A pharmaceutical composition for the treatment and / or prevention of p53-mutant ovarian cancer.

Abstract

This invention relates to a pharmaceutical composition for the treatment and / or prevention of p53-mutant ovarian cancer, the pharmaceutical composition comprising one or more of CDK8 inhibitors and CDC7 inhibitors. This invention identifies CDK8 and CDC7 as key dependent targets of p53-mutant ovarian cancer and proposes for the first time the combined use of CDK8 inhibitors and CDC7 inhibitors to synergistically inhibit the proliferation and drug resistance formation of p53-mutant ovarian cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition for the treatment and / or prevention of p53-mutant ovarian cancer. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive system, characterized by insidious onset, rapid progression, and poor prognosis. Current clinical treatment mainly includes surgical resection combined with platinum-based chemotherapy, but many patients still experience recurrence and drug resistance after treatment.

[0003] At the molecular level, the p53 gene is one of the most commonly mutated genes in ovarian cancer. Mutations in this gene can lead to abnormal cell cycle regulation, defective DNA damage repair, and abnormal proliferation of tumor cells. Studies have shown that p53 mutations are closely related to the development and progression of ovarian cancer, chemotherapy resistance, and poor prognosis.

[0004] Currently, treatment options for ovarian cancer mainly include chemotherapy drugs, PARP inhibitors, and some molecularly targeted drugs. However, existing treatment regimens primarily target patients with BRCA mutations or DNA repair deficiencies, lacking effective specific treatment strategies for p53-mutant ovarian cancer. Furthermore, existing drugs generally suffer from limited efficacy, easy development of drug resistance, and significant toxic side effects in clinical applications. This may be due to the abnormal activation of multiple pathways caused by p53 mutations, making single-target therapy difficult to achieve ideal results.

[0005] Therefore, developing a highly effective drug composition for p53-mutant ovarian cancer is of great significance for improving treatment efficacy, overcoming drug resistance, and improving patient prognosis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a pharmaceutical composition for the treatment and / or prevention of p53-mutant ovarian cancer.

[0007] The present invention provides a pharmaceutical composition for preparing a treatment and / or prevention of p53-mutant ovarian cancer, the pharmaceutical composition comprising one or more of CDK8 inhibitors and CDC7 inhibitors.

[0008] The CDK8 inhibitor includes RVU120; the CDC7 inhibitor includes TAK-931.

[0009] RVU120 is a selective CDK8 / CDK19 small molecule inhibitor that can regulate tumor cell transcription, cell cycle progression, and DNA damage response pathways by inhibiting Mediator kinase complex-related signal transduction, thereby enhancing the sensitivity of tumor cells to replication stress. The CDC7 inhibitor is TAK-931, a highly selective CDC7 kinase inhibitor that can inhibit DNA replication initiation, induce replication fork arrest and DNA damage accumulation, leading to tumor cell proliferation inhibition and apoptosis. Preferably, the combined use of RVU120 and TAK-931 can synergistically enhance tumor cell replication stress and DNA damage levels, inhibit tumor cell proliferation activity, and improve the killing effect on CCNE1 aberrantly amplified ovarian cancer cells.

[0010] The drugs include CDK8 inhibitors and CDC7 inhibitors.

[0011] The dosage of the CDK8 inhibitor is 50-80 mg / kg; the dosage of the CDC7 inhibitor is 60 mg / kg.

[0012] This invention provides the use of a CDK8 inhibitor or a CDC7 inhibitor in the preparation of drugs for the treatment and / or prevention of p53-mutant ovarian cancer.

[0013] This invention provides the application of a combination of CDK8 inhibitor and CDC7 inhibitor in the preparation of drugs for the treatment and / or prevention of p53-mutant ovarian cancer.

[0014] The CDK8 inhibitor includes RVU120; the CDC7 inhibitor includes TAK-931.

[0015] RVU120 and TAK-931 can be administered simultaneously, separately, or sequentially to inhibit the proliferation of p53 mutant ovarian cancer cells, induce tumor cell cycle arrest, and / or promote tumor cell apoptosis, thereby achieving the treatment and / or prevention of p53 mutant ovarian cancer.

[0016] The mass ratio of the CDK8 inhibitor to the CDC7 inhibitor is (5~8):6.

[0017] The dosage of the CDK8 inhibitor is 50-80 mg / kg; the dosage of the CDC7 inhibitor is 60 mg / kg.

[0018] The drug includes capsules, injections, or sustained-release formulations.

[0019] This invention employs a CRISPR-Cas9 whole-genome library screening method to identify genes critically dependent on cell proliferation and survival in p53 mutant ovarian cancer cells. The results revealed CDK8 and CDC7 as potential key targets. Based on this, the following pharmaceutical composition was developed:

[0020] CDK8 inhibitor (RVU120 (sel120)), dosage range 50 ~ 80 mg / kg, once every other day.

[0021] The CDC7 inhibitor (TAK-931) is administered at a dose range of 60 mg / kg every other day.

[0022] This composition can be prepared as an oral, injectable, or sustained-release formulation, and can be administered as a single-dose combination or multiple-dose regimen depending on the course of the disease and tolerability. Drug A (CDK8 inhibitor) can inhibit the abnormal proliferation signaling of p53 mutant ovarian cancer cells by blocking CDK8-mediated transcriptional regulation; Drug B (CDC7 inhibitor) can inhibit DNA replication initiation and S-phase processes, thereby enhancing the sensitivity of tumor cells to drug-induced DNA damage; the combined use of the two can synergistically block tumor proliferation pathways, significantly improve anti-tumor activity, and reduce the incidence of drug resistance.

[0023] The combination scheme of the present invention can be optimized by adjusting the dosage and administration regimen of each drug. At the same time, CDK8 inhibitors and CDC7 inhibitors can be replaced with similar compounds known in the art to form a variety of alternative combination schemes, ensuring the wide applicability and implementability of the present invention.

[0024] This invention is the first to propose the combined use of CDK8 inhibitors and CDC7 inhibitors to synergistically inhibit the proliferation and drug resistance formation of p53 mutant ovarian cancer cells.

[0025] Note: CDK8: Cyclin-Dependent Kinase 8, which regulates transcription and cell proliferation;

[0026] CDC7: Cell Division Cycle 7, involved in the initiation of DNA replication;

[0027] CRISPR-Cas9: A gene editing tool used for whole-genome library knockout screening;

[0028] p53-mutant ovarian cancer: Mutations in the TP53 gene in ovarian cancer cells lead to cell cycle dysregulation and increased drug resistance.

[0029] Beneficial effects

[0030] (1) Significantly improves treatment effect: The combined use of CDK8 inhibitor and CDC7 inhibitor can synergistically inhibit the proliferation of p53 mutant ovarian cancer cells and enhance the apoptosis rate, with better treatment effect than single drug use.

[0031] (2) Reduce the incidence of drug resistance: Block multiple proliferation and DNA repair-related pathways, effectively delaying or overcoming the problem of tumor drug resistance.

[0032] (3) Wide range of applications: The drug composition can be prepared as oral, injectable or sustained-release preparations, adaptable to a variety of administration methods, and the components can replace similar compounds, making it highly operable in clinical practice.

[0033] (4) Save R&D costs and accelerate translation: By screening targets through CRISPR, blind experiments can be reduced, drug development efficiency can be improved, and experimental resources and time can be saved.

[0034] (5) Social and medical value: It provides new treatment strategies for patients with p53-mutant ovarian cancer, which helps to improve prognosis and prolong survival. Attached Figure Description

[0035] Figure 1 A schematic diagram of the CRISPR-Cas9 library screening process;

[0036] Figure 2 This is a schematic diagram illustrating the mechanism of action of the drug composition.

[0037] Figure 3 The combination of CDC7 inhibitors and CDK8 inhibitors significantly inhibited the proliferation of p53 mutant ovarian cancer cells;

[0038] Figure 4 The combination of CDC7 inhibitors and CDK8 inhibitors significantly enhanced apoptosis in p53 mutant ovarian cancer cells;

[0039] Figure 5 The combination of CDC7 inhibitors and CDK8 inhibitors significantly prolonged the survival of p53-mutant ovarian cancer PDX. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0041] Example 1

[0042] CRISPR-Cas9 library screening identifies key targets.

[0043] 1. Cell model

[0044] Select p53 mutant ovarian cancer cell lines (such as OVCAR3, SKOV3-p53mut) and culture them in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0045] 2. Importing CRISPR-Cas9 libraries

[0046] A whole-genome CRISPR-Cas9 knockout library was used to introduce the library into ovarian cancer cells via lentiviral infection. The infection efficiency was controlled at 30%-40%, ensuring an average coverage of ≥500-fold for each target gene.

[0047] 3. Gene function screening

[0048] Infected cells were cultured for 7–14 days, and surviving cells were collected for high-throughput sequencing (NGS) analysis of sgRNA abundance changes.

[0049] By comparing genes whose sgRNA abundance decreased significantly before and after library import, key dependent genes CDK8 and CDC7 were identified.

[0050] Corresponding attached diagram: Figure 1 (Diagram of the CRISPR-Cas9 library screening process). For example... Figure 1 As shown: CDC7 was knocked down in p53 mutant ovarian cancer cell lines, and a CRISPR-Cas9 library was introduced for gene function screening, which identified key genes dependent on CDK8 and CDC7.

[0051] like Figure 1 The diagram illustrates the standard experimental procedure for CRISPR-Cas9 whole-genome / targeted library screening. First, an sgRNA library is designed and constructed according to the research objective. The library typically contains multiple sgRNA sequences targeting the whole genome or a specific gene set, and is packaged using a lentiviral vector. Subsequently, the virus infects the target cell population and is transduced under low multiple of infection (MOI) conditions to ensure that each cell typically integrates only a single sgRNA, thereby achieving independent perturbation of gene function. After infection, a stable cell population integrating sgRNA is obtained through antibiotic selection or fluorescent labeling, maintaining sufficient cell coverage to ensure library complexity.

[0052] During the screening phase, cells were divided into control and treatment groups according to the experimental design, such as drug treatment, gene stress, or microenvironmental stimulation, to simulate specific biological selection pressures. After a certain period of culture, surviving cell populations or those with specific phenotypes were collected, genomic DNA was extracted, and integrated sgRNA sequences were amplified. Subsequently, high-throughput sequencing technology was used to quantitatively analyze the abundance of sgRNAs.

[0053] Finally, bioinformatics analysis was used to compare the enrichment or deletion of sgRNAs in different groups, thereby identifying key functional genes affecting cell survival, proliferation, or specific phenotypes. Enriched sgRNAs typically correspond to potential tumor suppressor gene knockout effects, while significantly depleted sgRNAs suggest that the relevant genes play an important role in cell survival. This process can systematically analyze gene function and be used to screen potential therapeutic targets.

[0054] Example 2

[0055] Preparation and administration of pharmaceutical compositions

[0056] 1. Drug selection

[0057] a. CDK8 inhibitors: CDK8 small molecule inhibitors (RVU120 (sel120)) reported in the literature can be selected;

[0058] b. CDC7 inhibitors: Small molecule inhibitors of CDC7 (TAK-931) that have been reported in the literature can be selected.

[0059] 2. Drug combination preparation

[0060] a. Mix CDK8 inhibitors and CDC7 inhibitors in a certain ratio (e.g., 1:1 or optimized according to dosage);

[0061] b. It can be prepared as oral capsules, injections, or sustained-release formulations.

[0062] 3. Administration method

[0063] Oral administration: Dissolve the drug in a suitable solvent and administer to mouse models via gastric tube once daily or at intervals.

[0064] Example 3

[0065] Drug Mechanism of Action and Efficacy Verification

[0066] 1. In vitro experiments

[0067] a. Treat p53 mutant ovarian cancer cells with CDK8 inhibitors, CDC7 inhibitors, and their combination (drugs from Example 2) for 48 hours;

[0068] b. Cell proliferation assay: Cell viability was detected by MTT or CellTiter-Glo method;

[0069] c. Apoptosis detection: Annexin V / PI staining and flow cytometry were used to detect the apoptosis rate.

[0070] d. Results showed that cell proliferation was significantly inhibited and apoptosis rate was significantly increased in the combined group.

[0071] 2. In vivo experiments

[0072] a. Establish a mouse xenograft model of p53 mutant ovarian cancer;

[0073] b. Randomized grouping: control group, CDK8 inhibitor group, CDC7 inhibitor group, and combination therapy group;

[0074] c. Administer the drug continuously for 2–4 weeks, and monitor tumor volume and mouse survival rate;

[0075] d. Results showed that tumor growth was significantly inhibited and survival rate was improved in the combination therapy group.

[0076] like Figure 2 As shown in the diagram, the mechanism of action of the drug composition is as follows: CDK8 inhibitors act on transcriptional regulatory pathways; CDC7 inhibitors act on DNA replication initiation; the combination inhibits cell cycle arrest and induces apoptosis; the anti-tumor effect is enhanced and drug resistance is delayed. Figure 2 CDC7 inhibitors enhance intracellular replication stress by blocking DNA replication initiation activation and inhibiting MCM complex phosphorylation, leading to impaired replication origin "fire" and inducing replication fork arrest. CDK8 inhibitors, on the other hand, interfere with Mediator complex-mediated transcriptional regulation, reducing DNA damage repair and replication stress-related gene expression, while increasing transcriptional aberrations and the accumulation of RNA:DNA hybrids (R-loops), thus triggering transcription-replication conflict. When both are used in combination, the replication fork instability caused by CDC7 inhibition and the transcriptional impairment and decreased repair capacity caused by CDK8 inhibition overlap, resulting in the continuous accumulation of DNA double-strand breaks that cannot be effectively repaired. Ultimately, this activates the ATM / ATR-dependent DNA damage response pathway, triggering cell cycle arrest and replication catastrophe, thereby achieving a significant synergistic lethal effect.

[0077] like Figure 3 As shown, in p53 mutant ovarian cancer cells, the combination of CDC7 inhibitor and CDK8 inhibitor exhibited more significant inhibitory effects on cell proliferation compared to single-drug treatment or the control group. After combination therapy, cell viability significantly decreased, suggesting a possible synergistic effect between the two targeting pathways, thereby jointly interfering with tumor cell cycle progression and proliferation. Figure 3 The effect of a combination of CDC7 inhibitors and CDK8 inhibitors on the proliferation of p53 mutant ovarian cancer cells was detected using the CCK-8 assay. The results showed that, compared with the single-drug treatment group and the control group, the combination therapy group significantly inhibited the proliferative activity of p53 mutant ovarian cancer cells.

[0078] like Figure 4 As shown, the combination of CDC7 inhibitors and CDK8 inhibitors significantly induced apoptosis in p53-mutant ovarian cancer cells. Compared with single-drug treatment, the proportion of apoptosis in the combination therapy group was significantly increased, suggesting that this combination strategy not only inhibits cell proliferation but also further activates cell death programs, thereby enhancing the anti-tumor effect. Figure 4 Annexin V flow cytometry analysis results: The combination of CDC7 inhibitor and CDK8 inhibitor significantly enhanced apoptosis in p53 mutant ovarian cancer cells. Annexin V-FITC / PI double staining was used to detect the apoptosis level of p53 mutant ovarian cancer cells under different treatment conditions. Flow cytometry results showed that, compared with the single-drug treatment group, the combined treatment group of CDC7 inhibitor and CDK8 inhibitor significantly enhanced early apoptosis (Annexin V). ＋ / PI - ) and late apoptosis (Annexin V) ＋ / PI ＋ The significantly increased proportion of apoptosis in p53-mutant ovarian cancer cells suggests that the combined use of the two inhibitors can synergistically induce enhanced apoptosis. Data from each group are presented in the form of representative scatter plots and quantitative statistical graphs; statistical analysis showed that the differences were statistically significant.

[0079] like Figure 5 As shown, the effect of CDC7 inhibitor combined with CDK8 inhibitor on the survival of p53-mutant ovarian cancer PDX (patient-derived xenograft) model mice was investigated. Compared with the control group and the single-drug treatment group, the combination therapy group significantly prolonged the survival time of PDX model mice, suggesting that CDC7 inhibitor and CDK8 inhibitor have a synergistic anti-tumor effect. This indicates that the survival time of animals treated with CDC7 inhibitor combined with CDK8 inhibitor was significantly prolonged, showing a superior survival benefit compared to the control group and the single-drug treatment group, suggesting that this combination regimen also has clear anti-tumor activity and potential translational application value in in vivo models.

[0080] Example 4

[0081] Alternative drugs and dosage forms

[0082] Alternative compounds

[0083] a. CDK8 inhibitors can be replaced with CDK8 small molecule inhibitors known in the art;

[0084] b. CDC7 inhibitors can be replaced with CDC7 small molecule inhibitors known in the art;

[0085] c. Alternative solutions must ensure inhibitory activity and synergistic effects.

[0086] Dosage optimization

[0087] a. The ratio and dosage of CDK8 inhibitors to CDC7 inhibitors can be adjusted according to body weight, tolerance, and tumor type;

[0088] b. It can be optimized through in vitro IC50 determination and in vivo efficacy experiments.

[0089] Diversified dosage forms

[0090] c. It can be used to prepare oral, injectable, and sustained-release formulations to meet different clinical application needs.

Claims

1. A pharmaceutical composition for preparing a treatment and / or prevention of p53-mutant ovarian cancer, characterized in that, The drug includes one or more of CDK8 inhibitors and CDC7 inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that, The CDK8 inhibitor is RVU120; the CDC7 inhibitor is TAK-931.

3. The pharmaceutical composition according to claim 1, characterized in that, The drugs include CDK8 inhibitors and CDC7 inhibitors.

4. The pharmaceutical composition according to claim 1, characterized in that, The dosage of the CDK8 inhibitor is 50-80 mg / kg; the dosage of the CDC7 inhibitor is 60 mg / kg.

5. The use of a CDK8 inhibitor or CDC7 inhibitor in the preparation of a drug for the treatment and / or prevention of p53-mutant ovarian cancer.

6. The use of a combination of a CDK8 inhibitor and a CDC7 inhibitor in the preparation of a drug for the treatment and / or prevention of p53-mutant ovarian cancer.

7. The application according to claim 5 or 6, characterized in that, The CDK8 inhibitor is RVU120, and the CDC7 inhibitor is TAK-931.

8. The application according to claim 6, characterized in that, The mass ratio of the CDK8 inhibitor to the CDC7 inhibitor is (5~8):

6.

9. The application according to any one of claims 5-6, characterized in that, The drug includes capsules, injections, or sustained-release formulations.

Images