Use of a polypeptide in the treatment of psoriasis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a polypeptide in treatment of psoriasis. The amino acid sequence of the polypeptide provided in the application is SEQ ID NO. 1, which can effectively reduce the spleen-body ratio of a psoriasis mouse, reduce the inflammation level in the body, significantly reduce the epidermal thickness of the psoriasis mouse and the inflammatory factors in the lesion tissue, reduce the keratinocyte proliferation, and reduce the recruitment of inflammatory cells to the lesion to release inflammatory factors. The polypeptide provided in the application exhibits certain advantages in aspects of reducing inflammatory response and promoting tissue repair in treatment of psoriasis. The application provides a new method and means for treatment of psoriasis, and promotes the progress and development of medicine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a polypeptide in treatment of psoriasis. BACKGROUND

[0002] Psoriasis is a common polygenic hereditary skin disease, which is often manifested as localized or widely distributed scaly erythema or plaques. The cause of psoriasis is not clear, but it has been identified that it is caused by the joint action of genetic factors and environmental factors and many other factors. This disease is related to factors such as meridian, food, climate, season, family history, etc. Psoriasis has many types, the most common of which is the ordinary type, in addition to which there are arthritic type, pustular type and erythroderma type.

[0003] Psoriasis has diversity in symptoms, mainly including red plaques on the skin, usually covered with silver-white scales, skin itching, burning or pain, dry and cracked skin, and even bleeding, etc. Some patients may be accompanied by joint pain, swelling and abnormality of the nails (toes). Psoriasis is not contagious, and the transmission route usually includes family inheritance or environmental stimulation.

[0004] The main purpose of treating psoriasis is to control the disease and alleviate the symptoms of patients. Current treatment methods include drug therapy, phototherapy and traditional Chinese medicine therapy, etc. Polypeptide treatment of psoriasis has shown its unique advantages to some extent. Polypeptide drugs, such as placental polypeptide and spleen polypeptide, have significant immune regulation function. They can regulate the immune function of the body and inhibit the activation of T lymphocytes, thereby reducing the excessive immune response of psoriasis patients. This immune regulation helps to improve the pathological process of psoriasis and reduce skin damage and inflammatory reaction. Polypeptide drugs also have anti-inflammatory effects, which can reduce the inflammatory reaction of the skin of psoriasis patients. By inhibiting the release of inflammatory factors and reducing the infiltration of inflammatory cells, polypeptide drugs help to reduce symptoms such as redness, itching and pain of the skin, and improve the quality of life of patients. Polypeptide drugs also have the effect of promoting tissue repair. They can stimulate the proliferation and differentiation of skin cells and accelerate the healing process of skin lesions. This is of great significance to improve the skin damage of psoriasis and reduce recurrence.

[0005] Although polypeptide drugs may also have some side effects during treatment, overall they are relatively safe. Under the guidance of professional doctors, patients can adjust the dose and administration method according to their own condition to reduce the occurrence of adverse reactions. At the same time, polypeptide drugs usually do not cause serious side effects such as liver and kidney damage, which makes them a relatively safe treatment option for some patients.

[0006] However, polypeptide treatment of psoriasis is not without limitations. For example, its efficacy can vary from individual to individual, which is mainly due to the differences in physiological status, genetic factors, disease stages, and sensitivity and metabolic capacity to drugs of each person. Therefore, in order to more effectively utilize polypeptides for treatment and meet the needs of different patients, more polypeptide drugs need to be developed. SUMMARY

[0007] To solve the above problems, the present application provides a polypeptide which can effectively treat psoriasis, providing patients with more treatment options and meeting their individualized treatment needs.

[0008] In one aspect, the present application provides a polypeptide for use in the preparation of a medicament for preventing and / or treating psoriasis, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1.

[0009] Specifically, the administration dose of the medicament can be 1-5 mg / kg, such as 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, and any point value within the range.

[0010] Specifically, the polypeptide can further include one or more of a homing peptide, a cytotoxic payload, or a linker.

[0011] Specifically, the polypeptide can be the only or main active ingredient of the medicament.

[0012] Specifically, the medicament further includes a pharmaceutically acceptable excipient.

[0013] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifying agent, a preservative, an antioxidant, a buffering agent, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizing agent, a thickening agent, a stabilizing agent, a sweetener, and a flavoring agent.

[0014] Further preferably, the pharmaceutically acceptable excipient is at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, and mineral oil.

[0015] Specifically, the dosage form of the medicament includes, but is not limited to, tablets, liquid agents, capsules, powders, suppositories, granules, sprays, or liniments.

[0016] Preferably, the dosage form of the medicament can be a liquid agent.

[0017] Specifically, the administration mode of the drug is selected from oral administration, intravenous injection, local injection, intradermal injection or subcutaneous injection.

[0018] Preferably, the administration mode of the drug can be intravenous injection, intradermal injection or subcutaneous injection.

[0019] Specifically, the drug can also be used in combination with other drugs for preventing and / or treating psoriasis.

[0020] Further specifically, the other drugs for preventing and / or treating psoriasis are glucocorticoids, vitamin D3 derivatives, retinoids, calcineurin inhibitors, keratoplastic and keratolytic agents, immunosuppressants or antibiotic drugs.

[0021] Preferably, the glucocorticoids include but are not limited to halometasone ointment, desonide ointment, mometasone furoate ointment, clobetasol propionate ointment and compound dexamethasone acetate cream.

[0022] The vitamin D3 derivatives include but are not limited to calcipotriene ointment and tacalcitol ointment.

[0023] The retinoids include but are not limited to tretinoin cream, tazarotene cream and acitretin capsule.

[0024] The calcineurin inhibitors include but are not limited to tacrolimus ointment and pimecrolimus cream.

[0025] The keratoplastic and keratolytic agents include but are not limited to salicylic acid, sulfur and urea.

[0026] The immunosuppressants include but are not limited to methotrexate and cyclosporine soft capsules.

[0027] The retinoids include but are not limited to acitretin capsule.

[0028] The antibiotic drugs include but are not limited to amoxicillin and clindamycin.

[0029] Specifically, the combination can be simultaneous use or sequential use.

[0030] Specifically, the psoriasis includes but is not limited to psoriasis vulgaris, psoriasis arthropathica, pustular psoriasis or erythrodermic psoriasis.

[0031] Specifically, the psoriasis is psoriasis vulgaris.

[0032] Technical effects achieved by the present application:

[0033] (1) The polypeptide MrIC provided by the present application can effectively reduce the spleen-body ratio of psoriasis mice, i.e. reduce inflammation in vivo.

[0034] (2) The polypeptide MrIC provided by the application can significantly reduce the epidermal thickness of psoriasis mice.

[0035] (3) The polypeptide MrIC provided by the application can effectively reduce the level of inflammatory factors in the lesion tissue of psoriasis mice.

[0036] (4) The polypeptide MrIC provided by the application can reduce the proliferation of keratinocytes in psoriasis mice.

[0037] (5) The polypeptide MrIC provided by the application can reduce the recruitment of inflammatory cells to the lesion site to release inflammatory factors. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Disease severity score (PASI score) of the lesion skin of the control group and the experimental group mice.

[0039] Figure 2 Spleen to body size of the control group and the experimental group mice.

[0040] Figure 3 HE staining results of the lesion skin of the control group and the experimental group mice on the back, and the epidermal thickness evaluated according to the staining results.

[0041] Figure 4 qPCR detection of the expression level of inflammatory factors in the lesion tissue of the control group and the experimental group mice.

[0042] Figure 5 Immunofluorescence detection of the expression level of keratinocyte proliferation markers in the lesion tissue of the control group and the experimental group mice, wherein e in the figure represents the epidermis, and d represents the dermis.

[0043] Figure 6 Immunofluorescence detection of the expression level of angiogenesis markers and macrophage markers in the lesion tissue of the control group and the experimental group mice, wherein e in the figure represents the epidermis, and d represents the dermis.

[0044] Figure 7 Disease severity score results of the lesion skin of the control group mice on the back.

[0045] Figure 8 Spleen to body size of the control group mice.

[0046] Figure 9 HE staining results of the lesion skin of the control group mice on the back, and the epidermal thickness evaluated according to the staining results.

[0047] Figure 10 qPCR detection of the expression level of inflammatory factors in the lesion tissue of the control group mice. DETAILED DESCRIPTION

[0048] The application will be further described in conjunction with specific examples, the following examples are not used to limit the application, but only for the description of the application. The experimental methods used in the following examples are not specifically described, the experimental methods not noted in the examples are usually carried out under the conventional conditions, the materials, reagents used in the following examples are commercially available, unless otherwise specified.

[0049] The purchase source of the main reagents in the application is shown in Table 1:

[0050] Table 1

[0051]

[0052]

[0053] Example 1

[0054] Polypeptide MrIC sequence (SEQ ID NO. 1): PECCTHPACHVSNPELC.

[0055] 1.1 Animal modeling and administration

[0056] Animals: 6-week-old SPF C57 / Bl6j mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0057] By local imiquimod induction of psoriasis model, the exposed area of 2cm x 3cm size was formed on the central area of the back of C57BL / 6J mice, and imiquimod (IMQ) cream (about 62.5mg / one) was applied to the exposed skin of the back for 7 consecutive days. On the 8th day, the surface of the skin lesions was observed, and most of the mice had complete or incomplete scales on the surface of the skin lesions after modeling, the scales were flaky, the skin lesions were thickened, and the elevation was obvious.

[0058] Group setting: divided into 6 groups, 4 mice in each group. Including 2 control groups (blank group, MrIC 5mg / kg) and 4 experimental groups (IMQ, IMQ+MrIC concentration of 1, 3, 5mg / kg). Specifically, named: blank group, MrIC 5mg / kg, IMQ, IMQ+MrIC 1mg / kg, IMQ+MrIC 3mg / kg, IMQ+MrIC 5mg / kg.

[0059] Administration: the experimental group mice were modeled at the same time, and the polypeptide solution was injected intraperitoneally at the specified concentration (1, 3, 5mg / kg) every day, and the administration was continued for 7 days; the control group mice were applied with the same dose of vaseline cream. On the 8th day of modeling, the mice were sampled, and the overall lesion condition of the psoriasis mouse skin was evaluated by PASI score. The severity of skin inflammation (PASI score) was evaluated daily, including: measuring the skin thickness, scabbing, and erythema.

[0060] Score with 5-point scale (0-4) respectively:

[0061] Skin thickness:

[0062] 0: Smooth skin without wrinkles

[0063] 1: Slight wrinkles on the skin at the edge of the drug application area

[0064] 2: Slight wrinkles on the skin of the entire drug application area

[0065] 3: Further deepening of wrinkles in the drug application area

[0066] 4: On the basis of a score of 3, the mouse has conditions such as weight loss or poor condition

[0067] Crusting:

[0068] 0: Smooth skin without scales

[0069] 1: Slight scales on the skin of the drug application area

[0070] 2: The skin of the entire drug application area is covered with scales

[0071] 3: Further deepening of scales in the drug application area

[0072] 4: On the basis of a score of 3, the mouse has conditions such as weight loss or poor condition

[0073] Erythema:

[0074] 0: Smooth skin.

[0075] 1: Slight redness on the skin of the drug application area

[0076] 2: The skin of the entire drug application area is red

[0077] 3: Further deepening of redness in the drug application area

[0078] 4: On the basis of a score of 3, the mouse has conditions such as weight loss or poor condition

[0079] Total score: The sum of the scores of skin thickness, crusting and erythema.

[0080] 1.2 Detection index

[0081] On the 8th day of modeling, the mice were anesthetized and sacrificed after the back skin was scored by PASI. The skin lesions of the mice were taken, and the pathological changes of the lesions were observed by histopathology and immunofluorescence, and the expression of related inflammatory factors such as IL-1β, IL-6, TNF-α in the skin tissue of the lesions was detected by qPCR. The primer sequences of the inflammatory factors are shown in Table 2:

[0082] Table 2

[0083]

[0084] Histopathology and immunofluorescence The steps for observing the pathological changes in the lesions were as follows:

[0085] Histopathology experiment: The skin samples were fixed in a 4% paraformaldehyde solution, then embedded in paraffin and cut into 4 pm thick sections for hematoxylin-eosin (HE) staining. The obtained images were taken by an Olympus BX61 microscope at a magnification of 10 times. To evaluate the average epidermal thickness, ImageJ software was used to analyze three randomly selected regions for each mouse.

[0086] Immunofluorescence experiment:

[0087] First step: slide preparation

[0088] (1) Bake the slide at 60°C for 1-2 hours;

[0089] (2) Xylene I, II, III, each for 10 minutes;

[0090] (3) Gradient alcohol: 100% 2 x 5 minutes→95% 1 x 5 minutes→70% 1 x 5 minutes;

[0091] (4) Wash with water for 2 times, 5 minutes / time.

[0092] Second step: microwave treatment

[0093] (1) Microwave heating to boil the antigen repair solution, put the slide into the boiling water (about 3 minutes), then switch to medium / low heat for 15-20 minutes (pay attention to replenish the solution in the middle to prevent the slide from drying);

[0094] (2) Take out and cool naturally at room temperature;

[0095] (3) Distilled water washing for 3 minutes;

[0096] (4) 3% hydrogen peroxide treatment of the slide for 10-15 minutes;

[0097] (5) 1 x TBST (or PBS) washing for 2 times, 3 minutes / time.

[0098] Third step: blocking

[0099] (1) Wipe off the moisture around the tissue, and draw a water barrier circle with an immunohistochemical pen (note: to prevent the sample from drying);

[0100] (2) Drop blocking solution 1% bovine serum albumin (BSA) (about 100-200 μL per piece, according to the size of the tissue, as long as the tissue is completely covered), placed in a wet box at room temperature for 30 minutes.

[0101] Fourth step: primary antibody incubation

[0102] (1) Remove the blocking solution;

[0103] (2) Drop primary antibody working solution (about 100-200 μL per piece, according to the size of the tissue, as long as the tissue is completely covered), incubate at 4°C overnight;

[0104] (3) 1x TBST (or PBS) shake wash 3 times, 3 minutes each time.

[0105] Fifth step: HRP-labeled secondary antibody incubation

[0106] (1) Drop secondary antibody working solution, incubate at room temperature for 1 hour;

[0107] (2) 1x TBST (or PBS) wash 2 times, 3 minutes each time.

[0108] Sixth step: signal amplification

[0109] (1) Drop fluorescent dye 480 solution (100-150 μL) on the slide, incubate at room temperature for 10 minutes;

[0110] (2) 1x TBST (or PBS) shake wash 2 times, 3 minutes each time.

[0111] Seventh step: microwave treatment (remove the primary and secondary antibodies of the previous round)

[0112] (1) Microwave heating to boil antigen repair solution, put the slide in boiling water, then transfer to medium / low heat for 15 minutes (pay attention to supplement the liquid in the middle to prevent the slide from drying);

[0113] (2) Take out and cool naturally at room temperature;

[0114] (3) Distilled water wash for 3 minutes;

[0115] (4) 1x TBST (or PBS) wash 2 times, 3 minutes each time;

[0116] (5) Start the experiment from step three to step seven again, and replace the dye in the sixth step with fluorescent dye 570.

[0117] Eighth step: DAPI restain and mounting

[0118] (1) Drop DAPI working solution (about 100-150 μL) on the slide, immerse the tissue area as appropriate;

[0119] (2) Incubate in the wet chamber for 3-5 minutes at room temperature;

[0120] (3) Wash with 1x TBST (or PBS) for 5 minutes;

[0121] (4) Wash with distilled water for 3 minutes;

[0122] (5) Add fluorescent anti-quenching mounting medium to mount the coverslips, and use nail polish to fix the coverslips.

[0123] Step 9: Observe under a fluorescence microscope and collect images. Fluorescence images are acquired using an LSM980 microscope, and subsequent analysis is performed using ImageJ software.

[0124] Comparative Example

[0125] The comparative example was set up according to Example 1. The settings of the comparative example are shown in Table 3:

[0126] Table 3

[0127]

[0128] The concentration of ArIB[V11L, V16D] (hereinafter referred to as ArIB) used was 1 mg / kg, and the polypeptide sequence was DECCSNPACRLNNPHDCRRR (SEQ ID NO. 12). The present application also injected 5 mg / kg of MrIC intraperitoneally on the basis of Comparative Example 1, which is recorded as IMQ + ArIB 1 mg / kg + MrIC 5 mg / kg.

[0129] 1. Results and analysis of PASI score

[0130] The results of the disease severity score of the skin lesions on the back of the mice showed that MrIC could significantly reduce the PASI score value Figure 1 ). ArIB significantly increased the PASI score value, and the addition of MrIC significantly improved the disease aggravation phenomenon caused by ArIB Figure 7 ).

[0131] 2. Results and analysis of spleen / body ratio

[0132] The spleen / body ratio reflects the level of inflammation in the body. When MrIC was administered at a concentration of 3 mg / kg and 5 mg / kg, the spleen / body ratio was significantly lower than that of the IMQ group Figure 2 ).

[0133] ArIB significantly increased the spleen / body ratio of the mice, and the addition of MrIC significantly reduced the increase in the spleen / body ratio caused by ArIB Figure 8 ).

[0134] 3. Results and analysis of histopathology

[0135] HE staining results of mouse dorsal skin sections and epidermal thickness assessment based on the staining results showed that MrIC significantly reduced epidermal thickness at concentrations of 1, 3, and 5 mg / kg. Figure 3 ).

[0136] ArIB significantly increases epidermal thickness, while the addition of MrIC significantly improves the epidermal thickening phenotype caused by ArIB. Figure 9 ).

[0137] 4. Results and analysis of expression of psoriasis-related inflammatory factors

[0138] qPCR was used to detect the expression levels of inflammatory factors in mouse skin lesions. MrIC at a concentration of 5 mg / kg effectively reduced the levels of inflammatory factors in the skin lesions. Figure 4 ).

[0139] ArIB significantly increases the expression level of inflammatory factors, while the addition of MrIC significantly reduces the level of inflammatory factors in the lesion tissue. Figure 10 ).

[0140] 5. Immunofluorescence observation results and analysis

[0141] K17 is a marker of keratinocytes (staining red with immunofluorescence), and Ki67 is a marker of cell proliferation (staining green with immunofluorescence); both molecules are highly correlated with epidermal hyperplasia. Compared to the IMQ group, the red and green fluorescence intensities decreased after MrIC administration, indicating that MrIC can reduce keratinocyte proliferation. Figure 5 ).

[0142] CD31 is a marker of angiogenesis, and F4 / 80 is a marker of macrophages, both highly correlated with the severity of psoriasis. Compared to the IMQ group, the intensity of both red and green fluorescence decreased after MrIC administration, indicating that MrIC can reduce the recruitment of inflammatory cells to the lesion site to release inflammatory factors. Figure 6 ).

Claims

1. The use of a polypeptide in the preparation of a medicament for treating psoriasis vulgaris, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The dosage of the drug is 1-5 mg / kg.

3. The application according to claim 2, characterized in that, The polypeptide is the sole or main active ingredient of the drug.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, or stabilizers.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, calcium phosphate, alginate, gelatin, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, and propylparaben.

7. The application according to any one of claims 1-6, characterized in that, The drug is in liquid form.

8. The application according to claim 7, characterized in that, The administration method of the drug is selected from intravenous injection, local injection, intradermal injection, or subcutaneous injection.

9. The application according to claim 8, characterized in that, The drug is used in combination with other drugs for the prevention and / or treatment of psoriasis.

10. The application according to claim 9, characterized in that, Other medications used to prevent and / or treat psoriasis include glucocorticoids, vitamin D3 derivatives, retinoids, calcineurin inhibitors, keratolytic agents and keratolytic agents, immunosuppressants, or antibiotics.

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