APPLICATION OF PCSK9 INHIBITORS IN THE PREPARATION OF DRUGS FOR THE TREATMENT OF INFLAMMATORY IMMUNE DISEASES

Abstract

The invention belongs to the biopharmaceutical. It involves the role and mechanism of PCSK9 in inflammatory immune diseases, and the application of PCSK9 inhibitors to the preparation of drugs for the treatment of inflammatory immune diseases mediated by T cells. In particular, it involves the use of PCSK9 monoclonal antibody, PCSK9 interference RNA and PCSK9 small molecule inhibitors for treating psoriasis, atopic dermatitis, or urticaria. The invention uses psoriasis as an embodiment for the study of inflammatory and immune diseases. It is found that PCSK9 plays an important role in the treatment of inflammatory immune diseases. The PCSK9 monoclonal antibody, PCSK9 interference RNA, and PCSK9 small molecule inhibitor can be further developed for treating inflammatory immune-diseases, such as psoriasis with fewer adverse reactions, at low cost, and with good efficacy.

Claims

1. A method of treating an inflammatory immune disease by administration of a therapeutically effective amount of a PCSK9 inhibitor to a human in need thereof, wherein the inflammatory immune disease is selected from the group consisting of psoriasis, psoriatic arthritis, eczema, atopic dermatitis, urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, and lymphoma.

2. The method of claim 1, wherein the PCSK9 inhibitor is a PCSK9 monoclonal antibody, a PCSK9 small molecule interfering RNA or a PCSK9 small molecule inhibitor.

3. The method of claim 2, wherein the inflammatory immune disease is psoriasis, atopic dermatitis or urticaria, wherein the PCSK9 inhibitor is topically or systemically administered.

4. The method of claim 3, wherein the inflammatory immune disease is psoriasis.

5. The method of claim 2, wherein the PCSK9 inhibitor is a small molecule interference RNA (siPCSK9) that significantly inhibits the abnormal proliferation of human keratinocytes and promotes their apoptosis through NFkb pathway.

6. The method of claim 1, wherein the PCSK9 inhibitor is a PCSK9 vaccine.

Description

III. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1: Mild skin inflammation was observed in imiquimod cream (IMQ) treatment region of PCSK9 knockout mice. After 5 days of continuous coating, marked erythema, thickening of scales and lesions were observed and measured in the coating area of at the back of C57BL/6 mice. In PCSK9 knockout mice, there were only slight erythema, thickening of scales and lesions.

[0011] FIG. 2: The score of skin inflammation (erythema+scale+infiltration) in PCSK9 knockout mice were significantly lower than that in control group (P<0.05). A daily score was given on the skin lesions of the drug-coated area on the back of the mice, the erythema, scale, lesion thickening and total score of C57BL/6 mice were significantly higher than those of PCSK9 gene knockout mice (P<0.05).

[0012] FIG. 3: The histopathological changes of skin in the IMQ treatment region of PCSK9 knockout mice were closer to normal. After 5 days' coating, the skin tissue (treated and non-treated) of mice back were stained with HE. The skin of IMQ treatment area of C57BL/6 mice showed typical psoriasis pathological changes, such as skin thickening, dermatoid lengthening, thickening of spinous layer, hyperkeratosis with incomplete keratosis, Kogoj abscess and Munro abscess. However, the skin of PCSK9 gene knockout mice showed mild epidermis thickening and hyperkeratosis, no extension of dermatoid, thickening of spinous layer, hyperkeratosis with incomplete keratosis, and typical psoriatic pathological changes, such as Kogoj abscess and Munro abscess. The untreated skin of both groups showed normal skin structure.

[0013] FIG. 4: Immunofluorescence assay showed: Compared with PCSK9 knockout mice, the expression of PCSK9 in the control group was unregulated in the skin of the inflammatory area. The expression of PCSK9 in IMQ treated area of C57BL/6 mice was significantly higher than that in untreated area. However, there was no PCSK9 expression in the skin of IMQ treated and untreated regions of PCSK9 knockout mice.

[0014] FIG. 5: The expression of NF-kB in the skin of PCSK9 knockout mice was significantly lower than that of the control group. The expression of NF-kB in the skin of C57BL/6 mice and PCSK9 knockout mice was significantly higher than that of the untreated regions in the skin of C57BL/6 mice and PCSK9 knockout mice respectively. However, the expression of NF-kB in the skin of PCSK9 knockout mice was significantly lower than that of C57BL/6 mice.

[0015] FIG. 6: si-PCSK9 transfection significantly inhibited cell viability of human keratinocytes. The survival number of primary keratinocytes transfected with si-PCSK9 was significantly lower than that of si-Con transfection (P<0.001).

[0016] FIG. 7: si-PCSK9 transfection significantly promoted apoptosis and inhibited the proliferation of human keratinocytes. The apoptosis of primary keratinocytes transfected with si-PCSK9 increased significantly (P<0.05), while proliferating (the proportion of S+G2/M phase cells) decreased significantly (P<0.05).

[0017] FIG. 8: Immunohistochemically staining showed that the expression of PCSK9 in psoriatic lesions was significantly higher than that in psoriatic non-lesions and normal controls. PCSK9 positive expression cells were mainly distributed in epidermis, and dermis near the epidermis, but not in dermis vessels.

[0018] FIG. 9: The results of Q-PCR (quantative PCR) showed that the expression of PCSK9 in psoriatic lesions (PP) group was significantly higher than that in psoriatic non-lesion (PN) group and normal control (NN) group (P<0.001).

[0019] FIG. 10: The expression of PCSK9 in peripheral blood of patients with psoriasis, eczema and urticaria was significantly higher than that of normal controls (P<0.05).

[0020] FIG. 11: PCSK9 protein can significantly promote the secretion of IL-17, IL-2 and IFN-Gamma (P<0.01) by CD4 T cells isolated from peripheral blood of patients with psoriasis, eczema and urticaria.

[0021] FIG. 12: Subcutaneous injection of PCSK9 small molecule inhibitor and PCSK9 monoclonal antibody can reduce the score of erythema, scale and infiltration induced by IMQ in mice skin (P<0.01).

[0022] FIG. 13: External use of PCSK9 small molecule inhibitors and PCSK9 monoclonal antibodies can significantly reduce the score of skin erythema, scale, and infiltration of IMQ induced mice. The efficacy of PCSK9 small molecule inhibitor is better than that of PCSK9 monoclonal antibody (P<0.01).

[0023] FIG. 14: Compared with control siRNA(siCon), siPCSK9 could significantly reduce the expression of PCSK9 in mouse skin one day after the application, and the inhibition rate was more than 80%.

[0024] FIG. 15: siPCSK9 can inhibit the expression of NF kappa B, IL-17, IL-22, IL-23 and other inflammatory factors after a day of skin treatment.

[0025] FIG. 16: After induction of IMQ, the skin psoriasis lesion, erythema, scale and infiltration score of mice treated with siPCSK9 were significantly lower than those of the control group (P<0.01).

[0026] FIG. 17: On days 2-6 after induction of IMQ, compared with the control group, the skin erythema, scales and infiltration of BALB mice induced by IMQ were significantly inhibited by siPCSK9.

IV. DETAILED DESCRIPTION OF THE INVENTION

Technical Issues

[0027] The problems to be solved in the present invention: the role and mechanism of PCSK9 in the treatment of inflammatory immune diseases and the application of PCSK9 inhibitors in the preparation of drugs for the treatment of inflammatory immune diseases. The invention uses psoriasis as an example for the study of inflammatory immune diseases, and finds that PCSK9 plays an important role in the treatment of a variety of inflammatory immune diseases.

Technical Solution

[0028] We first found that PCSK9 plays an important role in the treatment of a variety of inflammatory immune diseases. We used PCSK9 knockout transgenic mice to establish psoriatic like inflammatory model induced by imiquimod, which proves that inhibition of PCSK9 has a very obvious therapeutic effect on psoriatic inflammatory lesions. A study on the mechanism of inhibiting PCSK9 in the treatment of inflammatory immune diseases by cultured human keratinocytes found that knock-down of PCSK9 expression by siRNA could significantly inhibit the abnormal proliferation of keratinocyte and promote its apoptosis through NFkb pathway.

[0029] To further confirm the role of PCSK9 in the pathogenesis of psoriasis, the invention collected lesions and non-lesions from 30 psoriasis patients, and skin samples from 30 normal human subjects. The immunohistochemistry results showed: the expression of PCSK9 in psoriatic lesions was significantly higher than that in non-lesions and normal controls (p<0.05). PCSK9 positive cells were mainly distributed in epidermis and dermis near the epidermis, but not in dermis vessels. Detected by real-time fluorescence quantitative nucleic acid amplification detection system (Q-PCR), it was found that the expression of PCSK9 was found in the skin of both patients and normal subjects, but it was significantly higher in the psoriasis patients than in the normal controls (P<0.01). No expression of PCSK9 was found in the peripheral blood mononuclear cells (PBMC) of patients with psoriasis, eczema, and urticaria and the control group by Q-PCR. However, the expression of PCSK9 in blood in patients with psoriasis, eczema and urticaria was significantly higher than that in the control group by ELISA (P<0.05).

[0030] Next, we isolated CD4 T cells from peripheral blood of patients with active psoriasis, eczema and urticaria and found PCSK9 protein could significantly promote the secretion of IL-17, IL-2 and IFN-Gamma by CD4 T cells, and increased the expression of NFkb, especially in psoriatic patients.

[0031] In all PCSK9 inhibitors, representative monoclonal antibodies, small interfering RNA and small molecular inhibitors were selected herein. Treated psoriasis-like mice model by subcutaneous injection (the dose is the same as the known LDL cholesterol experiment) in comparison with control group. The results showed that the therapeutic effect of PCSK9 monoclonal antibody group and PCSK9 small molecule inhibitor group was significantly better than that of control group. There were no obvious adverse reactions in PCSK9 monoclonal antibody group and PCSK9 small molecule inhibitor group. The results showed that the systemic use of PCSK9 small molecule inhibitors and PCSK9 monoclonal antibodies had obvious therapeutic effects on psoriasis-like inflammation and the therapeutic effect was significantly better than that of the control group.

[0032] In some embodiments, representative PCSK9 small molecule inhibitors and PCSK9 monoclonal antibodies were prepared for use as an external ointment (drug concentration 0.001-0.05%) and applied to IMQ induced inflammatory psoriasis like lesions in mice once a day in comparison with external ointment without drugs as controls. The results showed that the efficacy of PCSK9 monoclonal antibody group and PCSK9 small molecule inhibitor group was significantly better than that of control group. The effect of PCSK9 small molecule inhibitor group was better than that of PCSK9 monoclonal antibody group, and there was no obvious adverse reaction in each group. It was proved that topical use of PCSK9 small molecule inhibitor and PCSK9 monoclonal antibody had obvious therapeutic effect on psoriasis-like inflammation and their efficacy was better than that of control group.

[0033] In some embodiments, representative PCSK9 small molecule interference RNA (siPCSK9) was applied to IMQ induced psoriasis like inflammatory lesions in mice, once a day. Compared with the control group, the lesions of the siPCSK9 group were improved obviously, and no obvious adverse reaction was found.

[0034] For the first time, it was found that: 1. Both systemic and topical use of PCSK9 monoclonal antibody, PCSK9 small molecule inhibitor, and siPCSK9 have therapeutic effects on psoriatic lesions, which are significantly superior to those of the control group. In some embodiments, the invention relates to the treatment of psoriatic lesion by a PCSK9 inhibitor selected from the group consisting of representative PCSK9 monoclonal antibody, PCSK9 small molecule inhibitor and siPCSK9.

[0035] Because of its high inhibiting efficiency, selectivity and good efficacy, monoclonal antibody has been widely used in the research of new drugs. Our study also found that, small molecular inhibitors and small interfering RNA aimed at PCSK9 were more effective than monoclonal antibodies in the treatment of psoriasis-like inflammation. Generally, monoclonal antibodies have fewer side effects than small molecular inhibitors, and are more stable than small interfering RNA preparations. However, according to our data, small molecular PCSK9 inhibitors and siPCSK9 not only have a good efficacy in the treatment of psoriasis like inflammation, but also have mild side effects.

[0036] It is well known to one skilled in this field that, based on the above mechanism of PCSK9, PCSK9 inhibitors have therapeutic effect on other T cell mediated chronic immune diseases. These include, but are not limited to, psoriasis, psoriatic arthritis, eczema (atopic dermatitis), urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis, scleroderma, diabetes, chronic liver disease and lymphoma, etc. In some embodiments, the invention relates to the treatment of a T-cell medicated chronic immune disease, wherein the T-cell medicated chronic immune disease is selected from psoriasis, psoriatic arthritis, eczema (atopic dermatitis), urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, and lymphoma; wherein the PCSK9 inhibitor is a PCSK9 small molecule inhibitor, a PCSK9 monoclonal antibody or a siPCSK9; wherein the PCSK9 inhibitor can be used alone or in combination with other therapeutic agents, including traditional drugs and other new biological agents.

[0037] In an embodiment, a PCSK9 inhibitor is used for treating an inflammatory immune disease, wherein the inflammatory immune disease is selected from the group consisting of psoriasis, psoriatic arthritis, eczema, atopic dermatitis, urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, and lymphoma.

[0038] In an embodiment, the invention is related to a method of treating an inflammatory immune disease by administration of a therapeutically effective amount of a PCSK9 inhibitor to a human in need thereof, wherein the inflammatory immune disease is selected from the group consisting of psoriasis, psoriatic arthritis, eczema, atopic dermatitis, urticaria, glucocorticoid dependent dermatitis, rheumatoid arthritis, scleroderma, diabetes, chronic liver disease, and lymphoma.

[0039] In an embodiment, the PCSK9 inhibitor is selected from the group consisting of a PCSK9 monoclonal antibody, a siPCSK9, a PCSK9 small molecule inhibitor and a PCSK9 vaccine.

[0040] In an embodiment, the PCSK9 inhibitor is a PCSK9 monoclonal antibody.

[0041] In an embodiment, the PCSK9 inhibitor is a siPCSK9.

[0042] In an embodiment, the PCSK9 inhibitor is a PCSK9 small molecule inhibitor.

[0043] In an embodiment, the PCSK9 inhibitor is a PCSK9 vaccine.

[0044] In some embodiments, a PCSK9 inhibitor selected from a PCSK9 monoclonal antibody, a siPCSK9 and a PCSK9 small molecule inhibitor is used for treating psoriasis, atopic dermatitis or urticaria. In some embodiment, the PCSK9 inhibitor was applied topically on the skin.

[0045] In an embodiment, the invention relates to a method of treating psoriasis by topical use of a PCSK9 inhibitor, wherein the PCSK9 inhibitor is selected from the group consisting of a PCSK9 monoclonal antibody, a siPCSK9 and a PCSK9 small molecule inhibitor. In an embodiment, the PCSK9 inhibitor is a PCSK9 vaccine.

[0046] In an embodiment, the inhibition of PCSK9 expression by siPCSK9 can significantly inhibit the abnormal proliferation of human keratinocytes and promote their apoptosis through NFkb pathway. In an embodiment, the invention relates to a method of treating keratinocytes by the use of siPCSK9.

[0047] In an embodiment, the PCSK9 small molecule inhibitor is selected from the group consisting of

##STR00001##

[0048] In an embodiment, the PCSK9 small molecule inhibitor is selected from the compounds listed in Table 1. The patent application number cited in Table 1 are incorporated by reference in their entirety.

TABLE-US-00001 TABLE 1 Compound Patent Application No. Structural formula CAS No. 1 [00002] 1632250-49-7 WO2014170786 CN105143203 2 [00003] 1629166-02-4 US20150376139 WO2016107603 WO2016107602 WO2014150395 3 [00004] SBC115076 US2014022957 CN105228616 4 [00005] 1962177-03-2 WO2016107603 5 [00006] 1000339-97-8 WO2017034997 6 [00007] 7 [00008] 2087490-99-9 WO2017034994 8 [00009] 2087440-16-0 WO2017034990 WO2017034994 9 [00010] 423148-46-3 WO2017222953 10 [00011] 130305-64-5 EP372776

[0049] In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody selected from the group consisting of Alirocumab, Evolocumab, Bococizumab, DS-001, CA-001, MS-001, LY3015014, NS-001, and antigen binding fragments, variants, conjugates or biosimilars thereof.

[0050] In an embodiment, the PCSK9 inhibitor is Alirocumab or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:1 and SEQ ID NO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:1 and SEQ ID NO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:1 and SEQ ID NO:2. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:1 and SEQ ID NO:2.

[0051] In an embodiment, the PCSK9 inhibitor is Evolocumab or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0052] In an embodiment, the PCSK9 inhibitor is Bococizumab or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0053] In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15

[0054] In an embodiment, the PCSK9 inhibitor is DS-001 or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, DS-001 is a PCSK9 antibody comprising SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0055] In an embodiment, the PCSK9 inhibitor is CA-001 or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, CA-001 is a PCSK9 antibody comprising SEQ ID NO:20 and SEQ ID NO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:20 and SEQ ID NO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:20 and SEQ ID NO:21. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:20 and SEQ ID NO:21.

[0056] In an embodiment, the PCSK9 inhibitor is MS-001 or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, MS-001 is a PCSK9 antibody comprising SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, MS-001 is a PCSK9 antibody comprising SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0057] In an embodiment, the PCSK9 inhibitor is LY3015014 or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, LY3015014 is a PCSK9 antibody comprising SEQ ID NO:34 and SEQ ID NO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:34 and SEQ ID NO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:34 and SEQ ID NO:35. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:34 and SEQ ID NO:35.

[0058] In an embodiment, the PCSK9 inhibitor is NS-001 or antigen binding fragments, variants, conjugates or biosimilars thereof. In an embodiment, NS-001 is a PCSK9 antibody comprising SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 99% identical to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 95% identical to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41. In an embodiment, the PCSK9 inhibitor is a PCSK9 antibody comprising sequences at least 90% identical to SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

TABLE-US-00002 TABLE2 SEQ IDNO. Description Sequence 1 Alirocumab GluMetGlnLeuValGluSerGlyGlyGlyLeuValGlnProGly Variablezonein GlySerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSer heavychain SerHisTrpMetLysTrpValArgGlnAlaProGlyLysGlyLeu GluTrpValAlaAsnIleAsnGlnAspGlySerGluLysTyrTyrVal AspSerValLysGlyArgPheThrIleSerArgAspAsnAlaLys AsnSerLeuPheLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrTyrCysAlaArgAspIleValLeuMetValTyrAsp MetAspTyrTyrTyrTyrGlyMetAspValTrpGlyGlnGlyThr ThrValThrValSerSer 2 Alirocumab AspIleValMetThrGlnSerProLeuSerLeuProValThrProGly Variablezonein GluProAlaSerIleSerCysArgSerSerGlnSerLeuLeuHisSer lightchain AsnGlyAsnAsnTyrLeuAspTrpTyrLeuGlnLysProGlyGln SerProGlnLeuLeuIleTyrLeuGlySerAsnArgAlaSerGlyVal ProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeu LysIleSerArgValGluAlaGluAspValGlyValTyrTyrCysMet GlnThrLeuGlnThrProLeuThrPheGlyGlyGlyThrLysVal GluIleLys 3 Evolocumab GluValGlnLeuValGluSerGlyGlyGlyLeuValLysProGly Variablezonein GlySerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSer heavychain SerTyrSerMetAsnTrpValArgGlnAlaProGlyLysGlyLeu GluTrpValSerSerIleSerSerSerSerSerTyrIleSerTyrAla AspSerValLysGlyArgPheThrIleSerArgAspAsnAlaLys AsnSerLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrPheCysAlaArgAspTyrAspPheTrpSerAlaTyr TyrAspAlaPheAspValTrpGlyGlnGlyThrMetValThrVal SerSer 4 Evolocumab GlnSerValLeuThrGlnProProSerValSerGlyAlaProGlyGln Variablezonein ArgValThrIleSerCysThrGlySerSerSerAsnIleGlyAlaGly lightchain TyrAspValHisTrpTyrGlnGlnLeuProGlyThrAlaProLys LeuLeuIleSerGlyAsnSerAsnArgProSerGlyValProAsp ArgPheSerGlySerLysSerGlyThrSerAlaSerLeuAlaIleThr GlyLeuGlnAlaGluAspGluAlaAspTyrTyrCysGlnSerTyr AspSerSerLeuSerGlySerValPheGlyGlyGlyThrLysLeu ThrValLeu 5 Evolocumab GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly Variablezonein AlaSerValLysValSerCysLysAlaSerGlyTyrThrLeuThrSer heavychain TyrGlyIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrp MetGlyTrpValSerPheTyrAsnGlyAsnThrAsnTyrAlaGln LysLeuGlnGlyArgGlyThrMetThrThrAspProSerThrSer ThrAlaTyrMetGluLeuArgSerLeuArgSerAspAspThrAla ValTyrTyrCysAlaArgGlyTyrGlyMetAspValTrpGlyGln GlyThrThrValThrValSerSer 6 Evolocumab GlnSerAlaLeuThrGlnProAlaSerValSerGlySerProGlyGln Variablezonein SerIleThrIleSerCysThrGlyThrSerSerAspValGlyGlyTyr lightchain AsnSerValSerTrpTyrGlnGlnHisProGlyLysAlaProLys LeuMetIleTyrGluValSerAsnArgProSerGlyValSerAsnArg PheSerGlySerLysSerGlyAsnThrAlaSerLeuThrIleSerGly LeuGlnAlaGluAspGluAlaAspTyrTyrCysAsnSerTyrThr SerThrSerMetValPheGlyGlyGlyThrLysLeuThrValLeu 7 Bococizumab SerTyrTyrMetHis Heavychain: CDR1 8 Bococizumab GlyTyrThrPheThrSerTyrTyrMetHis Heavychain: CDR1 9 Bococizumab GlyTyrThrPheThrSerTyr Heavychain: CDR1 10 Bococizumab SerProPheGlyGlyArg Heavychain: CDR2 11 Bococizumab GluIleSerProPheGlyGlyArgThrAsnTyrAsnGluLysPhe Heavychain: LysSer CDR2 12 Bococizumab GluArgProLeuTyrAlaSerAspLeu Heavychain: CDR3 13 Bococizumab ArgAlaSerGlnGlyIleSerSerAlaLeuAla Lightchain: CDR1 14 Bococizumab SerAlaSerTyrArgTyrThr Lightchain: CDR2 15 Bococizumab GlnGlnArgTyrSerLeuTrpArgThr Lightchain: CDR3 16 DS-001 AlaSerAspGluGluIleGlnAspValSerGlyThrTrpTyrLeu LysAlaMetThrValAspArgPheLysIleAlaSerTrpProArgSer ValThrProMetThrLeuThrThrLeuGluGlyGlyAsnLeuGlu AlaLysValThrMetAsnTrpTrpGlyArgSerGlnGluValLys AlaValLeuGluArgThrAspGluProGlyLysTyrThrAlaGln GlyAspArgHisValAlaTyrIleIleArgSerProValLysAspHis TyrIlePheTyrSerGluGlyAsnLeuGlnGlyGluThrValPro GlyValTrpLeuValGlyArgAspProLysAsnAsnLeuGluAla LeuGluAspPheGluLysAlaAlaGlyAlaArgGlyLeuSerThr GluSerIleLeuIleProArgGlnSerGluThrSerSerProGly 17 DS-001 GlyAlaSerAspGluGluIleGlnAspValSerGlyThrTrpTyr LeuLysAlaMetThrValAspArgPheLysIleAlaSerTrpPro ArgSerValThrProMetThrLeuThrThrLeuGluGlyGlyAsn LeuGluAlaLysValThrMetAsnTrpTrpGlyArgSerGlnGlu ValLysAlaValLeuGluArgThrAspGluProGlyLysTyrThr AlaGlnGlyAspArgHisValAlaTyrIleIleArgSerHisValLys AspHisTyrIlePheTyrSerGluGlyAsnLeuGlnGlyGluThr ValProGlyValTrpLeuValGlyArgAspProLysAsnAsnLeu GluAlaLeuGluAspPheGluLysAlaAlaGlyAlaArgGlyLeu SerThrGluSerIleLeuIleProArgGlnSerGluThrSerSerPro Gly 18 DS-001 AlaSerAspGluGluIleGlnAspValSerGlyThrTrpTyrLeu LysAlaMetThrValAspArgPheLysIleAlaSerTrpProArgSer ValThrProMetThrLeuThrThrLeuGluGlyGlyAsnLeuGlu AlaLysValThrMetAsnTrpTrpGlyArgSerGlnGlnValLys AlaValLeuGluArgThrAspGluProGlyLysTyrThrAlaGln GlyAspArgHisValAlaTyrIleIleArgSerProValLysAspHis TyrIlePheTyrSerGluGlyAsnLeuGlnGlyGluThrValPro GlyValTrpLeuValGlyArgAspProLysAsnAsnLeuGluAla LeuGluAspPheGluLysAlaAlaGlyAlaArgGlyLeuSerThr GluSerIleLeuIleProArgGlnSerGluThrSerSerProGlyGly GlyGlySerLeuAlaGluAlaLysGluAlaAlaAsnAlaGluLeu AspSerTyrGlyValSerAspPheTyrLysArgLeuIleAspLys AlaLysThrValGluGlyValGluAlaLeuLysAspAlaIleLeu AlaAlaLeuProGly 19 DS-001 GlyAlaSerAspGluGluIleGlnAspValSerGlyThrTrpTyr LeuLysAlaMetThrValAspArgPheLysIleAlaSerTrpPro ArgSerValThrProMetThrLeuThrThrLeuGluGlyGlyAsn LeuGluAlaLysValThrMetAsnTrpTrpGlyArgSerGlnGlu ValLysAlaValLeuGluArgThrAspGluProGlyLysTyrThr AlaGlnGlyAspArgHisValAlaTyrIleIleArgSerHisValLys AspHisTyrIlePheTyrSerGluGlyAsnLeuGlnGlyGluThr ValProGlyValTrpLeuValGlyArgAspProLysAsnAsnLeu GluAlaLeuGluAspPheGluLysAlaAlaGlyAlaArgGlyLeu SerThrGluSerIleLeuIleProArgGlnSerGluThrSerSerPro GlyLysLeuGlyGlyGlyGlySerLeuAlaGluAlaLysGluAla AlaAsnAlaGluLeuAspSerTyrGlyValSerAspPheTyrLys ArgLeuIleAspLysAlaLysThrValGluGlyValGluAlaLeu LysAspAlaIleLeuAlaAlaLeuProGlyLysLeuAsn 20 CA-001 MetGlyArgGlnLeuAlaGlyCysGlyAspAlaGlyLysLysAla SerPheLysMetSerThrValHisGluIleLeuCysLysLeuSer LeuGluGlyAspHisSerThrProProSerAlaTyrGlySerValLys AlaTyrThrAsnPheAspAlaGluArgAspAlaLeuAsnIleGlu ThrAlaIleLysThrLysGlyValAspGluValThrIleValAsnIle LeuThrAsnArgSerAsnAlaGlnArgGlnAspIleAlaPheAla TyrGlnArgArgThrLysLysGluLeuAlaSerAlaLeuLysSer AlaLeuSerGlyHisLeuGluThrValIleLeuGlyLeuLeuLys ThrProAlaGlnTyrAspAlaSerGluLeuLysAlaSerMetLys GlyLeuGlyThrAspGluAspSerLeuIleGluIleIleCysSerArg ThrAsnGlnGluLeuGlnGluIleAsnArgValTyrLysGluMet TyrLysThrAspLeuGluLysAspIleIleSerAspThrSerGlyAsp PheArgLysLeuMetValAlaLeuAlaLysGlyArgArgAlaGlu AspGlySerValIleAspTyrGluLeuIleAspGlnAspAlaArg AspLeuTyrAspAlaGlyValLysArgLysGlyThrAspValPro LysTrpIleSerIleMetThrGluArgSerValProHisLeuGlnLys ValPheAspArgTyrLysSerTyrSerProTyrAspMetLeuGlu SerIleArgLysGluValLysGlyAspLeuGluAsnAlaPheLeu AsnLeuValGlnCysIleGlnAsnLysProLeuTyrPheAlaAsp ArgLeuTyrAspSerMetLysGlyLysGlyThrArgAspLysVal LeuIleArgIleMetValSerArgSerGluValAspMetLeuLysIle ArgSerGluPheLysArgLysTyrGlyLysSerLeuTyrTyrTyrIle GlnGlnAspThrLysGlyAspTyrGlnLysAlaLeuLeuTyrLeu CysGlyGlyAspAsp 21 CA-001 MetSerThrValHisGluIleLeuCysLysLeuSerLeuGluGly AspHisSerThrProProSerAlaTyrGlySerValLysAlaTyrThr AsnPheAspAlaGluArgAspAlaLeuAsnIleGluThrAlaIle LysThrLysGlyValAspGluValThrIleValAsnIleLeuThrAsn ArgSerAsnAlaGlnArgGlnAspIleAlaPheAlaTyrGlnArg ArgThrLysLysGluLeuAlaSerAlaLeuLysSerAlaLeuSer GlyHisLeuGluThrValIleLeuGlyLeuLeuLysThrProAla GlnTyrAspAlaSerGluLeuLysAlaSerMetLysGlyLeuGly ThrAspGluAspSerLeuIleGluIleIleCysSerArgThrAsnGln GluLeuGlnGluIleAsnArgValTyrLysGluMetTyrLysThr AspLeuGluLysAspIleIleSerAspThrSerGlyAspPheArg LysLeuMetValAlaLeuAlaLysGlyArgArgAlaGluAspGly SerValIleAspTyrGluLeuIleAspGlnAspAlaArgAspLeuTyr AspAlaGlyValLysArgLysGlyThrAspValProLysTrpIleSer IleMetThrGluArgSerValProHisLeuGluLysValPheAsp ArgTyrLysSerTyrSerProTyrAspMetLeuGluSerIleArgLys GluValLysGlyAspLeuGluAsnAlaPheLeuAsnLeuValGln CysIleGlnAsnLysProLeuTyrPheAlaAspArgLeuTyrAsp SerMetLysGlyLysGlyThrArgAspLysValLeuIleArgIle MetValSerArgSerGluValAspMetLeuLysIleArgSerGlu PheLysArgLysTyrGlyLysSerLeuTyrTyrTyrIleGlnGln AspThrLysGlyAspTyrGlnLysAlaLeuLeuTyrLeuCysGly GlyAspAsp 22 MS-001 MetGluLysMetLeuAlaGlyCysPheLeuLeuIleLeuGlyGln IleValLeuLeuProAlaGluAlaArgGluArgSerArgGlyArg SerIleSerArgGlyArgHisAlaArgThrHisProGlnThrAlaLeu LeuGluSerSerCysGluAsnLysArgAlaAspLeuValPheIleIle AspSerSerArgSerValAsnThrHisAspTyrAlaLysValLys GluPheIleValAspIleLeuGlnPheLeuAspIleGlyProAspVal ThrArgValGlyLeuLeuGlnTyrGlySerThrValLysAsnGlu PheSerLeuLysThrPheLysArgLysSerGluValGluArgAla ValLysArgMetArgHisLeuSerThrGlyThrMetThrGlyLeu AlaIleGlnTyrAlaLeuAsnIleAlaPheSerGluAlaGluGlyAla ArgProLeuArgGluAsnValProArgValIleMetIleValThrAsp GlyArgProGlnAspSerValAlaGluValAlaAlaLysAlaArg AspThrGlyIleLeuIlePheAlaIleGlyValGlyGlnValAspPhe AsnThrLeuLysSerIleGlySerGluProHisGluAspHisVal PheLeuValAlaAsnPheSerGlnIleGluThrLeuThrSerVal PheGlnLysLysLeuCysIleAsnIleMetCysSerThrLeuGlu HisAsnCysAlaHisPheCysIleAsnIleProGlySerTyrValCys ArgCysLysGlnGlyTyrIleLeuAsnSerAspGlnThrThrCys ArgIleGlnAspLeuCysAlaMetGluAspHisAsnCysGluGln LeuCysValAsnValProGlySerPheValCysGlnCysTyrSer GlyTyrAlaLeuAlaGluAspGlyLysArgCysValAlaValAsp TyrCysAlaSerGluAsnHisGlyCysGluHisGluCysValAsn AlaAspGlySerTyrLeuCysGlnCysHisGluGlyPheAlaLeu AsnProAspLysLysThrCysThrLysIleAspTyrCysAlaSer SerAsnHisGlyCysGlnHisGluCysValAsnThrAspAspSer TyrSerCysHisCysLeuLysGlyPheThrLeuAsnProAspLys LysThrCysArgArgIleAsnTyrCysAlaLeuAsnLysProGly CysGluHisGluCysValAsnMetGluGluSerTyrTyrCysArg CysHisArgGlyTyrThrLeuAspProAsnGlyLysThrCysSer ArgValAspHisCysAlaGlnGlnAspHisGlyCysGluGlnLeu CysLeuAsnThrGluAspSerPheValCysGlnCysSerGluGly PheLeuIleAsnGluAspLeuLysTyrCysSerArgValAspTyr CysLeuLeuSerAspHisGlyCysGluTyrSerCysValAsnMet AspArgSerPheAlaCysGlnCysProGluGlyHisValLeuArg SerAspGlyLysThrCysAlaLysLeuAspSerCysAlaLeuGly AspHisGlyCysGluHisSerCysValSerSerGluAspSerPhe ValCysGlnCysPheGluGlyTyrIleLeuArgGluAspGlyLys ThrCysArgArgLysAspValCysGlnAlaIleAspHisGlyCys GluHisIleCysValAsnSerAspAspSerTyrThrCysGluCys LeuGluGlyPheArgLeuAlaGluAspGlyLysArgCysArgArg LysAspValCysLysSerThrHisHisGlyCysGluHisIleCys ValAsnAsnGlyAsnSerTyrIleCysLysCysSerGluGlyPhe ValLeuAlaGluAspGlyArgArgCysLysLysCysThrGluGly ProIleAspLeuValPheValIleAspGlySerLysSerLeuGlyGlu GluAsnPheGluValValLysGlnPheValThrGlyIleIleAspSer LeuThrIleSerProLysAlaAlaArgValGlyLeuLeuGlnTyrSer ThrGlnValHisThrGluPheThrLeuArgAsnPheAsnSerAla LysAspMetLysLysAlaValAlaHisMetLysTyrMetGlyLys GlySerMetThrGlyLeuAlaLeuLysHisMetPheGluArgSer PheThrGlnGlyGluGlyAlaArgProLeuSerThrArgValPro ArgAlaAlaIleValPheThrAspGlyArgAlaGlnAspAspVal SerGluTrpAlaSerLysAlaLysAlaAsnGlyIleThrMetTyrAla ValGlyValGlyLysAlaIleGluGluGluLeuGlnGluIleAlaSer GluProThrAsnLysHisLeuPheTyrAlaGluAspPheSerThr MetAspGluIleSerGluLysLeuLysLysGlyIleCysGluAla LeuGluAspSerAspGlyArgGlnAspSerProAlaGlyGluLeu ProLysThrValGlnGlnProThrGluSerGluProValThrIleAsn IleGlnAspLeuLeuSerCysSerAsnPheAlaValGlnHisArg TyrLeuPheGluGluAspAsnLeuLeuArgSerThrGlnLysLeu SerHisSerThrLysProSerGlySerProLeuGluGluLysHis AspGlnCysLysCysGluAsnLeuIleMetPheGlnAsnLeuAla AsnGluGluValArgLysLeuThrGlnArgLeuGluGluMetThr GlnArgMetGluAlaLeuGluAsnArgLeuArgTyrArg 23 MS-001 MetGluLysMetLeuAlaGlyCysPheLeuLeuIleLeuGlyGln IleValLeuLeuProAlaGluAlaArgGluArgSerArgGlyArg SerIleSerArgGlyArgHisAlaArgThrHisProGlnThrAlaLeu LeuGluSerSerCysGluAsnLysArgAlaAspLeuValPheIleIle AspSerSerArgSerValAsnThrHisAspTyrAlaLysValLys GluPheIleValAspIleLeuGlnPheLeuAspIleGlyProAspVal ThrArgValGlyLeuLeuGlnTyrGlySerThrValLysAsnGlu PheSerLeuLysThrPheLysArgLysSerGluValGluArgAla ValLysArgMetArgHisLeuSerThrGlyThrMetThrGlyLeu AlaIleGlnTyrAlaLeuAsnIleAlaPheSerGluAlaGluGlyAla ArgProLeuArgGluAsnValProArgValIleMetIleValThrAsp GlyArgProGlnAspSerValAlaGluValAlaAlaLysAlaArg AspThrGlyIleLeuIlePheAlaIleGlyValGlyGlnValAspPhe AsnThrLeuLysSerIleGlySerGluProHisGluAspHisVal PheLeuValAlaAsnPheSerGlnIleGluThrLeuThrSerVal PheGlnLysLysLeuCysThrAlaHisMetCysSerThrLeuGlu HisAsnCysAlaHisPheCysIleAsnIleProGlySerTyrValCys ArgCysLysGlnGlyTyrIleLeuAsnSerAspGlnThrThrCys ArgIleGlnAspLeuCysAlaMetGluAspHisAsnCysGluGln LeuCysValAsnValProGlySerPheValCysGlnCysTyrSer GlyTyrAlaLeuAlaGluAspGlyLysArgCysValAlaValAsp TyrCysAlaSerGluAsnHisGlyCysGluHisGluCysValAsn AlaAspGlySerTyrLeuCysGlnCysHisGluGlyPheAlaLeu AsnProAspLysLysThrCysThrLysIleAspTyrCysAlaSer SerAsnHisGlyCysGlnHisGluCysValAsnThrAspAspSer TyrSerCysHisCysLeuLysGlyPheThrLeuAsnProAspLys LysThrCysArgArgIleAsnTyrCysAlaLeuAsnLysProGly CysGluHisGluCysValAsnMetGluGluSerTyrTyrCysArg CysHisArgGlyTyrThrLeuAspProAsnGlyLysThrCysSer ArgValAspHisCysAlaGluGluAspHisGlyCysGluGlnLeu CysLeuAsnThrGluAspSerPheValCysGlnCysSerGluGly PheLeuIleAsnGluAspLeuLysThrCysSerArgValAspTyr CysLeuLeuSerAspHisGlyCysGluTyrSerCysValAsnMet AspArgSerPheAlaCysGlnCysProGluGlyHisValLeuArg SerAspGlyLysThrCysAlaLysLeuAspSerCysAlaLeuGly AspHisGlyCysGluHisSerCysValSerSerGluAspSerPhe ValCysGlnCysPheGluGlyTyrIleLeuArgGluAspGlyLys ThrCysArgArgLysAspValCysGlnAlaIleAspHisGlyCys GluHisIleCysValAsnSerAspAspSerTyrThrCysGluCys LeuGluGlyPheArgLeuAlaGluAspGlyLysArgCysArgArg LysAspValCysLysSerThrHisHisGlyCysGluHisIleCys ValAsnAsnGlyAsnSerTyrIleCysLysCysSerGluGlyPhe ValLeuAlaGluAspGlyArgArgCysLysLysCysThrGluGly ProIleAspLeuValPheValIleAspGlySerLysSerLeuGlyGlu GluAsnPheGluValValLysGlnPheValThrGlyIleIleAspSer LeuThrIleSerProLysAlaAlaArgValGlyLeuLeuGlnTyrSer ThrGlnValHisThrGluPheThrLeuArgAsnPheAsnSerAla LysAspMetLysLysAlaValAlaHisMetLysTyrMetGlyLys GlySerMetThrGlyLeuAlaLeuLysHisMetPheGluArgSer PheThrGlnGlyGluGlyAlaArgProLeuSerThrArgValPro ArgAlaAlaIleValPheThrAspGlyArgAlaGlnAspAspVal SerGluTrpAlaSerLysAlaLysAlaAsnGlyIleThrMetTyrAla ValGlyValGlyLysAlaIleGluGluGluLeuGlnGluIleAlaSer GluProThrAsnLysHisLeuPheTyrAlaGluAspPheSerThr MetAspGluIleSerGluLysLeuLysLysGlyIleCysGluAla LeuGluAspSerAspGlyArgGlnAspSerProAlaGlyGluLeu ProLysThrValGlnGlnProThrValGlnHisArgTyrLeuPhe GluGluAspAsnLeuLeuArgSerThrGlnLysLeuSerHisSer ThrLysProSerGlySerProLeuGluGluLysHisAspGlnCys LysCysGluAsnLeuIleMetPheGlnAsnLeuAlaAsnGluGlu ValArgLysLeuThrGlnArgLeuGluGluMetThrGlnArgMet GluAlaLeuGluAsnArgLeuArgTyrArg 24 MS-001variable GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly zoneinheavy GluSerLeuLysIleSerCysLysGlySerGlyTyrSerPheThr chain AsnTyrTrpIleSerTrpValArgGlnMetProGlyLysGlyLeu GluTrpMetGlyIleIleTyrProGlyAspSerTyrThrAsnTyrSer ProSerPheGlnGlyGlnValThrIleSerAlaAspLysSerIleSer ThrAlaTyrLeuGlnTrpSerSerLeuLysAlaSerAspThrAla MetTyrTyrCysAlaArgAspTyrTrpTyrLysProLeuPheAsp IleTrpGlyGlnGlyThrLeuValThrValSerSer 25 MS-001variable AspIleValMetThrGlnSerProAspSerLeuAlaValSerLeu zoneinlightchain GlyGluArgAlaThrIleAsnCysArgSerSerGlnSerValLeuTyr SerSerAsnAsnLysAsnTyrLeuAlaTrpTyrGlnGlnLysPro GlyGlnProProLysLeuLeuIleTyrTrpAlaSerThrArgGluSer GlyValProAspArgPheSerGlySerGlySerGlyThrAspPhe ThrLeuThrIleSerSerLeuGlnAlaGluAspValAlaValTyrTyr CysGlnGlnTyrSerSerPheProIleThrPheGlyGlnGlyThr LysValGluIleLysArg 26 MS-001variable GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGly zoneinheavy GlySerLeuArgLeuSerCysLysAlaSerGlyTyrThrPheSer chain SerTyrGlyMetTyrTrpValArgGlnAlaProGlyLysGlyLeu GluTrpIleGlyTrpIleAspProGlySerGlyGlyThrLysTyrAsn GluLysPheLysGlyLysAlaThrIleSerArgAspAsnSerLys AsnThrLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrTyrCysAlaArgGluArgTyrGlyTyrTyrPheAsp TyrTrpGlyGlnGlyThrLeuValThrValSerSerAlaSer 27 MS-001variable GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGly zoneinheavy GlySerLeuArgLeuSerCysLysAlaSerGlyTyrThrPheSer chain SerTyrGlyMetTyrTrpValArgGlnAlaProGlyLysGlyLeu GluTrpIleGlyTrpIleAspProGlySerGlyGlyThrLysTyrAsn GluLysPheLysGlyLysAlaThrIleSerArgAspAsnSerLys AsnThrLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrTyrCysAlaArgGluArgTyrGlyTyrTyrPheAsp TyrTrpGlyGlnGlyThrLeuValThrValSerSer 28 MS-001variable GluIleValLeuThrGlnSerProAlaThrLeuSerLeuSerProGly zoneinlightchain GluArgAlaThrIleThrCysArgAlaSerGlnTyrValGlySerTyr LeuAsnTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu IleTyrAspAlaSerAsnArgAlaThrGlyIleProAlaArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu GluProGluAspPheAlaValTyrTyrCysGlnValTrpAspSer SerProProValValPheGlyGlyGlyThrLysValGluIleLys 29 MS-001variable GluValGlnLeuLeuGluSerGlyGlyGlyLeuValGlnProGly zoneinheavy GlySerLeuArgLeuSerCysLysAlaSerGlyPheThrPheThr chain SerTyrTyrMetHisTrpValArgGlnAlaProGlyLysGlyLeu GluTrpIleGlyArgIleAsnProAspSerGlySerThrLysTyrAsn GluLysPheLysGlyArgAlaThrIleSerArgAspAsnSerLys AsnThrLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrTyrCysAlaArgGlyGlyArgLeuSerTrpAspPhe AspValTrpGlyGlnGlyThrLeuValThrValSerSer 30 MS-001variab1e AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly zoneinlightchain AspArgValThrIleThrCysArgAlaSerGlnAspIleSerArgTyr LeuAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeu IleTyrAlaAlaSerSerLeuGlnSerGlyValProSerArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu GlnProGluAspPheAlaThrTyrTyrCysAlaAlaTyrAspTyr SerLeuGlyGlyTyrValPheGlyAspGlyThrLysValGluIle Lys 31 MS-001variable AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly zoneinlightchain AspArgValThrIleThrCysArgAlaSerGlnAspIleSerArgTyr LeuAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeu IleTyrAlaAlaSerSerLeuGlnSerGlyValProSerArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu GlnProGluAspPheAlaThrTyrTyrCysAlaAlaTyrAspTyr SerLeuGlyGlyTyrValPheGlyGlyGlyThrLysValGluIle Lys 32 MS-001variable GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly zoneinheavy SerSerValLysValSerCysLysAlaSerGlyGlyThrPheAsnSer chain HisAlaIleSerTrpValArgGlnAlaProGlyGlnGlyLeuGluTrp MetGlyGlyIleAsnProIleLeuGlyIleAlaAsnTyrAlaGlnLys PheGlnGlyArgValThrIleThrAlaAspGluSerThrSerThrAla TyrMetGluLeuSerSerLeuArgSerGluAspThrAlaValTyr TyrCysAlaArgHisTyrGluIleGlnIleGlyArgTyrGlyMetAsn ValTyrTyrLeuMetTyrArgPheAlaSerTrpGlyGlnGlyThr LeuValThrValSerSer 33 MS-001variable AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly zoneinlightchain AspArgValThrIleThrCysArgAlaSerGlnGlyIleArgSerAla LeuAsnTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeu IleTyrAsnGlySerThrLeuGlnSerGlyValProSerArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleSerSerLeu GlnProGluAspPheAlaValTyrTyrCysGlnGlnPheAspGly AspProThrPheGlyGlnGlyThrLysValGluIleLysArg 34 LY3015014 GluValGlnLeuValGluSerGlyGlyGlyLeuValLysProGly Heavychain GlySerLeuArgLeuSerCysAlaAlaSerGlyPheProPheSer LysLeuGlyMetValTrpValArgGlnAlaProGlyLysGlyLeu GluTrpValSerThrIleSerSerGlyGlyGlyTyrThrTyrTyrPro AspSerValLysGlyArgPheThrIleSerArgAspAsnAlaLys AsnSerLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAspThr AlaValTyrTyrCysAlaArgGluGlyIleSerPheGlnGlyGlyThr TyrThrTyrValMetAspTyrTrpGlyGlnGlyThrLeuValThr ValSerSerAlaSerThrLysGlyProSerValPheProLeuAlaPro CysSerArgSerThrSerGluSerThrAlaAlaLeuGlyCysLeu ValLysAspTyrPheProGluProValThrValSerTrpAsnSerGly AlaLeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSer GlyLeuTyrSerLeuSerSerValValThrValProSerSerSerLeu GlyThrLysThrTyrThrCysAsnValAspHisLysProSerAsn ThrLysValAspLysArgValGluSerLysTyrGlyProProCys ProProCysProAlaProGluAlaAlaGlyGlyProSerValPhe LeuPheProProLysProLysAspThrLeuMetIleSerArgThr ProGluValThrCysValValValAspValSerGlnGluAspPro GluValGlnPheAsnTrpTyrValAspGlyValGluValHisAsn AlaLysThrLysProArgGluGluGlnPheAsnSerThrTyrArg ValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGly LysGluTyrLysCysLysValSerAsnLysGlyLeuProSerSerIle GluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnVal TyrThrLeuProProSerGlnGluGluMetThrLysAsnGlnVal SerLeuThrCysLeuValLysGlyPheTyrProSerAspIleAlaVal GluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThr ProProValLeuAspSerAspGlySerPhePheLeuTyrSerArg LeuThrValAspLysSerArgTrpGlnGluGlyAsnValPheSer CysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLys SerLeuSerLeuSerLeuGly 35 LY3015014Light AspIleValMetThrGlnSerProLeuSerLeuProValThrProGly chain GluProAlaSerIleSerCysArgSerSerLysSerLeuLeuHisArg AsnGlyIleThrTyrSerTyrTrpTyrLeuGlnLysProGlyGlnSer ProGlnLeuLeuIleTyrGlnLeuSerAsnLeuAlaSerGlyVal ProAspArgPheSerGlySerGlySerGlyThrAspPheThrLeu LysIleSerArgValGluAlaGluAspValGlyValTyrTyrCysTyr GlnAsnLeuGluLeuProLeuThrPheGlyGlnGlyThrLysVal GluIleLysArgThrValAlaAlaProSerValPheIlePheProPro SerAspGluGlnLeuLysSerGlyThrAlaSerValValCysLeu LeuAsnAsnPheTyrProArgGluAlaLysValGlnTrpLysVal AspAsnAlaLeuGlnSerGlyAsnSerGlnGluSerValThrGlu GlnAspSerLysAspSerThrTyrSerLeuSerSerThrLeuThr LeuSerLysAlaAspTyrGluLysHisLysValTyrAlaCysGlu ValThrHisGlnGlyLeuSerSerProValThrLysSerPheAsn ArgGlyGluCys 36 NS-001variable GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly zoneinheavy AlaSerValLysValSerCysLysAlaSerGlyTyrThrPheSerThr chain MetTyrMetSerTrpValArgGlnAlaProGlyGlnGlyLeuGlu TrpMetGlyArgIleAspProAlaAsnGluHisThrAsnTyrAla GlnLysPheGlnGlyArgValThrMetThrArgAspThrSerIle SerThrAlaTyrMetGluLeuSerArgLeuThrSerAspAspThr AlaValTyrTyrCysAlaArgSerTyrTyrTyrTyrAsnMetAsp TyrTrpGlyGlnGlyThrLeuValThrValSerSer 37 NS-001variable GlnIleValLeuThrGlnSerProAlaThrLeuSerValSerProGly zoneinlightchain GluArgAlaThrLeuSerCysArgAlaSerGlnSerValSerTyr MetHisTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu IleTyrGlyValPheArgArgAlaThrGlyIleProAspArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleGlyArgLeu GluProGluAspPheAlaValTyrTyrCysLeuGlnTrpSerSer AspProProThrPheGlyGlnGlyThrLysLeuGluIleLys 38 NS-001variable GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly zoneinheavy AlaSerValLysValSerCysLysAlaSerGlyTyrThrPheSerThr chain MetTyrMetSerTrpValArgGlnAlaProGlyGlnGlyLeuGlu TrpMetGlyArgIleAspProAlaAsnGluHisThrAsnTyrAla GlnLysPheGlnGlyArgValThrMetThrArgAspThrSerIle SerThrAlaTyrMetGluLeuSerArgLeuThrSerAspAspThr AlaValTyrTyrCysAlaArgSerTyrTyrTyrTyrAsnMetAsp TyrTrpGlyGlnGlyThrLeuValThrValSerSer 39 NS-001variable GluIleValMetThrGlnSerProAlaThrLeuSerValSerProGly zoneinlightchain GluArgAlaThrLeuSerCysArgAlaSerGlnSerValSerTyr MetHisTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu IleTyrGlyValPheArgArgAlaThrGlyIleProAspArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleGlyArgLeu GluProGluAspPheAlaValTyrTyrCysLeuGlnTrpSerSer AspProProThrPheGlyGlnGlyThrLysLeuGluIleLys 40 NS-001variable GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGly zoneinheavy AlaSerValLysValSerCysLysAlaSerGlyTyrThrPheSerThr chain MetTyrMetSerTrpValArgGlnAlaProGlyGlnGlyLeuGlu TrpMetGlyArgIleAspProAlaAsnGluHisThrAsnTyrAla GlnLysPheGlnGlyArgValThrMetThrArgAspThrSerIle SerThrAlaTyrMetGluLeuSerArgLeuThrSerAspAspThr AlaValTyrTyrCysAlaArgSerTyrTyrTyrTyrAlaMetAspTyr TrpGlyGlnGlyThrLeuValThrValSerSer 41 NS-001variable GlnIleValLeuThrGlnSerProAlaThrLeuSerValSerProGly zoneinlightchain GluArgAlaThrLeuSerCysArgAlaSerGlnSerValSerTyr MetHisTrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeu IleTyrGlyValPheArgArgAlaThrGlyIleProAspArgPheSer GlySerGlySerGlyThrAspPheThrLeuThrIleGlyArgLeu GluProGluAspPheAlaValTyrTyrCysLeuGlnTrpSerSer AspProPro

Beneficial Effect

[0059] The invention provides a new and better treatment method for the treatment of inflammatory immune diseases. Through the disclosure of the invention, the system or topical PCSK9 inhibitor can be further prepared, and a new monomer drug or compound preparation containing various PCSK9 inhibitors can be developed. Furthermore, new monomers or compound formulations containing all kinds of PCSK9 inhibitors have been developed for the treatment of various types of inflammatory immune diseases, especially psoriasis, eczema and urticaria. Clinical trials have shown that these drugs, which contain PCSK9 inhibitors, are effective, have little adverse reactions, and are well tolerated in patients. In particular, topical use alone can significantly improve lesions in immune diseases such as psoriasis. It is very suitable for the current clinical demand and is expected to have great application prospect, which will bring more advantages to the patients with inflammatory and immunological diseases.

V. EXAMPLES

[0060] The following specific examples illustrate the embodiments of this invention. Technical personnel in this field can easily understand the other advantages and effects of the invention from the contents disclosed in this invention. The invention is not limited to the embodiments described herein. Before further describing the embodiments of this invention, it should be understood that the scope of protection of the invention is not limited to the following specific embodiment. It should be also understood that the term used in the specific examples of the invention is used to describe a specific embodiment rather than to limit the protection scope of the invention. In the description and claims of the invention, unless expressly stated in the text, the singular form one and this include plural forms.

[0061] It should be understood that when a numerical range is given in an example, unless stated by the invention, the two endpoints and any between the two endpoints of each numerical range can be selected. Unless defined herein, all technical and scientific terms used in the invention have the same meaning as those commonly understood by those in the technical field. Except for specific methods, equipment, materials used in the embodiment, according to the description of the invention, one skilled in the art would understand the use of the equivalents of the methods, equipment, and materials described herein.

Example 1

PCSK9 Knockout Significantly Alleviates Psoriasis-Like Inflammation Reaction Induced by IMQ in Mice

[0062] Reagents: 5% imiquimod cream (IMQ), PCSK9 antibody (abcam), NF-kB antibody (abcam)

[0063] Experimental animal: C57BL/6 (B6) mice, 7 male; C57BL/6-PCSK9-r-mice, 5 males and 5 females. The mice were purchased at the Jackson Institute in Maine, USA (The Jackson Laboratory).

[0064] Experimental methods:

[0065] 1. After two groups of mice with different genotypes were treated with back hair removal, 5% Imiquimod cream of 62.5 mg was applied daily for 5 days.

[0066] 2. Scores (the score of erythema, scales, skin lesions thickening and total score) were taken and archived (the score was obtained by two researchers respectively then average score was calculated) before and after the application.

[0067] 3. On the last day of the experiment, all the mice were eulogized and the skin tissue of the back (treated and untreated) was taken.

[0068] 4. The morphology of skin lesions in each group was observed by HE staining and the expression and distribution of PCSK9 and NF-kB in skin lesions of 4 groups were measured by immunofluorescence method.

[0069] Experimental results:

[0070] 1. After 5 days of continuous application, there were obvious erythema, scales and infiltration in the back of 7 C57BL/6 mice, which were in accordance with the appearance of psoriasis-like skin lesions. In 10 PCSK9 knockout mice, there were only slight erythema, scales and infiltration in the back coating area (See FIG. 1).

[0071] 2. A daily score was given on the skin lesions of the drug-coated area on the back of the mice, the score of erythema, scale, infiltration and total score of C57BL/6 mice were significantly higher than those of PCSK9 knockout mice (P<0.05).

[0072] 3. After 5 days of application, the skin tissues of the mice's back (treated and untreated) were taken for HE staining and pathological changes were observed. The skin of IMQ treatment area of C57BL/6 mice showed typical psoriasis pathological changes, such as skin thickening, dermatoid lengthening, thickening of spinous layer, hyperkeratosis with incomplete keratosis, Kogoj abscess and Munro abscess, etc. However, the skin of PCSK9 gene knockout mice showed mild epidermis thickening and hyperkeratosis, no typical pathological changes of psoriasis, such as extension of dermatoid, thickening of spinous layer, hyperkeratosis with incomplete keratosis, Kogoj abscess and Munro abscess. The untreated skin of both groups showed normal skin structure (see FIG. 3).

Example 2

[0073] Transfection of si-PCSK9 Enhances Apoptosis and Inhibits Proliferation of Human Keratinocytes.

[0074] Experimental materials and reagents:

[0075] (1) Human primary keratinocytes (Lifeline Cell Technology, FC-0064)

[0076] (2) DermaLife Culture medium of keratinocyte (Lifeline Cell Technology, LL-0007), si-PCSK9 (Santa Cruz,sc-45482), Lipofectamine 3000 transfection reagent (ThermalFisher, L-3000001), AnnexinV (BD), PI (BD).

[0077] Experimental methods:

[0078] 1. Cultured human keratinocytes were replanted in 6 wells plates. When the cell density was 60-70%, si-RNA (si-Con and siPCSK9) transfection was carried out. After 24 h/48 h/72 h of transfection, measured the cell activity of each pore by MTT method. 3 wells were selected at each time point for each group, the average value was taken and the cell activity curve was plotted.

[0079] 2. Cultured human keratinocytes were replanted in 6 wells plates. When the cell density was 60-70%, si-RNA (si-Con & si-PCSK9) transfection was carried out. After 24 h/48 h/72 h of transfection, cells were harvested respectively for AnnexinV and PI staining to detect cell apoptosis and cell cycle. 3 wells were selected at each time point for each group, the average value was taken.

[0080] Experimental results:

[0081] 1. The number of survival cells of human primary keratinocytes transfected with si-PCSK9 was significantly lower than that of si-Con transfection (P<0.05, See FIG. 6).

[0082] 2. The apoptosis of primary keratinocytes transfected with si-PCSK9 was significantly higher than that of si-Con transfection (P<0.05), while proliferating (the proportion of S+G2/M phase cells) decreased significantly (see FIG. 7).

Example 3

[0083] The Expression of PCSK9 in Psoriatic Lesions was Significantly Higher than that in Non-Lesions and Normal Controls.

[0084] Experiment materials: lesions and non-lesion from 30 psoriasis patients, skin samples from 30 normal people were treated with PCSK9 antibody (abcam).

[0085] Methods: immunohistochemistry, real time quantitative-PCR.

[0086] Results: the skin of patients with skin lesions and non-lesions was extracted by drilling method. The skin of normal people originated from the redundant skin of cosmetic surgery. The results of immunohistochemistry showed that the expression of PCSK9 in psoriatic lesions was significantly higher than that in non-lesions and normal controls. PCSK9 positive cells were mainly distributed in epidermis and dermis near epidermis, but not in dermis vessels (see FIG. 8). RNA was extracted from skin homogenate. Q-PCR detection showed that the expression of PCSK9 in psoriatic lesions group was higher than that in psoriatic non-lesion group and normal control group (P<0.05) (see FIG. 9). The results of immunohistochemistry and Q-PCR were consistent.

Example 4

[0087] Expression of PCSK9 in Peripheral Blood Mononuclear Cells and Plasma in Patients with Psoriasis, Eczema and Urticaria.

[0088] Materials: 5 ml peripheral blood from 30 cases of psoriasis, 30 cases of eczema, 30 cases of urticaria, and 30 cases of normal persons was collected respectively.

[0089] Empirical method:

(1) Separation of plasma and separation of peripheral PBMC by density gradient method.
(2) Detection of PCSK9 expression in PBMC by real-time quantitative Q-PCR.
(3) The expression of PCSK9 in plasma was detected by enzyme linked immunosorbent assay (Elisa) (PCSK9 Quantikine ELISA Kit, America R&D)
(4) Results: (1) No PCSK expression was detected in PBMC of the samples of psoriasis, eczema, urticarial and normal people.
(5) PCSK expression in the samples of psoriasis, eczema, and urticaria are much higher than that in the sample of the normal people. PCSK expression is the highest in the samples of psoriasis.

Example 5

[0090] Effect of PCSK9 Protein on the Secretion of IL-17 and IL-2 from Peripheral CD4+T Cells in Patients with Psoriasis, Eczema or Urticaria.

[0091] Materials: peripheral blood of 10 cases of psoriasis, 10 cases of eczema, 10 cases of urticaria, and 10 cases of normal people. ELISA kit was purchased from Raybiotech Company of USA.

[0092] Experimental methods:

[0093] (1) Isolation of CD4.sup.+T cells from peripheral blood:

[0094] After PBMC was isolated by Ficoll-Hypaque from human peripheral blood by density gradient centrifugation, washed with 10 times volume 1BD beads buffer. Then 50 ul BD IMag TM CD4 beads were added to each 107 cells. The beads were mixed well and incubated at room temperature for 30 minutes. 1 ml 1BD magnetic bead buffer solution was added, and the cells were transferred to the round bottom tube and placed in the magnetic frame for 8 to 10 minutes. And then the supernatant was discarded and removed the test tube from the magnetic field. After resuspension of cells attached to tube wall with 1 ml 1BD magnetic bead buffer, the tubes were placed in magnetic field for 2-4 min again. The supernatant was discarded removed it out of the magnetic field, resuspended the cells and placed the tube in the magnetic field for 2-4 min. The cells obtained from the supernatant can be used in subsequent experiments. BD IMag TM CD4 separation system was purchased from BD Biosciences Company.

[0095] (2) Determination of cytokines secreted by CD4.sup.+T cells in Peripheral Blood

[0096] The supernatant was collected after cell culture under different conditions. IFN-Gamma represented TH1 type cytokines, IL-4 represented TH2 type cytokines, and IL-17 represented TH17 cytokines. The detection of IFN-Gamma, IL-4, IL-17 was conducted by ELISA.

[0097] Results: Compared to the normal patients, PCSK9 protein can significantly promote the secretion of IL-17, IL-2 and IFN-Gamma (P<0.01) by CD4 T cells isolated from peripheral blood of patients with psoriasis, eczema and urticaria (FIG. 11).

Example 6

[0098] Subcutaneous Injection of PCSK9 Small Molecule Inhibitor and PCSK9 Monoclonal Antibody Significantly Alleviated Systemic Immune Abnormalities and Skin Lesions Induced by IMQ in Mice. The Efficacy of PCSK9 Small Molecule Inhibitor is Better than that of Subcutaneous Injection of PCSK9 Monoclonal Antibody.

[0099] Reagents:

The PCSK9 small molecule inhibitor used for this example has the formula of:

##STR00012##

[0100] The PCSK9 monoclonal antibody used for this example is Evolocumab. 40 SPF female BALB/c mice aged 6-8 weeks were randomly divided into normal control group, model group, PCSK9 small molecule inhibitor group and PCSK9 monoclonal antibody group. There were 10 mice for each group. After intraperitoneal injection of pentobarbital sodium 80 mg/kg, the back was shaved with an area of about 2 cm3 cm. The mice were feed separately for 1 day.

[0101] (1) In normal control group, Vaseline was applied locally. In model group, inhibitor group and monoclonal antibody group, 62.5 mg of 5% imiquimod cream was applied on the back every day for 6 consecutive days, photos were taken and the PASI score was evaluated.

[0102] (2) On the first day, normal saline was subcutaneously administrated to normal control group and model group, PCSK9 small molecule inhibitor was subcutaneously administrated (8 mg/kg, Purchased from Selleck Inc.) to inhibitor group, and PCSK9 monoclonal antibody was subcutaneously administrated to monoclonal antibody group (10 mg/kg, Purchased from abcam Inc.)

[0103] Experimental results:

[0104] (1) After 6 days of continuous application, there were marked erythema, scales and infiltration in the back of the mice in the model group. However, in PCSK9 small molecule inhibitor group and PCSK9 monoclonal antibody group, there was only mild erythema, scales and infiltration in the back coating area of mice. The effect of PCSK9 small molecule inhibitor group was better than that of PCSK9 monoclonal antibody group. No lesions were observed in the vaseline group (FIG. 12).

[0105] (2) A daily scoring was given on the skin lesions of the drug-coated area on the back of the mice. The score of erythema, scales, infiltration and total score of PCSK9 small molecule inhibitor group (erythema+scales+infiltration) were significantly lower than that of PCSK9 monoclonal antibody group (P<0.01).

Example 7

[0106] Both PCSK9 Small Molecule Inhibitor and PCSK9 Monoclonal Antibody Improved the IMQ Induced Psoriatic Lesions of Mice, and the Efficacy of Small Molecule Inhibitor was Better than that of Monoclonal Antibody

[0107] Reagents:

[0108] The PCSK9 small molecule inhibitor used for this example has the formula of:

##STR00013##

[0109] The PCSK9 monoclonal antibody used for this example is Evolocumab.

[0110] (1) 40 SPF female BALB/c mice aged 6-8 weeks were randomly divided into normal control group, model group, PCSK9 inhibitor group and PCSK9 monoclonal antibody group. There were 10 mice for each group. After intraperitoneal injection of pentobarbital sodium 80 mg/kg, the back was shaved with an area of about 2 cm3 cm. The mice were feed separately for 1 day.

[0111] (2) In normal control group, vaseline was applied locally. In model group, small molecule inhibitor group and monoclonal antibody group, 62.5 mg of 5% imiquimod cream was applied on the back every day for 6 consecutive days, and photos were taken and the PASI score was evaluated.

[0112] (3) Pretreatment: vaseline was applied on the back skin with mics in normal control group and model group, PCSK9 small molecule inhibitor cream (0.01% concentration) was applied to inhibitor group once daily. And, PCSK9 monoclonal antibody cream (0.01% concentration) was applied to monoclonal antibody group once daily.

[0113] (4) After 7 days pretreatment, vaseline was applied on the back skin with mice in normal control group. At the same time, 62.5 mg 5% imiquimod cream was regularly applied daily on the back with mice in model group, small molecule inhibitor group and monoclonal antibody group. PCSK9 small molecule inhibitor cream or PCSK9 monoclonal antibody cream was applied after one hour. For 6 consecutive days, photos were taken and the PASI score was evaluated.

[0114] Experimental results:

[0115] 1. After 6 days of continuous administration, erythema, scales and infiltration were observed in the coating areas at the back of the mics in the model group. However, there were only slight erythema, scales and infiltration in the back of the mice in the PCSK9 small molecule inhibitor group and the PCSK9 monoclonal antibody group. The effect of PCSK9 small molecule inhibitor group was better than that of PCSK9 monoclonal antibody group. No lesion was observed in the vaseline group (See FIG. 13).

[0116] 2. A daily score was given on the skin lesions of the drug-coated area on the back of the mice. The score of erythema, scales, infiltration and total score (erythema+scales+infiltration) in PCSK9 small molecule inhibitor group was significantly lower than that in PCSK9 monoclonal antibody group (P<0.01).

Example 8

[0117] External Use of siRNA for PCSK9 could Significantly Reduce Psoriatic Lesions Induced by IMQ in Mice.

[0118] Materials and methods:

[0119] 1. Animal models: two types of mice models, C57BL/6J (B6) and Balb/cByJ (BALB) were used in this study. The mice was purchased at the Jackson Institute in Maine, USA. (The Jackson Laboratory). The strain number was 000664, 001026 and 005993 respectively (www.jax.org). All the mice in this experiment were female, aged 3-8 months, 5 mice for each group.

[0120] 2. Psoriatic lesions induced by IMQ: the back hair of B 6 mice was removed one day before the experiment. During the experiment, 62.5 mg imiquimod (IMQ, 3 M pharmaceuticals) were smeared on the skin of each mouse with an area of 2 cm.sup.2 on the back. In some mice, right auricle was also smeared with 5 mg IMQ to detect skin thickening easily. After 24 hours, score of the skin color, thickness and scales degree was evaluated.

[0121] 3. Inhibition of PCSK9 by siRNA and its effect on psoriatic lesions induced by IMQ: Two siRNAs for PCSK9 (siPCSK91, 2) and a random sequence siRNA (siCon), used as a control trial, was synthesized by Sigma-Aldrich company (Sigma-Aldrich, USA). The sequence was shown in Table 3. Mixed siPCSK9-1 and 2 equally and diluted it to 20 M with normal saline. 12.5 l diluted siPCSK9 was evenly mixed with 7.5 l moisturizer (CVS Pharmacy, Baby Lotion), so it was done with siCon. Applied 20 l siRNA-moisturizer mixture to the skin 1 hour before IMQ was applied. PCSK9 monoclonal antibody was purchased from abcam Company and was evenly mixed with PCSK9 moisturizer (CVS pharmacy, Baby Lotion) to generate an equal concentration emulsion with siPCSK9 emulsion.

[0122] 4. From the first day, siCON was applied on the back skin of B6 mice in model group after 1 hour-vaseline treatment. B6 mice in siPCSK9 group were smeared with 5% imiquimod cream on their back, then 1 hour thereafter, siPCSK9 was applied once daily. In PCSK9 monoclonal antibody group, 5% imiquimod cream was applied on the back skin of B6 mice, then 1 hour thereafter, PCSK9 monoclonal antibody was applied once daily.

[0123] 5. Gene expression detection by semi quantitative (real-time PCR): Primer sequence was shown in Table 3

TABLE-US-00003 TABLE3 siRNAsequenceandmodification Gene 5-3 Sense 5-3 Antisense siPCSK9-1 GccuGGAGuuuA UUCCgAAuAAAC uucGGAAdT*dT UCcAGGCdT*dT siPCSK9-2 AGGuGuAucucc GUGUCuAGGAGA uAGAcAcdT*dT uAcACCUdT*dT siCON cuuAcGcuGAGu UCGAAGuACUcA AcuucGAdT*dT GCGuAAGdT*dT Note: lowercase letters denote 2-OMe modification. All sequences were connected with phosphorothioate at the end of the sequence.

TABLE-US-00004 TABLE4 Primersequence Gene ForwardSequence ReverseSequence PCSK9 TTGCAGCAGCTG CCGACTGTGATG GGAACTT ACCTCTGGA NFkB CTGGTGGACACA ATAGGCACTGTC TACAGGAAGAC TTCTTTCACCTC IL-17A TTTTCAGCAAGG TTCATTGTGGAG AATGTGGA GGCAGAC IL-22 TTTCCTGACCAA CTGGATGTTCTG ACTCAGCA GTCGTCAC IL-23 CACCTCCCTACT TGGGCATCTGTT AGGACTCAGC GGGTCT

[0124] Experimental results:

[0125] 1. siPCSK9 can significantly reduce the expression of PCSK9 in the skin of B6 mice one day after being applied to the skin (p<0.05); inhibition rate is over 80% (FIG. 14). A day after siPCSK9 treatment of skin, it can inhibit the expression of NF B, IL-17, IL-22, IL-23 and other immune factors (FIG. 15). The expression of these inflammatory factors is closely related to the pathogenesis and severity of psoriasis, suggesting that siPCSK9 can inhibit the occurrence of psoriasis and alleviate the pathological reaction.

[0126] 2. After 6 days of continuous application, there were significant erythema, scales and infiltration in the coating area of the model group, but only slight erythema, scale and infiltration in the siPCSK9 group (see FIG. 16). The therapeutic effect of siPCSK9 group on psoriasis lesions in mice was significantly better than that in control group (P<0.01), but no lesions were found in Vaseline group.

[0127] 3. Daily scoring of skin lesions in B6 mice showed that the scores of erythema, scale, infiltration and the total scores (erythema+scale+infiltration) in siPCSK9 group were significantly lower than those in control group (P<0.01) (FIG. 16).

[0128] 4. To further verify the inhibitory effect of siPCSK9 on psoriatic lesions induced by IMQ, we repeated the experiment in FIG. 16 in another wild mice BALB using the same treatment, and the results was very similar (FIG. 17).

[0129] The specific embodiments are described above in detail. Within the knowledge of the technical personnel in this field, various changes can also be made without departing from the concept of the invention.