NEW APPLICATION OF CHEMOKINE RECEPTOR CCR6 INHIBITOR IN PREVENTING RECURRENCE OF PSORIASIS

Abstract

Provided is a new application of a chemokine receptor CCR6 inhibitor in preventing the recurrence of psoriasis. Specifically, provided is a use of the chemokine receptor CCR6 inhibitor for preparing a preparation or composition that is administered to a subject so as to prevent the recurrence of psoriasis in the subject. Also provided is a pharmaceutical product that prevents the recurrence of psoriasis. Using the provided drug products may effectively prevent or alleviate the recurrence of psoriasis.

Claims

1-3. (canceled)

4. A pharmaceutical product for preventing the recurrence of psoriasis, comprising: (a) a first pharmaceutical composition comprising (a1) a chemokine receptor CCR6 inhibitor, and (a2) a first pharmaceutically acceptable carrier; and (b) a second pharmaceutical composition comprising (b1) an additional drug or active ingredient thereof useful for treatment of psoriasis, which is not an CCR6 inhibitor, and (b2) a second pharmaceutically acceptable carrier.

5. The pharmaceutical product of claim 4, wherein (b1) the additional drug or active ingredient thereof other than CCR6 inhibitor comprises: glucocorticoid, IL-17A antagonist, TNF-a antagonist, IL-12/IL-23 antagonist, calcineurin inhibitor, vitamin D3 derivative, and combinations thereof.

6. The pharmaceutical product of claim 4, wherein in the second pharmaceutical composition, the component (b1) is a glucocorticoid, and accounts for 0.01-50 wt %, preferably 0.01-10 wt %, and more preferably 0.1-5 wt % of the total weight of the second pharmaceutical composition.

7. The pharmaceutical product of claim 4 or 5, wherein in the second pharmaceutical composition, the component (b1) is an IL-17A antagonist, and has a concentration of 1-1,000 mg/ml, preferably 5-500 mg/ml, and more preferably 10-100 mg/ml in the second pharmaceutical composition.

8. A pharmaceutical product, comprising: (i) a first container, and a first pharmaceutical composition in the first container, comprising (a1) a chemokine receptor CCR6 inhibitor, and (a2) a first pharmaceutically acceptable carrier; and (ii) a second container, and a detection agent in the second container for detecting a substance selected from the group consisting of: IL-17, γδT17 cells, CD3+ T cells, CCL20, CCR6, and combinations thereof, in a sample derived from a subject.

9. The pharmaceutical product of claim 8, wherein the sample is selected from the group consisting of: blood, skin tissue, draining lymph node tissue, distal lymph node tissue, and combinations thereof.

10. The pharmaceutical product of claim 8, wherein the detection comprises qualitative detection and quantitative detection.

11. A method for preventing recurrence of psoriasis which comprises a step of: administering to a subject in need a chemokine receptor CCR6 inhibitor.

12. The method of claim 11, wherein the subject is a patient with psoriasis.

13. The method of claim 11, wherein the subject is a patient who has experienced psoriasis recurrence.

14. The method of claim 11, wherein the chemokine receptor CCR6 inhibitor is selected from the group consisting of an extracellular soluble fragment of CCR6, a blocking antibody, a small molecule compound, an antisense nucleic acid, and combinations thereof.

15. The method of claim 11, wherein the chemokine receptor CCR6 inhibitor is administered in combination with an additional drug or active ingredient thereof which is selected from the group consisting of glucocorticoid, IL-17A antagonist, TNF-a antagonist, IL-12/IL-23 antagonist, calcineurin inhibitor, vitamin D3 derivative, and combinations thereof.

16. The method of claim 15, wherein the chemokine receptor CCR6 inhibitor and the administered in combination with an additional drug or active ingredient thereof are administered to the subject simultaneously or sequentially.

Description

DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows results at 3 time points, i.e., before, after treatment with halometasone, and during psoriasis recurrence. Flow cytometry staining showed that the proportion of IL-17-producing cells in peripheral blood increased after treatment and decreased during recurrence; the proportion of IL-17-producing T cells (T17 cells) in skin lesions decreased after treatment and increased during recurrence. Transcriptome sequencing (RNA-seq) showed that the expression of various genes in the skin lesions of psoriasis patients was altered, and especially, the chemokines such as CCL20, CCL5, CCL2, CXCL10, etc., decreased after treatment and increased during recurrence.

[0051] FIG. 2 shows that in mice treated with halometasone topically for ear skin inflammation, the symptoms of skin psoriasis were significantly relieved, but the proportion of γδT17 cells in the draining and distal lymph nodes was significantly increased.

[0052] FIG. 3 shows that when psoriasis was re-induced, halometasone-treated mice relapsed rapidly, with a significant decrease in the proportion of γδT17 cells in the draining lymph nodes and distal lymph nodes.

[0053] FIG. 4 shows the simultaneous treatment of wild-type (WT) and CCR6.sup.−/− mice with GC. Compared with WT mice, CCR6.sup.−/− mice not only showed reduced symptoms of psoriasis, but also did not show an increase in the proportion of γδT cells in lymph nodes after halometasone treatment.

[0054] FIG. 5 shows that halometasone could quickly and effectively control psoriasis, but the disease was prone to recurrence. The expression of psoriasis-related genes decreased after treatment, but increased during recurrence.

[0055] FIG. 6 shows that the proportions of γδT cells and the subpopulations of Vγ4 and Vγ4Vδ4 T cells in distal lymph nodes were higher after halometasone treatment, and the proportion of IL-17 secretion after stimulation was also higher.

[0056] FIG. 7 shows that the proportion of γδT17 cells in lymph nodes of mice in halometasone treatment group was still significantly increased after drug administration was stopped.

[0057] FIG. 8 shows that the proportion of γδT17 cells in lymph nodes after stimulation of mice in halometasone treatment group was still significantly increased after drug administration was stopped.

[0058] FIG. 9 shows that in re-induced psoriasis, the proportion of Vγ4 and Vγ4Vδ4 T cells in the skin was higher in the GC group, but there was mild or no change in the lymph nodes.

[0059] FIG. 10 shows that when psoriasis recurred after GC treatment, CCR6 KO mice still showed milder symptoms of psoriasis compared with wild-type mice, and did not show a decrease of proportion of γδT17 cells in lymph nodes in the GC group.

[0060] FIG. 11 shows that the use of FTY720 prevented γδT17 cells from migrating from the draining lymph nodes, and mice in the GC group did not appear rapid recurrence of psoriasis.

[0061] FIG. 12 shows that when imiquimod was replaced by mannan for inducing recurrence of psoriasis, the mice in the GC group no longer had rapid disease recurrence.

DETAILED DESCRIPTION

[0062] After extensive and intensive research and massive screening, the inventors have developed a method for preventing recurrence of psoriasis for the first time.

[0063] Specifically, the inventors utilized an imiquimod (IMQ)-induced mouse psoriasis model to simulate the whole clinical process of topical glucocorticoid treatment in patients with psoriasis: administering of drugs—stop administering—recurrence of disease. It was found that after topical glucocorticoid (GC) treatment, the proportion of γδT cells secreting the pathogenic cytokine IL-17 (γδT17 cells) decreased in the skin, but increased in draining and distant lymph nodes. This change persisted until 2 weeks after GC treatment was discontinued. When IMQ was used again to cause psoriasis recurrence, the previously GC-treated mice exhibited more severe disease recurrence, including more rapid epidermal thickening and increased neutrophil infiltration. Correspondingly, the proportion of γδT17 cells in draining and distant lymph nodes was indistinguishable or even lower compared to the controls.

[0064] This indicates that the skin remission after GC treatment is probably due to the migration of γδT17 cells from the skin to the lymph nodes, and when the disease relapses, γδT17 cells can quickly migrate back from the lymph nodes to the skin, leading to the rapid development of psoriasis. That is, GC treatment may lead to psoriasis recurrence by affecting the redistribution of γδT17 cells in vivo.

[0065] The inventors found that GC treatment in CCR6 knockout mice did not affect the redistribution of γδT17 cells in vivo and thus did not lead to psoriasis recurrence. In a further research, it is found that such migratory γδT17 cells have the properties of memory cells. In conclusion, the inventors have found that inhibiting the redistribution of γδT17 cells plays a key role in controlling the recurrence of skin inflammation.

[0066] On this basis, the inventors have completed the present invention.

[0067] Pharmaceutical Composition and Administration Method Thereof

[0068] In the present invention, it provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: a chemokine receptor CCR6 inhibitor and additional drugs or active ingredient thereof for treating psoriasis.

[0069] As used herein, the term “effective amount” or “effective dose” refers to an amount that is functional or active in humans and/or animals and is acceptable to humans and/or animals.

[0070] As used herein, a “pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergy), i.e., a substance with a reasonable benefit/risk ratio. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, and comprises various excipients and diluents.

[0071] The pharmaceutical composition of the present invention comprises a safe and effective amount of the active ingredients of the present invention, and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, a pharmaceutical preparation should be matched to the administration method, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with normal saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0072] The effective amount of the active ingredients of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated of patients, patient's weights, patient's immune status, route of administration, and the like. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.

[0073] The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, and combinations thereof. The choice of carrier should be matched to administration method, as is well known to those skilled in the art.

[0074] The first active ingredient (a1) of a chemokine receptor CCR6 inhibitor in the present invention can be used in combination with the second active ingredient (b1) of a additional drug or active ingredient thereof useful for the treatment of psoriasis, which is not a CCR6 inhibitor. The second active ingredient (b1) is an drug already available in the prior art, including but not limited to: glucocorticoids, IL-17A antagonists, TNF-α antagonists, IL-12/IL-23 antagonists, calcineurin inhibitors, vitamin D3 derivatives, and combinations thereof.

[0075] The Present Invention Comprise the Following Main Beneficial Effects.

[0076] 1) Compared with general medicines for treating psoriasis, the present invention has specifically solved the problem of recurrence of psoriasis.

[0077] 2) It is discovered in the present invention for the first time that the redistribution of pathogenic cells is the mechanism of psoriasis recurrence.

[0078] 3) The present invention aims at the redistribution characteristics of pathogenic cells in the process of psoriasis recurrence, and has better effect than that of common therapeutic drugs.

[0079] 4) The present invention can effectively inhibit the redistribution of pathogenic cells, thereby effectively preventing or relieving the recurrence of psoriasis.

[0080] The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only to illustrate the invention, not to limit the scope of the invention. The experimental methods with no specific conditions described in the following examples are generally performed under the conventional conditions e.g., the conditions described by Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacture's instructions. Unless indicated otherwise, all percentage and parts are calculated by weight.

Example 1: Detection of Changes in Related Gene Expression in Patients with Psoriasis Recurrence

[0081] In this example, patients with psoriasis vulgaris were recruited and glucocorticoids (GC) were topically (externally) administered for 2 weeks of treatment. Thereafter, the administration were stopped until the disease relapsed, and further research on the mechanism of psoriasis recurrence was carried out.

[0082] Among the psoriasis patients recruited, 12 patients experienced disease recurrence after an effective GC therapy was stopped (FIG. 5a).

[0083] Transcriptome sequencing (RNA-seq) was performed on the skin lesions of these patients before GC treatment, after GC treatment, and after disease recurrence. The results showed that the expression of psoriasis-related genes such as IL-17 and IL-23 was down-regulated after effective GC treatment (FIG. 1a and FIG. 5b). Conversely, the same genes were upregulated when the disease relapsed (FIG. 1b and FIG. 5b).

[0084] Comparing gene expression profiles before treatment, after treatment, and during recurrence, it was revealed that up to 400 genes had altered expression (FIG. 1c). Notably, the gene expression changes of multiple chemokines and their receptors were more pronounced (FIG. 1d), including CCL20, CCL5, and CXCL10 (FIG. 1e). Skin tissue immunofluorescence further showed changes in CCL20 expression at different time points (FIG. 1f).

Example 2: Detection of T Cell Distribution in Patients with Psoriasis Recurrence

[0085] In this example, in psoriasis patients receiving GC treatment, the changes in the proportion of CD3+ T cells and T17 cells in the skin and peripheral blood before treatment, after treatment and during recurrence were detected.

[0086] The results showed that after effective GC treatment, the proportion of CD3+ T cells and dermal T17 cells infiltrated in the skin decreased (FIG. 1g), and the proportion of CD3+ T cells and dermal T17 cells in the skin increased when the disease relapsed. In contrast, T17 cells in the blood showed the opposite proportional change, i.e., the proportion increased after treatment and decreased during recurrence (FIG. 1h).

[0087] The above study suggests that the redistribution of T cells, especially the redistribution of T17 cells, plays a key role in the remission and recurrence of psoriasis.

Example 3: Detection of Changes in T Cell Distribution in GC-Treated Psoriasis Animal Model

[0088] In this example, an animal model to simulate clinical GC treatment for psoriasis was established (FIG. 2a). An imiquimod (IMQ)-induced mouse psoriasis model was utilized to simulate the whole clinical process of topical glucocorticoid treatment in patients with psoriasis: administering of drug—stop administering—recurrence of disease.

[0089] All mice were smeared with IMQ for three weeks. Among them, from the 6th day, GC or petrolatum (Vas) was applied to the skin of the mice, and all treatments were stopped on the 21st day. Mice entered a 2-week drug withdrawal period, after which IMQ was applied again for 7 consecutive days to induce psoriasis recurrence.

[0090] On day 21, it was seen that the ear thickness of GC-treated mice was significantly thinner than that of the control group (FIG. 2b), which was consistent with clinical remission of psoriasis after GC treatment. Consistent with previous studies, the skin thickness of TCRd.sup.−/− mice was significantly thinner than that of normal mice (WT) (FIG. 2b).

[0091] The results of skin H&E staining also confirmed the above results (FIG. 2c). GC treatment also significantly reduced the proportion of CD3+ T cells in the skin (FIG. 6a), which was consistent with the clinical results. Dermal γδT cells, especially Vγ4Vδ4 cells, were significantly reduced in the skin of GC-treated mice (FIG. 6a).

[0092] Immunofluorescence and flow staining demonstrated reduced neutrophil infiltration in the GC-treated skin. However, in TCRd.sup.−/− mice, there was no statistical difference between the experimental group and the control group.

Example 4: Detection of Changes in IL-17 Secretion Level in GC-Treated Psoriasis Animal Model

[0093] γδT cells are the main cells that secrete IL-17 in the skin. In this example, γδT17 cells in the skin and lymph nodes were detected.

[0094] Consistent with disease progression, the proportions of CD3, γδ, Vγ4, Vγ4Vδ4 that secreted IL-17 were reduced in the skin of GC-treated mice (FIG. 2e). In skin-draining lymph nodes, although the proportion of γδT cells was decreased in GC treatment group, the proportion of Vγ4 cells was increased (FIG. 6b).

[0095] Interestingly, Vγ4 cells and Vγ4Vδ4 cells secreted more IL-17 in mice of GC treatment group (FIG. 2f). This change was more significant in distant lymph nodes, wherein the proportions of γδT, Vγ4, Vγ4Vδ4 (FIG. 6c), and the proportions of IL-17 secretion thereof (FIG. 2g) were all increased in GC treatment group.

[0096] It is noticed that the proportions of IL1R expression in γδT, Vγ4, Vγ4Vδ4 cells in the skin were all increased in GC group, and IL1R expression was correlated with the memory ability of Vγ4 T17.

[0097] These data suggested that γδT17 cells in the skin after GC treatment might migrate to draining and distant lymph nodes, and had a memory cell phenotype.

Example 5: Detection of Changes in γδT17 Cell Level and IL-17 Secretion Level in Mice During Drug Withdrawal Period

[0098] In order to simulate the clinical withdrawal period, all mice in the experimental group and the control group were rested for 2 weeks without treatment.

[0099] After 2 weeks, the ear thickness of mice in Vas control group gradually recovered (FIG. 7a), and this result was also confirmed by H&E staining (FIG. 7b).

[0100] However, more neutrophils were infiltrated in the skin of mice in GC group than in the Vas control group; in TCRd.sup.−/− mice, there was no difference between GC group and Vas control group.

[0101] In the skin of mice in GC experimental group, the proportions of CD3, γδT, Vγ4Vδ4 were still lower than those in the control group (FIG. 8a), and the proportion of γδT17 was also lower than that in the control group (FIG. 7d). Although the proportions of γδT, Vγ4, Vγ4Vδ4 cells remained unchanged or slightly increased in the draining and distant lymph nodes, the proportion of IL-17-secreting cells was significantly increased (FIGS. 7e, 7f).

[0102] The above studies showed that 2 weeks after drug withdrawal, although the level of γδT17 cells in the dermis was still low in GC experimental group, the draining and distant lymph nodes still had high levels of γδT17 cells.

Example 6: Detection of γδT17 Cell Level and IL-17 Secretion Level in Psoriasis Recurrence Mouse Model

[0103] In this example, IMQ was applied to the mice in the experimental group and the control group again to simulate the recurrence of psoriasis after clinical withdrawal.

[0104] After using IMQ for 3 days, it could be observed that the ear thickening speed of the mice in GC group was faster than that in the control group (FIG. 3a), and the epidermal thickening fold in GC group was also higher than that in Vas group, but there was no difference in TCRd.sup.−/− mice between GC group and Vas group (FIG. 3a). H&E staining also confirmed the above results (FIG. 3b).

[0105] The infiltrated neutrophils in the skin of mice in GC group exceeded those in Vas control group, while the opposite results were shown in TCRd.sup.−/− mice between GC group and Vas group (FIG. 3C).

[0106] The proportions of CD3 and γδT cells were not significantly different between the two groups, while the proportions of Vγ4, Vγ4Vδ4 cells were higher in GC group (FIG. 9a). Although the proportions of Vγ4T17 and Vγ4Vδ4T17 cells were slightly decreased in GC group, the levels of IL-17 secreted by CD3 and γδT cells in the skin were not significantly different between the two groups (FIG. 3d).

[0107] At draining and distant sites, the proportions of γδT, Vγ4, Vγ4Vδ4 cells in lymph nodes varied slightly or did not change between the two groups (FIGS. 9b, 9c). There was no difference in γδT17 in skin draining lymph nodes between the two groups, but the proportions of IL-17 secreted by γδT, Vγ4, Vγ4Vδ4 cells were significantly lower in the distant lymph nodes of GC group (FIG. 3f). This contrasted sharply with the elevated proportion of IL-17 in draining and distant lymph nodes of GC group before IMQ re-challenge (FIG. 7f).

[0108] These studies suggested that elevated proportion of γδT17 in lymph nodes and distant sites would migrate back to the skin when the disease relapsed, leading to rapid skin inflammation.

Example 7: Study on the Relationship Between CCR6 Gene Knockout and γδT17 Cell Redistribution

[0109] Cytokine receptors CCR6 and CCR2 are known to mediate the migration of γδT17 cells in the occurrence of skin inflammation. In this example, the above-mentioned mechanism was studied in wild-type mice (WT), CCR6.sup.−/−, and CCR2.sup.−/− mice.

[0110] After GC treatment, although the ear thicknesses of the above three strains of mice were reduced, CCR6.sup.−/− mice showed differences from WT mice, while CCR2.sup.−/− mice showed a similar phenotype as WT mice (FIG. 4a). Both GC-treated WT and CCR2.sup.−/− mice showed significantly lower thickness than those in their corresponding controls, but there was no significant difference between CCR6.sup.−/− mice and the corresponding control (FIG. 4a).

[0111] After GC treatment of the three strains of mice, the levels of IL-17 secreted by CD3, γδT, Vγ4, Vγ4Vδ4 cells in the skin were all reduced (FIG. 4b), but unlike the WT group and CCR2.sup.−/− group, the increased levels of IL-17 secreted by γδT, Vγ4, Vγ4Vδ4 cells in draining and distant sites were not observed in CCR6.sup.−/− mice (FIG. 4c).

[0112] Similarly, WT and CCR2 mice showed faster disease recurrence after re-challenge with IMQ, but the recurrence was significantly suppressed in CCR6.sup.−/− mice. CCR6.sup.−/− mice exhibited thinner ear thickness (FIG. 10a). Neutrophils also showed similar changes (FIG. 10a).

[0113] In the skin, there was no significant difference in the levels of IL-17 secreted by CD3, γδT, Vγ4, Vγ4Vδ4 cells between GC treatment group and the corresponding control group of WT and CCR2.sup.−/− mice. However, in GC-treated CCR6.sup.−/− mice, the levels of IL-17 secreted by dermal γδT, Vγ4, Vγ4Vδ4 cells were significantly lower than those in the corresponding control group.

[0114] In addition, the proportions of γδT17, Vγ4T17, and Vγ4Vδ4T17 cells decreased in draining and distant lymph nodes in WT mice, but not in CCR6.sup.−/− mice (FIG. 10c).

[0115] The above results indicated that knockout of CCR6 gene would inhibit not only the migration of γδT17 from the skin to the lymph nodes, but also the migration of γδT17 from the lymph nodes to the skin.

[0116] All documents mentioned in the present invention are incorporated by reference herein as if each document were incorporated separately by reference. Furthermore, it should be understood that after reading the foregoing teachings of the invention, various changes or modifications may be made to the invention by those skilled in the art and that these equivalents also fall in the scope of the claims appended to this application.