Use of a ccr2 inhibitor for the manufacture of a medicament for the treatment of hemophagocytic lymphohistiocytosis

Abstract

This invention belongs to the field of biomedical technology and discloses the application of CCR2 inhibitors in the preparation of drugs for treating hemophagocytic lymphohistiocytosis (HLH). By establishing a mouse model of HLH, this invention reveals for the first time that CCR2 can serve as an effective drug target for treating HLH. Experiments showed that treatment with the CCR2-specific antagonist RS102895 significantly reduced splenomegaly, improved peripheral blood cell reduction, alleviated liver damage, corrected metabolic disorders in model mice, and effectively inhibited serum cytokine storm and abnormal activation of splenic macrophages. Based on these findings, CCR2 can serve as a novel target for HLH treatment, and its inhibitors can be used to prepare highly effective and low-toxicity drugs for treating HLH.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CCR2 inhibitors in the preparation of drugs for the treatment of hemophagocytic lymphohistiocytosis. Background Technology

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory syndrome caused by excessive activation of the immune system. Its clinical features include persistent high fever, hepatosplenomegaly, cytopenia, severe cytokine storm, and multiple organ failure, with an extremely high mortality rate.

[0003] Currently, the first-line standard treatment for HLH (such as the HLH-94 / 04 regimen) mainly relies on broad immunosuppression using glucocorticoids and cytotoxic drugs (such as etoposide). Although this regimen can induce remission in some patients, it has significant limitations: First, it is a non-specific treatment, which, while suppressing the pathological immune response, extensively impairs normal immune defense and hematopoietic function, leading to serious complications such as infection and bone marrow suppression; second, some patients have poor treatment response or are prone to relapse after discontinuation of the drug; finally, long-term use of these drugs has cumulative toxic side effects, affecting the long-term quality of life of patients. Therefore, there is an urgent clinical need to develop new treatment strategies with stronger targeting and better efficacy. Summary of the Invention

[0004] Through in-depth research, the inventors have discovered for the first time that abnormal activation of the CCR2 signaling pathway is a key factor specifically driving the polarization of splenic macrophages towards the pro-inflammatory M1 phenotype in HLH pathological conditions, and this mechanism plays a central driving role in the pathogenesis of HLH. Based on this new discovery, the inventors propose that targeting the CCR2 signaling pathway can fundamentally inhibit the pathological progression of HLH.

[0005] To verify the feasibility of CCR2 as a therapeutic target for HLH, the inventors constructed a CpG oligodeoxynucleotide-induced HLH mouse model and administered the CCR2-specific antagonist RS102895 intraperitoneally to the mice in the treatment group at the same time as the model was established.

[0006] Experimental results showed that, compared with the untreated model group, RS102895 treatment not only effectively inhibited M1 polarization of splenic macrophages, verifying the mechanism of action revealed in this invention, but more surprisingly, through intervention of a single target, RS102895 treatment simultaneously and significantly reversed multiple core pathological manifestations in model mice, including significantly reducing splenomegaly, improving peripheral blood cell reduction, alleviating liver damage, correcting metabolic disorders, and effectively inhibiting serum cytokine storm.

[0007] In summary, this invention provides the application of CCR2 inhibitors in the preparation of drugs for the treatment of hemophagocytic lymphohistiocytosis (HLH). The therapeutic strategy proposed in this invention stems from the intervention of a newly identified key target in the disease mechanism of HLH. By regulating a single target, a synergistic effect of improving symptoms in multiple systems can be achieved. This provides a novel solution for developing highly targeted new HLH therapies aimed at intervening at the source of the disease, effectively overcoming the shortcomings of existing treatments, such as limited efficacy, high relapse rate, and non-specific immunosuppression.

[0008] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides the use of CCR2 inhibitors in the preparation of medicaments for the treatment of hemophagocytic lymphohistiocytosis.

[0009] In conjunction with the first aspect of the present invention, in some embodiments, the CCR2 inhibitor is a small molecule antagonist RS102895 that selectively inhibits the function of the CCR2 protein.

[0010] In conjunction with the first aspect of the present invention, in some embodiments, the pathophysiological features of the hemophagocytic lymphohistiocytosis include one or more of the following: splenomegaly, liver damage, metabolic disorders, hematologic abnormalities, and cytokine storm.

[0011] Furthermore, the splenomegaly includes a significant increase in spleen weight and visible hemophagocytosis in the tissue; the liver damage includes elevated levels of serum aspartate aminotransferase and alanine aminotransferase; the metabolic disorder includes elevated levels of serum triglycerides; the hematologic abnormalities include a decrease in the number of red blood cells, white blood cells, hemoglobin, and platelets in peripheral blood; and the cytokine storm includes elevated levels of serum IFN-γ, IL-1β, and IL-6.

[0012] In conjunction with the first aspect of the invention, in some embodiments, the treatment of hemophagocytic lymphohistiocytosis comprises one or more of the following: (1) Reduce splenomegaly and decrease the proportion of hemophagocytic macrophages in splenic tissue; (2) Improve peripheral blood cell reduction, wherein improving peripheral blood cell reduction includes increasing the number of red blood cells and white blood cells; (3) Reduce liver damage, wherein reducing liver damage includes lowering serum levels of aspartate aminotransferase and alanine aminotransferase; (4) Improve metabolic disorders, including reducing serum triglyceride levels; (5) Suppressing cytokine storm, wherein suppressing cytokine storm includes reducing serum levels of IFN-γ, IL-1β and IL-6; In conjunction with the first aspect of the present invention, in some embodiments, the dosage form of the drug is an injection or an oral dosage form.

[0013] In conjunction with the first aspect of the invention, in some embodiments, the medicament includes a pharmaceutically acceptable excipient or carrier. Further, the excipient includes at least one selected from sweeteners, colorants, lubricants, and fillers.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a CCR2 inhibitor and a pharmaceutically acceptable carrier for treating hemophagocytic lymphohistiocytosis, wherein the CCR2 inhibitor is a small molecule antagonist RS102895.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention reveals for the first time that CCR2 is a novel therapeutic target for hemophagocytic lymphohistiocytosis (HLH). By targeting and inhibiting CCR2, macrophage function can be precisely regulated, simultaneously improving multi-system clinical symptoms such as splenomegaly, cytopenia, liver damage, and metabolic disorders, and also inhibiting cytokine storms at their source. This strategy is highly targeted and, compared to traditional chemotherapy regimens, is expected to reduce the risk of off-target effects, providing a new treatment option for HLH. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The therapeutic effect of the CCR2 inhibitor RS102895 provided in Example 1 on the HLH mouse model. Among them, Figure 1 A is a schematic diagram of the experimental procedure for RS102895 treatment in a CpG-induced HLH mouse model; Figure 1 B shows representative images of the spleen of mice in each group and quantitative analysis of spleen weight; Figure 1 C shows representative images of Liu staining of spleen cell smears from each group of mice and quantitative analysis of corresponding positive cells (n=6). The arrows in the figure indicate hemophagocytic cells. Figure 1 D represents the complete blood cell count analysis of mice in each group; Figure 1 E is a graph showing the statistical results of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and triglyceride (TG) levels in each group of mice. Figure 1 F represents the quantitative analysis of plasma IFN-γ, IL-1β, and IL-6 cytokine levels in each group of mice. Figure 1 G represents the CD45 concentration in the spleen of mice from different treatment groups. + F4 / 80 + CD80 + Macrophage ratio; Figure 1 H represents representative Western blot images and statistical analysis of related protein expression in splenic macrophages of mice in different treatment groups (n=6). Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] the term: Cytokine storm: Under severe inflammatory stimulation, immune cells are abnormally overactivated, releasing large amounts of ILs in a short period of time. 6. IFN γ, TNF Multiple pro-inflammatory cytokines, including alpha, trigger an uncontrolled systemic cascade of inflammatory responses. This response can further lead to a series of pathological changes, such as tissue damage, organ dysfunction, metabolic disorders, and hematologic abnormalities, and is the core pathophysiological process of hemophagocytic lymphohistiocytosis.

[0020] This invention, through the establishment of a mouse model of hemophagocytic lymphohistiocytosis (HLH), reveals for the first time that CCR2 can serve as an effective drug target for the treatment of HLH. In vivo experiments showed that treatment with the CCR2-specific antagonist RS102895 significantly reduced splenomegaly, improved peripheral blood cell reduction, alleviated liver damage, corrected metabolic disorders in the model mice, and effectively inhibited serum cytokine storm and abnormal activation of splenic macrophages.

[0021] Based on this, in a first aspect, the present invention provides the use of CCR2 inhibitors in the preparation of medicaments for the treatment of hemophagocytic lymphohistiocytosis.

[0022] In conjunction with the first aspect of the present invention, in some embodiments, the CCR2 inhibitor is RS102895, a small molecule antagonist that selectively inhibits the function of the CCR2 protein. The CCR2 inhibitor RS102895 selected in the embodiments of the present invention is a recognized, highly selective small molecule antagonist of CCR2, and its specific binding to the CCR2 receptor and its inhibitory activity have been well demonstrated in existing studies (see Mirzadegan T, et al.). J Biol Chem . 2000;275(34):25562-71). Based on this, Example 1 uses RS102895 as a tool compound to verify the therapeutic effect of targeted inhibition of CCR2 function on HLH.

[0023] In conjunction with the first aspect of the present invention, in some embodiments, the pathophysiological features of the hemophagocytic lymphohistiocytosis include one or more of the following: splenomegaly, liver damage, metabolic disorders, hematologic abnormalities, and cytokine storm.

[0024] Furthermore, the splenomegaly includes a significant increase in spleen weight and visible hemophagocytosis in the tissue; the liver damage includes elevated levels of serum aspartate aminotransferase and alanine aminotransferase; the metabolic disorder includes elevated levels of serum triglycerides; the hematologic abnormalities include a decrease in the number of red blood cells, white blood cells, hemoglobin, and platelets in peripheral blood; and the cytokine storm includes elevated levels of serum IFN-γ, IL-1β, and IL-6.

[0025] In conjunction with the first aspect of the invention, in some embodiments, the treatment of hemophagocytic lymphohistiocytosis comprises one or more of the following: (1) Reduce splenomegaly and decrease the proportion of hemophagocytic macrophages in splenic tissue: like Figure 1 As shown in Figure B, the spleen weight of mice in the RS102895 treatment group was significantly lower than that in the HLH model group; Figure 1 As shown in Figure C, compared to the HLH model group, the proportion of hemophagocytic macrophages in the spleen cell smears of mice treated with RS102895 was significantly reduced. These results indicate that the CCR2 inhibitor RS102895 can effectively alleviate HLH-related splenomegaly and hemophagocytosis in the spleen tissue.

[0026] (2) Improve peripheral blood cytopenia, wherein improving peripheral blood cytopenia includes increasing the number of red blood cells and white blood cells: like Figure 1As shown in Figure D, the levels of erythrocytes (RBCs), leukocytes (WBCs), and hemoglobin (Hb) in the peripheral blood of mice in the RS102895 treatment group were partially restored compared to the HLH model group. The results indicate that the CCR2 inhibitor RS102895 can improve peripheral blood cell reduction induced by HLH.

[0027] (3) Reduce liver damage, wherein reducing liver damage includes lowering serum aspartate aminotransferase and alanine aminotransferase levels: like Figure 1 As shown in Figure E, the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice treated with RS102895 were significantly lower than those in the HLH model group. These results indicate that the CCR2 inhibitor RS102895 can alleviate HLH-related liver damage.

[0028] (4) Improvement of metabolic disorders, including reduction of serum triglyceride levels: like Figure 1 As shown in Figure E, the serum triglyceride (TG) level in mice treated with RS102895 was significantly lower than that in the HLH model group. These results indicate that the CCR2 inhibitor RS102895 can improve the metabolic disorders associated with HLH.

[0029] (5) Suppressing cytokine storm, wherein suppressing cytokine storm includes reducing serum levels of IFN-γ, IL-1β and IL-6: like Figure 1 As shown in Figure F, the serum levels of key inflammatory factors IFN-γ, IL-1β, and IL-6 in mice treated with RS102895 were significantly lower than those in the HLH model group. These results indicate that the CCR2 inhibitor RS102895 can effectively suppress the cytokine storm characteristic of HLH.

[0030] In some embodiments, the treatment of hemophagocytic lymphohistiocytosis further includes inhibiting M1 macrophage polarization. Flow cytometry analysis showed ( Figure 1 G), F4 / 80 in the spleen of mice in the RS102895 treatment group + CD80 + The proportion of macrophages (M1 type) was significantly lower than that in the HLH model group. The results indicate that the CCR2 inhibitor RS102895 can inhibit the polarization of splenic macrophages towards the pro-inflammatory M1 type.

[0031] In conjunction with the first aspect of the present invention, in some embodiments, the dosage form of the drug is an injection or an oral dosage form. In Example 1, the CCR2 inhibitor RS102895 was administered via intraperitoneal injection, with an effective dose of 5 mg / kg, and the solvent used for the injection was physiological saline.

[0032] In conjunction with the first aspect of the invention, in some embodiments, the medicament includes a pharmaceutically acceptable excipient or carrier. Further, the excipient includes at least one selected from sweeteners, colorants, lubricants, and fillers.

[0033] In a second aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a CCR2 inhibitor and a pharmaceutically acceptable carrier for treating hemophagocytic lymphohistiocytosis, wherein the CCR2 inhibitor is a small molecule antagonist RS102895.

[0034] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to total protein extraction, protein sample preparation, molecular biology detection (such as ELISA, Western blot), cell biology techniques (such as flow cytometry, Liu staining), in vitro model construction, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met the general quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental and control groups were set up according to the grouping methods commonly used in the art.

[0035] The C57BL / 6J mice used in the following examples were purchased from Hubei Beinte Biotechnology Co., Ltd., and all animal experiments have been approved by the Ethics Committee of the Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Approval No.: 2024-IACUC-4881).

[0036] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data are expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. In the attached figures, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates no statistical difference.

[0037] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0038] Example 1: Therapeutic effect of CCR2 inhibitor RS102895 on HLH mouse model 1. Purpose To verify the therapeutic effect of the CCR2-specific antagonist RS102895 on hemophagocytic lymphohistiocytosis and to preliminarily explore its mechanism of action.

[0039] 2. Experimental materials and instruments Experimental animals: SPF-grade male C57BL / 6J mice, 6-8 weeks old, weighing approximately 23g, 50 mice in total, purchased from Hubei Beinte Biotechnology Co., Ltd. Main reagents: CpG oligodeoxynucleotides (CpG ODN) were purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: 2800643900; CCR2 antagonist RS102895 was purchased from MedChemExpress (MCE) in the United States, product number: HY-18611A; The IFN-γ, IL-1β and IL-6 cytokine quantitative enzyme-linked immunosorbent assay (ELISA) kits were all purchased from Thermo Fisher Scientific.

[0040] Main instrument: Flow cytometer (DxFLEX) purchased from Beckman Coulter, USA.

[0041] 3. Experimental Methods Male C57BL / 6J mice aged 6-8 weeks were randomly divided into three groups: a control group, an HLH model group, and an RS102895 treatment group. Except for the control group, the other two groups underwent HLH model induction via intraperitoneal injection of CpG oligodeoxynucleotides, administered every 3 days for a total of 4 injections. The treatment group received RS102895 (5 mg / kg) intraperitoneally immediately after each CpG injection, every 12 hours until the end of the experiment. The control and model groups received an equal volume of physiological saline. Mice were sacrificed 24 hours after the last administration, and serum, spleen, and peripheral blood samples were collected for the following tests.

[0042] (1) Spleen index: Weigh the spleen and calculate the spleen index (spleen weight / body weight × 100%). (2) Peripheral blood cell count: The levels of red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb) and platelets (PLT) were detected using a fully automated blood cell analyzer; (3) Serum biochemical indicators: The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and triglycerides (TG) were detected using a fully automated biochemical analyzer; (4) Serum inflammatory factors: The levels of IFN-γ, IL-1β and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA); (5) Analysis of spleen macrophages: F4 / 80 macrophages in spleen tissue were detected by flow cytometry. + CD80 +The proportion of macrophages (M1 type) and hemophagocytic macrophages; (6) Western blot: Splenic macrophages were sorted and the phosphorylation levels of PI3K and AKT were detected.

[0043] 4. Experimental Results The experimental procedure in this embodiment is as follows: Figure 1 As shown in Figure A, compared with the untreated HLH model group, the RS102895 treatment group showed significantly reduced splenomegaly, a significantly decreased spleen index (P<0.05), and a significantly reduced proportion of hemophagocytic macrophages in the spleen tissue. Figure 1 Peripheral blood tests showed that RBC, WBC, and Hb levels partially recovered after RS102895 treatment, while PLT count showed no significant change. Figure 1 D). Figure 1 Serum biochemical markers of E showed that the levels of AST, ALT, and TG in the treatment group were significantly lower than those in the model group (P<0.05). Serum inflammatory factor detection ( Figure 1 F) showed that RS102895 significantly inhibited the characteristic cytokine storm of HLH, with a significant decrease in IFN-γ, IL-1β, and IL-6 levels (P<0.05). Flow cytometry results ( Figure 1 G) showed that F4 / 80 in the spleen of the RS102895 treatment group + CD80 + The proportion of macrophages (M1 type) was significantly lower than that in the model group, indicating that M1 type polarization was suppressed. Western blot results ( Figure 1 H) showed that the phosphorylation levels of PI3K and AKT in splenic macrophages were significantly upregulated after RS102895 treatment.

[0044] 5. Conclusion The CCR2-specific antagonist RS102895 demonstrated a clear therapeutic effect in the HLH mouse model, effectively reducing splenomegaly, improving peripheral blood cell reduction and liver damage, correcting metabolic disorders, inhibiting systemic cytokine storm, and suppressing M1 polarization of splenic macrophages. This provides key preclinical experimental evidence for establishing CCR2 as a new therapeutic target for HLH.

[0045] The above results indicate that targeted inhibition of CCR2 can effectively improve various core clinical phenotypes of HLH, and its mechanism of action may be related to the regulation of the PI3K / AKT signaling pathway and the inhibition of abnormal macrophage activation. The CCR2 inhibitor used in this invention has a clear target and higher targeting selectivity compared to traditional chemotherapy drugs, possessing good clinical application potential and providing a new strategy and option for the treatment of hemophagocytic lymphohistiocytosis.

[0046] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0047] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0048] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of CCR2 inhibitors in the preparation of drugs for the treatment of hemophagocytic lymphohistiocytosis, characterized in that, The CCR2 inhibitor is RS102895, a small molecule antagonist that selectively inhibits the function of the CCR2 protein.

2. The application according to claim 1, characterized in that, The pathophysiological features of hemophagocytic lymphohistiocytosis include one or more of the following: splenomegaly, liver damage, metabolic disorders, hematologic abnormalities, and cytokine storm.

3. The application according to claim 2, characterized in that, The splenomegaly includes a significant increase in spleen weight and visible hemophagocytosis in the tissue.

4. The application according to claim 2, characterized in that, The liver damage includes elevated levels of serum aspartate aminotransferase and alanine aminotransferase.

5. The application according to claim 2, characterized in that, The metabolic disorder includes elevated serum triglyceride levels.

6. The application according to claim 2, characterized in that, The hematologic abnormalities include a decrease in the number of red blood cells, white blood cells, hemoglobin, and platelets in peripheral blood.

7. The application according to claim 2, characterized in that, The cytokine storm includes elevated levels of IFN-γ, IL-1β, and IL-6 in serum.

8. The application according to claim 1, characterized in that, The treatment for hemophagocytic lymphohistiocytosis includes one or more of the following: (1) Reduce splenomegaly; (2) Improves peripheral blood cell reduction; (3) Reduce liver damage; (4) Improves metabolic disorders; (5) Suppress cytokine storm.

9. The application according to claim 1, characterized in that, The drug is available in either injectable or oral form.

10. A pharmaceutical composition, characterized in that, The invention comprises an effective amount of a CCR2 inhibitor and a pharmaceutically acceptable carrier for the treatment of hemophagocytic lymphohistiocytosis, wherein the CCR2 inhibitor is the small molecule antagonist RS102895.

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