Annonaceous acetogenin compound and application thereof

Abstract

The invention relates to a drug combination of annona squamosa lactone compounds and sorafenib (SF), which is characterized in that four common human hepatoma cell lines HepG2, HuH7, MHCC97H and HCCLM3 are used as research objects, and the drug combination effect among different components is evaluated by using an MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 3-diphenyltetrazolium bromide) method and Synergy Finder software. The combination of the compound and SF shows an obvious synergistic inhibition effect on the growth of hepatoma carcinoma cells, and meanwhile, the pharmaceutical composition can synergistically reduce the ATP level of the hepatoma carcinoma cells and induce hepatoma carcinoma cell apoptosis. Pharmacodynamic experiments show that the compounds annonacin and SF can synergistically inhibit tumor growth and induce tumor cell apoptosis, and have no obvious toxicity to normal organs of nude mice at a low dosage. In addition, transcriptome analysis predicts that the anti-liver cancer target of the annonacin is possibly related to the SLC33A1 gene. The method can be used for preparing anti-hepatoma drugs and researching related mechanisms. The annona lactone compound disclosed by the invention is derived from annona montana Macf. Which is an annona plant, namely annona montana Macf.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry, and particularly relates to the use of annonaceous acetogenins in combination with sorafenib for the treatment of liver cancer. Background Art

[0002] Liver cancer is one of the common malignant tumors in clinical practice. In China, it is the fourth most common cancer (about 400,000 cases) and the second leading cause of cancer death (about 340,000 cases), and the incidence and mortality rates in men are 2-3 times higher than those in women. [1] Hepatitis B virus, hepatitis C virus, fatty liver, alcoholic cirrhosis, smoking, obesity, etc. may all be the inducing factors for the onset of liver cancer. The infection of hepatitis B virus in China is relatively serious, and the incidence of liver cancer shows an increasing trend year by year, posing a great threat to the national health of China. Liver cancer has a hidden onset and a poor prognosis. Most patients are diagnosed at the middle and advanced stages and are unable to undergo surgical treatment. Transarterial chemoembolization (TACE) and chemotherapy have become the common treatment options for advanced liver cancer. [2]

[0003] Sorafenib is a multi-kinase small molecule inhibitor and is currently still the first-line drug for the systemic chemotherapy of liver cancer in clinical practice. [3] Clinical data studies have shown that sorafenib is relatively effective in the treatment of middle and advanced liver cancer regardless of the disease etiology and disease burden. [4] However, the use of a large dose of sorafenib can cause intolerable systemic side effects in patients due to its poor accumulation at the tumor site and off-target effects, and the clinical treatment effect is difficult to achieve the expected level. A series of toxic and side effects will occur during the long-term use of sorafenib for the treatment of middle and advanced liver cancer, including hand-foot syndrome, drug rash, severe gastrointestinal reactions, etc. In addition, relevant clinical studies have shown that reducing the dose of sorafenib can reduce the toxic and side effects of the patients receiving the drug, but the treatment effect is difficult to reach that of the conventional dosing regimen. [5] Currently, in clinical practice, in order to enhance the efficacy of sorafenib or reduce its toxic side effects, the method of combined drug use is often adopted. [6-9] Although the in-study combined drug combinations reduce the dosing dose of sorafenib through combined administration, there is no obvious anti-tumor synergistic activity. Due to the limitations of the chemical structures and related therapeutic targets of many clinical chemotherapy drugs, it is difficult to combine them with sorafenib to achieve the goal of reducing toxicity and increasing efficacy. Therefore, it is of great research significance to explore drugs that can enhance the anti-liver cancer activity of sorafenib, especially to discover synergistic active molecules from natural products with rich structures and to explore the combined drug use strategy of natural products and sorafenib.

[0004] The seeds of Annona squamosa are rich in various bioactive natural compounds, and the annonaceous acetogenins isolated from plants of the Annonaceae family exhibit strong in vitro anti-tumor activity.

[10] Studies have shown that the anti-tumor activity of annonaceous acetogenins may be derived from the tetrahydrofuran ring on their fatty chains and the terminal unsaturated lactone ring, which act on mitochondrial complex I and NADH oxidase on the cytoplasmic membrane, thereby reducing the ATP level in cancer cells.

[11] Therefore, Annona squamosa seeds have become a potential source for synergistically enhancing the anti-hepatocellular carcinoma effect of sorafenib.

[0005] In this invention, four human hepatocellular carcinoma cell lines, HepG2, HuH7, MHCC97H, and HCCLM3, were used as the research objects, and the anti-hepatocellular carcinoma activity of compounds 1-7 isolated from Annona montana Macf. was tested for its synergistic effect with sorafenib. The test results showed that these compounds all had a certain synergistic activity. By constructing a xenograft model of human hepatocellular carcinoma LM3 cells subcutaneously, the in vivo anti-tumor activity of compound 1 combined with sorafenib was evaluated. Compound 1 had a certain anti-tumor activity alone, and when used in combination with sorafenib, it could synergistically enhance the anti-hepatocellular carcinoma activity of sorafenib alone. Immunohistochemical results showed that the combined use of drugs could promote tumor cell apoptosis and the combined use at a lower dose had good safety, and there were no obvious lesions in the main organs of the mice. Transcriptome analysis showed that compound 1 might act on the lipid metabolism pathway to produce anti-tumor effects. The potential action target of compound 1 might be related to SLC33A1.

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[0020]

[10] Bermejo, A.; Figadere, B.; Zafra-Polo, M.C.; Barrachina, I.; Estornell, E.; Cortes, D.

[0021] Acetogenins from Annonaceae: recent progress in isolation, synthesis and mechanisms of action.

[0022] Nat Prod Rep 2005, 22(2), 269 - 303.

[0023]

[11] Liaw, C.C.; Wu, T.Y.; Chang, F.R.; Wu, Y.C. Historic Perspectives on Annonaceous Acetogenins from the Chemical Bench to Preclinical Trials. Planta Med 2010, 76(13), 1390 - 1404. Summary of the Invention

[0024] I. Experimental Materials

[0025] Compounds 1 - 7 were all isolated from the chloroform fraction of the seeds of Annona montana Macf.

[0026] Sorafenib (MB1666, Dalian Meilun Biotechnology Co., Ltd.); DMEM medium (319 - 005 - CL), fetal bovine serum FBS (086 - 150), phosphate - buffered saline PBS (311 - 101 - CL) were all from Nanjing Vicent Biotechnology Co., Ltd.; Trypsin (2500 - 72, Gibco, USA); Penicillin - streptomycin mixture (KGY003) and Trypan Blue (KGY015) were both from Nanjing KeyGen Biotech Co., Ltd.; 3 - (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyltetrazolium bromide MTT (298 - 93 - 1, Biosharp); Dimethyl sulfoxide DMSO (RNBH8338, Merck, Germany); Matrigel high - concentration matrix (354248, Corning, USA); Hematoxylin - eosin staining solution (Shanghai Daixuan Biotechnology Development Co., Ltd.); TUNEL kit (Roche, Switzerland). Cell culture consumables were from Corning, USA. Other analytical - grade reagents were from Shanghai National Pharmaceutical Group Co., Ltd.

[0027] Human liver cancer cell lines HepG2, HuH - 7, MHCC97H and HCCLM3 were all provided by the Liver Cancer Research Institute of Fudan University for co - anti - tumor activity testing; human embryonic kidney cell line HEK293T was purchased from CTCC for cytotoxic activity testing.

[0028] BALB / c nude mice (18 - 22 g, half male and half female) were purchased from Shanghai SIPPR - BK Laboratory Animal Co., Ltd. for in - vivo pharmacodynamic evaluation.

[0029] II. Experimental methods

[0030] 2.1 Cell culture

[0031] Human liver cancer cell lines HepG2, HuH - 7, MHCC97H and HCCLM3, or human embryonic kidney cell line HEK293T were all inoculated at an appropriate concentration in DMEM medium containing 10 - 20% FBS. If there are no special requirements, DMEM medium containing 10% FBS is routinely used. The cells were cultured in a cell culture incubator at 37°C, 5% CO2 and saturated humidity. Passage and medium change were carried out according to the different growth conditions of the cells.

[0032] 2.2 MTT method for determining the inhibition of liver cancer cell proliferation by the combination of compounds and SF

[0033] Take cells in the logarithmic growth phase, digest and centrifuge them, then add complete medium to prepare a cell suspension with a specific concentration, and inoculate it in a 96-well plate. After culturing the cells for 12 h, administer the drugs. The final concentration gradients of sorafenib and the compound are set to 0, 1, 2, 4, 8, and 16 μM. After 48 h of drug intervention, add 20 μL of MTT solution to each well and continue to incubate for 4 h. Discard the supernatant, add 150 μL of DMSO, and measure the OD value at 490 nm. The experimental results are repeated three times.

[0034] 2.3 Evaluation of drug combination effects

[0035] Use mathematical modeling in the Synergy Finder software to evaluate the synergy index of combined drugs. Among the four mathematical models, ZIP, Loewe, HSA, and Bliss, analyze the synergy index (Synergy Score) for each concentration combination. An average synergy index greater than 5 is considered that the compound has synergy activity.

[0036] III. Results of synergy activity tests

[0037] In vitro synergy screening of annonaceous acetogenins 1-7 against hepatocellular carcinoma was carried out. Use the isobologram and Synergy Finder software to evaluate the synergy activity, and it was found that annonaceous acetogenins 1-7 showed certain synergy activity against different hepatocellular carcinoma cell lines.

[0038] 3.1 Annonaceous acetogenin synergizes with sorafenib against hepatocellular carcinoma HepG2 cells

[0039] The results of the synergy test showed that when annonaceous acetogenins 2, 3, 5, and 6 synergized with sorafenib against the proliferation of HepG2 cells, the combined inhibitory effect on cell proliferation was poor, and there was almost no synergistic effect against hepatocellular carcinoma. Compounds 3 and 5 showed certain synergistic anti-hepatocellular carcinoma effects only at high doses (16 μM), while antagonized sorafenib in most dosing combinations.

[0040] Compounds 1 and 4 showed certain synergistic anti-hepatocellular carcinoma activity, and most dosing combinations were able to inhibit the proliferation of hepatocellular carcinoma cells. However, the average value of the synergy index was less than 5, indicating that the ability of the compounds to synergistically inhibit HepG2 cells was poor.

[0041] 3.2 Annonaceous acetogenin synergizes with sorafenib against hepatocellular carcinoma HuH7 cells

[0042] The results of the synergy test showed that compounds 1 and 7 had certain synergy activity only at specific concentrations (1:1 = 16 μM, 7:SF = 1-4 μM), but some of the synergy indices were <5, indicating that compounds 1 and 7 had only weak synergy anti-hepatocellular carcinoma activity.

[0043] The synergy indices of Compounds 4, 5, and 6 are all greater than 5, indicating that Compounds 4, 5, and 6 have synergistic anti - liver cancer activity. Among them, Compounds 5 and 6 have better synergistic activity when sorafenib is at a lower dose (SF = 1 - 4 μM).

[0044] 3.3 Annonaceous acetogenins synergize with sorafenib against liver cancer MHCC97H cells

[0045] The synergy test results show that Compounds 2, 3, 4, and 6 have a certain ability to inhibit tumor growth in most drug combinations. Among them, the average synergy index of Compound 4 is greater than 17, indicating that Compound 4 has strong activity in synergizing with sorafenib against liver cancer MHCC97H cells

[0046] 3.4 Annonaceous acetogenins synergize with sorafenib against liver cancer HCCLM3 cells

[0047] The synergy test results show that in the drug combinations of compound concentrations (1 - 16 μM) and sorafenib (1 - 16 μM), Compounds 1 - 7 all have synergistic anti - liver cancer activity. At the same time, the synergy indices (Table 3 - 15) show that the synergy indices of Compounds 1 - 7 are all greater than 10, indicating that Compounds 1 - 7 are highly specific to LM3 and all have a good ability to synergize with sorafenib in inhibiting the growth of liver cancer cells.

[0048] In summary, annonaceous acetogenins 1 - 7 all synergistically inhibit the proliferation of liver cancer cells to varying degrees with sorafenib.

[0049] IV. Annonaceous acetogenins reduce the ATP level in liver cancer cells

[0050] The ATP experiment shows that both the compounds and sorafenib alone acting on liver cancer cells can reduce the intracellular ATP level. It is speculated that the compounds affect the cell growth metabolism, thereby causing cell apoptosis or necrosis, resulting in the reduction of the ATP level. When Compound 1 (8 μM) is used in combination with sorafenib, it can synergistically reduce the intracellular ATP level in all four types of liver cancer cells, while there is no significant difference when used in combination with sorafenib at a lower concentration. Compound 4 (4, 8 μM) used in combination with sorafenib can both produce a certain effect of reducing the intracellular ATP level, and the results are significantly different. The reduction of the ATP concentration is basically consistent with the MTT results.

[0051] V. Annonaceous acetogenins synergize with sorafenib to induce apoptosis of liver cancer LM3 cells

[0052] Flow cytometry experiments showed that compared with the blank group and the sorafenib single-drug group, after treating LM3 cells with acetogenin compounds 1 and 4 (4 μM) in combination with the same concentration of SF, the apoptosis rate in the combination drug group increased by approximately 3-4 times (P<0.05), indicating that the combination of compounds 1 and 4 with SF could synergistically induce apoptosis in LM3 cells. These findings were consistent with the results obtained from the cell MTT activity assay.

[0053] VI. Acetogenin synergizes with sorafenib to inhibit tumor growth

[0054] Nude mouse xenograft tumor experiments showed that compound 1 had good anti-tumor activity alone and synergistic anti-tumor activity.

[0055] Compound 1 at a concentration of 8 mg / kg had good anti-tumor activity alone and could inhibit tumor growth.

[0056] The tumor weights of the medium-high dose combination group (1:4 mg / kg, SF:32 mg / kg) and the high dose combination group (1:8 mg / kg, SF:32 mg / kg) decreased by approximately 4-6 times compared with the blank group (P<0.0001), and decreased by approximately 3-5 times compared with the sorafenib single-drug group (P<0.05, P<0.001), indicating that the combination of drugs could significantly enhance the inhibitory effect on nude mouse xenograft tumors of liver cancer. The corresponding tumor inhibition rates were calculated based on the average tumor weight, with the medium-high dose combination group and the high dose combination group being 71.1% and 82.0% respectively.

[0057] VII. Acetogenin synergizes with sorafenib to induce apoptosis in tumor cells

[0058] The apoptosis of tumor cells in the xenograft tumor model was detected by double staining of TUNEL / DAPI on the sliced tumor tissues. Different degrees of cell apoptosis occurred in all the test groups. Compared with the sorafenib single-administered group, the combination group had a stronger inhibitory effect on nude mouse xenograft tumors, and the apoptosis rate increased by 2-9 times. Compared with the compound single-administered group, the apoptosis rate increased by 2-3 times, and the results were significantly different (#p<0.05, p<0.001). Therefore, compound 1 and sorafenib could synergistically induce apoptosis in tumor cells.

[0059] VIII. Preliminary safety evaluation of acetogenin synergizing with sorafenib in anti-tumor

[0060] The biosafety of the combined administration of compound 1 and sorafenib was evaluated by HE staining of pathological sections of the main organs of nude mice. The HE results showed that there were no obvious lesions in all the test groups. In vitro experiments showed that compound 1 had certain cytotoxicity and showed a dose-dependent manner. Therefore, using a lower dose of the compound during combined drug use could reduce the toxic side effects brought by the acetogenin compound.

[0061] IX. Transcriptome analysis of the potential mechanism of action of annonaceous acetogenins

[0062] Transcriptome analysis showed that genes related to immune activation changed after co - administration, such as JAK - STAT, T - cell receptor, etc. At the same time, Compound 1 inhibited the lipid metabolism pathway up - regulated when sorafenib was used alone. Comparing with the predicted targets by Swiss TargetPrediction, it was speculated that the potential action target of Compound 1 might be related to SLC33A1.

[0063] X. Statistical analysis

[0064] The data were processed using Graphpad Prism 8.0.2 statistical software. One - way ANOVA was used for the differential comparison between two groups. * P < 0.05 indicates that the difference is statistically significant. Description of the drawings

[0065] Figure 1 . Structural formulas of annonaceous acetogenins 1 - 7

[0066] Figure 2 . Effects of the combination of Compounds 1 - 7 and SF on the proliferation of HepG2 cells.

[0067] (A, C, E, G, I, K, M are the dose - effect curves of the compounds and SF alone or in different concentration combinations after 48 - h treatment; B, D, F, H, J, L, N are the equivalent heat maps of combination drug use (ZIP model). Orange indicates synergistic effect, blue represents antagonistic effect, and the darker the color, the stronger the corresponding effect)

[0068] Figure 3 . Effects of the combination of Compounds 1 - 7 and SF on the proliferation of HuH7 cells.

[0069] (A, C, E, G, I, K, M are the dose - effect curves of the compounds and SF alone or in different concentration combinations after 48 - h treatment; B, D, F, H, J, L, N are the equivalent heat maps of combination drug use (ZIP model). Orange indicates synergistic effect, blue represents antagonistic effect, and the darker the color, the stronger the corresponding effect)

[0070] Figure 4 . Effects of the combination of Compounds 1 - 7 and SF on the proliferation of MHCC97H cells.

[0071] (A, C, E, G, I, K, M are the dose - effect curves of the compounds and SF alone or in different concentration combinations after 48 - h treatment; B, D, F, H, J, L, N are the equivalent heat maps of combination drug use (ZIP model). Orange indicates synergistic effect, blue represents antagonistic effect, and the darker the color, the stronger the corresponding effect)

[0072] Figure 5 . Effects of the combination of Compounds 1-7 and SF on the proliferation of HCCLM3 cells.

[0073] (A, C, E, G, I, K, M are the dose-effect curves after treatment with the compound and SF alone or in different concentration combinations for 48 h; B, D, F, H, J, L, N are the equivalent heat maps (ZIP model) of the combined drugs. Orange indicates synergistic effect, blue represents antagonistic effect, and the darker the color, the stronger the corresponding effect)

[0074] Figure 6 . Heat map of the synergy index of Compounds 1-7. (ZIP model, an average synergy index greater than 5 indicates that the compound has synergistic activity).

[0075] Figure 7 . Compound 1 synergistically reduces the ATP level of hepatoma cells with sorafenib. (HepG2: A-B; HuH7: C-D; 97H: E-F; LM3: G-H. Compound (8 μM): ** p < 0.01, *** p < 0.001, **** p < 0.0001; Compound (4 μM): ### p < 0.001, #### p < 0.0001)

[0076] Figure 8 . Compound 4 synergistically reduces the ATP level of hepatoma cells with sorafenib. (HepG2: A-B; HuH7: C-D; 97H: E-F; LM3: G-H. Compound (8 μM): * p < 0.05, ** p < 0.01, **** p < 0.0001; Compound (4 μM): ### p < 0.001, #### p < 0.0001)

[0077] Figure 9 . Effects of the combination of Compounds 1 and 4 and SF on the apoptosis of LM3 cells.

[0078] (A: Results after Annexin-FITC / PI staining of SF, Compounds 1 and 4 alone and in combination at a concentration of 4 μM. B: Histogram of the total apoptosis rate of cells. * p < 0.05, **** p < 0.0001)

[0079] Figure 10. Effects of different concentration combinations of Compound 1 and SF on the xenograft tumor model in nude mice (A: Line graph of tumor volume changes; B: Scatter plot of tumor weight; C: Line graph of nude mouse body weight changes; D: Tumor tissue diagram). **** p < 0.0001 vs. vehicle; # p < 0.05, ### p < 0.001 vs. SF)

[0080] Figure 11 . Effects of Compound 1 in combination with SF on tumor apoptosis in xenograft tumor-bearing nude mice.

[0081] (A: Confocal microscopy images of tumor tissue after double staining with TUNEL / DAPI; B: Percentage of TUNEL-positive cells in tumor tissue. * p < 0.05, *** p < 0.001 vs. vehicle; # p < 0.05, ## p < 0.01 vs. SF).

[0082] Figure 12 . HE-stained sections of major organs in nude mice.

[0083] (A: Confocal microscopy images of tumor tissue after double staining with TUNEL / DAPI; B: Percentage of TUNEL-positive cells in tumor tissue. * p < 0.05, *** p < 0.001 vs. vehicle; # p < 0.05, ## p < 0.01 vs. SF)

[0084] Figure 13 . Transcriptomic analysis of the combined inhibition of xenograft tumors by Compound 1 and SF. (A) Swiss TargetPrediction analysis of the top 15 targets of the interaction between Compound 1 and SF. (B) 41 predicted targets of the combined use of Compound 1 and sorafenib. (C) Volcano plot of differential gene expression, with red representing upregulated genes and green representing downregulated genes. (D) ssGSEA pathway expression difference map. (E) Gene set enrichment analysis of interferon α and E2F target genes. (F) M1-M6 modules identified in WGCNA analysis. (G) Pathway enrichment analysis of the M4 module. (H) 13 Hub genes identified by protein-protein interaction network. (I) Venn diagram of Compound 1 target genes. Detailed implementation methods

[0085] Example 1: Annonaceous acetogenin compounds synergistically inhibit the proliferation of hepatoma cells with sorafenib

[0086] I. Instruments and materials

[0087] 1.1 Cell lines

[0088] Human hepatoma cell lines HepG2, HuH-7, MHCC97H and HCCLM3 were all provided by the Liver Cancer Research Institute of Fudan University, and the human embryonic kidney cell line HEK293T was stored in our laboratory.

[0089] 1.2 Experimental instruments

[0090] Electronic balance (YP2001, Shanghai Liangping Instrument Co., Ltd.); CO₂ incubator (MCO-5AC, SANYO, Japan); Laminar flow cabinet (CA-920-3, Shanghai Shangjing Purification Equipment Co., Ltd.); Inverted microscope (IMT, OLYMPUS, Japan); ELISA reader (Multiskan FC, Thermo, USA); Electric thermostatic water bath (CU420, Shanghai Hengyi Technology Co., Ltd.); Autoclave (SYQ-DSX-280B, Shanghai Shen'an Medical Instrument Factory); Hemocytometer (XB-K-25, Shanghai Xinya Optical Instrument Factory); Low-speed tabletop centrifuge (KA-1000C, Shanghai Anting Co., Ltd.); Liquid nitrogen tank (YDS-30, Chengdu Jinfeng Liquid Nitrogen Container Co., Ltd.).

[0091] 1.3 Experimental reagents and materials

[0092] Sorafenib (MB1666, Dalian Meilun Biotechnology Co., Ltd.); DMEM medium (319-005-CL), fetal bovine serum FBS (086-150), phosphate buffer PBS (311-101-CL) were all from Nanjing Vincent Biotechnology Co., Ltd.; Trypsin (2500-72, Gibco, USA); Penicillin-streptomycin mixture (KGY003) and Trypan Blue (KGY015) were both from Nanjing KeyGen Biotech Co., Ltd.; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT (298-93-1, Biosharp); Dimethyl sulfoxide DMSO (RNBH8338, Merck, Germany); Cell culture consumables were from Corning, USA. Other analytical grade reagents were from Shanghai National Pharmaceutical Group Co., Ltd.

[0093] II. Experimental methods

[0094] 2.1 Cell culture

[0095] Cell culture and passage: Human hepatoma cell lines HepG2, HuH-7, MHCC97H and HCCLM3, or the human embryonic kidney cell line HEK293T were inoculated into DMEM medium containing FBS at appropriate concentrations. The cells were cultured in a CO₂ incubator at 37°C.

[0096] 2.2 Preparation of related solutions

[0097] 1) Sorafenib (SF): Dissolve it in DMSO to prepare the sorafenib stock solution.

[0098] 2) Test samples: Weigh a certain amount of the crude extract, dissolve it fully in DMSO to prepare the stock solution. Dilute it to the required concentration with complete medium before adding the drug.

[0099] 2.3 Test on the anti - liver cancer activity of the crude extract of Annona squamosa L. in combination with sorafenib

[0100] Setting of drug concentrations and grouping: The final concentration gradients of sorafenib and the compound are set as 0, 1, 2, 4, 8, 16 μM.

[0101] Adding drugs: Add 50 μL of single drugs and dual drugs with different concentrations into 96 - well plates. Add blank complete medium to the 0 - concentration group. The drug intervention is based on the final concentration as the dosing concentration. Set two replicates for each group of experiments.

[0102] Color development: After incubating with drugs for 48 h, add 20 μL of 5 mg / mL MTT solution to each well and continue culturing in the cell incubator for 4 h. Aspirate the supernatant with a 1 - mL disposable syringe and add 150 μL DMSO to dissolve it fully.

[0103] Detection: Measure the absorbance (OD) value at 490 nm using an enzyme - linked immunosorbent monitor.

[0104] Calculation: Cell Viability (%) = (ODdrug - administered group - ODblank group) / (ODcontrol group - ODblank group) * 100%

[0105] 2.4 Evaluation of the combined effect of drugs

[0106] Use mathematical modeling in the Synergy Finder software to evaluate the synergy index of the combined drugs. Among them, use the ZIP model to analyze the synergy index (Synergy Score) for each concentration combination. Orange represents a synergistic effect under this drug combination, and blue represents an additive / antagonistic effect under this drug combination. An average synergy index greater than 5 is considered that the compound has synergistic activity.

[0107] III. Results of synergistic activity screening

[0108] To study the anti - liver cancer effect of the combination of compounds and sorafenib, MTT assay was used to detect the cell viability after incubation with different drug combinations for 48 h, and Synergy Finder software was used for analysis. Four mathematical models, ZIP, Loewe, HSA and Bliss, were used to score the synergy index of the compound - synergistic drugs. For most compounds, the drug concentrations were set within the range of IC 50 range.

[0109] 3.1 Annona Compounds Synergize with Sorafenib against Hepatocellular Carcinoma HepG2 Cells

[0110] Using hepatocellular carcinoma HepG2 cells, the effects of the combination of compounds 1 - 7 and sorafenib on cell proliferation were studied ( Figure 2 , Table 1).

[0111] The MTT results showed that when annonaceous acetogenins 2, 3, 5, 6 synergized with sorafenib against HepG2 cell proliferation, the combined anti - liver cancer effect was poor, and there was almost no synergistic anti - liver cancer effect. Compounds 3 and 5 showed certain anti - liver cancer synergy only at high doses (16 μM), while antagonized sorafenib in most drug combinations. Compounds 1 and 4 showed certain synergistic anti - liver cancer activity, and most drug combinations could inhibit the proliferation of liver cancer cells. The average synergy index of compounds 1 and 4 was less than 5, indicating that the synergistic anti - liver cancer ability of the compounds was not strong.

[0112] Table 1. Synergy Index and Drug Sensitivity Index (CSS) of Compounds 1 - 7 against HepG2

[0113]

[0114] a The mean value of each medication combination.

[0115] b CSS was calculated based on the IC 50 of two drugs.

[0116] 3.2 Annona Compounds Synergize with Sorafenib against Hepatocellular Carcinoma HuH7 Cells

[0117] Using hepatocellular carcinoma HuH7 cells, the effects of the combination of compounds 1 - 7 and sorafenib on cell proliferation were studied ( Figure 3, (Table 2). The MTT results showed that the synergy indices of compounds 2 and 3 against liver cancer HuH7 cells were negative under most dosing combinations, and the mean ZIP index was negative. Therefore, compounds 2 and 3 did not have synergistic anti-liver cancer activity. Compounds 1 and 7 only had certain synergistic activity at specific concentrations (1:1 = 16 μM, 7:SF = 1 - 4 μM), but some of the synergy indices were < 5, indicating that compounds 1 and 7 only had weak synergistic anti-liver cancer activity. The average synergy indices of compounds 4, 5, and 6 were all greater than 5, indicating that 4, 5, and 6 had anti-liver cancer activity. Among them, compounds 5 and 6 had better synergistic activity when sorafenib was at a lower dose (SF = 1 - 4 μM).

[0118] Table 2. Synergy indices and drug sensitivity indices (CSS) of compounds 1 - 7 against HuH7

[0119]

[0120] a The mean value of each medication combination.

[0121] b CSS was calculated based on the IC 50 of two drugs.

[0122] 3.3 Antagonistic effects of annonaceous acetogenins in combination with sorafenib against liver cancer MHCC97H cells

[0123] Using liver cancer MHCC97H cells, the effects of the combination of compounds 1 - 7 and sorafenib on cell proliferation were studied ( Figure 4 , Table 3). The MTT results showed that compounds 1, 5, and 7 did not have or only had weak synergistic anti-liver cancer activity with sorafenib.

[0124] Compounds 2, 3, 4, and 6 had certain ability to inhibit liver cancer growth under most dosing combinations. Among them, compound 4 had the best activity, and the synergy index was greater than 15, indicating that compound 4 had strong synergistic anti-liver cancer activity against MHCC97H cells with sorafenib.

[0125] Table 3. Synergy indices and drug sensitivity indices (CSS) of compounds 1 - 7 against MHCC97H

[0126]

[0127] a The mean value of each medication combination.

[0128] b CSS was calculated based on the IC 50 of two drugs.

[0129] 3.4 Annonaceous compounds synergize with sorafenib against HCCLM3 hepatocellular carcinoma cells

[0130] Using HCCLM3 hepatocellular carcinoma cells, the effects of the combination of compounds 1 - 7 and sorafenib on cell proliferation were studied ( Figure 5 , Table 4). The MTT results showed that under the dosing combinations of compound concentrations (1 - 16 μM) and sorafenib (1 - 16 μM), compounds 1 - 7 all had synergistic anti - hepatocellular carcinoma activity. The average synergy index of compounds 1 - 7 was greater than 10, indicating that compounds 1 - 7 had strong specificity for LM3 and all had good ability to synergistically inhibit the growth of hepatocellular carcinoma with sorafenib.

[0131] Table 4. Synergy index and drug sensitivity index (CSS) of compounds 1 - 7 against HCCLM3

[0132]

[0133] a The mean value of each medication combination.

[0134] b CSS was calculated based on the IC 50 of two drugs.

[0135] Example 2: Annonaceous lactone compounds synergize with sorafenib to reduce the ATP level of hepatocellular carcinoma cells and induce apoptosis 1.1 The combination of annonaceous lactone and SF reduces the ATP level of hepatocellular carcinoma cells

[0136] According to the screening results of the MTT synergy experiment, corresponding compound (2, 4, 8 μM) and sorafenib (8, 16 μM) concentrations were selected for combination to detect the effect of their combined use on the ATP level of hepatocellular carcinoma cells.

[0137] The ATP experiment showed ( Figures 7 - 8) When the compound and sorafenib act on liver cancer cells alone, they can both reduce the ATP level in the cells. It is speculated that the compound affects the cell growth and metabolism, thereby causing apoptosis or necrosis of the cells, resulting in the reduction of the ATP level. When compound 1 (8 μM) is used in combination with sorafenib, it can synergistically reduce the ATP level in all four liver cancer cell lines, while there is no significant difference when used in combination with sorafenib at a lower concentration. When compound 4 (4, 8 μM) is used in combination with sorafenib, it can both produce a certain effect of reducing the ATP level in the cells, and the results are significantly different. The reduction of the ATP concentration is basically consistent with the MTT results.

[0138] 1.2 Annonaceous acetogenins and SF combination induce apoptosis of liver cancer LM3 cells

[0139] After the HCCLM3 cells were treated with drugs for 48 h, they were stained with Annexin-FITC / PI and analyzed by flow cytometry ( Figure 9 , Table 5). At a concentration of 4 μM, compounds 1 and 4 only had a slight effect on inducing cell apoptosis, and the apoptosis rates were 2.39% and 1.28% respectively. When the cells were treated with sorafenib alone at 4 μM, the apoptosis rate was 4.79%. When compounds 1 and 4 were used in combination with sorafenib, they could produce an obvious synergistic effect on inducing apoptosis of HCCLM3, and the apoptosis rates of the synergistic groups were 18.54% and 11.61% respectively, and the results were significantly different.

[0140] Table 5. Effects of the combination of compound 1 and SF on apoptosis of liver cancer LM3 cells

[0141]

[0142] Example 3. Annonaceous acetogenin compounds synergistically inhibit the growth of liver cancer xenografts with sorafenib

[0143] For cell-derived xenograft (CDX) of human tumor cell lines, human tumor cells are inoculated into immunodeficient mice by subcutaneous or orthotopic injection to form tumors in the mice, so as to observe the effects of the test substances on tumor growth. The in vivo synergistic anti-hepatocellular carcinoma activity of compound 1 with sorafenib was studied through the CDX subcutaneous xenograft tumor model.

[0144] I. Instruments and materials

[0145] 1.1 Animals and cell lines

[0146] BALB / c nude mice (18 - 22 g, half male and half female) were purchased from Shanghai SIPPR-BK Laboratory Animal Co., Ltd. The human hepatocellular carcinoma cell line HCCLM3 was provided by the Cancer Institute of Fudan University.

[0147] 1.2 Experimental instruments

[0148] Dehydrator (HP300, Shenzhen Dakewei Medical Equipment Co., Ltd.); Paraffin embedding machine (TKY-BMB, Hubei Taikang Medical Equipment Co., Ltd.); Tissue spreading machine (KD-P, Jinhua Cody Instrument and Equipment Co., Ltd.); Microtome (3000A, Shenzhen Dakewei Medical Equipment Co., Ltd.).

[0149] 1.3 Experimental reagents and materials

[0150] Hematoxylin-eosin staining solution (Shanghai Daixuan Biotechnology Development Co., Ltd.); TUNEL kit (Roche, Switzerland). Cell culture consumables are from Corning, USA. Other analytical grade reagents are from Shanghai National Pharmaceutical Group Co., Ltd.

[0151] II. Experimental methods

[0152] 2.1 The specific method of cell culture is the same as that in Example 1.

[0153] 2.2 Preparation of relevant solutions

[0154] 1. 0.5% CMC-Na solution: 0.5 g of carboxymethyl cellulose sodium is dissolved in 100 mL of ultrapure water, and after full swelling, it is autoclaved (121 °C for 30 min) and then reserved for use.

[0155] 2. Sorafenib solution: Weigh an appropriate amount of sorafenib, add DMSO to prepare a stock solution for standby. Before administration, it is prepared according to the body weight of nude mice and diluted into suspension of different concentrations required for the experiment according to the formula of DMSO-Tween-80-0.5% CMC-Na = 5:5:90.

[0156] 3. Compound 1 solution: Weigh an appropriate amount of compound 1, add DMSO to prepare a stock solution for standby. Before administration, it is prepared according to the body weight of nude mice and diluted into suspension of different concentrations required for the experiment according to the formula of DMSO-Tween-80-0.5% CMC-Na = 5:5:90.

[0157] 2.3 Xenograft subcutaneous transplantation experiment of human tumor cell lines

[0158] Establish a xenograft tumor model of human tumor cell lines. Nude mice about 4 weeks old are raised in a SPF-class animal room and inoculated with HCCLM3 cells after 3-4 days of adaptive culture.

[0159] Preparation of cell suspension: Take LM3 cells in the logarithmic growth phase, add 0.25% trypsin for digestion. After centrifugation, the supernatant is aspirated and a cell suspension with a density of 5 - 10*10 7 cells / mL is prepared. Each nude mouse is inoculated with 0.1 mL under the armpit.

[0160] Observe and record the growth status of the tumor. When the tumor grows to the corresponding volume, group the animals (n = 7, including 4 female mice and 3 male mice), and simultaneously administer the corresponding drugs.

[0161]

[0162] a The blank group was given a 0.5% CMC-Na solution containing 5% DMSO and 5% Tween-80.

[0163] The administration method was intragastric administration, and each nude mouse was administered every other day at a dose of 0.1 mL / 10 g. Euthanasia was performed 24 h after the last administration, and the tumors and corresponding organs were photographed and dissected.

[0164] Tumor volume calculation formula: V = 0.52 * L * W 2 (L is the long diameter, W is the short diameter)

[0165] Relative Tumor Volume (RTV): RTV = V n / V0 (V n is the tumor volume on the nth day, and V0 is the initial tumor volume)

[0166] Tumor inhibition rate (%) = (average tumor weight of the blank group - average tumor weight of the drug administration group) / average tumor weight of the blank group * 100%. Or the tumor inhibition rate (%) = (RTV vehicle -RTV treatment) / RTV vehicle ) * 100% represents the tumor inhibition rate.

[0167] 2.4 Histopathological examination of tumor tissues

[0168] After euthanasia of the nude mice, the tumor tissues were dissected, rinsed twice with PBS, and then fixed in 4% paraformaldehyde solution. Impregnation with paraffin and embedding were performed, and the sections were cut into 5 - 8 μm thin slices. After dewaxing, hematoxylin-eosin (HE) staining and TUNEL / DAPI staining were used, and dehydration and mounting were carried out, followed by scanning and observation of the pathological sections.

[0169] 2.5 Transcriptome analysis of tumor tissues with reference

[0170] After euthanasia of the nude mice, the tumor tissues were dissected, rinsed twice with PBS, and then quickly frozen in liquid nitrogen. Transcriptome analysis was performed on the blank group, sorafenib group, high-dose compound 1 group, and high-dose combination group. Differentially expressed genes were identified based on the gene expression levels in different samples, and functional analysis of the selected differentially expressed genes such as Pathway analysis was carried out.

[0171] 2.6 Safety evaluation

[0172] When the animal experiment cycle reached the end point, the nude mice were euthanized and their hearts, livers, spleens, lungs, and kidneys were removed. After being rinsed twice with PBS, they were fixed with 4% paraformaldehyde solution. They were then infiltrated with wax, embedded, and cut into thin sections of 5 - 8 μm. After dewaxing, hematoxylin - eosin (HE) staining was performed. After dehydration and sealing, the sections were scanned and observed.

[0173] III. In vivo pharmacodynamic evaluation of annonaceous acetogenins combined with sorafenib

[0174] 3.1 Synergistic inhibition of the growth of hepatocellular carcinoma xenografts by the combination of compound 1 and sorafenib

[0175] By constructing a human hepatocellular carcinoma LM3 cell xenograft subcutaneous tumor model, intragastric administration was carried out every two days, and the body weight and tumor diameter of the nude mice were measured. The change curve of the nude mice's body weight was plotted, and at the same time, the rough tumor volume was calculated and plotted into the corresponding tumor growth curve to investigate the effects of compound 1, sorafenib alone or in combination on tumor growth during the administration period ( Figure 10 ). At the end of the administration period, the tumors were dissected, weighed, and the tumor weight scatter plot was drawn, and the corresponding tumor inhibition rate was calculated.

[0176] Compared with the blank group, all experimental groups had certain anti - tumor activities in inhibiting tumor growth. The trend of tumor volume change at a compound 1 concentration of 4 mg / kg was similar to that of sorafenib used alone, and the average tumor volume at the end of the administration was basically the same, but there were large differences among groups of compound 1 (4 mg / kg). Compound 1 at a concentration of 8 mg / kg had good anti - tumor activity alone and could inhibit tumor growth. The inhibitory effects of the low - dose synergistic group and the medium - low - dose synergistic group on tumor volume growth were comparable. It was speculated that the anti - tumor activity of the compound at low doses was not obvious. The tumor weights of the medium - high - dose synergistic group and the high - dose synergistic group had better tumor inhibitory effects compared with the blank group (P < 0.0001), and also had better anti - tumor activities compared with the group using sorafenib alone (P < 0.05, P < 0.001). Compared with the medium - dose group, the combined - drug volume of the medium - high - dose synergistic group was smaller (P < 0.05), indicating that the combined drug could significantly enhance the inhibitory effect on hepatocellular carcinoma xenografts in nude mice. The corresponding tumor inhibition rates were calculated through the average tumor weight, among which the medium - high - dose synergistic group and the high - dose synergistic group were 71.1% and 82.0% respectively.

[0177] Table 7. Effects of different concentration combinations of compound 1 and SF on the nude mouse xenograft tumor model

[0178]

[0179] 3.2 Synergistic promotion of tumor cell apoptosis and safety evaluation by the combination of compound 1 and sorafenib

[0180] The apoptosis of the transplanted tumor model was detected by double staining of sliced tumor tissues with TUNEL / DAPI( Figure 11 ). DAPI staining can penetrate the cell membrane and bind to double-stranded DNA in the nucleus to play a labeling role, while TUNEL staining can detect the cleavage of nuclear DNA during apoptosis. Through microscopic observation and analysis of fluorescence intensity by Image J, different degrees of cell apoptosis occurred in the drug administration groups. The apoptosis rate of the compound alone treatment group increased with the increase of the compound concentration, and the apoptosis rates were 12.5% and 29.0% respectively. Compared with the sorafenib alone administration group, the synergistic group had a stronger inhibitory effect on the transplanted tumors of nude mice. Moreover, obvious cell apoptosis occurred when the compound administration concentration was 4 mg / kg and 8 mg / kg, and the results were significantly different (#p < 0.05, p < 0.001). Therefore, compound 1 and sorafenib have a certain synergistic anti-tumor effect.

[0181] Table 8. Apoptosis rate of tumor cells in the combination of compound 1 and SF

[0182]

[0183] 3.3 Preliminary safety evaluation of the combination of compound 1 and sorafenib

[0184] The biosafety of the co-administration of compound 1 and sorafenib was evaluated by HE staining of pathological sections of the main organs of nude mice( Figure 12 ). The HE results showed that there were no obvious lesions in the hearts of all the tested groups. The hepatic lobule structure was clear, and the hepatic sinusoids were not significantly compressed, narrowed or disappeared. The red and white pulp structures of the spleen were relatively clear. In the lungs, the alveolar wall was not significantly thickened, the alveolar structure was clear, and there were no obvious lesions; the glomerular morphology was normal in the drug administration groups, and the renal tubular epithelial cells were not edematous. The above experimental results indicate that low-dose compound 1 has no obvious cytotoxicity.

[0185] 3.4 Effects of the combination of compound 1 and sorafenib on the transcriptome genes of transplanted tumors

[0186] To explore the molecular mechanism of the action of compound 1 and sorafenib, we predicted the potential targets of compound 1 using the Swiss TargetPrediction website. As Figure 13 shown in A, 40% of the top 50 targets were related to the regulation of protein phosphorylation (30% were kinases and 10% were phosphatases), which was consistent with the classical mechanism of sorafenib. At the same time, compound 1 may play an anti-tumor role by affecting the remaining 60% of the targets related to protein phosphorylation. Among them, about 2% of the targets were major active transporters. Among the 41 predicted targets, it was found that compound 1 and sorafenib may affect processes such as cellular immune response and energy metabolism regulation( Figure 13B).

[0187] To further explore the interaction between sorafenib and compound 1 treatment, transcriptomic analysis was performed on four groups of xenograft tumor groups (blank group, sorafenib group, compound 1 single drug group, and combination group). Comparing the differential gene expression after drug administration ( Figure 13 C), 341 genes were found to have consistent changes, among which 118 genes were upregulated (red) and 223 genes were downregulated (green). The effect of combination drug administration on gene sets was analyzed using ssGSEA, and significant immune activation was shown after combination drug administration, such as JAK-STAT, T cell receptor, etc. ( Figure 13 D). However, compound 1 significantly inhibited various energy metabolisms that were slightly elevated after sorafenib administration, including lipid metabolism processes such as PPAR signaling, oxidative phosphorylation, and glycerolipid metabolism. GSEA analysis of the HALLMARK gene set showed that the interferon-α response was significantly activated, while pathways such as E2F targets were inhibited. ( Figure 13 E) A co-expression network was constructed through WGCNA analysis to determine the potential targets of compound 1 and sorafenib treatment. We identified a total of six modules (M1-M6, Figure 13 F). The M4 module in the compound 1 single drug group was negatively enriched, indicating that this module was inhibited, and the degree of inhibition increased significantly in the combination drug administration group. Pathway enrichment analysis showed that the genes in the M4 module were significantly enriched in lipid metabolism-related pathways, especially cholesterol metabolism ( Figure 13 G). PPI interaction network analysis identified 13 Hub genes ( Figure 13 H). By analyzing these 13 Hub genes and the predicted 41 targets, we identified that SLC33A1 (also known as acetyl-CoA transporter 1) might be a potential target for compound 1 in the treatment of advanced liver cancer ( Figure 13 I).

Claims

1. An acetogenin compound, characterized in that 2. The compound according to claim 1, wherein it synergistically inhibits the growth of human liver cancer cells with sorafenib; the concentration of the sorafenib is 1 - 16 μM or 32 mg / kg; the human liver cancer cells include HepG2, HuH7, MHCC97H, HCCLM3.

3. The compound according to claim 2, wherein Within the range of 1 - 16 μM action concentration, it has the effect of increasing the inhibition of liver cancer cells by sorafenib to varying degrees.

4. The compound according to claim 3, wherein, It has the effect of reducing the ATP level of liver cancer and / or inducing apoptosis of liver cancer cells, and the effective action concentration is 1 - 16 μM.

5. The compound according to claim 4, characterized in that, It has synergistic anti-tumor activity with sorafenib, and the effective action concentration is 1 - 8 mg / kg.

6. The compound according to claim 5, wherein The anti-liver cancer action target is related to the SLC33A1 gene.

7. The compound according to any one of claims 1-6, characterized in that, It is derived from plant isolation, including being obtained by chromatographic separation from Annona montana Macf.

8. Use of an acetogenin compound in the manufacture of an anti-liver cancer drug composition.

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