Application of small-molecule compound in preparation of ARVC (arachidonic acid-associated virus C) medicine

Abstract

The invention relates to an application of a small molecule compound in preparation of ARVC drugs. The small molecule compound is a CD44 inhibitor. Experiments prove that the CD44 inhibitor can relieve the fibrosis degree of PKP2 < + / -> human-derived heart organs, and a new direction is provided for treatment of ARVC and research and development of related drugs.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule drugs, and particularly relates to the application of a small molecule compound in the preparation of ARVC drugs. Background Art

[0002] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a chronic progressive familial genetic disease, which is mainly manifested by the replacement of right ventricular myocardial tissue with fibrous tissue and adipose tissue to varying degrees. The onset of ARVC is relatively insidious, and its patients will experience symptoms such as right ventricular dysfunction, ventricular arrhythmias, heart failure, and sudden cardiac death. Studies have shown that more than 40% of ARVC patients have genetic mutations in PKP2. PKP2 mutations can destroy the normal connection between myocardial cells, degenerate desmosome structures, cause compensatory fibrous tissue infiltration and proliferation of myocardial cells, and replace myocardial cells with fibrofatty tissue, leading to adverse remodeling and arrhythmias. At present, the drug treatment of ARVC is mainly antiarrhythmic drugs, but their therapeutic effect is still unsatisfactory. So far, there are still no approved small molecule drugs for the process of myocardial tissue fibrosis in ARVC. Therefore, it is of great significance to develop a small molecule drug that can reverse myocardial tissue fibrosis for ARVC patients with PKP2 mutations.

[0003] CD44 is a complex transmembrane adhesion glycoprotein expressed in a variety of human cells, including embryonic stem cells, differentiated cells, and cancer cells. Because it plays an important role in cell proliferation, adhesion, and migration, it has long been considered a marker for tumor stem cells. In 2021, the Center for Drug Evaluation (CDE) of the China National Medical Products Administration approved HP558, an inhibitor that specifically targets the splice variant CD44v6 of the CD44 family, for clinical research in metastatic esophageal squamous cell carcinoma (ESCC). However, in the cardiovascular field, especially in the hearts of ARVC patients with PKP2 mutations, it is still unknown what role CD44 plays. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of a small molecule compound in the preparation of ARVC drugs, thereby providing a new direction for the treatment of ARVC and the research and development of related drugs.

[0005] The present invention provides an application of a small molecule compound in the preparation of an ARVC drug, wherein the small molecule compound is a CD44 inhibitor.

[0006] Furthermore, the CD44 inhibitor includes but is not limited to Compound-6 (Antitumor agent-109).

[0007] Furthermore, the effects of the CD44 inhibitor include reducing the degree of cardiac fibrosis in ARVC.

[0008] Furthermore, the ARVC drug is targeted at ARVC patients with PKP2 gene mutations and heart failure patients without gene mutations.

[0009] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0010] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0011] The present invention also provides a pharmaceutical composition for ARVC, comprising a therapeutically effective amount of a CD44 inhibitor.

[0012] Furthermore, the dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

[0013] Beneficial Effects

[0014] The present invention proves through experiments that CD44 inhibitors can reduce PKP2 + / - The degree of fibrosis in human cardiac organoids provides a new direction for the treatment of ARVC and the development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the present invention.

[0016] Figure 2 Schematic diagram of cardiac organoid differentiation and microscopic observation of PKP2-WT and PKP2 on different days + / - Morphological picture of the heart organ.

[0017] Figure 3a -b for PKP2 + / - Results of combined transcriptomic and proteomic analysis at different stages of development of WT and PKP2-WT cardiac organoids (day 14 and day 28); comparison of mRNA and protein changes in cardiac organoids on day 14 (A) and day 28 (D); B and E, KEGG pathway classification histograms on day 14 and day 28; C and F, GO pathway classification histograms on day 14 and day 28; G, bubble plot of KEGG pathways on day 14; H, PPI analysis gene map; I, ​​protein interaction analysis between CD44 and PKP2.

[0018] Figure 4These are the results of immunofluorescence experiments on human heart organoids on days 14 and 28.

[0019] Figure 5 PKP2 + / - The results of the cardiac organoid inhibition CD44 immunofluorescence experiment. Compound-6 (Antitumoragent-109) is a CD44 inhibitor.

[0020] Figure 6 Coupound-6, a CD44 inhibitor, in PKP2 + / - Results of cell proliferation assay (CCK-8) in cardiac organoids. Results are expressed as percentage of mean ± SEM (n≥4). DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0022] Example 1

[0023] Discovery of CD44 as a key factor in ARVC disease progression in patients with PKP2 gene mutations

[0024] In order to explore the potential molecular mechanism of PKP2 in ARVC, the applicants constructed + / - and PKP2-WT cardiac organoids. Organoid morphology is shown in Figure 2 , and its ARVC phenotype has been confirmed. For related phenotypic identification, please refer to the patent application: application number 2024108672975.

[0025] Subsequently, ARVC heart organoid samples at different developmental stages (days 14 and 28) were taken for transcriptome and proteome analysis, and differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were jointly analyzed. According to different trends, DEGs were divided into four groups (Figures 3A and D): RNA decrease and protein increase (4 genes in part 1), RNA decrease and protein decrease (25 genes in part 7), RNA increase and protein increase (8 genes in part 3), and RNA increase and protein decrease (6 genes in part 9). Next, the applicant performed KEGG (Figures 3B, E, and G) and GO analysis (Figures 3C and F) on the above 43 genes. The results showed that during the development of the heart of ARVC patients with PKP2 mutations, PKP2 affects processes such as cell growth and death, cell bioadhesion, immune system, lipid metabolism, and signal transduction. It is worth noting that CD44, as a key factor of the immune system, is enriched in both GO and KEGG analysis. Next, the applicant performed a protein-protein interaction (PPI) network analysis on PKP2 (Figure 3H), and further determined that CD44 was the core gene of the PPI network by calculating the Betweenness value (pTM (0.65) + ipTM (0.31) > 0.75) (Figure 3H). The applicant further predicted the protein interaction between CD44 and PKP2 by AlphaFold3, and used pymol to simulate the interaction between protein complexes (Figure 3I). The results showed that PKP2 can bind to CD44 at the hydrophilic binding site and form 5 hydrogen bonds (ASP-164 and ARG-24, TYR-156 and TYR-25, ASP-155 and TYR-62, ASP-155- and ALA-82, ASP-155-ILE-79) (Figure 3I-Table 1).

[0026] To verify this result, further analysis was performed using Western Blot and Immunofluorescence Assay. The results showed that compared with the PKP2-WT group, PKP2 + / - The expression of CD44 in cardiac organoids increased significantly on days 14 and 28. These results suggest that CD44 is abnormally increased in the hearts of ARVC patients ( Figure 4 ).

[0027] Example 2

[0028] CD44 inhibitors for the treatment of ARVC with PKP2 gene mutations

[0029] The applicant previously used CD44 inhibitor (Antitumor agent-109, Compound 6) in PKP2 mutant heart organoids. PKP2 was incubated at concentrations of 0.1 μM, 0.3 μM, and 1 μM. + / - The effect of CD44 inhibition on ARVC was verified by immunofluorescence experiments. The results showed that with the increase of CD44 inhibitor concentration, the marker of ARVC cardiac organoid fibroblasts α-SMA showed a gradual decrease trend ( Figure 5 ), and within the concentration range used, CD44 inhibitors did not affect the activity of cardiac organoids ( Figure 6 ).

[0030] The above results indicate that CD44 inhibitors can reduce the degree of fibrosis in ARVC hearts without affecting the activity of cardiac organoids.

[0031] In conclusion, CD44 inhibitors can be used to treat ARVC with PKP2 gene mutations.

Claims

1. Use of a small molecule compound in the preparation of an ARVC drug, characterized in that: The small molecule compound is a CD44 inhibitor.

2. The use according to claim 1, characterized in that: The CD44 inhibitors include but are not limited to Compound-6.

3. The use according to claim 1, characterized in that: The effects of the CD44 inhibitor include reducing the degree of cardiac fibrosis in ARVC.

4. The use according to claim 1, characterized in that: The ARVC drug is targeted at ARVC patients with PKP2 gene mutations and heart failure patients without gene mutations.

5. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

6. The use according to claim 1, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

7. A pharmaceutical composition for ARVC, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of the CD44 inhibitor according to claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

Images