Use of btk protac compound nx-5948

Abstract

The present application relates to the technical field of biological medicine, in particular to a new application of a BTK PROTAC compound NX-5948, especially to a new application of a Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 in a drug for treating secondary hemophagocytic syndrome (HLH). In the present application, the BTK PROTAC compound NX-5948 inhibits the activation of the macrophage BTK / NF-κB axis signaling pathway through targeting, on the one hand, reduces the number of activated macrophages by inhibiting the proliferation and survival of macrophages, on the other hand, induces the polarization of macrophages from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages through the immunoregulatory effect on macrophages, and then inhibits the release of cytokines and the occurrence and development of HLH, so that the effect of the new application of the present application is that it can inhibit the abnormal activation of macrophages and improve the clinical symptoms or laboratory indexes existing in patients with secondary hemophagocytic syndrome.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, in particular to a new application of a BTK PROTAC compound NX-5948, in particular to a new application of a Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 in a drug for treating secondary hemophagocytic syndrome (HLH). BACKGROUND

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a fatal syndrome triggered by overactivation of the immune system, characterized by persistent fever, hepatosplenomegaly, cytopenia and multi-organ failure, with extremely poor clinical prognosis.

[0003] Currently, the standard treatment regimen (such as the HLH-1994 / 2004 regimen) for HLH relies on the chemotherapy strategy of etoposide combined with dexamethasone, but about 25%-50% of patients do not respond to treatment, and the 5-year survival rate is only 54%;

[0004] Although the introduction of targeted drugs (such as JAK inhibitor ruxolitinib, interferon gamma antibody emapalumab) in recent years has partially improved the prognosis of patients, the problems of insufficient depth of disease remission and recurrence are still major challenges in clinical practice, therefore, there is an urgent need to explore the pathogenesis of HLH and develop new treatment strategies;

[0005] Studies have shown that abnormal activation of macrophages is a key driver of the pathological process of HLH, however, under the inflammatory microenvironment, macrophages polarize to the pro-inflammatory M1 phenotype, release a large amount of cytokines (such as IFN-γ, TNF-α, IL-6), intensify their own activation through a positive feedback loop, and further activate cytotoxic T cells (CTL) and natural killer (NK) cells, ultimately leading to a "cytokine storm" and multi-organ failure, therefore, inhibition of abnormal activation of macrophages is a key target for improving the prognosis of HLH;

[0006] Bruton's tyrosine kinase (BTK) as a core kinase of the B cell receptor (BCR) signaling pathway, has been found in recent years to regulate the proliferation, activation and inflammatory response of macrophages, that is, in HLH, BTK is activated by cytokines (such as IFN-γ, IL-6) through the TLR-mediated signaling pathway, and then phosphorylates the downstream transcription factor NF-κB, driving the expression of pro-inflammatory genes, forming a "BTK / NF-κB axis" vicious cycle, although the first generation of BTK inhibitors (such as ibritumomab) has shown certain potential in the treatment of HLH, it only inhibits the kinase activity of BTK, and cannot completely block the NF-κB signal transduction, and the clinical efficacy is limited by the residual effects of the cytokine storm;

[0007] In recent years, the PROTAC technology realizes the complete degradation of pathogenic proteins through the ubiquitin-proteasome pathway, providing a new idea to overcome the limitations of traditional inhibitors.

[0008] Studies have shown that BTK PROTAC can efficiently degrade BTK protein, block its downstream signaling, and exhibit significant efficacy in B-cell malignancies and autoimmune diseases. However, the application of BTK PROTAC in HLH treatment has not been reported, and whether it can improve the pathological process of HLH by regulating macrophage polarization and inhibiting cytokine storm remains unknown. SUMMARY

[0009] The present application aims to address the deficiencies of existing HLH treatment regimens. The present application proposes a new application of Bruton's tyrosine kinase protein degradation targeting chimera (BTK PROTAC) compound NX-5948 in the preparation of a drug for treating secondary hemophagocytic syndrome (HLH). This application can inhibit abnormal activation of macrophages and improve the clinical symptoms or laboratory indicators present in patients with secondary hemophagocytic syndrome.

[0010] To achieve the above-mentioned purpose, the present application specifically adopts the following technical solutions:

[0011] The present application proposes the application of Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 or its pharmaceutically acceptable salt as a pharmaceutical active ingredient in the preparation of a drug for treating secondary hemophagocytic syndrome.

[0012] As a preferred technical solution of the present application, the application can inhibit abnormal activation of macrophages.

[0013] As a preferred technical solution of the present application, the application can improve at least one of the following clinical symptoms or laboratory indicators in patients with secondary hemophagocytic syndrome:

[0014] Clinical symptoms: fever, hepatosplenomegaly, central nervous system dysfunction, liver and kidney function failure;

[0015] Laboratory indicators: cytopenia, hypertriglyceridemia and / or hypofibrinogenemia, elevated ferritin, elevated soluble interleukin 2 receptor (sCD25), bone marrow hemophagocytosis, and pro-inflammatory cytokine storm.

[0016] As a preferred technical solution of the present application, the compound structure of the BTK PROTAC compound NX-5948 is:

[0017]

[0018] is 3-((4-(1-((1-(6-(((S)-2,6-dioxopiperidin-3-yl)carbamoyl)pyridin-3-yl)piperidin-4-yl)methyl)piperidin-4-yl)phenyl)amino)-5-((R)-3-(3-yl-2-oxoimidazolidin-1-yl)piperidin-1-yl)pyrazine-2-carboxylic acid.

[0019] As a preferred technical solution of the present application, the administration mode of the drug is at least one of oral administration, intravenous injection, subcutaneous injection or intrathecal injection.

[0020] As a preferred technical solution of the present application, the administration mode of the drug is at least one of oral administration, intravenous injection, subcutaneous injection or intrathecal injection.

[0021] The present application also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for treating secondary hemophagocytic syndrome.

[0022] The dosage form of the pharmaceutical composition is oral preparation, injection or sustained-release preparation.

[0023] In addition, the present application also provides a method for treating secondary hemophagocytic syndrome, comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to a patient in need thereof.

[0024] The beneficial effects of the present application are as follows:

[0025] In the present application, the BTK PROTAC compound NX-5948 targets and inhibits the activation of the BTK / NF-κB axis signaling pathway of macrophages, on the one hand, reduces the number of activated macrophages by inhibiting the proliferation and survival of macrophages, and on the other hand, induces the polarization of macrophages from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages through immune regulation of macrophages, thereby inhibiting the release of cytokines and the occurrence and development of HLH. Therefore, the application of the Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutical active ingredient in the preparation of a drug for treating secondary hemophagocytic syndrome is feasible, so that the present application has the effect of being able to inhibit the abnormal activation of macrophages and improve the clinical symptoms or laboratory indicators existing in patients with secondary hemophagocytic syndrome. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a detection result graph of the detection of the expression level of BTK protein in the cell after the NX-5948 treatment of the RAW264.7 cell line in Experiment 1 by Western blot method;

[0027] Figure 2 Figure 2 is a graph showing the results of the CCK-8 method for detecting the activity of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the RAW264.7 cell line in Experiment 2.

[0028] Figure 3 Figure 3 is a graph showing the results of the CCK-8 method for detecting the activity of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the JeKo-1 cell line in Experiment 3.

[0029] Figure 4 Figure 4 is a graph showing the results of flow cytometry for detecting the apoptosis of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the HLH in vitro cell model in Experiment 4.

[0030] Figure 5 Figure 5 is a graph showing the results of flow cytometry for detecting the intracellular reactive oxygen species (ROS) level of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the HLH in vitro cell model in Experiment 5.

[0031] Figure 6 Figure 6 is a graph showing the results of PI staining for detecting the cell cycle of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the HLH in vitro cell model in Experiment 6.

[0032] Figure 7 Figure 7 is a graph showing the results of ELISA for detecting the concentration of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the supernatant of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the HLH in vitro cell model in Experiment 7.

[0033] Figure 8 Figure 8 is a graph showing the results of flow cytometry for detecting the phenotype of macrophage polarization of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the HLH in vitro cell model in Experiment 8.

[0034] Figure 9 Figure 9 is a graph showing the results of weighing the body weight of mice and the weight of liver and spleen tissues of mice after administration of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 in mice with HLH syndrome in Experiment 9.

[0035] Figure 10Figure 10 is a graph showing the results of blood routine test of peripheral blood of mice with HLH syndrome after administration of Ruxolitinib, Ibrutnib, Zanubrutinib and NX-5948 in Experiment 10.

[0036] Figure 11 Figure 11 is a graph showing the results of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and uric acid (UA) test of peripheral blood serum of mice with HLH syndrome after administration of Ruxolitinib, Ibrutnib, Zanubrutinib and NX-5948 in Experiment 11.

[0037] Figure 12 Figure 12 is a graph showing the results of cytokine concentration test of peripheral blood serum of mice with HLH syndrome after administration of Ruxolitinib, Ibrutnib, Zanubrutinib and NX-5948 in Experiment 12.

[0038] Figure 13 Figure 13 is a graph showing the results of serum ferritin and IL-2R level test of peripheral blood serum of mice with HLH syndrome after administration of Ruxolitinib, Ibrutnib, Zanubrutinib and NX-5948 in Experiment 13.

[0039] Figure 14 Figure 14 is a structural diagram of the compound structure of BTK PROTAC compound NX-5948. DETAILED DESCRIPTION

[0040] The raw materials, equipment and reagents used in the specific embodiments of the present application are known products, which are obtained by purchasing commercially available products.

[0041] In recent years, the targeted protein degradation chimera (PROTAC) technology has made breakthrough progress in the field of drug research and development. This technology "hijacks" E3 ubiquitin ligase, and realizes specific degradation of target proteins by using the ubiquitin-proteasome system.

[0042] Lim et al. (Oral bioavailability of BTK PROTACs against wild-type and C481-mutated BTK in a human lymphoma CDX mouse model. Blood Adv 7, 92-105 (2023)) and Sun et al. (Potential of PROTACs for degradation of Bruton’s tyrosine kinase mutants for the treatment of ibrtinib-resistant non-Hodgkin’s lymphoma. Leukemia 33, 2105-2110 (2019)) studies show that the “event-driven” mechanism of PROTAC-based can effectively overcome the resistance limitations of traditional BTK inhibitors and improve the efficiency of target inhibition;

[0043] Huang et al. (Ibritumib-based BTK PROTACs have in vivo anti-inflammatory efficacy by inhibiting NF-κB activation. European Journal of Medicinal Chemistry 259, 115664 (2023)) and Zhang et al. (Structure-feature analysis strategy of oral bioavailability PROTACs against Bruton’s tyrosine kinase for lymphoma treatment. Journal of Medicinal Chemistry 65, 9096-9125 (2022)) further verified its in vitro and in vivo activity;

[0044] Currently, the BTK PROTAC compound NX-5948 developed by Nurix Therapeutics has entered phase I clinical trials, and its compound structure is as follows:

[0045] , which is 3-((4-(1-((1-(6-(((S)-2,6-dioxopiperidin-3-yl)carbamoyl)pyridin-3-yl)piperidin-4-yl)methyl)piperidin-4-yl)phenyl)amino)-5-((R)-3-(3-yl-2-oxoimidazolidin-1-yl)piperidin-1-yl)pyrazine-2-carboxylic acid. Preclinical data show its therapeutic potential in B-cell malignancies and autoimmune diseases. However, the application of BTK PROTAC in the treatment of secondary hemophagocytic syndrome (HLH) has not been reported, and its mechanism of action in regulating macrophage polarization and inhibiting cytokine storm to improve the pathological process of HLH is still unknown;

[0046] Therefore, the application first proposes the application of the Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutically active ingredient in the preparation of a drug for treating secondary hemophagocytic syndrome; the pre-experiment of the application takes the RAW264.7 mononuclear macrophage cell line as the research object, gives CPG+IFN-γ stimulation to simulate the HLH inflammation of macrophages, and compares the intervention effects of BTK inhibitors (Ibrutinib, Zanubrutinib), the BTK PROTAC compound NX-5948 and JAK inhibitors (Ruxolitinib, positive control) on cell proliferation, activation and inflammatory factor release, so as to systematically evaluate the conversion value of the BTK PROTAC compound NX-5948 in the treatment of HLH.

[0047] The application in the application can inhibit abnormal activation of macrophages and improve at least one of the following clinical symptoms or laboratory indexes of patients with secondary hemophagocytic syndrome:

[0048] Clinical symptoms: fever, hepatosplenomegaly, central nervous system dysfunction, liver and kidney function failure;

[0049] Laboratory indexes: cytopenia, hypertriglyceridemia and / or hypofibrinogenemia, elevated ferritin, elevated soluble interleukin 2 receptor (sCD25), bone marrow hemophagocytosis, and proinflammatory cytokine storm.

[0050] Experiment 1:

[0051] Experimental basis: RAW264.7 cell line (mouse mononuclear macrophage cell line) is treated with different concentrations of NX-5948 for 24 hours;

[0052] Detection method: Western blot method is used to detect the expression level of intracellular BTK protein, and housekeeping protein GAPDH is used as an internal reference standardization control;

[0053] Detection result: as Figure 1Results: The results showed that the gray ratio of BTK and GAPDH protein in the control group (only adding PBS), 1 picomole per liter group, 10 picomole per liter group, 100 picomole per liter group, 1 nanomole per liter group, 10 nanomole per liter group, 100 nanomole per liter group, 1 micromole per liter group was 1.289, 0.234, 0.125, 0.067, 0.072, 0.032, 0.019, 0.013 respectively. NX-5948 can significantly degrade BTK target protein, and the degradation effect shows a concentration-dependent characteristic. The gray ratio of target protein (BTK) to internal reference protein (GAPDH) in NX-5948 treatment group is significantly lower than that in the control group, which proves that BTK PROTACH compound NX-5948 realizes effective targeted clearance of BTK protein through the ubiquitin proteasome system.

[0054] The experimental background of experiments 2, 3, 4, 5 and 6 described below is as follows: to explore the effect and mechanism of BTK PROTAC compound NX-5948 on inhibiting cell proliferation;

[0055] Experiment 2:

[0056] Experimental basis: RAW264.7 cell lines were treated with different concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948.

[0057] Detection method: CCK-8 method was used to detect the activity of RAW264.7 cell lines.

[0058] Detection results: As shown in Figure 2 Results: The IC50 value of NX-5948 treated RAW264.7 cell lines for 5 days was 0.14 uM. Under the same conditions, the above cells were treated with different concentrations of Ruxolitninb, Ibrutinib and Zanubrutinib. The results showed that the IC50 of Ruxolitninb, Ibrutinib and Zanubrutinib were significantly higher than that of NX-5948. Therefore, NX-5948 has a significant antiproliferative effect on RAW264.7 cells in vitro, and is significantly better than Ruxolitninb, Ibrutinib and Zanubrutinib.

[0059] Experiment 3:

[0060] Experimental background: Based on the mature research system of Ibrutinib, Zanubrutinib and Ruxolitinib in JeKo-1 cell lines.

[0061] Experimental basis: Jeko-1 cell lines were treated with different concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948;

[0062] Detection method: CCK-8 method was used to detect JeKo-1 cell line activity;

[0063] Detection results: as shown in the results Figure 3 Results: The IC50 value of NX-5948 treated Jeko-1 cell line for 5 days was 1.32uM, under the same conditions, different concentrations of Ruxolitninb, Ibrutinib and Zanubrutinib were used to treat the above cells, and the results showed that the IC50 of Ruxolitninb, Ibrutinib and Zanubrutinib were significantly higher than that of NX-5948, thus NX-5948 had a significant in vitro anti-proliferation effect on Jeko-1 cell line, and was significantly better than Ruxolitninb, Ibrutinib and Zanubrutinib;

[0064] The conclusion of experiments 2 and 3 is that NX-5948 has a significantly superior anti-proliferation activity on Jeko-1 and RAW264.7 cell lines;

[0065] Experiment 4:

[0066] Experimental basis: 1uM concentration of Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948 was used to treat the HLH in vitro cell model constructed by RAW 264.7+CPG(10uM)+IFN-γ(40ng / ml);

[0067] Detection method: Flow cytometry was used to detect cell apoptosis;

[0068] Detection results: as shown in the results Figure 4 As shown in the results, after treatment with 1uM concentration of NX-5948 for 24 hours, the apoptosis rate of RAW264.7 cell line was 60.5%, which was significantly higher than that of the control group, the model group, the Ruxolitninb group, the Ibrutinib group and the Zanubrutinib group, which were 6.45%, 7.3%, 14.59% and 7.36% respectively. Thus, NX-5948 can induce RAW264.7 cell apoptosis, and is significantly better than Ruxolitninb, Ibrutinib and Zanubrutinib;

[0069] Conclusion: NX-5948 can promote macrophage apoptosis;

[0070] Experiment 5:

[0071] Experimental basis: The HLH in vitro cell model constructed by RAW 264.7 + CPG (10 uM) + IFN-γ (40 ng / ml) was treated with 1 uM Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948 for 24 hours;

[0072] Detection method: Intracellular reactive oxygen species (ROS) levels were detected by flow cytometry;

[0073] Test results: such as Figure 5 The results showed that after treatment with 1 μM NX-5948 for 24 hours, the average fluorescence intensity of ROS in RAW264.7 cells was 856, which was significantly higher than that of the control group, model group, ruxolitninib group, ibrutinib group, and zanubrutinib group (454, 501, 619, and 594, respectively). This indicates that NX-5948 can promote ROS generation in RAW264.7 cells and is significantly superior to ruxolitninib, ibrutinib, and zanubrutinib.

[0074] Conclusion: NX-5948 can significantly increase the level of reactive oxygen species (ROS) in RAW264.7 cells;

[0075] Experiment 6:

[0076] Experimental basis: The HLH in vitro cell model constructed by RAW 264.7 + CPG (10 uM) + IFN-γ (40 ng / ml) was treated with 1 uM Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948 for 24 hours;

[0077] Detection method: PI staining method to detect the cell cycle of HLH cell model;

[0078] Test results: such as Figure 6 The results showed that after treatment with 1 μM NX-5948 for 24 hours, the ratio of RAW264.7 cells in S phase + G2 phase to G1 phase was 0.922, which was significantly higher than that in the control group, model group, ruxolitninb group, ibrutinib group, and zanubrutinib group (0.626, 0.519, 0.659, 0.723, and 0.575, respectively). This indicates that NX-5948 can arrest the cell cycle of RAW264.7 cells in S phase + G2 phase, and is significantly superior to ruxolitninb, ibrutinib, and zanubrutinib.

[0079] Conclusion: NX-5948 can arrest RAW264.7 cells in S and G2 phases;

[0080] Experiments 2, 3, 4, 5, and 6 prove that the BTK PROTAC compound NX-5948 can effectively inhibit the proliferation of macrophages, and its effect is better than that of traditional BTK inhibitors (Ibrutinib, Zanubrutinib) and JAK inhibitors (Ruxolitinib) under the same conditions.

[0081] The experimental background of the following experiments 7 and 8 is to explore the effect and mechanism of the BTK PROTAC compound NX-5948 on the inhibition of the release of cytokines by macrophages in vitro.

[0082] Experiment 7:

[0083] Experimental basis: use 1uM concentration of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948 to treat the HLH in vitro cell model constructed by RAW 264.7+CPG (10uM)+IFN-γ (40ng / ml) for 24 hours.

[0084] Detection method: detect the concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the cell supernatant by ELISA.

[0085] Detection results: Figure 7Results The average concentrations of TNF-a, IL-6 and IL-1β in the control group were 117.32, 152.17 and 2.19 pg / ml, respectively. After stimulation with CPG+IFN-γ, RAW 264.7 cells were activated and released a large amount of proinflammatory cytokines, and the average concentrations of TNF-a, IL-6 and IL-1β were 1822.09, 1648.05 and 27.78 pg / ml, respectively. NX-5948 could significantly inhibit the release of the above three cytokines, and the average concentrations of TNF-a, IL-6 and IL-1β were 1031.61, 302.48 and 3.22 pg / ml, respectively. In terms of improving TNF-a, the NX-5948 group was significantly better than the Ruxolitinib group (1803.88 pg / ml), and there was no significant difference with the Ibrutinib group (1103.42 pg / ml) and the Zanubrutinib group (1149.27 pg / ml). In terms of improving IL-6, the NX-5948 group was significantly better than the Ruxolitinib group (494.08 pg / ml), the Ibrutinib group (466.20 pg / ml) and the Zanubrutinib group (1498.46 pg / ml). In terms of improving IL-1β, the NX-5948 group was significantly better than the Ruxolitinib group (30.26 pg / ml) and the Zanubrutinib group (11.02 pg / ml), and there was no significant difference with the Ibrutinib group (3.20 pg / ml).

[0086] Conclusion: NX-5948 can improve the release of proinflammatory cytokines in RAW264.7 cell line.

[0087] Experiment 8:

[0088] Experimental basis: RAW 264.7+CPG (10 uM)+IFN-γ (40 ng / ml) HLH in vitro cell model was constructed using 1 uM concentration of Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948 for 24 hours;

[0089] Detection method: Flow cytometry was used to detect the phenotype of macrophage polarization in HLH cell model, and RAW264.7 cell surface M1 macrophage biomarker CD86 and M2 macrophage CD163 were detected. The results showed that the cells in the second quadrant were M1 macrophages, and the cells in the fourth quadrant were M2 macrophages.

[0090] Detection results: As shown in the results Figure 8Results showed that the proportion of RAW 264.7 cell polarization to M1 macrophages increased to 61.4% after stimulation by CPG+IFN-γ, NX-5948 can significantly inhibit the proportion of M1 macrophage polarization to 35.44%, and the effect is better than Ruxolitninb, Ibrutinib and Zanubrutinib 52.23%, 56.43%, 58.32%;

[0091] Conclusion: NX-5948 inhibits the polarization of macrophages to M1 phenotype in RAW264.7 cell line;

[0092] Experiments 7, 8 proved that BTK PROTAC compound NX-5948 can effectively inhibit the release of cytokines by macrophages, and its effect is better than that of traditional BTK inhibitors (Ibrutinib, Zanubrutinib) and JAK inhibitors (Ruxolitinib) under the same conditions.

[0093] Experiments 1-8 above are all in vitro experiments, which prove that BTK PROTAC compound NX-5948 has significant anti-HLH activity. In order to further verify the pharmacodynamics of NX-5948 in HLH, in the following experiments, we use CpG-ODN1826 to repeatedly stimulate TLR9, simulate autoimmune diseases, and construct secondary HLH mouse models for in vivo experiments;

[0094] The experimental background in the following experiments is the treatment of BTK PROTAC compound NX-5948 on CpG-induced secondary hemophagocytic syndrome HLH model, focusing on exploring its intervention efficiency in improving cytokine storm and related hematological indicators in vivo;

[0095] Experiment 9:

[0096] Experimental basis: 6-8 week old C57BL / 6 mice were used, and CpG-ODN1826 (50ug / time) was injected intraperitoneally every other day from day 0 (D0) to induce secondary HLH syndrome in mice. From day 4 (D4), Ruxolitninb, Ibrutinib, Zanubrutinib and NX-5948 were given at a drug dose of 50mg Bid by gavage. The mice were divided into 6 groups, namely control group, model group, Ruxolitninb group, Ibrutinib group, Zanubrutinib group and NX-5948 group;

[0097] Detection method: After the end of administration, the mice were weighed using an animal weight scale, and the liver and spleen tissues were dissected and weighed;

[0098] Detection results: As Figure 9Results showed that the liver and spleen accounted for 4.03% and 0.17% of the body weight in the control group, respectively. The model group showed significant hepatosplenomegaly, with liver and spleen accounting for 8.70% and 1.88% of the body weight, respectively. The liver and spleen of the NX-5948 group were significantly smaller than those of the control group, with liver and spleen accounting for 7.13% and 1.13% of the body weight, respectively. In terms of improving liver enlargement, the NX-5948 group was significantly better than the Ruxolitninb group (7.55%), the Ibrutinib group (7.82%), and the Zanubrutinib group (7.76%). In terms of improving splenomegaly, the NX-5948 group was significantly better than the Ibrutinib group (1.00%) and the Zanubrutinib group (1.69%), and there was no significant difference compared with the Ruxolitninb group (1.56%);

[0099] Conclusion: NX-5948 has a significant effect on improving liver and spleen enlargement.

[0100] Experiment 10:

[0101] Experimental basis: The same as the experimental basis of Experiment 9.

[0102] Detection method: After administration, peripheral blood of mice was taken and blood routine was detected using a full-automatic blood routine detector:

[0103] Detection results: As Figure 10 Results showed that the platelet count of the control group was 1099*10 9 / L, and the model group showed significant thrombocytopenia, with a platelet count of 378*10 9 / L. The NX-5948 group significantly improved the abnormal platelet count, with a platelet count of 523*10 9 / L. In terms of improving platelet count, the NX-5948 group was significantly better than the Ruxolitinib group (426*10 9 / L) and the Ibrutinib group (455*10 9 / L), and there was no significant difference compared with the Zanubrutinib group (518*10 9 / L). There was no significant difference in the effect of all drug groups on hemoglobin and white blood cells.

[0104] Conclusion: NX-5948 has a significant effect on improving thrombocytopenia.

[0105] Experiment 11:

[0106] Experimental basis: The same as the experimental basis of Experiment 9.

[0107] Detection method: after the end of administration, the mouse peripheral serum was taken, and the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and uric acid (UA) were detected by using the automatic blood biochemical detector;

[0108] The detection results are as follows: Figure 11 As shown in the results, the ALT, AST and UA of the control group were 30.85 units per liter (U / L), 82.25 U / L and 72.92 micromoles per liter (umol / L), respectively. Compared with the control group, the model group showed significant increases in ALT, AST and UA, which were 156.85 U / L, 300.19 U / L and 171.81 umol / L, respectively, indicating impaired liver and kidney function. Compared with the model group, the ALT, AST and UA of the NX-5948 group were significantly reduced, which were 71.95 U / L, 212.98 U / L and 21.12 umol / L, respectively, indicating that NX-5948 could improve the liver and kidney function of HLH mice. In terms of improving ALT, the NX-5948 group was significantly better than the Ibrutinib group (107.71 U / L) and the Zanubrutinib group (138.04 U / L), and had no significant difference compared with the Ruxolitinib group (48.68 U / L). In terms of improving AST, the NX-5948 group had no significant difference compared with the Ruxolitinib group (174.68 U / L), the Ibrutinib group (215.38 U / L) and the Zanubrutinib group (232.57 U / L). In terms of improving UA, the NX-5948 group was significantly better than the Ruxolitinib group (130.48 U / L), and had no significant difference compared with the Ibrutinib group (34.41 U / L) and the Zanubrutinib group (20.06 U / L);

[0109] Conclusion: NX-5948 has a significant improvement effect on liver and kidney function;

[0110] Experiment 12:

[0111] Experimental basis: same as experiment 9;

[0112] Detection method: after the end of administration, the mouse peripheral serum was taken, and the cytokine concentration was detected by using the ELISA kit;

[0113] The detection results are as follows: Figure 12Results: The average concentrations of TNF-a, IL-6 and IL-1β in the control group were 24.97, 19.07 and 10.31 pg / ml, respectively. The levels of the three cytokines in the model group were significantly higher than those in the control group, with average concentrations of 42.32, 497.42 and 68.57 pg / ml, respectively. NX-5948 significantly inhibited the release of the three cytokines, with average concentrations of 21.91, 285.84 and 13.6 pg / ml, respectively. In terms of improving TNF-a, the NX-5948 group was significantly better than the Ruxolitinib group (29.67 pg / ml), with no significant difference from the Ibrutinib group (21.44 pg / ml) and the Zanubrutinib group (21.2 pg / ml). In terms of improving IL-6, the NX-5948 group was significantly better than the Ibrutinib group (404.84 pg / ml) and the Zanubrutinib group (362.70 pg / ml), with no significant difference from the Ruxolitinib group (301.31 pg / ml). In terms of improving IL-1β, the NX-5948 group was significantly better than the Ibrutinib group (39.78 pg / ml) and the Zanubrutinib group (35.48 pg / ml), with no significant difference from the Ruxolitinib group (16.76 pg / ml).

[0114] Conclusion: NX-5948 significantly improved the serum cytokine levels in mice.

[0115] Experiment 13:

[0116] Experimental basis: Same as the experimental basis of Experiment 9.

[0117] Detection method: After the administration, the mouse peripheral serum was taken, and the serum ferritin and IL-2R levels were detected using the ELISA kit.

[0118] Detection results: As Figure 13Results showed that the average concentrations of ferritin and IL-2R in the control group were 165.69 and 1.34 ng / ml, respectively. Compared with the control group, the model group showed a significant increase in serum ferritin and IL-2R, with average concentrations of 1556.10 and 10.85 ng / ml, respectively. NX-5948 can significantly correct the above abnormalities, with average concentrations of ferritin and IL-2R being 404.45 and 4.26 ng / ml, respectively. In terms of improving ferritin, the NX-5948 group was significantly better than the Ibrutinib group (1302.99 ng / ml) and the Zanubrutinib group (1029.41 ng / ml), and there was no significant difference with the Ruxolitinib group (524.50 ng / ml). In terms of improving IL-2R, the NX-5948 group had no significant difference with the Ruxolitinib group (3.88 ng / ml), the Ibrutinib group (5.07 ng / ml), and the Zanubrutinib group (3.58 ng / ml).

[0119] Conclusion: NX-5948 significantly improves the levels of serum ferritin and soluble CD25 (IL-2R) in mice.

[0120] Experiments 9-13 show that the BTK PROTAC compound NX-5948 has a significant effect on the treatment of CpG-induced secondary hemophagocytic syndrome HLH model.

[0121] The results of experiments 1-13 show that the BTK PROTAC compound NX-5948 targets and inhibits the activation of the macrophage BTK / NF-κB axis signaling pathway, on the one hand, by inhibiting the proliferation and survival of macrophages, reducing the number of activated macrophages, and on the other hand, by inducing macrophages to polarize from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages through immune regulation of macrophages, thereby inhibiting cytokine release and the occurrence and development of HLH. Therefore, the application of the Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 or its pharmaceutically acceptable salt as a pharmaceutical active ingredient in the preparation of a drug for treating secondary hemophagocytic syndrome is feasible, and thus the new application of the present application has the effect of being able to inhibit the abnormal activation of macrophages and improve the clinical symptoms or laboratory indicators present in patients with secondary hemophagocytic syndrome.

Claims

1. Use of Bruton's tyrosine kinase protein degradation targeting chimera BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutically active ingredient in the preparation of a medicament for treating secondary hemophagocytic syndrome; The compound structural formula of the BTK PROTAC compound NX-5948 is: 。 2. Use according to claim 1, characterized in that: The dosage form of the medicament is a tablet, a capsule, a freeze-dried powder injection or an oral solution, and contains a pharmaceutically acceptable carrier and / or an auxiliary agent.

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