Chemokine receptor 8 antagonistic polypeptide compounds, compositions, kits, and uses

Abstract

The present application provides a CCR8 antagonistic polypeptide compound, a pharmaceutical composition thereof, a kit and their uses. The CCR8 antagonistic polypeptide compound can be used for preventing or treating cancer or tumor, inflammatory disease, viral infection, IgG4-related disease, skin immune disease or its related symptoms.

Description

Chemokine receptor 8 antagonist polypeptide compound, composition, kit and use Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to a chemokine receptor 8 (CCR8) antagonist polypeptide compound, a pharmaceutical composition and a kit containing the same, and their uses, especially for treating and / or preventing cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, immune skin diseases or related symptoms thereof (e.g., immune skin diseases related to the CCL1-CCR8 signaling pathway or related symptoms thereof). Background Art

[0002] CCR8 (Chemokine receptor 8) is a seven-transmembrane G protein-coupled receptor that belongs to the CC subfamily of chemokine receptors. Although the gene encoding CCL1 is located within this chemokine gene cluster, it is the only CC chemokine in the cluster that specifically binds to CCR8; in other words, it has a single ligand / receptor relationship. In vitro studies have shown that IgE binding and cross-linking on mast cells leads to significant upregulation of the inflammatory chemokine CCL1, whose specific receptor, CCR8, is expressed on a small subset of circulating T cells and abundantly on interstitial dendritic cells, in vitro-generated Langerhans cells, and their monocyte precursors. Although dendritic cells retain their CCR8 status during maturation, transient activation of circulating T cells recruits CCR8 from cytoplasmic reserves to the cell surface.

[0003] Immune skin diseases, also known as immune skin diseases, are a type of skin disease that causes skin damage due to immune imbalance, including, for example, inflammatory skin diseases (a group of diseases caused by immune system disorders leading to skin tissue destruction, including eczema, atopic dermatitis, psoriasis, chronic urticaria, etc.), autoimmune skin diseases (due to autoimmune disorders, the immune system no longer protects the body from bacteria and viruses, but instead attacks its own tissues, thereby causing various skin diseases, such as connective tissue diseases (lupus erythematosus, scleroderma, dermatomyositis, etc.), skin vasculitis (allergic vasculitis, white atrophy, allergic purpura, urticarial vasculitis), autoimmune bullous diseases (pemphigoid, bullous pemphigoid, dermatitis herpetiformis, epidermolysis bullosa, etc.)).

[0004] Atopic dermatitis (AD) is a chronic, relapsing skin disease with a steadily increasing prevalence in children, reaching 10%-20%. Skin-infiltrating T cells, dendritic cells (DCs), and mast cells play a key role in its pathogenesis. Expression of the CC chemokine CCL1 (I-309) is significantly and selectively upregulated in atopic dermatitis compared with psoriasis, cutaneous lupus erythematosus, or normal skin. Serum levels of CCL1 are significantly higher in patients with atopic dermatitis than in healthy individuals. Studies have shown that CCL1-CCR8 connects adaptive and innate immune functions that play a role in the initiation and amplification of atopic dermatitis.

[0005] Atopic dermatitis has a complex pathophysiology and is the basis of multiple clinical phenotypes. AD remains difficult to treat due to limited response to existing therapies. Based on the mechanism by which the CCL1-CCR8 axis promotes the progression of atopic dermatitis, this application develops a peptide that specifically binds to CCR8 to block CCL1-CCR8 signaling, thereby achieving the effect of treating / preventing atopic dermatitis. The peptide is highly safe for humans or animals and is more suitable for atopic dermatitis, a chronic disease that requires long-term medication. Summary of the Invention

[0006] The present application provides a polypeptide compound that can specifically bind to CCR8 and block the CCL1 (ligand)-CCR8 (receptor) signaling pathway, thereby being used as an active ingredient in a therapeutic and / or preventive agent for cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, and skin immune diseases.

[0007] In one aspect, the present application provides a CCR8 antagonist polypeptide compound, comprising an amino acid sequence selected from the following or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto:

[0008] (1) H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-CONH2 (SEQ ID NO: 1),

[0009] wherein Xaa1 is Val, Ala, Leu, or Ile, Xaa2 is Leu or Val, Xaa3 is Leu, Val, or Ile, Xaa4 is Thr, Ser, Ala, or Ile, Xaa5 is Ser, Thr, or Ala, and Xaa6 is Ile, Val, Leu, AIB, Cys, Nle, Ala, Met, or Abu (e.g., Xaa6 is Val, Leu, AIB, Cys, Nle, Ala, Met, or Abu; or Xaa6 is Ile, Val, AIB, Cys, Nle, Ala, Met, or Abu);

[0010] (2)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Ile-Pro-Asn-Ile-Xaa7-Xaa8-Xaa9-Xaa 10 -Xaa 11 -CONH2 (SEQ ID NO: 2),

[0011] Among them, Xaa7 is Leu or Val, Xaa8 is Leu, Val or Ile, Xaa9 is Thr, Ala or Ser, Xaa 10 Thr, Ala or Ser, Xaa 11 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu;

[0012] (3)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 12 -Xaa 13 -Xaa 14 -Xaa 15 -Xaa 16 -CONH2 (SEQ ID NO: 3),

[0013] Among them, Xaa 12 Leu or Val, Xaa 13 Leu, Val or Ile, Xaa 14 Thr, Ala or Ser, Xaa 15 Thr, Ala or Ser, Xaa 16 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu;

[0014] (4)H2N-AIB-Arg-Pro-Xaa 17-Xaa 18 -Xaa 19 -Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Xaa 20 -Xaa 21 -Abu-CONH2 (SEQ ID NO: 4),

[0015] Among them, Xaa 17 -Xaa 18 -Xaa 19 Asp-Lys-Ala or Glu-Orn-Ser, Xaa 20 Thr, Ala or Ser, Xaa 21 is Thr or Ser; or

[0016] (5)H2N-AIB-Arg-Pro-Glu-Orn-Ser-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 22 -Xaa23-Xaa 24 -Xaa 25 -Xaa 26 -CONH2 (SEQ ID NO: 5),

[0017] Among them, Xaa 22 Leu or Val, Xaa 23 Leu, Val or Ile, Xaa 24 Thr, Ala or Ser, Xaa 25 Thr, Ala or Ser, Xaa 26 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu.

[0018] In one aspect, the present application provides a polynucleotide encoding the CCR8 antagonist polypeptide compound.

[0019] In one aspect, the present application provides a pharmaceutical composition comprising the above-mentioned polypeptide compound and pharmaceutically acceptable excipients.

[0020] In one aspect, the present application provides a kit comprising the above-mentioned polypeptide compound, or a pharmaceutical composition thereof.

[0021] In one aspect, the present application provides the use of the aforementioned polypeptide compound, pharmaceutical composition, or kit in the preparation of a medicament for preventing or treating cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, skin immune diseases, or related symptoms thereof. Alternatively, the present application provides the use of the aforementioned polypeptide compound, pharmaceutical composition, or kit in the preparation of a preparation for blocking the CCL1-CCR8 signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 shows the mass spectrum of the purified TCR10 sample, wherein the HPLC purity of the polypeptide compound can reach 95.62%.

[0023] FIG. 2 is a diagram showing the expression of CCR8 on the surface of interstitial dendritic cells detected by flow cytometry in Experimental Example 2. FIG.

[0024] FIG3 is a diagram showing the Transwell chemotaxis assay of Experimental Example 2 to detect the migration ability of iDCs in the presence or absence of TCR10.

[0025] 4 is a graph showing the statistical results of the number of migrating cells in Experimental Example 2 (the graph shows the results of the blank control group, the positive control group, the test compound group, and the antibody control group from left to right).

[0026] 5 is a graph showing changes in mouse body weight over time in the drug efficacy screening test of Test Example 3. FIG.

[0027] 6 is a graph showing the degree of ear swelling of mice in the drug efficacy screening test of Test Example 3. FIG.

[0028] 7 is a graph showing the skin lesion scores on the back of mice in the drug efficacy screening test of Test Example 3. FIG.

[0029] FIG8 shows the liver, kidney, and spleen indexes of mice in the drug efficacy screening test of Test Example 3 (from left to right).

[0030] FIG9 shows the analysis of pathological sections of mouse back skin in the drug efficacy screening test of Experimental Example 3.

[0031] FIG. 10 shows the analysis of mouse ear pathological sections in the drug efficacy screening test of Experimental Example 3. FIG.

[0032] Figure 11 shows the skin conditions of mice in the dose-effect test of Experimental Example 4: (A) control group; (B) model group; (C) 0.1% mometasone furoate group; (D) 20 mg crisaborole group; (E) TCR09 (1 mg) group; (F) TCR10 (1 mg) group; (G) TCR09 (3 mg) group; (H) TCR10 (3 mg) group.

[0033] FIG. 12 shows changes in mouse body weight over induction time in the gel preparation treatment experiment of Test Example 5.

[0034] FIG. 13 shows the trend of body weight change (%) of mice in the gel preparation treatment test of Test Example 5.

[0035] FIG. 14 shows the skin lesion scores of mice in the gel preparation treatment test of Test Example 5 as a function of induction time.

[0036] FIG. 15 shows the skin lesion scores of mice at the time of the final administration (day 14) in the gel preparation treatment study of Test Example 5 (**: p<0.01; ***: p<0.001).

[0037] FIG. 16 shows the ear swelling degree of mice in the gel preparation treatment test of Test Example 5 according to the induction time.

[0038] FIG. 17 shows the ear swelling degree of mice at the time of the last administration (day 14) in the gel preparation treatment study of Test Example 5 (***: p<0.001).

[0039] FIG. 18 shows the cytokine IL-4 content per mg of skin (pg / mg skin) of mice in the gel preparation treatment experiment of Test Example 5 (*: p<0.05; **: p<0.01; ***: p<0.001).

[0040] FIG. 19 shows the HE scores of mice in the gel preparation treatment test of Test Example 5 (*: p<0.05; ***: p<0.001).

[0041] FIG. 20 shows the epidermal thickness of mice in the gel preparation treatment test of Test Example 5 (*: p<0.05; ***: p<0.001).

[0042] FIG. 21 shows the mast cell counts of mice in the gel preparation treatment test of Test Example 5 (*: p<0.05; ***: p<0.001).

[0043] FIG22 shows the mouse skin pathological sections of the normal control group, model group, and gel matrix group in the gel preparation treatment experiment of Experimental Example 5 (HE staining on the left and toluidine blue staining on the right).

[0044] 23 shows mouse skin pathological sections of the mometasone furoate group, crisaborole group, and 2% TCR10 gel group in the gel preparation treatment experiment of Test Example 5 (HE staining on the left, toluidine blue staining on the right).

[0045] FIG24 shows pathological sections of mouse skin in the 5% TCR10 gel group in the gel preparation treatment experiment of Test Example 5 (HE staining on the left, toluidine blue staining on the right).

[0046] FIG25 shows the skin conditions of mice in the gel preparation treatment experiment of Experimental Example 5, from left to right: normal control group, model group, gel matrix control group, mometasone furoate gel group, crisaborole ointment group, 2% TCR10 gel group, and 5% TCR10 gel group. DETAILED DESCRIPTION

[0047] Next, the present application will be described in more detail to facilitate understanding of the present invention by those skilled in the art. However, it should be noted that the scope of protection of the present application is not limited thereto. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0048] In this application, unless otherwise indicated, the term "polypeptide compound" refers to a compound formed by multiple amino acids linked by peptide bonds. The polypeptides of the present disclosure may contain natural amino acids, synthetic amino acids (e.g., 2-aminobutyric acid (Abu), 2-aminoisobutyric acid (AIB), norleucine (Nle)) or amino acid analogs, as well as chemical modifications (e.g., phospholipid modifications).

[0049] As used herein, and unless otherwise indicated, the term "treat," ...

[0050] As used herein, unless otherwise indicated, all numbers expressing amounts of ingredients, measurements, or reaction conditions are to be understood as being modified in all instances by the term "about" to account for possible measurement errors. For example, when used in conjunction with a percentage, the term "about" can mean a variation within a range of ±1%, such as ±0.5%, of the value of the object to which it is applied.

[0051] In this document, unless otherwise indicated, singular terms encompass plural referents and vice versa. Similarly, the term "or" is intended to include "and" and vice versa unless the context clearly indicates otherwise.

[0052] As used herein, unless otherwise indicated, the terms "comprise, comprise, and comprising" or their equivalents (e.g., contain, containing, include, including) are open-ended expressions and should be understood to mean "including but not limited to," meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0053] The term "X" and "Xaa" herein are equivalent and refer to unspecified amino acids. The scope of the term is specified by the definition in the relevant expression. In order to distinguish multiple "X" in the same amino acid sequence, the multiple Xs that appear successively are numbered separately (i.e., written as X n ) and define the scope they cover respectively.

[0054] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0055] The term "subject" encompasses any vertebrate, e.g., mammals and non-mammals, such as humans, non-human primates (e.g., chimpanzees, rhesus monkeys, cynomolgus monkeys, etc.), sheep, dogs, cats, horses, cows, chickens, pigs, mice, etc., preferably humans.

[0056] CCR8 antagonistic polypeptide compounds

[0057] In the first aspect of the present disclosure, the present application provides a CCR8 antagonist polypeptide compound, comprising an amino acid sequence selected from the following: or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto:

[0058] (1) H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-CONH2 (SEQ ID NO: 1),

[0059] wherein Xaa1 is Val, Ala, Leu, or Ile, Xaa2 is Leu or Val, Xaa3 is Leu, Val, or Ile, Xaa4 is Thr, Ser, Ala, or Ile, Xaa5 is Ser, Thr, or Ala, and Xaa6 is Ile, Val, Leu, AIB, Cys, Nle, Ala, Met, or Abu;

[0060] (2)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Ile-Pro-Asn-Ile-Xaa7-Xaa8-Xaa9-Xaa 10 -Xaa 11 -CONH2 (SEQ ID NO: 2),

[0061] Among them, Xaa7 is Leu or Val, Xaa8 is Leu, Val or Ile, Xaa9 is Thr, Ala or Ser, Xaa 10 Thr, Ala or Ser, Xaa 11 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu;

[0062] (3)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 12 -Xaa 13 -Xaa 14 -Xaa 15 -Xaa 16 -CONH2 (SEQ ID NO: 3),

[0063] Among them, Xaa 12 Leu or Val, Xaa 13 Leu, Val or Ile, Xaa 14 Thr, Ala or Ser, Xaa 15 Thr, Ala or Ser, Xaa 16 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu;

[0064] (4)H2N-AIB-Arg-Pro-Xaa 17 -Xaa 18 -Xaa 19-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Xaa 20 -Xaa 21 -Abu-CONH2 (SEQ ID NO: 4),

[0065] Among them, Xaa 17 -Xaa 18 -Xaa 19 Asp-Lys-Ala or Glu-Orn-Ser, Xaa 20 Thr, Ala or Ser, Xaa 21 is Thr or Ser; or

[0066] (5)H2N-AIB-Arg-Pro-Glu-Orn-Ser-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 22 -Xaa23-Xaa 24 -Xaa 25 -Xaa 26 -CONH2 (SEQ ID NO: 5),

[0067] Among them, Xaa 22 Leu or Val, Xaa 23 Leu, Val or Ile, Xaa 24 Thr, Ala or Ser, Xaa 25 Thr, Ala or Ser, Xaa 26 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu.

[0068] In some embodiments, Xaa1-Xaa2-Xaa3 are Val-Leu-Leu. Further, Xaa4 is Thr, Ser, or Ala. Further, Xaa5 is Ser, Thr, or Ala. Still further, Xaa6 is Ile, Val, Leu, AIB, Cys, Nle, Ala, Met, or Abu.

[0069] In some preferred embodiments, Xaa1-Xaa2-Xaa3 is Val-Leu-Leu, Xaa4 is Thr or Ser, Xaa5 is Ser, and Xaa6 is Ile, Val, AIB, Cys, Nle, Ala, Met, or Abu. In some alternative preferred embodiments, Xaa1-Xaa2-Xaa3 is Val-Leu-Leu, Xaa4 is Thr or Ser, Xaa5 is Ser, Thr, or Ala, and Xaa6 is Ile.

[0070] In some embodiments, Xaa1-Xaa2-Xaa3-Xaa4 is Val-Leu-Leu-Thr, Xaa5 is Ser, Thr or Ala, and Xaa6 is Val, Leu, AIB, Cys, Nle, Ala, Met or Abu. In some embodiments, Xaa1-Xaa2-Xaa3-Xaa4 is Val-Leu-Leu-Ser, Xaa5 is Ser or Thr, and Xaa6 is Ile, Val, AIB, Cys, Nle, Ala, Met or Abu.

[0071] In some embodiments, the CCR8 antagonist polypeptide compound comprises the following amino acid sequence: H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Xaa 27 -Ser-Val-CONH2 (SEQ ID NO: 6), wherein Xaa 27 It is Thr or Ser.

[0072] In some embodiments, the CCR8 antagonist polypeptide compound comprises the following amino acid sequence: H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Ser-Ser-Xaa 28 -CONH2 (SEQ ID NO: 206), wherein Xaa 28 It is Ile or Val.

[0073] In some embodiments, the CCR8 antagonist polypeptide compound comprises an amino acid sequence selected from any one of SEQ ID NO: 7 to SEQ ID NO: 205. In some preferred embodiments, the amino acid sequence of the CCR8 antagonist polypeptide compound is shown in SEQ ID NO: 16 or SEQ ID NO: 15.

[0074] In some embodiments, Xaa7-Xaa8 are Leu-Leu. Further, Xaa9 is Thr or Ser, Xaa 10 Ser or Thr, Xaa 11 For Val.

[0075] In some embodiments, Xaa 12 -Xaa 13 is Leu-Leu. Further, Xaa 14 Thr or Ser, Xaa 15 Ser or Thr, Xaa 16 For Val.

[0076] In some embodiments, Xaa 17 -Xaa 18 -Xaa 19 Asp-Lys-Ala, Xaa 20 Thr or Ser, Xaa 21 It is Ser or Thr.

[0077] In some embodiments, Xaa 22 -Xaa 23 is Leu-Leu. Further, Xaa 24 Thr or Ser, Xaa 25 Ser or Thr, Xaa 26 For Val.

[0078] In the present context, each of the above-mentioned CCR8 antagonistic polypeptide compounds and variants thereof retains the ability to bind to CCR8 and block the CCL1-CCR8 signaling pathway.

[0079] In some embodiments, the present application provides a polynucleotide encoding the above-mentioned CCR8 antagonist polypeptide compound.

[0080] It is known to those skilled in the art that, due to codon degeneracy, each amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the polypeptide of the present disclosure can be synthesized using methods well known in the art (eg, de novo solid phase DNA synthesis, PCR amplification).

[0081] The polypeptides disclosed herein are preferably synthesized using solid phase synthesis.

[0082] Pharmaceutical compositions and kits

[0083] In a second aspect, the present application provides a pharmaceutical composition comprising the aforementioned polypeptide compound and a pharmaceutically acceptable excipient. Alternatively, the present application provides a kit comprising the aforementioned polypeptide compound or a pharmaceutical composition thereof.

[0084] In some embodiments, the pharmaceutically acceptable pharmaceutical excipients may be selected from, for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants, diluents, etc. For other available pharmaceutically acceptable pharmaceutical excipients, see, for example, Handbook of Pharmaceutical Excipients (4th edition), written by RC Luo et al., translated by Zheng Junmin, 2005, Chemical Industry Press.

[0085] In some embodiments, the pharmaceutical composition further comprises additional therapeutic agents, including but not limited to glucocorticoids, anti-inflammatory drugs, antipruritic drugs, antiviral drugs, antihistamines, immunosuppressants, temperature-lowering drugs, anticancer drugs, anti-fibrosis drugs, etc.

[0086] In some embodiments, the pharmaceutical composition further comprises another atopic dermatitis therapeutic agent, such as glucocorticoids (such as hydrocortisone, hydrocortisone butyrate, triamcinolone acetonide, mometasone, mometasone furoate, etc.), calcineurin inhibitors (such as tacrolimus, pimecrolimus), PDE4 inhibitors (such as crisaborole, roflumilast), JAK inhibitors (such as ruxolitinib, upadacitinib), antibiotics (such as mupirocin, fusidic acid, erythromycin, quinolone antibiotics (including norfloxacin, ofloxacin), , pefloxacin, enoxacin, ciprofloxacin, fleroxacin, gatifloxacin, moxifloxacin), tetracycline), antipruritic drugs (such as doxepin, flurbiprofen, diclofenac diethylamine, diclofenac, ibuprofen), wet compresses (such as aluminum acetate solution, potassium permanganate solution), calamine lotion or zinc cream, antihistamines (such as loratadine, desloratadine, mizolastine, ebastine), antiviral drugs (such as acyclovir, valacyclovir, penciclovir), immunosuppressants (such as cyclosporine, azathioprine, methotrexate), etc.

[0087] In some embodiments, the pharmaceutical composition of the present disclosure can be in the form of a powder, granules, a film, a cream, an ointment, a gel, a lotion, a foam, a patch, a liniment, an elixir, an aerosol, an emulsion, a solution, or a suspension.

[0088] In some embodiments, the kit may further include instructions for use. In some embodiments, the kit may further include reagents for diagnosing patients.

[0089] In some embodiments, the kit further comprises tools that can be used to administer the drug, such as a syringe, a catheter, a swab, a cotton swab, and the like.

[0090] use

[0091] In the third aspect of the present disclosure, the present application provides the use of the polypeptide compound described in the first aspect of the present disclosure or the pharmaceutical composition or kit described in the second aspect in the preparation of a drug for preventing or treating cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, skin immune diseases or related symptoms thereof.

[0092] In some embodiments, the present application relates to a method for preventing or treating cancer or tumor, inflammatory disease, viral infection, IgG4-related disease, skin immune disease or related symptoms thereof in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polypeptide compound described in the first aspect of the present disclosure or the pharmaceutical composition or kit described in the second aspect.

[0093] In some embodiments, the present application relates to a polypeptide compound described in the first aspect of the present disclosure or a pharmaceutical composition or kit described in the second aspect for preventing or treating cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, skin immune diseases or related symptoms thereof.

[0094] In some embodiments, the present application relates to use of the polypeptide compound described in the first aspect of the present disclosure or the pharmaceutical composition or kit described in the second aspect in preparing a preparation for blocking the CCL1-CCR8 signaling pathway.

[0095] In some embodiments, the present application relates to a method for blocking the CCL1-CCR8 signaling pathway in a subject in need thereof, comprising administering to the subject an effective amount of the polypeptide compound described in the first aspect of the present disclosure or the pharmaceutical composition or kit described in the second aspect.

[0096] In some embodiments, the present application relates to the polypeptide compound described in the first aspect of the present disclosure or the pharmaceutical composition or kit described in the second aspect for blocking the CCL1-CCR8 signaling pathway.

[0097] In some embodiments, the tumor is selected from a solid tumor, more preferably multiple myeloma, lymphoma or melanoma; the cancer is selected from esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, kidney cancer, bladder cancer, cutaneous fibrosarcoma, osteosarcoma, pancreatic cancer or prostate cancer.

[0098] In some embodiments, the inflammatory disease is selected from asthma and irritable bowel disease.

[0099] In some embodiments, the IgG4-related disease is selected from sialadenitis.

[0100] In some embodiments, the skin immune disease or related symptoms thereof is a skin immune disease or related symptoms thereof associated with the CCL1-CCR8 signaling pathway.

[0101] In some embodiments, the skin immune disease or its related symptoms are selected from eczema, atopic dermatitis, psoriasis, lupus erythematosus, scleroderma, dermatomyositis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, bullous epidermolysis or their related symptoms. In some preferred embodiments, the skin immune disease or its related symptoms are selected from atopic dermatitis or its related symptoms (such as dry skin, itchy skin, eczematous skin lesions (including erythema, papules, redness, blisters, skin erosion, epidermal thickening), xeroderma related symptoms, palmar dermatitis related symptoms, white skin scratch disease, Hertoghe sign, infraorbital wrinkles, orbital black halo (darkening around the eyes), low hairline). Preferably, the atopic dermatitis is infantile atopic dermatitis, childhood atopic dermatitis or youth and adult atopic dermatitis. Or preferably, the atopic dermatitis is mild, moderate or severe atopic dermatitis. Currently, there are many known methods for evaluating the severity of atopic dermatitis. For example, the following indicators can be used clinically to judge the severity of atopic dermatitis: mild atopic dermatitis is a rash area of ​​less than 5%; moderate atopic dermatitis is 5% to 10%, or the rash recurs; severe atopic dermatitis is skin lesions exceeding 10% of the body surface area, or the dermatitis is persistent, and the severe itching affects sleep.

[0102] In some embodiments, the pharmaceutical composition may be in the form of a sterile aqueous solution, microemulsion, liposome, or powder.

[0103] In some embodiments, the pharmaceutical composition may be in the form of a unit dosage to facilitate administration to a patient according to a desired dose.

[0104] As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount that can produce a desired response (including preventing or slowing the occurrence of a disease, reducing the severity of a disease, improving the patient's condition, inducing an immune response, delaying or at least slowing the progression of a disease, or curing) in a subject or patient in need thereof, and can be determined by those skilled in the art based on the age, sex, weight, physical condition, family history, risk of disease, sensitivity to drugs, etc. of the subject or patient in combination with the contents of this disclosure. An effective dosage can be administered to a subject in a single dosage, or divided into multiple dosages of the same or different doses. The dosage range of the pharmaceutical composition described herein can be determined empirically by a clinician based on the mode of administration (including administration time, administration interval, administration route), the patient's age, weight, sex or pathological condition, diet, excretion rate, and sensitivity to drugs, etc.

[0105] The above-mentioned polypeptide, pharmaceutical composition or kit can be administered (e.g., by injection or infusion) to a subject in need thereof by conventional routes of administration in the art (e.g., parenteral, intravenous, intramuscular, intraarterial, intraperitoneal, subcutaneous, transdermal, topical administration). The above-mentioned polypeptide or pharmaceutical composition of the present application can be formulated into the form of an injection, a sterile aqueous solution, an emulsion, a suspension, a liposome, a gel or a powder.

[0106] The CCR8 antagonist polypeptide compounds described herein can exhibit excellent ability to block the CCL1-CCR8 signaling pathway, and thus it can be expected that they can show good therapeutic effects on diseases involving the CCL1-CCR8 signaling pathway and their related symptoms (such as atopic dermatitis) in test animals.

[0107] Example

[0108] Next, the present application will be further described in detail through examples, but the protection scope of the present application is not limited to these examples.

[0109] Unless otherwise specified, the reagents, materials and instruments used in the following examples are those conventionally used in the art and can be purchased commercially or synthesized by conventional methods known in the art.

[0110] Example 1 Composition and Synthesis of Polypeptide Compounds

[0111] The polypeptide compounds having inhibitory function against chemokine receptor 8 (CCR8) used in the following examples and test examples comprise an amino acid sequence represented by any one of SEQ ID NO: 7 to SEQ ID NO: 205, and are numbered TCR01 to TCR153 and TCR155 to TCR200, respectively, as described in the table below.

[0112] The polypeptide compounds disclosed herein can be synthesized and confirmed by the following process.

[0113] 1. Synthesis of polypeptide compounds

[0114] The abbreviations used in this disclosure have the following meanings:

[0115] The sources of the reagents used in this example are as follows:

[0116] Fmoc-Rink Amide-AM resin was purchased from Xi'an Lanxiao; Fmoc-AA-OH amino acid was purchased from Chengdu Zhengyuan Biochemical; DMF, DCM, and TFA were purchased from Tianjin Concord; DIEA, DIC, Oxyma, and DTT were purchased from Suzhou Haofan; Tis was purchased from Shaoyuan Reagent; unless otherwise specified, other reagents were purchased from Sinopharm or Aladdin.

[0117] Peptide synthesis

[0118] (1) Using solid-phase synthesis, select the carrier resin according to the structure of the peptide chain to be synthesized. When the C-terminus is an amide, select Fmoc-Rink Amide-AM resin;

[0119] (2) Coupling of the first amino acid on the resin (the coupling method of Fmoc-Rink Amide-AM resin is consistent with the amino acid coupling method): After swelling the resin in DCM, add 1.5eq Fmoc-AA-OH and 7.5eq DIEA, use DCM as solvent, and react at room temperature for 30-60 min. Drain the reaction solution and wash. Block the remaining sites on the support with a reaction solution of MeOH:DIEA:DCM = 2:1:17 (volume ratio), react at room temperature for 10-30 min, and wash.

[0120] (3) Amino acid coupling: add 10 times the V / m equivalent of 20% piperidine / DMF solution to the swollen resin, react at room temperature for 5 to 10 minutes, then drain the reaction solution and wash three times with 10 times the V / m equivalent of DMF and DCM, respectively, to obtain a resin with exposed amino groups after drying; use the amino acid condensation reaction solution (its composition is Fmoc-AA-OH 3eq, Oxyma 3eq and DIC 3eq), use 10 times the V / m equivalent of DMF as solvent, and couple at room temperature for 30 to 120 minutes; after the reaction is completed, add 10 times the V / m equivalent of DMF to wash the resin, and the ninhydrin test is negative;

[0121] (4) Repeat the operation of (3) and sequentially couple the Fmoc-AA-OH at the subsequent sites to the target peptide resin according to the amino acid sequence, remove the Fmoc protecting group of the last amino acid at the end of the resin, wash and shrink, and then dry the peptide resin to constant weight;

[0122] (5) The peptide chain was cleaved from the resin using a cleavage reagent (TFA:TIS:DTT:Mesph:Meoph:H2O=80:1:5:5:5:4, by volume). After filtering the resin, the resin was washed with TFA (1.2 eq by volume / m), and the combined filtrates were evaporated to remove some TFA.

[0123] (6) Add methyl tert-butyl ether to the filtrate obtained in the above step (5) at a volume ratio of filtrate: methyl tert-butyl ether = 1:5, centrifuge, wash the filter cake with methyl tert-butyl ether, centrifuge three times, and vacuum dry to obtain the target linear polypeptide;

[0124] (7) The crude polypeptide was dissolved (or the reaction solution after cyclization was taken), filtered through a 0.22 μm microporous filter, and then separated and purified by preparative liquid chromatography using gradient elution. Mobile phase A was 0.1% TFA and water, and mobile phase B was acetonitrile. The flow rate was 1.0 mL / min. The target polypeptide compound was separated and prepared, and the pure product was lyophilized.

[0125] 2. Confirmation of polypeptide compounds

[0126] The polypeptide compound synthesized in item 1 above was identified by HPLC and structurally analyzed using a Thermo Fisher Scientific LTQ XL linear ion trap mass spectrometer. The chromatographic mobile phase consisted of acetonitrile and water. Mobile phase A consisted of 0.1% TFA and water, and mobile phase B consisted of acetonitrile. The elution gradient was adjusted based on the desired product. The peak ejaculation time was consistent with that of the crude product, and the purity of the polypeptides prepared in this example was greater than 90%. For example, the analysis results of TCR10 on the LTQ XL linear ion trap mass spectrometer are shown in Figure 1. Detailed information about the polypeptide compound TCR10 synthesized in this example is shown in Table 1.

[0127] Table 1 Final product information of peptide compound TCR10

[0128] The HPLC identification was performed and the structure analysis was performed by LTQ XL linear ion trap mass spectrometer. The HPLC purity of the prepared TCR01-TCR09 and TCR11-TCR200 was greater than 90%.

[0129] Next, the activity of the prepared exemplary CCR8 antagonistic polypeptide compound was verified by the following test examples.

[0130] Atopic dermatitis is a chronic, relapsing skin disease with a steadily increasing prevalence among children, reaching 10%-20%. Skin-infiltrating T cells, dendritic cells, and mast cells are believed to play a crucial role in its pathogenesis. Expression of the CC chemokine CCL1 (I-309) has been reported to be significantly and selectively upregulated in atopic dermatitis compared with psoriasis, cutaneous lupus erythematosus, or normal skin. Serum levels of CCL1 are significantly higher in patients with atopic dermatitis than in healthy individuals. Dendritic cells (DCs), mast cells, and dermal endothelial cells are abundant sources of CCL1 in atopic dermatitis and during allergen challenge. Patients with atopic dermatitis exhibit disrupted skin barrier function, frequent allergic reactions to allergens, defects in antimicrobial immune defenses, and a genetic predisposition. Clinical observations in AD, combined with recent scientific findings, suggest that atopic dermatitis undergoes amplification cycles. These amplification processes perpetuate inflammatory responses within the skin and contribute to the development of the AD phenotype. Study demonstrates that the CCL1-CCR8 axis connects adaptive and innate immune functions that play a role in the initiation and amplification of atopic dermatitis [1-2] .

[0131] In the following Test Examples 1-2, SPR was used to determine the ability of the polypeptide compound to bind to the CCR8 receptor, and a transwell chemotaxis assay was used to evaluate whether the polypeptide compound could inhibit cell migration by blocking the CCL1-CCR8 axis. In the following Test Examples 3-5, female Balb / c mice weighing 18 to 22 g were used; and a 75% ethanol solution of 50 mM MC903 (calcipotriol) was used as a modeling agent. MC903, or calcipotriol, is a low-calcium analog of vitamin D3 that is widely used in the treatment of psoriasis. Topical application of MC903 can stimulate the high expression of TSLP in mouse epithelial keratinocytes, promote the differentiation and activation of CD4+ T cells into Th2 cells and the release of inflammatory cytokines, causing mice to develop an atopic dermatitis phenotype.

[0132] Experimental Example 1: In vitro SPR affinity determination of polypeptide compounds and CCR8 protein

[0133] A human CCR8 full-length protein (VLP) chip was prepared using an amino-coupling method. The full-length CCR8 protein (VLP) was immobilized onto two channels of a CM5 chip. This assay was used to test the affinity of the peptide compound for the CCR8 protein and confirm its ability to bind to the CCR8 receptor.

[0134] (1) Experimental plan

[0135] Vehicle for test compounds (TCR10 and TCR09): PBS solution (containing 0.05% Tween 20).

[0136] The above polypeptide compound was prepared into a 1 mM solution as a drug stock solution, and then diluted with the above PBS solution to a concentration of 2.56×10 -2 Working solutions of various concentrations were prepared, including 0.128 nM, 0.64 nM, 3.2 nM, 16 nM, 80 nM, 400 nM, 2 μM, and 10 μM, with the aforementioned PBS solution serving as a blank control. The working solutions at these concentrations were captured on the Fc3 and Fc4 channels of the CCR8(VLP)-CM5 chip using the aforementioned PBS solution. The peptide compound samples at each concentration and the blank control were flowed through the test channels (Fc3 and Fc4) at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 120 seconds. After each test, the chip was regenerated with Gly-HCl pH 1.5 buffer.

[0137] (2) Test results

[0138] The test results are shown in Table 2. The binding affinities of TCR10 and TCR09 to human CCR8 protein were 17.17 nM and 228.8 nM, respectively.

[0139] Table 2 Affinity of polypeptide compounds to human CCR8 protein

[0140] Experimental Example 2: Transwell Chemotaxis Assay to Evaluate the Effect of Peptide Compounds on Cell Migration

[0141] Induce the differentiation of PBMC-derived interstitial dendritic cells, and use a transwell chemotaxis assay to count the number of interstitial dendritic cells that migrate in the presence or absence of the peptide compound. This assay evaluates whether the peptide compound blocks interstitial dendritic cell migration via the CCL1-CCR8 axis.

[0142] (1) Experimental plan

[0143] Vehicle for test compound (TCR10): PBS solution + RPMI 1640.

[0144] Antibody control: anti-CCR8 antibody (purchased from Jiman Biotechnology, GM-30913AB); solvent: RPMI 1640.

[0145] CD14 was isolated from human PBMC using magnetic beads +Monocytes were cultured for 6 days in the presence of GM-CSF and IL-4 to obtain interstitial dendritic cells, and the expression of CD209, CD1α, and CCR8 was detected by flow cytometry. The differentiated interstitial dendritic cells were collected and added to the upper chamber of the transwell. The samples were distributed to the upper and lower chambers of each chamber according to Table 3. Among them, only cells were added to the blank control group (referred to as the "blank group"), CCL1 and CXCL12 were added to the positive control group, the test compound group was added with the test compound and CCL1 and CXCL12, and the antibody control group was added with anti-CCR8 antibody and CCL1 and CXCL12; each group was tested in duplicate. Migrate for 5 hours in a 37°C, 5% CO2 incubator. After formaldehyde fixation and crystal violet staining, the non-migrated cells in the chamber were gently wiped off with a cotton swab. The cells that migrated through the chamber were counted using a microscope.

[0146] Table 3 Group settings and drug addition

[0147] (2) Test indicators: the number of interstitial dendritic cells migrating.

[0148] (3) Test results:

[0149] Flow cytometry results showed that interstitial dendritic cells expressed CCR8 on their surface (see Figure 2). As shown in Figures 3 and 4, interstitial dendritic cells in the positive control group responded to chemotaxis and migrated significantly compared to the blank group. After the addition of TCR10 or anti-CCR8 antibodies, the amount of interstitial dendritic cell (iDC) migration decreased. In particular, the reduction in cell migration was more significant after the addition of TCR10, indicating that TCR10 can block the CCL1-CCR8 axis, thereby affecting the migration of interstitial dendritic cells.

[0150] Experimental Example 3: Efficacy screening based on the calcipotriol-induced mouse atopic dermatitis model (primary screening)

[0151] After shaving the back skin of mice, MC903 was applied once daily (50 μL on a 2 cm*2 cm area of ​​back skin and 20 μL on the ears) to induce atopic dermatitis in the mice. This test was used to test the efficacy of the CCR8 antagonist polypeptide disclosed herein in preventing atopic dermatitis.

[0152] (1) Experimental plan

[0153] Vehicle for test compounds (TCR10 and TCR09): normal saline.

[0154] Positive control drugs: 0.3 wt% mometasone gel (glucocorticoid) and KMC420 (JAK inhibitor, 10 mM; vehicle: normal saline).

[0155] Mice in the normal control group were treated with 75% ethanol solution for 14 days without MC903 induction. Mice in the model group were only smeared with MC903 every day for 14 consecutive days. The dosage is shown in the following table. During the administration period, mice in the positive control group and the test compound (TCR9 and TCR10) groups were first smeared with the positive control drug (50 μL of mometasone gel and KMC420 were applied to the back and 20 μL to the ears (10 μL each inside and outside)) or the test compound (TCR10 and TCR09, both at a concentration of 20 mg / mL were applied to the back 50 μL, and both at a concentration of 50 mg / mL were applied to the inside and outside of the ears 10 μL each). After drying, the modeling agent was applied (50 μL was applied to the back skin each time and 20 μL was applied to the ears (10 μL each inside and outside)) once a day for 14 consecutive days.

[0156] Table 4 Test plan for drug efficacy screening test compounds

[0157] Before MC903 modeling, body weight, remaining food intake, and ear thickness were measured in each group of mice. Subsequently, body weight, remaining food intake, and ear thickness were measured before daily dosing. Ear and back skin were photographed and scored for lesion severity at regular intervals. After the final dose, ear and back skin, liver, kidney, and spleen were collected and fixed in formalin for pathological examination.

[0158] (2) Experimental indicators: mouse body weight, ear swelling, skin lesion score, and pathological analysis.

[0159] Table 5 Scoring criteria for mouse skin lesions

[0160] (3) Test results

[0161] The test results are shown in Figures 5-10. Compared to the model group, the CCR8 antagonist polypeptide compound administration group significantly improved weight loss in mice, achieving results substantially comparable to those in the normal control group (see Figure 5). Compared to the model group, the CCR8 antagonist polypeptide compound administration group significantly reduced ear swelling, essentially reaching the level of normal mice (see Figure 6). The back skin lesion score was close to that of normal mice (see Figure 7), and the liver, kidney, and spleen indices were comparable or substantially similar to those of normal mice (see Figure 8).

[0162] As shown in Figure 9, pathological analysis of the mouse back skin revealed that the epidermis of the skin tissue of the normal control group mice was intact, with a clear structure and visible keratinized layer. The collagen fibers in the dermis were neatly arranged, and the hair follicles of the skin appendages were scattered. The subcutaneous tissue, located beneath the dermis, was composed of loose connective tissue, adipose tissue, and muscular layer, and showed no obvious abnormalities (Figure 9(a)). In the model group mice, local keratinocyte hyperplasia (black arrow), moderate epidermal thickening (yellow arrow), and scattered lymphocyte and neutrophil infiltration (red arrow) were observed in the dermis (Figure 9(b)). In the 0.3% mometasone gel-treated group mice, local keratinocyte hyperplasia (black arrow), extensive epidermal thickening (yellow arrow), and localized epidermal thickening (red arrow) were observed (Figure 9(c)). In the KMC420-treated group mice, extensive epidermal thickening (black arrow) was observed in the tissue, and a small amount of lymphocyte infiltration (yellow arrow) was observed in the dermis (Figure 9(d)). In the TCR09-treated mice, parakeratosis was rare in the stratum corneum (black arrow), the epidermis was moderately thickened (yellow arrow), and scattered lymphocyte and neutrophil infiltration (red arrow) was observed in the epidermis and dermis (Figure 9(e)). In the TCR10-treated mice, extensive epidermal thickening (black arrow) and scattered lymphocyte and neutrophil infiltration (yellow arrow) were observed in the dermis (Figure 9(f)).

[0163] As shown in Figure 10, pathological analysis of mouse ears revealed that the epidermis of the ear tissue of mice in the normal control group was intact, with a clear structure and visible keratinized layer. The collagen fibers in the dermis were neatly arranged, and the hair follicles and sebaceous glands of the skin appendages were scattered. The subcutaneous tissue was located beneath the dermis and consisted of loose connective tissue, adipose tissue, and muscle layer, with no obvious abnormalities (Figure 10(a)). In the model group, the keratin layer was mostly hyperplastic (black arrow), with local parakeratosis (yellow arrow), moderate thickening of the epidermis (red arrow), and a large number of lymphocytes and neutrophils infiltrated in the dermis (blue arrow), with a small amount of connective tissue hyperplasia (green arrow) and a small amount of neovascularization (brown arrow) (Figure 10(b)). In the 0.3% mometasone gel-treated mice, extensive epidermal thickening (black arrows), numerous epidermal cell necrosis and nuclear dissolution (yellow arrows), numerous lymphocyte and neutrophil infiltration in the surrounding dermis (red arrows), and a small amount of venous stasis and dilation (blue arrows) were observed (Figure 10(c)). In the KMC420-treated mice, multiple stratum corneum thickening (black arrows) and large abscesses (yellow arrows) were observed locally. Extensive epidermal thickening (red arrows) was observed in the tissues, and moderate lymphocyte and neutrophil infiltration (blue arrows) was observed in the dermis, accompanied by mild connective tissue hyperplasia (green arrows) (Figure 10(d)). In the TCR09-treated mice, extensive mild epidermal thickening was observed in the tissues, with more pronounced thickening in some areas (black arrows), and mild connective tissue hyperplasia (yellow arrows) was observed in some areas of the epidermis, accompanied by lymphocyte and neutrophil infiltration (red arrows) (Figure 10(e)). In the TCR10-treated mice, the epidermis of the ear tissue was intact, with a clear structure and a visible keratinized layer. The collagen fibers in the dermis were neatly arranged, and the skin appendages, hair follicles, and sebaceous glands were scattered. The subcutaneous tissue was located beneath the dermis and consisted of loose connective tissue, adipose tissue, and muscle layer, with no obvious abnormalities observed (Figure 10(f)).

[0164] Test Example 4: Dose-effect test

[0165] After shaving the back skin of mice, MC903 was applied once daily (50 μL applied to an area of ​​2 cm x 2 cm on the back skin, and 10 μL applied to the inside and outside of the ears) to induce atopic dermatitis in the mice. For the treated groups (including the positive control group and the test compound group), the drug's preventive effect on atopic dermatitis was tested by pre-applying the drug before each MC903 application.

[0166] (1) Experimental plan

[0167] Test compound solution: Use DMSO to dissolve the drug to an appropriate concentration as a stock solution (stock solution for back: 200 mg / mL; stock solution for ear: 500 mg / mL), then prepare the working solution according to the following formula (by volume ratio) and store at 4°C: (1) 10% DMSO stock solution + 40% PEG300 + 5% Tween 80 + 45% NS (normal saline), the drug concentration in the final working solution obtained is: 20 mg / mL for the working solution for the back; 50 mg / mL for the working solution for the ear; (2) 30% DMSO stock solution + 40% PEG300 + 5% Tween 80 + 25% NS (normal saline), the drug concentration in the final working solution obtained is: 60 mg / mL for the working solution for the back; 150 mg / mL for the working solution for the ear.

[0168] Positive control drugs: 0.1 wt% mometasone furoate and 2 wt% crisaborole ointment.

[0169] The mice in the normal control group were treated with 75% ethanol solution only for 14 days, and no MC903 was used to induce modeling. The mice in the model group were only smeared with MC903 every day for 14 consecutive days, and the dosage was shown in the following table. During the administration period, the mice in the positive control drug group and the test compound (TCR9 and TCR10) group were first smeared with the positive control drug (0.1% mometasone furoate was applied in an amount of 50 μL on the back and 20 μL on the ear (10 μL each on the inside and outside); 2% crisaborole ointment was applied in an amount of 20 mg on the back and 20 mg on the ear (10 mg each on the inside and outside)) or the test polypeptide compound (TCR09 (1 mg) and TCR10 (1 mg) at a concentration of 20 mg / mL) every day. Apply 50 μL on the back and 10 μL each on the inside and outside of the ear at a concentration of 50 mg / mL; apply 50 μL on the back of TCR09 (3 mg) and TCR10 (3 mg) at a concentration of 60 mg / mL, and 10 μL each on the inside and outside of the ear at a concentration of 150 mg / mL). After it is completely dry, apply the modeling agent (50 μL on the back skin each time and 20 μL on the ear (10 μL each on the inside and outside)) once a day for 14 consecutive days.

[0170] Table 6 Dose-effect test compound testing plan

[0171] After the last administration, the back skin of mice in each group was photographed.

[0172] (2) Test indicators

[0173] Observation and monitoring of skin lesions in mice.

[0174] (3) Test results

[0175] The experimental results of the dose-effect test are shown in Figure 11.

[0176] As shown in Figure 11, compared with the model group, both the 3mg TCR09 group and the 3mg TCR10 group could significantly improve the skin lesions on the back of mice, among which the 3mg TCR10 group had the best improvement effect.

[0177] Test Example 5: Evaluation of the efficacy of gel preparation in treatment models

[0178] After shaving the back skin of the mice, MC903 was applied once a day (50 μL was applied to an area of ​​2 cm*2 cm of back skin). The success of the model was confirmed by the degree of skin lesions on the back of the mice, thereby obtaining a mouse atopic dermatitis model.

[0179] (1) Experimental plan

[0180] Drug solution stock solution: Weigh the required amount of drug (TCR10), add DMSO to fully dissolve it, and obtain the stock solution. The concentration of the stock solution for back is 200 mg / mL; the concentration of the stock solution for ear is 500 mg / mL.

[0181] Gel Matrix: Weigh 1.5 wt% of HPMC (hydroxypropyl methylcellulose) based on the final gel matrix, add water and stir, and allow to swell overnight. Add 20% glycerol and 0.5% Tween 80 based on the total weight of the gel matrix, and an appropriate amount of triethanolamine (adjust the pH of the system to 7-8), and stir evenly.

[0182] Gel preparation: The above-mentioned drug solution mother solution was mixed with an appropriate amount of gel matrix, water was added to the full amount, and the gel was stirred to make it uniform to obtain 2wt% and 5wt% TCR10 gel preparations.

[0183] Positive control drugs: 0.1 wt% mometasone furoate gel and 2 wt% crisaborole ointment.

[0184] The mice in the normal control group (Normal group) were treated with 75% ethanol solution only for 14 days, once a day, without inducing modeling with MC903. The mice in the model group (Model group) were applied with MC903 once a day for 14 consecutive days, and the dosage was shown in the following table. After 7 days of continuous application of MC903 to establish the model, the mice in the following groups were given medication every day after applying MC903: for the gel matrix control group, 50 mg of gel matrix was applied to the back skin, and 25 mg of gel matrix was applied to the ear skin (12.5 mg each inside and outside), twice a day for 7 consecutive days; for the mometasone furoate gel group, 50 μL of 0.1% mometasone furoate gel was applied to the back skin, and 17 μL of 0.1% mometasone furoate gel was applied to the ear skin (inside and outside), For the crisaborole ointment group, 20 mg of 2% crisaborole ointment was applied to the back skin, and 7 mg of 2% crisaborole ointment was applied to the ear skin (3.5 mg each for the inside and outside), twice a day for 7 consecutive days; for the TCR10 gel group, 50 mg of 2% and 5% TCR10 gel were applied to the back skin, and 25 mg of each were applied to the ear skin (12.5 mg each for the inside and outside), twice a day for 7 consecutive days.

[0185] Table 7 Dosage regimen for gel formulation treatment model

[0186] Before MC903 modeling, body weight, remaining food intake, and ear thickness were measured in each group of mice. Subsequently, body weight, remaining food intake, and ear thickness were measured before daily dosing. Dorsal skin was photographed and the severity of skin lesions was scored regularly. After the final dose, dorsal skin was collected to measure cytokine levels, epidermal thickness, and skin mast cell counts. Skin samples were fixed in formalin for pathological examination.

[0187] (2) Test indicators

[0188] Mouse body weight, mouse skin lesion score (scoring criteria are the same as in Experimental Example 1), mouse ear swelling, cytokine content (IL-4) per mg of mouse skin, mouse HE score, mouse epidermal thickness, mouse skin mast cell count, and histopathological sections.

[0189] Table 8 HE scoring criteria

[0190] (3) Test results

[0191] The experimental results of the gel formulation treatment model are shown in Figures 12 to 25. Compared with the model group mice, the TCR10 gel group mice showed improved weight loss (see Figures 12 and 13), significantly reduced skin lesion scores (see Figures 14 and 15), significantly improved ear swelling (see Figures 16 and 17), a significant decrease in the level of IL-4, an inflammatory cytokine associated with atopic dermatitis (see Figure 18), significantly improved HE scores (see Figure 19), and reduced epidermal thickness and mast cell counts (see Figures 20 and 21).

[0192] As shown in Figures 22-24, the pathological conditions of mice in the TCR10 gel group were significantly improved. Specifically, pathological HE staining showed that the skin of mice in the normal control group was normal, while the skin of mice in the model group showed obvious thickening, hyperkeratosis / incompleteness, extension of epidermal epithelial horns and dermal congestion, inflammatory cell infiltration, fibrosis and other symptoms. Compared with the model group, the skin scores of mice in the gel matrix control group (50 mg back + 25 mg ear, BID), mometasone furoate gel group (50 μL back + 17 μL ear, QD) and crisaborole ointment group (20 mg back + 7 mg ear, BID) did not show a significant decrease. The skin scores of the 2% TCR10 gel group (50 mg back + 25 mg ear, BID) and the 5% TCR10 gel group (50 mg back + 25 mg ear, BID) were significantly reduced, with p values ​​of p < 0.05 and p < 0.001, respectively. It can be seen that all tested concentrations of TCR10 gel preparations can significantly alleviate the pathological symptoms of mice, among which the 5% TCR10 gel group (50 mg back + 25 mg ear, BID) has the best therapeutic effect (see the left sub-figures of Figures 22-24).

[0193] Pathological epidermal thickness: Compared with the normal control group, the epidermis of mice in the model group was significantly thickened (p < 0.001). Compared with the model group, the epidermal thickness of mice in the gel matrix control group (50 mg back + 25 mg ear, BID) was essentially the same. The epidermal thickness of mice in the mometasone furoate gel group (50 μL back + 17 μL ear, QD) and crisaborole ointment group (20 mg back + 7 mg ear, BID) showed a decreasing trend, but the difference was not significant. The epidermal thickness of mice in the 2% TCR10 gel group (50 mg back + 25 mg ear, BID) and 5% TCR10 gel group (50 mg back + 25 mg ear, BID) was significantly reduced, with p values ​​of p < 0.05 and p < 0.05, respectively, indicating a significant improvement. Compared with the gel matrix control group, the TCR10 gel group showed a decreasing trend after treatment, indicating that the TCR10 gel preparation can improve epidermal thickening (see Figure 20).

[0194] As shown in FIG25 , the crisaborole ointment group, the 2% TCR10 gel group, and the 5% TCR10 gel group could all improve the skin lesions on the back of the mice, among which the 5% TCR10 gel group had the best improvement effect.

[0195] When tested according to the above method, TCR01 to TCR08, TCR11 to TCR153, and TCR155 to TCR200 all showed effects on improving atopic dermatitis that were substantially equivalent to those of TCR09 and TCR10, demonstrating reduced skin lesion scores, improved weight loss, and a significant decrease in the levels of atopic dermatitis-related inflammatory cytokines (IL-4). After treatment, a keratinized layer was visible in the back epidermis, and the collagen fibers in the dermis were neatly arranged. The subcutaneous tissue was located below the dermis and was composed of loose connective tissue, adipose tissue, and muscle layer, with no obvious abnormalities observed.

[0196] References

[0197] 1.Gombert M.et al.,CCL1-CCR8 interactions:an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation.J Immunol.2005Apr 15;174(8):5082-91.

[0198] 2.Homey,B.et al.,Modulation of Chemokines by Staphylococcal Superantigen in Atopic Dermatitis.Chem Immunol Allergy.2007,93:181-194.

[0199] For the purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their disclosure predates the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications become part of the common general knowledge in the art in any country.

[0200] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific implementation modes and examples described above, but that various modifications, replacements, or recombinations can be made without departing from the spirit of the present application, which all fall within the scope of protection of the present application.

Claims

1. A CCR8 antagonist polypeptide compound, comprising an amino acid sequence selected from the following or an amino acid sequence having at least 80% identity thereto: (1) H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-CONH2 (SEQ ID NO: 1), Among them, Xa a1 is Val, Ala, Leu or Ile, Xaa2 is Leu or Val, Xaa3 is Leu, Val or Ile, Xaa4 is Thr, Ser, Ala or Ile, Xaa5 is Ser, Thr or Ala, Xaa6 is Ile, Val, Leu, AIB, Cys, Nle, Ala, Met or Abu; (2)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Ile-Pro-Asn-Ile-Xaa7-Xaa8-Xaa9-Xaa 10 -No 11 -CONH2(SEQ ID NO:2), Among them, Xaa7 is Leu or Val, Xaa8 is Leu, Val or Ile, Xaa9 is Thr, Ala or Ser, Xaa 10 Thr, Ala or Ser, Xaa 11 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu; (3)H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 12 -No 13 -No 14 -No 15 -No 16 -CONH2(SEQ ID NO:3), Among them, Xaa 12 Leu or Val, Xaa 13 Leu, Val or Ile, Xaa 14 Thr, Ala or Ser, Xaa 15 Thr, Ala or Ser, Xaa 16 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu; (4) H2N-AIB-Arg-Pro-Xaa 17 -No 18 -No 19 -Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Xaa 20 -No 21 -Abu-CONH2(SEQ ID NO:4), Among them, Xaa 17 -Xaa 18 -Xaa 19 Asp-Lys-Ala or Glu-Orn-Ser, Xaa 20 Thr, Ala or Ser, Xaa 21 is Thr or Ser; or (5)H2N-AIB-Arg-Pro-Glu-Orn-Ser-Ala-Leu-Gly-Tyr-Asn-Orn-Arg-Pro-Ile-Pro-Asn-Ile-Xaa 22 -No23-No 24 -No 25 -No 26 -CONH2(SEQ ID NO:5), Among them, Xaa 22 Leu or Val, Xaa 23 Leu, Val or Ile, Xaa 24 Thr, Ala or Ser, Xaa 25 Thr, Ala or Ser, Xaa 26 is Leu, Val, AIB, Cys, Nle, Ala, Met or Abu.

2. The polypeptide compound according to claim 1, wherein Xaa1-Xaa2-Xaa3 is Val-Leu-Leu; and / or Xaa4 is Thr, Ser or Ala; and / or Xaa5 is Ser, Thr or Ala; and / or Xaa6 is Ile, Val, Leu, AIB, Cys, Nle, Ala, Met or Abu.

3. The polypeptide compound according to claim 1 or 2, wherein: Xaa1-Xaa2-Xaa3 are Val-Leu-Leu, Xaa4 are Thr or Ser, Xaa5 are Ser, and Xaa6 are Ile, Val, AIB, Cys, Nle, Ala, Met or Abu; Alternatively, Xaa1-Xaa2-Xaa3 are Val-Leu-Leu, Xaa4 is Thr or Ser, Xaa5 is Ser, Thr or Ala, and Xaa6 is Ile.

4. The polypeptide compound according to claim 1 or 2, wherein: Xaa1-Xaa2-Xaa3-Xaa4 are Val-Leu-Leu-Thr, Xaa5 are Ser, Thr or Ala, and Xaa6 are Val, Leu, AIB, Cys, Nle, Ala, Met or Abu; Alternatively, Xaa1-Xaa2-Xaa3-Xaa4 are Val-Leu-Leu-Ser, Xaa5 is Ser or Thr, and Xaa6 is Ile, Val, AIB, Cys, Nle, Ala, Met or Abu.

5. The polypeptide compound according to any one of claims 1 to 4, wherein The polypeptide compound comprises the following amino acid sequence: H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Xaa 27 -Ser-Val-CONH2 (SEQ ID NO: 6), wherein Xaa 27 is Thr or Ser; or H2N-AIB-Arg-Pro-Asp-Lys-Ala-Ala-Leu-Gly-Tyr-Gln-Lys-Arg-Pro-Leu-Pro-Gln-Val-Leu-Leu-Ser-Ser-Xaa 28 -CONH2 (SEQ ID NO: 206), wherein Xaa 28 It is Ile or Val.

6. The polypeptide compound according to claim 1, wherein The polypeptide compound comprises an amino acid sequence shown in any one selected from SEQ ID NO: 7 to SEQ ID NO: 205; preferably, the amino acid sequence of the polypeptide compound is shown in SEQ ID NO: 16 or SEQ ID NO:

15.

7. The polypeptide compound according to claim 1, wherein (1) Xaa7-Xaa8 are Leu-Leu; preferably, Xaa9 is Thr or Ser, and Xaa 10 Ser or Thr, Xaa 11 for Val; (2)Xaa 12 -Xaa 13 is Leu-Leu; preferably, Xaa 14 Thr or Ser, Xaa 15 Ser or Thr, Xaa 16 for Val; (3)Xaa 17 -Xaa 18 -Xaa 19 Asp-Lys-Ala, Xaa 20 Thr or Ser, Xaa 21 is Ser or Thr; or (4)Xaa 22 -Xaa 23 is Leu-Leu; preferably, Xaa 24 Thr or Ser, Xaa 25 Ser or Thr, Xaa 26 For Val.

8. A pharmaceutical composition comprising the polypeptide compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutically acceptable pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants, and diluents; Preferably, the pharmaceutical composition is a powder, granules, a film-coating, a cream, an ointment, a gel, a lotion, a foam, a patch, a liniment, an elixir, an aerosol, an emulsion, a solution or a suspension.

10. The pharmaceutical composition according to claim 8 or 9, wherein The pharmaceutical composition further comprises another atopic dermatitis therapeutic agent, which is selected from glucocorticoids, calcineurin inhibitors, PDE4 inhibitors, JAK inhibitors, antibiotics, antipruritic drugs, wet compresses, calamine lotion or zinc cream, antihistamines, antiviral drugs, and immunosuppressants.

11. A kit comprising the polypeptide compound according to any one of claims 1 to 7, or the pharmaceutical composition according to any one of claims 8 to 10.

12. A polypeptide compound according to any one of claims 1 to 7, a pharmaceutical composition according to any one of claims 8 to 10, or a kit according to claim 11 for preventing or treating cancer or tumors, inflammatory diseases, viral infections, IgG4-related diseases, skin immune diseases, or symptoms related thereto.

13. The polypeptide compound, pharmaceutical composition or kit for use as claimed in claim 12, wherein: The tumor is selected from solid tumors, more preferably multiple myeloma, lymphoma or melanoma; the cancer is selected from esophageal cancer, lung cancer, non-small cell lung cancer, gastric cancer, head and neck squamous cell carcinoma, liver cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, kidney cancer, bladder cancer, cutaneous fibrosarcoma, osteosarcoma, pancreatic cancer or prostate cancer, the inflammatory disease is selected from asthma or allergic enteritis, the IgG4-related disease is selected from sialadenitis, the skin immune disease or its related symptoms is a skin immune disease related to the CCL1-CCR8 signaling pathway or its related symptoms; more preferably, the skin immune disease or its related symptoms is selected from eczema, atopic dermatitis, psoriasis, lupus erythematosus, scleroderma, dermatomyositis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, epidermolysis bullosa or their related symptoms.

14. The polypeptide compound, pharmaceutical composition or kit for use according to claim 12 or 13, wherein: The skin immune disease or its related symptoms are selected from atopic dermatitis or its related symptoms; More preferably, the atopic dermatitis is infantile atopic dermatitis, childhood atopic dermatitis, or adolescent and adult atopic dermatitis, or the atopic dermatitis is mild, moderate or severe atopic dermatitis.

15. A polypeptide compound according to any one of claims 1 to 7, a pharmaceutical composition according to any one of claims 8 to 10, or a kit according to claim 11 for blocking a CCL1-CCR8 signaling pathway.

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