A compound containing as (iii), darinaparsin, and its use

Abstract

The application belongs to the technical field of new use of medicines, and particularly discloses an As(III)-containing compound Darinaparsin and application thereof, and the As(III)-containing compound Darinaparsin is applied to preparation of a medicine for treating inflammatory bowel disease. The As(III)-containing compound Darinaparsin and application thereof are used to solve the defects of low response rate and easy drug resistance of the existing medicines for treating inflammatory bowel disease, and provide a new use of the As(III)-containing compound Darinaparsin, which is used for treating inflammatory bowel disease, and provide a new treatment scheme with definite curative effect and clear mechanism.

Description

Technical Field

[0001] This invention relates to the field of new uses of pharmaceuticals, and in particular to Darinaparsin, an As(III)-containing compound, and its applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, nonspecific inflammatory bowel diseases, mainly including ulcerative colitis (UC) and Crohn's disease (CD). Its etiology involves multiple aspects such as genetics, environment, and immune response. Clinical treatment focuses on controlling inflammation and relieving symptoms, but existing drugs have significant limitations: aminosalicylic acid drugs, glucocorticoids, and immunosuppressants have limited efficacy, and anti-TNF antibody drugs are effective in only about 30% of patients, and long-term use easily leads to drug resistance and decreased treatment effectiveness.

[0003] NOD2 (Nucleotide-binding oligomerization domain 2), as a cytoplasmic pattern recognition receptor, participates in the innate immune response by recognizing bacterial muramyl dipeptide (MDP). Its overactive signaling is closely related to various inflammatory diseases such as IBD. NOD2 signaling transduction depends on XIAP protein (X-linked inhibitor of apoptosis protein). The BIR2 domain of XIAP binds to the kinase domain of RIPK2 and ubiquitinates RIPK2 through the RING domain. This process is a necessary step in NOD2 signaling transduction. Therefore, targeting XIAP to inhibit the NOD2 signaling pathway is a potential strategy for the treatment of inflammatory diseases.

[0004] Darinaparsin is an organic arsenic compound belonging to the mitochondrial-targeting drug class. It is known to have anticancer activity and is mainly used to treat hematologic malignancies and solid tumors. Its mechanism of action includes disrupting mitochondrial function, increasing reactive oxygen species production, and regulating cell signal transduction pathways. However, there are currently no reports or applications of its use in the treatment of inflammatory bowel disease. Summary of the Invention

[0005] The purpose of this invention is to provide an application of Darinaparsin, a compound containing As(III), which addresses the shortcomings of existing drugs for treating inflammatory bowel disease, such as low response rates and easy drug resistance. This invention provides a new use for Darinaparsin, a compound containing As(III), in the treatment of inflammatory bowel disease, offering a new treatment option with definite efficacy and a clear mechanism of action.

[0006] To achieve the above objectives, the present invention provides a compound containing As(III), Darinaparsin, with the following structural formula: .

[0007] The present invention also provides an application of the As(III)-containing compound Darinaparsin in the preparation of a drug for treating inflammatory bowel disease.

[0008] Preferably, inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

[0009] Preferably, the drug exerts its therapeutic effect by inhibiting the NOD2-RIPK2 signaling pathway.

[0010] Preferably, the mechanism of action of the drug includes: Darinaparsin binds to the BIR2 and RING domains of the XIAP protein, releases zinc ions from the XIAP domain, disrupts the interaction between XIAP and RIPK2, inhibits the ubiquitination and phosphorylation of RIPK2, and thereby inhibits downstream inflammatory pathways.

[0011] The present invention also provides a pharmaceutical composition for treating inflammatory bowel disease, comprising Darinaparsin, a compound containing As(III), or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0012] Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, granules, powders, syrups, oral liquids, suppositories, or ointments.

[0013] Preferably, the dosage of the As(III)-containing compound Darinaparsin is 0.01 mg to 100 mg / kg, and the administration method includes once or multiple times a day, or at intervals of several days.

[0014] The advantages and beneficial effects of this invention using Darinaparsin, an As(III)-containing compound, and its applications are as follows: 1. This study expanded the application of Darinaparsin by using it for the first time in the treatment of inflammatory bowel disease, filling a gap in the use of this compound in the treatment of inflammatory diseases.

[0015] 2. The mechanism of action is clear. It works by targeting XIAP to inhibit the NOD2-RIPK2 signaling pathway, thus exerting its effect at the source of the inflammatory pathogenesis, and the efficacy is definite.

[0016] 3. Animal experiments have confirmed that it can significantly alleviate inflammatory symptoms in IBD mice, reduce the expression of pro-inflammatory factors, and improve intestinal tissue damage, providing a reliable basis for clinical application.

[0017] 4. The drug composition has a variety of dosage forms, flexible administration, and is suitable for the needs of different patients, and has broad clinical application prospects.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 The results of the thermostability experiments of XIAP protein and its domains are shown. A represents the full length of XIAP, B represents the BIR2 domain, C represents the RING domain, and Western blot bands in the control group (Ctrl) and Darinaparsin (DAR) treatment group are shown. The right side corresponds to the temperature-band intensity quantification curve. D represents the MALDI-TOF-MS result of the BIR2 domain, and E represents the detection result of the RING domain. Figure 2 The effect of Darinaparsin targeting the BIR2 and RING domains on zinc ions, where A is the BIR2 domain and B is the RING domain; Figure 3 To show that Darinaparsin inhibits the NOD2-RIPK2 pathway activated by MDP, A represents the effect of DAR on the interaction between XIAP and RIPK2, B represents the detection of RIPK2 ubiquitination level, C represents the detection of phosphorylation level of key proteins in the signaling pathway by Western blot, D represents HCT116 cells, E represents THP1 cells, and F represents THP1 cells. Figure 4 The therapeutic effect of Darinaparsin (DAR) in a DSS-induced inflammatory bowel disease (IBD) mouse model is shown in Figure A, where A is a schematic diagram of the experimental procedure, B is the body weight change curve, C is the Disease Activity Index (DAI) score, D is a comparison of colon length, E is a histopathological section of HE-stained colon tissue, and F is the colon tissue of mice in different treatment groups. Il6 , Il1b , Tnfα The transcriptional level is shown in G, which represents the CETSA blots of the colon and liver of mice in different treatment groups. The lower side corresponds to the temperature-band intensity quantification curve. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] The following examples are not intended to limit the invention, but are only for illustration. Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Example 1 Darinaparsin (DAR), a compound containing As(III), has the following structural formula: .

[0024] The application of Darinaparsin, an As(III)-containing compound, is discussed in the preparation of a drug for treating inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. The drug exerts its therapeutic effect by inhibiting the NOD2-RIPK2 signaling pathway. The mechanism of action involves Darinaparsin binding to the BIR2 and RING domains of the XIAP protein, releasing zinc ions from the XIAP domain, disrupting the interaction between XIAP and RIPK2, inhibiting RIPK2 ubiquitination and phosphorylation, and thereby suppressing downstream inflammatory pathways.

[0025] A pharmaceutical composition for treating inflammatory bowel disease comprises Darinaparsin, an As(III)-containing compound, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical composition is tablets, capsules, granules, powders, syrups, oral solutions, suppositories, or ointments. The dosage of Darinaparsin, an As(III)-containing compound, is 0.01 mg to 100 mg / kg, administered once or multiple times daily, or at intervals over several days.

[0026] The core technical solution of this invention is to use Darinaparsin to prepare a drug for treating inflammatory bowel disease. Specifically, its effectiveness and mechanism of action are demonstrated through the following experiments: Targeted binding validation: CETSA assay showed that Darinaparsin can reduce the thermostability of XIAP, BIR2 and RING proteins in cells; MALDI-TOF-MS assay confirmed that the purified BIR2 domain can bind two or more Darinaparsin molecules and the RING domain can bind four or more Darinaparsin molecules, indicating that Darinaparsin can directly bind XIAP protein in cells and in vitro.

[0027] Mechanism of action investigation: Zinc release experiments show that Darinaparsin can replace Zn in the BIR2 and RING domains of XIAP. 2+This allows for targeted action against XIAP; co-IP and WB experiments confirmed that Darinaparsin can disrupt the interaction between XIAP and RIPK2, inhibiting ubiquitination and phosphorylation of RIPK2 in the MDP-activated NOD2 signaling pathway; q-RT-PCR experiments showed that Darinaparsin can significantly reduce inflammatory factors after MDP stimulation. IL8 , TNFα The transcriptional levels of TNFα-stimulated inflammatory factors (such as MDP) were significantly lower than those of TNFα-stimulated inflammatory factors under the same conditions. This indicates that the inhibitory effect of naringin on the NF-κB signaling pathway is specifically targeted at NOD2 inhibition.

[0028] Animal experiments validated that intraperitoneal injection of 100 mg / kg Darinaparsin into IBD mice with DSS modeling resulted in a significantly lower disease activity index (DAI) and a significantly longer colon in the treatment group compared to the model group. The intestinal mucosa remained intact, and pro-inflammatory factors (DIA) were also reduced. Il6 , Il1b , Tnfα The transcriptional level was significantly reduced, confirming its significant anti-inflammatory effect in vivo.

[0029] The present invention also provides a pharmaceutical composition comprising Darinaparsin, the composition comprising a therapeutically effective amount of Darinaparsin or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, which can be prepared into conventional dosage forms such as tablets, capsules, and granules, and the dosage is adjusted according to the patient's condition, weight, disease severity, etc., ranging from 0.01 mg to 100 mg / kg.

[0030] Example 2 The effects of Darinaparsin on the thermostability of each protein were detected by overexpressing XIAP-Flag, BIR2-HA, and RING-HA in cells using CETSA. The binding of Darinaparsin to each protein was observed by MALDI-TOF-MS after purifying BIR2 and RING proteins in vitro.

[0031] like Figure 1 As shown in Figure A, the CETSA blot represents the binding of Darinaparsin to XIAP. HEK293t cells overexpressing XIAP-Flag were pretreated with Darinaparsin for 4 hours and then subjected to gradient heating. The soluble full-length XIAP-Flag protein in the sample supernatant was detected by Western blotting using the Flag antibody. The melting temperature shift between the treated sample and the control group was measured based on the CETSA melting curve. △Tm ).

[0032] like Figure 1 As shown in Figure B, the CETSA blot represents the binding of Darinaparsin to BIR2. HEK293t cells overexpressing BIR2-HA were pretreated with Darinaparsin for 4 hours and then subjected to gradient heating. The soluble BIR2-HA protein in the sample supernatant was detected by Western blotting using HA antibody. The melting temperature shift between the treated sample and the control group was measured based on the CETSA melting curve. △Tm ).

[0033] like Figure 1 As shown in Figure C, the CETSA blot represents the binding of Darinaparsin to RING. HEK293t cells overexpressing RING-HA were pretreated with Darinaparsin for 4 hours and then subjected to gradient heating. The soluble RING-HA protein in the sample supernatant was detected by Western blotting using HA antibody. The melting temperature shift between the treated sample and the control group was measured based on the CETSA melting curve. △Tm ),like Figure 1 As shown in Figure D, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) was used to detect the interaction between Darinaparsin and the BIR2 domain. Apo-BIR2 (120-240) was pre-incubated with a gradient of Darinaparsin, or without a gradient of Darinaparsin. The binding molar ratio between Darinaparsin and the BIR2 domain was approximately 2:1 or higher. Figure 1 As shown in Figure E, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry was used to detect the interaction between Darinaparsin and the RING domain. Apo-RING (429-471) was pre-incubated with gradient Darinaparsin or without gradient Darinaparsin. The binding molar ratio between Darinaparsin and the RING domain was approximately 4:1 or higher.

[0034] Based on the above, it can be seen that Darinaparsin can reduce the thermal stability of XIAP, BIR2 and RING proteins in cells. BIR2 and RING proteins purified in vitro can bind 2 or 4 or even more Darinaparsin molecules, which indicates that Darinaparsin can bind XIAP both in cells and in vitro.

[0035] Example 3 The effect of Darinaparsin addition on the Zn content in the BIR2 and RING domains of XIAP was detected by 4-(2-pyridiniazo)resorcinol (PAR).2+ The impact.

[0036] like Figure 2 China A and Figure 2 As shown in Figure B, the UV absorption spectra of 100 μM PAR with Zn-BIR2 and Zn-RING in solutions containing 5 mM glutathione are obtained with or without Darinaparsin. Based on the above, it can be concluded that Darinaparsin can replace Zn in the BIR2 and RING domains. 2+ To achieve the effect of targeting XIAP.

[0037] Example 4 First, XIAP-Flag and RIPK2-HA were overexpressed in cells. Then, different concentrations of Darinaparsin were added to observe its effect on the binding of XIAP and RIPK2. Next, after MDP was added to cells to activate the NOD2 signaling pathway, the effects of different concentrations of Darinaparsin on RIPK2 phosphorylation and ubiquitination, as well as on downstream inflammatory signaling pathways, were observed. Finally, the effect of Darinaparsin on the transcriptional levels of inflammatory factors after MDP or TNFα stimulation was observed.

[0038] like Figure 3 As shown in Figure A, after overexpressing XIAP-flag and RIPK2-3xHA in HEK293t, the cells were treated with Darinaparsin and then pulled down using a flag gel. The reaction was stopped by adding an additional 5x SDS loading buffer. Figure 3 As shown in Figure B, Ub-HA and RIPK2-6xHis were overexpressed in HEK293t, treated with Darinaparsin, stimulated with MDP, and pulled down using His gel. The reaction was stopped by adding an additional 5xSDS loading buffer during the experiment. Figure 3 After HCT116 was activated by C MDP, Darinaparsin at varying concentrations was added to observe the phosphorylation of p65 and RIPK2. The total protein levels of p65 and RIPK2 were measured, with β-actin as an internal control. Figure 3 After HCT116 was activated by MDP, Darinaparsin at varying concentrations was added to observe the transcriptional levels of IL8 and TNFα, with GAPDH as an internal control. Figure 3 After THP1 was activated by E MDP, Darinaparsin at different concentrations was added to observe the transcriptional levels of IL8 and TNFα, with GAPDH as an internal control. Figure 3 After THP1 was activated by TNFα, Darinaparsin at varying concentrations was added to observe the transcriptional levels of IL8 and TNFα, with GAPDH as an internal control.

[0039] Based on the above, it can be seen that Darinaparsin can disrupt the interaction between XIAP and RIPK2, and Darinaparsin can inhibit the NOD2 signaling pathway activated by MDP, including inhibiting RIPK2 ubiquitination and phosphorylation, as well as downstream inflammatory pathways such as NF-κB.

[0040] Example 5 Male C57BL / 6 mice aged 6-8 weeks were selected. The control group was fed purified water, while the model group and the treatment group were fed purified water containing 5% sodium dextran sulfate (DSS) for seven days, while drug intervention was also administered. Mice in the control and model groups were injected intraperitoneally with physiological saline, while mice in the treatment group were injected intraperitoneally with Darinaparsin (100 mg / kg). During the drug administration process, the mice's weight changes, fecal characteristics, and fecal blood loss were observed and recorded daily, and scores were assigned to calculate the Disease Activity Index (DAI).

[0041] The scoring rules and calculation method are as follows: DAI = (Weight Score + Diarrhea Score + Occult Blood Score) / 3. Where: Weight Score: 0 (weight increase or no change), 1 (weight decrease 1-5%), 2 (weight decrease 6-10%), 3 (weight decrease 11-15%), 4 (weight decrease more than 15%); Diarrhea Score: 0 (normal stool), 2 (loose stool), 4 (stool sticking to the anus or diarrhea); Occult Blood Score: 0 (negative occult blood), 2 (positive occult blood), 4 (visible bleeding).

[0042] Figure 4 In diagram A, we see experimental colitis in mice induced by DSS modeling. Figure 4 China B and Figure 4 In the middle C, the body weight and DAI score of mice in different treatment groups are shown. Figure 4 In the middle, D represents the length of the colon tissue of mice in different treatment groups; Figure 4 E represents pathological sections of colon tissue from mice in different treatment groups; Figure 4 F represents the colon tissue of mice in different treatment groups. Il6 , Il1b , Tnfα transcriptional levels; Figure 4 G represents the CETSA imprint of the colon of mice in different treatment groups.

[0043] Comparing the differences in DAI scores among the groups, it can be seen that Darinaparsin has a therapeutic effect on inflammatory bowel disease. The DAI score of the treated group was significantly lower than that of the model group. Comparing the colon length among the groups, the colon length of the treated group was significantly longer than that of the model group. Mid-segments of colon tissue from each group were selected, fixed overnight in formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). It was found that the intestinal mucosa morphology of the treated group was intact compared to the model group, indicating a significant therapeutic effect. Furthermore, the transcriptional levels of pro-inflammatory factors were significantly reduced in the treated group compared to the model group. CETSA experiments on colon tissue showed that Darinaparsin targets XIAP, indicating that Darinaparsin exerts its anti-inflammatory effect in mouse tissues through XIAP. Therefore, the compound Darinaparsin shows promise as a drug for the prevention or treatment of inflammatory bowel disease.

[0044] Therefore, this invention utilizes Darinaparsin, an As(III)-containing compound, and its applications. Its mechanism of action is clearly defined; it targets XIAP to inhibit the NOD2-RIPK2 signaling pathway, exerting its effect at the source of the inflammatory pathogenesis, resulting in definite therapeutic efficacy. Animal experiments have confirmed that it can significantly alleviate inflammatory symptoms in IBD mice, reduce the expression of pro-inflammatory factors, and improve intestinal tissue damage, providing a reliable basis for clinical application. This invention also provides pharmaceutical compositions containing Darinaparsin, which contain a therapeutically effective amount of Darinaparsin or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. These compositions can be prepared into conventional dosage forms such as tablets, capsules, and granules. The dosage is adjusted according to the patient's condition, weight, and disease severity, ranging from 0.01 mg to 100 mg / kg.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A compound containing As(III), Darinaparsin, characterized in that, Its structural formula is: .

2. The application of Darinaparsin, an As(III)-containing compound according to claim 1, is characterized in that: It is used in the preparation of drugs for treating inflammatory bowel disease.

3. The application of Darinaparsin, an As(III)-containing compound according to claim 2, characterized in that: Inflammatory bowel diseases include Crohn's disease and ulcerative colitis.

4. The application of Darinaparsin, an As(III)-containing compound according to claim 2, characterized in that: The drug exerts its therapeutic effect by inhibiting the NOD2-RIPK2 signaling pathway.

5. The application of Darinaparsin, an As(III)-containing compound according to claim 1, characterized in that: The mechanism of action of the drug includes: Darinaparsin binds to the BIR2 and RING domains of the XIAP protein, releases zinc ions from the XIAP domain, disrupts the interaction between XIAP and RIPK2, inhibits RIPK2 ubiquitination and phosphorylation, and thereby inhibits downstream inflammatory pathways.

6. A pharmaceutical composition for treating inflammatory bowel disease, characterized in that: It comprises Darinaparsin, a compound containing As(III) as described in claim 1, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is tablets, capsules, granules, powders, syrups, oral liquids, suppositories, or ointments.

8. The pharmaceutical composition according to claim 6, characterized in that: The dosage of Darinaparsin, a compound containing As(III), is 0.01 mg to 100 mg / kg, and the administration method includes once or multiple times a day, or at intervals of several days.

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