Use of a cdk4 / 6 inhibitor in combination with oxaliplatin in the preparation of a therapeutic drug for chemotherapy-resistant colorectal cancer

Abstract

The application discloses application of a CDK4 / 6 inhibitor combined with oxaliplatin in preparation of a treatment drug for chemotherapy-resistant colorectal cancer. The combined use of the CDK4 / 6 inhibitor combined with oxaliplatin has excellent therapeutic effect on locally advanced rectal cancer which is resistant to a first-line chemotherapy scheme, can overcome the drug resistance of the rectal cancer patient to the chemotherapy drug, so that the tumor proliferation is inhibited, and the apoptosis of the cancer cell is promoted. The application provides a combined drug treatment scheme for neoadjuvant therapy of locally advanced rectal cancer chemotherapy resistance, can achieve the purpose of effectively treating or assisting in treating the rectal cancer, and provides a new way for treating, diagnosing or predicting the rectal cancer patient.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical applications. More specifically, it relates to the use of CDK4 / 6 inhibitors in combination with oxaliplatin in the preparation of therapeutic drugs for chemotherapy-resistant colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the leading cancers threatening the lives and health of Chinese residents, causing a severe social burden. According to the latest data released by the National Cancer Center, in 2015, there were 387,600 new cases of colorectal cancer in China, accounting for 9.87% of all malignant tumors; and 187,100 deaths were caused by colorectal cancer, accounting for 8.01% of all malignant tumor deaths. How to effectively reduce the disease burden of colorectal cancer in my country is a major public health issue that urgently needs to be addressed. The development of colorectal cancer mostly follows an "adenoma-carcinoma" sequence, generally taking 5-10 years from precancerous lesions to cancer, providing an important time window for early diagnosis and clinical intervention. The 5-year relative survival rate for stage I colorectal cancer is 90%, while the 5-year relative survival rate for stage IV colorectal cancer with distant metastasis is only 14%. Currently, the etiology of colorectal cancer is unclear, but a large amount of research evidence suggests that its occurrence and development are the result of multiple factors, including genetics, environment, and lifestyle. Current research has identified risk factors such as: family history of colorectal cancer, inflammatory bowel disease, red and processed meat intake, diabetes, obesity, and heavy alcohol consumption.

[0003] The diagnosis and treatment of rectal cancer is extremely challenging. Due to its anatomical structure, rectal cancer surgery is difficult, with a high incidence of complications and a high local recurrence rate, which also significantly impacts quality of life. In particular, locally advanced rectal cancer (LARC) has historically had poor treatment outcomes.

[0004] Locally advanced rectal cancer is a relative concept, referring to rectal cancer relative to early-stage rectal cancer (which can achieve a very low local recurrence rate and a good 5-year survival rate simply through total mesorectal resection or even local resection) and late-stage rectal cancer (with metastases to distant organs throughout the body). Locally advanced rectal cancer is defined as rectal cancer located within 12 cm of the anus, where the primary tumor, as detected by imaging or pathological examination, has invaded the muscular layer of the intestinal wall and reached surrounding structures (c / pT3-4b) or has lymph node metastasis within the mesentery and true pelvis (c / pN1-2), but without distant metastasis (M0).

[0005] Neoadjuvant therapy is currently the standard treatment for locally advanced rectal cancer, and it can reduce tumor stage and improve efficacy. With the establishment of neoadjuvant therapy as the standard treatment strategy for locally advanced rectal cancer, a series of clinical studies have been conducted on the application of different chemotherapy drugs during radiotherapy, including fluorouracil (5-FU) monotherapy and combination chemotherapy regimens such as oxaliplatin or irinotecan.

[0006] However, some patients develop resistance to neoadjuvant therapy (chemotherapy) and have extremely poor prognoses. Failure of neoadjuvant therapy in locally advanced rectal cancer is often due to resistance to chemotherapy drugs, leading to uncontrolled tumor growth and ultimately, an inability to control the disease. Currently, there is a lack of effective treatment options for patients resistant to neoadjuvant chemotherapy, and there is an urgent need to develop new combination therapy regimens to improve prognosis.

[0007] Therefore, elucidating the molecular mechanisms of resistance to neoadjuvant chemotherapy in rectal cancer, discovering new targets, developing new treatment regimens, and effectively improving the current treatment status of locally advanced rectal cancer are urgent problems that need to be solved. Summary of the Invention

[0008] To address the aforementioned technical problems, the first objective of this invention is to provide the application of CDK4 / 6 inhibitors in combination with oxaliplatin in the preparation of therapeutic drugs for chemotherapy-resistant colorectal cancer. This approach can overcome the resistance of rectal cancer to oxaliplatin, thereby inhibiting tumor proliferation and promoting tumor cell apoptosis, achieving the goal of effectively treating or adjuvantly treating rectal cancer, and providing a new approach for the treatment, diagnosis, or prediction of rectal cancer patients.

[0009] The second objective of this invention is to provide the application of CDK4 / 6 inhibitors in combination with oxaliplatin in the preparation of neoadjuvant therapy for chemotherapy-resistant colorectal cancer.

[0010] A third objective of this invention is to provide the use of TEAD4 as a biomarker for predicting the sensitivity of chemotherapy-resistant colorectal cancer patients to treatment regimens combining CDK4 / 6 inhibitors with oxaliplatin.

[0011] A fourth objective of this invention is to provide the application of reagents for detecting TEAD4 in the preparation of products.

[0012] The fifth objective of this invention is to provide a kit for predicting the sensitivity of chemotherapy-resistant colorectal cancer patients to a treatment regimen combining a CDK4 / 6 inhibitor and oxaliplatin.

[0013] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0014] The inventors' team has conducted long-term and in-depth research on neoadjuvant therapy for chemotherapy-resistant colorectal cancer and locally advanced rectal cancer. Through screening, testing, and validation of a large number of drugs, the results show that CDK4 / 6 inhibitors combined with oxaliplatin have excellent therapeutic effects on locally advanced rectal cancer resistant to first-line chemotherapy regimens. This combination can overcome the resistance of rectal cancer patients to chemotherapy drugs, thereby inhibiting tumor proliferation and promoting tumor cell apoptosis. Furthermore, by integrating transcriptome sequencing and histone immunoprecipitation sequencing, the inventors discovered that CDK4 / 6 inhibitors can regulate the expression of homologous recombination repair-related genes by inhibiting TEAD4, further enhancing the DNA damage effect of oxaliplatin on rectal cancer cells and synergistically inhibiting tumor growth. This invention provides a combination drug therapy strategy for neoadjuvant therapy of chemotherapy-resistant locally advanced rectal cancer, achieving the goal of effective treatment or adjuvant therapy for rectal cancer.

[0015] Therefore, this invention claims protection for the following:

[0016] Application of CDK4 / 6 inhibitors in combination with oxaliplatin in the preparation of therapeutic drugs for chemotherapy-resistant colorectal cancer.

[0017] Furthermore, the application of CDK4 / 6 inhibitors in combination with oxaliplatin in the preparation of neoadjuvant therapy for chemotherapy-resistant colorectal cancer.

[0018] Furthermore, the application of TEAD4 as a biomarker for predicting the sensitivity of chemotherapy-resistant colorectal cancer patients to CDK4 / 6 inhibitor combined with oxaliplatin therapy.

[0019] Furthermore, the reagent for detecting TEAD4 is used in the preparation of a product that can predict the sensitivity of chemotherapy-resistant colorectal cancer patients to CDK4 / 6 inhibitors combined with oxaliplatin.

[0020] Preferably, the chemotherapy-resistant colorectal cancer is locally advanced rectal cancer that has developed resistance to first-line chemotherapy regimens.

[0021] Preferably, the locally advanced rectal cancer is TEAD4-positive locally advanced rectal cancer.

[0022] Preferably, the CDK4 / 6 inhibitor is one or more of palbociclib, ribociclib, abemaciclib, or dalcilib (SHR6390).

[0023] Preferably, the reagent for detecting TEAD4 is an anti-TEAD4 antibody (Abcam, ab97460).

[0024] Furthermore, a kit for predicting the sensitivity of chemotherapy-resistant colorectal cancer patients to a CDK4 / 6 inhibitor combined with oxaliplatin therapy, including a reagent for detecting TEAD4, should also be within the scope of protection of this application.

[0025] Preferably, the reagent for detecting TEAD4 is an anti-TEAD4 antibody (Abcam, ab97460).

[0026] The present invention has the following beneficial effects:

[0027] This invention presents a combination therapy regimen of CDK4 / 6 inhibitors and oxaliplatin, which demonstrates excellent therapeutic efficacy for locally advanced rectal cancer resistant to first-line chemotherapy. This regimen overcomes the resistance of rectal cancer patients to chemotherapy drugs, thereby inhibiting tumor proliferation and promoting cancer cell apoptosis. This invention provides a combination drug therapy strategy for neoadjuvant therapy of chemotherapy-resistant locally advanced rectal cancer, achieving effective treatment or adjuvant therapy for rectal cancer. It offers a novel approach to inhibiting neoadjuvant therapy in locally advanced rectal cancer and has promising application prospects. Attached Figure Description

[0028] Figure 1 To verify the inhibitory effect of CDK4 / 6 inhibitors combined with oxaliplatin on colorectal cell lines. Among them, Figure 1 Schematic diagram of the screening experiment for drug A in China; Figure 1 In the figure, B represents the ratio of cell viability after combination therapy versus single-drug treatment; Figure 1 The diagram in C represents candidate target drugs for the DLD1 and WiDr cell lines. Figure 1 The results of secondary screening of 11 target drugs (small molecule inhibitors) are shown in Figure D. Figure 1 E in the text represents the combination index of the drugs used in combination. Figure 1 F represents the cell proliferation index results for different treatment groups.

[0029] Figure 2 TEAD4 can be used as a test to predict the sensitivity of rectal cancer to chemotherapy. Among other things, Figure 2 China A and Figure 2 In section B, the experiment involves protein-protein interactions. Figure 2 In the middle C, the effect of si-RNA knockdown of TEAD4 on oxaliplatin resistance was observed. Figure 2 The expression of TEAD4 in tissue samples from chemotherapy-sensitive and chemotherapy-resistant patients is shown in Figure D. Figure 2 E represents the expression of TEAD4 in tissue samples from patients with primary colorectal cancer and recurrent metastatic tumors.

[0030] Figure 3The study describes the inhibitory effect of a CDK4 / 6 inhibitor combined with oxaliplatin on a mouse tumor-bearing model. Among these, Figure 3 China A and Figure 3 In Figure B, the effect of different treatment groups on the subcutaneous tumor volume in mice is shown. Figure 3 C and Figure 3 D represents the effect of different treatment groups on tumor volume in a chemotherapy-sensitive PDX model; Figure 3 China E to Figure 3 In the figure, H represents the effect of different treatment groups on tumor volume in a chemotherapy-resistant PDX model. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Example 1: Combined Drug Screening Trial

[0034] Combination therapy screening experiment: The inventors constructed a small molecule inhibitor library containing 130 growth cycle-related inhibitors and conducted drug screening experiments on oxaliplatin combination therapy in two drug-resistant colorectal cancer cell lines, DLD1 and WiDr. Figure 1 Figure A illustrates a drug screening experiment. DLD1 or WiDr cells were plated and treated with drugs, including 130 small molecule inhibitors related to cell cycle progression and oxaliplatin, 24 hours after plating. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Kit (#G7573, Promega, Madison, WI) 96 hours after treatment. A control group was also set up using only the 130 small molecule inhibitors related to cell cycle progression.

[0035] By calculating the ratio of cell viability after treatment with 130 small molecule inhibitors in combination with oxaliplatin to the cell viability after treatment with a single drug in the control group, the optimal drug combination was determined. Figure 1 Figure B shows the ratio of cell activity after treatment with 130 small molecule inhibitors in combination with oxaliplatin to cell activity after treatment with a single drug. Drug screening results revealed that CDK4 / 6 inhibitors (including palbociclib, ribociclib, and abemaciclib) were the best drugs for reversing oxaliplatin resistance.

[0036] Further integration and comparison of drug screening results in the two drug-resistant cell lines, DLD1 and WiDr, led to the identification of candidate target drugs; such as Figure 1 As shown in Figure C, this is a schematic diagram of candidate target drugs for two drug-resistant cell lines, DLD1 and WiDr. At the same time, the analysis of the combined drug screening experiment of the above-mentioned DLD1 and WiDr drug-resistant cells showed that 11 target drugs had good effects on both cell lines.

[0037] Further Figure 1 Eleven target drugs (small molecule inhibitors) from the Chinese drug C underwent secondary screening and validation (the procedure is as described above), such as... Figure 1 As shown in Figure D, CDK4 / 6 inhibitors can effectively reverse oxaliplatin resistance.

[0038] Furthermore, cell proliferation and colony formation assays were used to further verify the combined efficacy of CDK4 / 6 inhibitors and oxaliplatin in colorectal cancer cells. Cell proliferation assays were performed using the CellTiter-Glo Luminescent CellViability kit (#G7573, Promega, Madison, WI) at specific time points. The values ​​at each time point were compared with those on the first day to calculate the cell proliferation curve. Colony formation assays were performed by platening colorectal cancer cells in 6-well plates. After 24 hours, the cells were treated with CDK4 / 6 inhibitors combined with oxaliplatin (Comb group), DMSO group, oxaliplatin group (OXA group), and palbociclib group (Palb group), respectively. The plates were stained 7-10 days later to observe the colony formation in each treatment group.

[0039] like Figure 1 As shown in Figure E, the combination index of CDK4 / 6 inhibitors (including palbociclib, ribociclib, and abemaciclib) in combination with oxaliplatin for DLD1 and WiDr resistant cell lines was less than 1, indicating that the combination of CDK4 / 6 inhibitors and oxaliplatin has a synergistic effect.

[0040] like Figure 1As shown in Figure F, for both DLD1 and WiDr cell lines, the cell proliferation index gradually increased over time in all four treatment groups. Specifically, the cell proliferation index of the CDK4 / 6 inhibitor combined with oxaliplatin (Comb group) was significantly lower than that of the DMSO group, the oxaliplatin group (OXA group), and the palbociclib group (Palb group). The cell proliferation index of the oxaliplatin group (OXA group) and the palbociclib group (Palb group) showed little difference, with the palbociclib group (Palb group) slightly lower than the oxaliplatin group (OXA group). The DMSO group had the highest cell proliferation index. These results indicate that the combined use of CDK4 / 6 inhibitors and oxaliplatin has a synergistic effect, and is more effective in inhibiting the clonal proliferation of DLD1 and WiDr resistant cells compared to using oxaliplatin or palbociclib alone.

[0041] Example 2: TEAD4 used to predict chemotherapy sensitivity in rectal cancer

[0042] The expression of TEAD4 in patient samples was detected by immunohistochemistry (IHC) using an anti-TEAD4 antibody (Abcam, ab97460). The specific procedure was as follows: paraffin sections were dewaxed by immersion in xylene, followed by gradient hydration with alcohol (100%-95%-80%-60%-H2O), and then microwave antigen retrieval was performed (prepared with 10mM sodium citrate, 0.05% Tween 20, pH 6.0, microwaved on high for 5 minutes, then on medium-low for 20 minutes), and cooled to room temperature. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 10 minutes at room temperature, followed by blocking with sheep serum at 37°C for 30 minutes. The primary antibody (anti-TEAD4, Abcam, ab97460) was diluted 1:300 and incubated overnight at 4°C, followed by incubation with the secondary antibody at 37°C for 30 minutes, and then stained with diaminobenzidine (DAB). Rabbit IgG was used concurrently as a negative control instead of the primary antibody.

[0043] Through the protein interaction experiments described above, such as Figure 2 China A and Figure 2 As shown in Figure B, the results demonstrate that RB1 and TEAD4 bind to each other. Figure 2 As shown in Figure C, knocking down TEAD4 expression with siRNA can also reverse oxaliplatin resistance in patients, as described above. Figure 2 As shown in Figures A through C, RB1 mediates chemotherapy resistance by regulating TEAD4.

[0044] Based on tumor regression before and after chemotherapy, patients with tumor regression exceeding 50% were defined as chemotherapy-sensitive, and those with tumor regression less than 50% were defined as chemotherapy-resistant. The expression of TEAD4 was detected in tissue samples from chemotherapy-sensitive and chemotherapy-resistant patients (the specific procedures are as described above). Figure 2As shown in Figure D, increased TEAD4 expression was found in the tumor tissue of chemotherapy-resistant patients.

[0045] In addition, the expression of TEAD4 was detected in tissue samples from patients with primary colorectal cancer and recurrent metastatic tumors (the specific procedures are as described above). Figure 2 As shown in Figure E, TEAD4 is highly expressed in recurrent and metastatic tumors. These results demonstrate that TEAD4 can serve as a biomarker for the prognostic efficacy of chemotherapy in rectal cancer, and can predict the sensitivity of rectal cancer to chemotherapy.

[0046] Example 3: Effects of CDK4 / 6 inhibitors and oxaliplatin on mouse tumor-bearing models

[0047] In the 2D clonogenic assay, drug-resistant colorectal cancer cells (DLD1, WiDr) were digested and dispersed to form a single-cell suspension. Cells were counted using a cell counting chamber, and a certain number of cells (10,000-20,000 cells / well) were seeded into 6-well plates and shaken well. The medium containing the drug was replaced the next day, and thereafter the medium was replaced every 3 days for 8 to 10 days. After observing satisfactory colony growth (approximately 90% of the control cell density) under a microscope, the 6-well plates were removed, washed once with PBS, and the cells were fixed with methanol solution. Finally, the plates were stained with crystal violet (0.5% crystal violet, 20% methanol), rinsed with water, dried, and photographed.

[0048] In the cell proliferation assay, cells were digested and dispersed to form a single-cell suspension. Cells were counted using a cell counting chamber, and a certain number of cells (1000-2000 cells / well) were seeded into 96-well plates, with 3 accessory wells allocated to each treatment group. The medium containing the drug was replaced the next day, and the cells were incubated at 37°C for 4 days. The number of viable cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Working solution was prepared according to the CellTiter-Glo Luminescent Assay kit, and 50 μL of working solution was added to each well (96-well plate). The plates were then placed in the dark on a shaker for 3 minutes, incubated for 10 minutes, and the values ​​were read using a microplate reader.

[0049] In animal experiments to assess drug efficacy, female BALB / c immunodeficient mice (nude mice, 5-8 weeks old) were housed in an animal testing center for 5-7 days. Sufficient cells were cultured simultaneously. On the day of cell seeding, the cells were digested and washed three times with PBS solution, at a ratio of 5 x 10⁻⁶ cells / day. 7 DLD1 cells were resuspended at / mL, and each mouse was subcutaneously injected with 0.1mL of PBS containing DLD1 cells into the dorsal subcutaneous tissue. When the subcutaneous tumor reached 70mm... 3According to the randomization principle, mice with subcutaneous tumors were stratified and grouped according to tumor size, and divided into control and drug treatment groups: the control group was treated with DMSO solvent (intraperitoneal injection) (DMSO group); the drug treatment groups were: palbociclin group (Palb group) (33 mg / kg, once every three days, intraperitoneal injection), oxaliplatin group (OXA group) (3 mg / kg, once every three days, intraperitoneal injection), and CDK4 / 6 inhibitor combined with oxaliplatin group (Comb group) (drug dosage and administration time were the same as the single drug groups). Mouse weight and tumor size were measured every two days to monitor the mice's response to the drugs and tumor progression. The size of the subcutaneous tumor was measured using calipers, and the calculation formula was: volume = (tumor width² x tumor length) / 2 (V = W² x L / 2). No experimental mice were excluded in this experiment; one mouse in the combined treatment group died unexpectedly. Mice were monitored for approximately 3-5 weeks after drug treatment.

[0050] like Figure 3 China A and Figure 3 As shown in Figure B, for mice with subcutaneous tumors, the volume of subcutaneous tumors gradually increased over time in all four treatment groups. At day 21, the subcutaneous tumor volume in the CDK4 / 6 inhibitor combined with oxaliplatin group (Comb group) was significantly smaller than that in the DMSO group, oxaliplatin group (OXA group), and palbocicillin group (Palb group); while the difference in subcutaneous tumor volume between the oxaliplatin group (OXA group) and the palbocicillin group (Palb group) was not significant; the DMSO group had the largest subcutaneous tumor volume. This indicates that the combined use of CDK4 / 6 inhibitors and oxaliplatin achieved better anti-tumor effects, and that the combined use of CDK4 / 6 inhibitors and oxaliplatin can effectively reverse oxaliplatin resistance.

[0051] Furthermore, three PDX models derived from colorectal cancer patient tumor tissue were constructed (a chemotherapy-sensitive PDX model (CRC6), and chemotherapy-resistant PDX models (CRC7 and CRC8)). For example... Figure 3 C and Figure 3 As shown in Figure D, there was no significant difference in tumor volume between the CDK4 / 6 inhibitor combined with oxaliplatin group (Comb group) and the oxaliplatin group (OXA group). This indicates that no combined effect of CDK4 / 6 inhibitor and oxaliplatin was observed in the chemotherapy-sensitive PDX model.

[0052] In chemotherapy-resistant PDX models, such as Figure 3 As shown in Figures E to H, the subcutaneous tumor volume in the CDK4 / 6 inhibitor combined with oxaliplatin (Comb group) was significantly smaller than that in the DMSO group, oxaliplatin group (OXA group), and palbociclin group (Palb group). This indicates that the combination of CDK4 / 6 inhibitors and oxaliplatin is significantly effective, suggesting that CDK4 / 6 inhibitors can be clinically applied to chemotherapy-resistant colorectal cancer patients.

[0053] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The application of CDK4 / 6 inhibitors combined with oxaliplatin in the preparation of therapeutic drugs for chemotherapy-resistant colorectal cancer; characterized in that, The CDK4 / 6 inhibitor is one or more of palbociclin, ribociclib, and abecilib.

2. The application of CDK4 / 6 inhibitors combined with oxaliplatin in the preparation of adjuvant therapy drugs for chemotherapy-resistant colorectal cancer, characterized in that, The CDK4 / 6 inhibitor is one or more of palbociclin, ribociclib, and abecilib.

3. The application according to claim 1 or 2, characterized in that, Chemotherapy-resistant colorectal cancer refers to locally advanced rectal cancer that has developed resistance to first-line chemotherapy regimens.

4. The application according to claim 3, characterized in that, The locally advanced rectal cancer mentioned is TEAD4-positive locally advanced rectal cancer.

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