Use of a combination of an hdac8 inhibitor and a parp inhibitor for the manufacture of a medicament for the treatment of ovarian cancer, hepatocellular carcinoma

Abstract

The application discloses application of an HDAC8 inhibitor in preparation of a medicine for enhancing the curative effect of a PARP inhibitor or reversing / improving the drug resistance thereof, and relates to the technical field of biological medicines.The scheme of combining the HDAC8 inhibitor with the PARP inhibitor can significantly enhance the curative effect of the PARP inhibitor, reverse / improve the drug resistance of the PARP inhibitor, and effectively solve the core problem in the clinical application of the PARP inhibitor.The application provides a new strategy capable of effectively overcoming the drug resistance of the PARP inhibitor, improving the treatment sensitivity and being safe by means of the combined inhibition of the HDAC8 and the PARP, and makes up for the deficiencies of the prior art in the targeting, safety and curative effect stability, and provides a new feasible path for solving the long-existing technical defects in the field.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of HDAC8 inhibitors in the preparation of drugs for enhancing the efficacy of PARP inhibitors or reversing / improving their resistance. Background Technology

[0002] Abnormal DNA damage repair is one of the important molecular bases for tumor development, progression, and prognosis. Poly(ADP-ribose) polymerase (PARP) plays a crucial role in the recognition and repair of single-stranded DNA damage. Based on the principle of "synthetic lethality," PARP inhibitors have been widely used in the clinical treatment of tumors associated with BRCA1 / 2 mutations or homologous recombination repair deficiency (HRD), and have shown certain efficacy in various tumors such as ovarian cancer, breast cancer, and prostate cancer.

[0003] However, current research and clinical practice indicate that PARP inhibitors still have significant limitations in practical application. On the one hand, a considerable proportion of patients respond poorly to initial treatment with PARP inhibitors, exhibiting insufficient treatment sensitivity; on the other hand, some patients gradually develop acquired resistance after initial benefit, significantly weakening the long-term therapeutic effect of PARP inhibitors.

[0004] To improve the efficacy of PARP inhibitors, various combination therapies or sensitization strategies have been proposed in existing technologies, such as combining with DNA damage inducers, cell cycle regulator inhibitors, or other epigenetic regulatory drugs. However, these strategies still have the following shortcomings: First, some combination regimens lack clear and stable molecular targets, resulting in significant individual differences in treatment effects; second, some combination therapies may significantly increase toxic side effects, limiting their clinical application; and third, existing research on how to systematically enhance PARP inhibitor sensitivity by regulating chromatin state or the function of DNA damage repair-related proteins is still insufficient, and a mature and widely applicable technical solution has not yet been developed.

[0005] Histone deacetylases (HDACs) are important epigenetic regulators that play a crucial role in chromatin structure regulation, gene transcription regulation, and DNA damage repair. Although there are reports of anti-tumor studies targeting HDAC inhibitors, these studies mainly focus on broad-spectrum HDAC inhibitors. Their mechanisms of action are complex, their target specificity is insufficient, and their application in combining with other key cancer therapy inhibitors to improve sensitivity remains unclear and lacks clear and systematic technical insights.

[0006] Currently, there is an urgent need in this field to develop a technology with a clear target and stable effect without excessively increasing toxic side effects, so as to effectively enhance the therapeutic sensitivity of tumor cells to PARP inhibitors and overcome or delay the occurrence of drug resistance. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to develop a technical means with a clear target and stable effect without excessively increasing toxic side effects, so as to effectively enhance the therapeutic sensitivity of tumor cells to PARP inhibitors and overcome or delay the occurrence of drug resistance.

[0008] To address the above problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides the use of HDAC8 inhibitors in the preparation of drugs for enhancing the efficacy of PARP inhibitors or for reversing / improving PARP inhibitor resistance.

[0009] Furthermore, the HDAC8 inhibitor is a selective HDAC8 inhibitor.

[0010] Furthermore, the selective HDAC8 inhibitor is PCI-34051 or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the PARP inhibitor is selected from olaparib, niraparib, rucaparib, talazoparib, or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the PARP inhibitor is olaparib or a pharmaceutically acceptable salt thereof.

[0013] Furthermore, the tumor is ovarian cancer, hepatocellular carcinoma, breast cancer, prostate cancer, or pancreatic cancer.

[0014] Furthermore, the tumor is ovarian cancer.

[0015] Furthermore, the tumor is hepatocellular carcinoma.

[0016] In a second aspect, the present invention provides the use of an HDAC8 inhibitor in the preparation of a medicament for use in combination with a PARP inhibitor, wherein the combination enhances the antitumor efficacy of the PARP inhibitor or reverses tumor resistance to the PARP inhibitor.

[0017] Thirdly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an HDAC8 inhibitor and a PARP inhibitor, and / or comprising a pharmaceutically acceptable carrier or excipient.

[0018] Furthermore, the pharmaceutical composition is used to treat or prevent tumors that have acquired resistance to PARP inhibitors.

[0019] Fourthly, the present invention provides a pillbox comprising: (i) a first pharmaceutical unit containing an HDAC8 inhibitor; and (ii) a second pharmaceutical unit containing a PARP inhibitor; The first and second drug units are configured for combined administration to enhance the antitumor efficacy of PARP inhibitors or reverse tumor resistance to PARP inhibitors.

[0020] Compared with the prior art, the technical effects achieved by the present invention include: This invention provides the application of HDAC8 inhibitors in the preparation of drugs for enhancing the efficacy of PARP inhibitors or for reversing / improving PARP inhibitor resistance. The combination of HDAC8 inhibitors and PARP inhibitors in this invention significantly enhances the efficacy of PARP inhibitors and reverses / improves PARP inhibitor resistance, effectively solving a core challenge in the clinical application of PARP inhibitors. On the one hand, the solution provided by this invention selectively inhibits the well-defined target HDAC8, producing a synergistic effect with PARP inhibition, thereby significantly reversing acquired resistance in tumors and overcoming the shortcomings of existing broad-spectrum epigenetic regulation schemes, such as unclear targets and unstable efficacy. On the other hand, while achieving significant efficacy enhancement, this combination scheme has not shown significant toxicity additiveness and has been proven effective in various tumor models (such as ovarian cancer and liver cancer), simultaneously overcoming the high safety risks of combination therapy in existing technologies and the problems of poor primary treatment response or limited applicability of PARP inhibitors themselves. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1This figure presents an analysis of the combined effect of the HDAC8 inhibitor PCI-34051 and olaparib in PARP inhibitor-resistant ovarian cancer cells. Figures A and B show the three-dimensional response surface of cell viability after treatment with different concentrations of olaparib and PCI-34051, either alone or in combination, in PARP inhibitor-resistant ovarian cancer cell lines A2780-OlaR and OVCAR8-OlaR, respectively. X-axis: Olaparib concentration (μM); Y-axis: PCI-34051 concentration (μM); Z-axis: Cell viability (% of Control). The color gradient represents the combined effect index calculated based on the drug interaction model; blue areas indicate synergistic effects, red areas indicate antagonistic effects, and the color gradient from red to blue indicates a gradual increase from antagonism to synergy. This figure demonstrates that HDAC8 inhibition and PARP inhibition have synergistic antitumor activity in vitro.

[0023] Figure 2 This study aims to demonstrate the in vivo antitumor effect of HDAC8 inhibition on PARP inhibitors in a PARP-resistant ovarian cancer xenograft model. In the figure, A is a schematic diagram of the experimental procedure; B is a representative tumor image of each group at the treatment endpoint; C is the dynamic growth curve of tumor volume during treatment, with data expressed as mean ± SEM; D is a quantitative statistical graph of tumor weight in each group at the treatment endpoint; E is an immunohistochemical staining image of representative Ki67 and Cleaved Caspase-3 in tumor tissue; F is a bar chart of Ki67 staining H-score quantitative analysis; and G is a bar chart of Cleaved Caspase-3 staining H-score quantitative analysis.

[0024] This series of images systematically demonstrates that, in in vivo models, combination therapy can synergistically inhibit tumor growth, reduce proliferation, induce apoptosis, and effectively reverse PARP inhibitor resistance.

[0025] Figure 3 Results showing the enhancement of hepatocellular carcinoma's sensitivity to PARP inhibitors by HDAC8 inhibitors. In the figure, A is a schematic diagram of the establishment and experimental analysis process of the orthotopic hepatocellular carcinoma model; B is a schematic diagram of the drug treatment timeline and grouping; C is a quantitative curve showing the change in bioluminescence intensity of tumors in each group over time during treatment; D is a gross photograph of the orthotopic liver tumors in each group of mice at the treatment endpoint; E is a representative image of H&E staining in tumor tissue; F is a representative image of Ki67 and Cleaved Caspase-3 immunohistochemical staining in tumor tissue; G is a bar chart of quantitative analysis of the Cleaved Caspase-3 and Ki67 positive cell rates (H-score). Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0028] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0029] Example 1: Validation of the synergistic effect of HDAC8 inhibitors and PARP inhibitors in PARP-resistant cells in vitro 1.1 Cell lines and reagents Human ovarian adenocarcinoma cell line A2780 and its resistant subline A2780-OlaR induced by long-term exposure to olaparib, as well as human ovarian cancer cell line OVCAR8 and its resistant subline OVCAR8-OlaR, were used to construct PARP inhibitor resistance models. The HDAC8-specific inhibitor was PCI-34051, and the PARP inhibitor was olaparib.

[0030] 1.2 Experimental Methods A2780-OlaR and OVCAR8-OlaR cells were seeded at appropriate densities in 96-well plates. After cell attachment, different concentrations of Olaparib (concentration gradient: 0, 0.078, 0.156, 0.313, 0.625, 1.25 μM) and PCI-34051 (concentration gradient: 0, 0.625, 1.25, 2.5, 5, 10 μM) were administered as single or combined treatments. Cell viability was assessed using the CCK-8 assay after 72 hours of treatment. Cell viability was normalized to "% of Control" with the DMSO treatment group as a control. Drug interactions were analyzed using the Chou-Talalay combination index model and CompuSyn software, generating a three-dimensional response surface plot.

[0031] 1.3 Experimental Results like Figure 1 As shown in Figures A and B, in two PARP inhibitory-resistant cell lines, the combination of low-dose PCI-34051 and olaparib showed a significant synergistic inhibitory effect in the medium-to-high dose range (e.g., olaparib 0.313-1.25 μM, PCI-34051 2.5-10 μM) (blue area in the figure). The three-dimensional response surface plot visually shows that the cell survival rate in the combination treatment group was significantly lower than that in each single-drug treatment group, and the interaction model calculations indicate that this region represents a synergistic effect. These results demonstrate that the HDAC8 inhibitor PCI-34051 can significantly enhance the killing effect of the PARP inhibitor olaparib on drug-resistant tumor cells, partially reversing their resistance.

[0032] Example 2: HDAC8 inhibitors enhance the in vivo efficacy of PARP inhibitors in a drug-resistant ovarian cancer xenograft model 2.1 Animal Model Establishment and Grouping A2780 (sensitive) cells and A2780-OlaR (resistant) cells were subcutaneously inoculated into BALB / c nude mice. Tumors were allowed to grow to approximately 100 mm in size. 3 At that time, tumor-bearing mice were randomly divided into four groups (n=5 in each group): (1) solvent control group (DMSO); (2) Olaparib monotherapy group (50 mg / kg); (3) PCI-34051 monotherapy group (25 mg / kg); (4) Olaparib and PCI-34051 combination therapy group (dose same as monotherapy group). The drugs were administered via intraperitoneal injection every two days for 16 days. The experimental procedure is as follows: Figure 2 As shown in A.

[0033] 2.2 Tumor growth monitoring and endpoint analysis During treatment, tumor volume was dynamically monitored and calculated over time (mm). 3 ), plot the tumor volume growth curve, and perform tumor sampling after the treatment cycle ( Figure 2 (BD), while simultaneously embedding and sectioning the tumor tissue in paraffin.

[0034] 2.3 Histological and Immunohistochemical (IHC) Analysis Tumor sections were stained with H&E, Ki67 (a proliferation marker), and Cleaved Caspase-3 (an apoptosis marker). Representative images are shown below. Figure 2 As shown in E. The staining intensity of Ki67 and Cleaved Caspase-3 was scored using the H-score semi-quantitative method. Figure 2 (FG).

[0035] 2.4 In vivo experimental results In the A2780-OlaR resistance model, olaparib monotherapy showed limited tumor-suppressive effect, while combined with PCI-34051, tumor growth was significantly inhibited, with tumor volume and endpoint weight significantly lower than the monotherapy group and the control group. Figure 2 (BD), showing a synergistic anti-tumor effect.

[0036] IHC analysis showed that the Ki67 positive signal in the tumor tissue of the combination therapy group was significantly reduced ( Figure 2 F), while the positive signal of Cleaved Caspase-3 was significantly enhanced (F). Figure 2 (G) indicates that the combination therapy can effectively inhibit tumor cell proliferation and promote apoptosis, and the effect is more prominent in the drug-resistant model.

[0037] Example 3: HDAC8 inhibitors enhance the antitumor effect of PARP inhibitors in an orthotopic hepatocellular carcinoma model 3.1 Establishment and Treatment of Orthotopic Hepatocellular Carcinoma Model RIL-175 hepatocellular carcinoma cells carrying the luciferase reporter gene were injected into the liver parenchyma of C57BL / 6 mice to establish an orthotopic hepatocellular carcinoma model. Figure 3 (A). Monitored by an in vivo imaging system (IVIS), when the tumor volume reaches 120 ± 15 mm. 3 Mice were randomly divided into four groups (n=5 per group): (1) control group; (2) PCI-34051 monotherapy group; (3) Olaparib monotherapy group; (4) combination therapy group. The dosing regimens were as follows: Figure 3 As shown in B, the treatment lasted for 17 days.

[0038] 3.2 In vivo imaging and endpoint analysis IVIS imaging was performed regularly during treatment to quantitatively analyze the bioluminescence intensity of the tumor region. Figure 3 (C). After treatment, the liver was harvested for gross observation. Figure 3 The tumor tissue was subjected to H&E staining and IHC staining for Ki67 and Cleaved Caspase-3. Figure 3 EF), and perform quantitative scoring ( Figure 3 (G).

[0039] 3.3 Experimental Results Figure 3 The C-line plot in the figure shows the tumor bioluminescence intensity (×10) after inhibitor injection. 4 The change in photons per second over time, combined with IVIS imaging, shows ( Figure 3(B) The tumor bioluminescence intensity in the combination therapy group was significantly lower than that in each single-drug group and the control group at all time points, indicating that the combination therapy had the strongest tumor-suppressing effect.

[0040] Histological results showed that the tumor burden was lowest in the combination therapy group (GMT+8) of the liver. Figure 3 H&E staining and IHC analysis further confirmed that combination therapy most effectively inhibited tumor cell proliferation (lowest Ki67 expression) and induced apoptosis (highest Cleaved Caspase-3 expression), with significantly better effects than any single-drug treatment. Figure 3 EG).

[0041] The above embodiments provide strong preclinical evidence demonstrating that the present invention can re-enhance the sensitivity of drug-resistant tumors to PARP inhibitors through HDAC8 inhibition. Specifically, by introducing an HDAC8 inhibitor, the regulatory processes related to DNA damage repair within tumor cells are intervened, thereby effectively enhancing the therapeutic response of tumor cells to PARP inhibitors. These results highlight the significant translational application potential of dual-targeting PARP1 / HDAC8 inhibition as a potential therapeutic strategy to overcome PARP1 resistance in ovarian cancer and other human malignancies.

[0042] In summary, this invention provides a technical solution for the application of HDAC8 inhibitors in the preparation of drugs for enhancing the efficacy of PARP inhibitors or for reversing / improving PARP inhibitor resistance. This solution intervenes in the regulatory processes related to DNA damage repair within tumor cells, aiming to effectively enhance the therapeutic response of tumor cells to PARP inhibitors.

[0043] In existing technologies, to address the common problems of insufficient therapeutic sensitivity and drug resistance associated with PARP inhibitors, non-specific combination strategies or broad-spectrum epigenetic regulation methods are often employed, which frequently fail to balance improved efficacy and safety. This invention creatively selects HDAC8, a specific epigenetic regulatory subtype, as the intervention target, achieving synergistic enhancement of PARP inhibitor efficacy through its selective inhibition. This approach significantly differs from previous broad-spectrum HDAC inhibitors or non-targeted combination regimens, improving therapeutic specificity and controllability while overcoming the technical limitations of "broad-spectrum regulation—high toxicity—unstable efficacy," demonstrating the non-obviousness of the inventive concept.

[0044] This regimen not only effectively enhances the inhibitory effect of PARP inhibitors on tumor cell proliferation at the in vitro cellular level, but also demonstrates a clear improvement in efficacy in tumor models that have developed resistance to or are initially insensitive to PARP inhibitors. Further animal experiments confirmed that this combination strategy significantly inhibits tumor growth without observing a significant increase in toxic side effects, indicating that it achieves improved efficacy while maintaining good safety. Furthermore, this regimen shows application potential against various PARP inhibitors and multiple tumor types associated with abnormal DNA damage repair, possessing a broad range of applications and promising prospects for clinical translation.

[0045] This invention provides a novel strategy that effectively overcomes PARP inhibitor resistance, improves treatment sensitivity, and has good safety by targeting the combined inhibition of HDAC8 and PARP. It makes up for the shortcomings of existing technologies in terms of targeting, safety and efficacy stability, and provides a new feasible path to solve the long-standing technical defects in this field.

[0046] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0047] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The combination of an HDAC8 inhibitor and a PARP inhibitor is used in the preparation of a drug for treating ovarian cancer and hepatocellular carcinoma; wherein the HDAC8 inhibitor is PCI-34051 or a pharmaceutically acceptable salt thereof; and the PARP inhibitor is olaparib or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition, characterized in that, It contains a therapeutically effective amount of PCI-34051 or a pharmaceutically acceptable salt thereof, olaparib or a pharmaceutically acceptable salt thereof, and / or contains a pharmaceutically acceptable carrier or excipient.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is used to treat ovarian cancer and hepatocellular carcinoma.

4. A medicine box, characterized in that, The medicine box includes: (i) A first pharmaceutical unit containing PCI-34051 or a pharmaceutically acceptable salt thereof; and (ii) A second pharmaceutical unit containing olaparib or a pharmaceutically acceptable salt thereof; The first drug unit and the second drug unit are configured for combined administration for the treatment of ovarian cancer and hepatocellular carcinoma.

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