Method of determining compositions stimulating skin stem cell formation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. A composition for stimulating the skin stem cell production containing interleukin-1 alpha and a dermatologically acceptable diluent or carrier.

EFFECT: invention provides improving the stem cell stimulation.

2 ex

 

The technical field

[0001] This invention relates to a drug designed to stimulate production of stem cells in the skin, and the method of their identification.

The level of technology

[0002] a Stem cell is a cell that has two properties: (1) retains the ability to heal itself by dividing mitotic cells while maintaining the undifferentiated state and (2) has activity to differentiate into any type of specialized cell. Stem cells can be identified by specific markers of stem cells. Such markers are well known in the prior art (Rao R, et al., Biology of Reproduction, 71: 1772-1778, 2004). Usually markers of stem cells represent gene products that functionally linked to the maintenance of the undifferentiated state of stem cells, maintaining prepotential, multipotential or sometimes unipotential. An example of a reliable marker of stem cells is HOST, which is often called OSC/4 or Pou5f1. It is a transcription factor of the POU family is very important to ensure the undifferentiated state of stem cells and prepotential (Watson CM, et al., Cell Struct. Func 90, 26: 123-129, 2001; Pesce M et al.. Stem Cells, 19: 271-278, 2001).

[0003] In Mature skin revealed at least three populations of endogenous stem cells. Population of stem cells the detection is provided in the convex part 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). It is known that another population of stem cells located in mezhvolokonnom basal layer of the epidermis (Tumbar T et al. Science 303, 359-363, 2004; Taylor G et al. Cell 102, 451-461, 2000). Another population of stem cells is a mesenchymal cell. This population is able to form many cell types in the laboratory and even capable of generating layer of the epidermis composed of cells of different types in the 3-dimensional skin model (Crigler L, et al.. FASEB Journal. 2007 21: 2050-2063). Because of the ability of endogenous stem cells for skin regeneration there is a need for substances that can stimulate the production of stem cells in the skin to activate skin regeneration and renewal process under adverse circumstances, such as injury, illness or aging. However, reliable identification of such substances is limited by the lack of suitable methods.

[0004] the Purpose of this invention is to provide a medicine designed to stimulate production of stem cells in the skin, and the method of their identification.

Detailed description of the invention

[0005] This invention provides a method for identifying a drug candidate designed to stimulate production of stem cells in the skin include the s: (a) the application of the test drug on the skin of the animal; and (b) determining, improving test drug level markers of stem cells in the skin of the animal, identifying thus the test drug as intended to stimulate production of stem cells in the skin, if the test drug increases levels of markers of stem cells in the skin.

[0006] additionally, this invention provides a drug designed to stimulate production of stem cells in the skin, with the specified drug is identified by the method comprising: (a) the application of the test drug on the skin of the animal; and (b) determining, improving test drug level markers of stem cells in the skin of the animal, identifying thus the test drug as a drug designed to stimulate production of stem cells in the skin, if the test drug increases levels of markers of stem cells in the skin.

[0006] additionally, this invention provides a composition for stimulating the production of stem cells in the skin, containing a drug designed to stimulate production of stem cells in the skin, with the specified drug is identified by the method comprising: (a) the application of the test drug on the skin of the animal; and (b) determining, improving test drug level markers of stem cells in the skin zivotnog is, identifying thus the test drug as a drug designed to stimulate production of stem cells in the skin, if the test drug increases levels of markers of stem cells in the skin; and acceptable for cosmetic purposes diluent or carrier.

[0008] Preferably, the animal according to the present invention has been mammals. More preferably, the mammal was a mouse.

[0009] the Term "stem cell" refers to a cell that has two properties: (1) regeneration and (2) activity, where the term self-healing refers to the ability of cells to undergo numerous cycles of cell division while maintaining the undifferentiated state and where the term activity refers to the ability of cells to differentiate into any type of specialized cells.

[0008] Preferably, the stem cells according to this invention were poly potent, multipotent or unipotent stem cells. More preferably, the stem cells according to this invention were poly potent stem cells. The term poly potent refers to a stem cell that can differentiate into cells derived from any of the three layers of the skin, namely, the mesoderm, endoderm and the ectoderm, from which arise all cells of the body. The term "multi-pot the percentage" refers to a stem cell, that can occur only cells of a closely-related family of cells. The term "unipotent" refers to a stem cell that can differentiate only into cells of the same type.

[0010] the Term "stem cell marker" refers to any marker that recognizes poly potent, multipotent or unipotent cells in the skin. This invention is not limited anyhow specific marker for stem cells, but applies to all such markers, currently known or open or hereafter developed. Specialist in the art knows how to identify specific suitable marker of stem cells for use in the method according to this invention. However, the preferred marker of stem cells according to this invention is selected from the group of marker gene expression, consisting of

More preferably, the stem cell marker according to this invention was OCT. In the practical application of this invention, the stem cell marker can be detected by the method known from the prior art. Such methods include, inter alia, Northern blotting, Western blotting, immunohistochemistry, flow cytometry, ELISA analysis with the enzyme label and immune electron spectroscopy.

[0011] the Term "germ teologichesky acceptable diluent or carrier" refers to one or more compatible solid or liquid diluents or carriers of cells fillers, suitable for introduction into any part of the human skin and is compatible with the product suitable to stimulate production of stem cells. Examples of such carriers include, inter alia, distilled or deionized water, propylene glycol, glycerin and oil.

[0012] Preferably, the drug is suitable to stimulate the production of stem cells, which can be identified by the method according to this invention, was selected from the group consisting of interleukins and growth factors.

[0013] More preferably, the interleukin this invention was selected from a group comprising 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 for this invention was selected from a group including CSF, GM-CSF, VEGF, EGF, FGF, IGF and PDGF.

[0015] the compositions of this invention suitable for regulating the condition of skin, visible and/or tangible inhomogeneities of the skin (especially on the surface of the skin; such heterogeneity is usually undesirable). Such heterogeneity can the be induced or caused by internal and/or external factors and include signs of skin aging, described in this application. The term "regulating skin condition" includes preventive regulation and/or therapeutic regulating skin condition, including visible and/or tangible heterogeneity in the skin. In the context of this application preventive regulating skin condition includes delay, minimizing and/or preventing visible and/or tangible inhomogeneities in the skin. In the context of this application therapeutic regulation of skin condition involves reducing the intensity of symptoms, i.e. reducing, minimizing and/or remove inhomogeneities in the skin. The regulating skin condition includes improvement of skin appearance and/or tactile sensations. In addition, the compositions of this invention are particularly useful in the treatment of acne or other skin inflammations.

[0016] the compositions of this invention are particularly useful for regulating signs of skin aging, mainly highly visible and/or tangible discontinuities in the texture of the skin associated with aging. "Regulation of signs of skin aging includes preventive regulation and/or therapeutic regulation of one or more of these signs (similarly, the regulation of this sign of aging, i.e. wrinkles, wrinkles or pores, includes preventive regulation and/or therapeu the practical regulation of this topic). In the context of this application of preventive regulation of this trait includes the delay, minimizing and/or preventing signs of aging. In the context of this application therapeutic regulation of such evidence involves reducing the intensity of symptoms, i.e. reducing, minimizing and/or removing signs of aging.

[0017] the Signs of aging skin include, without limitation, all symptoms that are visible to the eye or detectable by touch, as well as all other macro - or microeffects caused by skin aging. Such signs may be induced or caused by internal factors or external factors, i.e. age ageing and/or the influence of environmental conditions. These signs may result from processes which include, inter alia, the development of inhomogeneities textures, such as wrinkles, including fine surface wrinkles, and large deep wrinkles, skin folds, fractures, tumors, large pores (i.e., associated with accessory structures, such as ducts of sweat glands, sebaceous glands, or hair follicles), flaking and dandruff and/or other forms of unevenness or roughness, loss of skin elasticity (loss and/or inactivation of functional elastin in the skin), sagging (including puffiness in the eye area and cheeks), the loss of platnost the skin, the loss of the tightening of the skin, loss of skin return after deformation, discoloration (including circles), spotting, discoloration, skin with hyperpigmentation, such as aging spots or freckles, corneal tumors, pathological differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the stratum corneum, dermis, epidermis, vascular system of the skin (i.e. telangiectasia or spider veins), and underlying tissues, especially those located close to the skin.

[0018] the Percentage of the drug in this invention is from 0.0000001%to 10%, preferably from 0.000001 to 0,00001% by weight of the composition.

[0019] the compositions of this invention typically are prepared so that the pH was about 4.5 and about to 9.5, more preferably, he was about 5 and about 7.5.

[0020] For proofs of the invention are the following examples. Examples are given only for explanation and are in no way limit the scope of the invention.

Example 1

[0021] This example demonstrates the identification methods of preparation, suitable to stimulate production of stem cells in the skin.

Males white mouse BALB/C mice aged 5 days randomly divided into experimental group (group size n=10) and control groups is (n=10). All the experiments were carried out in accordance with the Principles of the observations of laboratory animals, formulated by the NIH. The cream containing the test drug applied to the skin of mice in the experimental group once a day for 5 weeks. Mice of the control group received the drug environment. At the end of the experiment, samples of the skin exposed to the drug were obtained from mice under anesthesia. Was made immunohistochemical analysis of skin samples for marker stem cell levels OST using goat polyclonal antibodies OST (laboratory Santa Cruz Biotechnology, Santa Cruz, California in a solution of 1:8000). Briefly, skin samples were fixed in 10% formalin and then paraffin according to the standard procedure. Paraffin sections were cut at 4 µm and dewaxed. Endogenous peroxidases were blocked with 3% hydrogen peroxide. Then the sections were heated for 5 min in 10 mm citrate buffer, to destroy nucleomegaly, were treated with 1% BSA at room temperature for 40 min and were placed in a thermostat at night at 4°C with primary antibodies OST. The next day, the sections were incubated (step 1) with biotinylated anti-goat immunoglobulin (laboratory Santa Cruz Biotechnology, Santa Cruz, CA)used at 1:200 solution and (step 2) avidin-biotinyl complex, conyuge ofanim with peroxidase from horseradish. Was performed staining with 3-amino-ethyl-carbazole and hydrogen peroxide. And in the end a contrast staining with Mayer hematoxylin. Painted by immunohistochemical image slices of the skin was examined under an optical microscope. Estimated number OST-positive cells / mm2. The results are expressed in crednerite the density increases OST-positive cells compared with the control group. The difference between groups was considered significant when p<0.05 (unidirectional ANOVA).

Identification of a drug candidate. The purpose of the testing was to identify drug candidate suitable to stimulate production of stem cells in the skin. We used five drug candidates. The cream containing the test drug or the environment, was made in the usual way. The cream was applied to the skin of mice, as described in method. The density of stem cells in the skin was evaluated by immunohistochemical method, as described above. When processing four test drugs no effect on the production of stem cells was not detected. One test drug, interleukin 1 alpha, showed a significant and 1.9-fold (p<0.05) increase in the level of stem cells in the skin compared with the control group. Thus, interleukin 1 alpha was identified as a drug candidate, brightneedle stimulate production of stem cells in the skin.

Example 2

[0022] This example shows the composition to stimulate production of stem cells in the skin

The percentage of ingredients, in weight%

Interleukin 1 alpha 0,000001, Citrate buffer as necessary to obtain a pH of 5.5, distilled water to 100

Manufacturing solution: the ingredients are mixed in the usual manner for the manufacture of the composition. Way to stimulate the production of stem cells in the skin: 1 ml of the composition is locally applied to the scalp, leave it on the skin for a period of time not less than about 15 minutes.

Composition to stimulate production of stem cells in the skin, including interleukin 1 alpha and dermatologically-acceptable diluent or carrier.



 

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