Pharmaceutical composition and application thereof in preparation of medicines for treating tumors

Abstract

The invention provides a pharmaceutical composition and application thereof in preparation of medicines for treating tumors. The pharmaceutical composition comprises the orbitrazine fumarate, a second active agent and pharmaceutically acceptable auxiliary materials. The second active agent comprises at least one of tamoxifen, irinotecan hydrochloride, sorafenib, cis-platinum, doxorubicin hydrochloride, paclitaxel and etoposide. The scheme provided by the invention has a remarkable synergistic anti-tumor effect, and a combined medication scheme is expected to provide a safer and more effective treatment choice for tumor patients, so that the pharmaceutical composition has an important clinical application value.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a pharmaceutical composition and its application in preparing a drug for treating tumors. Background Art

[0002] Cancer is one of the major diseases that pose a serious threat to human health worldwide, and its treatment has always been a key research focus in the medical field. Traditional cancer treatments include surgery, radiotherapy, and chemotherapy, but these methods often have certain limitations, such as the inability to completely remove tumor cells due to surgery, damage to normal tissues caused by radiotherapy, and the significant toxic side effects and drug resistance of chemotherapy drugs. In recent years, targeted therapy and immunotherapy have made significant progress, but further optimization is still needed. With the in-depth study of the mechanisms of tumor occurrence and development, the strategy of combining drugs to treat tumors has gradually attracted attention.

[0003] Caspase-3 is a key executor of apoptosis, and its precursor form is Procaspase-3. Procaspase-3 normally exists in the cytoplasm as an zymogen. Numerous studies have shown that Procaspase-3 is overexpressed in various tumor cells and tissues. Orbitazine fumarate, chemically known as (4-benzyl-[1,4]diazepan-1-yl)-acetyl(3-allyl-2-hydroxy-methylenephenyl)hydrazine fumarate, has the following chemical formula: It is a procaspase-3 activator that specifically activates procaspase-3, converting it into active caspase-3, thereby inducing apoptosis in tumor cells and exerting anti-tumor effects. Compared with traditional chemotherapy drugs, orbitazine fumarate has less impact on normal cells and has fewer toxic side effects. For example, literature reports (e.g., CN101503394B) indicate that homopiperazine acetylhydrazine derivatives (including orbitazine fumarate) have demonstrated good anti-tumor activity.

[0004] In addition, existing studies (CN117357533A) have shown that the combination of orbitazine fumarate and temozolomide exhibits good safety and efficacy in the treatment of recurrent high-grade gliomas. However, for other types of tumors, the combination of orbitazine fumarate with various chemotherapy drugs and targeted drugs and their potential therapeutic effects have not been fully studied and applied. Summary of the Invention

[0005] In order to solve the above problems, the present application provides a pharmaceutical composition and its use in preparing a drug for treating tumors.

[0006] On the one hand, the present application provides a pharmaceutical composition comprising: orbitazine fumarate; a second active agent comprising at least one of tamoxifen, irinotecan hydrochloride, sorafenib, cisplatin, doxorubicin hydrochloride, paclitaxel, and etoposide; and pharmaceutically acceptable excipients.

[0007] In some embodiments, the concentration of orbitazine fumarate is 0.0549 μM to 40 μM.

[0008] In some embodiments, the second active agent comprises tamoxifen and the tamoxifen concentration is 0.003 μM to 20 μM.

[0009] In some embodiments, the concentration ratio of tamoxifen to orbitazine fumarate is 1:0.2-162.

[0010] In some embodiments, the second active agent comprises irinotecan hydrochloride at a concentration of 0.003 μM to 6.667 μM.

[0011] In some embodiments, the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 1:5-162.

[0012] In some embodiments, the second active agent comprises sorafenib and the concentration of sorafenib is 0.003 μM to 2.222 μM.

[0013] In some embodiments, the concentration ratio of sorafenib to orbitazine fumarate is 1:18-162.

[0014] On the other hand, the present application provides a use of a pharmaceutical composition in the preparation of a drug for treating tumors, wherein the pharmaceutical composition is any of the above-mentioned pharmaceutical compositions.

[0015] In some embodiments, the tumor comprises at least one of pancreatic cancer, liver cancer, gastric cancer, multiple myeloma, ovarian cancer, malignant embryonal rhabdoid tumor, and breast cancer.

[0016] In some embodiments, the medicament comprises an effective amount of a pharmaceutical composition.

[0017] In some embodiments, the order of administration of the drugs is simultaneous or sequential.

[0018] In some embodiments, orbitazine fumarate is used to induce apoptosis in tumor cells, and the second active agent is used to kill tumor cells by other mechanisms.

[0019] In some embodiments, the drug is contacted with a tumor.

[0020] In some embodiments, the dosage forms of the drug include oral preparations, injections and other dosage forms.

[0021] In some embodiments, oral preparations include tablets, capsules, granules, sustained-release preparations, oral solutions, and other oral dosage forms.

[0022] This application provides a composition comprising orbitazine fumarate and a chemotherapeutic agent, a targeted drug, or an immunotherapy agent, as well as the use of this composition in the preparation of a medicament for treating tumors. This combination therapy has significant synergistic anti-tumor effects, and is expected to provide a safer and more effective treatment option for cancer patients, with significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without any creative effort.

[0024] Figure 1 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:40 μM.

[0025] Figure 2 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:13.333 μM.

[0026] Figure 3 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:4.444 μM.

[0027] Figure 4 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 6.667 μM:40 μM.

[0028] Figure 5 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 2.222 μM:40 μM.

[0029] Figure 6 The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 0.741 μM:40 μM.

[0030] Figure 7The figure is a curve diagram showing the relationship between the concentration of tamoxifen and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 0.247 μM:40 μM.

[0031] Figure 8 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:40 μM.

[0032] Figure 9 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:13.333 μM.

[0033] Figure 10 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:4.444 μM.

[0034] Figure 11 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 6.667 μM:40 μM.

[0035] Figure 12 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 2.222 μM:40 μM.

[0036] Figure 13 The figure is a curve diagram showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 0.741 μM:40 μM.

[0037] Figure 14 The graph is a graph showing the relationship between the concentration of orbitazine fumarate and the inhibition rate of human breast cancer cells when the concentration ratio of tamoxifen to orbitazine fumarate is 0.247 μM:40 μM.

[0038] Figure 15 The figure is a curve diagram showing the relationship between the concentration of tamoxifen alone and the inhibition rate of human breast cancer cells.

[0039] Figure 16 The figure is a curve diagram showing the relationship between the concentration of single-drug orbitazine fumarate and the inhibition rate of human breast cancer cells.

[0040] Figure 17The graph shows the relationship between the concentration of irinotecan hydrochloride and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 6.667 μM:40 μM.

[0041] Figure 18 The graph shows the relationship between the concentration of irinotecan hydrochloride and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 2.222 μM:40 μM.

[0042] Figure 19 The graph shows the relationship between the concentration of irinotecan hydrochloride and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.741 μM:40 μM.

[0043] Figure 20 The graph shows the relationship between the concentration of irinotecan hydrochloride and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.247 μM:40 μM.

[0044] Figure 21 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 6.667 μM:40 μM.

[0045] Figure 22 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 2.222 μM:40 μM.

[0046] Figure 23 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.741 μM:40 μM.

[0047] Figure 24 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human pancreatic cancer cells when the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.247 μM:40 μM.

[0048] Figure 25 The figure is a curve diagram showing the relationship between the concentration of irinotecan hydrochloride alone and the inhibition rate of human pancreatic cancer cells.

[0049] Figure 26 The graph shows the relationship between the concentration of orbitazine fumarate alone and the inhibition rate of human pancreatic cancer cells.

[0050] Figure 27The graph shows the relationship between the concentration of sorafenib and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 2.222 μM:40 μM.

[0051] Figure 28 The graph shows the relationship between the concentration of sorafenib and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 0.741 μM:40 μM.

[0052] Figure 29 The graph shows the relationship between the concentration of sorafenib and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 0.247 μM:40 μM.

[0053] Figure 30 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 2.222 μM:40 μM.

[0054] Figure 31 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 0.741 μM:40 μM.

[0055] Figure 32 The graph shows the relationship between the concentration of orbitazine fumarate and the inhibition rate of human liver cancer cells when the concentration ratio of sorafenib to orbitazine fumarate is 0.247 μM:40 μM.

[0056] Figure 33 The figure is a curve diagram showing the relationship between the concentration of sorafenib alone and the inhibition rate of human liver cancer cells.

[0057] Figure 34 The figure is a curve diagram showing the relationship between the concentration of a single drug of orbitazine fumarate and the inhibition rate of human liver cancer cells. DETAILED DESCRIPTION

[0058] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Unless otherwise specified, the kit materials used in the following examples are all commercially available.

[0059] The present application provides orbitazine fumarate with good anti-cancer effects and exhibits good synergistic effects when used in combination with various drugs. This application uses the CellTiter-Glo detection method as an example to illustrate the specific application.

[0060] Experimental methods

[0061] 1. Cell plating

[0062] (1) Prepare complete culture medium and mix thoroughly.

[0063] (2) Cell recovery and passaging, specifically including: taking out the cell cryopreservation tube from the liquid nitrogen tank, quickly thawing it in a 37°C water bath, transferring the cell suspension to a 15mL centrifuge tube with a pipette, and adding 10mL of complete culture medium; centrifuging at 1000rpm for 5 minutes, discarding the supernatant, resuspending the cell pellet with 5mL of complete culture medium, transferring it to a T75 culture flask, adding 15mL of culture medium, and culturing it in a 37°C, 5% carbon dioxide incubator; passaging the cells twice, and selecting cell lines with good growth status.

[0064] (3) Take the cell culture flask out of the incubator.

[0065] (4) Treat adherent cells, specifically including: aspirating the culture medium, washing with trypsin, discarding the waste liquid, and adding 3 mL of fresh trypsin to the culture flask for digestion. When the cells are loose and about to detach from the flask wall, add 9 mL of complete culture medium to stop the trypsin digestion and gently mix. Use a pipette to transfer the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Suspended cells: Aspirate the cell suspension and transfer it into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0066] (5) Discard the supernatant.

[0067] (6) Add an appropriate volume of culture medium to the centrifuge tube and gently pipette to resuspend the cells evenly.

[0068] (7) Count cells using a Vi-Cell XR cell counter.

[0069] (8) Adjust the cell suspension to an appropriate concentration.

[0070] (9) Add the cell suspension to a 96-well plate at 100 μL / well and place the plate in a CO2 incubator overnight.

[0071] 2. Preparation and addition of compound plates

[0072] (1) On the second day after seeding, prepare the compound solution and add it to the above cell wells using the HPD300 automatic dosing device according to the required concentration.

[0073] (2) Incubate in a 37°C 5% carbon dioxide incubator for 120 hours.

[0074] 3. Reagent preparation and testing

[0075] (1) Thaw CellTiter-Glo Buffer at room temperature and equilibrate the lyophilized CellTiter-Glo substrate to room temperature.

[0076] (2) Add CellTiter-Glo Buffer to CellTiter Glo substrate and mix thoroughly.

[0077] (3) Take out the cell plate and equilibrate it to room temperature.

[0078] (4) Add 100 μL of the mixed CellTiter Glo reagent to each well, shake in the dark for 10 minutes, and incubate for 10 minutes.

[0079] (5) Place the culture plate in a plate reader to detect the luminescent signal intensity of each well; calculate the inhibition rate according to the following formula: Inhibition rate (%) = (1-(RLU compound -RLU blank ) / (RLU DMSO –RLU blank ))×100%.

[0080] (6) Use XLFit to draw the drug efficacy inhibition rate curve and calculate IC 50 The values ​​were obtained using a 4-parameter model [fit = (A + ((BA) / (1 + ((C / x)^D))))].

[0081] Example 1

[0082] In this example, the test cells were human breast cancer cells (ZR-75-30), seeded at a density of 5600 cells / well, and the compound solutions were tamoxifen in dimethyl sulfoxide (DMSO) and orbitazine fumarate (SM-1) in DMSO. Following the above experimental method, the following 96-well plate layout was obtained, as shown in Table 1.1.

[0083] Table 1.1 Layout of Tamoxifen and Orbitazine Fumarate Combined Action on ZR-75-30 Cells

[0084]

[0085]

[0086] As can be seen from Table 1.1, the 96-well plate is divided into rows AH and columns 1-12.

[0087] The first column of row A is the blank control (Blank), that is, the composition in the well is culture medium.

[0088] The composition of each well in row A, columns 2-10 is culture medium, ZR-75-30 cells, 0.2% DMSO by volume, a fixed concentration of 40 μM orbitazine fumarate, and tamoxifen. Specifically, the concentrations of tamoxifen in row A are 20.0000 μM, 6.6667 μM, 2.2222 μM, 0.7407 μM, 0.2469 μM, 0.0823 μM, 0.0274 μM, 0.0091 μM, and 0.0030 μM from columns 2 to 10. In other words, the concentration of tamoxifen in row A is diluted from 20.0000 μM to 0.0030 μM in a 3-fold dilution manner from columns 2 to 10. Therefore, rows A, columns 2-10 can test the effect of the combination of orbitazine fumarate and tamoxifen.

[0089] The composition of the wells in row A, column 11 is culture medium, ZR-75-30 cells, 0.2% DMSO by volume, and 40 μM orbitazine fumarate. Therefore, the effect of orbitazine fumarate alone can be tested in row A, column 11.

[0090] The 12th column of row A is the DMSO control, that is, the composition in the well is ZR-75-30 cells, culture medium, and 0.2% by volume of DMSO.

[0091] The concentration of orbitazine fumarate in row B, columns 2-10 is 13.3333 μM. The layout of the wells in row B is otherwise identical to that in row A. The concentration of orbitazine fumarate in row C, columns 2-10 is 4.4444 μM. The layout of the wells in row C is otherwise identical to that in row A. The concentration of orbitazine fumarate in row D, columns 2-10 is 1.4815 μM. The layout of the wells in row D is otherwise identical to that in row A. The concentration of orbitazine fumarate in row E, columns 2-10 is 0.4938 μM. The layout of the wells in row E is otherwise identical to that in row A. The concentration of orbitazine fumarate in row F, columns 2-10 is 0.1646 μM. The layout of the wells in row F is otherwise identical to that in row A. The concentration of orbitazine fumarate in columns 2-10 of row G is 0.0549 μM. Other than that, the layout of the wells in row G is exactly the same as that in row A.

[0092] The wells in row H, columns 1-10 and row H, column 12 do not contain orbitazine fumarate. Otherwise, the layout of the wells in row H, columns 1-10 and row H, column 12 is identical to that of row A. The composition of row H, column 11 is identical to that of column 12. Therefore, row H can be used to test the effect of tamoxifen alone.

[0093] The luminescence intensity of the 96-well plate having the layout of Table 1.1 above was detected to obtain the following luminescence intensity Table 1.2.

[0094] Luminous intensity table 1.2

[0095]

[0096] From Table 1.2 above, we can calculate the average luminous intensity of the first column (blank control group) Blank is 32558, with a standard deviation of SD Blank The coefficient of variation CV is 12917.62. Blank is 39.68%, median Blank It is 31885.

[0097] From Table 1.2 above, we can calculate the average luminescence intensity of column 12 (DMSO control group) DMSO 9301319, standard deviation SD DMSO The coefficient of variation CV is 302783.98. DMSO 3.26%, median DMSO It is 9355096.

[0098] Based on the above data, the signal window SW can be further calculated as follows: DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / SD DMSO =27.48.

[0099] Based on the above data, we can further calculate Z Factor = ((avrage DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / (avrage DMSO -avrage Blank )=0.90.

[0100] Therefore, the signal window SW indicates that the experimental results are highly sensitive and can distinguish drug effects from background noise. The Z Factor indicates that the experimental data are reliable and the experimental system is stable, suitable for high-throughput screening.

[0101] Based on the luminescence intensity of each well in the luminescence intensity table 1.2 and the formula for calculating the inhibition rate, inhibition = (1-(RLU compound -RLU blank ) / (RLU DMSO -RLUblank ))×100% can be used to obtain the compound inhibition rate of each well in the presence of orbitazine fumarate and / or tamoxifen (columns 2-11) in Table 1.3.

[0102] Compound inhibition rate Table 1.3

[0103]

[0104] As can be seen from Table 1.3, columns 2-10 of rows AG show that the combination of orbitazine fumarate and tamoxifen has a good inhibitory effect on ZR-75-30 cells. Columns 2-10 of row H show the inhibitory effect of tamoxifen alone on ZR-75-30 cells. Column 11 shows that orbitazine fumarate alone also has a good inhibitory effect on ZR-75-30 cells.

[0105] In order to obtain the half-inhibitory concentration IC50 and the combination index (CI) of the combined drug, the data in Table 1.3 were partially extracted to obtain the following partial compound inhibition rate Table 1.4.

[0106] Table 1.4 Inhibition rates of some compounds

[0107]

[0108] As can be seen from Table 1.4, the inhibition rate of the combination of 20 μM tamoxifen and 40 μM orbitazine fumarate on ZR-75-30 cells is 100.14%. After the concentration of this combination is diluted 3 times, the inhibition rate of ZR-75-30 cells is 99.74%. Similarly, each subsequent dilution of the combination by 3 times will lead to a decrease in the inhibition rate of ZR-75-30 cells. After 6 dilutions, the inhibition rate of the combination of 0.0274 μM tamoxifen and 0.0549 μM orbitazine fumarate on ZR-75-30 cells is 2.07%.

[0109] Similarly, the inhibition rate of the combined use of 20 μM tamoxifen and 13.333 μM orbitazine fumarate on ZR-75-30 cells was 100.14%. After 5 dilutions with 3 times each time, the inhibition rate of the combined use of 0.0823 μM tamoxifen and 0.0549 μM orbitazine fumarate on ZR-75-30 cells was 4.71%.

[0110] Similarly, the inhibition rate of the combined use of 20 μM tamoxifen and 4.444 μM orbitazine fumarate on ZR-75-30 cells was 100.07%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combined use of 0.2469 μM tamoxifen and 0.0549 μM orbitazine fumarate on ZR-75-30 cells was 1.18%.

[0111] Similarly, the inhibition rate of the combined use of 6.667 μM tamoxifen and 40 μM orbitazine fumarate on ZR-75-30 cells was 100.10%. After 6 dilutions with 3 times each time, the inhibition rate of the combined use of 0.0091 μM tamoxifen and 0.0549 μM orbitazine fumarate on ZR-75-30 cells was -1.25%.

[0112] Similarly, the inhibition rate of the combined use of 2.222 μM tamoxifen and 40 μM orbitazine fumarate on ZR-75-30 cells was 100.03%. After 6 dilutions with 3-fold dilution each time, the inhibition rate of the combined use of 0.0030 μM tamoxifen and 0.0549 μM orbitazine fumarate on ZR-75-30 cells was -1.02%.

[0113] Similarly, the inhibition rate of the combined use of 0.741 μM tamoxifen and 40 μM orbitazine fumarate on ZR-75-30 cells was 99.87%. After 5 dilutions with 3 times each time, the inhibition rate of the combined use of 0.0030 μM tamoxifen and 0.1646 μM orbitazine fumarate on ZR-75-30 cells was 0.54%.

[0114] Similarly, the inhibition rate of the combined use of 0.247 μM tamoxifen and 40 μM orbitazine fumarate on ZR-75-30 cells was 99.90%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combined use of 0.0030 μM tamoxifen and 0.4938 μM orbitazine fumarate on ZR-75-30 cells was 16.28%.

[0115] The data in row H (the inhibition rate of tamoxifen monotherapy on ZR-75-30 cells) and column 11 (the inhibition rate of orbitazine fumarate monotherapy on ZR-75-30 cells) in Table 1.3 and the data in Table 1.4 were processed using the 4-parameter model [fit = (A + ((BA) / (1 + ((C / x) ^ D))))] in XLFit to obtain the data processing table 1.5 and Figures 1-16 .

[0116] Data processing table 1.5

[0117]

[0118]

[0119] like Figure 1As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of tamoxifen on ZR-75-30 cells, Corrected AbsIC50, which is 0.419 in Table 1.5.

[0120] like Figure 2 As shown in Table 4, the six inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:13.333 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 1.134 in Table 1.5.

[0121] like Figure 3 As shown in Table 4, the five inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:4.444 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 3.121 in Table 1.5.

[0122] like Figure 4 As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 6.667 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 0.160 in Table 1.5.

[0123] like Figure 5 As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 0.067 in Table 1.5.

[0124] like Figure 6 As shown in Table 4, the six inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 0.022 in Table 1.5.

[0125] like Figure 7 As shown in Table 4, the five inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 0.007 in Table 1.5.

[0126] like Figure 8 As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on ZR-75-30 cells, which is 0.839 in Table 1.5.

[0127] like Figure 9 As shown in Table 4, the six inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:13.333 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on ZR-75-30 cells, which is 0.756 in Table 1.5.

[0128] like Figure 10 As shown in Table 4, the five inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 20 μM:4.444 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on ZR-75-30 cells, which is 0.694 in Table 1.5.

[0129] like Figure 11 As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 6.667 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on ZR-75-30 cells, which is 0.962 in Table 1.5.

[0130] like Figure 12 As shown in Table 4, the seven inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of orbitazine fumarate on ZR-75-30 cells Corrected Abs IC50, which is 1.202 in Table 1.5.

[0131] like Figure 13 As shown in Table 4, the six inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 1.212 in Table 1.5.

[0132] like Figure 14As shown in Table 4, the five inhibition rate points with a concentration ratio of tamoxifen to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of tamoxifen on ZR-75-30 cells, which is 1.134 in Table 1.5.

[0133] like Figure 15 As shown, after the 9 inhibition rate points of tamoxifen in Table 3 are fitted into a curve, the half-inhibitory concentration of tamoxifen on ZR-75-30 cells can be obtained, which is 8.415 in Table 1.5.

[0134] like Figure 16 As shown, after the seven inhibition rate points of fumaric acid orbitazine in Table 3 are fitted into a curve, the half-inhibitory concentration of fumaric acid orbitazine on ZR-75-30 cells can be obtained, which is 1.337 in Table 1.5.

[0135] The half-inhibitory concentration data at different concentrations in Table 1.5 were extracted to calculate the combination index, thereby obtaining the combination index Table 1.6.

[0136] Joint Index Table 1.6

[0137]

[0138] As can be seen from Table 1.6, the half-inhibitory concentration IC50 of tamoxifen alone on ZR-75-30 cells a It is 8.4153μM.

[0139] The half-inhibitory concentration IC50 of orbitazine fumarate monotherapy on ZR-75-30 cells b It is 1.3373μM.

[0140] When the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:40 μM, the half-inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 0.4194 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.677.

[0141] When the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:13.333 μM, the half inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 1.1340 μM. b / ab is 0.7560 μM. Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.700.

[0142] When the concentration ratio of tamoxifen to orbitazine fumarate is 20 μM:4.444 μM, the half inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 3.1208μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.889.

[0143] When the concentration ratio of tamoxifen to orbitazine fumarate is 6.667 μM:40 μM, the half-inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 0.1604μM. b / ab Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.738.

[0144] When the concentration ratio of tamoxifen to orbitazine fumarate is 2.222μM:40μM, the half-inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 0.0668 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.907.

[0145] When the concentration ratio of tamoxifen to orbitazine fumarate is 0.741μM:40μM, the half-inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 0.0224 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.909.

[0146] When the concentration ratio of tamoxifen to orbitazine fumarate is 0.247 μM:40 μM, the half inhibitory concentration IC50 of tamoxifen on ZR-75-30 cells is a / ab The half inhibitory concentration IC50 of orbitazine fumarate on ZR-75-30 cells is 0.0070μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.849.

[0147] Therefore, when the concentration ratio of tamoxifen and orbitazine fumarate is 1:0.2-162, the combination index CI is less than 1, and the combined use of tamoxifen and orbitazine fumarate produces a synergistic effect and enhances the anti-tumor effect.

[0148] In addition, the combination of tamoxifen and orbitazine fumarate can reduce the dose of a single drug, reduce toxic side effects, and reduce the risk of drug resistance by inducing cell apoptosis through multiple pathways.

[0149] Example 2

[0150] In this example, the test cells were human pancreatic cancer cells (AsPC-1), seeded at a density of 3000 cells / well. The compound solutions were irinotecan hydrochloride in dimethyl sulfoxide (DMSO) and orbitazine fumarate (SM-1) in DMSO. Following the above experimental method, the following 96-well plate layout was obtained, as shown in Table 2.1.

[0151] Table 2.1 Layout of the combined action of irinotecan hydrochloride and orbitazine fumarate on AsPC-1 cells

[0152]

[0153]

[0154] As can be seen from Table 2.1, the 96-well plate is divided into rows AH and columns 1-12.

[0155] The first column of row A is the blank control (Blank), that is, the composition in the well is culture medium.

[0156] The composition of each well in row A, columns 2-10 is culture medium, AsPC-1 cells, 0.2% DMSO by volume, orbitazine fumarate fixed at 40 μM, and irinotecan hydrochloride. Specifically, the concentrations of irinotecan hydrochloride in row A are 20.0000 μM, 6.6667 μM, 2.2222 μM, 0.7407 μM, 0.2469 μM, 0.0823 μM, 0.0274 μM, 0.0091 μM, and 0.0030 μM from columns 2 to 10. In other words, the concentration of irinotecan hydrochloride in row A is diluted from 20.0000 μM to 0.0030 μM in a 3-fold dilution manner from columns 2 to 10. Therefore, row A, columns 2-10 can test the effect of the combination of orbitazine fumarate and irinotecan hydrochloride.

[0157] The composition of the wells in row A, column 11 is culture medium, AsPC-1 cells, 0.2% by volume of DMSO, and 40 μM orbitazine fumarate. Therefore, the effect of orbitazine fumarate alone can be tested in row A, column 11.

[0158] The 12th column of row A is the DMSO control, that is, the composition in the well is AsPC-1 cells, culture medium, and 0.2% by volume of DMSO.

[0159] The concentration of orbitazine fumarate in row B, columns 2-10 is 13.3333 μM. The layout of the wells in row B is otherwise identical to that in row A. The concentration of orbitazine fumarate in row C, columns 2-10 is 4.4444 μM. The layout of the wells in row C is otherwise identical to that in row A. The concentration of orbitazine fumarate in row D, columns 2-10 is 1.4815 μM. The layout of the wells in row D is otherwise identical to that in row A. The concentration of orbitazine fumarate in row E, columns 2-10 is 0.4938 μM. The layout of the wells in row E is otherwise identical to that in row A. The concentration of orbitazine fumarate in row F, columns 2-10 is 0.1646 μM. The layout of the wells in row F is otherwise identical to that in row A. The concentration of orbitazine fumarate in columns 2-10 of row G is 0.0549 μM. Other than that, the layout of the wells in row G is exactly the same as that in row A.

[0160] The wells in row H, columns 1-10 and row H, column 12 do not contain orbitazine fumarate. Otherwise, the layout of the wells in row H, columns 1-10 and row H, column 12 is identical to that of row A. The composition of row H, column 11 is identical to that of column 12. Therefore, row H can be used to test the effect of irinotecan hydrochloride alone.

[0161] The luminescence intensity of the 96-well plate having the layout of Table 2.1 above was detected, and the following luminescence intensity Table 2.2 was obtained.

[0162] Luminous intensity table 2.2

[0163]

[0164] From Table 2.2 above, we can calculate the average luminous intensity of the first column (blank control group) Blank 169029, standard deviation SD Blank The coefficient of variation CV is 53031.52. Blank is 31.37%, median Blank It is 184739.

[0165] From Table 2.2 above, we can calculate the average luminescence intensity of column 12 (DMSO control group) DMSO is 19926773, with a standard deviation of SD DMSO The coefficient of variation CV is 887120.74. DMSO 4.45%, median DMSO It is 19627067.

[0166] Based on the above data, the signal window SW can be further calculated as follows: DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / SD DMSO =19.09.

[0167] Based on the above data, we can further calculate Z Factor = ((avrage DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / (avrage DMSO -avrage Blank )=0.86.

[0168] Therefore, the signal window SW indicates that the experimental results are highly sensitive and can distinguish drug effects from background noise. The Z Factor indicates that the experimental data are reliable and the experimental system is stable, suitable for high-throughput screening.

[0169] Based on the luminescence intensity of each well in the luminescence intensity table 2.2 and the formula for calculating the inhibition rate, inhibition = (1-(RLU compound -RLU blank ) / (RLU DMSO -RLU blank ))×100% can be used to obtain the compound inhibition rate of each well in the presence of orbitazine fumarate and / or irinotecan hydrochloride (columns 2-11) (Table 2.3).

[0170] Compound inhibition rate Table 2.3

[0171]

[0172] As can be seen from Table 2.3, columns 2-10 of rows AG show that the combination of orbitazine fumarate and irinotecan hydrochloride has a good inhibitory rate on AsPC-1 cells. Columns 2-10 of row H show the inhibitory rate of irinotecan hydrochloride alone on AsPC-1 cells. Column 11 shows that orbitazine fumarate alone also has a good inhibitory rate on AsPC-1 cells.

[0173] In order to obtain the half-inhibitory concentration IC50 and the combination index (CI) of the combined drug, the data in Table 2.3 were partially extracted to obtain the following partial compound inhibition rate Table 2.4.

[0174] Table 2.4 Inhibition rates of some compounds

[0175]

[0176]

[0177] As can be seen from Table 2.4, the inhibition rate of the combination of 20 μM irinotecan hydrochloride and 40 μM obitazine fumarate on AsPC-1 cells is 91.16%. After the concentration of this combination is diluted 3 times, the inhibition rate of AsPC-1 cells is 72.72%. Similarly, each subsequent dilution of the combination by 3 times will lead to a decrease in the inhibition rate of AsPC-1 cells. After 6 dilutions, the inhibition rate of the combination of 0.0274 μM irinotecan hydrochloride and 0.0549 μM obitazine fumarate on AsPC-1 cells is 0.25%.

[0178] Similarly, the inhibition rate of the combination of 20 μM irinotecan hydrochloride and 13.333 μM orbitazine fumarate on AsPC-1 cells was 76.77%. After 5 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0823 μM irinotecan hydrochloride and 0.0549 μM orbitazine fumarate on AsPC-1 cells was 4.88%.

[0179] Similarly, the inhibition rate of the combination of 20 μM irinotecan hydrochloride and 4.444 μM orbitazine fumarate on AsPC-1 cells was 71.64%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.2469 μM irinotecan hydrochloride and 0.0549 μM orbitazine fumarate on AsPC-1 cells was 4.58%.

[0180] Similarly, the inhibition rate of the combination of 6.667 μM irinotecan hydrochloride and 40 μM orbitazine fumarate on AsPC-1 cells was 90.31%. After 6 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0091 μM irinotecan hydrochloride and 0.0549 μM orbitazine fumarate on AsPC-1 cells was 1.17%.

[0181] Similarly, the inhibition rate of the combination of 2.222 μM irinotecan hydrochloride and 40 μM obitazine fumarate on AsPC-1 cells was 91.88%. After 6 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM irinotecan hydrochloride and 0.0549 μM obitazine fumarate on AsPC-1 cells was 1.78%.

[0182] Similarly, the inhibition rate of the combination of 0.741 μM irinotecan hydrochloride and 40 μM orbitazine fumarate on AsPC-1 cells was 90.14%. After 5 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM irinotecan hydrochloride and 0.1646 μM orbitazine fumarate on AsPC-1 cells was 1.41%.

[0183] Similarly, the inhibition rate of the combination of 0.247 μM irinotecan hydrochloride and 40 μM obitadine fumarate on AsPC-1 cells was 91.50%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM irinotecan hydrochloride and 0.4938 μM obitadine fumarate on AsPC-1 cells was 5.95%.

[0184] The data in row H (the inhibition rate of irinotecan hydrochloride monotherapy on AsPC-1 cells) and column 11 (the inhibition rate of orbitazine fumarate monotherapy on AsPC-1 cells) in Table 2.3 and the data in Table 2.4 were processed using the 4-parameter model [fit = (A + ((BA) / (1 + ((C / x) ^ D))))] in XLFit to obtain the data processing table 2.5 and Figures 17-26 .

[0185] Data processing table 2.5

[0186]

[0187] like Figure 17 As shown in Table 4, the seven inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 6.667 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells, Corrected Abs IC50, which is 0.442 in Table 2.5.

[0188] like Figure 18 As shown in Table 4, the seven inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration Corrected Abs IC50 of irinotecan hydrochloride on AsPC-1 cells, which is 0.144 in Table 2.5.

[0189] like Figure 19 As shown in Table 4, the six inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells, Corrected Abs IC50, which is 0.050 in Table 2.5.

[0190] like Figure 20 As shown in Table 4, the five inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells, Corrected Abs IC50, which is 0.017 in Table 2.5.

[0191] like Figure 21 As shown in Table 4, the seven inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 6.667 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on AsPC-1 cells, which is 2.655 in Table 2.5.

[0192] like Figure 22 As shown in Table 4, the seven inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on AsPC-1 cells, which is 2.598 in Table 2.5.

[0193] like Figure 23 As shown in Table 4, the six inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells, Corrected Abs IC50, which is 2.712 in Table 2.5.

[0194] like Figure 24 As shown in Table 4, the five inhibition rate points with a concentration ratio of irinotecan hydrochloride to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells, Corrected Abs IC50, which is 2.763 in Table 2.5.

[0195] like Figure 25 As shown, after the 9 inhibition rate points of irinotecan hydrochloride in Table 3 are fitted into a curve, the half-inhibitory concentration of irinotecan hydrochloride on AsPC-1 cells can be obtained, the Corrected Abs IC50, which is 11.539 in Table 2.5.

[0196] like Figure 26 As shown, after the 7 inhibition rate points of fumaric acid orbitazine in Table 3 are fitted into a curve, the half-inhibitory concentration of fumaric acid orbitazine on AsPC-1 cells can be obtained, the Corrected Abs IC50, which is 2.863 in Table 2.5.

[0197] The half-inhibitory concentration data at different concentrations in Table 2.5 were extracted to calculate the combination index, thereby obtaining the combination index Table 2.6.

[0198] Joint Index Table 2.6

[0199]

[0200] As can be seen from Table 2.6, the half-inhibitory concentration IC50 of irinotecan hydrochloride alone on AsPC-1 cells a It is 11.5394μM.

[0201] The half-inhibitory concentration IC50 of orbitazine fumarate monotherapy on AsPC-1 cells bIt is 2.8628μM.

[0202] When the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 6.667 μM:40 μM, the half-inhibitory concentration IC50 of irinotecan hydrochloride on AsPC-1 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on AsPC-1 cells is 0.442 μM. b / ab Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.966.

[0203] When the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 2.222μM:40μM, the half-inhibitory concentration IC50 of irinotecan hydrochloride on AsPC-1 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on AsPC-1 cells is 0.144 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.920.

[0204] When the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.741μM:40μM, the half-inhibitory concentration IC50 of irinotecan hydrochloride on AsPC-1 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on AsPC-1 cells is 0.050 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.952.

[0205] When the concentration ratio of irinotecan hydrochloride to orbitazine fumarate is 0.247 μM:40 μM, the half-inhibitory concentration IC50 of irinotecan hydrochloride on AsPC-1 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on AsPC-1 cells is 0.017 μM. b / ab Therefore, at this concentration ratio, the combined index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b=0.967.

[0206] Therefore, when the concentration ratio of irinotecan hydrochloride and obitazine fumarate is 1:5-162, the combination index CI is less than 1, and the combined use of irinotecan hydrochloride and obitazine fumarate produces a synergistic effect and enhances the anti-tumor effect.

[0207] In addition, the combination of irinotecan hydrochloride and orbitazine fumarate can reduce the dose of a single drug, reduce toxic side effects, and reduce the risk of drug resistance by inducing cell apoptosis through multiple pathways.

[0208] Example 3

[0209] In this example, the test cells were human hepatocellular carcinoma cells (HuH-7), seeded at a density of 3,000 cells / well. The compound solutions were a dimethyl sulfoxide (DMSO) solution of sorafenib and a DMSO solution of orbitazine fumarate (SM-1). Following the above experimental method, the following 96-well plate layout (Table 3.1) was obtained.

[0210] Table 3.1 Layout of sorafenib combined with orbitazine fumarate in HuH-7 cells

[0211]

[0212]

[0213] As can be seen from Table 3.1, the 96-well plate is divided into rows AH and columns 1-12.

[0214] The first column of row A is the blank control (Blank), that is, the composition in the well is culture medium.

[0215] The composition of each well in row A, columns 2-10, is culture medium, HuH-7 cells, 0.2% DMSO by volume, a fixed concentration of 40 μM orbitazine fumarate, and sorafenib. Specifically, the concentrations of sorafenib in row A, from columns 2 to 10, are 20.0000 μM, 6.6667 μM, 2.2222 μM, 0.7407 μM, 0.2469 μM, 0.0823 μM, 0.0274 μM, 0.0091 μM, and 0.0030 μM. In other words, the concentration of sorafenib in row A is diluted from 20.0000 μM to 0.0030 μM in a 3-fold dilution pattern from columns 2 to 10. Therefore, rows A, columns 2-10 can test the effect of the combination of orbitazine fumarate and sorafenib.

[0216] The composition of the wells in row A, column 11 is culture medium, HuH-7 cells, 0.2% by volume of DMSO, and 40 μM orbitazine fumarate. Therefore, the effect of orbitazine fumarate alone can be tested in row A, column 11.

[0217] The 12th column of row A is the DMSO control, that is, the composition in the well is HuH-7 cells, culture medium, and 0.2% by volume of DMSO.

[0218] The concentration of orbitazine fumarate in row B, columns 2-10 is 13.3333 μM. The layout of the wells in row B is otherwise identical to that in row A. The concentration of orbitazine fumarate in row C, columns 2-10 is 4.4444 μM. The layout of the wells in row C is otherwise identical to that in row A. The concentration of orbitazine fumarate in row D, columns 2-10 is 1.4815 μM. The layout of the wells in row D is otherwise identical to that in row A. The concentration of orbitazine fumarate in row E, columns 2-10 is 0.4938 μM. The layout of the wells in row E is otherwise identical to that in row A. The concentration of orbitazine fumarate in row F, columns 2-10 is 0.1646 μM. The layout of the wells in row F is otherwise identical to that in row A. The concentration of orbitazine fumarate in columns 2-10 of row G is 0.0549 μM. Other than that, the layout of the wells in row G is exactly the same as that in row A.

[0219] The wells in row H, columns 1-10 and row H, column 12 do not contain orbitazine fumarate. Otherwise, the layout of the wells in row H, columns 1-10 and row H, column 12 is identical to that of row A. The composition of row H, column 11 is identical to that of column 12. Therefore, row H can be used to test the effect of sorafenib alone.

[0220] The luminescence intensity of the 96-well plate having the layout of Table 3.1 above was detected, and the following luminescence intensity Table 3.2 was obtained.

[0221] Luminous intensity table 3.2

[0222]

[0223] From Table 3.2 above, we can calculate the average luminous intensity of the first column (blank control group) Blank is 33404, with a standard deviation of SD Blank The coefficient of variation CV is 9762.94. Blank is 29.23%, median Blank It is 36221.

[0224] From Table 3.2 above, we can calculate the average luminescence intensity of column 12 (DMSO control group)DMSO is 17466634, with a standard deviation of SD DMSO The coefficient of variation CV is 717674.56. DMSO The median is 4.11%. DMSO It is 17709705.

[0225] Based on the above data, the signal window SW can be further calculated as follows: DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / SD DMSO =21.25.

[0226] Based on the above data, we can further calculate Z Factor = ((avrage DMSO -3×SD DMSO )-(avrage Blank +3×SD Blank )) / (avrage DMSO -avrage Blank )=0.87.

[0227] Therefore, the signal window SW indicates that the experimental results are highly sensitive and can distinguish drug effects from background noise. The Z Factor indicates that the experimental data are reliable and the experimental system is stable, suitable for high-throughput screening.

[0228] Based on the luminescence intensity of each well in the luminescence intensity table 3.2 and the formula for calculating the inhibition rate, inhibition = (1-(RLU compound -RLU blank ) / (RLU DMSO -RLU blank ))×100% can be used to obtain the compound inhibition rate of each well in the presence of orbitazine fumarate and / or sorafenib (columns 2-11) in Table 3.3.

[0229] Table 3.3: Compound inhibition rates

[0230]

[0231] As can be seen from Table 3.3, columns 2-10 of rows AG show that the combination of orbitazine fumarate and sorafenib has a good inhibitory rate on HuH-7 cells. Columns 2-10 of row H show the inhibitory rate of sorafenib alone on HuH-7 cells. Column 11 shows that orbitazine fumarate alone also has a good inhibitory rate on HuH-7 cells.

[0232] In order to obtain the half-inhibitory concentration IC50 and the combination index (CI) of the combined drug, the data in Table 3.3 were partially extracted to obtain the following partial compound inhibition rate Table 3.4.

[0233] Table 3.4 Inhibition rates of some compounds

[0234]

[0235] As can be seen from Table 3.4, the inhibition rate of the combination of 20μM sorafenib and 40μM orbitazine fumarate on HuH-7 cells is 100.07%. After the concentration of this combination is diluted 3 times, the inhibition rate of HuH-7 cells is 97.20%. Similarly, each subsequent dilution of the combination by 3 times will lead to a decrease in the inhibition rate of HuH-7 cells. After 6 dilutions, the inhibition rate of the combination of 0.0274μM sorafenib and 0.0549μM orbitazine fumarate on HuH-7 cells is -9.37%.

[0236] Similarly, the inhibition rate of the combination of 20 μM sorafenib and 13.333 μM orbitazine fumarate on HuH-7 cells was 99.98%. After 5 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0823 μM sorafenib and 0.0549 μM orbitazine fumarate on HuH-7 cells was -6.08%.

[0237] Similarly, the inhibition rate of the combination of 20 μM sorafenib and 4.444 μM orbitazine fumarate on HuH-7 cells was 99.77%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.2469 μM sorafenib and 0.0549 μM orbitazine fumarate on HuH-7 cells was 0.59%.

[0238] Similarly, the inhibition rate of the combination of 6.667 μM sorafenib and 40 μM orbitazine fumarate on HuH-7 cells was 99.99%. After 6 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0091 μM sorafenib and 0.0549 μM orbitazine fumarate on HuH-7 cells was -16.72%.

[0239] Similarly, the inhibition rate of the combination of 2.222 μM sorafenib and 40 μM orbitazine fumarate on HuH-7 cells was 99.09%. After 6 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM sorafenib and 0.0549 μM orbitazine fumarate on HuH-7 cells was -13.08%.

[0240] Similarly, the inhibition rate of the combination of 0.741 μM sorafenib and 40 μM orbitazine fumarate on HuH-7 cells was 98.09%. After 5 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM sorafenib and 0.1646 μM orbitazine fumarate on HuH-7 cells was -15.00%.

[0241] Similarly, the inhibition rate of the combination of 0.247 μM sorafenib and 40 μM orbitazine fumarate on HuH-7 cells was 97.98%. After 4 dilutions with 3-fold dilution each time, the inhibition rate of the combination of 0.0030 μM sorafenib and 0.4938 μM orbitazine fumarate on HuH-7 cells was -8.58%.

[0242] The data in row H (the inhibition rate of sorafenib monotherapy on HuH-7 cells) and column 11 (the inhibition rate of orbitazine fumarate monotherapy on HuH-7 cells) in Table 3.3 and the data in Table 3.4 were processed using the 4-parameter model [fit = (A + ((BA) / (1 + ((C / x) ^ D))))] in XLFit to obtain data processing Table 3.5 and Figures 27-34 .

[0243] Data Processing Table 3.5

[0244]

[0245]

[0246] like Figure 27 As shown in Table 4, the seven inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of sorafenib on HuH-7 cells, which is 0.083 in Table 3.5.

[0247] like Figure 28 As shown in Table 4, the six inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of sorafenib on HuH-7 cells, which is 0.029 in Table 3.5.

[0248] like Figure 29 As shown in Table 4, the five inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of sorafenib on HuH-7 cells, which is 0.010 in Table 3.5.

[0249] like Figure 30 As shown in Table 4, the seven inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 2.222 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration (IC50) of orbitazine fumarate on HuH-7 cells, which is 1.495 in Table 3.5.

[0250] like Figure 31 As shown in Table 4, the six inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 0.741 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of sorafenib on HuH-7 cells, which is 1.571 in Table 3.5.

[0251] like Figure 32 As shown in Table 4, the five inhibition rate points with a concentration ratio of sorafenib to orbitazine fumarate of 0.247 μM:40 μM are fitted into a curve to obtain the half-inhibitory concentration CorrectedAbs IC50 of sorafenib on HuH-7 cells, which is 1.584 in Table 3.5.

[0252] like Figure 33 As shown, after the 9 inhibition rate points of sorafenib in Table 3 are fitted into a curve, the half-inhibitory concentration of sorafenib on HuH-7 cells can be obtained, which is 2.434 in Table 3.5.

[0253] like Figure 34 As shown, after the seven inhibition rate points of orbitazine fumarate in Table 3 are fitted into a curve, the half-inhibitory concentration of orbitazine fumarate on HuH-7 cells can be obtained, which is 1.659 in Table 3.5.

[0254] The half-inhibitory concentration data at different concentrations in Table 3.5 were extracted to calculate the combination index, thereby obtaining the combination index Table 3.6.

[0255] Joint Index Table 3.6

[0256]

[0257] As can be seen from Table 3.6, the half-inhibitory concentration IC50a of sorafenib alone on HuH-7 cells is 2.4343 μM.

[0258] The half-inhibitory concentration IC50 of orbitazine fumarate monotherapy on HuH-7 cells b It is 1.6594μM.

[0259] When the concentration ratio of sorafenib to orbitazine fumarate is 2.222μM:40μM, the half-inhibitory concentration IC50 of sorafenib on HuH-7 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on HuH-7 cells is 0.083 μM. b / ab Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.935.

[0260] When the concentration ratio of sorafenib to orbitazine fumarate is 0.741μM:40μM, the half-inhibitory concentration IC50 of sorafenib on HuH-7 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on HuH-7 cells is 0.029 μM. b / ab Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.959.0.947

[0261] When the concentration ratio of sorafenib to orbitazine fumarate is 0.247 μM:40 μM, the half-inhibitory concentration IC50 of sorafenib on HuH-7 cells is a / ab The half-inhibitory concentration IC50 of orbitazine fumarate on HuH-7 cells is 0.010 μM. b / ab Therefore, at this concentration ratio, the combination index CI = IC50 a / ab / IC50 a +IC50 b / ab / IC50 b =0.959.

[0262] Therefore, when the concentration ratio of sorafenib and orbitazine fumarate is 1:18-162, the combination index CI is less than 1, and the combination of sorafenib and orbitazine fumarate produces a synergistic effect and enhances the anti-tumor effect.

[0263] In addition, the combination of sorafenib and orbitazine fumarate can reduce the dose of a single drug, reduce toxic side effects, and reduce the risk of drug resistance by inducing cell apoptosis through multiple pathways.

[0264] In summary, this application provides a composition comprising orbitazine fumarate and a chemotherapeutic agent, a targeted drug, or an immunotherapy agent, as well as the use of this composition in the preparation of a medicament for treating tumors. This combination therapy has significant synergistic anti-tumor effects, and the combination therapy is expected to provide a safer and more effective treatment option for cancer patients, with significant clinical application value.

[0265] The above are only some embodiments of the present application and are not intended to limit the scope of patent protection of the present application. It should be understood that those skilled in the art can make modifications within the spirit and scope of the present application.

Claims

1. A pharmaceutical composition, characterized in that include: Orbitazine fumarate; A second active agent comprises at least one of tamoxifen, irinotecan hydrochloride, sorafenib, cisplatin, doxorubicin hydrochloride, paclitaxel, and etoposide; and Pharmaceutically acceptable excipients.

2. The pharmaceutical composition according to claim 1, characterized in that The concentration of orbitazine fumarate is 0.0549 μM to 40 μM.

3. The pharmaceutical composition according to claim 2, characterized in that The second active agent includes tamoxifen, and the tamoxifen concentration is 0.003 μM to 20 μM.

4. The pharmaceutical composition according to claim 3, characterized in that The concentration ratio of the tamoxifen to the orbitazine fumarate is 1:0.2-162.

5. The pharmaceutical composition according to claim 2, characterized in that The second active agent includes irinotecan hydrochloride, and the concentration of irinotecan hydrochloride is 0.003 μM to 6.667 μM.

6. The pharmaceutical composition according to claim 5, characterized in that The concentration ratio of the irinotecan hydrochloride to the orbitazine fumarate is 1:5-162.

7. The pharmaceutical composition according to claim 2, characterized in that The second active agent includes sorafenib, and the concentration of sorafenib is 0.003 μM to 2.222 μM.

8. The pharmaceutical composition according to claim 7, characterized in that The concentration ratio of the sorafenib to the orbitazine fumarate is 1:18-162.

9. Use of a pharmaceutical composition in preparing a drug for treating tumors, characterized in that: The pharmaceutical composition is the pharmaceutical composition according to any one of claims 1 to 8.

10. The use according to claim 9, characterized in that The tumor includes at least one of pancreatic cancer, liver cancer, gastric cancer, multiple myeloma, ovarian cancer, malignant embryonal rhabdoid tumor, and breast cancer.

11. The use according to claim 9, characterized in that The medicament comprises an effective amount of the pharmaceutical composition.

12. The use according to claim 9, characterized in that The order of administration of the drugs is simultaneous or sequential administration.

13. The use according to claim 9, characterized in that The orbitazine fumarate is used to induce apoptosis of tumor cells, and the second active agent is used to kill the tumor cells through other mechanisms.

14. The use according to any one of claims 9 to 13, characterized in that: The drug contacts the tumor.

15. The use according to claim 9, characterized in that The dosage forms of the drug include oral preparations, injections and other dosage forms.

16. The use according to claim 15, characterized in that The oral preparations include tablets, capsules, granules, sustained-release preparations, oral solutions and other oral dosage forms.

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