluripotent" refers to a stem cell that can differentiate into cells derived from any of the three germ layers viz. mesoderm, endoderm, and ectoderm from which all the cells of the body arise. The term "multipotent" refers to a stem cell that can produce only cells of a closely related family of cells. The term "unipotent" refers to a stem cell that can differentiate into only one cell type.

[0010] The term "stem cell marker" refers to any marker recognizing pluripotent, multipotent, or unipotent cells in skin. The present invention is not limited in any way to specific stem cell marker but is applicable to all such markers now known or subsequently discovered or developed. A skilled artisan recognizes how to determine a particular suitable stem cell marker for the use in the method of the present invention. Nonetheless, a preferred stem cell marker of the present invention is selected from the group of gene expression markers consisting of OCT4, NANOG, SOX2, TDGF1 , DNMT3B, GABRB3, GDF3, BRIX, CD9, COMMD3, CRABP2, EBAF, FGF4, FGF5, FOXD3, GAL, GBX2, GRB7, IFITM1 , IFITM2, IL6ST, IMP2, KIT, LEFTB, LIFR, LIN28, NODAL, NOG, NR5A2, NR6A1 , PODXL, PTEN, REST, SEMA3A, SFRP2, SSEA-1 , SSEA-3, SSEA-4, TRA-1 -60, TRA-1 -81 , TERT, TFCP2L1 , UTF1 , ABCG2, Notch-1 , Zpf206, LeftyA, LeftyB, c-Myc, Klf4, Xist and ZFP42. More preferably, the stem cell marker of the present invention is OCT4. In practicing the present invention the stem cell marker can be detected by methods known in the art. Such methods include, but are not limited to, northern blotting, western blotting, immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay, and immunoelectron spectroscopy.

[0011] The term «dermatologically acceptable diluent or carrier" refers to one or more compatible solid or liquid filler diluents or carriers, which are suitable for administration to any portion of the human skin, and are compatible with the compound suitable for stimulating stem cell production. Examples of such carriers include, but are not limited to, distilled or deionized water, propyleneglycol, glycerol, and oil.

[0012] Preferably, the compound suitable for stimulating stem cell production, said compound identifiable by the method of the present invention is selected from the group consisting of interleukins and growth factors.

[0013] More preferably, the interleukin of the present invention is selected from the group consisting of interleukin 1 alpha, interleukin 1 family member 5 (delta), interleukin 1 family member 6 (epsilon), interleukin 1 family member 7 (zeta), interleukin 1 family member 8 (eta), interleukin 1 family member 9, interleukin 1 family member 10 (theta), interleukin 2, interleukin 3, interleukin 4, interleukin 6, interleukin 8, and interleukin 18.

[0014] More preferably, the growth factor of the present invention is selected from the group consisting of CSF, GM-CSF, VEGF, EGF, FGF, IGF, and PDGF.

[0015] The compositions of the present invention are useful for regulating the skin condition, visible and/or tactile discontinuities in skin (especially the skin surface; such discontinuities are generally undesirable). Such discontinuities may be induced or caused by internal and/or external factors, and include the signs of skin aging described herein. The term "regulating skin condition" includes prophylactically regulating and/or therapeutically regulating skin condition, including visible and/or tactile discontinuities in skin. As used herein, prophylactically regulating skin condition includes delaying, minimizing and/or preventing visible and/or tactile discontinuities in skin. As used herein, therapeutically regulating skin condition includes ameliorating, e.g., diminishing, minimizing and/or effacing, discontinuities in skin. Regulating skin condition involves improving skin appearance and/or feel. Also, the compositions of the present invention are particularly advantageous for treatment of acne and other skin disorders.

[0016] The compositions of the present invention are particularly advantageous for regulating signs of skin aging, more especially visible and/or tactile discontinuities in skin texture associated with aging.

"Regulating the signs of skin aging" includes prophylactically regulating and/or therapeutically regulating one or more of such signs (similarly, regulating a given sign of skin aging, e.g., lines, wrinkles or pores, includes prophylactically regulating and/or therapeutically regulating that sign). As used herein, prophylactically regulating such signs includes delaying, minimizing and/or preventing signs of skin aging. As used herein, therapeutically regulating such signs includes ameliorating, e.g., diminishing, minimizing and/or effacing signs of skin aging.

[0017] "Signs of skin aging" include, but are not limited to, all outward visibly and tactilely perceptible manifestations as well as any other macro or micro effects due to skin aging. Such signs may be induced or caused by intrinsic factors or extrinsic factors, e.g., chronological aging and/or environmental damage. These signs may result from processes which include, but are not limited to, the development of textural discontinuities such as wrinkles, including both fine superficial wrinkles and coarse deep wrinkles, skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), scaliness, flakiness and/or other forms of skin unevenness or roughness, loss of skin elasticity (loss and/or inactivation of functional skin elastin), sagging (including puffiness in the eye area and jowls), loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration (including undereye circles), blotching, sallowness, hyperpigmented skin regions such as age spots and freckles, keratoses, abnormal differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the stratum corneum, dermis, epidermis, the skin vascular system (e.g., telangiectasia or spider vessels), and underlying tissues, especially those proximate to the skin.

[0018] The content of the compound of the invention is in the range from 0.0000001 to 10 %, preferably 0.000001 to 0.00001 % by the weight of the composition.

[0019] The compositions of the present invention are usually formulated to have a pH from about 4.5 to 9.5, more preferably from about 5 to about 7.5. [0020] The following examples are presented to demonstrate the invention. The examples are illustrative only and are not intended to limit the scope of the invention in any way.

Example 1

[0021] This example demonstrates the methods of identifying a compound suitable for stimulating stem cell production in skin

Method. 5-day old male BALB/C mice were randomly allocated to experimental groups (n=10 per group) and control group (n=10). All experiments were carried out in accordance with the "Principle of laboratory animal care" formulated by NIH. A cream comprising a test compound was applied onto skin of mice from experimental groups once- a-day for five weeks. Control mice received vehicle. At the end of the experiment, samples of skin contacted with the test compounds were obtained from all mice under anesthesia. Stem cell marker OCT4 levels in skin samples were analyzed immunohistochemically using goat polyclonal OCT4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1 :8000). Briefly, skin samples were fixed in 10% formalin and then paraffined by standard procedure. Paraffined sections were cut at 4 μm and deparaffinized. Endogenous peroxidases were quenched with 3% hydrogen peroxide. Then, sections were heated for 5 min in 10 mM citrate buffer to destroy nuclear membranes, treated with 1 % BSA at room temperature for 40 min, and were incubated overnight at 4°C with the OCT4 primary antibodies. The following day, the sections were incubated with (step 1 ) biotinilated anti-goat immunoglobulin B (Santa Cruz Biotechnology, Santa Cruz, CA) used at 1 :200 dilutions and (step 2) horseradish peroxidase-conjugated avidin-biotin complex. Staining was executed with 3-amino-ethyl-carbazole and hydrogen peroxide. Finally, counterstaining was performed with Mayer's hematoxylin. Immunohistochemically stained skin sections were examined by light microscopy. Number of OCT4-positive cells per mm² was counted. Results are expressed in fold increase in mean density of OCT4-positive cells compared with control. Difference between groups were considered significant at p<0.05 (one-way ANOVA).

Identifying a candidate compound. The aim of the testing was to identify a candidate compound suitable for stimulating stem cell production in skin. Five test compounds were used. A cream comprising a test compound or vehicle was prepared by conventional methods. The cream was applied onto mice skin as described under the method. Density of stem cells in skin was assessed immunohistochemically as described above. No effect on stem cell production was found upon treatment with the four tested compounds. One test compound, interleukin 1 alpha, demonstrated significant 1.9-fold (p<0.05) increase in stem cell levels in skin as compared to control. Thus, interleukin 1 alpha was identified as the candidate compound suitable for stimulating stem cell production in skin.

Example 2

[0022] This example demonstrates the composition for stimulating stem cells production in skin

Ingredient Content, wt.%

Interleukin 1 alpha 0.000001 Citrate buffer qs to pH 5.5 Distilled water to 100

The solution preparation: ingredients are mixed in the conventional manner to prepare the composition. The method of stimulating stem cell production in skin: 1 ml of the composition is topically applied to the facial skin, and is preferably left on the skin for a period of at least about 15 minutes.

Claims

Patent Claims

1. A method for identifying a candidate compound suitable for stimulating stem cell production in skin, said method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the candidate compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin.

2. The method of claim 1 , wherein the animal is a mouse.

3. The method of claim 1 , wherein said stem cell marker is selected from the group of gene expression markers consisting of OCT4, NANOG, SOX2, TDGF1 , DNMT3B, GABRB3, GDF3, BRIX, CD9, COMMD3, CRABP2, EBAF, FGF4, FGF5, FOXD3, GAL, GBX2, GRB7, IFITM1 , IFITM2, IL6ST, IMP2, KIT, LEFTB, LIFR, LIN28, NODAL, NOG, NR5A2, NR6A1 , PODXL, PTEN, REST, SEMA3A, SFRP2, SSEA-1 ,

SSEA-3, SSEA-4, TRA-1 -60, TRA-1 -81 , TERT, TFCP2L1 , UTF1 , ABCG2, Notch-1 , Zpf206, LeftyA, LeftyB, c-Myc, Klf4, Xist and ZFP42.

4. The method of claim 3, wherein the stem cell marker is OCT4.

5. A compound suitable for stimulating stem cell production in skin, said compound identifiable by a method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin.

6. The compound of claim 5, wherein the animal is a mouse.

7. The compound of claim 5, wherein the stem cell marker is selected from the group of gene expression markers consisting of OCT4, NANOG, SOX2, TDGF1 , DNMT3B, GABRB3, GDF3, BRIX, CD9,

COMMD3, CRABP2, EBAF, FGF4, FGF5, FOXD3, GAL, GBX2, GRB7, IFITM1 , IFITM2, IL6ST, IMP2, KIT, LEFTB, LIFR, LIN28, NODAL, NOG, NR5A2, NR6A1 , PODXL, PTEN, REST, SEMA3A, SFRP2, SSEA-1 , SSEA-3, SSEA-4, TRA-1 -60, TRA-1-81 , TERT, TFCP2L1 , UTF1 , ABCG2, Notch-1 , Zpf206, LeftyA, LeftyB, c-Myc, Klf4,

Xist and ZFP42.

8. The compound of claim 7, wherein the stem cell marker is OCT4.

9. The compound of claim 5, wherein said compound is selected from the group consisting of interleukins and growth factors.

10. The compound of claim 9, wherein said interleukin is selected from the group consisting of interleukin 1 alpha, interleukin 1 family member 5 (delta), interleukin 1 family member 6 (epsilon), interleukin 1 family member 7 (zeta), interleukin 1 family member 8 (eta), interleukin 1 family member 9, interleukin 1 family member 10 (theta), interleukin 2, interleukin 3, interleukin 4, interleukin 6, interleukin 8, and interleukin 18.

11. The compound of claim 9, wherein said growth factor is selected from the group consisting of CSF, GM-CSF, VEGF, EGF, FGF, IGF, and PDGF.

12.A composition for stimulating stem cell production in skin comprising a compound suitable for stimulating stem cell production in skin, said compound identifiable by a method comprising: (a) contacting a test compound with the skin of an animal; and (b) determining whether the test compound increases a level of a stem cell marker in the skin of the animal, thereby identifying the test compound as the compound suitable for stimulating stem cell production in skin if the test compound increases the level of the stem cell marker in the skin; and a dermatologically acceptable diluent or carrier.

13. The composition of claim 12, wherein the stem cell marker is selected from the group of gene expression markers consisting of OCT4, NANOG, SOX2, TDGF1 , DNMT3B, GABRB3, GDF3, BRIX, CD9, COMMD3, CRABP2, EBAF, FGF4, FGF5, FOXD3, GAL, GBX2,

GRB7, IFITM1 , IFITM2, IL6ST, IMP2, KIT, LEFTB, LIFR, LIN28, NODAL, NOG, NR5A2, NR6A1 , PODXL, PTEN, REST, SEMA3A, SFRP2, SSEA-1 , SSEA-3, SSEA-4, TRA-1 -60, TRA-1-81 , TERT, TFCP2L1 , UTF1 , ABCG2, Notch-1 , Zpf206, LeftyA, LeftyB, c-Myc, Klf4, Xist and ZFP42.

14. The composition of claim 12, wherein said compound is selected from the group consisting of interleukins and growth factors.

15. The composition of claim 14, wherein said interleukin is selected from the group consisting of interleukin 1 alpha, interleukin 1 family member 5 (delta), interleukin 1 family member 6 (epsilon), interleukin 1 family member 7 (zeta), interleukin 1 family member 8 (eta), interleukin 1 family member 9, interleukin 1 family member 10 (theta), interleukin 2, interleukin 3, interleukin 4, interleukin 6, interleukin 8, and interleukin

18.

16. The composition of claim 14, wherein said growth factor is selected from the group consisting of CSF, GM-CSF, VEGF, EGF, FGF, IGF, and PDGF.