A pharmaceutical combination of navitoclax and a ca9 inhibitor and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a combined application of Navitoclax and a CA9 inhibitor in preparation of a medicine for treating renal clear cell carcinoma. It is found that Navitoclax and a CA9 inhibitor SLC-0111 are combined to use, can significantly inhibit the proliferation and activity of renal clear cell carcinoma cells through a synergistic effect. In vivo, the growth of a tumor is effectively inhibited, and the survival period of a tumor-bearing individual is significantly prolonged. A new effective drug combination scheme is provided for treating renal clear cell carcinoma, and especially for overcoming Navitoclax single-drug insensitivity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug combination of Navitoclax and CA9 inhibitor SLC-0111 and its application. Background Technology

[0002] Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of renal cell carcinoma in adults. Its development and progression are closely related to characteristic mechanisms such as activation of hypoxic signals, metabolic reprogramming, and apoptosis tolerance. Although targeted therapy and immunotherapy have made progress, there is still a lack of highly effective treatment strategies for some patients, especially those who are insensitive to existing therapies or have developed resistance.

[0003] Among numerous potential targets, apoptosis pathways and tumor metabolism are two key areas. Navitoclax is an orally effective small molecule inhibitor targeting both BCL-2 and BCL-XL. Its mechanism of action involves mimicking endogenous pro-apoptotic proteins, binding to the anti-apoptotic proteins BCL-2 and BCL-XL, thereby releasing the bound pro-apoptotic proteins (such as BIM and BAX), ultimately inducing increased mitochondrial outer membrane permeability and cytochrome C release, initiating the intrinsic apoptotic program of tumor cells. However, in preclinical and clinical studies of solid tumors (such as clear cell renal cell carcinoma), Navitoclax monotherapy often results in resistance due to adaptive survival mechanisms such as metabolic reprogramming and upregulation of other anti-apoptotic proteins (such as MCL-1), limiting its efficacy. Summary of the Invention

[0004] This invention provides a drug combination of Navitoclax and CA9 inhibitor SLC-0111 and its application, in order to address the problem of limited efficacy of Navitoclax monotherapy in the treatment of clear cell renal cell carcinoma.

[0005] The inventors prepared Navitoclax and the CA9 (Carbonic Anhydrase 9) inhibitor SLC-0111 into separate dosage forms and conducted experiments by combining them. Both in vitro and in vivo experimental results showed that the combined use could significantly enhance the anti-tumor effect.

[0006] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides a pharmaceutical combination for treating clear cell renal cell carcinoma, comprising a therapeutically effective amount of Navitoclax or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of SLC-0111 or a pharmaceutically acceptable salt thereof.

[0007] In conjunction with the first aspect of the invention, in some embodiments, the Navitoclax and the CA9 inhibitor are separate formulations, with a weight ratio of 1.30:1 to 1.35:1.

[0008] Secondly, the present invention provides the use of the above-mentioned drug combination in the preparation of a medicament for treating clear cell renal cell carcinoma in patients.

[0009] In conjunction with the second aspect of the invention, in some embodiments, the patient is a patient who is not responsive to Navitoclax monotherapy.

[0010] In conjunction with a second aspect of the invention, in some embodiments, the drug is used in at least one of the following applications: (1) Inhibits the proliferation of tumor cells; (2) Inhibit tumor growth in the subjects; (3) Prolong the survival of subjects suffering from tumors.

[0011] In conjunction with a second aspect of the invention, in some embodiments, the drug is formulated into a pharmaceutically acceptable dosage form. Further, the dosage form is an injection or an oral formulation.

[0012] Compared with the prior art, the present invention has at least the following beneficial effects: This study is the first to propose and validate a novel strategy for the treatment of clear cell renal cell carcinoma using a combination of Navitoclax and the CA9 inhibitor SLC-0111. In vitro cell experiments and in vivo animal model studies confirmed that this combination therapy produces a significant synergistic anti-tumor effect. Specifically, at the cellular level, it effectively inhibits tumor cell proliferation and exhibits a strong synergistic killing effect on cells insensitive to Navitoclax monotherapy; in vivo, it significantly inhibits tumor growth; and, more importantly, it substantially prolongs the survival of tumor-bearing individuals. This combination therapy provides a novel drug combination with proven efficacy and clinical application potential, addressing the limitations of Navitoclax monotherapy. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The dose-response curve of cell viability as a function of drug concentration after treating human clear cell renal cancer OS-RC-2 cells with Navitoclax as a single drug.

[0015] Figure 2 The dose-response curve of cell viability as a function of drug concentration after treating human clear cell renal carcinoma OS-RC-2 cells with SLC-0111 as a single drug.

[0016] Figure 3 This study aims to obtain the dose-response curves of combined drug therapy after treating OS-RC-2 cells with different concentrations of SLC-0111 for 48 hours under a fixed final concentration of 500 nM Navitoclax. The DMSO group represents the control group, which did not add Navitoclax but only added SLC-0111 at the same concentration gradient and an equal volume of DMSO. The Navitoclax group represents the experimental group, which added 500 nM Navitoclax in combination with SLC-0111 at the same concentration gradient.

[0017] Figure 4 To investigate the effect of combined drug administration regimens on the growth of xenografts in mice.

[0018] Figure 5 Statistical analysis of tumor volume in each group at the experimental endpoint.

[0019] Figure 6 To investigate the effect of combined drug administration regimens on the survival of mice. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0021] As described in the background section, existing treatment regimens for clear cell renal cell carcinoma suffer from limitations such as limited efficacy of single-agent targeted drugs, weak effects of metabolic targeted drugs, and a lack of effective combination strategies. Through in vitro cell experiments and in vivo animal model (PDX) studies, the inventors discovered and validated the synergistic anti-tumor mechanism of Navitoclax combined with a CA9 inhibitor (such as SLC-0111). The inventors believe that this combination regimen, by disrupting tumor metabolic homeostasis through CA9 inhibitors, significantly enhances the sensitivity of tumor cells to Navitoclax-induced apoptosis, and can serve as an effective new synergistic treatment strategy. Corresponding drug combinations have been developed to address these clinical treatment challenges, providing a novel and effective solution.

[0022] In summary, in a first aspect, the present invention provides a pharmaceutical combination for treating clear cell renal cell carcinoma, comprising a therapeutically effective amount of Navitoclax or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of SLC-0111 or a pharmaceutically acceptable salt thereof.

[0023] In Example 3, in an in vivo animal model, mice were simultaneously injected intraperitoneally with Navitoclax (50 mg / kg) and SLC-0111 (38 mg / kg) in DMSO solution, once every 2 days for 2 weeks. The survival status of the mice was then observed. Figure 6 The results showed that the median survival of mice in the combination therapy group was significantly longer than that of either single-drug group or the control group (P<0.001), exhibiting the highest long-term survival rate. Example 3 confirmed that the combination of effective amounts of Navitoclax and SLC-0111 can treat renal cell carcinoma.

[0024] In conjunction with the first aspect of the invention, in some embodiments, the Navitoclax and the CA9 inhibitor are separate formulations, with a weight ratio of 1.30:1 to 1.35:1.

[0025] It should be noted that the optimal synergistic concentration ratio determined in the in vitro cell experiments of this invention differs from the dosage weight ratio used in the in vivo animal experiments. The in vitro experimental results of Example 1 ( Figure 3 The results showed that when the combined administration achieved a 50% inhibitory effect, Navitoclax and SLC-0111 exhibited a significant synergistic effect at a molar ratio of approximately 1:21.5 (weight ratio of approximately 1:8.8) (CI<1). However, to further determine the therapeutic effect and optimal dose ratio of the combined Navitoclax and SLC-0111, the inventors established a xenograft mouse model derived from patients with clear cell renal cell carcinoma. The results of Examples 2 and 3 showed that the combined administration of Navitoclax and the CA9 inhibitor SLC-0111 produced a significant combined antitumor effect in vivo, significantly inhibiting tumor growth and prolonging the survival of the model mice. Figures 4-6 ).

[0026] Those skilled in the art will understand that this difference in the optimal ratio between in vitro and in vivo is primarily due to the different pharmacokinetic (PK) processes of the drug in vivo compared to the direct cellular exposure environment in vitro. The in vivo dosing ratio needs to be adjusted based on the bioavailability, metabolic clearance, and tissue distribution characteristics of the two drugs. In the in vivo experiments of this invention, the effective dose of Navitoclax in the combined dosing group was 50 mg / kg, and the effective dose of SLC-0111 was 38 mg / kg, with the two being independent formulations at a weight ratio of 1.31:1. This in vivo dosing ratio has been experimentally verified to achieve the expected synergistic therapeutic effect while ensuring safety.

[0027] Secondly, the present invention provides the use of the above-mentioned drug combination in the preparation of a medicament for treating renal cell carcinoma.

[0028] In conjunction with a second aspect of the invention, in some embodiments, the patient is a patient who is insensitive to Navitoclax monotherapy.

[0029] Figure 3 The results showed that when the SLC-0111 concentration was 0 μM, the cell viability in the Navitoclax monotherapy group was close to 100%, indicating that the OS-RC-2 cell line had intrinsic resistance to Navitoclax monotherapy. With increasing SLC-0111 concentration, cell viability decreased significantly in a concentration-dependent manner, confirming that SLC-0111 could effectively reverse the resistance of OS-RC-2 cells to Navitoclax. Therefore, the patients treated with the combination of Navitoclax and SLC-0111 in this invention were those who were insensitive to Navitoclax monotherapy.

[0030] In conjunction with a second aspect of the invention, in some embodiments, the renal cell carcinoma is clear cell renal carcinoma.

[0031] The cell material used in the in vitro experiments in Example 1 was the human clear cell renal cell carcinoma line OS-RC-2, while the in vivo experiments in Examples 2 and 3 used a patient-derived xenograft mouse model of clear cell renal cell carcinoma. Both in vitro cell experiments and in vivo animal model studies demonstrated that the combined use of Navitoclax and the CA9 inhibitor SLC-0111 significantly enhanced the anti-tumor effect. Therefore, the drug combination provided by this invention can treat renal cancer, particularly clear cell renal cell carcinoma.

[0032] In conjunction with a second aspect of the invention, in some embodiments, the drug is used in at least one of the following applications: (1) Inhibits the proliferation of tumor cells: The results of the in vitro cell experiments in Example 1 showed that ( Figure 3The combined use of Navitoclax and the CA9 inhibitor SLC-0111 significantly enhanced the inhibitory effect on the viability of OS-RC-2 clear cell renal cell carcinomas. Specifically, starting from a SLC-0111 concentration of 1 µM, the cell viability in the combined treatment group was significantly lower than that in the corresponding SLC-0111 monotherapy group, and this synergistic inhibitory effect increased with increasing SLC-0111 concentration, indicating that the drug combination can effectively inhibit the proliferation of tumor cells.

[0033] (2) Inhibits tumor growth in the subjects: The results of the in vivo efficacy verification experiment in mice in Example 2 showed that ( Figure 4 In a clear cell renal cell carcinoma PDX model, compared with the control group or the Navitoclax / SLC-0111 monotherapy group, the combination of Navitoclax and SLC-0111 produced the strongest tumor growth inhibition. At the experimental endpoint ( Figure 5 The tumor volume in the combined drug group was significantly lower than that in the single drug group and the control group, with a clear statistical difference, indicating that the drug combination can effectively inhibit tumor growth in the subjects.

[0034] (3) Prolonging the survival of subjects suffering from tumors: The survival benefit verification results of Example 3 show that ( Figure 6 In a clear cell renal cell carcinoma PDX model, Kaplan-Meier survival analysis showed significant differences in overall survival among the groups (log-rank test, P < 0.0001), with the group receiving the combined Navitoclax and SLC-0111 showing a significantly longer median survival than the other groups. This indicates that the combined strategy not only inhibits tumor growth but also substantially prolongs survival with the tumor, demonstrating that the drug combination can effectively extend the survival of subjects with tumors.

[0035] In conjunction with the second aspect of the invention, in some embodiments, the drug is formulated into a pharmaceutically acceptable dosage form. Further, the dosage form is an injection or an oral formulation. In Example 1, the cell administration method was suspension drip infusion, with RPMI-1640 medium (containing 1‰ DMSO) as the solvent. When administered alone, Navitoclax and SLC-0111 achieved 50% inhibition rates of 2.76 μM and 57.91 μM, respectively. In Examples 2 and 3, the mouse administration method was intraperitoneal injection. Navitoclax and SLC-0111 were formulated into injections, with the dose of Navitoclax injection being 50 mg / kg and the dose of SLC-0111 injection being 38 mg / kg, with DMSO solution as the solvent.

[0036] It should be noted that SLC-0111 alone also has a certain inhibitory effect on ccRCC cells: in in vitro cell experiments, under a DMSO background, SLC-0111 showed a concentration-dependent inhibition of OS-RC-2 cell viability. Figure 3 (blue curve); In in vivo animal model experiments, compared with the control group, the tumor volume of the SLC-0111 monotherapy group was significantly reduced ( Figure 4 and Figure 5 These results indicate that SLC-0111 possesses certain in vitro and in vivo antitumor activity in ccRCC.

[0037] In the combined dosing regimens of Examples 2 and 3, SLC-0111 was administered via intraperitoneal injection at a dose of 38 mg / kg in DMSO.

[0038] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture, cell biology techniques (such as cell proliferation and apoptosis detection), animal modeling, data collection, analysis, and statistical methods. All experimental reagents used were commercially available and meet general quality standards in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental and control groups were set up according to conventional grouping methods in the art.

[0039] The renal cell carcinoma line OS-RC-2 used in the following examples was purchased from Wuhan Pronosai Biotechnology Co., Ltd. Its cell identity was confirmed by STR typing, and the mycoplasma test result was negative. The NCG mice (NOD / ShiLtJGpt-) used in the following examples... Prkdc em26Cd52 Il2rg em26Cd22 The animals (600g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and were housed in an SPF-grade environment. All animal experiments have been approved by the Ethics Committee of the Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Approval No.:

[2024] 5198).

[0040] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data were expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference.

[0041] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0042] Example 1: Synergistic inhibitory effect of Navitoclax combined with CA9 inhibitor on the viability of clear cell renal cell carcinoma. 1. Purpose This embodiment aims to evaluate the effects of Navicotclax and the CA9 inhibitor SLC-0111, alone and in combination, on the viability of human renal clear cell carcinoma OS-RC-2 cells, and to calculate the combination index (CI) based on data from fixed-concentration Navicotclax combined with SLC-0111 gradient treatment to verify whether the two have a synergistic inhibitory effect.

[0043] 2. Experimental Materials Cell line: Human kidney clear cell carcinoma line OS-RC-2 (Wuhan Pronosai Biotechnology Co., Ltd., CL-0177). Investigational drugs: Navitoclax (MedChemExpress, HY-10087), CA9 inhibitor SLC-0111 (MedChemExpress, HY-13513); Main reagent: Cell Counting Kit-8 reagent kit (MedChemExpress, HY-K0301).

[0044] 3. Experimental Methods 3.1 Cell Culture and Seeding: OS-RC-2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Logarithmic growth phase cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 5 × 10⁶ cells / well in 96-well plates and pre-cultured in an incubator for 24 hours. Before the experiment, the cells were digested, counted, and thoroughly mixed, then seeded at a density of 5 × 10⁶ cells / well. 3 Cells were seeded at a density of [number] cells per well in 96-well cell culture plates. After seeding, the cells were pre-cultured in a cell culture incubator for 24 hours to allow them to adhere and reach a stable growth state before drug treatment. The cell seeding density, culture medium volume, and solvent volume were kept consistent across all wells to minimize the impact of non-drug factors on the test results.

[0045] 3.2 Drug treatment: 3.2.1 Single-drug dose-response experiment To evaluate the effects of Navitoclax and SLC-0111 on OS-RC-2 cell viability, Navitoclax and SLC-0111 were used as single-drug treatment groups. The Navitoclax treatment group had concentration gradients of 0, 0.1, 0.5, 1, 2, 4, and 10 μM; the SLC-0111 treatment group had concentration gradients of 0, 0.5, 1, 2, 5, 10, 25, 50, 100, and 200 μM. All drug concentrations were treated under the same culture conditions, and an equal volume of DMSO solvent was included as a control group. Each concentration was used in five replicates, and the treatment time was 48 hours.

[0046] After treatment, CCK-8 reagent was added to each well according to the CCK-8 kit instructions. After further incubation, the absorbance was measured at 450 nm using a microplate reader. Using the solvent control group as a 100% cell viability reference, the relative cell viability at each drug concentration was calculated, and dose-response curves for Navitoclax and SLC-0111 were plotted accordingly. Figure 1 and Figure 2 .

[0047] 3.2.2 Combined drug dose-response experiment To evaluate whether the combined use of Navitoclax and SLC-0111 could enhance the inhibitory effect on OS-RC-2 cell viability, the final concentration of Navitoclax was fixed at 500 nM in the combination drug experiment, and SLC-0111 concentration gradients were set at 0, 0.5, 1, 2, 5, 10, 25, 50, and 100 μM. Two parallel treatment systems were also set up in the experiment. DMSO + SLC-0111 group (DMSO control group): Equal amounts of OS-RC-2 cells were seeded, and gradient concentrations of SLC-0111 (0, 0.5, 1, 2, 5, 10, 25, 50, 100 μM) were added to each well. At the same time, DMSO solution with the same volume as Navitoclax added in the experimental group was added to ensure that the final culture volume of each well was consistent. Navitoclax + SLC-0111 group (experimental group): Equal amounts of OS-RC-2 cells were seeded and treated with gradient concentrations of SLC-0111 (0, 0.5, 1, 2, 5, 10, 25, 50, 100 μM) and Navitoclax at a final concentration of 500 nM.

[0048] For each concentration in each group, five replicates were set up. After 48 hours of drug treatment, CCK-8 reagent was added to each well, and incubation continued for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0049] 3.3 Data Processing and Statistical Analysis 3.3.1 Plotting the dose-response curve Cell viability (%) = [(OD value of experimental wells - OD value of blank wells) / (OD value of solvent control wells - OD value of blank wells)] × 100%. Wherein, blank wells are wells containing an equal volume of culture medium and DMSO, but without cells or drugs; solvent control wells are wells containing cells and an equal volume of DMSO, but without the test drug. In the combined drug assay, the DMSO control wells with a SLC-0111 concentration of 0 μM and without Navitoclax were used as the normalized reference for cell viability.

[0050] The experiment was independently repeated three times. Data are expressed as mean ± standard deviation. Dose-response curves were plotted using GraphPad Prism software, and the half-maximal inhibitory concentrations (IC50) of Navitoclax and SLC-0111 monotherapy were calculated. 50 The combined drug effect was calculated using the Chou-Talalay method, which yielded the combined index (CI).

[0051] 3.3.2 Calculation of the Joint Index (CI) The CI value was calculated using the Chou-Talalay method, with the formula: CI = D1 / Dx1 + D2 / Dx2. Where D1 and D2 are the actual concentrations of Navitoclax and SLC-0111 used when the combined drug achieved a 50% inhibition rate, respectively; and Dx1 and Dx2 are the individual concentrations (i.e., IC50) of Navitoclax and SLC-0111 when each drug achieved a 50% inhibition rate. 50 CI < 0.9 indicates synergistic effect, 0.9 to 1.1 indicates additive effect, and > 1.1 indicates antagonistic effect.

[0052] Statistical analysis was performed using two-way ANOVA, combined with Sidak's multiple comparison test.

[0053] 4. Results 4.1 Dose-response relationship of monotherapy like Figure 1 and Figure 2 As shown, both Navitoclax and SLC-0111 monotherapy treatment of OS-RC-2 cells for 48 hours exhibited concentration-dependent cell viability inhibition. The IC50 of Navitoclax monotherapy was calculated. 50 The IC50 of SLC-0111 monotherapy is 2.76 μM (i.e., Dx1). 50The concentration was 57.97 μM (i.e., Dx2). Notably, low concentrations of Navitoclax (e.g., 100 nM) did not significantly inhibit cell viability, indicating that the cell line was not sensitive to low-dose Navitoclax monotherapy.

[0054] 4.2 Quantitative verification of synergistic effects (combination index, CI) like Figure 3 As shown, in the DMSO background, SLC-0111 exhibited concentration-dependent inhibition of OS-RC-2 cell viability (blue curve). At 0 µM and 0.5 µM, cell viability was close to that of the control group (approximately 100%), but gradually decreased with increasing concentrations to 2 µM and above. When Navitoclax (500 nM) was used in combination with different concentrations of SLC-0111 (red curve), starting from a SLC-0111 concentration of 1 µM, the cell viability in the combination therapy group was significantly lower than that in the corresponding SLC-0111 monotherapy group. As the SLC-0111 concentration further increased, the difference between the two curves (i.e., the synergistic effect) became more significant.

[0055] When the combined drug therapy produces a 50% inhibitory effect, the required Navitoclax concentration is 0.5 μM (D1), and the required SLC-0111 concentration is 10.73 μM (D2). According to the CI calculation formula: CI = D1 / Dx1 + D2 / Dx2 = (0.5 / 2.76) + (10.73 / 57.97) = 0.181 + 0.185 = 0.366. The CI value is much less than 0.9, indicating that Navitoclax and SLC-0111 have a strong synergistic effect under these conditions.

[0056] 5. Conclusion This embodiment demonstrates that both Navitoclax and SLC-0111 monotherapy have inhibitory effects on OS-RC-2 cells, but the cell line is not sensitive to low doses of Navitoclax (100 nM). When the two are used in combination, the CI value is calculated to be 0.366 (<0.5) under the condition of achieving 50% inhibition, which quantitatively confirms that there is a strong synergistic effect between Navitoclax and SLC-0111.

[0057] Example 2: Validation experiment on the antitumor effect of combined drug administration in mice 1. Purpose This embodiment aims to evaluate the antitumor effect of Navitoclax combined with the CA9 inhibitor SLC-0111 in vivo using a patient-derived xenograft (PDX) mouse model of clear cell renal cell carcinoma, providing key evidence for the preclinical efficacy of the drug combination.

[0058] 2. Experimental Materials Experimental animals: NCG mice, four weeks old, male, weighing 18±2g, a total of 32 mice; Investigational drugs: Navitoclax (MedChemExpress, HY-10087) and CA9 inhibitor SLC-0111 (MedChemExpress, HY-13513).

[0059] 3. Experimental Methods The establishment of the human tumor xenograft (PDX) model was carried out after approval by the ethics committee and informed consent from the patients. Freshly removed tumor tissue was aseptically obtained and rapidly transported in an ice bath containing dual antibiotics. Necrotic and adipose tissue was removed in a laminar flow hood, and a 2×2×2 mm tissue block was subcutaneously inoculated into the axillary region of 4-week-old SPF-grade NCG-immunodeficient mice. Once the primary xenograft reached a suitable size, the tumor tissue was aseptically dissected and passaged continuously to the F2 or F3 generation using the same method to obtain a stable PDX model. When the tumor volume reached 100 mm², the model was considered successful. 3 Drug treatment was initiated at that time, and the tumor-bearing mice were randomly divided into four groups, as detailed below: Control group (i.e. DMSO group): NCG mice were injected intraperitoneally with an equal volume of DMSO solution; Navitoclax monotherapy group (i.e. Navi_1 group): NCG mice were intraperitoneally injected with Navitoclax at a dose of 50 mg / kg; CA9 inhibitor monotherapy group (i.e. SLC-0111_1 group): NCG mice were intraperitoneally injected with SLC-0111 at a dose of 38 mg / kg; Combo group (i.e., Combo_1 group): NCG mice were injected intraperitoneally with Navitoclax and SLC-0111 at doses of 50 mg / kg and 38 mg / kg, respectively.

[0060] Each group was given the medication once every 2 days for 2 consecutive weeks.

[0061] Measure the tumor's long diameter (L) and short diameter (W) every two days using calipers, according to the formula V = L × W. 2 / 2 Calculate the tumor volume, where V: tumor volume, in mm. 3 L: Longest diameter of the tumor (mm); W: Widest diameter of the tumor (perpendicular to the long diameter) (mm). Mice were euthanized 36 days after tumor inoculation, following animal ethics guidelines.

[0062] 4. Results In vivo efficacy studies showed that the combined administration group exhibited the strongest tumor growth inhibition ability. For example... Figure 4 As shown, compared with the control group, Navitoclax monotherapy, CA9 inhibitor monotherapy, and combination therapy all significantly delayed tumor growth, with the combination therapy group (Combo_1 group) showing the most significant inhibitory effect. At the experimental endpoint, the tumor volume in the Combo_1 group was significantly lower than that in the monotherapy group and the control group, with a clear statistical difference, further confirming the synergistic tumor-suppressive effect of combination therapy. Figure 5 ).

[0063] 5. Conclusion This embodiment demonstrates, using a PDX animal model, that the combined administration of Navitoclax and the CA9 inhibitor SLC-0111 produces a synergistic anti-tumor effect in vivo, significantly inhibiting tumor growth. These results provide solid in vivo experimental evidence for the effectiveness of this drug combination in treating clear cell renal cell carcinoma.

[0064] Example 3: Validation Experiment of Survival Benefit of Combination Therapy 1. Purpose This embodiment aims to evaluate and verify the significant benefit of combining Navitoclax with the CA9 inhibitor SLC-0111 in prolonging overall survival in tumor-bearing mice using a patient-derived xenograft (PDX) mouse model of clear cell renal cell carcinoma.

[0065] 2. Experimental materials and animal model establishment The cell lines, animals, sources of the test drugs, and methods for establishing the PDX model used in the experiment were the same as in Example 2.

[0066] 3. Experimental Methods 3.1 Grouping and Dosing Control group (i.e., Group=DMSO group): NCG mice were injected intraperitoneally with an equal volume of DMSO solution; Navitoclax monotherapy group (i.e. Navi group): NCG mice were intraperitoneally injected with Navitoclax at a dose of 50 mg / kg; CA9 inhibitor monotherapy group (i.e. SLC-0111 group): NCG mice were injected intraperitoneally at a dose of 38 mg / kg; Combined administration group (i.e. dual drug combination group): NCG mice were injected intraperitoneally with Navitoclax and SLC-0111 at doses of 50 mg / kg and 38 mg / kg, respectively.

[0067] Each group was given the medication once every 2 days for 2 consecutive weeks.

[0068] 3.2 Observation endpoints and indicators In this embodiment, overall survival was the sole primary endpoint. Survival time in mice was recorded from the first administration, with natural death or death due to excessive tumor burden (tumor volume > 1000 mm²) as the endpoint. 3 The endpoint is the point at which euthanasia must be performed due to ethical endpoints such as severe ulceration or cachexia, so as to reflect the true impact of the treatment plan on survival to the greatest extent.

[0069] 4. Results Survival analysis results as follows Figure 6 As shown in the figure, both Navitoclax and SLC-0111 monotherapy significantly prolonged the median survival of mice compared to the control group. The median survival of mice in the combination therapy group was significantly longer than that of either monotherapy group or the control group (P<0.001), exhibiting the highest long-term survival rate. These results indicate that the combination of Navitoclax and SLC-0111 has a synergistic effect in prolonging overall survival, demonstrating a significant survival benefit.

[0070] 5. Conclusion This embodiment demonstrates that in a clear cell renal cell carcinoma PDX model, the combined administration of Navitoclax and the CA9 inhibitor SLC-0111 significantly prolongs the overall survival of tumor-bearing mice, showing superior efficacy compared to monotherapy. This provides direct and compelling evidence for the clinical application of this drug combination in improving patient survival outcomes.

[0071] The above results indicate that the combined use of Navitoclax and CA9 inhibitors produces a synergistic anti-tumor effect. This regimen is highly effective and has a clear mechanism, providing a solid theoretical and practical foundation for the development of new drug compositions targeting clear cell renal cell carcinoma.

[0072] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0073] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0074] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A pharmaceutical combination for treating clear cell renal cell carcinoma, characterized in that, It contains a therapeutically effective amount of Navitoclax or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of SLC-0111 or a pharmaceutically acceptable salt thereof.

2. The drug combination according to claim 1, characterized in that, The Navitoclax and SLC-0111 are independent formulations, with a weight ratio of 1.30:1 to 1.35:

1.

3. Use of the pharmaceutical combination of claim 1 or 2 in the preparation of a medicament for treating clear cell renal cell carcinoma in a patient.

4. The use according to claim 3, characterized in that, The patient in question was unresponsive to Navitoclax monotherapy.

5. The use according to claim 3, characterized in that, The drug is used in at least one of the following applications: (1) Inhibits the proliferation of tumor cells; (2) Inhibit tumor growth in the subjects; (3) Prolong the survival of subjects suffering from tumors.

6. The use according to claim 3, characterized in that, The drug is formulated into a pharmaceutically acceptable dosage form, which is either an injection or an oral preparation.

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