Application of dextro-streptanoate in preparation of anti-tumor drugs

Abstract

The invention belongs to the field of biomedicine, and particularly relates to an application of dextro-strepton in preparation of antitumor drugs, and a chemical formula of a strepton derivative is shown as a formula 1. According to the application disclosed by the invention, the dextro-strepton for treating epilepsy can be used for reducing the activity of tumor cells such as pancreatic cancer, reducing the tumor volume and prolonging the survival time of tumor mice, and has a remarkable anti-tumor effect; the dextro-strepton is combined with a chemotherapeutic drug or a radiotherapy mode for use, so that the anti-tumor effect can be remarkably enhanced. In addition, the invention also finds that the effect of dextro-streptanophore is obviously better than that of levorotatory streptanophore, and a new thought is provided for the treatment of pancreatic cancer and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an application of dextrorotatory-slipanto in the preparation of anti-tumor drugs. Background Art

[0002] STIRIPENTOL, also known as stirpentol, is a new drug for the treatment of epilepsy that was launched in 2007. Its chemical name is 4,4-dimethyl-1-(3,4-methylenedioxyphenyl)-1-penten-3-ol. French researchers reported that the addition of STIRIPENTOL to sodium valproate and clobazam can effectively treat severe myoclonic epilepsy in infants and young children; STIRIPENTOL is a cytochrome P450 inhibitor. When used in combination with clobazam and sodium valproate, STIRIPENTOL has a therapeutic effect on severe myoclonic epilepsy (SMEI). The School of Medicine at Washington University in Missouri, USA, believes that it is difficult to determine whether STIRIPENTOL reduces the frequency of epileptic seizures in patients in this combination drug experiment, because the mechanism of action of STIRIPENTOL is to increase the blood concentration of other drugs such as clobazam and sodium valproate.

[0003] The mechanism of action of Silipentol is quite unique. It can increase GABA release and inhibit the activity of certain enzymes, thereby increasing the blood concentration of other anti-epileptic drugs. Silipentol was approved in the European Union in 2001 for the adjuvant treatment of severe infantile epilepsy myoclonic seizures. Stiripentol analogs can be used to target lactate dehydrogenase to treat epilepsy. Energy metabolism can regulate the excitability of neurons, so special dietary treatments may inhibit drug-resistant epilepsy. According to a report in the journal Science (DOI: 10.1126 / science.aaa129), studies have found that stiripentol, an anti-epileptic drug, is a lactate dehydrogenase A (LDHA) inhibitor. By modifying the chemical structure of stiripentol, the research team discovered a previously unknown LDH inhibitor that can effectively inhibit epilepsy in vivo. In summary, stiripentol analogs - LDH inhibitors are expected to become an emerging class of anti-epileptic drugs.

[0004] Prior art WO2014115764A1 discloses that stiripentol is a lactate dehydrogenase (such as LDH-5) inhibitor, and then they found that it actually has an anti-cancer effect. Lactate dehydrogenase LDH-5 is a protein in the body that participates in the proliferation of cancer cells, and inhibiting its activity can inhibit the proliferation of cancer cells. It is easy to speculate that "stiripentol is used as an anticancer agent (secondary medical use)", and it has also been confirmed that it actually has an anti-cancer effect. Stiripentol can be a mixture of two stereoisomers (racemate), or a purified product of either stereoisomer. Effective growth inhibition of leukemia cells Jurkat and multiple myeloma cells KMS11 and KMS12PE was observed.

[0005] Prior art CN 118045081 A found that stiripentol used to treat epilepsy can reduce the activity of tumor cells such as non-small cell lung cancer, colorectal cancer, and gastric cancer, reduce tumor volume, and prolong the survival time of tumor mice, and has a significant anti-tumor effect; stiripentol combined with chemotherapy drugs or radiotherapy can significantly enhance the anti-tumor effect, and stiripentol combined with chemotherapy drugs or radiotherapy has a synergistic effect. A new use of stiripentol has been discovered, expanding the application range of stiripentol.

[0006] Prior art CN 116763776A discloses the use of stiripentol in the preparation of a drug for treating ovarian cancer. It is first discovered that the anti-epileptic drug stiripentol (ST) has good anti-tumor activity against ovarian cancer cell lines and can significantly inhibit the proliferation and migration abilities of ovarian cancer cell lines. It has the same effect as an anti-lactic acid metabolism drug sodium oxamate (SO) and has the effect of significantly inhibiting the growth of human xenograft ovarian cancer tumors.

[0007] However, the above-mentioned prior art has limited exploration of cancer types and does not distinguish between the effects of left-handed and right-handed rotation. Therefore, it is urgent to further explore the application scenarios of Slipanto. Therefore, the present invention provides an application of right-handed Slipanto in the preparation of anti-tumor drugs. Summary of the invention

[0008] The present invention first provides the use of a slipanto derivative in the preparation of an anti-tumor drug; optionally, the chemical formula of the slipanto derivative is as shown in Formula 1,

[0009]

[0010] The present invention also provides the use of a slipanto derivative in preparing a drug for reducing and / or reversing tumor drug resistance; optionally, the chemical formula of the slipanto derivative is as shown in Formula 1.

[0011] In certain embodiments, the tumor drug resistance refers to the resistance of the tumor to chemotherapeutic drugs.

[0012] Chemotherapy drugs include common chemotherapy regimens, such as oxaliplatin + fluorouracil; oxaliplatin + capecitabine; irinotecan + fluorouracil, etc.

[0013] The present invention also provides the use of a slipantoyan derivative in the preparation of a drug for enhancing the effect of tumor radiotherapy; optionally, the chemical formula of the slipantoyan derivative is as shown in Formula 1.

[0014] In some embodiments, the radiotherapy comprises ionizing radiation, X-knife or gamma knife, or radioactive particles.

[0015] The present invention also provides the use of a slipanto derivative in combination with a chemotherapeutic drug in the preparation of an anti-tumor drug; optionally, the chemical formula of the slipanto derivative is as shown in Formula 1.

[0016] In certain embodiments, the tumor comprises non-small cell lung cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0017] In certain embodiments, the chemotherapeutic drug is a DNA damaging chemotherapeutic drug.

[0018] In certain embodiments, the DNA damaging chemotherapy drug comprises cisplatin, oxaliplatin, doxorubicin, fluorouracil or cyclophosphamide.

[0019] Finally, the present invention provides an anti-tumor combination drug, which includes a slipanto derivative and a chemotherapeutic drug; optionally, the chemical formula of the slipanto derivative is as shown in Formula 1.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] (1) It was found that Dextro-Slipanto, which is used to treat epilepsy, can reduce the activity of pancreatic cancer and other tumor cells, reduce tumor volume, and prolong the survival time of tumor mice, showing significant anti-tumor effects. Dextro-Slipanto combined with chemotherapy drugs or radiotherapy can significantly enhance the anti-tumor effect.

[0022] (2) It was found that the effect of dextro-Slipanto was significantly better than that of levo-Slipanto, which provided a new idea for the treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Cell experiment results diagram; Figure 1 A, R-slipanto + chemotherapy group; Figure 1 B stands for R-Slipanto; Figure 1 C is S-slipanto + chemotherapy group; Figure 1 D is the S-Slipanto group; Figure 1 E is the chemotherapy group; Figure 1 F is the racemization + chemotherapy group; Figure 1G is the result of the racemic group;

[0024] Figure 2 IC50 experimental results of different experimental groups;

[0025] Figure 3 Experimental results in mice; Figure 3 A, R-slipanto + chemotherapy group; Figure 3 B, S-slipanto + chemotherapy group; Figure 3 C is the R-Slipanto group; Figure 3 D is the S-Slipanto group; Figure 3 E is the chemotherapy group; Figure 3 F, Silipanto + chemotherapy group; Figure 3 G is the Slipanto group.

[0026] Figure 4 The results of in vitro tumor tissue size in mice. From top to bottom, they are: racemic slipanto, S-slipanto, R-slipanto, chemotherapy alone, S-slipanto + chemotherapy, racemic slipanto, R-slipanto + chemotherapy.

[0027] Figure 5 Experimental results with melanoma organoids; Figure 5 A, R-slipanto + chemotherapy group; Figure 5 B stands for R-Slipanto; Figure 5 C is S-slipanto + chemotherapy group; Figure 5 D is the S-Slipanto group; Figure 5 E is the chemotherapy group; Figure 5 F is the racemization + chemotherapy group; Figure 5 G is the result of the racemic group;

[0028] Figure 6 Experimental results on breast cancer organoids; Figure 6 A, R-slipanto + chemotherapy group; Figure 6 B stands for R-Slipanto; Figure 6 C is S-slipanto + chemotherapy group; Figure 6 D is the S-Slipanto group; Figure 6 E is the chemotherapy group; Figure 6 F is the racemization + chemotherapy group; Figure 6 G is the result of the racemic group;

[0029] Figure 7 Experimental results on esophageal cancer organoids; Figure 7 A, R-slipanto + chemotherapy group; Figure 7 B stands for R-Slipanto; Figure 7 C is S-slipanto + chemotherapy group; Figure 7 D is the S-Slipanto group; Figure 7 E is the chemotherapy group; Figure 7 F is the racemization + chemotherapy group; Figure 7 G is the result of the racemic group;

[0030] Figure 8 Experimental results with pancreatic cancer organoids; Figure 8 A, R-slipanto + chemotherapy group; Figure 8 B stands for R-Slipanto; Figure 8 C is S-slipanto + chemotherapy group; Figure 8 D is the S-Slipanto group; Figure 8 E is the chemotherapy group; Figure 8 F is the racemization + chemotherapy group; Figure 8 G is the result of the racemic group; DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] 1 Reagents:

[0033] PI (propidium iodide) HY-D0815, calcein AM, DMEM (Dulbecco's Modified Eagle Medium) or RPMI 1640, FBS (fetal bovine serum), Penicillin-Streptomycin, GlutaMAX TM , HEPES buffer, EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), Insulin, Hydrocortisone, Ampicillin, Gentamicin, Amphotericin B, Bcl-2 inhibitor, Z-VAD-FMK; Trypsin-EDTA, PBS (phosphate buffered saline), and DMSO (dimethyl sulfoxide) were all purchased from commercial companies.

[0034] Dextrorotatory-Slipanto is prepared by our company. The starting materials piperonal and methyl tert-butyl ketone are condensed and hydrogenated to generate Slipanto; they are esterified with chiral organic acid resolution reagent, recrystallized and resolved, and the ester is hydrolyzed to generate R-Slipanto.

[0035] The preparation of R-Slipanto will be patented separately.

[0036]

[0037] 2 The main equipment is shown in Table 1;

[0038] Table 1 Main equipment

[0039] instrument Part Number brand CO2 Incubator 371 Thermo Inverted microscope TS2-S-SM NIKON Biological Safety Cabinets HR40-IIA2(KY) Qingdao Haier Biomedical Co., Ltd. Fluorescence microscopy M7000 Invitrogen Centrifuge L530R Xiangyi

[0040] Example 1 Cell experiment

[0041] 1. Purpose of the experiment

[0042] To verify the effects of racemic slipanto, R-slipanto and S-slipanto on the treatment of pancreatic cancer with conventional chemotherapy drugs.

[0043] 2. Experimental Methods

[0044] HPAF-II was purchased and cultured. According to the purpose of the experiment, multiple groups were set up, namely: observation group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, experimental group 6, and experimental group 7. The observation group was normal in vitro cell culture, experimental group 1 was routinely treated with drugs (oxaliplatin + irinotecan), experimental group 2 was added with R-slipanto on the basis of routine drug addition, experimental group 3 was added with only R-slipanto, experimental group 4 was added with S-slipanto on the basis of routine drug addition, and experimental group 5 was added with only S-slipanto; experimental group 6 was added with Slipanto (racemic) on the basis of routine drug addition, and experimental group 7 was added with Slipanto (racemic). Each group was repeated 5 times.

[0045] 3. Experimental steps

[0046] Proliferation culture: HPAF-II cells were expanded in a CO2 incubator under standard culture conditions (37°C, 5% CO2). When the cells grew to about 80-90% confluence, the cells were digested with trypsin and re-seeded in a 96-well plate, with a certain number of cells inoculated per well (e.g., 5000-10000 cells / well). The cells were allowed to attach and culture in the 96-well plate for 24 hours.

[0047] Adding drugs: Add drugs to cells, set 5 replicates for each group, and set a control group (no drug treatment). Continue to culture in a CO2 incubator for 72 hours.

[0048] Cell number photography: After 72 hours, take pictures using a cell imaging system and count the cells.

[0049] IV. Experimental Results

[0050] The results are as follows Figure 1-Figure 2 As shown, the results showed that the combination of racemic Silipanto, R-Slipanto and S-Slipanto with conventional drugs can enhance the inhibition of pancreatic cancer cells at the cellular level, and R-Slipanto showed a significantly better effect than S-Slipanto (cell counting data of four repeated experiments, student's t test P value = 0.000134).

[0051] Example 2 Mouse Experiment

[0052] 1. Purpose of the experiment

[0053] Based on the above cell experiment results, the verification scope and methods were expanded to further explore the effects of racemic slipanto, R-slipanto and S-slipanto on the treatment of pancreatic cancer with conventional chemotherapy drugs.

[0054] 2. Experimental Methods

[0055] Pancreatic cancer tumor-bearing mice were used to set up 8 experimental units, namely: observation group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, experimental group 6, and experimental group 7. The observation group was normal cultured organoid tissue, experimental group 1 was given conventional medication (oxaliplatin + irinotecan), experimental group 2 was given R-slipanto on the basis of conventional medication, experimental group 3 was given R-slipanto, experimental group 4 was given S-slipanto on the basis of conventional medication, and experimental group 5 was given S-slipanto; experimental group 6 was given Slipanto (racemic) on the basis of conventional medication, and experimental group 7 was given Slipanto (racemic).

[0056] 3. Experimental steps

[0057] Tumor formation in mice: Nude mice were selected and 100 μL of pancreatic cancer cell suspension was subcutaneously inoculated into the right back or abdomen of each mouse using sterile operation. After injection, the injection site was gently massaged to ensure uniform distribution and promote tumor formation. The mice were observed daily for tumor formation, and obvious tumor formation was found within 14 days.

[0058] Drug treatment: After 14 days, the tumor volume reached 100-200mm 3 , drug treatment was started; the drug administration frequency was 2-3 times a week for 4 weeks. During the drug administration, the drug dose (mg / kg) was calculated and the drug volume was adjusted according to the mouse body weight; the drug was dissolved in an appropriate solvent and administered using a gavage needle.

[0059] Record tumor volume: After 4 weeks, record the condition of the mice by images; humanely kill the mice, remove the tumor tissue, and measure the tumor size.

[0060] IV. Experimental Results

[0061] The results are as follows Figure 3 , 4 As shown, the results showed that the tissue size of the R-stiripentol + chemotherapy group was the smallest, both in terms of tumor size in vivo and in vitro, indicating that in animal experiments, the reorganization showed significantly better effects in the treatment of pancreatic cancer.

[0062] Example 3 Organoid Experiment

[0063] 1. Purpose of the experiment

[0064] Verify the sensitizing effect of R-Slipanto on chemotherapy drugs of human tissue, and expand the scope of verification from pancreatic cancer to malignant melanoma, breast cancer, and esophageal cancer; at the same time, test the killing effect on tumor organoids.

[0065] 2. Experimental Methods

[0066] Common tumor organoids of melanoma, breast cancer, esophageal cancer, and pancreatic cancer were used for culture, and four groups of organoid tissues were set up, and each group of organoids was further divided into 7 experimental units: observation group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, experimental group 6, and experimental group 7. The observation group was normal cultured organoid tissue, experimental group 1 was routinely treated with drugs (melanoma: paclitaxel + cisplatin; breast cancer: epirubicin + cyclophosphamide; esophageal cancer: oxaliplatin + fluorouracil; pancreatic cancer: oxaliplatin + irinotecan), experimental group 2 added R-slipanto on the basis of routine drug addition, experimental group 3 only added R-slipanto, experimental group 4 added S-slipanto on the basis of routine drug addition, experimental group 5 only added S-slipanto; experimental group 6 added Slipanto (racemic) on the basis of routine drug addition, and experimental group 7 only added Slipanto (racemic).

[0067] 3. Principle of CalceinAM / PI live-dead staining experiment

[0068] By staining cells with Calcein-AM (calcein) and Propidium Iodide (PI), live and dead cells in the same cell culture dish can be observed simultaneously under a fluorescence microscope.

[0069] Green fluorescence is CalceinAM live cell staining. CalceinAM itself has no fluorescence. After entering the cell, it is hydrolyzed by endogenous esterase in the cell to generate a polar molecule calcein (Calcein) with a strong negative charge that cannot penetrate the cell membrane, and is thus retained in the cell. Calcein can emit strong green fluorescence. Since dead cells lack esterase, CalceinAM is often used for viability testing and short-term labeling of living cells.

[0070] The red fluorescence is PI dead cell staining. PI is a cell nucleus staining reagent that can stain DNA and is commonly used in cell apoptosis detection. Its full name is Propidium Iodide. It is an analog of ethidium bromide and releases red fluorescence after being embedded in double-stranded DNA. Although PI cannot pass through the membrane of living cells, it can pass through damaged cell membranes and stain the nucleus.

[0071] IV. Experimental Procedure

[0072] Plating: Digest and centrifuge the organoids that are in good growth condition, resuspend the cell pellet with 1 ml of washing solution, and take 10 ul for counting; after centrifugation again, add matrix gel to resuspend the cell pellet according to the counting results.

[0073] Glue dispensing: 12-well plate, 2 wells for the control group (1 drop of gel per well, 2.5ul / drop, 6000cell / drop); 2 wells for the experimental group (2 drops of gel per well, 2.5ul / drop, 6000cell / drop); add 800ul of organoid-specific culture medium to each well.

[0074] The next day, add medicine.

[0075] The control group did not need to add drugs, and the three-well experimental groups were added with a combination of chemotherapy drugs, slipanto (racemic), S-slipanto, and R-slipanto.

[0076] Photography: Take photos under an optical microscope (4X, 10X) at 0h, 24h, 72h, 120h, and 144h respectively.

[0077] Live and dead staining photography: At 72 hours, take out one well of the control and experimental groups, use calcein-AM and PI dye solution for live and dead staining, place in an incubator in the dark for incubation for 30 minutes, and then take fluorescent calcein-AM green light and PI red light photos (4X, 10X).

[0078] At 144 hours, one well of the control and one well of the test group were taken out, and live-dead staining was performed in the same manner, and photos were taken (4X, 10X).

[0079] V. Experimental Results

[0080] The photo results are as follows Figure 5-Figure 8 As shown in Table 2, the results of cell activity counting are shown in Table 2. According to the results of the two levels, it can be concluded that R-Slipanto has the best effect. Among them, due to the special composition of the organ culture substrate, the color imaging system of pancreatic cancer organoids has a certain color brightness difference compared with the other three groups.

[0081] The cell count was statistically analyzed using multi-group difference analysis (KW test), and the result showed P = 0.0007689, indicating that the comparison of all categories showed that there were differences between the groups. Then the overall four groups (R + chemotherapy, S + chemotherapy, racemization + chemotherapy, chemotherapy group) were extracted for multi-group difference analysis, and the result showed P = 0.02211, indicating that there were still differences between the groups. However, the difference analysis (student's t test) of the above four groups was found to be different only between the R + chemotherapy group and the other three groups (the P values ​​of R + chemotherapy compared with S + chemotherapy, single chemotherapy, and racemization + chemotherapy were 0.002522, 0.001416, and 0.002368, respectively), and the P values ​​of the other groups were all greater than 0.1 and there was no difference. Through statistical analysis, it can be seen that R-Slipanto + chemotherapy has the best killing effect on tumor cells.

[0082] Table 2 Cell activity count table

[0083] Group Melanoma Breast cancer Esophageal cancer Pancreatic cancer R+chemotherapy 1640 2312 2106 1703 R Group 9134 8903 9413 9635 S+chemotherapy 3103 3211 3912 2980 Group S 9023 8991 9375 9758 Chemotherapy alone 3387 3108 3841 3005 Racemization + chemotherapy 2741 2995 3453 3001 Racemic group 9204 9022 9464 9233

[0084] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a slipanto derivative in the preparation of an anti-tumor drug; Optionally, the chemical formula of the slipanto derivative is as shown in Formula 1, 2. Use of a slipanto derivative in the preparation of a drug for reducing and / or reversing tumor drug resistance; Optionally, the chemical formula of the slipanto derivative is as shown in Formula 1.

3. The use according to claim 2, characterized in that The tumor drug resistance refers to the resistance of the tumor to chemotherapy drugs.

4. Use of slipantoyan derivatives in the preparation of drugs for enhancing the effect of tumor radiotherapy; Optionally, the chemical formula of the slipantoyan derivative is as shown in Formula 1.

5. The use according to claim 4, characterized in that The radiotherapy methods include ionizing radiation, X-knife or gamma knife, and radioactive particles.

6. Use of a slipanto derivative in combination with a chemotherapeutic drug in the preparation of an anti-tumor drug; optionally, the chemical formula of the slipanto derivative is as shown in Formula 1.

7. The use according to any one of claims 1 to 6, characterized in that: The tumor includes one or more of pancreatic cancer, melanoma, ovarian cancer, breast cancer, and esophageal cancer.

8. The use according to claim 3 or 6, characterized in that: The chemotherapeutic drug is a DNA damaging chemotherapeutic drug.

9. The use according to claim 8, characterized in that The DNA damaging chemotherapy drugs include cisplatin, oxaliplatin, doxorubicin, fluorouracil or cyclophosphamide.

10. An anti-tumor combination drug, characterized in that: It includes a Silipanto derivative and a chemotherapeutic drug; optionally, the chemical formula of the Silipanto derivative is as shown in Formula 1.

Images