Drug-combined anti-tumor composition, anti-tumor combined product and application of drug-combined anti-tumor composition and anti-tumor combined product

Abstract

The invention provides a drug-combined anti-tumor composition, an anti-tumor combined product and application of the drug-combined anti-tumor composition and the anti-tumor combined product. The anti-tumor composition simultaneously comprises the following active ingredients: the lenvatinib and the veratramine. According to the invention, veratramine is added for combination on the basis of a first-line targeted drug of the liver cancer, namely the lenvatinib, so that the tumor killing effect of the drug is enhanced in an in-vitro organoid level and in an in-vivo mouse tumor formation experiment.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and this application claims the priority of Chinese invention patent application No. CN202310794503.X filed on June 30, 2023, which is incorporated herein by reference in its entirety. Specifically, it relates to an anti-tumor composition for combined use, an anti-tumor combination product and its application. Background Art

[0002] Primary liver cancer (PLC) ranks fourth in incidence and second in mortality among the most common malignant tumors in my country. Its treatment options are limited, the effective drug response rate is low, and the mortality rate of patients is high. Although targeted drugs have shown good efficacy in liver cancer, they are usually only effective for some patients. The lack of accurate diagnosis and treatment options for liver cancer has become a bottleneck problem.

[0003] Lenvatinib is a first-line drug commonly used in the clinical treatment of primary liver cancer. Lenvatinib is a tyrosine kinase (RTK) receptor inhibitor that can inhibit the kinase activity of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR) and VEGFR3 (FLT4). It can also inhibit pathological angiogenesis in other tumors, inhibit tumor growth and disease progression. It is suitable for patients with unresectable liver cancer in stages IIb, IIIa, IIIb and Child-Pugh A in liver function in the China liver cancer staging (CNLC) scheme. In 2018, guidelines such as the National Comprehensive Cancer Network (NCCN), the European Society for Medical Oncology (ESMO), the European Association for the Study of the Liver (EASL), and the Chinese Society of Clinical Oncology (CSCO) recommended lenvatinib as a first-line standard treatment for advanced hepatocellular carcinoma. The clinical efficacy of lenvatinib in the treatment of primary liver cancer is 24.1%, which is not applicable to most patients. Therefore, there is an urgent need to develop an effective combination drug regimen for lenvatinib to enhance its tumor-killing effect.

[0004] Veratramine is a known natural steroidal alkaloid isolated from plants of the lily family, such as Veratrum, which has the effects of lowering blood pressure, antagonizing Na+ channel activity and acting as a 5-HT agonist. Veratramine is also an inhibitor of transcription factor AP-1, which can specifically bind to the sequence (TRE 5′-TGACTCA-3′) and inhibit AP-1 transcription of downstream target genes. Under normal physiological conditions, AP-1 regulates cell proliferation, differentiation and apoptosis by regulating a series of downstream target genes. Overactivation of AP-1 can lead to tumor formation, inflammation and infection. The c-Jun protein belongs to the activator protein-1 (AP-1) transcription complex and is the most transcriptionally active transcription factor in the AP-1 complex. Veratramine has a good inhibitory effect on the activity of c-Jun and can inhibit the transcription of its downstream target genes.

[0005] Current status of anti-tumor research on veratramine: After blocking the transcription of AP-1, veratramine can induce cell apoptosis, block the G0 / G1 phase of the cell cycle, inhibit cell proliferation, and play a certain tumor inhibitory effect in liver cancer cell lines. However, as a traditional Chinese medicine, veratramine has limited combination drug regimens, and veratramine has not been applied in the clinical treatment of tumors. Summary of the invention

[0006] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide an anti-tumor composition for combined use, an anti-tumor combination product and its application. The present invention can significantly enhance the drug sensitivity of lenvatinib in liver cancer organoids and mice through the combined use of lenvatinib and veratramine, and achieve better tumor treatment effects. Therefore, the combined drug regimen has good application prospects.

[0007] Before describing the present invention, the terms used in this article are defined as follows:

[0008] The term "JUN" refers to: a gene on chromosome 1, HGNC number 6204, which is the transforming gene of avian sarcoma virus 17 and encodes a protein that is highly similar to the viral protein and directly interacts with specific target DNA sequences to regulate gene expression.

[0009] The term "c-JUN" refers to the protein encoded by JUN.

[0010] The term "Lenvatinib" refers to: 4-[3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide, English name Lenvatinib, alias lenvatinib, Lenvatinib, and the active ingredient herein includes its lenvatinib free base; lenvatinib mesylate, and lenvatinib base.

[0011] The term "veratramine" refers to: (2S,3R,5S)-5-methyl-2-[(1S)-1-[(3S,6AR,11AS,11BR)-2,3,4,6,6A,11,11A,11B-octahydro-3-hydroxy-10,11B-dimethyl-1H-benzo[A]fluoren-9-yl]ethyl]-3-piperidinol, English name: Veratramine, also known as veratramine; veratramine (veratramine); veratramine.

[0012] The term "control" means: blank control.

[0013] The term "HCC" refers to: Hepatocellular carcinoma.

[0014] The term "HE" refers to: Hematoxylin-eosin staining.

[0015] The term "PLC" refers to: primary liver cancer.

[0016] The term "EpCAM" refers to: Epithelial cell adhesion molecule, which is a transmembrane glycoprotein with a molecular weight of 40kDa encoded by the GA-733-2 gene. As a homophilic calcium-independent epithelial cell adhesion molecule, it plays a role in the process of epithelial carcinogenesis.

[0017] The term "AFP" refers to alpha-fetoprotein, which belongs to the albumin family and is mainly synthesized by fetal liver cells and yolk sac.

[0018] The term "Hepatocyte" means: liver cell.

[0019] To achieve the above objectives, the first aspect of the present invention provides an anti-tumor composition for combined use, which comprises: lenvatinib and veratramine as active ingredients, and one or more pharmaceutically acceptable carriers.

[0020] The second aspect of the present invention provides an anti-tumor combination product, which includes active ingredients lenvatinib and veratramine, respectively present in the form of independent preparations; and one or more pharmaceutically acceptable carriers contained in the independent preparations.

[0021] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, the concentration ratio of the two active ingredients lenvatinib and the veratramine is 1:0.1-10, preferably 1:0.2-5, more preferably 1:0.5-2, and most preferably 1:1.

[0022] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, the administration method of the anti-tumor composition or the anti-tumor combination product is selected from one or more of the following: injection, oral administration, sublingual administration, and rectal instillation.

[0023] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, when the administration method is oral administration, the dosage form of the independent preparation in the anti-tumor composition or the anti-tumor combination product is selected from one or more of the following: powder, tablet, capsule, granule, syrup, pill, soft capsule and oral solution.

[0024] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, wherein the administration method is injection administration:

[0025] The dosage form of the anti-tumor composition or anti-tumor combination product is selected from one or more of the following: solution injection, emulsion injection, suspension; and / or

[0026] The injection administration is selected from one or more of the following: intravenous injection, intramuscular injection, and acupoint injection.

[0027] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, the one or more pharmaceutically acceptable carriers are selected from: fillers, wetting agents, binders, disintegrants, lubricants, solvents, solubilizers, preservatives, flavoring agents, colorants, pH regulators, and antioxidants.

[0028] According to the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect of the present invention, the dosage form of the anti-tumor composition and the anti-tumor combination product is a solution, a suspension or an aerosol.

[0029] The third aspect of the present invention provides the use of the anti-tumor composition of the first aspect or the anti-tumor combination product of the second aspect in the preparation of a drug for treating cancer;

[0030] Preferably, the cancer is liver cancer or bile duct cancer, most preferably liver cancer.

[0031] According to the third aspect of the present invention, the liver cancer is selected from one or more of the following: primary liver cancer, hepatocellular carcinoma, and intrahepatic bile duct carcinoma.

[0032] According to a specific embodiment of the present invention, in order to overcome the problem of low efficacy of existing liver cancer targeted drugs, the purpose of the present invention is to provide a new combination drug regimen of lenvatinib. The combination drug regimen can fill the gap in the field of accurate diagnosis and treatment of liver cancer and improve the treatment effect of liver cancer.

[0033] The first aspect of the present invention: It is found that high expression of JUN is associated with lenvatinib resistance

[0034] The specific screening process is:

[0035] The inventors collected surgical specimens from multiple patients with primary liver cancer, sampled at multiple points, and successfully established a liver cancer organoid biobank covering hundreds of sites through a three-dimensional culture method. The inventors' team confirmed that the organoids retained the pathological characteristics of the patients through hematoxylin-eosin (HE) staining. Through immunofluorescence and immunohistochemical staining of organoids and corresponding patient specimens, it was found that the molecular markers of liver cancer organoids were also consistent with those of clinical liver cancer patients.

[0036] Nearly 100 sites were selected for exon and transcriptome sequencing of tissues and corresponding organoids, and the genomic characteristics of tissues and corresponding organoids were analyzed. The molecular characteristics of tissue samples highly preserved in the organoid biobank were verified from multiple dimensions such as somatic mutations, copy number variations, and transcriptome similarities. In addition, the large-scale liver cancer tissue and organoid cohorts established by the team are consistent with previous liver cancer studies and have high-incidence mutations in liver cancer. In addition, the inventors' liver cancer data reflect varying degrees of inter-tumor and intra-tumor heterogeneity, providing a basis for studying the heterogeneity of liver cancer drug sensitivity.

[0037] The present invention selected lenvatinib, a commonly used liver cancer drug in clinical practice, and conducted large-scale drug screening on the organoid biobank. The inventors compared the drug sensitivity of the organoids with the clinical treatment effects and found that their drug sensitivity effects were highly consistent.

[0038] In order to study the key resistance gene of lenvatinib, a first-line targeted drug for liver cancer, transcriptome sequencing was performed on multiple organoid sites, and through bioinformatics analysis, JUN was identified as the key resistance gene of lenvatinib.

[0039] The present invention is suitable for patients with liver cancer who are resistant to lenvatinib, and is expected to form a new combined medication regimen for patients who are resistant to lenvatinib, thereby achieving better therapeutic effects.

[0040] The present invention directly provides a combined medication regimen of c-Jun inhibitors veratramine and lenvatinib, and provides verification at the cell and mouse levels, which can more directly guide clinical liver cancer medication.

[0041] The anti-tumor composition and combination product of the combined drug administration method of the present invention may have but are not limited to the following beneficial effects:

[0042] 1. The key drug resistance gene JUN of lenvatinib was identified, and the combined use of lenvatinib and c-Jun inhibitor veratramine was determined. This can improve the therapeutic effect of lenvatinib and provide clues for the use of lenvatinib and the precise treatment of liver cancer.

[0043] 2. The present invention adds veratramine to the first-line targeted drug for liver cancer, lenvatinib, and enhances the drug's tumor-killing effect at the in vitro organoid level and in vivo mouse tumor formation experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0045] Figure 1 The organoids of patients with hepatocellular carcinoma (HCC) retain the pathological characteristics and tissue markers of the patients; wherein, Figure 1 A shows HE and immunohistochemical staining of different sites in HCC patients; Figure 1 B shows the microscopic status and HE staining of HCC patient organoids; Figure 1 C shows immunofluorescence staining of organoids; Figure 1 D shows immunohistochemical staining of organoids.

[0046] Figure 2 The exon and transcriptome characteristics of organoid-retaining patients are shown; among them, Figure 2 A shows that exome sequencing reveals that organoids retain the mutational signature of patients; Figure 2 B shows that transcriptome sequencing shows that organoids retain the transcriptome characteristics of patients.

[0047] Figure 3 Organoid drug susceptibility testing is shown; wherein, Figure 3 A shows the drug sensitivity curve of lenvatinib; Figure 3 B shows the in vitro drug sensitivity test of organoids, which is consistent with the drug response of clinical patients.

[0048] Figure 4 The lenvatinib resistance target interaction map is shown.

[0049] Figure 5 The figure shows the combined effect of lenvatinib and veratramine at the organoid level; the horizontal axis shows different drug concentrations of lenvatinib and veratramine (added at a concentration ratio of 1:1), and the vertical axis shows the survival rate of the organoids at this concentration; among them, Figure 5 A shows the killing effects of lenvatinib, veratramine and combination therapy (concentration ratio 1:1) on human organs of patient 1 with primary liver cancer at different concentrations; Figure 5B shows the killing effect of lenvatinib, veratramine and combination therapy (concentration ratio 1:1) on human organs of primary liver cancer patient 2 at different concentrations; Figure 5 C shows the killing effect of lenvatinib, veratramine and combined use (concentration ratio 1:1) on human organs of primary liver cancer patient 3 at different concentrations; Figure 5 D shows the half inhibition rate, i.e., IC50, of organoids in a large-scale drug sensitivity test of lenvatinib, veratramine, and combination therapy (concentration ratio 1:1). The half inhibition rate of the combination therapy was lower than that of the lenvatinib monotherapy group (P<0.05) and the veratramine monotherapy group (P<0.05), and the difference was statistically significant.

[0050] Figure 6 The combined effect of lenvatinib and veratramine in mice is shown. The horizontal axis shows the number of days (calculated from the subcutaneous implantation of organoids), and the vertical axis shows the tumor volume; Figure 6 A shows that the organoids of patient 1 with primary liver cancer were implanted subcutaneously in Balb / c mice, and lenvatinib, veratramine, combined treatment, and saline control were given for 14 days, and the tumor volume was continuously monitored; Figure 6 B shows that the organoids of patient 2 with primary liver cancer were implanted subcutaneously in Balb / c mice, and lenvatinib, veratramine, combined treatment, and saline control were given for 14 days, and the tumor volume was continuously monitored; Figure 6 C shows that the organoids from patient 3 with primary liver cancer were implanted subcutaneously in Balb / c mice, and lenvatinib, veratramine, combination therapy, and saline control were administered for 14 days, and the tumor volume was continuously monitored.

[0051] Figure 7 The synergistic effect of lenvatinib and veratramide was shown in 8 liver cancer organoids; among them, Figure 7 A shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P94C2) is 1.21; Figure 7 B shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (number P20C2) is 2.11; Figure 7 C shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P107C2) is 3.76; Figure 7 D shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P74C3) is 3.39; Figure 7 E shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P104C3) is 5.61; Figure 7F shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P108C1) is 7.67; Figure 7 G shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P101C3) is 9.03; Figure 7 H shows that the synergistic coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P102C2) is 9.42. DETAILED DESCRIPTION

[0052] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0053] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0054] The reagents and instruments used in the following examples are as follows:

[0055] Reagents:

[0056] Lenvatinib was purchased from MCE, catalog number HY-10981.

[0057] Veratramine was purchased from MCE Company, product number HY-N0837.

[0058] DMEM / F12 culture medium was purchased from Corning, catalog number 10-092-CVRC.

[0059] Double antibody, purchased from Gibco, catalog number 15140122.

[0060] Glutathione was purchased from Gibco, catalog number 35050061.

[0061] Buffer, purchased from Tocris, catalog number 3173.

[0062] B27 nutrient solution was purchased from Life Company, catalog number 12587-010.

[0063] N2 nutrient solution was purchased from Life Company, catalog number 17502048.

[0064] N-acetylcysteine ​​was purchased from Sigma, product number A9165-5g.

[0065] Nicotinamide was purchased from Sigma, product number N0636.

[0066] Gastrin was purchased from Meilunbio, catalog number MB10217.

[0067] Recombinant human epidermal growth factor was purchased from Peprotech, product number AF-100-15.

[0068] Recombinant human fibroblast growth factor 10 was purchased from Peprotech, catalog number 100-26.

[0069] Recombinant human hepatocyte growth factor was purchased from Peprotech, catalog number 100-39.

[0070] Forskolin was purchased from Tocris Bioscience, catalog number 1099 / 10.

[0071] Matrigel was purchased from Corning, catalog number 354234.

[0072] CCK-8 cell proliferation detection kit was purchased from Beyotime, catalog number C0040.

[0073] Example 1

[0074] This example is used to illustrate the screening of lenvatinib drug sensitivity markers:

[0075] In this example, a liver cancer organoid biobank covering 376 sites was successfully established through a 3D culture method. Taking the surgical specimens of patients with hepatocellular carcinoma (HCC) as an example, the inventors used HE staining to confirm that the organoids retained the pathological characteristics of the patients ( Figure 1 A, 1B). By performing immunofluorescence and immunohistochemistry staining on organoids and corresponding patient specimens, it was found that HCC diagnostic markers AFP and hepatocyte were positive in HCC patients and their organoids ( Figure 1 C, 1D), while the bile duct cancer marker EPCAM was negative, which is consistent with the molecular markers of liver cancer patients in clinic. This shows that the organoid system established in this example well retains the pathological characteristics of the patient.

[0076] This example selects 99 sites to sequence the exons and transcriptomes of tissues and corresponding organoids, and analyzes the genomic characteristics of tissues and corresponding organoids. The organoid biobank highly retains the molecular characteristics of tissue samples from multiple dimensions such as somatic mutations, copy number variations, and transcriptome similarity. The large-scale liver cancer tissue and organoid cohort established by the inventor's team is consistent with previous liver cancer studies, with high-incidence mutations in liver cancer ( Figure 2 A). At the transcriptome level, organoids and corresponding patient tissues are also highly correlated ( Figure 2 B).

[0077] In this example, 376 organoids were tested for drug sensitivity to the first-line drug lenvatinib for liver cancer. This example plotted the drug sensitivity curve of the first-line drug lenvatinib ( Figure 3 A), and the drug sensitivity of the patient's organoids is generally consistent with the actual clinical drug effects of the patient ( Figure 3 B).

[0078] In this example, lenvatinib resistance genes were further analyzed. Based on the interaction map of resistance genes, JUN, a gene located in the core region, was selected as the key target of resistance ( Figure 4 ).

[0079] Example 2

[0080] This example is used to illustrate the effect of lenvatinib combined with veratramine at the organoid level.

[0081] The inventors randomly selected three lenvatinib-resistant organoids (numbered P1, P2, and P3, corresponding to Figure 5 Lenvatinib alone (1 nM to 50 μM), veratramine alone (1 nM to 50 μM), and lenvatinib combined with veratramine (1 nM to 50 μM) were cultured in organoids for 4 days. The concentration of lenvatinib combined with veratramine (lenvatinib to combined veratramine concentration ratio 1:1) varied from 1 nM to 50 μM, and the organoids were then tested for drug sensitivity.

[0082] Figure 5 A shows the killing effects of lenvatinib, veratramine and combination therapy (concentration ratio 1:1) on human organs of patient 1 with primary liver cancer at different concentrations; Figure 5 B shows the killing effect of lenvatinib, veratramine and combination therapy (concentration ratio 1:1) on human organs of primary liver cancer patient 2 at different concentrations; Figure 5 C shows the killing effect of lenvatinib, veratramine and combination therapy (concentration ratio 1:1) on human organs of patient 3 with primary liver cancer at different concentrations. Figure 5It can be seen that lenvatinib combined with veratramine (concentration ratio 1:1) significantly reduced cell survival rate compared with lenvatinib (P<0.001). The inventors found that the cell activity of the lenvatinib and veratramine combination group was significantly lower than that of the lenvatinib monotherapy group. It can be seen that in lenvatinib-resistant organoids, the combination of lenvatinib and veratramine can significantly enhance the drug sensitivity of lenvatinib (refer to Figure 5 Compared with lenvatinib alone, it has a significant tumor killing effect (P<0.05). Figure 5 It can be seen that after the inventors found the effect of combined drug treatment in these three patients, they further treated organoids with lenvatinib alone, veratramine alone, and lenvatinib combined with veratramine in the entire organoid biobank, and detected the half inhibition rate, i.e., IC50 (half maximal inhibitory concentration) of 376 organoids. It was found that the half inhibition rate of the combination drug group was lower than that of the lenvatinib monotherapy group (P<0.05) and veratramine monotherapy group (P<0.05), and the differences were statistically significant.

[0083] Example 3

[0084] This example is used to illustrate the synergistic effect of lenvatinib and veratramine at the organoid level under different concentration gradients and ratios.

[0085] In this embodiment, a concentration gradient of lenvatinib and veratramide was established, and each drug was 1nM, 10nM, 100nM, 1000nM, and 10000nM, respectively. The two drugs were arranged in different concentration gradients to treat 8 liver cancer organoids (numbered: P94C2, P20C2, P107C2, P74C3, P104C3, P108C1, P101C3 and P102C2) for 96 hours. As shown in Table 1, the killing effect on organoids under the arrangement and combination concentrations was calculated. The group without drug addition was used as a control. Cell activity inhibition rate (%) = cell absorbance at a certain lenvatinib and veratramide combination concentration / absorbance of the control group without drug addition × 100%.

[0086] The synergistic effect of the two drugs at different concentrations was observed through the ZIP (Zero Interaction Potency) synergistic coefficient, and ZIP score>0 was synergistic.

[0087] Figure 7 The synergistic effect of lenvatinib and veratramide was shown in 8 liver cancer organoids; among them, Figure 7 A shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P94C2) is 1.21; Figure 7B shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (number P20C2) is 2.11; Figure 7 C shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P107C2) is 3.76; Figure 7 D shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P74C3) is 3.39; Figure 7 E shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P104C3) is 5.61; Figure 7 F shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P108C1) is 7.67; Figure 7 G shows that the synergy coefficient of lenvatinib and veratramide at different concentrations in the organoid (No. P101C3) is 9.03; Figure 7 H shows that the synergistic coefficient of lenvatinib and veratramide at different concentrations in the organoid (code P102C2) is 9.42. Figure 7 As shown in the figure, in 8 liver cancer organoids (numbered as P94C2, P20C2, P107C2, P74C3, P104C3, P108C1, P101C3 and P102C2), lenvatinib and veratramide have a relatively good synergistic effect. The ZIP score is >0.

[0088] Table 1 Cell activity inhibition rate of lenvatinib and veratramine at different concentration ratios

[0089]

[0090] Table 1 shows the cell activity inhibition rates of lenvatinib and veratramine at different concentration ratios in three organoids. As can be seen from Table 1, when the concentration ratio of lenvatinib to veratramine is 1:1, the cell activity inhibition rate is the largest, which is 65.35%, 62.33%, and 49.05%, respectively. This shows that different lenvatinib and veratramine concentration ratios have tumor cell activity inhibition rates.

[0091] Example 4

[0092] This example is used to illustrate the effect of the combination of lenvatinib and veratramine at the mouse model level.

[0093] Based on the concentration gradient combination data screened by the inventors at the organoid level, a concentration ratio of 1:1 has a good tumor killing effect in vitro. Therefore, this example directly selected a 1:1 ratio of lenvatinib and veratramine in a mouse model.

[0094] The inventors used liver cancer organoids (the liver cancer organoids used in this example are the same as those in Example 2, such as Figure 6 The tumors (hepatoma subcutaneous tumors) were implanted in Balb / c nude mice. 3 At 4 pm, the nude mice were randomly divided into 4 groups, 5 mice in each group. In the next 3 weeks, lenvatinib (10 mg kg -1 , oral administration), veratramine (10 mg kg -1 , oral administration), lenvatinib combined with veratramine oral administration (lenvatinib 10 mg kg -1 + Veratramine 10mg kg -1 , gavage) treated mice.

[0095] Tumor volume was measured daily and quantified every five days using the formula: tumor volume = 1 / 2 length × width 2 , analyzed by ANOVA, Figure 6 It can be seen that compared with lenvatinib, lenvatinib combined with veratramine significantly reduced the size of mouse tumors (P<0.001). The inventors found that the tumor volume of the lenvatinib and veratramine combined group was significantly smaller than that of the lenvatinib monotherapy group. It can be seen that when the organoids are subcutaneously implanted into the mouse model, the combination of lenvatinib and veratramine can significantly reduce the volume of subcutaneous tumors in mice ( Figure 6 ). Compared with lenvatinib alone, it had a significant effect on shrinking tumors (P<0.05).

[0096] summary :

[0097] Based on the liver cancer combination drug regimen based on lenvatinib and veratramine as exemplified in Examples 2-5 above, the inventors verified that lenvatinib and veratramine have superior tumor treatment effects than lenvatinib at the organoid and mouse tumor formation levels (P<0.05).

[0098] Specifically, in Example 2, the inventors randomly selected 3 lenvatinib-resistant organoids from the liver cancer organoid biobank. Lenvatinib, veratramine, and lenvatinib combined with veratramine (1:1 concentration) were cultured in organoids for 4 days, and then the organoids were tested for drug sensitivity. It was found that the cell activity of the combination group was significantly lower than that of the lenvatinib monotherapy group.

[0099] In Example 3, the inventors set up a concentration gradient for lenvatinib and veratramide in this example, and each drug was 1nM, 10nM, 100nM, 1000nM, and 10000nM to treat 8 liver cancer organoids (numbered: P94C2, P20C2, P107C2, P74C3, P104C3, P108C1, P101C3 and P102C2) for 96 hours. The group without drug addition was used as a control. The synergistic effect of the two drugs at different concentrations was observed by the ZIP (Zero Interaction Potency) synergy coefficient. It was found that all 8 organoids showed synergy of lenvatinib and veratramide (ZIP score>0).

[0100] Subsequently, in Example 4, the inventors implanted the organoids subcutaneously in Balb / c nude mice. 3 At 4 pm, the nude mice were randomly divided into groups, with 5 mice in each group. In the following 3 weeks, lenvatinib (10 mg kg -1 , oral administration), veratramine (10 mg kg -1 , oral administration), lenvatinib combined with veratramine (10 mg kg -1 The tumor volume was measured every day and quantified every five days using the formula: tumor volume = 1 / 2 length × width 2 Through ANOVA analysis, the tumor volume of the combination group was significantly smaller than that of the lenvatinib monotherapy group.

[0101] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. An anti-tumor composition for combined use, characterized in that: The anti-tumor composition comprises: lenvatinib and veratramine as active ingredients, and one or more pharmaceutically acceptable carriers.

2. An anti-tumor combination product, characterized in that: The anti-tumor combination product comprises active ingredients lenvatinib and veratramine, which are respectively present in the form of independent preparations; and one or more pharmaceutically acceptable carriers contained in the independent preparations.

3. The antitumor composition according to claim 1 or the antitumor combination product according to claim 2, characterized in that: The concentration ratio of the two active ingredients, lenvatinib and the veratramine, is 1:0.1-10, preferably 1:0.2-5, more preferably 1:0.5-2, and most preferably 1:

1.

4. The antitumor composition according to claim 1 or the antitumor combination product according to claim 2, characterized in that: The administration method of the anti-tumor composition or anti-tumor combination product is selected from one or more of the following: injection, oral administration, sublingual administration, and rectal instillation.

5. The antitumor composition or antitumor combination product according to claim 4, characterized in that: When the administration method is oral administration, the dosage form of the anti-tumor composition or the independent preparation in the anti-tumor combination product is selected from one or more of the following: powder, tablet, capsule, granule, syrup, pill, soft capsule and oral solution.

6. The antitumor composition or antitumor combination product according to claim 4, characterized in that: When the administration method is injection: The dosage form of the anti-tumor composition or anti-tumor combination product is selected from one or more of the following: solution injection, emulsion injection, suspension; and / or The injection administration is selected from one or more of the following: intravenous injection, intramuscular injection, and acupoint injection.

7. The antitumor composition according to claim 1 or the antitumor combination product according to claim 2, characterized in that: The one or more pharmaceutically acceptable carriers are selected from the group consisting of fillers, wetting agents, binders, disintegrants, lubricants, solvents, solubilizers, preservatives, flavoring agents, colorants, pH adjusters, and antioxidants.

8. The antitumor composition according to claim 1 or the antitumor combination product according to claim 2, characterized in that: The dosage form of the anti-tumor composition and the anti-tumor combination product is solution, suspension or aerosol.

9. Use of the antitumor composition or antitumor combination product according to any one of claims 1 to 8 in the preparation of a drug for treating cancer; Preferably, the cancer is liver cancer or bile duct cancer, most preferably liver cancer.

10. The use according to claim 9, characterized in that: The liver cancer is selected from one or more of the following: primary liver cancer, hepatocellular carcinoma, and intrahepatic bile duct carcinoma.

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