Composition containing mCCL6 / hCCL15-CCR1 signaling pathway blocker and application thereof

Abstract

The invention provides an application of a composition containing an mCCL6 / hCCL15-CCR1 signal channel blocking agent in preparation of a medicine for treating diseases related to inflammatory bowel diseases. According to the application, the mechanism that expression of mCCL6 / hCCL15 in intestinal fibrosis is increased, intestinal fibroblast activation and collagen generation are promoted, and then intestinal fibrosis progress can be promoted is found for the first time. In a mouse intestinal fibrosis model, the intestinal fibrosis can be effectively relieved by blocking a CCL6-CCR1 signal transduction pathway by using an mCCL6 neutralizing antibody and a CCR1 small molecule antagonist. According to the application, the specific target spot is found, the CCL6-CCR1 signal channel is inhibited, and the intestinal fibrosis is specifically treated, so that the application has breakthrough significance on health improvement of patients with the intestinal fibrosis and even intestinal stenosis, and a new idea is provided for treatment of the intestinal fibrosis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of a composition comprising an mCCL6 / hCCL15-CCR1 signaling pathway blocker in the preparation of a drug for treating inflammatory bowel disease-related diseases. Background Art

[0002] In recent years, with the changes in people's lifestyle and dietary structure, the incidence of inflammatory bowel disease (IBD) has increased year by year. About 30% of Crohn's disease (CD) patients and 5% of ulcerative colitis (UC) patients will eventually develop intestinal obstruction due to intestinal fibrosis, which seriously threatens the life and health of patients. Intestinal fibrosis is formed by excessive deposition of extracellular matrix (ECM) derived from activated mesenchymal cells in the intestinal wall, mainly in the submucosal layer and muscularis propria. Intestinal fiber formation is driven by a variety of cells, mainly including local fibroblast proliferation and activation, epithelial-mesenchymal transition (EMT), recruitment and differentiation of fibroblasts, and endothelial cell transformation (Endo-MT), among which myofibroblasts are the most critical factor. Since the occurrence of IBD is caused by inflammation, people have high expectations for effective anti-inflammatory therapies developed in the past 20 years, hoping that these therapies can fight fibrosis and stenosis. However, this expectation has not been realized. Currently used anti-inflammatory drugs such as biologics can significantly relieve intestinal inflammation, but the incidence of intestinal stenosis has not been substantially reduced. Therefore, there is an urgent need to develop specific therapeutic drugs for intestinal fibrosis, which is also one of the issues that need urgent attention in the field of intestinal fibrosis research.

[0003] Chemokines are a class of cytokines that are locally expressed at sites of inflammation and regulate the recruitment of leukocytes and lymphocytes to injured tissues. In addition to classical chemotaxis, chemokines can also directly interact with fibroblasts in fibrotic diseases. Mouse mCCL6 is highly homologous to human hCCL15 and can activate CCR1. It has been reported that mCCL6 plays an important role in many inflammatory disease processes, including enteritis, pulmonary fibrosis, allergic bronchopulmonary aspergillosis, sepsis, and experimental demyelinating diseases. However, the exact function of mCCL6 / hCCL15 in intestinal fibrosis is still unclear.

[0004] Currently, there is no effective treatment plan or medicine for complications related to inflammatory bowel disease. Anti-inflammatory drugs are mostly used clinically, but they have failed to truly reduce the incidence of intestinal stenosis. Summary of the invention

[0005] In view of the above-mentioned technical limitations, the present application proposes the use of a composition comprising an mCCL6 / hCCL15-CCR1 signaling pathway blocker in the preparation of a drug for treating inflammatory bowel disease-related diseases; it overcomes the deficiencies and defects mentioned in the background technology.

[0006] Prior art reports that mCCL6 is expressed in cells of the mouse neutrophil and macrophage lineages, and the receptor is CCR1. The inventors have found that mCCL6 is highly expressed in intestinal fibrotic colon tissue and is also significantly highly expressed in MDSCs. In the intestinal tissue of patients with intestinal stenosis, the expression of hCCL15 and its receptor CCR1 is also higher than that of inflammatory non-stenotic and healthy tissues. Therefore, it is further inferred that mCCL6 / hCCL15 in mouse intestinal fibrotic colon tissue may be closely related to the severity of intestinal fibrosis.

[0007] To achieve the above objectives, based on the previous research results, this application adopts the following technical solutions:

[0008] The inventive point of the present application is to provide the use of a composition comprising an mCCL6 / hCCL15-CCR1 signaling pathway blocker in the preparation of a drug for treating inflammatory bowel disease-related diseases.

[0009] Optionally, in the above application, the mCCL6 / hCCL15-CCR1 signaling pathway blocker is selected from one or more of mCCL6 neutralizing antibodies, hCCL15 antibodies and CCR1 antagonists.

[0010] Mouse mCCL6 has high homology to human hCCL15 and can activate CCR1.

[0011] Optionally, in the above application, the inflammatory bowel disease-related diseases are selected from one or more of intestinal fibrosis and intestinal stenosis.

[0012] Optionally, in the above application, the mCCL6 neutralizing antibody is selected from one or more of AF487, AB487NA and MAB487, and the hCCL15 antibody is selected from one or more of MAB363, AF363 and MAB3631.

[0013] Optionally, in the above application, the CCR1 antagonist is selected from one or more of BX471, J-113863, BMS-817399, AZD-4818, BMS-457 and CCX354.

[0014] The preferred mCCL6 neutralizing antibody MAB487 is selected from R&D Company, and the preferred CCR1 antagonist BX471 is selected from MCE Company.

[0015] BX471 is a potent and selective antagonist of CCR1. It can displace CCR1 ligands with high affinity and inhibit a large number of CCR1-mediated effects. It is an effective functional antagonist.

[0016] BX471 has the structural formula (I):

[0017]

[0018] Optionally, in the above application, the dosage of the mCCL6 neutralizing antibody is 2-8 μg / mouse; the dosage of the hCCL15 antibody is 0.4-3 mg / kg, preferably 1 mg / kg.

[0019] Optionally, in the above application, the dosage of the CCR1 antagonist is 3-20 mg / kg.

[0020] Optionally, in the above-mentioned application, the administration method of the drug includes oral administration, parenteral administration or a combination thereof; the parenteral administration includes intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intramedullary administration, intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intraosseous administration, intrathecal administration or a combination thereof.

[0021] The mCCL6 / hCCL15-CCR1 signaling pathway blockers provided in the present application are small molecule compounds or antibodies that can inhibit the function of mCCL6 / hCCL15 or CCR1 proteins, and can reduce or eliminate the expression of mCCL6 / hCCL15 or CCR1 proteins. These small molecule compounds or antibodies also include a series of modifications while maintaining this ability. Modified antibodies can be used to improve in vitro or in vivo properties, such as use effect, stability, activity and bioavailability.

[0022] The mCCL6 / hCCL15-CCR1 signaling pathway blocker of the present application can be provided in the form of a pharmaceutical composition. Pharmaceutical compositions suitable for use in the present invention include compositions containing active ingredients in an amount effective for achieving the intended purpose. The determination of an effective dose is entirely within the ability of those skilled in the art to be effective in treatment. The dose refers to the amount of active ingredient (e.g., an antibody or small molecule antagonist of the present invention) sufficient to treat a specific condition. The exact dosage will be determined by the physician according to factors related to the subject in need of treatment.

[0023] Compared with the prior art, this application has the following advantages:

[0024] At present, there is no specific therapeutic drug for the symptoms of patients with intestinal fibrosis. This application discovered for the first time that the expression of mCCL6 / hCCL15 in intestinal fibrosis increases, and promotes intestinal fibroblast activation and collagen production, which can promote the progression of intestinal fibrosis. In the mouse intestinal fibrosis model, intestinal fibrosis was effectively alleviated by blocking the CCL6-CCR1 signal transduction pathway using mCCL6 neutralizing antibodies and CCR1 small molecule antagonists. This application specifically treats intestinal fibrosis through specific targets, which has a groundbreaking significance for the improvement of the health of patients with intestinal fibrosis and even intestinal stenosis. The technology of this application can achieve the purpose of treating intestinal fibrosis by inhibiting the CCL6-CCR1 signaling pathway, providing a new idea for the treatment of intestinal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It was shown that the expression of mCCL6 in the colon tissue of intestinal fibrosis model mice was significantly increased.

[0026] Figure 2 The results showed that the expression of hCCL15 in the intestinal tissues of patients with clinical intestinal stenosis was significantly increased.

[0027] Figure 3 Shown is an in vivo evaluation of the therapeutic effect of mCCL6 neutralizing antibody, MAB487, on intestinal fibrosis in mice.

[0028] Figure 4 Shown is an in vivo evaluation of the therapeutic effect of CCR1 small molecule antagonist BX471 on intestinal fibrosis in mice.

[0029] Figure 5 Shown is a schematic diagram of the inhibitory effect of the mCCL6 neutralizing antibody, MAB487.

[0030] Figure 6 Shown is a schematic diagram of the inhibitory effect of hCCL15 neutralizing antibody - MAB363.

[0031] Figure 7 Shown is an in vivo evaluation of the therapeutic effect of the combination of (mCCL6 neutralizing antibody - MAB487 + CCR1 small molecule antagonist - BX471) on intestinal fibrosis in mice.

[0032] Figure 8 Shown is an in vivo evaluation of the therapeutic effect of another combination drug (mCCL6 neutralizing antibody - AF487 + CCR1 small molecule antagonist - J-113863) on intestinal fibrosis in mice. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0034] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0035] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0036] Example 1

[0037] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a recurrent gastrointestinal inflammatory disease. Intestinal fibrosis is a common complication of IBD, with more than 50% of CD patients and 10% of UC patients experiencing significant stenosis or even obstruction, which seriously threatens the patients' lives and health.

[0038] Studies have shown that intestinal mesenchymal cells, immune cells, cytokines, microbial products, and mesenteric adipocytes are jointly involved in the pathogenesis of intestinal fibrosis. The fibrogenic process is driven by a variety of cells, among which fibroblast activation is the direct cause of intestinal fibrosis. Regarding the treatment of intestinal fibrosis, there is currently no specific therapeutic drug, and it is mainly treated with anti-inflammatory drugs. However, recent reports show that despite the increasing anti-inflammatory ability of biological agents, the incidence of intestinal stenosis has not been substantially reduced in clinical practice. Inflammation and fibrosis are both closely linked and relatively independent. Clinical studies have shown that endoscopically healed UC mucosa still has persistent abnormalities, including ECM remodeling, fibrotic factor production, and activation of the TGF-β1 signaling pathway. Although chronic inflammation is the initiator of intestinal fibrosis, the existing therapeutic drugs have limited efficacy against intestinal fibrosis, which is also a major challenge faced clinically. Therefore, there is an urgent need to develop specific therapeutic drugs for intestinal fibrosis, which is also one of the issues that need urgent attention in the field of intestinal fibrosis research.

[0039] Based on the existing clinical treatment difficulties, the present application provides the use of a composition comprising an mCCL6 / hCCL15-CCR1 signaling pathway blocker in the preparation of a drug for treating inflammatory bowel disease-related diseases.

[0040] The mCCL6 / hCCL15-CCR1 signaling pathway blocker is selected as one or more of mCCL6 neutralizing antibodies, hCCL15 antibodies and CCR1 antagonists; mouse mCCL6 has a high homology with human hCCL15 and can activate CCR1.

[0041] IBD-related diseases target intestinal fibrosis or intestinal strictures.

[0042] The mCCL6 neutralizing antibody can be selected from all factors, antibodies, preparations, proteins and nucleic acid drugs that have an inhibitory effect on mCCL6. In the present application, one or more of AF487, AB487NA and MAB487 are selected; MAB487 is preferably used.

[0043] Similarly, hCCL15 antibodies can also be selected from all factors, antibodies, preparations, proteins and nucleic acid drugs that have an inhibitory effect on hCCL15. In the present application, one or more of MAB363, AF363 and MAB3631 are selected; MAB363 is preferably used.

[0044] CCR1 antagonists can also be selected from all factors, antibodies, preparations, proteins and nucleic acid drugs that have an inhibitory effect on CCR1. In this application, one or more of BX471, J-113863, BMS-817399, AZD-4818, BMS-457 and CCX354 are selected; BX471 is preferably used.

[0045] MAB487 was selected from R&D Company, and BX471 was selected from MCE Company.

[0046] The dosage of the mCCL6 neutralizing antibody for the mouse model is 2-8 μg / mouse; the dosage of the hCCL15 antibody for human patients is 0.4-3 mg / kg, preferably 1 mg / kg.

[0047] The CCR1 antagonist is administered at a dose of 3-20 mg / kg.

[0048] The administration of the drug includes oral administration, parenteral administration or a combination thereof; the parenteral administration includes intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intramedullary administration, intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intraosseous administration, intrathecal administration or a combination thereof.

[0049] Example 2

[0050] First, a TNBS intestinal fibrosis mouse model was established. The specific method is as follows:

[0051] Eight-week-old Balb / c female mice were selected, and the model group mice were presensitized on the first day: acetone and olive oil were mixed in a volume ratio of 4:1, and then the mixture was mixed with 5% (wt / vol) TNBS in a volume ratio of 4:1 to obtain 1% TNBS. An electric shaver was used to shave the hair of about 1.5 cm long and wide on the back and neck of the mouse, and 150 μL 1% TNBS was dripped on the exposed area after exposing the skin. The mice were enemaed every week for seven weeks: the mice were fasted for 24 hours, and then anesthetized by intraperitoneal injection of 80 mg / kg sodium pentobarbital. A 3.5F catheter was lubricated with glycerol, connected to a 1mL syringe, and gently inserted into the mouse anus about 4 cm deep. Then 80 μL TNBS enema was slowly injected and hung upside down for 30 seconds. The concentrations of TNBS were 0.75%, 1.5%, 2.5%, 2.5%, 2.5%, and 2.5%, respectively, for a total of six times.

[0052] Second, TNBS was used to repeatedly induce the establishment of a mouse intestinal fibrosis model. After the model was established, the mouse colon tissues were collected and the expression level of CCL6 was detected by ELISA. The test results were as follows: Figure 1 shown.

[0053] Third, clinical intestinal tissue samples were collected from patients with intestinal stricture, including healthy tissue, inflammatory non-stricture tissue, and stricture tissue. Intestinal tissue RNA was extracted, reverse transcribed, and hCCL15 mRNA levels were detected. The test results were as follows: Figure 2 shown.

[0054] Fourth, for the mouse intestinal fibrosis model, the mCCL6 neutralizing antibody (MAB487) was used alone in the mouse intestinal fibrosis model. MAB487 was intraperitoneally injected at a dose of 4 μg / mouse, 3 times a week, and continued for 6 cycles. IgG2B was used as a negative control. After the treatment model was established, the mouse colon tissue was collected, and the colon tissue was pathologically stained and statistically analyzed. The staining and statistical results are shown in the figure. Figure 3 shown.

[0055] Fifth, for the mouse intestinal fibrosis model, the CCR1 small molecule antagonist (BX471) was used alone in the body to treat mouse intestinal fibrosis. The small molecule antagonist BX471 was intraperitoneally injected at a dose of 20 mg / kg every day for 6 cycles. After the treatment model was established, the mouse colon tissue was collected, and the colon tissue was pathologically stained and statistically analyzed. The staining and statistical results are shown in the figure. Figure 4 shown.

[0056] The inhibitory effect of the commercial mCCL6 neutralizing antibody MAB487 is as follows Figure 5 shown.

[0057] The inhibitory effect of the commercial hCCL15 neutralizing antibody MAB363 is as follows Figure 6 shown.

[0058] Sixth, for the mouse intestinal fibrosis model, the mCCL6 neutralizing antibody (MAB487) and the CCR1 small molecule antagonist (BX471) were used in combination to treat mouse intestinal fibrosis. The mice in the MAB487 group were intraperitoneally injected with MAB487 at a dose of 4 μg / mouse, 3 times a week, for 6 cycles. For the mice in the BX471 treatment group, BX471 was intraperitoneally injected at a dose of 20 mg / kg every day for 6 cycles. For the mice in the combined treatment group of MAB487 and BX471, BX471 was intraperitoneally injected at a dose of 10 mg / kg every day, and MAB487 was intraperitoneally injected at a dose of 2 μg / mouse, 3 times a week, for 6 cycles. After the treatment model was constructed, the mouse colon tissues were collected, and the hydroxyproline content of the colon tissues was detected. The results of the hydroxyproline content detection and analysis are as follows. Figure 7 shown.

[0059] Seventh, for the mouse intestinal fibrosis model, the mCCL6 neutralizing antibody (AF487) and the CCR1 small molecule antagonist (J-113863) were used in combination to treat mouse intestinal fibrosis. The mice in the AF487 group were intraperitoneally injected with AF487 at a dose of 8 μg / mouse, 3 times a week, for 6 cycles. For the mice in the J-113863 treatment group, J-113863 was intraperitoneally injected at a dose of 6 mg / kg every day for 6 cycles. For the mice in the AF487 and J-113863 combined treatment group, J-113863 was intraperitoneally injected at a dose of 3 mg / kg every day, and AF487 was intraperitoneally injected at a dose of 4 μg / mouse, 3 times a week, for 6 cycles. After the treatment model was constructed, the mouse colon tissues were collected, and the hydroxyproline content of the colon tissues was detected. The results of the hydroxyproline content detection and analysis are as follows. Figure 8 shown.

[0060] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of a composition comprising an mCCL6 / hCCL15-CCR1 signaling pathway blocker in the preparation of a medicament for treating inflammatory bowel disease-related diseases.

2. The use according to claim 1, characterized in that: The mCCL6 / hCCL15-CCR1 signaling pathway blocker is selected from one or more of mCCL6 neutralizing antibodies, hCCL15 antibodies and CCR1 antagonists.

3. The use according to claim 1 or 2, characterized in that: The inflammatory bowel disease-related diseases are selected from one or more of intestinal fibrosis and intestinal stenosis.

4. The use according to claim 1 or 2, characterized in that: The mCCL6 neutralizing antibody is selected from one or more of AF487, AB487NA and MAB487, and the hCCL15 antibody is selected from one or more of MAB363, AF363 and MAB3631.

5. The use according to claim 1 or 2, characterized in that: The CCR1 antagonist is selected from one or more of BX471, J-113863, BMS-817399, AZD-4818, BMS-457 and CCX354.

6. The use according to claim 1 or 2, characterized in that: The dosage of the mCCL6 neutralizing antibody is 2 μg / mouse to 8 μg / mouse, and the dosage of the hCCL15 antibody is 0.4 mg / kg to 3 mg / kg.

7. The use according to claim 1 or 2, characterized in that: The dosage of the CCR1 antagonist is 3-20 mg / kg.

8. The use according to claim 1 or 2, characterized in that: The administration mode of the drug includes oral administration, parenteral administration or a combination thereof; the parenteral administration includes intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intramedullary administration, intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intraosseous administration, intrathecal administration or a combination thereof.

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