Use of a KRAS inhibitor in combination with an AKR inhibitor in combating tumor cell resistance

Abstract

The application provides application of a KRAS inhibitor and an AKR inhibitor in resisting drug resistance of tumor cells, and the KRAS inhibitor and the aldehyde-ketone reductase inhibitor can be combined to be used for treating various cancers, especially as a compound medicine for treating malignant tumors such as pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer and melanoma. The compound medicine can greatly improve the treatment effect of the existing KRAS inhibitor, can reverse the drug resistance of the chemotherapy medicine, can reduce the dosage of the KRAS inhibitor in the anti-tumor treatment, can not only avoid the increase of the dosage of the KRAS inhibitor due to the drug resistance, but also can correspondingly reduce the risk of the increase of the toxic side effect due to the increase of the dosage after the drug resistance, and has positive pharmaceutical value and wide social significance. The application further provides a compound anti-tumor medicine which comprises an aldehyde-ketone reductase inhibitor, a KRAS inhibitor and a pharmaceutically acceptable carrier.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and in particular to the application of the combination of KRAS inhibitors and AKR inhibitors in combating drug resistance in tumor cells. Background Technology

[0002] RAS (rat sarcoma) oncogene mutations are the most common activating mutations in human cancers, occurring in 30% of human tumors. RAS proteins, encoded by retroviral oncogenes, are among the earliest discovered proteins regulating cell growth. There are three main RAS isoforms: HRAS, KRAS, and NRAS. KRAS, through RNA splicing, expresses two distinct proteins, KRAS4A and KRAS4B (Tsai et al., 2015). These proteins share 85% amino acid sequence similarity, but subtle differences have been found. Each RAS protein includes four domains: an effector region comprising two switch domains and an allosteric region forming the G domain, where GTP binding and GTPase activity occur; a hypervariable region (HVR) containing the CAAX motif; and the most variable region among each RAS isoform. HRAS, KRAS, and NRAS proteins are widely expressed. KRAS is expressed in almost all cell types and is essential, but neither HRAS nor NRAS are essential for normal mouse development (Reck et al., 2021). Of these three proteins, KRAS is more universally expressed. Normal function of RAS proteins requires post-translational modifications, primarily to localize the protein to the inner surface of the cell membrane. If RAS proteins are mislocalized, they become inactive. Oncogenic mutations in the RAS family are most common at codons 12, 13, and 61. For KRAS… ， The vast majority (83%) of cancer-related mutations occur in codon 12 (Hobbs et al., 2016a) with five mutations: G12D, G12V, G12C, G13D, and Q61R (Prioret et al., 2020).

[0003] According to global cancer statistics, 19.29 million new cancer cases and 9.96 million cancer deaths occurred in 2020. Breast cancer had the highest incidence rate, becoming the leading cause of cancer death; lung cancer had the highest mortality rate, with 1.8 million deaths; and colorectal cancer caused 900,000 deaths. The persistently high mortality rates of lung and colorectal cancer are related to the KRAS gene mutation, which is common in these two cancers. Previous studies have shown that cancer patients with this mutation have a poorer prognosis. From November 2016 to July 2019, sequencing data from 11,951 tumor samples collected by multiple centers in China were analyzed for KRAS mutation status, and KRAS mutations were observed in 1,978 samples (16.6%). The frequency of KRAS mutations varies by cancer type, with an incidence of 81.5% in pancreatic cancer, 48.9% in colorectal cancer, and 23.5% in biliary tract cancer. In China, the incidence of KRAS mutations is 56.8% in pancreatic cancer, 33.5% in colorectal cancer, 17.3% in lung cancer, and 5.9% in gastric cancer. KRAS G12C mutations account for 2% of all cancer patients (Amin H. Nassar et al., 2021; Kim et al., 2020). To date, several KRAS G12C inhibitors have been reported in clinical trials. AMG 510 has become the first inhibitor to receive FDA approval, while some candidates, such as MRTX 849, are still in clinical trial research stages.

[0004] Drug resistance to targeted therapies is common, and resistance is more likely to develop with long-term use of single-target drugs. (KRAS) G12C Targeted therapies are no exception. Drug resistance limits the long-term efficacy of KRAS inhibitors (KRASi), making the comprehensive characterization of KRASi resistance mechanisms paramount. Previous research has reported on two newly developed KRAS inhibitors undergoing clinical trials for evaluation. G12C Inhibitors: Resistance to ARS-1620 and Compound 4. Studies have found that these two inhibitors can significantly inhibit KRAS. G12C Growth of two cancer cell lines, mutant PDAC and NSCLC, IC 50The fact that dose-treated cells regained their proliferative capacity after 7 days demonstrates the development of drug resistance. To investigate how cells respond to KRASi and develop resistance, researchers used TMT-labeled quantitative mass spectrometry to compare proteomic changes at different time points after inhibitor addition in PDAC and NSCLC cells. Proteomic analysis revealed similar pathway enrichment in the proteomes of different tumor cell lines (lung cancer and pancreatic cancer) after KRASi treatment. PPI and GO analysis of the top 300 upregulated proteins (24h and 7d) showed similarities in the upregulated pathways between the two tumor cell lines. At the 24h time point, analysis of overlapping functions between cell lines included: metabolism (PPP and lipid metabolism), cytokine signaling, and activation of the PI3K / AKT signaling pathway; while at the 7d time point, long-term adaptation and alterations were mainly related to: antigen presentation, response to oxidative stress, and lysosomal pathways. Data analysis was used to further investigate intracellular pathway alterations induced by short-term and long-term KRASi treatment, using proteomics data from 24 hours and 7 days (Xue et al., 2020). Tumor cells exhibit different acute and long-term adaptation mechanisms to KRASi. In the acute adaptation mechanism, cell cycle pathways, transcription, and translation are downregulated, while lipid metabolism and the TCA cycle are upregulated. Long-term KRASi treatment upregulates cell cycle pathways, transcription, DNA repair, mitochondrial respiration, and lysosomal metabolism, while significantly reducing mTOR signaling. This provides a theoretical basis for addressing resistance to KRAS-targeted therapy (Misale et al., 2019).

[0005] Aldehyde reductase inhibitors (AKRis) are reversible, non-competitive inhibitors of AKRs, selectively inhibiting them. AKRs participate in redox reactions using nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme. These enzymes have a broad substrate range, reducing carbonyl substrates such as aldehydes, ketone steroids, ketone prostaglandins, retinaldehyde, and lipid peroxidation products. Animal experiments have shown that AKRis inhibitors can significantly inhibit the accumulation of sorbitol in the sciatic nerve, erythrocytes, and retina of diabetic rat models, improving motor nerve conduction velocity and autonomic nerve function. Recent studies have shown that the AKRi family is highly expressed during tumor development, and that AKRis inhibitors can inhibit the growth and metastasis of breast cancer basal-like cells (chenfang Dong et al. Journal of experimental medicine (2017)). However, there are no reports on the role of AKRis inhibitors in tumor-targeted drug resistance, nor on their role in KRASi resistance.

[0006] Researchers have found that EMT activates the PI3K pathway in the presence of KRAS G12C inhibitors, contributing to both intrinsic and acquired resistance (Adachi et al., 2020). KRAS expression and activation are associated with sensitivity to KRAS G12C inhibitors. Epithelial-to-mesenchymal transition (EMT) inducement leads to both intrinsic and acquired resistance to KRAS G12C inhibition. In EMT-induced cells, PI3K remains activated in the presence of KRAS G12C inhibitors and is primarily regulated by the IGFR-IRS1 pathway. Researchers found that SHP2 plays a minimal role in PI3K pathway activation, in contrast to its crucial role in MAPK pathway activation. The combination of KRAS G12C inhibitors, PI3K inhibitors, and SHP2 inhibitors resulted in tumor regression in a mouse model of acquired AMG510 resistance. Takamasa's 2021 study found that AMG510 resistance is associated with secondary KRAS mutations.

[0007] Furthermore, Sotorasib (AMG-510), which was approved by the FDA in May 2021, has a recommended daily dose of up to 960 mg. Its irreversible covalent binding makes the small drug molecules more difficult to degrade in the body, which may lead to off-target effects and serious side effects.

[0008] Therefore, it is necessary to reduce drug resistance to drugs that inhibit KRAS-mutant tumors, thereby reducing the dosage of KRAS inhibitors, thus reducing the aforementioned adverse reactions, improving drug resistance, and addressing the shortcomings of existing technologies. Summary of the Invention

[0009] To address the aforementioned problems and fundamentally reverse drug resistance, thereby enhancing the efficacy of chemotherapy drugs and reducing their toxic side effects, this invention provides an application of combining a KRAS inhibitor and an AKR inhibitor in combating tumor cell drug resistance.

[0010] The inventors of this application, through extensive experimentation, discovered that KRAS inhibitors induce resistance in both KRAS-mutant cells and mice. Based on multi-omics data analysis and biochemical experiments, this invention demonstrates that resistance to KRAS inhibitors is generated during their application, and that the combined use of KRAS inhibitors and aldehyde-ketone reductase inhibitors in resistant cells can effectively reduce KRAS inhibitor resistance.

[0011] Therefore, the combination of KRAS inhibitors and aldehyde-ketone reductase inhibitors can be used to treat various tumors, such as lung cancer, lymphoma, esophageal cancer, ovarian cancer, pancreatic cancer, rectal cancer, glioma, cervical cancer, urothelial carcinoma, gastric cancer, endometrial cancer, liver cancer, bile duct cancer, breast cancer, colon cancer, leukemia, and melanoma. Pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer, and melanoma are preferred treatments.

[0012] It is preferred as a combination drug for treating malignant tumors such as pancreatic cancer, colorectal cancer, multiple myeloma, lung cancer, and melanoma. It can greatly improve the therapeutic effect of existing KRAS inhibitors, reverse the resistance of chemotherapy drugs, and reduce the dosage of KRAS inhibitors in anti-tumor treatment. It can not only avoid the need for increased dosage after the emergence of KRAS inhibitor resistance, but also reduce the risk of increased toxic side effects due to increased dosage after resistance. It has positive pharmaceutical value and broad social significance.

[0013] The above-mentioned objective of the present invention is achieved by the following technical solution: the application of the combined use of aldehyde reductase inhibitor and KRAS inhibitor in the preparation of anticancer drugs.

[0014] This invention provides a combination of an aldosterone reductase inhibitor and a KRAS inhibitor, wherein the aldosterone reductase inhibitor (AKRis) is selected from epalrestat, flufenamic acid butyl ester, methoxypropionic acid, medroxyprogesterone acetate, sodium meclomethasone, sulindac, methyl jasmonate, indomethacin, sulindac, progesterone, flufenamic acid, methyl jasmonate, 2'-hydroxyflavone, chloroxalam, epalrestat, or tolrestat.

[0015] This invention provides a combination of an aldosterone reductase inhibitor and a KRAS inhibitor, wherein the KRAS inhibitor is selected from AMG510 (Sotorasib), MRTX849 (Adagrasib), SiG12D-LODER (Silenseed Ltd), ARS-3248, GDC-6036, AZD-4785, mRNA-5671, B1 1701963, JAB-3312, RMC-4630, RAS-001, TG-03, RT11-i, TK-kRAS, PM-004, CWG-71b, pGBl-161, CWG-89, LY3537982, LY3499446, ISIS-2503, or III-121C.

[0016] The present invention also provides a compound antitumor drug comprising an aldosterone reductase inhibitor, a KRAS inhibitor, and a pharmaceutically acceptable carrier.

[0017] Furthermore, the present invention also provides the use of aldosterone reductase inhibitors and KRAS inhibitors in the preparation of medicaments for use in combination with cancer chemotherapy, radiotherapy, targeted therapy, immunotherapy, endocrine therapy, metabolic therapy, or in combination with other therapies.

[0018] Furthermore, the carrier is any one or a mixture of two or more of the following: sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, and lubricant.

[0019] Furthermore, the compound anticancer drug is any one of the following: topical preparation, oral preparation, and injectable preparation.

[0020] Furthermore, the oral formulation is any one of granules, capsules, and tablets.

[0021] The beneficial effects of the combined use of the KRAS inhibitor and AKR inhibitor described in this invention in combating tumor cell drug resistance are as follows:

[0022] 1. In KRAS-mutant cells and mice, resistance to KRAS inhibitors was found. Based on multi-omics data analysis and biochemical experiments, this invention demonstrates that resistance to KRAS inhibitors is generated during their action. Furthermore, the combined use of KRAS inhibitors and aldehyde-ketone reductase inhibitors in resistant cells can effectively reduce KRAS inhibitor resistance.

[0023] 2. In KRAS-mutated drug-resistant cell lines, the IC50 of KRAS inhibitors acting alone on drug-resistant cells was analyzed. 50 It is 10-20 times that of the combined use of KRAS inhibitors and aldosterone reductase inhibitors, indicating that the combined use of aldosterone reductase inhibitors can effectively inhibit the drug resistance caused by KRAS inhibitors. Attached Figure Description

[0024] Figure 1 This indicates the IC50 values ​​for KRASi alone, AKRi alone, and the combination of KRASi and AKRi in KRASi-resistant cell lines. 50 . Detailed Implementation

[0025] Certain preferred embodiments of the present invention are illustrated in the following non-limiting embodiments.

[0026] Experimental methods in the test examples of this invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0027] Aldehyde reductase inhibitor: epalrestat, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; KRAS inhibitor: AMG-510, prepared in-house. Experimental cells were obtained from ATCC. Cell culture was conducted in a CO2 incubator at 37℃.

[0028] Example 1 Biological Evaluation: Cell Viability Assay Method

[0029] KRASi resistant cells were cultured in 96-well plates, with 5 × 10⁶ cells per well. 3 Cells were cultured for 12 hours and then treated with a KRAS inhibitor and / or an aldosterone reductase inhibitor. Three treatments were used: KRAS inhibitor alone, aldosterone reductase inhibitor alone, and a combination of KRAS inhibitor and aldosterone reductase inhibitor. The concentration of the KRAS inhibitor group was 1×10⁻⁶. -3 μM, 1×10 -2 μM, 1×10 -1 The concentrations of the aldehyde reductase inhibitor groups were 1×10 μM, 1 μM, 10 μM, 100 μM, and DMSO, respectively. -3 μM, 1×10 -2 μM, 1×10 -1 The groups receiving μM, 1μM, 10μM, 100μM, and DMSO, and those using a combination of KRAS inhibitor and aldosterone reductase inhibitor at doses of 1×10⁻⁶, were given treatments. -3 μM, 1×10 -2 μM, 1×10 -1 The microplate culture groups were 1 μM, 1 μM, 10 μM, 100 μM, and DMSO, with three replicates for each condition. After 72 h of incubation, cck8 (purchased from Beyotime Biotechnology) was added and incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader. The results are shown in Tables 1-3 below.

[0030]

[0031]

[0032]

[0033] The statistical results are attached. Figure 1 As shown, the results indicate that in drug-resistant cells, the IC50 of KRASi alone is significantly lower. 50 The value is 35.05, using the KRASi + AKRi IC together. 50 The value is 1.696, using KRASi IC alone. 50 The value was 20.7 times that of the combined drug treatment, indicating that the drug resistance of resistant cells can be greatly reduced after the combined use of KRASi + AKRi.

[0034] For those skilled in the art, this disclosure is not limited to the foregoing illustrative embodiments and can be embodied in other specific forms without departing from its essential attributes. Therefore, it is intended that all aspects be considered illustrative rather than restrictive, that references be made to the appended claims rather than the foregoing embodiments, that references be made only to the appended claims and not to the foregoing examples, and that all variations falling within the meaning and scope of claim equivalence are therefore intended to be included herein.

[0035] All patents, patent applications, and references listed in this specification are incorporated herein by reference in their entirety. In case of inconsistencies, this disclosure, including its definitions, will be considered more persuasive.

Claims

1. The use of a combination composition of a KRAS inhibitor and an AKR inhibitor in the preparation of an antitumor drug, wherein the tumor is resistant to the KRAS inhibitor; wherein, The KRAS inhibitor is AMG510, the AKR inhibitor is epalrestat, and the tumor is pancreatic cancer.

2. A compound antitumor drug, characterized in that, It comprises an aldosterone reductase inhibitor, a KRAS inhibitor, and a pharmaceutically acceptable carrier; wherein the KRAS inhibitor is AMG510, and the aldosterone reductase inhibitor is epalrestat.

3. The compound antitumor drug according to claim 2, characterized in that, The carrier is any one or a mixture of two or more of the following: sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, and lubricant.

4. The compound antitumor drug according to claim 2, characterized in that, The compound antitumor drug is any one of the following: topical preparation, oral preparation, and injectable preparation.

5. The compound antitumor drug according to claim 4, characterized in that, The oral preparation is any one of granules, capsules, and tablets.

6. The compound antitumor drug according to claim 2, wherein the tumor is pancreatic cancer.

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