WO2002048136A1

WO2002048136A1 - Keratinocyte proliferation inhibitors

Info

Publication number: WO2002048136A1
Application number: PCT/JP2001/010928
Authority: WO
Inventors: Masayuki Tsuchiya; Toshihiko Ohtomo
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2000-12-14
Filing date: 2001-12-13
Publication date: 2002-06-20
Also published as: JP2004292314A; AU2002222628A1

Abstract

Keratinocyte proliferation inhibitors containing one or more zearalenones as the active ingredient; remedies or preventives for skin diseases in association with the abnormal proliferation of keratinocytes; a method of inhibiting the proliferation of keratinocytes which comprises using one or more zearalenones in an efficacious dose; a method of treating or preventing skin diseases in association with the abnormal proliferation of keratinocytes which comprises administering one or more zearalenones in an efficacious dose to patients with a need therefor; kits for inhibiting the proliferation of keratinocytes which consist of one or more zearalenones in an efficacious dose and a manual; kits for treating or preventing skin diseases in association with the abnormal proliferation of keratinocytes; and utilization of one or more zearalenones for producing keratinocyte proliferation inhibitors or remedies or preventives for skin diseases in association with the abnormal proliferation of keratinocytes.

Description

Specification

Keratinocyte proliferation inhibitor Technical field

The present invention provides a keratinocyte proliferation inhibitor containing zearalenone as an active ingredient, a therapeutic or prophylactic agent for a skin disease associated with abnormal proliferation of keratinocytes, containing zearalenone as an active ingredient, and administering an effective amount of zearalenone. A method of treating and preventing a skin disease associated with abnormal proliferation of keratinocytes, which comprises administering an effective amount of zelarenones, the use of zelarenones for producing a keratinocyte proliferation inhibitor, and the use of keratinocytes. Use of zearalenones for the manufacture of a therapeutic or prophylactic agent for a skin disease associated with abnormal growth, an effective amount of zearalenones, a kit for inhibiting the growth of keratinocytes including instructions for use, and an effective amount of zearalenones Abnormal growth of keratinocytes, including Cormorants relates to a kit for the treatment or prevention of skin disorders. Background art

Under normal physiological conditions, the proliferation of keratinocytes, which are skin epithelial cells, is strictly controlled. However, in many skin diseases such as psoriasis, immune and allergic skin diseases, and chronic wounds, the keratinocyte cell proliferation control mechanism has been disrupted, and skin thickening due to pathological abnormal proliferation of skin epithelial cells has been observed. . Excessive production of cytokins such as epidermal growth factor and other growth factors such as leukin 1, 4, 6, and 8 Although recognized (Gen. Pharmac., (1998) 5, 619-622), the detailed mechanism of keratinocyte proliferation in the above-mentioned diseases is unknown. Therefore, if a substance that suppresses the pathological cell proliferation of keratinocytes is found, it can be expected to be used as a therapeutic agent for various skin diseases characterized by abnormal proliferation of keratinocytes. Furthermore, any substance that does not affect the proliferation of stromal cells such as fibroblasts is expected to be useful in reducing side effects.

Various psoriasis treatments have been reported so far, but their effects are satisfactory. There is a need for more effective psoriasis treatments.

By the way, various compounds of zearalenones have been reported so far, and various actions, for example, inhibition of production of cytokins, particularly IL-11 (Japanese Patent Application Laid-Open No. 8-48093) , European Patent No. 664044A), Tyrosine kinase inhibition (WO96 / 13259), T cell activation based on MEK1 kinase inhibition and proliferation accompanying activation Inhibition (Biochemistry, 37; 9579-9585, 1998), inhibition of JNK / p38 activity (Biochem. Biophys. Res. Commun., 257; 19-23, 1999), inhibition of MEK (J. Antibiotics , 52; 1086- 1094, 1999), inhibition of cytokin (IL-1, IL-6, TNF-H) production after LPS stimulation (Int. J. ImunopharmacoL, 21; 799-814, 1999), LPS, Cytokine after ILN-r stimulation (IL-1 / 3, suppression of TNF-α production (Cytokine, 8; 751-761, 1996)) has also been reported.

However, it has not been known so far that zearalenones have an action of suppressing the proliferation of keratinocytes. Disclosure of the invention

An object of the present invention is to provide a keratinocyte proliferation inhibitor. Another object of the present invention is to provide an agent for treating or preventing a skin disease associated with keratinocyte proliferation.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that certain zearalenones have a keratinocyte-specific cell growth inhibitory action, and completed the present invention based on this finding. .

That is, the present invention provides a keratinocyte proliferation inhibitor comprising a zearalenone as an active ingredient. Further, the present invention provides a therapeutic or preventive agent for a skin disease associated with abnormal proliferation of keratinocytes, comprising a zearalenone as an active ingredient.

Furthermore, the present invention provides a method for inhibiting keratinocyte proliferation, which comprises administering an effective amount of a zearalenone to a patient in need thereof. Further, the present invention provides a method for treating and preventing a skin disease associated with abnormal proliferation of keratinocytes, which comprises administering an effective amount of zearalenones to a patient in need thereof.

In addition, the present invention relates to zearalenone for producing a keratinocyte proliferation inhibitor. Provides a kind of use. The present invention also provides use of zearalenones for producing a therapeutic or prophylactic agent for a skin disease associated with keratinocyte overgrowth. In addition, the present invention provides a kit for inhibiting the growth of keratinocytes, comprising an effective amount of zearalenones and instructions for use. The present invention also provides a kit for treating or preventing a skin disease associated with keratinocyte overgrowth, comprising an effective amount of zearalenones and instructions for use. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the inhibitory effect of compound (1) on human keratinocyte proliferation. FIG. 2 is a graph showing the inhibitory effect of compound (1) on human keratinocyte proliferation. FIG. 3 is a graph showing the effect of compound (1) on human keratinocyte. It is a graph which shows cytotoxicity.

FIG. 4 is a graph showing the effect of compound (1) on cell proliferation of human fibroblasts. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, zearalenones include zearalenone and its derivatives, for example, as shown below:

Z60I / I0df / I3d

9 £ m / Z0 OAV

Compounds (1) to (14) are compounds known per se and described in the literature (eg, Int. J. Imunopharmacol., 21: 799-814, 1999; J. Org. Chem., 43: 2339-2343, 1978; Chem. Pharm. Bull. 41: 373-375, 1993; J. Antibiotics, 52: 1077-1085, 1999; Biochem. Biophys. Res. Commun., 257: 19-23, 1999; Cytokine, 8: 751. -761, 1996; Pharmacol. Commun., 7: 301-308, 1996; Japanese Patent Publication No. 8-40893; European Patent Publication No. 664044A; Biochemistry, 37: 9579- 9585,

1998 etc.).

As the zearalenones, compounds (1) to (3) are preferable, and compound (1) is particularly preferable.

Skin diseases associated with abnormal proliferation of keratinocytes include the failure of the cell proliferation control mechanism of keratinocytes and the pathological abnormal proliferation of skin epithelial cells. Includes various diseases with thickening. Non-limiting examples of such skin diseases include psoriasis, immune skin diseases, allergic skin diseases, inflammatory skin diseases, chronic wounds, and the like. Keratinocyte proliferation inhibitors are expected to be effective as therapeutic or prophylactic agents. The administration form of the medicament containing the keratinocyte proliferation inhibitor of the present invention is not particularly limited, and may be oral administration, parenteral administration, systemic administration, or local administration. The medicament of the present invention can be generally administered parenterally, can be administered transdermally as a spray, talmes, lotions, ointments, etc., or intravenously or intramuscularly as an injection Or it can be administered subcutaneously.

The dose can be appropriately selected depending on the patient's body type, age, physical condition, type and degree of the disease, elapsed time after onset, and the like.In the case of parenteral administration, the dose is generally from 0.01 to 0.1 gg. A dose of O mgZ body weight kg / day, and in the case of oral administration, a dose of 1 ng to 10 g / body weight kgZ day can generally be expected to be effective.

In the present invention, zearalenones may be used alone or in combination of two or more.

The keratinocyte proliferation inhibitor and the therapeutic or prophylactic agent for a skin disease associated with keratinocyte abnormal growth of the present invention comprise one or more pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersants, and auxiliary agents. The composition can be administered as a pharmaceutical composition appropriately containing an agent, a preservative, a buffer, a binder, a stabilizer and the like in an appropriate form depending on the intended administration route.

Further, the kit of the present invention includes a pharmaceutical composition comprising zearalenones, a diluent, and any one or more carriers including the various carriers exemplified above, and instructions for use. Example

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

(Example 1) Effect of the compound of the present invention on cell proliferation of normal human keratinocytes Normal human keratinocytes (manufactured by Sanko Junyaku Co., Ltd.) from a newborn are seeded on a 96-well plate at 2 × 10 ³ cells / well using KGM-2 medium, and after the cells have adhered, compound (1) is finalized. concentration 10- ⁹ ~: It) 'was added to a ⁵ mol / L. After culturing for 3 days, add 1/10 volume of the medium for measuring the number of viable cells SF (WST-8, manufactured by Nacalai Tesque), culture at 37 ° C for 1 hour, and add 0.1 mol / L HC1 to the medium. The reaction was stopped by adding 1/10 volume. Immediately thereafter, the absorbance at 450 nm (reference wavelength: 655 nm) was measured using a microplate reader. A background obtained by performing the same treatment on a medium containing no cells was used as a background, and a value obtained by subtracting the background from the measured value was calculated.

The results are shown in Figure 1. In FIG. 1, each plot shows mean soil SD (n = 4). * * * Indicates P <0.001 in Dunnett's multiple comparison (comparison with control group).

Compound (1) is ^{^{10- 7 ~: L0- 5 mol /}} L concentration significantly inhibited the growth of Hitokerachinosai Bok of (multiple comparisons Dunnett, <0.00l), the IC _5. The value was 4.8Χ10 · ⁸ mol / L.

Subsequently, the growth inhibitory effect of normal human keratinocytes was evaluated by the incorporation of [ ³ H] thymidine. That is, seeded with KGM-2 medium in a 96-well plate at 2 X 10 ³ per newborn from normal human keratinocytes 1 Ueru compound after the cells against wear (1) to a final concentration of 10- ⁹ to : L0- ⁵ was added so as to be in mol / L. After culturing for 3 days, [ ³ H] thymidine (Amersham 'Pharmacia) was added, followed by further cultivation, and the uptake of [ ³ H] thymidine into cells was measured with a liquid scintillation counter.

The result is shown in figure 2. In FIG. 2, each plot shows mean soil SD (n = 5). ** In Dunnett's multiple comparison (comparison with the group of compound (1) at 0 mol / L concentration) ) Shows that it is 0.001.

As a result, compound (1) is ^{^{10 · 7 ~: L0- 5 mol}} / at a concentration of L significantly inhibited ^[3 H] thymidine uptake of human keratinocytes (multiple comparisons Dumiett, p rather 0.001), its IC50 The value was 3.2 × 10 ′ ⁸ mol / L. Similarly, compound (2) and compound

Experiments on (3) showed that the IC50 values were 2.9Χ10 · ⁷ mol / L and ぴ It was 2.6 X 10 " ⁶ mol / L.

(Example 2) Examination of cytotoxicity of the compound of the present invention in normal human keratinocytes The cytotoxicity of compound (1) was evaluated by measuring the amount of LDH leaked out of cells. That is, seeded with KGM-2 medium normal human keratinocytes in a 96-well plate at 2X 10 ³ per 1 Ueru, final concentration 10, ^9-10, ⁵ Compound (1) after the cells have adhered mol / L. After culturing for 3 days, LDH activity leaked into the medium was measured by LDH-cytotoxicity test ヮ KO (manufactured by Wako Pure Chemical Industries, Ltd.) using 50 L of 200 L of the culture supernatant. As a positive control, the final concentration of the control was 0.1 ° /. Tween 20 was added to the mixture, and the mixture was treated at 37 ° C. for 15 minutes. The LDH activity of the medium without cells alone was used as the background, and the value obtained by subtracting the background from the measured value was calculated.

The results are shown in Figure 3. In FIG. 3, each plot shows mean soil SD (n = 5 in each group; n = l in Tween 20 group). ** indicates that p <0.001 in Dunnett's multiple comparison (comparison with control group).

As a result, Compound (1) showed cytotoxicity to human keratinocytes at concentrations of 10- ⁵ mol / L (multiple comparisons Dunnett, p <0.001), was almost completely inhibited keratinocyte proliferation concentrations ( At 10 · ⁷ to 10 · ⁶ mol / L), no cytotoxicity was observed.

(Example 3) Investigation of the effect of the compound of the present invention on the cell proliferation of normal human fibroblasts A 96-well microplate was prepared so that the number of human fibroblast cell lines WI-38 cells (ATCC) was 2 x 10 ³ cells / well. (Falcon) using DMEM medium containing 10% fetal bovine serum. After overnight incubation to allow the cells to be attached thereto were added the compound (1) in such a way that ^{^{10- 9 ~ 10 6 mol / L}} , or DMSO as a control such that the final concentration of 1%. After culturing for 3 days, a viable cell counting reagent SF (WST-8, manufactured by Nacalai Tesque) was added to 1/10 of the medium. After culturing at 37 ° C for 1 hour, the absorbance at 450 nm (reference wavelength: 620 nm) was measured, and the same treatment was applied to the medium containing no cells as the background. The calculated relative value was calculated. Statistical processing was performed by Dunnett's multiple comparison.

Fig. 4 shows the results. In FIG. 4, each plot shows mean soil SD (n = 6 for each group, n = 4 for the control group).

As a result, a decrease in absorbance of about 25% was observed only under the condition where compound (1) was added so as to have a concentration of 1 × 10 · ⁶ mol / L. No effect was observed on the growth, and no remarkable effect such as the growth inhibitory effect of keratinocytes was observed. Therefore, it was confirmed that the cell growth inhibitory action of compound (1) was keratinocyte-specific. Industrial applicability

According to the present invention, since it has an activity of specifically suppressing the cell proliferation action of keratinocytes, various diseases characterized by abnormal proliferation of keratinocytes (for example, psoriasis, inflammatory, allergic skin diseases, chronic wounds, etc.) ) Is expected to be useful as a therapeutic or prophylactic agent. Further, since it does not substantially affect the proliferation of stromal cells such as fibroblasts, it is expected to be useful as an agent for treating or preventing the above-mentioned diseases with few side effects.

Claims

The scope of the claims

1. A keratinocyte growth inhibitor comprising one or more zearalenones as an active ingredient.

2. A therapeutic or preventive agent for a skin disease associated with abnormal proliferation of keratinocytes, comprising one or more zearalenones as an active ingredient.

3. The therapeutic or preventive agent according to claim 2, wherein the skin disease is psoriasis, an immune skin disease, an allergic skin disease, an inflammatory skin disease, or a chronic wound.

4. Zaralenones are the following compounds (1) to (3)

The inhibitor, the therapeutic agent or the prophylactic agent according to claim 1, which is selected from:

5. A method for inhibiting the growth of keratinocytes, comprising using an effective amount of one or more zearalenones.

6. A method for treating or preventing a skin disease associated with keratinocyte overgrowth, comprising administering to a patient in need of an effective amount of one or more zearalenones.

7. An effective amount of one or more zearalenones and a package containing instructions for use Kit for suppressing proliferation of latinocytes.

8. A kit for treating or preventing a skin disease associated with abnormal proliferation of keratinocytes, comprising an effective amount of one or more zearalenones and instructions for use.

9. Use of one or more zearalenones for producing a keratinocyte proliferation inhibitor.

10. Use of one or more zelarenones for producing a therapeutic or prophylactic agent for a skin disease associated with abnormal proliferation of keratinocytes.