Application of vorinostat in preparation of external skin preparation for treating psoriasis

Abstract

The invention provides an application of vorinostat in preparation of a skin external preparation for treating psoriasis, the external vorinostat can inhibit proliferation of keratinocytes, promote differentiation of keratinocytes, inhibit inflammation of keratinocytes and effectively improve skin lesion symptoms of psoriasis, and no obvious side effect is found in the treatment process of the external vorinostat. The safety is high, and long-term use is facilitated. The external medicine for psoriasis provided by the invention is stable in curative effect, small in side effect, good in patient tolerance and relatively short in treatment cycle, and can better meet the treatment requirements of psoriasis patients.

Description

Technical Field

[0001] The present invention relates to the technical field of psoriasis treatment drugs, and in particular to the use of vorinostat in preparing a skin external preparation for treating psoriasis. Background Art

[0002] Psoriasis, commonly known as "psoriasis", is a chronic, recurrent, inflammatory skin disease. It has a long course of disease, is prone to recurrence, and is relatively difficult to treat. The main characteristics of psoriasis are red papules or plaques on the skin, covered with multiple layers of silvery white scales, with clear boundaries, and accompanied by varying degrees of itching. In addition, some patients may also have damage to the nails, joints and other parts.

[0003] The pathogenesis of psoriasis is not yet fully understood, but it is generally believed to be related to multiple factors, including genetic factors, environmental factors, and immune factors. Genetic factors play an important role in the onset of psoriasis, and people with a family history of the disease are more likely to develop the disease. At the same time, environmental factors such as environmental pollution, smoking, and pesticides, as well as mental factors such as mental stress and mood swings, may also increase the risk of psoriasis.

[0004] There are many treatments for psoriasis, including topical medication, systemic medication, physical therapy, and traditional Chinese medicine. Topical medications include hormone drugs, vitamin D3 derivatives, and keratin peels. Hormone drugs are one of the first choice for topical treatment of psoriasis. They can eliminate local inflammation and relieve symptoms such as scaling and itching, but long-term use may cause side effects such as skin atrophy and pigmentation. Vitamin D3 derivatives, such as calcipotriol, can inhibit the excessive proliferation of skin cells, thereby reducing skin lesions. Their side effects are relatively small, but they are slow to take effect and are prone to cause eczema. For patients with thicker scales, keratin peels, such as salicylic acid, can be used to remove the excessively thick stratum corneum and promote drug absorption. Systemic medications include phototherapy, immunosuppressants, retinoic acid drugs, and biological agents. Phototherapy uses ultraviolet light to treat psoriasis, which can inhibit the excessive proliferation of epidermal keratinocytes, and also has anti-inflammatory and bactericidal effects. The side effects are relatively small. The main adverse reactions include local itching, erythema, pigmentation, etc., and there are fewer systemic adverse reactions. It has good long-term treatment effects and is one of the first choices for long-term treatment of psoriasis. It can greatly reduce the psychological burden of patients caused by long-term use of drugs. The disadvantages are that the treatment cost is relatively expensive, and the phototherapy course is generally long, and multiple treatments are required to see obvious results. Immunosuppressants can inhibit cell proliferation and immune inflammation, and have obvious therapeutic effects on psoriasis; at the same time, the side effects are also obvious. Immunosuppressants also have inhibitory and damaging effects on the body's normal tissue cells and immune functions, which may lead to leukopenia and thrombocytopenia, oral ulcers, gastrointestinal ulcers, abdominal pain, nausea, vomiting, abnormal liver function, hair loss and other side effects. Retinoic acid drugs can treat excessive proliferation of keratinocytes by interfering with cell differentiation and proliferation, and have a certain effect on psoriasis; but they may cause discomfort: long-term use may cause dry skin, desquamation and other discomfort symptoms; long-term use of retinoic acid drugs requires monitoring of liver function to prevent drugs from damaging the liver. Biological agents have a good effect on psoriasis, with a fast onset of action and the ability to quickly control the disease; and biological agents can be targeted for treatment, with strong specificity, can reduce side effects, and can significantly improve the quality of life; but the price of biological agents is relatively high, and long-term use will increase the economic burden; in addition, biological agents may have some adverse reactions, such as infection, allergies, etc.; for some patients, biological agents may affect the immune system and increase the risk of other diseases.

[0005] Clinically, the symptoms of psoriasis patients vary in severity, so the treatment plan also needs to be individualized according to the condition. For a large number of patients with mild symptoms, topical medication is the preferred and effective treatment method. At the same time, even for severe patients, topical medications are often used as an auxiliary treatment method in combination with other treatment methods such as oral medications and phototherapy to achieve better therapeutic effects. As mentioned above, traditional topical medications for the treatment of psoriasis usually include the use of topical medications such as glucocorticoids, vitamin D3 derivatives, retinoic acid drugs, and calcineurin inhibitors. The mechanism of action of these topical drugs is relatively broad, targeting the specific pathological process of psoriasis, and through different mechanisms of action, such as anti-inflammatory, anti-proliferative, and immunomodulatory, they can improve the skin lesions and symptoms of psoriasis patients to a certain extent. However, the above-mentioned topical drugs may affect other normal physiological processes, which leads to poor specificity of traditional drug treatments and prone to side effects, such as skin atrophy and pigmentation. In addition, different patients have different responses to drugs. Some patients are insensitive to traditional drugs, resulting in poor treatment effects of traditional topical drugs. This is related to individual differences, the activity of drug-metabolizing enzymes and other factors, and it is difficult to improve sensitivity through simple methods. Long-term use of traditional drugs may lead to serious side effects, such as skin atrophy and dependence, such as glucocorticoid dependence and eczematosis of vitamin D3 derivatives. These side effects not only affect the quality of life of patients, but may also increase medical costs.

[0006] Therefore, for patients with mild symptoms, topical drug treatment is the first choice because it has the advantages of local effect, few side effects, and easy use. For patients with severe symptoms, although systemic treatments such as biological agents are more commonly used, topical drugs are still used as auxiliary treatments to help alleviate symptoms and control the disease. Although existing treatment options such as topical hormone drugs and vitamin D3 derivatives have achieved certain therapeutic effects, there are still some defects, including but not limited to unstable efficacy, obvious side effects, poor patient tolerance, and long treatment cycles. Therefore, it is particularly important to develop new treatment options, especially treatment options for topical drugs, to address the shortcomings of existing technologies.

[0007] Vorinostat is a histone deacetylase inhibitor (HDACi), also known as suberoylanilide hydroxamic acid (SAHA). It works by inducing cell differentiation, blocking the cell cycle, and inducing cell regulation. It is mainly used to treat cutaneous T-cell lymphoma (CTCL) that is exacerbated, persistent, and recurrent or ineffective after treatment with two systemic drugs. In addition, vorinostat has also shown good therapeutic effects in the treatment of other tumors and AIDS. Systemic application of vorinostat has many side effects, including but not limited to fatigue, nausea, vomiting, diarrhea, loss of appetite, thrombocytopenia, anemia, hypokalemia, and potential liver and kidney damage. However, there are currently no research reports on the use of vorinostat in the treatment of psoriasis. Summary of the invention

[0008] In order to overcome at least one problem existing in the existing topical medication treatment for psoriasis, the present invention has developed a topical vorinostat for the treatment of psoriasis, which provides a topical medication with stable efficacy, few side effects, good patient tolerance and a relatively short treatment cycle to better meet the treatment needs of psoriasis patients.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention is to provide the use of vorinostat in preparing a skin external preparation for treating psoriasis.

[0011] Further, the chemical structure of the vorinostat (SAHA) is as follows:

[0012]

[0013] Furthermore, the skin topical preparation is a skin topical preparation having at least one of the following effects: inhibiting the activity of histone deacetylase, interfering with the deacetylation process, affecting gene expression and cell differentiation; reducing keratinocyte proliferation, promoting keratinocyte differentiation, reducing the expression of inflammatory factors in keratinocytes; improving the skin lesions of psoriasis, etc.

[0014] Furthermore, the above effects include but are not limited to: inhibiting the viability of HaCaT / NHEK cells, reducing the proliferation of HaCaT and NHEK cells, upregulating the expression of differentiation markers of keratinocytes (KRT1, KRT10 and FLG), reducing the expression of inflammatory factors (CXCL1, CXCL5, Il-1β, ICAM1, CCL5, CCL17 and CCL22) in HaCaT / NHEK cells; reducing the psoriasis-like phenotype and PASI score, reducing the thickness of lesions and inhibiting the expression of Ki67 in lesions, increasing the thickness of the granular layer, increasing the expression of Flg, Krt1 and Krt10, etc.

[0015] Furthermore, the dosage form of the skin external preparation includes: liquid, solid, emulsion, etc. Preferably, the skin external preparation is an emulsion.

[0016] Furthermore, in the above application, vorinostat is used as an active ingredient and is placed in an auxiliary carrier to prepare the skin external preparation.

[0017] Furthermore, the auxiliary carrier is simple cream.

[0018] Furthermore, in the skin external preparation, the final concentration of vorinostat is 10 to 50 μmol / g, preferably 40 μmol / g (about 1%).

[0019] It is understandable that the type and amount of the auxiliary carrier selected are also adaptively adjusted based on the different dosage forms, but it will not affect the efficacy of vorinostat. The above auxiliary carriers are all conventional reagents commonly used in the field.

[0020] Furthermore, the skin external preparation can be directly applied to the skin lesions or can be attached to a medium and applied to the skin lesions. For example, it can be attached to a medium such as gauze or a band-aid for application.

[0021] Furthermore, the skin topical preparation is used alone or in combination. For example, in combination, the skin topical preparation can be applied first, and after it is absorbed, other topical preparations for treating psoriasis (including Chinese medicine preparations or Western medicine preparations) can be applied, and the order of application can be changed; the skin topical preparation can also be used in combination with oral medications, phototherapy, etc.

[0022] The second aspect of the present invention is to provide a skin external preparation for treating psoriasis, wherein the active ingredient is vorinostat.

[0023] Furthermore, the dosage form of the skin external preparation includes: liquid, solid, emulsion, etc. Preferably, the skin external preparation is an emulsion.

[0024] Furthermore, the skin topical preparation is prepared by placing vorinostat in an auxiliary carrier; wherein the auxiliary carrier is a simple cream; in the skin topical preparation, the final concentration of vorinostat is 10 to 50 μmol / g, preferably 40 μmol / g (about 1%).

[0025] The third aspect of the present invention is to provide a method for preparing a skin topical preparation for treating psoriasis as described in any one of the second aspects, which comprises the steps of weighing a predetermined mass of simple cream, dissolving SAHA in DMSO to obtain a SAHA solution of a predetermined concentration, mixing a predetermined volume of the SAHA solution with the simple cream to obtain a skin topical preparation.

[0026] Furthermore, the SAHA concentration in the SAHA solution is 400mmol / L; wherein, the volume of the SAHA solution = the mass of the simple cream (g) × the SAHA concentration in the skin topical preparation ÷ 400mmol / L, the SAHA concentration in the skin topical preparation is 0.01-0.05mmol / g, and the mass of the simple cream is 25mg.

[0027] It is understandable that, depending on the specifications of the external skin preparation (emulsion), the quality of the simple cream used, the concentration of the SAHA solution and its volume, etc. can be adaptively adjusted according to actual conditions.

[0028] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0029] The present invention treats psoriasis by topical vorinostat (SAHA, a histone deacetylase inhibitor), which solves the problems of unstable efficacy and obvious side effects of topical drugs in the prior art. The specific advantages are as follows:

[0030] (1) Unique pharmacological mechanism: Vorinostat inhibits the activity of histone deacetylase, interferes with the deacetylation process, affects gene expression and cell differentiation, and thus plays a role in treating psoriasis. This mechanism is different from existing drugs and provides a new treatment approach.

[0031] (2) Topical administration: Topical administration allows the drug to be directly applied to the affected area, thus avoiding the systemic side effects that may be caused by oral medications and improving the patient's tolerance and compliance.

[0032] (3) High safety: Animal experiments have shown that long-term topical use of vorinostat to treat psoriasis has no obvious side effects and is highly safe and suitable for long-term use.

[0033] (4) Significant and stable efficacy: Animal experiments have shown that topical vorinostat can significantly improve the skin lesions of psoriasis, with significant and stable efficacy that is better than or at least comparable to existing topical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1The figure is a schematic diagram of the results of HDAC inhibitors (vorinostat (SAHA) and trichostatin A (TSA)) regulating keratinocyte proliferation, differentiation and inflammation in vitro in one embodiment of the present invention; wherein, Part A: CCK8 method is used to analyze the cell viability of HaCaT cells after treatment with different concentrations of SAHA or TSA; Part B: CCK8 method is used to analyze the cell viability of NHEK cells after treatment with different concentrations of SAHA or TSA; Part C: EdU method is used to analyze the proliferation of HaCaT cells after treatment with SAHA or TSA; Part D: EdU method is used to analyze the proliferation of NHEK cells after treatment with SAHA or TSA; Part E: SAHA or TSA Part A: Morphological observation of HaCaT cells under an optical microscope after treatment; Part F: Morphological observation of NHEK cells under an optical microscope after treatment with SAHA or TSA; Part G: qRT-PCR detection of differentiation markers of HaCaT cells after treatment with SAHA or TSA; Part H: qRT-PCR detection of differentiation markers of NHEK cells after treatment with SAHA or TSA; Part I: qRT-PCR detection of inflammatory factors in HaCaT cells after treatment with SAHA or TSA; Part J: qRT-PCR detection of inflammatory factors in NHEK cells after treatment with SAHA or TSA; All cells were treated with 10 ng / mL M5 mixture 24 hours before harvesting; Scale bar, 100 μm (CD part); Scale bar, 50 μm (EF part); NS, not significant; *p<0.05, **p<0.01, ***p<0.001.

[0036] Figure 2 The results of the safety of topical application of HDAC inhibitors (SAHA and TSA) to treat psoriasis-like mouse model skin lesions in one embodiment of the present invention are shown in the figure; wherein: Part A: experimental study design; Part B: photos of the mouse back skin at the end of the evaluation; Part C: HE staining of the mouse back skin at the end of the evaluation; Part D: changes in the weight of the mice during the evaluation after topical treatment with SAHA, * compared with the DMSO group, P < 0.05; Part E: changes in the weight of the mice during the evaluation after topical treatment with TSA, * compared with the DMSO group, P < 0.05; the scale bar represents 100 μm (Part C);

[0037] Figure 3The figure is a schematic diagram of the results of the efficacy of local SAHA in treating skin lesions in a psoriasis-like mouse model in one embodiment of the present invention; wherein, Part A: experimental study design of local SAHA in treating IMQ-induced psoriasis-like skin inflammation; Part B: photos of mouse skin lesions on day 9; Part C: PASI scores of skin lesions from day 4 to day 9; Part D: HE staining of skin; Part E: measurement of epidermal thickness of mouse skin lesions; Part F: Ki67 staining of skin lesions; Part G: qRT-PCR detection of differentiation markers in skin lesion tissue; Part H: qRT-PCR detection of inflammatory factors in skin lesion tissue; scale bar, 50 μm (Part D); scale bar represents 100 μm (Part F); compared with the DMSO group, *p<0.05, **p<0.01, #p<0.05 in the SAHA 0.5 μmol group. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0039] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0040] The experimental methods used in the following examples are basically conventional methods in the art, as exemplified below:

[0041] (1) Construction of psoriasis mouse model;

[0042] The animal experiments were approved by the Ethics Committee of Shanghai Dermatology Hospital (2023-49 (animal)). C57BL / 6 mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were used to establish a psoriasis mouse model. Imiquimod (IMQ) cream (5%) (H20030129, Sichuan Mingxin Pharmaceutical Co., Ltd.) was evenly applied to the back of the mice daily for 5 consecutive days. The Psoriasis Area and Severity Index (PASI) score of the psoriasis-like mouse model was evaluated daily. At the end of the experiment, skin samples were collected for histological analysis and gene expression profiling.

[0043] (2) Cell culture and treatment;

[0044] Human keratinocytes HaCaT cells (Shanghai Fuheng Biotechnology Co., Ltd.) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Newborn neonatal epidermal keratinocytes (NHEKs) were obtained from ATCC and cultured in DermaCult TM Keratinocytes were cultured in Keratinocyte Expansion Medium (100-0501 & 100-0502, STEMCELL, USA). To simulate the inflammatory environment, cells were incubated with 10 ng / mL M5 mixture containing tumor necrosis factor (TNF)-α, interleukin (IL)-22, IL-1α, tumor inhibitor M and IL-17A for 24 hours. Keratinocytes were treated with vorinostat (SAHA, HY-10221, MedChemExpress) and trichostatin A (TSA, MB5445-1, Meilun Bio, China).

[0045] (3) Cell viability assay;

[0046] Cell viability was determined using the Cell Counting Kit-8 (CCK-8). Cells were seeded in 96-well plates and then treated with HDACi (SAHA, TSA). After 24h and 48h, 10μL of CCK-8 solution was added to each well, and the absorbance at 450nm was recorded to indicate the relative viability of the cells.

[0047] (4) 5-ethyl-2'-deoxyuridine (EdU) assay;

[0048] Cells were seeded in 24-well plates and treated with HDACi (SAHA, TSA) or transfected. After 48 hours, BeyoClick containing AlexaFluor 555 (Beyotime, China) was used to TM EdU cell proliferation kit was used to perform EdU detection according to the manufacturer's instructions. The EdU positive rate indicates the relative proliferation capacity of keratinocytes.

[0049] (5) qRT-PCR detection;

[0050] Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen) and analyzed using PrimeScript TM Reverse transcription was performed using SYBR Green Premium Ex Taq TM Quantitative PCR was performed using a Takara kit and specific primer pairs. GAPDH was used as an internal reference and 2 -ΔΔCt Methods Data analysis.

[0051] (6) Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC);

[0052] Tissue samples were fixed, dehydrated, embedded and sliced. Hematoxylin and eosin solution was used for HE staining. Ki-67 (27309-1-AP, Proteintech) primary antibody and HRP-conjugated secondary antibody were used for IHC staining. The staining of tissue sections was observed under an optical microscope and photographed, and analyzed using ImageJ software.

[0053] (7) Statistical data;

[0054] All experiments were performed at least three times, and the data are presented as mean ± standard deviation (SD). Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, USA). Student's t-test was used to compare differences between two groups, one-way ANOVA was used for multiple comparisons, and Newman-Keuls test was used. p value < 0.05 was considered statistically significant.

[0055] Example 1 - Preparation of a skin external preparation containing an HDAC inhibitor

[0056] In this embodiment, a skin external preparation containing SAHA or TSA was prepared, and the specific steps are briefly described as follows:

[0057] Skin topical preparation containing SAHA: Weigh a certain mass of simple cream on an electronic balance, add a certain volume of 400mmol / L SAHA (dissolved in DMSO); SAHA volume = simple cream mass (g) × SAHA concentration (0-0.05mmol / g) ÷ 400mmol / L; fully mix, centrifuge, and obtain topical creams with different concentrations of SAHA. Preferably, the concentration of SAHA in the topical cream for practical application is 40μmol / g (about 1%).

[0058] Skin topical preparation containing TSA: Weigh a certain mass of simple cream on an electronic balance, add a certain volume of 100mmol / L TSA (dissolved in DMSO); TSA volume = simple cream mass (g) × TSA concentration (0-0.01mmol / g) ÷ 100mmol / L. Mix thoroughly and centrifuge to obtain topical creams with different concentrations of TSA.

[0059] Example 2 - HDAC inhibitors regulate keratinocyte proliferation, differentiation and inflammation in vitro

[0060] SAHA and TSA are commonly used HDAC inhibitors that can inhibit the function of HDAC1 / 2 / 3. Therefore, this example selects SAHA and TSA to study the effect of inhibiting HDAC1 / 2 / 3 on psoriatic keratinocytes. SAHA and TSA are both dissolved in DMSO and added to the cell culture medium to treat the cells, specifically including the following steps:

[0061] (1) HaCaT cells were treated with 0.5μM, 1.0μM, 2.0μM, and 4.0μM SAHA; HaCaT cells were treated with 0.125μM, 0.25μM, 0.5μM, and 1.0μM TSA; NEHK cells were treated with 2μM, 4μM, 8μM, and 16μM SAHA; NEHK cells were treated with 0.5μM, 1μM, 2μM, and 4μM TSA; DMSO was used as the control group in each group. After the experiment, the cell viability was measured using the CCK-8 kit. Figure 1 As shown in parts A and B. CCK8 assay showed that 1 μM SAHA and 0.25 μM TSA significantly inhibited the viability of HaCaT cells, while 4 μM SAHA and 1 μM TSA significantly inhibited the viability of NHEK cells.

[0062] (2) HaCaT cells were treated with 2 μM SAHA or 0.5 μM TSA, NHEK cells were treated with 8 μM SAHA or 2 μM TSA, and the control group was treated with DMSO. After the experiment, the effects of SAHA or TSA treatment on keratinocyte proliferation, differentiation, and inflammation were detected.

[0063] like Figure 1 As shown in parts C and D of Figure 3, EdU detection showed that SAHA and TSA could reduce the proliferation of HaCaT and NHEK cells. Figure 1 As shown in parts E and F of the figure, the keratinocytes became elongated and polygonal in shape, and displayed long black tentacles. Figure 1 As shown in parts G and H of Figure 3, differentiation markers of keratinocytes, including KRT1, KRT10, and FLG, were upregulated after SAHA and TSA treatment. Figure 1 As shown in parts I and J of the data, the expression of inflammatory factors CXCL1, CXCL5, IL-1β, ICAM1, CCL5, CCL17, and CCL22 in HaCat cells and / or NHEK cells was significantly reduced after SAHA treatment; however, TSA had no anti-inflammatory effect on keratinocytes and may even increase the expression of some inflammatory factors.

[0064] The above experimental results show that both SAHA and TSA treatment can inhibit the proliferation of keratinocytes in vitro and promote the differentiation of keratinocytes in vitro, and SAHA treatment can also inhibit the inflammation of keratinocytes in vitro.

[0065] Example 3 - Safety verification of topical HDAC inhibitors in the treatment of psoriasis-like mouse model lesions

[0066] This example detects the local and systemic toxicity of SAHA and TSA to mice. The experimental design is as follows: Figure 2 As shown in Part A, SAHA or TSA is first dissolved in DMSO, and then pure cream is used as a base to prepare creams of different concentrations as described in Example 1. The specific steps are briefly described as follows:

[0067] Experimental groups: C57BL / 6 mice (SAHA group: SAHA 0μmol (DMSO) group, SAHA 0.25μmol group, SAHA0.5μmol group, SAHA 1.0μmol group; TSA group: TSA 0μmol (DMSO) group, TSA 0.0625μmol group, TSA 0.125μmol group, TSA 0.25μmol group).

[0068] SAHA and TSA treatment: The mass of topical cream (25 mg) in each group remained the same, and the amount of cream (25 mg) = drug amount (SAHA 0-1.0 μmol; TSA 0-0..25 μmol) ÷ different drug concentrations. For example, if the drug amount is 1.0 μmol SAHA, the drug concentration in the cream is 40 μmol / g. The drug was applied to the back of the mice once a day. For details, see Figure 2 Part A of

[0069] The mice were weighed during the experiment; the evaluation ended on the 10th day or when obvious rash or significant weight loss appeared; at the end of the experiment, the skin lesions of the mice were photographed, their laboratory indicators were tested, and skin samples were collected for histological analysis and gene expression profiling analysis.

[0070] Depend on Figure 2 As shown in Part B of the Figure, there was no rash on the mouse skin after topical application of SAHA, whereas topical application of TSA induced an inflammatory rash with erythema, scaling, and even erosion. Figure 2 As shown in Part C of the study, no histopathological changes were found after treatment with SAHA at all doses; however, treatment with 0.0625 μmol TSA resulted in skin thickening, and 0.25 μmol TSA resulted in skin thickening, ulceration, epidermal neutrophil infiltration, and dermal lymphocyte infiltration. As shown in the laboratory examinations in Table 1, topical application of SAHA and TSA did not cause significant abnormalities in laboratory indicators such as blood routine, liver and kidney function, and C-reactive protein. Figure 2 As shown in parts D and E of the Figure 3, topical application of SAHA did not lead to significant changes in the body weight of mice, but topical application of TSA led to a significant reduction in the body weight of mice.

[0071] Table 1 - Comparison of laboratory tests after topical HDACi application

[0072]

[0073]

[0074] WBC: white blood cell; PLT: platelet; RBC: red blood cell; HGB: hemoglobin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALB: plasma albumin; CR: creatinine; GLU: glucose; CRP: c-reactive protein. There was no significant difference in the laboratory test results between the experimental group and the DMSO group (N = 4 in each group).

[0075] From the above experimental results, it can be seen that no obvious side effects were found in topical use of vorinostat, and it is highly safe.

[0076] Example 4 - Verification of the efficacy of topical SAHA in treating skin lesions in a psoriasis-like mouse model

[0077] In this example, IMQ was used to establish a psoriasis-like mouse model, and SAHA was topically applied to the skin. The experimental design is as follows: Figure 3 As shown in Part A, SAHA is first dissolved in DMSO, and then used as a base of simple cream to prepare a cream for use. For details, see Example 3, which is briefly described as follows:

[0078] Experimental groups: SAHA 0μmol (DMSO) group, SAHA 0.5μmol group, SAHA 1.0μmol group; psoriasis-like mouse model was induced by applying 62.5mg imiquimod on the back once a day, see Figure 3 Part A of

[0079] SAHA treatment: The mass of topical cream (25 mg) in each group remained the same, and the drug dosage (25 mg) = drug dosage (SAHA 0-1.0 μmol) ÷ different drug concentrations. The drug was applied to the back of the mice once a day, as shown in the table below. Figure 3Part A of

[0080] During the experiment, the Psoriasis Area and Severity Index (PASI) score of the psoriasis-like mouse model was evaluated every day. After the experiment, the mice were killed by cervical dislocation, and the back lesions were cut off for HE staining, immunohistochemical staining, RNA extraction and other experiments.

[0081] like Figure 3 As shown in Parts B and C of the study, topical SAHA was found to significantly reduce the psoriasis-like phenotype and PASI scores. Figure 3 As shown in parts D and F of Figure 3, SAHA significantly reduced lesion thickness and inhibited Ki67 expression in lesions, corresponding to the proliferation inhibition observed in vitro. Figure 3 As shown in part D of the figure, SAHA increased the thickness of the granular layer, which occurs in the late stage of keratinocyte differentiation and is reduced or absent in psoriasis. Figure 3 As shown in Figure G, it was confirmed that the expression of Flg, Krt1, and Krt10 was increased in skin lesions after SAHA treatment, and these data correspond to the effects of HDAC inhibitors on promoting differentiation observed in vitro. Figure 3 As shown in part H, the expressions of inflammatory factors CXCL1, IL-1β, and ICAM1 were also significantly reduced, which also confirmed the anti-inflammatory effect of SAHA in vivo.

[0082] From the above experimental results, it can be seen that SAHA can improve the skin lesions of psoriasis and is a potential candidate drug for the local treatment of psoriasis.

[0083] It can be seen from the experimental results of the above embodiments that the method of treating psoriasis with topical vorinostat in the present invention has the following advantages: (1) The present invention significantly improves the therapeutic effect of psoriasis by using topical vorinostat and utilizing its unique pharmacological mechanism of action; compared with traditional topical drugs, vorinostat can more deeply affect cell differentiation and gene expression, thereby more effectively controlling the disease and alleviating skin lesions; (2) Due to the use of topical administration, the present invention avoids the systemic side effects that may be caused by oral drugs; during use, the risk of adverse reactions in patients is greatly reduced, thereby improving the safety of treatment and the comfort of patients; (3) The present invention not only brings new treatment hope to psoriasis patients, but also promotes the development of scientific research in dermatology and related fields. It provides a new idea for studying the pathogenesis and treatment methods of psoriasis, and lays a foundation for the development of more effective therapeutic drugs and methods; (4) Vorinostat has been developed for decades and is inexpensive. If it can be used in clinical practice, it will greatly reduce medical costs.

[0084] In summary, the present invention, through the innovative scheme of external use of vorinostat for treating psoriasis, brings about many beneficial effects such as significantly improving efficacy, reducing side effects, promoting scientific research progress, and reducing medical costs. The above effects work together on psoriasis patients, bringing them a better treatment experience and higher quality of life.

[0085] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Use of vorinostat in the preparation of a skin external preparation for treating psoriasis.

2. The use according to claim 1, characterized in that: The skin topical preparation has at least one of the following effects: inhibiting the activity of histone deacetylase, interfering with the deacetylation process, affecting gene expression and cell differentiation; reducing keratinocyte proliferation, promoting keratinocyte differentiation, and reducing the expression of inflammatory factors in keratinocytes; and improving the skin lesions of psoriasis.

3. The use according to claim 1, characterized in that: The dosage forms of the skin external preparation include: liquid, solid and emulsion.

4. The use according to claim 1, characterized in that: Vorinostat is used as an active ingredient and is placed in an auxiliary carrier to prepare the skin external preparation; wherein the skin external preparation is an emulsion.

5. The use according to claim 4, characterized in that: The auxiliary carrier is a simple cream; in the skin external preparation, the final concentration of vorinostat is 10 to 50 μmol / g.

6. A skin external preparation for treating psoriasis, characterized in that: The active ingredient of the skin external preparation is vorinostat.

7. The external skin preparation for treating psoriasis according to claim 6, characterized in that: The skin external preparation is prepared by placing vorinostat in an auxiliary carrier, and the skin external preparation is an emulsion; Wherein, the auxiliary carrier is a simple cream; in the skin external preparation, the final concentration of vorinostat is 10 to 50 μmol / g.

8. A method for preparing the external skin preparation for treating psoriasis according to claim 6 or 7, characterized in that: The method comprises the following steps: weighing a predetermined mass of simple cream, dissolving SAHA in DMSO to obtain a SAHA solution of a predetermined concentration, and mixing a predetermined volume of the SAHA solution with the simple cream to obtain a skin external preparation.

9. The preparation method according to claim 8, characterized in that: The SAHA concentration in the SAHA solution is 400mmol / L; wherein, the volume of the SAHA solution = the mass of the simple cream (g) × the SAHA concentration in the skin topical preparation ÷ 400mmol / L, the SAHA concentration in the skin topical preparation is 0.01-0.05mmol / g, and the mass of the simple cream is 25mg.

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