Skin external medicine containing sirolimus for treating skin sclerosis in systemic scleroderma

Abstract

The present disclosure finds that skin sclerosis can be ameliorated by applying an externally applied skin medicine containing sirolimus to the skin in systemic scleroderma, and thus finds a novel medicine for treating or preventing skin sclerosis in systemic scleroderma.

Description

Technical Field

[0001] The present invention relates to a skin external medicine containing sirolimus for treating skin sclerosis in systemic scleroderma. Background Art

[0002] Systemic Sclerosis is a disease in which extracellular matrices such as collagen accumulate in the skin and organs throughout the body, resulting in fibrosis and hardening of the skin and organs. Systemic Sclerosis is designated as an intractable disease, and there is no fundamental treatment. When systemic Sclerosis develops, the flexion contracture caused by skin hardening and the limited range of motion of the joints lead to a significant decrease in QOL (Quality of Life). There is no proven useful treatment for skin hardening, and many patients take large amounts of adrenocortical hormones (steroids) orally, so various side effects become a problem. Therefore, an effective treatment method with fewer side effects is desired.

[0003] It has been disclosed that oral administration of sirolimus (also known as rapamycin) to systemic scleroderma patients improves symptoms associated with systemic scleroderma, such as skin fibrosis (Non-Patent Document 1). In addition, it has been disclosed that intraperitoneal injection of sirolimus (hereinafter also referred to as rapamycin) inhibits skin fibrosis in scleroderma model mice (bleomycin-induced scleroderma model mice and tight-skinned mice), that is, the proliferation of fibroblasts and collagen production are inhibited in a dose-dependent manner (Non-Patent Document 2).

[0004] Sirolimus-containing gel (Nobelpharma Co., Ltd., product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g) is an external medicine suitable for skin lesions such as angiofibroma associated with tuberous sclerosis. Tuberous sclerosis is a disease in which mTOR (mechanistic target of rapamycin) signaling is constantly activated due to gene mutation. By applying a sirolimus-containing gel externally to skin lesions such as angiofibroma associated with tuberous sclerosis, mTOR signaling is inhibited, thereby exerting a therapeutic effect.

[0005] On the other hand, conventionally, there is no sirolimus topical drug that can be used clinically, except for topical drugs that are suitable for skin lesions such as angiofibroma associated with tuberous sclerosis. Therefore, there are no reports on the use of sirolimus as a topical drug for skin application to study the improvement effect of skin sclerosis in systemic sclerosis, and there are no reports on the therapeutic effect of further using a topical drug for skin application containing sirolimus for skin sclerosis in systemic sclerosis.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent literature 1: Arthritis Rheum., 2009; 60: 3821-3830

[0009] Non-patent document 2: Arthritis Rheum., 2010: 62: 2476-2487

[0010] Non-patent document 3: Arthritis Rheum., 2000; 43: 2455-2463

[0011] Non-patent literature 4: Arthritis Rheum., 2004; 50: 1918-1927

[0012] Non-patent literature 5: Ann Rheum Dis., 2005; 64: 1233-1235

[0013] Non-patent literature 6: N Engl J Med., 2006; 354: 2655-2666

[0014] Non-patent literature 7: J Transplant., 2009; 2009: 701464 Summary of the invention

[0015] Problems to be solved by the invention

[0016] The technical problem of the present invention is to provide a medicine for treating or preventing skin sclerosis in systemic scleroderma.

[0017] Solutions for solving problems

[0018] In order to solve the above technical problems, intensive studies were conducted and as a result, it was found that a significant improvement effect of skin sclerosis was obtained by applying a skin topical medication containing sirolimus to scleroderma model mice (hereinafter also referred to as scleroderma-like skin sclerosis model mice) prepared by subcutaneous administration of bleomycin, which led to the present invention.

[0019] That is, the present invention is as follows.

[0020] [1] A skin topical drug comprising sirolimus, wherein the skin topical drug is used to treat or prevent skin sclerosis in systemic scleroderma.

[0021] [2] The skin external application medicine according to [1], wherein the treatment or prevention refers to the inhibition of early stage skin fibrosis.

[0022] [3] The skin external medicine according to [1] or [2], wherein the dosage form of the skin external medicine is a gel, cream, ointment, liquid, emulsion or patch.

[0023] [4] The skin external preparation according to any one of [1] to [3], wherein sirolimus is contained in a ratio of 0.2% by mass or more based on the total amount of the skin external preparation.

[0024] [5] The skin external medicine according to any one of [1] to [4], wherein the skin external medicine inhibits collagen production in the skin of the subject to which the medicine is applied.

[0025] [6] The skin external medicine according to any one of [1] to [5], wherein the skin external medicine suppresses inflammation in the skin of the subject to which the medicine is applied.

[0026] [7] A method for treating or preventing skin sclerosis in systemic sclerosis, comprising: applying sirolimus to the skin of a subject in need thereof.

[0027] [8] Sirolimus, wherein the sirolimus is used for the treatment or prevention of skin sclerosis in systemic scleroderma by administration to the skin.

[0028] [9] Use of sirolimus in the manufacture of a topical skin medication for treating or preventing skin sclerosis in systemic scleroderma.

[0029] Effects of the Invention

[0030] Skin sclerosis can be improved by applying a skin external application containing sirolimus to the skin of systemic sclerosis. Therefore, skin sclerosis in systemic sclerosis, for which there is currently no effective treatment, can be treated or prevented by a method with few systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the schedule of applying sirolimus gel to the skin in the early stage of fibrosis in scleroderma-like skin sclerosis model mice.

[0032] Figure 2 This is a microscopic photograph showing the inhibitory effect of sirolimus gel on skin fibrosis in scleroderma-like skin sclerosis model mice. In the figure, the portion indicated by the arrow represents the thickness of the skin.

[0033] Figure 3 This is a graph showing the inhibitory effect of sirolimus gel on skin fibrosis in scleroderma-like skin sclerosis model mice. The graph shows the results of measuring the skin thickness of each mouse.

[0034] Figure 4 This is a graph showing the inhibitory effect of sirolimus gel on skin fibrosis in scleroderma-like skin sclerosis model mice. It shows the results of measuring the amount of soluble collagen in the skin tissue of each mouse.

[0035] Figure 5 This is a graph showing the progress of sirolimus gel application to fibrotic skin in scleroderma-like skin sclerosis model mice.

[0036] Figure 6 This is a graph showing the therapeutic effect of sirolimus gel on skin fibrosis in scleroderma-like skin sclerosis model mice. It shows the results of measuring the amount of soluble collagen in the skin tissue of each mouse.

[0037] Figure 7 This is a microscopic photograph showing the effect of sirolimus gel on inflammation in skin fibrosis sites in scleroderma-like skin sclerosis model mice. CD3-positive cells and CD68-positive cells in the skin tissue of each mouse were stained. The scale bar is 50 μm.

[0038] Figure 8 This is a graph showing the effect of sirolimus gel on inflammation in skin fibrosis sites in scleroderma-like skin sclerosis model mice. It shows the results of measuring the number of CD3-positive cells in the skin tissue of each mouse.

[0039] Fig. 9 This is a graph showing the effect of sirolimus gel on inflammation in skin fibrosis sites in scleroderma-like skin sclerosis model mice. It shows the results of measuring the number of CD68-positive cells in the skin tissue of each mouse.

[0040] Fig.10 This is a graph showing the effect of sirolimus gel on inflammation in skin fibrosis sites in scleroderma-like skin sclerosis model mice. It shows the results of measuring the expression level of TNF-α in the skin tissue of each mouse.

[0041] Fig.11 This is a graph showing the effect of sirolimus gel on inflammation in skin fibrosis sites in scleroderma-like skin sclerosis model mice. It shows the results of measuring the expression level of TGF-β in the skin tissue of each mouse.

[0042] Fig.12 This is a microscopic photograph showing the effect of sirolimus gel on the expression of phosphorylated mTOR in skin fibrosis sites in scleroderma-like skin sclerosis model mice. Phosphorylated mTOR-expressing cells in the skin tissue of each mouse were stained.

[0043] Fig.13This is a graph showing the effect of sirolimus gel on the expression of phosphorylated mTOR in skin fibrosis sites in scleroderma-like skin sclerosis model mice. It shows the results of measuring the number of CD68-positive cells in the skin tissue of each mouse.

[0044] Fig.14 The photographs show the effect of sirolimus gel on the expression of phosphorylated mTOR in skin fibrosis sites in scleroderma-like skin sclerosis model mice. The photographs show the results of confirming the expression level of phosphorylated mTOR by Western blotting of skin tissue samples derived from each mouse. DETAILED DESCRIPTION

[0045] One embodiment of the present invention is a topical skin medicine comprising sirolimus, which is used for treating and / or preventing skin sclerosis in systemic scleroderma.

[0046] Sirolimus is a macrolide compound. Sirolimus can be represented by the IUPAC name, for example, (3S, 6R, 7E, 9R, 10R, 12R, 14S, 15E, 17E, 19E, 21S, 23S, 26R, 27R, 34aS)-9, 10, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 32, 33, 34, 34a-hexadecanoic-9, 27-dihydroxy-3-[(1R)-2-[(1 S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazatridecene-1,5,11,28,29(4H,6H,31H)-pentanone, and can also be represented by CAS No. 53123-88-9.

[0047] Sirolimus may be a derivative thereof as long as it does not hinder the effect of the present invention, and may be a compound in which the functional group of sirolimus is arbitrarily modified. Examples of sirolimus derivatives include esters, ethers, amides, carbonates, carbamates, sulfonates, oximes, hydrazones, and hydroxylamine derivatives.

[0048] As sirolimus, commercially available sirolimus may be used, or sirolimus produced by, for example, Streptomyces hygroscopicus as a bacterium by a method known to those skilled in the art may be used, or chemically synthesized sirolimus may be used.

[0049] The skin external medicine of this embodiment is used to treat and / or prevent skin sclerosis in systemic sclerosis. The degree and stage of skin sclerosis in systemic sclerosis are not particularly limited, and for example, it can be used in the early stage of skin fibrosis, and can also be used after the skin fibrosis has intensified.

[0050] In this embodiment, treatment may refer to the complete disappearance of skin sclerosis, or may refer to the improvement of skin sclerosis after the application of the skin external medicine of this embodiment even if the skin sclerosis has not completely disappeared. For example, after the application of the skin external medicine of this embodiment, the thickness of the skin at the lesion site is thinned, the amount of soluble collagen in the skin tissue is reduced, the inflammation in the skin tissue is suppressed, the expression of phosphorylated mTOR in the skin tissue is reduced, etc., compared with before the application of the skin external medicine, it can be judged that the skin sclerosis is improved. In addition, for example, after the application of the skin external medicine of this embodiment, the thickness of the skin at the lesion site, the amount of soluble collagen in the skin tissue, the value indicating the degree of inflammation (such as the number of CD3 positive cells, the number of CD68 positive cells, the expression of TNF-α, the expression of TGF-β, etc.), the expression of phosphorylated mTOR, etc. are low values ​​compared with a predetermined cutoff value, it can be judged that the skin sclerosis is improved. The therapeutic effect of skin sclerosis can be judged based on at least one of these indicators.

[0051] In this embodiment, prevention may refer to completely inhibiting the occurrence of skin sclerosis from a state where skin sclerosis has not occurred, or may refer to a reduction in the degree of aggravation or aggravation of skin sclerosis. For example, by comparing the subject after the application of the skin external medicine of this embodiment with the subject to which the skin external medicine has not been applied, the thickness of the skin at the lesion site is thin, the amount of soluble collagen in the skin tissue is small, the degree of inflammation in the skin tissue is mild, the expression of phosphorylated mTOR in the skin tissue is small, etc., it can be judged that the degree of aggravation or aggravation of skin sclerosis is reduced. In addition, for example, after the application of the skin external medicine of this embodiment, when the thickness of the skin at the lesion site, the amount of soluble collagen in the skin tissue, the value indicating the degree of inflammation (such as the number of CD3 positive cells, the number of CD68 positive cells, the expression of TNF-α, the expression of TGF-β, etc.), the expression of phosphorylated mTOR, etc. are low values ​​compared to a predetermined cutoff value, it can be judged that the degree of aggravation or aggravation of skin sclerosis is reduced. The preventive effect of skin sclerosis can be judged based on at least one of these indicators.

[0052] The thickness of the skin can be measured by methods known to those skilled in the art. For example, the thickness of the skin can be measured by staining the skin tissue collected from the subject with hematoxylin & eosin (HE) and observing it under a microscope.

[0053] The amount of soluble collagen in skin tissue can be measured by methods known to those skilled in the art. For example, it can be measured by Sircol assay using skin tissue collected from a subject.

[0054] The determination of the value indicating the degree of inflammation in the skin tissue can be performed by methods known to those skilled in the art. For example, the skin tissue taken from the subject can be immunostained and observed under a microscope to determine the number of cells related to inflammation, such as the number of CD3-positive cells and the number of CD68-positive cells, thereby determining the value indicating the degree of inflammation. In addition, the expression level of cytokines such as TNF-α expression level and TGF-β expression level, etc., which are related to inflammation, can be determined by using the skin tissue taken from the subject and performing methods such as real-time PCR, thereby determining the value indicating the degree of inflammation.

[0055] The phosphorylated mTOR expression level in skin tissue can be measured by methods known to those skilled in the art, for example, by Western blotting using skin tissue collected from a subject.

[0056] The shape of the skin external medicine of the present embodiment is not particularly limited, and can be made into a gel, cream, ointment, liquid, emulsion, or patch. These shapes can be appropriately selected according to the purpose. Regardless of the shape, since it is an external medicine, it is different from an oral medicine and can exert its pharmacological effect only locally after administration, so it can be used for treatment with few systemic side effects.

[0057] As for the skin topical medicine of the present embodiment, as long as it contains sirolimus as an active ingredient and does not hinder the effect of the skin topical medicine of the present embodiment, other ingredients are not particularly limited and can be appropriately selected according to the purpose, etc. For example, when the skin topical medicine is a gel, it can contain carboxyvinyl polymer, ethanol, 2,2',2"-nitrilotriethanol, etc. as a base material, and can further contain other arbitrary ingredients as needed. As for other arbitrary ingredients, for example, pharmacologically acceptable solvents, additives, etc. can be listed. As pharmacologically acceptable solvents, water, physiological saline, buffer solutions, etc. can be listed. As additives, stabilizers, pH adjusters, thickeners, antioxidants, isotonic agents, buffers, dissolution aids, suspending agents, preservatives, antifreeze agents, cryoprotectants, freeze-drying protectants, antibacterial agents, etc. can be listed.

[0058] The skin external medicine of this embodiment can be manufactured by a method known to those skilled in the art, or can be manufactured by a method including the step of mixing the above-mentioned various components. The concentration of the various components, the temperature and time during the manufacture, the pH of the skin external medicine, etc. can be appropriately prepared as needed.

[0059] In addition, the skin external medicine of this embodiment can use the commercial skin external medicine. For example, when using a gel, it can be manufactured by a method known to those skilled in the art. As a commercially available gel containing sirolimus, for example, the product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g (Nobelpharma Co., Ltd.) can be used.

[0060] The content of sirolimus in the skin external medicine of the present embodiment is not particularly limited as long as it does not hinder the therapeutic effect of the skin external medicine of the present embodiment. For example, relative to the overall skin external medicine, the content of sirolimus can be more than 0.1 mass%, can be more than 0.2 mass%, can be more than 0.3 mass%, can be more than 0.4 mass%, can be more than 0.5 mass%, can be more than 1.0 mass%, can be more than 2.0 mass%, can be more than 5.0 mass%, can be more than 10.0 mass%. In addition, relative to the overall skin external medicine, the content of sirolimus can be less than 30.0 mass%, can be less than 20.0 mass%, can be less than 15.0 mass%, can be less than 10.0 mass%, can be less than 5.0 mass%, can be less than 4.0 mass%, can be less than 3.0 mass%, can be less than 2.0 mass%, can be less than 1.0 mass%. These upper and lower limits can be combined to form a fixed range. For example, the sirolimus content can be 0.1% to 30.0% by mass, 0.2% to 20.0% by mass, or 0.2% to 5.0% by mass relative to the entire external skin medication.

[0061] The dosage of the skin topical drug of the present embodiment is not particularly limited as long as it does not hinder the effect of the composition, and can be appropriately adjusted according to the age, sex, weight, degree and stage of skin hardening of the subject. For example, when applying the skin topical drug containing the above-mentioned sirolimus content to the skin of an adult (weight 60 kg), 0.1 g to 10.0 g of the skin topical drug can be applied daily, 0.3 g to 5.0 g of the skin topical drug can be applied daily, 0.4 g to 2.0 g of the skin topical drug can be applied daily, and 0.4 g to 1.0 g of the skin topical drug can be applied daily. Specifically, for example, when a gel containing 0.2% by mass of sirolimus is applied as a skin topical medication to the skin of an adult (weighing 60 kg), 0.1 g to 10.0 g of the skin topical medication can be applied daily, 0.3 g to 5.0 g of the skin topical medication can be applied daily, 0.4 g to 2.0 g of the skin topical medication can be applied daily, and 0.4 g to 1.0 g of the skin topical medication can be applied daily.

[0062] The application may be performed once a day, once to twice a day, or once to three times a day.

[0063] The application site of the external skin medicine of this embodiment is not particularly limited as long as it is a site where skin sclerosis in systemic sclerosis occurs or a site that is prone to skin sclerosis, and examples thereof include the skin of fingers, forearms, and face.

[0064] The target animal of the external skin medicine of the present embodiment is not particularly limited as long as it is a mammal that develops skin sclerosis in systemic sclerosis, and examples thereof include humans and mice.

[0065] The skin external medicine of the present embodiment may be a skin external medicine that suppresses collagen production in the skin of an administration subject.

[0066] The inhibition of collagen production may refer to the complete disappearance of collagen production at the lesion site of the subject's skin hardening, or may refer to a decrease in the amount of soluble collagen after the administration of the skin topical medication of this embodiment, even if it does not completely disappear. For example, after the administration of the skin topical medication of this embodiment, it can be determined that collagen production is inhibited by, for example, a decrease in the amount of soluble collagen in the skin tissue compared to before the administration of the skin topical medication. In addition, for example, after the administration of the skin topical medication of this embodiment, when the amount of soluble collagen in the skin tissue is a low value compared to a predetermined cutoff value, it can be determined that collagen production is inhibited.

[0067] Alternatively, the inhibition of collagen production may refer to a situation in which collagen production is completely inhibited from occurring in the skin of the subject, particularly in skin tissue that may develop skin sclerosis, from a state in which collagen production does not occur, or may refer to an inhibition of an increase in collagen production. For example, by comparing the amount of soluble collagen in the skin tissue of a subject after administration of the skin topical medication of the present embodiment with that of a subject to which the skin topical medication has not been administered, it can be determined that collagen production is inhibited. In addition, for example, after administration of the skin topical medication of the present embodiment, when the amount of soluble collagen in the skin tissue is a low value compared to a predetermined cutoff value, it can be determined that collagen production is inhibited.

[0068] The skin external medicine of the present embodiment may be a skin external medicine that suppresses inflammation in the skin of an administration subject.

[0069] The suppression of inflammation may refer to the complete disappearance of inflammation at the lesion site of the subject's skin sclerosis, or may refer to the reduction of inflammation after the application of the skin topical medication of this embodiment, even if it has not completely disappeared. For example, after the application of the skin topical medication of this embodiment, the inflammation can be judged to be suppressed by reducing the value indicating the degree of inflammation in the skin tissue (e.g., the number of CD3-positive cells, the number of CD68-positive cells, the expression level of TNF-α, the expression level of TGF-β, etc.) compared to before the application of the skin topical medication. In addition, for example, after the application of the skin topical medication of this embodiment, when the value indicating the degree of inflammation in the skin tissue is a low value compared to a predetermined cutoff value, the inflammation can be judged to be suppressed.

[0070] Alternatively, the inhibition of inflammation may refer to the complete inhibition of inflammation from a non-inflammation state in the skin of the subject, especially in the skin tissue where skin sclerosis may occur, or may refer to the inhibition of the increase of inflammation. For example, by comparing the value indicating the degree of inflammation in the skin tissue of the subject after the application of the skin topical medication of the present embodiment with the subject to which the skin topical medication has not been applied, it can be judged that the inflammation is suppressed. In addition, for example, after the application of the skin topical medication of the present embodiment, when the value indicating the degree of inflammation in the skin tissue is a low value compared to a predetermined cutoff value, it can be judged that the inflammation is suppressed.

[0071] The external skin medicine of the present embodiment preferably exerts the above-mentioned therapeutic or preventive effect on the lesion site of skin sclerosis in a subject with systemic scleroderma by inhibiting the mTOR signaling by sirolimus.

[0072] The inhibition of the mTOR signal may refer to the complete disappearance of the mTOR signal at the lesion site of the subject's skin sclerosis, or may refer to the reduction of the mTOR signal after the administration of the skin topical medication of the present embodiment, even if it does not completely disappear. For example, after the administration of the skin topical medication of the present embodiment, the mTOR signal may be determined to be inhibited by, for example, a decrease in the expression of phosphorylated mTOR in the skin tissue compared to before the administration of the skin topical medication. In addition, for example, after the administration of the skin topical medication of the present embodiment, when the expression of phosphorylated mTOR in the skin tissue is a low value compared to a predetermined cutoff value, the mTOR signal may be determined to be inhibited.

[0073] Alternatively, the inhibition of the mTOR signal may refer to a situation in which the generation of the mTOR signal is completely inhibited from being generated in the skin of the subject, especially in the skin tissue where skin sclerosis may occur, from a state in which the mTOR signal is not generated, or may refer to the inhibition of the increase of the mTOR signal. For example, by comparing the expression of phosphorylated mTOR in the skin tissue of the subject after the administration of the skin topical medication of the present embodiment with that of the subject not administered with the skin topical medication, it can be determined that the mTOR signal is inhibited. In addition, for example, after the administration of the skin topical medication of the present embodiment, when the expression of phosphorylated mTOR in the skin tissue is a low value compared to a predetermined cutoff value, it can be determined that the mTOR signal is inhibited.

[0074] As other examples of this embodiment, the following contents can be cited, for example:

[0075] A method for treating or preventing skin sclerosis in systemic sclerosis, comprising: applying sirolimus to the skin of a subject in need thereof.

[0076] Sirolimus, wherein the sirolimus is used for use in the treatment or prevention of skin sclerosis in systemic scleroderma achieved by application to the skin.

[0077] Use of sirolimus in the manufacture of a topical skin medicine for treating or preventing skin sclerosis in systemic scleroderma.

[0078] Example

[0079] The present invention will be described in more detail by the following examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0080] <Example 1: Effect of Sirolimus Gel on Inhibition of Skin Fibrosis in Systemic Scleroderma>

[0081] By applying sirolimus gel (Nobelpharma Co., Ltd., product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g) to the skin in the early stage of fibrosis, it was studied whether skin fibrosis is inhibited. The scleroderma-like skin sclerosis model mice were prepared by the following method: C57BL / 6 mice (Japan SLC, 8 weeks old, female) were subcutaneously injected with 300 μg of bleomycin (Nippon Kayaku) daily. In addition, as control mice, mice were subcutaneously injected with PBS instead of bleomycin. In the following, the first day of bleomycin administration is set as day 1.

[0082] In Example 1, in order to confirm the effect of sirolimus on early skin fibrosis, the patients were treated with Figure 1 The external application of sirolimus gel was also started at the application schedule shown. Sirolimus gel was applied to the skin in an amount equivalent to 0.16 mg (80 mg of gel) of sirolimus per day. It should be noted that the external application of sirolimus-containing gel and the control sirolimus-free gel (a gel containing a base with exactly the same composition as the base in the sirolimus-containing gel, and a gel different from the sirolimus-containing gel only in that it does not contain sirolimus; that is, a placebo gel; provided by Nobelpharma Co., Ltd.) were used. 14 days after the start of bleomycin administration (day 14), skin tissue was collected from each model mouse and HE staining was performed by a method known to those skilled in the art, and the results are shown in Figures 2-3 . Figure 2 A representative example of the results of microscopic observation is shown in FIG. Figure 3 The graph related to skin thickness is shown in the figure (One-way ANOVA and Tukey's test; n=3-5; in the figure, ** indicates p<0.01, and the error bar indicates Mean+SEM (standard error)). As a result, it was confirmed that the skin thickness of the scleroderma-like skin sclerosis model mice treated with placebo gel became thicker, but the skin thickness of the scleroderma-like skin sclerosis model mice treated with sirolimus gel was thinner than that of the placebo gel. Moreover, Figure 4As shown, the amount of soluble collagen in the skin tissue of the lesion part of each mouse was measured by using the specific binding of collagen and Sirius red using the Sircol assay (Sircol Soluble Collagen Assay Kit, biocolor), which is a method known to those skilled in the art. The results showed that in scleroderma-like skin sclerosis model mice that were topically treated with sirolimus gel, the collagen content was suppressed compared to the case where a placebo gel was topically treated (one-way ANOVA and Tukey's test; n=3-5; in the figure, ** indicates p<0.01, and the error bar indicates Mean+SEM (standard error)). The above shows that skin fibrosis is suppressed by topically applying sirolimus gel from the early stage of skin fibrosis.

[0083] It should be noted that the average weight of C57BL / 6 mice (8 weeks old, female) used in each embodiment of the present disclosure is 20.1 g on average. After applying the amount of sirolimus equivalent to 0.16 mg (gel 80 mg) to the skin of the mice per day, the effects of inhibiting skin fibrosis can be confirmed. If the amount is converted to the amount of a person weighing 60 kg, it is about 0.48 g when converted to the amount equivalent to the gel. The same is true in the following embodiments.

[0084] <Example 2: Therapeutic effect of sirolimus gel on skin fibrosis in systemic scleroderma>

[0085] The study investigated whether skin fibrosis can be treated by applying sirolimus gel to the skin after fibrosis. The scleroderma-like skin sclerosis model mice were prepared by the following method: C57BL / 6 mice (Japanese SLC, 8 weeks old, female) were subcutaneously injected with 300 μg of bleomycin daily. In addition, as control mice, mice were subcutaneously injected with PBS instead of bleomycin. Hereinafter, the first day of bleomycin administration was set as day 1.

[0086] In Example 2, in order to confirm the effect of sirolimus on fibrotic skin, the skin was treated with Figure 5The application schedule shown also starts with external application of sirolimus gel (Nobelpharma Co., Ltd., product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g). The sirolimus gel is applied to the skin in an amount equivalent to 0.16 mg of sirolimus per day (80 mg of gel). It should be noted that the external application of sirolimus-containing gel and the control sirolimus-free gel (a gel containing a base with exactly the same composition as the base in the sirolimus-containing gel, and a gel that differs from the sirolimus-containing gel only in that it does not contain sirolimus; that is, a placebo gel; provided by Nobelpharma Co., Ltd.) were used. 14 days after the start of bleomycin administration (day 14), skin tissue was collected from each model mouse. As Figure 6 As shown, the amount of soluble collagen in the skin tissue of the lesion part of each mouse was measured by using the specific binding of collagen and Sirius red through the Sircol assay, which is a method known to those skilled in the art. As a result, it was shown that in the scleroderma-like skin sclerosis model mice to which sirolimus gel was applied externally, the collagen content was reduced compared with the case where a placebo gel was applied externally (one-way analysis of variance and Tukey's test; n=3-5; in the figure, ** indicates p<0.01, and the error bar indicates Mean+SEM (standard error)). The above shows that skin fibrosis can be treated by applying sirolimus gel externally to the fibrotic skin.

[0087] <Example 3: Effect of sirolimus gel on inflammation of skin fibrosis sites in systemic scleroderma>

[0088] It is known that serum levels of various cytokines, especially Th2 cytokines, Th17 cytokines, and TGF-β are increased in patients with systemic sclerosis (Non-patent Documents 3, 4). In addition, abnormal activation of immune cells such as T cells, B cells, NK cells, and macrophages has been confirmed in systemic sclerosis (Non-patent Documents 4, 5). In addition, it is known that immune activation leads to fibrosis via stimulation of collagen production by fibroblasts (Non-patent Document 6). That is, it is suggested that immune abnormalities are involved in the pathology of systemic sclerosis.

[0089] Here, it was studied whether the effect of inflammation on the skin fibrosis site can be obtained in the case of external use of sirolimus. The preparation of scleroderma-like skin sclerosis model mice was carried out in the same manner as in Example 1. That is, it was carried out by the following method: C57BL / 6 mice (Japanese SLC, 8 weeks old, female) were subcutaneously injected with 300μg of bleomycin every day. In addition, as control mice, mice that were subcutaneously injected with PBS instead of bleomycin were used. Hereinafter, the first day of bleomycin administration was set as day 1.

[0090] In Example 3, from day 1, Figure 1 The application schedule shown also starts with external application of sirolimus gel (Nobelpharma Co., Ltd., product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g). The sirolimus gel is applied to the skin in an amount equivalent to 0.16 mg of sirolimus per day (80 mg of gel). It should be noted that the external application of sirolimus-containing gel and the control sirolimus-free gel (a gel containing a base having exactly the same composition as the base in the sirolimus-containing gel, a gel that differs from the sirolimus-containing gel only in that it does not contain sirolimus; that is, a placebo gel; provided by Nobelpharma Co., Ltd.)

[0091] Fourteen days after the start of bleomycin administration (day 14), skin tissues were collected from each model mouse and immunostained with anti-CD3 antibody (anti-CD3 antibody, Abcam) and anti-CD68 antibody (anti-CD68 antibody, Bio-Rad) by methods known to those skilled in the art. The results are shown in FIG. Figures 7 to 9 . Figure 7 A representative example of the results of microscopic observation is shown in FIG. Figures 8-9 A graph related to the number of CD3-positive cells and CD68-positive cells is shown in (one-way ANOVA and Tukey's test; n=2-7; 6-7 areas were randomly measured in each mouse; in the figure, ** indicates p<0.01, * indicates p<0.05, and the error bar indicates Mean+SEM (standard error)). As a result, it was confirmed that: in scleroderma-like skin sclerosis model mice that applied placebo gel externally, the number of CD3-positive cells and CD68-positive cells increased significantly, and in scleroderma-like skin sclerosis model mice that applied sirolimus gel externally, the increase in the number of CD3-positive cells and CD68-positive cells was significantly suppressed compared with the case where the placebo gel was applied externally. Moreover, as Figures 10-11As shown, the expression levels of TNF-α and TGF-β in the skin of the lesion part of each mouse were measured by real-time PCR, which is a method known to those skilled in the art. As for the primers used in the real-time PCR method, the one related to the measurement of the expression level of TNF-α was TNFαmouse TNF-αForward (sigma): 5'-GCCTCTTCTCATTCCTGCTTG-3' (sequence number 1) as the forward primer and TNFαmouse TNF-αReverse (sigma): 5'-CTGATGAGAGGGAGGCCATT-3' (sequence number 2) as the reverse primer. In addition, in relation to the measurement of TGF-β expression, MA148599-F (Tgfb1) (takara): 5'-TACGGCAGTGGCTGAACCAA-3' (SEQ ID NO. 3) was used as a forward primer, and MA148599-R (Tgfb1) (takara): 5'-CGGTTCATGTCATGGATGGTG-3' (SEQ ID NO. 4) was used as a reverse primer. The results showed that in scleroderma-like skin sclerosis model mice treated with sirolimus gel, the expression of TNF-α was significantly reduced compared with the case of the placebo gel, and the expression of TGF-β was slightly reduced, although no significant difference was shown (one-way ANOVA and Tukey's test; n = 2 to 7; in the figure, ** indicates p < 0.01, and the error bar indicates Mean + SEM (standard error)). The above shows that external application of sirolimus gel to skin fibrosis sites in systemic scleroderma suppresses the infiltration of inflammatory cells (i.e., CD3-positive T cells and CD68-positive macrophages) produced in the sites, and also shows that the amount of inflammatory cytokines such as TNF-α is suppressed.

[0092] <Example 4: Effect of sirolimus gel on the expression of phosphorylated mTOR at skin fibrosis sites in systemic scleroderma>

[0093] It has been confirmed that sirolimus, an mTOR inhibitor, inhibits the signal transduction mechanism that depends on IL-2 and other cytokine receptors by inhibiting the activation of both T cells and B cells (Non-Patent Document 7), and that fibrosis-inducing inflammatory cytokines (IL-4, IL-61, IL-17, TGF-β1) are suppressed by intraperitoneal administration of sirolimus (Non-Patent Document 2). In other words, it is suggested that sirolimus has an immunosuppressive effect.

[0094] Here, it was studied whether the mTOR inhibitory effect can also be obtained in the case of topical sirolimus. The preparation of scleroderma-like skin sclerosis model mice was carried out in the same manner as in Example 1. That is, it was carried out by the following method: C57BL / 6 mice (Japanese SLC, 8 weeks old, female) were subcutaneously injected with 300 μg of bleomycin every day. In addition, as control mice, mice were subcutaneously injected with PBS instead of bleomycin. Hereinafter, the first day of bleomycin administration was set as day 1.

[0095] In Example 4, from day 1, Figure 1 The application schedule shown also starts with external application of sirolimus gel (Nobelpharma Co., Ltd., product name: RAPALIMUS (registered trademark) gel 0.2%: 2 mg of sirolimus in 1 g). The sirolimus gel is applied to the skin in an amount equivalent to 0.16 mg of sirolimus per day (80 mg of gel). It should be noted that the external application of the sirolimus-containing gel and the control sirolimus-free gel (a gel containing a base having exactly the same composition as the base in the sirolimus-containing gel, and a gel that differs from the sirolimus-containing gel only in that it does not contain sirolimus; that is, a placebo gel) are compared.

[0096] 14 days after the start of bleomycin administration (day 14), skin tissues were collected from each model mouse and immunostained with anti-p-mTOR antibody (Cell Signaling) by a method known to those skilled in the art. The results are shown in FIG. Figures 12-14 . Fig.12 A representative example of the results of microscopic observation is shown in FIG. Fig.13 A graph related to the number of phosphorylated mTOR (p-mTOR) expressing cells is shown in (one-way ANOVA and Tukey's test; n=2-6; 6 regions were randomly measured in each mouse; in the figure, ** indicates p<0.01, * indicates p<0.05, and the error bars indicate Mean+SEM (standard error)). As a result, it was confirmed that the number of p-mTOR expressing cells significantly increased in scleroderma-like skin sclerosis model mice that applied placebo gel externally, and the increase in the number of p-mTOR expressing cells was significantly suppressed in scleroderma-like skin sclerosis model mice that applied sirolimus gel externally compared to the case where the placebo gel was applied externally. Moreover, as Fig.14As shown, the expression of p-mTOR in the skin of the lesion of each mouse was confirmed by Western blotting, which is a method known to those skilled in the art. The primary antibody used was Phospho-mTOR (Ser2448) Antibody #2971 (Cell Signaling), and the secondary antibody was Anti-rabbit IgG, HRP-linked Antibody #7074 (Cell Signaling). As a result, it was shown that in the scleroderma-like skin sclerosis model mice to which sirolimus gel was applied externally, the expression of p-mTOR was suppressed compared with the case where a placebo gel was applied externally. The above shows that by applying sirolimus gel externally to the skin fibrosis site in systemic scleroderma, the hyperexpression of p-mTOR at the site is suppressed.

[0097] These results suggest that a therapeutic effect can be obtained by externally applying a sirolimus-containing gel to skin sclerosis in patients with systemic scleroderma. The present invention is expected to provide a new treatment method with fewer side effects and high safety for skin sclerosis of scleroderma, for which there is no effective and safe treatment method.

Claims

1. A skin external medicine, wherein: The topical skin medication comprises sirolimus, The external skin medicine is used for treating or preventing skin sclerosis in systemic scleroderma.

2. The skin external medicine according to claim 1, wherein The treatment or prevention refers to inhibiting early stage skin fibrosis.

3. The skin external medicine according to claim 1 or 2, wherein: The dosage form of the skin external medicine is a gel, cream, ointment, liquid, emulsion or patch.

4. The skin external medicine according to claim 1 or 2, wherein The sirolimus is contained in a ratio of 0.2 mass % or more based on the entirety of the external skin preparation.

5. The skin external medicine according to claim 1 or 2, wherein: The external skin medication suppresses collagen production in the skin of an administration subject.

6. The skin external medicine according to claim 1 or 2, wherein: The external skin medicine suppresses inflammation in the skin of an administration subject.

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