New application of BTK PROTAC compound NX-5948

Abstract

The invention relates to the technical field of biological medicines, in particular to a novel application of a BTK PROTAC compound NX-5948, and in particular relates to a novel application of a BTK PROTAC compound NX-5948 as a target chimera for degrading a Bruton's tyrosine kinase protein in a medicine for treating a secondary hemophagocytic syndrome (HLH). The BTK PROTAC compound NX-5948 in the invention inhibits activation of a BTK / NF-kappa B axis signal channel of macrophages in a targeted manner, on one hand, reduces the number of activated macrophages by inhibiting proliferation and survival of the macrophages, and on the other hand, induces polarization of the macrophages from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages by virtue of an immunoregulation effect on the macrophages, so that the anti-inflammatory effect of the BTK PROTAC compound NX-5948 is improved, and the anti-inflammatory effect of the BTK PROTAC compound NX-5948 is improved. Therefore, the new application has the effects that the abnormal activation of macrophages can be inhibited, and clinical symptoms or laboratory indexes existing in patients with the secondary hemophagocytic syndrome can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a new application of a BTK PROTAC compound NX-5948, and more particularly to a new application of a Bruton's tyrosine kinase protein degradation-targeted chimera BTK PROTAC compound NX-5948 in a drug for treating secondary hemophagocytic hyperplasia (HLH). Background Art

[0002] Hemophagocytic lymphohistiocytosis (HLH) is a fatal syndrome caused by an overactive immune system, characterized by persistent fever, hepatosplenomegaly, cytopenia, and multiple organ failure, with a very poor clinical prognosis. Currently, the standard treatment for HLH (such as the HLH-1994 / 2004 regimen) relies on a chemotherapy strategy of etoposide combined with dexamethasone, but approximately 25%-50% of patients do not respond to treatment, and the 5-year survival rate is only 54%; Although the introduction of targeted drugs in recent years (such as the JAK inhibitor ruxolitinib and the interferon gamma antibody emalimumab) has partially improved patient prognosis, insufficient disease remission depth and relapse remain major clinical challenges. Therefore, there is an urgent need to explore the pathogenesis of HLH and develop new treatment strategies. Studies have shown that abnormal macrophage activation is the core driving factor in the pathological process of HLH. However, in the inflammatory microenvironment, macrophages polarize toward the pro-inflammatory M1 phenotype, releasing large amounts of cytokines (such as IFN-γ, TNF-α, and IL-6). This exacerbates their own activation through a positive feedback loop and further activates cytotoxic T cells (CTLs) and natural killer (NK) cells, ultimately leading to a "cytokine storm" and multiple organ failure. Therefore, inhibiting abnormal macrophage activation is a key target for improving the prognosis of HLH. Bruton's tyrosine kinase (BTK), a core kinase in the B cell receptor (BCR) signaling pathway, has recently been found to regulate macrophage proliferation, activation, and inflammatory responses. Specifically, in HLH, BTK is activated by cytokines (such as IFN-γ and IL-6) through TLR-mediated signaling pathways, which in turn phosphorylates the downstream transcription factor NF-κB, driving pro-inflammatory gene expression and forming a vicious cycle called the "BTK / NF-κB axis." Although first-generation BTK inhibitors (such as ibrutinib) have shown some potential in the treatment of HLH, they only inhibit BTK kinase activity and cannot completely block NF-κB signaling. Furthermore, their clinical efficacy is limited by the residual effects of the cytokine storm. In recent years, the targeted protein degradation chimera (PROTAC) technology achieves complete degradation of pathogenic proteins through the ubiquitin-proteasome pathway, providing a new approach to overcome the limitations of traditional inhibitors; Studies have shown that BTK PROTAC can efficiently degrade BTK protein, block its downstream signal transduction, and exhibit significant therapeutic effects in B-cell malignancies and autoimmune diseases. However, the application of BTK PROTAC in the treatment of HLH 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 of the Invention

[0003] The purpose of the present invention is to address the shortcomings of existing HLH treatment options and propose a novel application of a Bruton's tyrosine kinase protein degradation targeted chimera (BTK PROTAC) compound NX-5948 in the preparation of a drug for treating secondary hemophagocytic hyperplasia (HLH). The application can inhibit abnormal macrophage activation and improve clinical symptoms or laboratory indicators in patients with secondary hemophagocytic hyperplasia (HLH).

[0004] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions: The present invention provides a Bruton's tyrosine kinase protein degradation-targeted 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.

[0005] As a preferred technical solution of the present invention, the application can inhibit abnormal activation of macrophages.

[0006] As a preferred technical solution of the present invention, the application can improve at least one of the following clinical symptoms or laboratory indicators of patients with secondary hemophagocytic syndrome: Clinical symptoms: fever, hepatosplenomegaly, central nervous system dysfunction, liver and kidney failure; Laboratory indicators: cytopenia, hypertriglyceridemia and / or hypofibrinogenemia, elevated ferritin, elevated soluble interleukin-2 receptor (sCD25), bone marrow hemophagocytosis, and proinflammatory cytokine storm.

[0007] As a preferred technical solution of the present invention, the compound structural formula of the BTK PROTAC compound NX-5948 is:

[0008] That 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-carboxamide.

[0009] As a preferred technical solution of the present invention, the drug is administered by at least one of oral administration, intravenous injection, subcutaneous injection or intrathecal injection.

[0010] As a preferred technical solution of the present invention, the drug is administered by at least one of oral administration, intravenous injection, subcutaneous injection or intrathecal injection.

[0011] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned BTKPROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, for treating secondary hemophagocytic syndrome; The dosage form of the pharmaceutical composition is an oral preparation, an injection or a sustained-release preparation.

[0012] In addition, the present invention 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.

[0013] The beneficial effects of the present invention are as follows: The BTK PROTAC compound NX-5948 in the present invention inhibits the activation of the BTK / NF-κB axis signaling pathway in macrophages by targeting. On the one hand, it reduces the number of activated macrophages by inhibiting the proliferation and survival of macrophages. On the other hand, it induces the polarization of macrophages from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages through the immunomodulatory effect on macrophages, thereby inhibiting cytokine release and the occurrence and development of HLH. Therefore, the use of the Bruton's tyrosine kinase protein degradation-targeted chimera BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutical active ingredient in the preparation of a drug for the treatment of secondary hemophagocytic syndrome is feasible. Therefore, the new application of the present invention has the effect of inhibiting the abnormal activation of macrophages and improving the clinical symptoms or laboratory indicators of patients with secondary hemophagocytic syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the result of Western blot analysis of BTK protein expression in RAW264.7 cells after treatment with NX-5948 in Experiment 1. Figure 2 This is the result of the CCK-8 assay in Experiment 2 to detect the viability of RAW264.7 cell lines using Ruxolitinb, Ibrutnib, Zanubrutinib, and NX-5948; Figure 3This is the result of the CCK-8 assay in Experiment 3 to detect the activity of the JeKo-1 cell line using Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948.

[0015] Figure 4 This is the flow cytometry result of detecting the apoptosis of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 in the HLH in vitro cell model in Experiment 4.

[0016] Figure 5 This is the result of flow cytometry detection of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the level of intracellular reactive oxygen species (ROS) in the HLH in vitro cell model in Experiment 5.

[0017] Figure 6 This is the result of the PI staining method in Experiment 6 to detect the effects of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the cell cycle of the HLH in vitro model.

[0018] Figure 7 This is the result of ELISA testing of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the cell supernatant of an in vitro HLH cell model in Experiment 7.

[0019] Figure 8 This is the flow cytometry test result of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 on the phenotype of macrophage polarization in the HLH in vitro cell model in Experiment 8.

[0020] Figure 9 This is a graph showing the test results of weighing the mice and their liver and spleen tissue weights after Ruxolitinb, Ibrutnib, Zanubrutinib, and NX-5948 were administered to mice with HLH syndrome in Experiment 9.

[0021] Figure 10 This is the test result of taking peripheral blood from mice with HLH syndrome after Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 were administered to them in Experiment 10, and then using a fully automatic blood routine tester to test the blood routine.

[0022] Figure 11This is the test result chart of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and uric acid (UA) in peripheral serum of mice after administration of Ruxolitinb, Ibrutnib, Zanubrutinib and NX-5948 to mice with HLH syndrome in Experiment 11, using a fully automatic blood biochemistry analyzer to test the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and uric acid (UA).

[0023] Figure 12 This figure shows the test results of cytokine concentrations detected using ELISA kits in peripheral serum of mice after administration of Ruxolitinb, Ibrutnib, Zanubrutinib, and NX-5948 to mice with HLH syndrome in Experiment 12.

[0024] Figure 13 This is the test result of collecting peripheral serum from mice with HLH syndrome after administration of Ruxolitinb, Ibrutnib, Zanubrutinib, and NX-5948 in Experiment 13 and detecting serum ferritin and IL-2R levels using ELISA kits.

[0025] Figure 14 This is the structural formula diagram of the BTK PROTAC compound NX-5948. DETAILED DESCRIPTION

[0026] The raw materials, equipment and reagents used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0027] In recent years, PROTAC technology has made breakthrough progress in the field of drug development. This technology “hijacks” E3 ubiquitin ligases and uses the ubiquitin-proteasome system to achieve specific degradation of target proteins. Lim et al. (Oral bioavailability of BTK PROTAC in human lymphoma CDX mouse models with wild-type and C481 mutant BTK. Blood Progress 7, 92–105 (2023)) and Sun et al. (Potential for treating ibrutinib-resistant non-Hodgkin lymphoma by PROTAC degradation of Bruton's tyrosine kinase mutants. Leukemia 33, 2105–2110 (2019)) studies have shown that the "event-driven" mechanism of action based on PROTAC can effectively overcome the resistance limitations of traditional BTK inhibitors and improve the efficiency of target inhibition; Huang et al. (discovered that ibrutinib-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. (Structural characterization strategy for orally bioavailable PROTACs targeting Bruton's tyrosine kinase for lymphoma treatment. Journal of Medicinal Chemistry 65, 9096–9125 (2022)) further verified its in vitro and in vivo activity; Currently, the BTK PROTAC compound NX-5948 developed by Nurix Therapeutics has entered Phase I clinical trials. Its compound structure is: , 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-carboxamide. Preclinical data show its therapeutic potential in B-cell malignancies and autoimmune diseases. Despite this, the application of BTK PROTAC in the treatment of secondary hemophagocytic syndrome (HLH) has not been reported, and its mechanism of action in improving the pathological process of HLH by regulating macrophage polarization and inhibiting cytokine storm remains unknown. Therefore, the present invention proposes for the first time the use of a Bruton's tyrosine kinase protein degradation-targeted chimera BTKP ROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutical active ingredient in the preparation of a drug for the treatment of secondary hemophagocytic syndrome; in a preliminary experiment of the present invention, the RAW264.7 mononuclear macrophage cell line was used as the research object, and CPG+IFN-γ stimulation was given to simulate HLH inflammation of macrophages. By comparing the intervention effects of BTK inhibitors (Ibrutinib, Zanubrutinib), BTK PROTAC compound NX-5948 and JAK inhibitor (Ruxolitinib, positive control) on cell proliferation, activation and inflammatory factor release, the translational value of BTK PROTAC compound NX-5948 in the treatment of HLH was systematically evaluated.

[0028] The application of the present invention can inhibit abnormal macrophage activation and improve at least one of the following clinical symptoms or laboratory indicators of patients with secondary hemophagocytic syndrome: Clinical symptoms: fever, hepatosplenomegaly, central nervous system dysfunction, liver and kidney failure; Laboratory indicators: cytopenia, hypertriglyceridemia and / or hypofibrinogenemia, elevated ferritin, elevated soluble interleukin-2 receptor (sCD25), bone marrow hemophagocytosis, and proinflammatory cytokine storm.

[0029] Experiment 1: Experimental basis: RAW264.7 cells (a mouse monocyte-macrophage cell line) were treated with various concentrations of NX-5948 for 24 hours. Detection method: Western blot was used to detect the expression level of BTK protein in cells, and the housekeeping protein GAPDH was used as an internal reference for standardization; Test results: Figure 1 The results showed that the grayscale ratios of BTK and GAPDH proteins in the control group (only PBS added), 1 pmol / L group, 10 pmol / L group, 100 pmol / L group, 1 nanomol / L group, 10 nanomol / L group, 100 nanomol / L group, and 1 micromol / L group were 1.289, 0.234, 0.125, 0.067, 0.072, 0.032, 0.019, and 0.013, respectively. NX-5948 can significantly degrade the BTK target protein, and the degradation effect is concentration-dependent. The grayscale ratio of the target protein (BTK) / the internal reference protein (GAPDH) in the NX-5948-treated group was significantly lower than that in the control group, demonstrating that the BTK PROTACH compound NX-5948 achieves effective targeted clearance of BTK protein through the ubiquitin proteasome system.

[0030] The experimental backgrounds of the following experiments 2, 3, 4, 5, and 6 are: to explore the effect and mechanism of BTK PROTAC compound NX-5948 in inhibiting cell proliferation; Experiment 2: Experimental basis: RAW264.7 cell line was treated with different concentrations of Ruxolitinib, Ibrutinib, Zanubrutinib, and NX-5948. Detection method: CCK-8 assay was used to detect the viability of RAW264.7 cell line; Test results: Figure 2The results showed that the IC50 value of NX-5948 in the RAW 264.7 cell line after 5-day treatment was 0.14uM. Under the same conditions, the cells were treated with different concentrations of Ruxolitninb, Ibrutinib, and Zanubrutinib. The results showed that the IC50 values ​​of Ruxolitninb, Ibrutinib, and Zanubrutinib were significantly higher than those of NX-5948. This indicates that NX-5948 has a significant in vitro anti-proliferative effect on RAW264.7 cells and is significantly superior to Ruxolitninb, Ibrutinib, and Zanubrutinib. Experiment 3: Background: Ibrutinib, Zanubrutinib, and Ruxolitinib have established a mature research system in the JeKo-1 cell line. Experimental basis: Jeko-1 cell line was treated with different concentrations of Ruxolitinib, Ibrutinib, Zanubrutinib, and NX-5948. Detection method: CCK-8 assay was used to detect the viability of JeKo-1 cell line; Test results: Figure 3 The results showed that the IC50 value of NX-5948 in the Jeko-1 cell line after 5 days of treatment was 1.32uM. Under the same conditions, the cells were treated with different concentrations of Ruxolitninb, Ibrutinib, and Zanubrutinib. The results showed that the IC50 values ​​of Ruxolitninb, Ibrutinib, and Zanubrutinib were significantly higher than those of NX-5948. This shows that NX-5948 has a significant in vitro anti-proliferative effect on the Jeko-1 cell line and is significantly superior to Ruxolitninb, Ibrutinib, and Zanubrutinib. The conclusions of Experiments 2 and 3 were that NX-5948 had significantly superior antiproliferative activity against Jeko-1 and RAW264.7 cell lines; Experiment 4: Experimental basis: An in vitro HLH cell model was constructed by treating RAW 264.7 cells with 1 uM concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948, plus CPG (10 uM) and IFN-γ (40 ng / ml). Detection method: Flow cytometry was used to detect cell apoptosis; Test results: Figure 4The results showed that after 24 hours of treatment with 1uM concentration of NX-5948, the apoptosis rate of RAW264.7 cell line was 60.5%, which was significantly higher than 6.45%, 7.3%, 14.59% and 7.36% in the control group, model group, ruxolitinib group, ibrutinib group and zanubrutinib group, respectively. This shows that NX-5948 can induce apoptosis in RAW264.7 cells and is significantly better than ruxolitinib, ibrutinib and zanubrutinib; Conclusion: NX-5948 can promote macrophage apoptosis; Experiment 5: Basis: A HLH in vitro cell model constructed by RAW 264.7 cells, CPG (10 μM), and IFN-γ (40 ng / ml) was treated with 1 μM concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948 for 24 hours. Detection method: Flow cytometry was used to detect the level of intracellular reactive oxygen species (ROS); Test results: Figure 5 The results showed that after 24 hours of treatment with 1uM concentration of NX-5948, the average fluorescence intensity of ROS in RAW264.7 cells was 856, which was significantly higher than 454, 501, 619, and 594 in the control group, model group, ruxolitinib group, ibrutinib group, and zanubrutinib group, indicating that NX-5948 can promote ROS generation in RAW264.7 cells and is significantly better than ruxolitinib, ibrutinib, and zanubrutinib. Conclusion: NX-5948 can significantly increase the level of reactive oxygen species (ROS) in RAW264.7 cells; Experiment 6: Basis: A HLH in vitro cell model constructed by RAW 264.7 cells, CPG (10 μM), and IFN-γ (40 ng / ml) was treated with 1 μM concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948 for 24 hours. Detection method: PI staining method to detect the cell cycle of HLH model; Test results: Figure 6The results showed that after 24 hours of treatment with 1uM concentration of NX-5948, the ratio of RAW264.7 cell line (S phase + G2 phase) / G1 phase cells was 0.922, which was significantly higher than 0.626, 0.519, 0.659, 0.723 and 0.575 in the control group, model group, ruxolitinib group, ibrutinib group and zanubrutinib group, respectively. This shows that NX-5948 can arrest the RAW264.7 cell cycle in the S phase + G2 phase, and is significantly better than ruxolitinib, ibrutinib and zanubrutinib; Conclusion: NX-5948 can arrest RAW264.7 cells in the S and G2 phases; Experiments 2, 3, 4, 5, and 6 demonstrated that the BTK PROTAC compound NX-5948 can effectively inhibit macrophage proliferation, and under the same conditions, its effect is better than traditional BTK inhibitors (Ibrutinib, Zanubrutinib) and JAK inhibitors (Ruxolitinib).

[0031] The experimental backgrounds of the following experiments 7 and 8 are: to explore the effect and mechanism of BTK PROTAC compound NX-5948 in inhibiting the release of cytokines by macrophages in vitro; Experiment 7: Basis: A HLH in vitro cell model constructed by RAW 264.7 cells, CPG (10 μM), and IFN-γ (40 ng / ml) was treated with 1 μM concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948 for 24 hours. Detection method: The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in cell supernatants were detected by ELISA; Test results: Figure 7The results showed that the average concentrations of TNF-α, IL-6 and IL-1β in the control group were 117.32, 152.17 and 2.19 pg / ml, respectively. After CPG+IFN-γ stimulation, RAW 264.7 cells were activated and released a large amount of pro-inflammatory cytokines, with the average concentrations of TNF-α, IL-6 and IL-1β being 1822.09, 1648.05 and 27.78 pg / ml, respectively. NX-5948 could significantly inhibit the release of the above three cytokines, with the average concentrations of TNF-α, IL-6 and IL-1β being 1031.61, 302.48 and 3.22 pg / ml, respectively. In terms of improving TNF-α, the NX-5948 group was significantly better than the Ruxolitinib group (1803.88 pg / ml), and compared with the Ibrutinib group (1103.42 pg / ml) and the Zanubru There was no significant difference between the NX-5948 group and the ruxolitinib 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), but had no significant difference with the ibrutinib group (3.20 pg / ml). Conclusion: NX-5948 can improve the release of proinflammatory cytokines in RAW264.7 cell line.

[0032] Experiment 8: Basis: A HLH in vitro cell model constructed by RAW 264.7 cells, CPG (10 μM), and IFN-γ (40 ng / ml) was treated with 1 μM concentrations of Ruxolitninb, Ibrutinib, Zanubrutinib, and NX-5948 for 24 hours. Detection method: Flow cytometry was used to detect the phenotype of macrophage polarization in the HLH cell model. The surface biomarkers of M1 macrophages CD86 and M2 macrophages CD163 were detected on RAW264.7 cells. The results show that the cells in the second quadrant are M1 macrophages, and the cells in the fourth quadrant are M2 macrophages. Test results: Figure 8The results showed that after CPG+IFN-γ stimulation, the proportion of RAW 264.7 cells polarized into M1 macrophages increased to 61.4%. NX-5948 significantly inhibited the polarization of M1 macrophages to 35.44%, and the effect was superior to 52.23%, 56.43%, and 58.32% of Ruxolitinib, Ibrutinib, and Zanubrutinib, respectively. Conclusion: NX-5948 inhibits the polarization of macrophages toward the M1 phenotype in the RAW264.7 cell line; Experiments 7 and 8 demonstrated that the BTK PROTAC compound NX-5948 can effectively inhibit the release of cytokines by macrophages, and under the same conditions, its effect is better than traditional BTK inhibitors (Ibrutinib, Zanubrutinib) and JAK inhibitors (Ruxolitinib).

[0033] Experiments 1-8 above are all in vitro experiments, demonstrating that the BTK PROTAC compound NX-5948 has significant anti-HLH activity. To further validate the pharmacodynamics of NX-5948 in HLH, in the following experiments, we used CpG-ODN1826 to repeatedly stimulate TLR9, mimicking autoimmune disease, and construct a secondary HLH mouse model for in vivo experiments. The following experiments all used the BTK PROTAC compound NX-5948 to treat a CpG-induced secondary hemophagocytic hyperlipidemia (HLH) model, focusing on exploring its efficacy in improving cytokine storms and related hematological indicators in vivo. Experiment 9: Experimental basis: Six- to eight-week-old C57BL / 6 mice were intraperitoneally injected with 50 μg of CpG-ODN1826 (CpG-ODN1826 can induce secondary HLH syndrome in mice) starting on day 0 (D0). Ruxolitninb, ibrutinib, zanubrutinib, and NX-5948 were administered orally at a dose of 50 mg Bid starting on day 4 (D4). The mice were divided into six groups: control group, model group, Ruxolitninb group, ibrutinib group, zanubrutinib group, and NX-5948 group. Detection method: After the administration, the mice were weighed using an animal scale, and the liver and spleen tissues were dissected and weighed; Test results: Figure 9The results showed that the liver and spleen in the control group accounted for 4.03% and 0.17% of body weight, respectively. The model group showed significant hepatosplenomegaly, with the liver and spleen accounting for 8.70% and 1.88% of body weight, respectively. The liver and spleen in the NX-5948 group were significantly smaller than those in the control group, with the liver and spleen accounting for 7.13% and 1.13% of 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 spleen enlargement, 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%). Conclusion: NX-5948 has a significant effect in improving hepatomegaly and splenomegaly; Experiment 10: Experimental basis: The same as that of Experiment 9; Detection method: After administration, peripheral blood was collected from mice and blood routine was detected using a fully automatic blood routine detector: Test results: Figure 10 The results showed that the platelet count in the control group was 1099*10 9 / L, compared with the control group, the model group showed obvious thrombocytopenia, with the platelet count of 378*10 9 / L, NX-5948 group could significantly improve the abnormal platelet count, with the platelet count being 523*10 9 / L, and in terms of improving platelet count, the NX-5948 group was significantly better than the Ruxolitinib group (426*10 9 / L) and Ibrutinib group (455*10 9 / L), compared with the Zanubrutinib group (518*10 9 There was no significant difference in the effects of all drug groups on hemoglobin and leukocytes; Conclusion: NX-5948 has a significant effect in improving thrombocytopenia; Experiment 11: Experimental basis: The same as that of Experiment 9; Detection method: After the administration, peripheral serum of mice was collected and alanine aminotransferase (ALT), aspartate aminotransferase (AST) and uric acid (UA) were tested using a fully automatic blood biochemistry analyzer; Test results: Figure 11The results showed that the ALT, AST and UA in 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 ALT, AST and UA in the model group were significantly increased, which were 156.85 U / L, 300.19 U / L and 171.81 μmol / L, respectively, indicating impaired liver and kidney function. Compared with the model group, the ALT, AST and UA in the NX-5948 group were significantly decreased, which were 71.95 U / L, 212.98 U / L and 21.12 μmol / L, respectively, indicating that NX-5948 can 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), but there was no significant difference compared with the Ruxolitinib group (48.68 U / L). In terms of improving AST, the NX-5948 group showed 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), but showed no significant difference compared with the Ibrutinib group (34.41 U / L) and the Zanubrutinib group (20.06 U / L). Conclusion: NX-5948 has a significant effect on improving liver and kidney function; Experiment 12: Experimental basis: The same as that of Experiment 9; Detection method: After administration, peripheral serum of mice was collected and cytokine concentrations were detected using ELISA kits; Test results: Figure 12The results showed that the average concentrations of TNF-α, IL-6 and IL-1β in the control group were 24.97, 19.07 and 10.31 pg / ml, respectively. The cytokine levels in the modeling group were significantly increased compared with the control group, with the average concentrations of TNF-α, IL-6 and IL-1β being 42.32, 497.42 and 68.57 pg / ml, respectively. NX-5948 could significantly inhibit the release of the above three cytokines, with the average concentrations of TNF-α, IL-6 and IL-1β being 21.91, 285.84 and 13.6 pg / ml, respectively. In terms of α, the NX-5948 group was significantly better than the Ruxolitinib group (29.67 pg / ml), and had 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), and had 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), and had no significant difference from the Ruxolitinib group (16.76 pg / ml). Conclusion: NX-5948 significantly improves serum cytokine levels in mice; Experiment 13: Experimental basis: The same as that of Experiment 9; Detection method: After administration, peripheral serum of mice was collected and serum ferritin and IL-2R levels were detected using ELISA kits; Test results: Figure 13The results 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 the average concentrations of ferritin and IL-2R being 1556.10 and 10.85 ng / ml, respectively. NX-5948 can significantly correct the above abnormalities. The average concentrations of ferritin and IL-2R were 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 had 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). Conclusion: NX-5948 significantly improves the levels of serum ferritin and soluble CD25 (IL-2R) in mice.

[0034] 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.

[0035] The experimental results of Experiments 1-13 illustrate that the BTK PROTAC compound NX-5948 inhibits the activation of the macrophage BTK / NF-κB axis signaling pathway by targeting, on the one hand, by inhibiting the proliferation and survival of macrophages and reducing the number of activated macrophages; on the other hand, through the immunomodulatory effect on macrophages, it induces the polarization of macrophages from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, thereby inhibiting cytokine release and the occurrence and development of HLH. Therefore, the use of the Bruton's tyrosine kinase protein degradation-targeted chimera BTK PROTAC compound NX-5948 or a pharmaceutically acceptable salt thereof as a pharmaceutical active ingredient in the preparation of a drug for the treatment of secondary hemophagocytic syndrome is feasible. Therefore, the new application of the present invention has the effect of inhibiting the abnormal activation of macrophages and improving the clinical symptoms or laboratory indicators of patients with secondary hemophagocytic syndrome.

Claims

1. Use of a 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 the treatment of secondary hemophagocytic syndrome.

2. The use according to claim 1, characterized in that: The application can inhibit abnormal activation of macrophages.

3. The use according to claim 1, characterized in that: The application can improve at least one of the following clinical symptoms or laboratory indicators of patients with secondary hemophagocytic syndrome: Clinical symptoms: hepatosplenomegaly, liver and kidney failure; Laboratory indicators: cytopenia, hypertriglyceridemia and / or hypofibrinogenemia, elevated ferritin, elevated soluble interleukin-2 receptor (sCD25), bone marrow hemophagocytosis, and proinflammatory cytokine storm.

4. The use according to claim 1, characterized in that: The compound structural formula of the BTK PROTAC compound NX-5948 is: 。 5. The use according to claim 1, characterized in that: The drug is administered by at least one of oral, intravenous, subcutaneous or intrathecal injection.

6. The use according to claim 1, characterized in that: The dosage form of the drug is tablet, capsule, lyophilized powder injection or oral solution, and contains pharmaceutically acceptable carriers and / or adjuvants.

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of the BTK PROTAC compound NX-5948 according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, and is used to treat secondary hemophagocytic syndrome.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is an oral preparation, an injection or a sustained-release preparation.

9. A method for treating secondary hemophagocytic syndrome, characterized in that: The method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 8 or 9 to a patient in need thereof.

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