Pharmaceutical composition for inhibiting drug resistance of lenvatinib and application of pharmaceutical composition in liver cancer

Abstract

The invention discloses a pharmaceutical composition for inhibiting drug resistance of lenvatinib and application of the pharmaceutical composition in liver cancer, and belongs to the technical field of biological medicine, the active ingredients of the pharmaceutical composition comprise pavatinib and lenvatinib, and the molar ratio of the pavatinib to the lenvatinib is (15-20): 6. According to the present invention, the expression of the PRMT1 is inhibited so as to inhibit the expression of the drug resistance regulation factor ALYREF of the lenvatinib, and the expression of the downstream target gene ACLY of the ALYREF, such that the liver cancer fatty acid metabolism disorder can be improved, the liver cancer cell in-vivo proliferation can be significantly inhibited, and the drug sensitivity of the lenvatinib can be recovered so as to significantly improve the anti-liver cancer proliferation drug activity of the lenvatinib.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a drug composition for inhibiting lenvatinib resistance and its application in liver cancer. BACKGROUND

[0002] Primary liver cancer is a malignant tumor originating from the liver, which is common worldwide, and is more common in middle-aged male groups. Patients often have persistent abdominal pain, right rib pain, fatigue and weight loss, and the incidence and mortality rates are at a high level. Primary liver cancer is not difficult to treat in the early stage of onset, but obvious symptoms usually do not appear at this stage, so many patients have entered the middle and late stages when diagnosed, and the treatment difficulty has increased significantly, which seriously threatens life safety.

[0003] Lenvatinib is an oral multi-receptor tyrosine kinase inhibitor (TKI) that can inhibit tumor growth and spread by blocking multiple oncogenic sites. It was approved by FDA in 2018 for first-line treatment of advanced hepatocellular carcinoma (HCC), becoming the first new HCC systemic treatment drug in more than ten years since sorafenib. Its clinical efficacy is significantly better than sorafenib, especially for hepatitis B virus (HBV) related liver cancer. However, about 60% of patients develop resistance within 1 year of treatment, resulting in a median overall survival (OS) of only about 1 year and an objective response rate (ORR) of 40%, and the drug resistance problem seriously restricts its clinical application. Current research suggests that its drug resistance mechanism involves abnormal activation of proliferation-related pathways (EGFR / FGFR / WNT, etc.), drug transport disorders, and remodeling of the tumor microenvironment, but the specific mechanism has not been clearly defined. Therefore, analyzing the molecular mechanism of lenvatinib resistance and developing targeted treatment strategies are key to improving the clinical efficacy of HCC.

[0004] RNA binding protein Aly / REF export factor (ALYREF) is the only protein with a classic RNA binding function region in the TREX (TRanscription-EXport) complex, which plays an important role as a nuclear linker protein in the process of mRNA nuclear export. As an RNA m5C methylation recognition enzyme, it regulates gene expression at the post-transcriptional level. Studies have found that ALYREF is a cancer-promoting gene that regulates liver cancer proliferation and has some relevance to lenvatinib resistance in liver cancer.

[0005] Paullazidine is an orally active Furamidine precursor, and the drug is currently approved by FDA for the treatment of pulmonary pneumocystis pneumonia; Furamidine is a selective and cell-permeable protein arginine methyltransferase 1 (PRMT1) inhibitor. However, there is currently insufficient evidence to support its routine use in lung cancer treatment in clinical practice. SUMMARY

[0006] In view of the defects and shortcomings of the prior art, the present application provides a drug composition for inhibiting lenvatinib resistance and its application in liver cancer, which inhibits the function of ALYREF protein by combining pafuramidine with lenvatinib, increases the anti-liver cancer activity of lenvatinib, and provides an important basis for the development of new drugs for treating lenvatinib-resistant liver cancer.

[0007] A drug composition for inhibiting lenvatinib resistance, wherein the active ingredients of the drug composition comprise pafuramidine and lenvatinib.

[0008] Further, the molar ratio of the pafuramidine to the lenvatinib is (15-20):6.

[0009] Further, the molar ratio of the pafuramidine to the lenvatinib is 17:6. For example, the concentration of pafuramidine is 17 muM, and the concentration of lenvatinib is 6 muM.

[0010] Further, the dosage form of the drug composition is one of a liquid preparation, a tablet, and an injection.

[0011] Further, the drug composition further comprises a pharmaceutically acceptable excipient.

[0012] The present application also provides the use of the above-mentioned drug composition in the preparation of a drug for treating liver cancer.

[0013] Further, the pafuramidine inhibits the drug resistance of liver cancer cells to lenvatinib by inhibiting the expression of PRMT1.

[0014] Further, the pafuramidine inhibits the expression of PRMT1, thereby inhibiting the expression of the lenvatinib resistance regulatory factor ALYREF and the expression of the downstream target gene ACLY of ALYREF.

[0015] Further, the liver cancer is lenvatinib-resistant liver cancer.

[0016] Further, the liver cancer is primary liver cancer.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application uses pafuramidine combined with lenvatinib for treating lenvatinib-resistant liver cancer, which inhibits the expression of PRMT1, thereby inhibiting the expression of the lenvatinib resistance regulatory factor ALYREF and the expression of the downstream target gene ACLY of ALYREF, so as to improve the fatty acid metabolism disorder of liver cancer, significantly inhibit the in-vivo proliferation of liver cancer cells, restore the drug sensitivity of lenvatinib, and thus significantly improve the drug activity of lenvatinib against liver cancer proliferation. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0020] Figure 1 The changes of the protein expression amounts of β-actin, ALYREF and ACLY for different concentrations of parifalidine in Example 1;

[0021] Figure 2 The IC value changes of Huh7 and Huh7LR under the combination of lenvatinib monotherapy and parifalidine + lenvatinib in the CCK-8 experiment in Example 2; 50

[0022] Figure 3 The staining changes of Huh7 and Huh7LR after being dyed by Giemsa staining solution under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in the clonogenicity experiment in Example 2;

[0023] Figure 4 The cell proliferation changes of Huh7 and Huh7LR under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in the clonogenicity experiment in Example 2;

[0024] Figure 5 The staining changes of Huh7 and Huh7LR after being dyed by oil red O staining solution after being induced by high-fat model under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in Example 3;

[0025] Figure 6 The appearance and weight of the subcutaneous ectopic tumors of the liver cancer ectopic subcutaneous tumor mice under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in Example 4;

[0026] Figure 7 The volume change curve of the subcutaneous ectopic tumors of the liver cancer ectopic subcutaneous tumor mice under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in Example 4;

[0027] Figure 8 The weight change curve of the liver cancer ectopic subcutaneous tumor mice under the combination of lenvatinib monotherapy, parifalidine monotherapy and parifalidine + lenvatinib in Example 4;

[0028] Figure 9 ​The expression amount of the proliferation-related Marker protein Ki67 in the tumor tissue of the liver cancer ectopic subcutaneous transplantation mouse under the administration of lenvatinib monotherapy, pafuramidine monotherapy, and pafuramidine + lenvatinib combination in Example 4.

[0029] Wherein: DMEM is DMEM medium; Huh7 is a liver cancer cell; Huh7 LR is a lenvatinib-resistant liver cancer cell; Lenvatinib is a lenvatinib monotherapy administration group; Pafuramidine is a pafuramidine monotherapy administration group; Lenvatinib + Pafuramidine is a pafuramidine + lenvatinib combination administration group; and Saline is a physiological saline control group. DETAILED DESCRIPTION

[0030] In order to make the objects, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the claimed present application, but merely represents a selection of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0031] The present embodiment provides a pharmaceutical composition for inhibiting lenvatinib resistance and its application in liver cancer. The active ingredients of the pharmaceutical composition include pafuramidine and lenvatinib. By inhibiting the expression of PRMT1, the expression of lenvatinib resistance regulatory factor ALYREF is inhibited, and the expression of ACLY, a target gene downstream of ALYREF, is inhibited, so that the disorder of fatty acid metabolism in liver cancer can be improved, the in vivo proliferation of liver cancer cells can be significantly inhibited, and the drug sensitivity of lenvatinib can be restored, thereby significantly improving the drug activity of lenvatinib against liver cancer proliferation. The specific implementation is as follows:

[0032] A pharmaceutical composition for inhibiting lenvatinib resistance, wherein the active ingredients of the pharmaceutical composition include pafuramidine and lenvatinib.

[0033] Pafuramidine (Pafuramidine): CAS No. 186953-56-0, molecular formula C 20 H 20N4O3, an experimental prodrug of the orally active metabolite DB75, is an effective antiparasitic compound with antimalarial activity, which can be used for studying parasitic infections and sleeping sickness. As an orally active Furamidine precursor, Parfuramide can inhibit PRMT1, thereby inhibiting the ability of ALYREF to bind mRNA and promote mRNA nuclear export, inhibit tumor cell proliferation, improve the drug sensitivity of Rovratinib, and has good in vivo safety and human tolerance.

[0034] Furamidine is a selective and cell-permeable protein arginine methyltransferase 1 (PRMT1) inhibitor with a CAS number of 73819-26-8, and ALYREF is a key component of the mRNA export protein complex TREX. PRMT1-mediated protein arginine methylation of ALYREF plays a key role in the function of ALYREF as a nuclear export factor. Furamidine can inhibit PRMT1, thereby inhibiting the ability of ALYREF to bind mRNA and promote mRNA nuclear export. In addition, experimental studies have found that Furamidine can effectively inhibit glioblastoma-derived GSC proliferation and tumor sphere formation, as well as the tumorigenic ability of hepatocarcinoma initiating cells and the proliferation of tumor cells.

[0035] As a preferred embodiment, the molar ratio of the Parfuramide to the Rovratinib is (15-20):6.

[0036] As a preferred embodiment, the molar ratio of the Parfuramide to the Rovratinib is 17:6.

[0037] As a preferred embodiment, the dosage form of the pharmaceutical composition is one of a liquid preparation, a tablet, and an injection.

[0038] As a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0039] The application also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating hepatocarcinoma.

[0040] As a preferred embodiment, the Parfuramide inhibits the drug resistance of hepatocarcinoma cells to Rovratinib by inhibiting the expression of PRMT1.

[0041] As a preferred embodiment, the Parfuramide inhibits the expression of the Rovratinib drug resistance regulatory factor ALYREF and the expression of the ALYREF downstream target gene ACLY by inhibiting the expression of PRMT1.

[0042] As a preferred embodiment, the hepatocarcinoma is a Rovratinib-resistant hepatocarcinoma.

[0043] As a preferred embodiment, the liver cancer is primary liver cancer.

[0044] The technical solutions disclosed in the present application will be further described in conjunction with the following examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure, and should not be regarded as limiting the scope of the present disclosure. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained from the market.

[0045] Experimental materials: fetal bovine serum (FBS), DMEM medium (purchased from Gibco); dimethyl sulfoxide (DMSO) (purchased from Sigma); 4% neutral formaldehyde (purchased from Beijing Solabio Technology Co., Ltd.); CCK-8 reagent (purchased from Yakeyin Biological Company); sodium palmitate / sodium oleate kit (purchased from Xi'an Quanchuang Technology Co., Ltd.); oil red O staining kit (purchased from Beijing Solabio Co., Ltd.); total RNA column extraction kit (purchased from Xinsaimai Biological Technology Co., Ltd.); PrimeScript™ RT Master Mix, SYBR® Premix Ex Taq™ II and RNase-free water (purchased from Dalian Takara Company); RIPA cell lysis buffer, protease phosphatase inhibitor, PMSF (purchased from Biyun Tian); BCA kit (purchased from Thermo Fisher); 5x SDS protein loading buffer (purchased from Solabio); antibodies (purchased from CST); ECL luminescent liquid (purchased from Xinsaimai); electrophoresis, membrane transfer equipment, gel preparation kit are BIO-RAD.

[0046] The pharmaceutical excipients include pharmaceutically acceptable non-toxic diluents, wetting agents, binders, disintegrants, preservatives, solubilizers, flavoring agents or lubricants, and the active ingredients of the pharmaceutical composition include the combination of lenvatinib and parafluramide.

[0047] The specific steps will be described below in conjunction with specific examples.

[0048] Example 1

[0049] This example is to verify the inhibitory effect of parafluramide on the key target ALYREF and its downstream metabolic enzyme ACLY.

[0050] Huh7 and Huh7 LR cells were cultured in vitro and treated with 10 μM, 20 μM, 30 μM of parfuramide. The proteins were extracted by conventional experimental techniques in the art, and β-actin antibody (CST 4970) was used as an internal reference protein to verify whether parfuramide affects the expression of ALYREF and downstream target ACLY. The results showed that with the increase of parfuramide drug concentration, the expression of ALYREF and ACLY decreased compared with the control group, indicating that parfuramide inhibits the expression of ALYREF and downstream target ACLY (see Figure 1 ).

[0051] Example 2

[0052] This example shows that parfuramide can significantly promote the anti-hepatoma activity of lenvatinib.

[0053] CCK-8 experiment: 2000 cells / well of Huh7 and Huh7 LR were inoculated in a 96-well plate, and cultured with DMEM medium containing 100 U / mL penicillin, 100 U / mL streptomycin and 10% fetal bovine serum, and incubated in a 37°C, 5% CO2 incubator for 24h. After 24h, 200μL of gradient concentration (0μM, 0.25μM, 0.5μM, 1μM, 2μM, 4μM, 8μM, 16μM, 32μM, 64μM) lenvatinib, 17μM parfuramide medium was added, and the cells were incubated in a cell incubator for 72h. After the stimulation of the drug composition, the culture medium was carefully aspirated, and CCK-8 working solution (10% 5 mg / mL CCK-8 stock solution + 90% DMEM base medium) was added at a volume of 100μL per well, and then the culture plate was placed in a 37°C incubator for 30min. The absorbance was measured at 450nm wavelength, and the relative viability and IC50 value of the cells were calculated. The results showed that the combination of parfuramide and lenvatinib can significantly reduce the IC50 value of lenvatinib (see Figure 2 ).

[0054] Clonogenic assay: (1) Cell plating: 200~400 live cells / well of Huh7 cells treated with 15 μM of lenvatinib, 17 μM of parfuramidine, 15 μM of lenvatinib and 17 μM of parfuramidine were plated in 6-well plates and cultured until each colony contained more than 5 cells. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then the fixing solution was removed and 1 mL of ready-to-use Giemsa staining solution was added for 15 min at room temperature. The results of the clonogenic assay showed that parfuramidine can increase the drug sensitivity of liver cancer cells to lenvatinib (see Figures 3-4 ).

[0055] Example 3

[0056] This example is about the improvement of fatty acid metabolism disorder in liver cancer by parfuramidine combined with lenvatinib.

[0057] Huh7 and lenvatinib-resistant liver cancer cells Huh7 LR were cultured in vitro, and DMEM complete medium containing 600 μM sodium oleate / 300 μM sodium palmitate was used. The cells were incubated in a 37°C, 5% CO2 incubator for 48 h to induce a high-fat model. After 48 h, the cells were fixed with 4% paraformaldehyde for 15 min, and then 60% isopropanol was used for 10 s, and then oil red O staining solution was added for 15 min. Excess dye was removed by adding 60% isopropanol, and then distilled water was used for washing. Then, hematoxylin was added for 30 s, and then distilled water was used for washing. An inverted microscope was used to observe the results. The results showed that the lipid accumulation in the parfuramidine combined with lenvatinib group was significantly reduced compared with the lenvatinib alone group (see Figure 5 ).

[0058] Example 4

[0059] This example is about the treatment regimen of parfuramidine combined with lenvatinib, which can significantly inhibit the proliferation of liver cancer in vivo, has good biological safety and drug tolerance.

[0060] Mouse liver cancer cells Hepa1-6 were inoculated subcutaneously in the left dorsal part of C57BL / 6 mice (1×106 cells / mouse) to construct liver cancer ectopic subcutaneous xenografts and to use them as research objects. After successful modeling, parfuramidine and lenvatinib were used alone or in combination for anti-tumor treatment. During the administration process, the changes in mouse body weight and subcutaneous xenograft volume were recorded. After the experiment, the subcutaneous xenograft tissues were collected, weighed and fixed. The expression of Ki-67 was detected by immunohistochemistry to observe the improvement of the treatment regimen of parfuramidine combined with lenvatinib on the anti-liver cancer activity of lenvatinib and to evaluate the biological safety of the combined regimen. From the appearance of the subcutaneous ectopic tumors, body weight and change curve of tumor volume during the entire treatment period, it can be seen that parfuramidine combined with lenvatinib has obvious anti-tumor proliferation activity (see Figures 6-7), and there was no significant difference in the body weight of mice in each experimental group during the administration (see Figure 8 The immunohistochemical experiment was used to detect the expression of the proliferation-related Marker Ki67 in the tumor tissue, and it was found that the combination of paraflavidine and lenvatinib could significantly inhibit the proliferation of hepatocellular carcinoma in vivo, as compared with lenvatinib alone (see Figure 9

[0061] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A pharmaceutical composition for inhibiting lenvatinib resistance, characterized in that, The active ingredients of the pharmaceutical composition include palfuratel and lenvatinib.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of palfuratel to lenvatinib is (15~20):

6.

3. The pharmaceutical composition according to claim 2, characterized in that, The molar ratio of palfuratel to lenvatinib is 17:

6.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The dosage form of the pharmaceutical composition is one of liquid preparation, tablet, or injection.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for treating liver cancer.

7. The application according to claim 6, characterized in that, The palfuratel inhibits the resistance of liver cancer cells to lenvatinib by suppressing PRMT1 expression.

8. The application according to claim 7, characterized in that, The palfuraidine inhibits the expression of lenvatinib resistance regulator ALYREF by inhibiting the expression of PRMT1, and also inhibits the expression of ALYREF downstream target gene ACLY.

9. The application according to claim 6, characterized in that, The liver cancer in question is lenvatinib-resistant liver cancer.

10. The application according to claim 6, characterized in that, The liver cancer mentioned is primary liver cancer.

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