Application of polypeptide in treatment of psoriasis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of polypeptide in treatment of psoriasis. The amino acid sequence of the polypeptide provided by the invention is SEQ ID NO.1, so that the spleen-body ratio of a psoriasis mouse can be effectively reduced, and the inflammation level in the body can be reduced; the epidermal thickness of psoriasis mice and inflammatory factors in skin lesion tissues can be remarkably reduced, keratinocyte proliferation is reduced, and inflammatory factors released by inflammatory cells recruited to skin lesion positions are reduced. The polypeptide provided by the invention shows certain advantages in the aspects of relieving inflammatory response, promoting tissue repair and the like when being used for treating psoriasis. The invention provides a new method and means for treating psoriasis, and promotes the progress and development of medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of a polypeptide in treating psoriasis. Background Art

[0002] Psoriasis is a common multi-gene hereditary skin disease, often manifested as localized or widespread scaly erythema or plaques. The cause of psoriasis is still unclear, but it has been identified as a combination of genetic and environmental factors. The disease is related to meridians, food, climate season, family history and other factors. There are many types of psoriasis, the most common is vulgaris, in addition to arthritis, pustular and erythrodermic types.

[0003] The symptoms of psoriasis are diverse, mainly including red patches on the skin, usually covered with silvery white scales, itching, burning or pain, dry, cracked skin, and even bleeding. Some patients may also experience joint pain, swelling, and abnormal nails. Psoriasis is non-infectious and is usually transmitted through family inheritance or environmental stimulation.

[0004] The main purpose of treating psoriasis is to control the disease and relieve the patient's symptoms. Current treatment methods include drug therapy, phototherapy and traditional Chinese medicine. Peptide treatment of psoriasis has shown its unique advantages to a certain extent. Peptide drugs, such as placental peptides and spleen peptides, have significant immunomodulatory functions. They can regulate the body's immune function and inhibit the activation of T lymphocytes, thereby reducing the excessive immune response of patients with psoriasis. This immunomodulatory effect helps to improve the pathological process of psoriasis and reduce skin damage and inflammatory response. Peptide drugs also have anti-inflammatory effects and can reduce skin inflammatory responses in patients with psoriasis. By inhibiting the release of inflammatory factors and reducing the infiltration of inflammatory cells, peptide drugs help reduce symptoms such as redness, itching and pain of the skin and improve the quality of life of patients. Peptide 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 for improving skin damage and reducing recurrence of psoriasis.

[0005] Although peptide drugs may also cause some side effects during treatment, overall, they are relatively safe. Under the guidance of professional doctors, patients can adjust the dosage and administration method according to their own conditions to reduce the occurrence of adverse reactions. At the same time, peptide 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, peptide treatment of psoriasis is not without limitations. For example, its efficacy may vary from person to person, mainly due to differences in each person's physiological state, genetic factors, disease stage, sensitivity to drugs, and metabolic capacity. Therefore, in order to more effectively utilize peptides for treatment and meet the needs of different patients, more peptide drugs need to be developed. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a polypeptide that can effectively treat psoriasis, providing patients with more treatment options and meeting their personalized treatment needs.

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

[0009] Specifically, the dosage of the drug 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 within the range.

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

[0011] In particular, the polypeptide may be the sole or main active ingredient of the drug.

[0012] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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

[0015] Specifically, the dosage form of the drug includes, but is not limited to, tablets, liquids, capsules, powders, suppositories, granules, sprays or ointments.

[0016] Preferably, the dosage form of the drug may be a liquid.

[0017] Specifically, the drug is administered by a method selected from oral, intravenous, topical, intradermal or subcutaneous injection.

[0018] Preferably, the drug can be administered by 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] More specifically, the other drugs for preventing and / or treating psoriasis are glucocorticoids, vitamin D3 derivatives, retinoic acid drugs, calcineurin inhibitors, keratinogens and keratolytics, immunosuppressants or antibiotics.

[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: calcipotriol ointment and tacalcitol ointment;

[0023] The retinoic acid drugs include but are not limited to: tretinoin cream, tazarotene cream and acitretin capsules.

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

[0025] The keratolytic 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 retinoic acid drugs include but are not limited to: Acitretin capsules.

[0028] The antibiotics include but are not limited to: amoxicillin and clindamycin.

[0029] Specifically, the combination can be used simultaneously or sequentially.

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

[0031] Specifically, the psoriasis is psoriasis vulgaris.

[0032] The technical effects achieved by the present invention are:

[0033] (1) The polypeptide MrIC provided by the present invention can effectively reduce the spleen-to-body ratio of psoriasis mice, that is, reduce inflammation in the body.

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

[0035] (3) The polypeptide MrIC provided by the present invention can effectively reduce the level of inflammatory factors in the skin lesions of psoriasis mice.

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

[0037] (5) The polypeptide MrIC provided by the present invention can reduce the recruitment of inflammatory cells to skin lesions and the release of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The disease severity of the lesional skin of mice in the control group and the experimental group was scored (PASI score).

[0039] Figure 2 The spleen sizes of mice in the control group and the experimental group are shown in Table 2.

[0040] Figure 3 Shown are the results of HE staining of skin sections from the back lesions of mice in the control group and the experimental group, as well as the epidermal thickness evaluated based on the staining results.

[0041] Figure 4 qPCR was used to detect the expression levels of inflammatory factors in the skin lesions of mice in the control group and the experimental group.

[0042] Figure 5 Immunofluorescence detection of the expression levels of keratinocyte proliferation markers in the skin lesion tissues of mice in the control group and the experimental group, where e represents the epidermis and d represents the dermis.

[0043] Figure 6 Immunofluorescence was used to detect the expression levels of angiogenesis markers and macrophage markers in the skin lesion tissues of mice in the control group and the experimental group, where e represents the epidermis and d represents the dermis.

[0044] Figure 7 This is the disease severity scoring result of the skin lesions on the back of control mice.

[0045] Figure 8 The spleen size is the ratio of the control mice.

[0046] Fig. 9 Shown are the results of HE staining of skin sections from the back lesions of control mice, as well as the epidermal thickness assessed based on the staining results.

[0047] Fig.10 For comparison, qPCR was used to detect the expression levels of inflammatory factors in the skin lesions of mice. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0049] The purchase sources of the main reagents in the present invention are 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 drug administration

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

[0057] The psoriasis model was induced by topical imiquimod. The central area of ​​the back of C57BL / 6J mice was depilated to form an exposed area of ​​2cm×3cm. Imiquimod (IMQ) cream (about 62.5mg / mouse) was applied to the exposed skin on the back for 7 consecutive days. The surface of the lesions was observed on the 8th day. After modeling, most of the lesions of the mice were completely or incompletely covered with scales, which were flaky, and the lesions were thick and prominent.

[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 concentrations of 1, 3, and 5mg / kg respectively). 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 injected with a peptide solution at a specified concentration (1, 3, 5 mg / kg) intraperitoneally every day for 7 consecutive days while the control group mice were smeared with the same dose of vaseline cream. Samples were collected from the mice on the 8th day after modeling, and the overall lesions of the skin of psoriasis mice were evaluated by PASI score. The severity of skin inflammation (PASI score) was evaluated daily, including: measuring skin thickness, scabs, and erythema.

[0060] The scores are given on a 5-point scale (0-4):

[0061] Skin thickness:

[0062] 0: Smooth skin without wrinkles

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

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

[0065] 3: The wrinkles in the application area are further deepened

[0066] 4: Based on the score of 3, the mice showed weight loss or poor condition.

[0067] Scabbing:

[0068] 0: Smooth skin without scaling

[0069] 1: Slight scaling of the skin in the application area

[0070] 2: The skin in the application area is completely covered with scales

[0071] 3: The degree of scaling in the application area deepens further

[0072] 4: Based on the score of 3, the mice showed weight loss or poor condition.

[0073] erythema:

[0074] 0: Smooth skin.

[0075] 1: The skin in the application area appears slightly red

[0076] 2: The skin in the application area turns red

[0077] 3: The red color of the applied area deepens further

[0078] 4: Based on the score of 3, the mice showed weight loss or poor condition.

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

[0080] 1.2 Detection indicators

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

[0082] Table 2

[0083]

[0084] The steps for histopathology and immunofluorescence observation of pathological changes in skin lesions are as follows:

[0085] Histopathology: Skin samples were fixed in 4% paraformaldehyde solution, then embedded in paraffin and cut into 4 μm thick slices for hematoxylin-eosin (HE) staining. The images obtained were taken at 10x magnification using an Olympus BX61 microscope. To assess the average epidermal thickness, three randomly selected areas of each mouse were analyzed using ImageJ software.

[0086] Immunofluorescence experiments:

[0087] Step 1: Slide Preparation

[0088] (1) Bake at 60℃ for 1-2 hours;

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

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

[0091] (4) Wash twice with water, 5 minutes each time.

[0092] Step 2: Microwave Treatment

[0093] (1) Microwave the antigen retrieval solution to boil, put the slide in and boil (about 3 minutes), then turn to medium / low heat and heat for 15-20 minutes (pay attention to refilling the solution during the process to prevent the slide from drying out);

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

[0095] (3) Wash with distilled water for 3 minutes;

[0096] (4) Treat the slides with 3% hydrogen peroxide for 10-15 minutes;

[0097] (5) Wash twice with 1× TBST (or PBS), 3 min each time.

[0098] Step 3: Closure

[0099] (1) Wipe away the moisture around the tissue and draw a water-blocking circle with an immunohistochemistry pen (note: to prevent the sample from drying out);

[0100] (2) Add a blocking solution of 1% bovine serum albumin (BSA) (about 100-200 μL / sheet, depending on the size of the tissue, to completely cover the tissue) and place in a humidified box for blocking at room temperature for 30 minutes.

[0101] Step 4: Primary Antibody Incubation

[0102] (1) Remove the blocking solution;

[0103] (2) Add primary antibody working solution (about 100-200 μL / sheet, depending on the size of the tissue, preferably to completely cover the tissue) and incubate overnight at 4°C;

[0104] (3) Wash three times with 1× TBST (or PBS) with shaking, 3 min each time.

[0105] Step 5: Incubation with HRP-labeled secondary antibody

[0106] (1) Add the secondary antibody working solution and incubate at room temperature for 1 hour;

[0107] (2) Wash twice with 1× TBST (or PBS), 3 min each time.

[0108] Step 6: Signal Amplification

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

[0110] (2) Wash twice with 1× TBST (or PBS) with shaking for 3 minutes each time.

[0111] Step 7: Microwave treatment (to remove the primary and secondary antibodies from the previous round)

[0112] (1) Microwave the antigen retrieval solution to boil, put the slide in and boil, then turn to medium / low heat and heat for 15 minutes (pay attention to replenish the solution during the process to prevent the slide from drying out);

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

[0114] (3) Wash with distilled water for 3 minutes;

[0115] (4) Wash twice with 1× TBST (or PBS), 3 min each time;

[0116] (5) The experiment is restarted from step 3 to step 7, wherein the dye in step 6 is replaced by fluorescent dye 570.

[0117] Step 8: DAPI counterstaining and sealing

[0118] (1) Add DAPI working solution (about 100-150 μL) to the slide to immerse the tissue area;

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

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

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

[0122] (5) Add fluorescent antifade sealing agent to seal the slide and fix the coverslip with nail polish.

[0123] Step 9: Observe and collect images under a fluorescence microscope. Fluorescence images were obtained using an LSM980 microscope and subsequently analyzed using ImageJ software.

[0124] Comparative Example

[0125] A comparative example is set with reference to Example 1. The comparative example is set as shown in Table 3:

[0126] Table 3

[0127]

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

[0129] 1. PASI score results and analysis

[0130] The results of disease severity scores of the back lesions of mice showed that MrIC could significantly reduce the PASI score value ( Figure 1 ). ArIB significantly increases the PASI score, and the addition of MrIC can significantly improve the disease aggravation caused by ArIB ( Figure 7 ).

[0131] 2. Spleen-body ratio results and analysis

[0132] The spleen-to-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-to-body ratio was significantly reduced compared with the IMQ group ( Figure 2 ).

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

[0134] 3. Histopathological results and analysis

[0135] The results of HE staining of mouse back skin sections and the evaluation of epidermal thickness based on the staining results showed that MrIC could significantly reduce epidermal thickness when administered at concentrations of 1, 3, and 5 mg / kg ( Figure 3 ).

[0136] ArIB significantly increased epidermal thickness, while the addition of MrIC could significantly improve the epidermal thickening phenotype caused by ArIB ( Fig. 9 ).

[0137] 4. Expression results and analysis of psoriasis-related inflammatory factors

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

[0139] ArIB significantly increased the expression level of inflammatory factors, while the addition of MrIC significantly reduced the level of inflammatory factors in skin lesions ( Fig.10 ).

[0140] 5. Immunofluorescence observation results and analysis

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

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

Claims

1. Use of a polypeptide in the preparation of a drug for preventing and / or treating psoriasis, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

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

3. The use according to claim 1, characterized in that: The polypeptide further comprises one or more of a homing peptide, a cytotoxic payload, or a linker.

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

5. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

8. The use according to any one of claims 1 to 7, characterized in that: The dosage form of the drug is tablet, liquid, capsule, powder, suppository, granule, spray or ointment.

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

10. The use according to claim 9, characterized in that: The drug is used in combination with other drugs for preventing and / or treating psoriasis.

11. The use according to claim 10, characterized in that: The other drugs for preventing and / or treating psoriasis include glucocorticoids, vitamin D3 derivatives, retinoic acid drugs, calcineurin inhibitors, keratinogens and keratolytics, immunosuppressants or antibiotics.

12. The use according to claim 10 or 11, characterized in that: The combined use is simultaneous use or sequential use.

13. The use according to claim 1, characterized in that: The psoriasis is psoriasis vulgaris, psoriasis arthritis, pustular psoriasis or erythrodermic psoriasis.

14. The use according to claim 13, characterized in that The psoriasis is psoriasis vulgaris.

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