Combination drug for treating kras-mutated ovarian cancer and use thereof

Abstract

The present application relates to a kind of combination drug for treating kras mutant ovarian cancer and its application.The present application proves by experiment that RB7LP can inhibit the proliferation of ovarian cancer cells, further, can promote the senescence and apoptosis of part kras mutant ovarian cancer cells, simultaneously, it also proves that KRAS inhibitor or EGFR inhibitor can synergistically Palbociclib combined lethal KRAS mutant ovarian cancer, compared with the treatment effect of Palbociclib or sotoracib alone, it is superior, the combination drug of the present application can synergistically induce kras mutant ovarian cancer cell apoptosis, increase cancer cell inhibition rate, curative effect is good, with very strong clinical application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a combination drug for treating kras-mutant ovarian cancer and its application. Background Technology

[0002] Ovarian cancer is a malignant tumor that occurs in the ovary. The most common type is the epithelial tumor, which develops from epithelial cells and accounts for 90% of all ovarian tumors. Medically, malignant tumors originating from epithelial tissue are collectively referred to as cancer. When epithelial cells undergo oncogenic gene mutations, the cells proliferate uncontrollably, leading to tumor formation. In some cases, the tumor may invade surrounding tissues and metastasize to other parts of the body. Common sites of metastasis for ovarian tumors are the abdominal cavity, bladder, or colon; these metastatic tumors are called metastatic tumors or secondary tumors.

[0003] Because ovarian cancer is asymptomatic in its early stages, it is quite difficult to differentiate its histological type and whether it is benign or malignant. During exploratory laparotomy, only 30% of ovarian cancer cases are found to be confined to the ovary; most have already spread to the bilateral adnexa of the uterus, the greater omentum, and various pelvic organs. Therefore, ovarian cancer is a major challenge in both diagnosis and treatment.

[0004] KRAS is one of the most common oncogenes in solid tumors, with approximately 30% of tumors showing KRAS mutations. The KRAS protein it controls is a relatively small protein located on the inner side of the cell membrane, and it is closely related to cell growth, differentiation, and proliferation. Tumors caused by KRAS gene mutations are very diverse, commonly including lung cancer, colorectal cancer, breast cancer, pancreatic cancer, liver cancer, and thyroid cancer. Although its mutation rate is low in almost any cancer type, mutation rates exceeding 10% are characteristic only of ovarian cancer and endometrial cancer.

[0005] Palbociclib is a CDK4 / 6 inhibitor approved in many countries for the treatment of advanced breast cancer. It can disrupt the proliferation of cancer cells. Palbociclib is an oral medication for treating advanced breast cancer. The medical community considers palbociclib, a CD4 / 6-targeting drug, and trastuzumab (Herceptin), a HER2-targeting drug, to be among the better targeted therapies for breast cancer today.

[0006] Currently, chemotherapy remains the primary treatment for ovarian cancer, while precise targeted therapy is rarely used in this field. Chinese Patent CN112912387A, published on June 4, 2021, discloses a binding protein and a high-affinity recombinant T-cell receptor (TCR) that specifically bind to the KRASG12V or Her2-ITD neoantigen. It also provides a composition encoding and / or expressing the binding protein and / or the high-affinity recombinant TCR, and recombinant host cells. These compositions and recombinant host cells can be used to treat subjects with non-small cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, or biliary tract cancer, where the KRASG12V neoantigen is a therapeutic target, or where the Her2-ITD neoantigen is a therapeutic target. Related vaccines, vaccine therapies, and vaccination regimens are also provided. Foreign literature (Sheng J, Kohno S, Okada N, Okahashi N, Teranishi K, Matsuda F, Shimizu H, Linn P, Nagatani N, Yamamura M, Harada K, Horike SI, Inoue H, Yano S, Kumar S, Kitajima S, Ajioka I, Takahashi C. Treatment of Retinoblastoma1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4 / 6 Inhibitor Combination Therapy. Hepatology. 2021 Oct; 74(4): 1971-1993.) has demonstrated that CDK4 / 6 inhibitors, when combined with appropriate kinase inhibitors, have the potential to treat a variety of RB1-related cancers, including hepatocellular carcinoma.

[0007] Currently, there are no reports on the combination drug for treating kras-mutant ovarian cancer as described in this invention, nor on its application. Summary of the Invention

[0008] The objective of this invention is to address the shortcomings of existing technologies by providing a combination drug for treating kras-mutant ovarian cancer and its application.

[0009] On the one hand, it provides information on the application of RB7LP in the preparation of drugs for treating ovarian cancer.

[0010] As a preferred example, the ovarian cancer described is a kras-mutant ovarian cancer.

[0011] As a preferred example, the RB7LP described herein can inhibit the proliferation of kras-mutant ovarian cancer cells.

[0012] Secondly, a combination therapy for treating kras-mutant ovarian cancer is provided, the combination therapy comprising a KRAS / EGFR target inhibitor and RB7LP / Palbociclib.

[0013] More preferably, the KRAS / EGFR target inhibitors are lazertinib and sotoracib, respectively.

[0014] Thirdly, the application of the aforementioned combination drugs in the preparation of drugs for treating kras-mutant ovarian cancer is provided.

[0015] Fourthly, an anti-ovarian cancer drug formulation with synergistic effects is provided, comprising the combination drug as described in any one of claims 3-5 and a pharmaceutically acceptable carrier.

[0016] The advantages of this invention are:

[0017] It has been confirmed that KRAS inhibitors or EGFR inhibitors can synergistically kill KRAS-mutant ovarian cancer with palbociclib, and the treatment effect is better than that of palbociclib or sotoracib alone. The combination drug of the present invention can synergistically induce apoptosis of KRAS-mutant ovarian cancer cells and increase the cancer cell inhibition rate, with good efficacy and strong clinical application value. Attached Figure Description

[0018] Appendix Figure 1 RB7LP inhibits the proliferation of ovarian cancer cells.

[0019] Appendix Figure 2 RB7LP promotes senescence and apoptosis in some kras-mutant ovarian cancer cells.

[0020] Appendix Figure 3 To mimic the role of RB7LP in kras-mutant ovarian cancer cells using Palbociclib.

[0021] Appendix Figure 4 Palbociclib, in conjunction with sotoracib, is lethal to KRAS-mutant ovarian cancer cells. Detailed Implementation

[0022] The present invention will be further described 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 description 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.

[0023] Example 1: Cell Experiment

[0024] 1. Experimental Materials

[0025] Ovarian cancer cell lines were purchased from the American College of Cell Bank (ATCC); high-glucose DMEM medium, RPMI-1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, dimethyl sulfoxide, and MTT powder were purchased from Gibco; trypsin-EDTA digestion solution was purchased from Sigma-Aldrich; RNA extraction reagent TRIzol, reverse transcription kit, and SYBR quantitative PCR reagent were purchased from Invitogen; RB7LP, pENTR-GFP, and pTRE3G-puro-DEST vectors were purchased from Addgene; other transfection reagents were purchased from Life Technologies; other commonly used reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd.; an ELISA reader was purchased from Bio-Rad; a real-time quantitative PCR instrument was purchased from ABI; a flow cytometer was purchased from BD; and a clean bench was purchased from Suzhou Sutai Purification Equipment Engineering Co., Ltd. Compound libraries were obtained from MCE. The Annexin V-Alexa Fluor apoptosis kit was purchased from Thermo Fisher Scientific.

[0026] 2 Experimental Methods

[0027] 2.1 Cell Culture

[0028] HEY cells were cultured in petri dishes with a medium containing 10% fetal bovine serum and 90% RPMI-1640 medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator. The medium was changed every other day, and the cells were passaged every 2-3 days.

[0029] 2.2 Lentiviral Preparation

[0030] 293T cells were transduced with 3gpTRE3G-puro-RB7LP-GFP and packaging plasmids (3μgpCMV-VSVG and 3μgpCMV-dR8.91). HEY cells were infected with filtered culture supernatant from 293T cells in the presence of 10μg / ml polybrene. After one week of culture in the presence of 1μg / ml doxycycline (631311, TaKaRa), GFP-positive HEY cells were sorted using a FACSAria cell sorter (BD). Single-cell clones were selected and expanded using limiting dilution. During passage, the resulting HEY cells were cultured in tetracycline-free medium containing 10% Tet System-approved FBS (631106, Clontech) and 1% penicillin-streptomycin solution (16823291, Wako) until further experiments were required.

[0031] 2.3 Real-time PCR detection

[0032] mRNA was extracted from HEY and ovcar8 cells using TRIzol reagent, and cDNA was synthesized using PCR-specific primers. Real-time PCR was performed to detect the target gene. Reaction conditions: 50℃ for 1 min, 95℃ for 30 s, 56℃-60℃ for 50 s, 72℃ for 35 s, for a total of 35 cycles. GAPDH was used as a reference. Data collection mainly involved calculating the initial cycle number (Ct) for all standards and samples using the quantitative PCR instrument software, and a standard curve was plotted based on the Ct values ​​of the standards. Then, based on 2... -△△Ct Quantitative analysis was performed using this method.

[0033] 2.4 Western blot detection

[0034] Whole cells were lysed, and the processed cell lysate was added to protein loading buffer. The mixture was heated at 95°C for 5 min, separated by 10% SDS-PAGE protein electrophoresis, and transferred to a nitrocellulose membrane. The membrane was sealed with 5% skim milk powder at room temperature for 2 h to remove non-specific background. The membrane was incubated with primary antibody at 4°C overnight, washed with PBST, and the corresponding rabbit secondary antibody was added. The membrane was incubated at room temperature in the dark for 30 min, washed with PBST, developed with ECL, and exposed to X-ray for imaging.

[0035] 2.5 MTT assay for cell proliferation rate

[0036] The induced pTRE3G-puro-RB7LP-GFP-HEY cells and the uninduced cells were digested and counted, and seeded at 3000 cells / well in 96-well plates. 20 μL of MTT solution (5 g / L) was added to each well, and the plates were incubated for 4 h. The culture medium was discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min until the purple crystals were fully dissolved. The absorbance (A) was measured at 492 nm using a microplate reader. The cell proliferation rate (%) was calculated as (Aexperimental group / Asolvent control group - 1) × 100%. Each group had 5 replicates, and the experiment was repeated 3 times.

[0037] 2.6 Assay of aging-related β-galactosidase

[0038] Cells were washed with PBS, fixed with PBS containing 4% formaldehyde at room temperature for 10 minutes, washed three more times with PBS, and then incubated with PBS containing 1 mg / mL 5-bromo-4-chloro-3-indoly, β-D-galactoside, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM magnesium chloride (pH 6.0) at 37°C for 24 hours. These images were obtained using a CKX41 microscope (Olympus).

[0039] 2.7 High-throughput drug screening:

[0040] The test cells (induced pTRE3G-puro-RB7LP-GFP-HEY cells and uninduced cells) were placed in 96-well plates (20,000 cells / well) containing kinase inhibitors and FDA library compounds to a final concentration of 1 μM. After incubation for 24 hours in the absence or presence of 1 μg / ml doxycycline, cell viability was assessed using cell counting reagent SF (07553-44, Nacalai tesque) on an Infinite F200Pro Tecan plate reader. The sensitivity index of DOX(+) cells was determined by calculating the ratio of A value to B value, where A value represents the viability of DOX(+) cells and B value represents the viability of DOX(-) cells.

[0041] 2.8 Flow cytometry detection of apoptosis

[0042] The induced pTRE3G-puro-RB7LP-GFP-HEY cells and uninduced cells were collected using 0.25% trypsin. All cells were washed once with pre-chilled PBS (0.1 mol / L, pH 7.4) at 4°C, and the cell count was adjusted to approximately 1 × 10⁶ / mL. Cells were treated according to the Annexin V-FITC apoptosis detection kit: 500 μL of Binding Buffer was added to suspend the cells; 5 μL of Annexin V-FITC was added and mixed; then 5 μL of LPI was added and gently mixed; the cells were incubated at room temperature for 20 min in the dark. Apoptosis was detected by flow cytometry within 1 h, with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The data were analyzed using FCSExpress 3.0 software.

[0043] 3 Experimental Results

[0044] 3.1 RB7LP inhibits the proliferation of kras-mutant ovarian cancer cells.

[0045] An inducible RB7LP virus was constructed and introduced into the ovarian cancer cell line HEY. Stable cell lines were obtained after puro and GFP selection. Overexpression rate was detected by Western blot and real-time PCR. The effect on cell senescence was detected by senescence-associated β-galactosidase staining. The effect on cell proliferation was detected by real-time PCR. Results showed that RB7LP inhibited the proliferation of ovarian cancer cells (see...). Figure 1 ).

[0046] 3.2 RB7LP promotes senescence and apoptosis in some kras-mutant ovarian cancer cells.

[0047] The effect of RB7LP on apoptosis in ovarian cancer cells was detected by Western blot. The effect on cell senescence was assessed using real-time PCR and senescence-associated β-galactosidase staining. Results showed that RB7LP only promoted senescence and apoptosis in some ovarian cancer cells.

[0048] 3.3 The role of Palbociclib mimicking RB7LP in kras-mutant ovarian cancer cells

[0049] Ovarian cancer cells were treated with palbociclib, and the effects of palbociclib on the proliferation and senescence of ovarian cancer cells were detected using colony formation assays, real-time PCR, and senescence-associated β-galactosidase staining. The results showed that palbociclib mimicked the role of RB7LP in kras-mutant ovarian cancer cells, promoting partial senescence and apoptosis in ovarian cancer cells. However, palbociclib alone was insufficient to kill ovarian cancer cells.

[0050] 3.4KRAS inhibitors or EGFR inhibitors can be combined with palbociclib to kill KRAS-mutant ovarian cancer.

[0051] To identify drugs that induce stronger apoptosis in RB7LP or Palbociclib, thereby killing ovarian cancer cells, we conducted high-throughput compound screening experiments. We found that the EGFR inhibitor (lazertinib) and the KRAS inhibitor (sotoracib) synergistically killed ovarian cancer cells with RB7LP or Palbociclib. Flow cytometry was then used to examine the effects of combination therapy or monotherapy on apoptosis. The results showed that combination therapy was significantly more effective than either Palbociclib or sotoracib alone.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of a combination drug in the preparation of a drug for treating HEY ovarian cancer cells with KRAS mutation, characterized in that, The combination drugs are sotorasib and palbociclib.

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