USE OF CD200 PROTEIN AND CD200 FUSION PROTEIN IN PREPARING A DRUG FOR TREATING PSORIASIS

Abstract

A use of a CD200 extracellular domain protein or a fusion protein formed from a CD200 extracellular domain protein and an Fc fragment in preparing a drug for treating psoriasis is disclosed.

Claims

1. Use of a CD200 extracellular domain protein or a fusion protein formed by a CD200 extracellular domain protein and an Fc fragment in preparation of drugs for treating psoriasis.

2. The use according to claim 1, wherein a nucleotide sequence of the CD200 extracellular domain protein is shown in SEQ ID NO: 1.

3. The use according to claim 1, wherein an amino acid sequence of the CD200 extracellular domain protein is shown in SEQ ID NO: 2.

4. The use according to claim 1, wherein Fc is an Fc fragment of human IgG1, IgG2 or IgG3, wherein a nucleotide sequence of the Fc fragment of the IgG1, IgG2 or IgG3 is shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 respectively.

5. The use according to claim 4, wherein an amino acid sequence of the Fc fragment of the IgG1, IgG2 or IgG3 is shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 respectively.

6. A complex, wherein the complex is obtained by adding one or more pharmaceutically acceptable excipients to the fusion protein according to claim 1.

7. The complex according to claim 6, wherein the excipients comprise a diluent, a filler, an adhesive, a wetting agent, an absorption enhancer, a surfactant, a lubricant and a stabilizer that are conventional in the pharmaceutical field.

8. Use of the complex according to claim 6 in preparation of drugs for treating psoriasis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] FIGS. 1A and 1B are diagrams of a transwell chamber for culture of CD200 and macrophages, wherein FIG. 1A shows a control group, and FIG. 1B shows a treatment group with a CD200 protein or a CD200-Fc fusion protein;

[0027] FIGS. 2A-2D are diagrams showing the inhibition of macrophage migration in vitro by CD200 protein and CD200-Fc fusion protein, wherein FIG. 2A shows the function of CD200 protein, FIG. 2B shows the function of a CD200 IgG1 fusion protein, FIG. 2C shows the function of a CD200 IgG2 fusion protein, and FIG. 2D shows the function of a CD200 IgG3 fusion protein;

[0028] FIGS. 3A-3L show results of ELISA for inflammatory factors IL-1β, IL-6 and INF-α after treatment with CD200 protein and CD200-Fc fusion protein, wherein FIG. 3A-3C show the function of CD200 protein, FIG. 3D-3F show the function of CD200 IgG1 fusion protein, FIG. 3G-3I show the function of CD200 IgG2 fusion protein, and FIG. 3J-3L show the function of CD200 IgG3 fusion protein;

[0029] FIGS. 4A-4H show quantitative PCR results of an impact of CD200 protein and CD200-Fc fusion protein on the proliferation of keratinocytes, wherein FIG. 4A-4B show the function of CD200 protein. FIG. 4C-4D show the function of CD200 IgG1 fusion protein, FIG. 4E-4F show the function of CD200 IgG2 fusion protein, and FIG. 4G-4H show the function of CD200 IgG3 fusion protein;

[0030] FIGS. 5A-5C show NF-κB protein detection results after treatment with CD200-Fc fusion protein, wherein FIG. 5A shows a western blot result diagram, and FIG. 5B-5C are diagrams showing a fold change compared with a reference gene GAPDH;

[0031] FIGS. 6A and 6B are diagrams showing results of a model of imiquimod-induced mouse psoriasis, wherein FIG. 6A is a diagram showing changes in a skin state of psoriasis mice, and FIG. 6B is a graph showing changes in the body weight of the psoriasis mice;

[0032] FIGS. 7A-7D are diagrams of a PASI score of imiquimod-induced skin psoriasis in mice, wherein FIG. 7A shows an infiltration score, FIG. 7B shows a skin lesion area score. FIG. 7C shows an erythema score, and FIG. 7D shows a total score;

[0033] FIG. 8 shows HE staining results of imiquimod-induced skin psoriasis in mice;

[0034] FIGS. 9A-9D are diagrams of a PASI score after treatment with CD200-Fc fusion protein, wherein FIG. 9A shows an infiltration score. FIG. 9B shows a skin lesion area score, FIG. 9C shows an erythema score, and FIG. 9D shows a total score; and

[0035] FIGS. 10A-10C show results of ELISA for inflammatory factors IL-1β, IL-6 and TNF-α in mice with psoriasis after treatment with CD200-Fc fission protein, wherein FIG. 10A shows IL-1β, FIG. 10B shows IL-6, and FIG. 10C shows TNT-α.

DETAILED DESCRIPTION

[0036] The following examples illustrate the present invention in detail: The examples are implemented on the premise of taking the present invention as the technical solution, and a detailed implementation solution and process are given, but the scope of the present invention is not limited to the following examples. The conditions and methods that are not indicated in the following embodiments are implemented conventionally.

Example 1

[0037] Inhibition of Macrophage Migration by CD200 Protein and CD200-Fc Fusion Protein

[0038] Macrophages play an important role in the occurrence and progression of psoriasis, and CD200 protein and fusion protein can inhibit the migration of the macrophages. A specific method and results are as follows:

[0039] Method: Peritoneal macrophages needed to be obtained first. Mice were injected intraperitoneally with 1 nil of autoclaved 5% thioglycolate broth every day. After three consecutive days, the mice were killed, ascites of the mice was sucked out by using a syringe and washed with PBS, and the cells were cultured in DMEM containing penicillin (100 U/ml) streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS). After 5 hours, the suspended cells were aspirated and washed three times with cold PBS to obtain adherent cells. The adherent macrophages were digested with 0.25% trypsin. The cell suspension in the DMEM medium was placed in an upper chamber of Matrigel-coated transwells (8 μm), and each insert contained 10.sup.5 cells. CD200 protein or CD200-Fc fusion protein was added into a lower chamber, as shown in FIGS. 1A and 1B. After incubation at 37° C. for 9 hours, the macrophages migrated to the lower chamber were counted.

[0040] Results: As shown in FIGS. 2A-2D and Tables 1 to 4, transwell experiments showed that peritoneal macrophages in a control group could migrate to the other side of the transwell, while the migration of macrophages treated with CD200 protein and CD200-Fc fusion protein was significantly inhibited (P 0.05, Student's t-test).

TABLE-US-00001 TABLE 1 Effect of CD200 protein on macrophage migration Group Item 1 2 3 4 5 Mean ± SEM P value Number control 73 90 397 175 96 166.20 ± 60.319 0.202 of cells CD200 116 40 94 125 8  76.60 ± 22.631 treatment Migration rate 62.9% 44.4% 23.6% 71.4% 8.3%  42.12 ± 11.792 —

TABLE-US-00002 TABLE 2 Effect of CD200 IgG1 fusion protein on macrophage migration Group Item 1 2 3 4 5 Mean ± SEM P value Number control 234 105 250 113 109 162.20 ± 32.701 0.016 of cells CD200 112 35 63 125 8  49.20 ± 17.825 treatment Migration rate 47.9% 32.4% 23.2% 21.2% 11.0%  27.14 ± 13.874 —

TABLE-US-00003 TABLE 3 Effect of CD200 IgG2 fusion protein on macrophage migration Group Item 1 2 3 4 5 Mean ± SEM P value Number control 102 106 221 319 282 206.00 ± 44.489 0.014 of cells CD200 1 56 61 74 101  58.60 ± 16.379 treatment Migration rate 0.9% 52.8% 27.6% 23.1% 35.8%  28.04 ± 18.937 —

TABLE-US-00004 TABLE 4 Effect of CD200 IgG3 fusion protein on macrophage migration Group Item 1 2 3 4 5 Mean ± SEM P value Number control 245 108 198 219 43 162.60 ± 37.755 0.013 of cells CD200 45 43 34 42 11 35.00 ± 6.285 treatment Migration rate 18.4% 39.8% 17.2% 19.2% 25.6% 24.04 ± 9.392 —

Example 2

[0041] Effect of Treatment with CD200 Protein and CD200-Fc Fusion Protein on Release of Inflammatory Factors

[0042] Inflammatory factors secreted by macrophages can aggravate the inflammatory response of psoriasis, while CD200 protein and fusion protein can inhibit the release of inflammatory factors from macrophages. A specific method and results are as follows:

[0043] Method: Peritoneum of normal mice and a model group were extracted and cultured. The process is as shown in Example 1. The cells were divided into three groups: a normal group, a treatment group with CD200 protein or CD200-Fc fusin protein, and a model group. After culture for 24 hours, the cell supernatant was collected for ELISA. A kit was purchased from Tianjin Anoric Biotechnology, and levels of IL-1β, IL-6 and TNF-α were detected.

[0044] Results: After treatment with CD200 protein or CD200-Fc fusion protein, the levels of IL-1β (F (2, 12)=12.28, P=0.0012, Newman-Keuls' test), IL-6 (F (2, 12)=28.21. P<0.0001, Newman-Keuls' test) and TNF-α (F (2, 12)=17.09, P=0.0003, Newman-Keuls' test) were lower than those of the model group, as shown in FIGS. 3A-3L and Tables 5 to 8, indicating that CD200 protein and CD200-Fc fusion protein could reduce the release of inflammatory factors.

TABLE-US-00005 TABLE 5 Effect of CD200 protein on release of inflammatory factors Group Item 1 2 3 4 5 Mean ± SEM P value TNF-α control 545.4 577.4 653.4 617.4 535.4 585 80 ± 22.15  0.013 CD200 432.4 538.4 435.4 588.4 485.4 496.00 ± 30.14  treatment IL-6 control 289.0 317.7 272.7 270.5 342.7 298.50 ± 13.93  0.121 CD200 246.5 297.0 194.0 259.0 292.7 258.00 ± 18.73  treatment IL-1β control 222.8 255.3 289.0 290.3 277.8 267.00 ± 12.72  0.027 CD200 250.3 214.0 236.5 290.3 221.5 223.50 ± 9.42  treatment

TABLE-US-00006 TABLE 6 Effect of CD200 IgG1 fusion protein on release of inflammatory factors Group Item 1 2 3 4 5 Mean ± SEM P value TNF-α control 585.2 523.7 598.4 601.3 555.4 574.80 ± 14.72  0.001 CD200 422.6 438.2 415.3 499.4 492.2 453.54 ± 17.68  treatment IL-6 control 278.2 297.4 298.4 267.4 276.5 285.58 ± 6.12  0.001 CD200 232.5 247.0 234.0 248.2 232.6 238.86 ± 3.58  treatment IL-1β control 290.5 288.5 258.0 291.3 267.4 279.14 ± 6.88  0.005 CD200 241.3 236.0 215.5 265.3 211.2 233.86 ± 9.74  treatment

TABLE-US-00007 TABLE 7 Effect of CD200 IG2 fusion protein on release of inflammatory factors Group Item 1 2 3 4 5 Mean ± SEM P value TNF-α control 568.4 597.4 523.4 549.4 621.3 571.98 ± 17.26  0.003 CD200 482.4 501.5 435.7 496.5 485.4 480.30 ± 17.68  treatment IL-6 control 286.0 297.7 292.3 299.5 317.5 298.60 ± 5.27  0.001 CD200 241.5 257.4 224.5 259.0 262.4 248.96 ± 7.09  treatment IL-1β control 232.8 275.3 279.6 295.3 287.8 274.16 ± 0.89  0.002 CD200 198.3 209.0 236.4 250.3 239.4 226.80 ± 9.77  treatment

TABLE-US-00008 TABLE 8 Effect of CD200 IgG3 fusion protein on release of inflammatory factors Group Item 1 2 3 4 5 Mean ± SEM P value TNF-α control 635.4 568.4 612.7 578.5 575.4 594.08 ± 12.85  0.002 CD200 499.5 513.4 515.4 516.0 545.2 517.90 ± 7.46  treatment IL-6 control 256.0 303.5 272.0 260.5 342.2 286.84 ± 16.13  0.002 CD200 196.5 197.5 194.0 199.0 292.7 215.94 ± 19.21  treatment IL-1β control 234.4 275.3 288.7 267.5 301.8 273.54 ± 17.40  0.0029 CD200 200.3 244.0 227.3 205.3 242.0 223.78 ± 9.07  treatment

Example 3

[0045] Effect of Macrophages on Keratinocyte Proliferation after Inhibition by CD200 Protein and CD200-Fc Fusion Protein

[0046] CD200 protein and CD200 fusion protein can indirectly inhibit the excessive proliferation of keratinocytes by inhibiting the activation of macrophages. Markers for keratinocyte proliferation used in this experiment were S100A7 and S100A8. A specific method and results are as follows:

[0047] Methods: Keratinocytes needed to be obtained first: Skin tissues were separated from neonatal mice and cut into pieces. The cut skin tissues were digested overnight with 25 U/ml neutral protease, and then digested with 0.05% trypsin-EDTA for 15 minutes. The cut skin tissues were washed with cold PBS, and suspended cells were incubated with a 154 CF medium. The keratinocytes were co-cultured with the macrophages treated with CD200 protein or CD200-Fc fusion protein in the remaining medium as the treatment group, and the keratinocytes were co-cultured with the untreated macrophages as the control group. After 48 hours, VCR detection was performed on the expression of S100A7 and S100A8 of keratinocytes. Primers used:

TABLE-US-00009 S100A7: 5′-GTACTCAGGTCATGGTTCTG-3′(upstream) 5′-GGTATTCAAGCAAGGTATCAC-3′(downstream) S100A8:  5′-GGAGTTCCTTGCGATGGTGAT-3′(upstream) 5′-TCCTTGTGGCTGTCTTTGTGA-3′(downstream).

[0048] Results: As shown in FIGS. 4A-4H and Tables 9 to 12, it was found that the expression of S100A7 (P<0.01, Student's t-test) and S100A8 (P<1, Student's t-test) of keratinocytes after co-culture of keratinocytes and macrophages treated with CD200 protein and CD200-Fc fusion protein was significantly lower than that of the cells in the control group, indicating that CD200 protein and CD200-Fc fusion protein inhibited the proliferation of keratinocytes by inhibiting the activation of macrophages.

TABLE-US-00010 TABLE 9 Effect of CD200 protein on proliferation of keratinocytes Group Item 1 2 3 4 5 Mean ± SEM P value S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.016 CD200 0.16 0.45 0.83 0.52 0.72 0.540 ± 0.116 treatment S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.003 CD200 0.44 0.32 0.58 0.71 0.27  0.46 ± 0.082 treatment

TABLE-US-00011 TABLE 10 Effect of CD200 IgG1 fusion protein on proliferation of keratinocytes Group Item 1 2 3 4 5 Mean ± SEM P value S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.002 CD200 0.34 0.23 0.54 0.68 0.32  0.42 ± 0.082 treatment S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.001 CD200 0.23 0.31 0.34 0.56 0.61  0.41 ± 0.074 treatment

TABLE-US-00012 TABLE 11 Effect of CD200 IgG2 fusion protein on proliferation of keratinocytes Group Item 1 2 3 4 5 Mean ± SEM P value S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.001 CD200 0.45 0.23 0.38 0.59 0.49  0.43 ± 0.060 treatment S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.002 CD200 0.67 0.16 0.30 0.42 0.38  0.39 ± 0.084) treatment

TABLE-US-00013 TABLE 12 Effect of CD200 IgG3 fusion protein on proliferation of keratinocytes Group Item 1 2 3 4 5 Mean ± SEM P value S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.002 CD200 0.71 0.32 0.41 0.54 0.32  0.48 ± 0.065 treatment S100A7 control 1 1 1 1 1 1.00 ± 0.00 0.002 CD200 0.48 0.27 0.54 0.56 0.67  0.50 ± 0.066) treatment

Example 4

[0049] CD200-Fc Fusion Protein Inhibits Activation of Macrophages by Using an NT-κB Pathway.

[0050] CD200 protein and CD200 fusion protein can inhibit the activation of macrophages by using the NF-κB pathway, thereby indirectly inhibiting excessive proliferation of keratinocytes. A specific method and results are as follows:

[0051] Method: Some macrophages obtained from mouse ascites fell into two groups for culture, where CD200-Fc fusion protein was added into one group of medium as a CD200 treatment group, and one group using a normal medium was a control group. After culture for 48 hours, a half of the cells were taken for protein extraction, and the other half of the cells were taken for WB detection. Protein samples from the cells were isolated h SDS-PA E. and transferred to a poly vinylidene fluoride (PVDF) membrane. After being blocked in a 5% BSA TBST solution, the membrane was treated with a rabbit anti-mouse antibody specific for CD200RL GAPDH, NT-κB p50, and then HRP-bound goat anti-rabbit IgG was incubated as a secondary antibody. The membrane was washed and exposed to an X-ray film using an enhanced chemiluminescence reaction.

[0052] Results: As shown in FIGS. 5A-5C, compared with that of the cells in the control group, the protein expression of NF-κB p50 in the cells treated with CD200-Fc fusion protein was significantly reduced, indicating that CD200/CD200R1 is involved in reducing the expression of NF-κB p50 to inhibit macrophage activation.

Example 5

[0053] Establishment and Verification of a Psoriasis Model on the Back of Mice Induced by Imiquimod (IMQ)

[0054] To verify therapeutic effect of CD200 protein and CD200-Fc fusion protein in mice, a mouse psoriasis model was established and evaluated. A specific method and results are as follows:

[0055] 5.1 Establishment of a Psoriasis Model on the Back of Mice

[0056] Methods: Ten 5-6 week-old male BALB/c mice weighing 17-20 g were selected, including five as a control group and five as a model group. The back hair of the mice was removed, and a dose of 62.5 mg of a commercially available 5% INN cream was administered by application for 7 consecutive days, with vaseline as negative control. The skin state of the mice was recorded daily by photographing. The severity of back skin inflammation was measured by using PAST scoring criteria. PAST scores included erythema, scales, infiltration, and total score. The erythema, scales, and infiltration scores ranged from 0 to 4, which represented none, slight, mild, obvious, very obvious, respectively.

[0057] Results: Compared with mice of the control group, IMQ-stimulated mice lost weight from the second day, as shown in FIGS. 6A-6B. The severity of the skin injury was assessed by PAST scores and confirmed as deepening, as shown in FIGS. 7A-7D.

[0058] 5.2 Validation of Skin Lesions of Psoriasis with HE Staining

[0059] Methods: The skin was taken out from the back of mice, fixed with 4% paraformaldehyde solution and embedded in paraffin. Paraffin-embedded (5-10 μm) sections were prepared and stained with Rh and examined under an optical microscope.

[0060] Results: As shown in FIG. 8, HE staining showed acanthosis, epidermal shedding, hyperkeratosis and cuticular layer thickening (P=0.01, Student's t-test). Based on the foregoing results, the appropriate time course, frequency and dose of IMQ stimulation were determined to successfully establish an IMQ-induced psoriasis-like mouse model.

Example 6

[0061] Effect of CD200-Fc Fusion Protein on IMQ-Induced Psoriasis Symptoms

[0062] The effect of CD200 protein and CD200-Fc fusion protein on the in vivo treatment of psoriasis was evaluated. A specific method and results are as follows:

[0063] Methods: From the 3.sup.rd day of IMQ-induced psoriasis inflammation, CD200 fusion protein was used for treatment (based on the results of the in vitro experiment, CD200 IgG2 Fc fission protein was selected as a experimental group for the in vivo experiment). CD200 fusion protein was injected once every two days by subcutaneous injection at the back. A dosage regimen is shown in Table 13.

TABLE-US-00014 TABLE 13 Therapeutic scheme for subcutaneous injection of CD200 fusion protein Group Item control CD200 treatment IMQ-mouse-model Model material Vaseline Imiquimod Imiquimod Type of Normal saline CD200-Fc Normal saline administration Administration Subcutaneous Subcutaneous Subcutaneous mode injection injection injection Administration 200 μL 200 μL (1 mg/kg) 200 μL dosage Administration Once every Once every Once every frequency two days two days two days Number in each 5 5 5 group

[0064] Results: As shown in FIGS. 9A-9D, compared with that of the model group, the skin lesions were significantly improved and the body weight loss was significantly reduced in the CD200 treatment group. In addition, PASI scores confirmed that infiltration, scales, and erythema were also diminished in mice in the CD200 treatment group.

Example 7

[0065] Effect of CD200-Fc Fusion Protein on the Expression of Skin Inflammatory Factors

[0066] Method: The back skin of 200 mg mice was isolated and placed in a four-fold volume of PBS. After the skin was ground and centrifuged, the supernatant was collected for ELISA. A kit was purchased from Tianjin Anoric Biotechnology, and levels of IL-1β, TNF-α, and IL-6 were detected according to the specification.

[0067] Results: As shown in FIGS. 10A-10C Table 14 and Table 15, the levels of IL-1β (F (2, 12)=12.28, P=0.0012. Newman-Keuls' test), IL-6 (F (2, 12)=28.21, P<0.0001. Newman-Keuls' test) and TNT-α (F (2, 123=17.09, P=0.0003, Newman-Keuls' test) were lower than those of the model group, indicating that CD200-Fc fusion protein could reduce the release of inflammatory factors.

TABLE-US-00015 TABLE 14 Effect of CD200 fusion protein on the expression of inflammatory factors of mouse skin Group Item 1 2 3 4 5 Mean ± SEM TNF-α control 202.4 284.4 220.4 181.4 68.4 187.20 ± 35.16  CD200 447.4 456.4 138.4 134.4 201.4 275.60 ± 72.96  treatment IMQ- 487.4 694.4 540.4 653.4 559.4 584.00 ± 84.84  mouse- model IL-6 control 86.50 74.00 75.25 77.75 76.50 78.0 ± 4.95 CD200 84.00 99.00 112.75 75.25 144.00 103.00 ± 27.03  treatment IMQ- 186.5 179.0 136.5 164.0 191.5 171.50 ± 22.15  mouse- model IL-1β control 109.00 111.50 112.75 114.00 127.75 115.00 ± 7.36  CD200 156.50 171.50 200.25 117.75 146.50 158.50 ± 30.49  treatment IMQ- 295.25 187.75 175.25 200.25 235.25 218.75 ± 48.27  mouse- model