Application of benzisoselenazole derivative and platinum medicine in preparation of medicine for treating tumor and postoperative tumor recurrence
阅读说明：本技术 苯并异硒唑衍生物与铂类药物联合用于制备***药物与术后肿瘤复发药物中的应用 (Application of benzisoselenazole derivative and platinum medicine in preparation of medicine for treating tumor and postoperative tumor recurrence ) 是由 曾慧慧 尹汉维 于 2018-08-06 设计创作，主要内容包括：本发明属于肿瘤治疗技术领域,公开了苯并异硒唑衍生物与铂类药物联合用于制备治疗肿瘤药物与术后肿瘤复发药物中的应用。所述苯并异硒唑衍生物具有如式A所示的结构,所述铂类抗癌药物选自顺铂、卡铂、奥沙利铂、奈达铂等中的至少一种,所述苯并异硒唑衍生物与铂类抗癌药物的摩尔比为(1～99):(1～99)。二者联用,可有效降低毒性高的铂类抗癌药物给药剂量,提升抗癌用药的安全性；可以通过降低Bcl-2/Bax蛋白表达比例来诱导Bel-7402细胞凋亡,可以协同抑制肿瘤组织内TrxR的表达,明显降低术后肿瘤细胞的增殖率,提升对术后肿瘤细胞的生长抑制率。<Image he="265" wi="700" file="DDA0001756036260000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the technical field of tumor treatment, and discloses an application of a benzisoselenazole derivative and a platinum drug in preparation of a drug for treating tumor and a drug for postoperative tumor recurrence. The benzisoselenazole derivative has a structure shown as a formula A, the platinum anticancer drugs are at least one of cisplatin, carboplatin, oxaliplatin, nedaplatin and the like, and the molar ratio of the benzisoselenazole derivative to the platinum anticancer drugs is (1-99) to (1-99). The two are used together, so that the administration dosage of the platinum anti-cancer drugs with high toxicity can be effectively reduced, and the safety of the anti-cancer drugs is improved; bel-7402 cell can be induced by reducing Bcl-2/Bax protein expression ratioApoptosis can synergistically inhibit the expression of TrxR in tumor tissues, obviously reduce the proliferation rate of postoperative tumor cells and improve the growth inhibition rate of the postoperative tumor cells.)
1. The benzisoselenazole derivative and platinum anticancer drugs are combined to prepare the drugs for treating tumors.
2. The use according to claim 1, wherein the benzisoselenazole derivative has a structure represented by formula A, and is selected from at least one of a compound represented by formula A, a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug, and a solvate thereof,
wherein R is1、R2Identical or different, independently of one another, from H or the following radicals: c1-12Alkyl radical, C3-20A cycloalkyl group;
wherein R is selected from C1-12Alkylene, phenylene, biphenylene, triphenylene, or
3. Use according to claim 1 or 2, wherein in the compound of formula a, R is1、R2Identical or different, independently of one another, from H, -CH3、-CH2CH3、-CH(CH3)2、-C(CH3)3、-CH(CH2)4or-CH (CH)2)5(ii) a R is selected from-CH2-、-C2H4-、-C4H8-, phenylene-C6H4-；
Preferably, the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -butane, the structure of which is shown as follows:
4. the use according to any one of claims 1 to 3, wherein the platinum anticancer drug is at least one selected from Cisplatin (CDDP), Carboplatin (CBP), oxaliplatin (1-OHP), nedaplatin (CDGP), etc.
5. The use according to any one of claims 1 to 4, wherein the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is (1-99): 1-99);
preferably, the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6);
more preferably, the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is 1:1, 2:1 (in vitro combination) or 60:1, 70:1, 80:1 (in vivo combination);
wherein the tumor comprises solid tumor and non-solid tumor, and can be benign or malignant; the tumor is an initial tumor; preferably, the tumor includes, but is not limited to: liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, gastric cancer, skin cancer, bladder cancer, ovarian cancer, prostate cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, lymph cancer, oral epithelial cancer, epithelial cervical cancer or chronic myelogenous leukemia.
6. The use according to any one of claims 1 to 4, wherein the benzisoselenazole derivative is used in combination with a platinum-based anticancer drug for inhibiting tumor cell proliferation;
preferably, the tumor cells include, but are not limited to: human liver cancer cell HepG2, human liver cancer cell Bel-7402, human liver cancer cell Huh7721, human colorectal cancer cell LoVo, human colorectal cancer cell RKO, human colorectal cancer cell SW480, human lung cancer cell A549, human lung cancer cell H1299, human lung cancer cell SPCA-1, human epithelial cervical cancer cell HeLa, human breast cancer cell MCF-7, human chronic myelogenous leukemia cell k562, human esophageal cancer cell KYSE150, human esophageal cancer cell KYSE450 or human esophageal cancer cell KYSE 510.
7. A method for inhibiting tumor cell proliferation by combining benzisoselenazole derivatives and platinum anticancer drugs comprises the steps of enabling the benzisoselenazole derivatives and the platinum anticancer drugs to act on tumor cells according to a ratio;
preferably, the formulation has the meaning as defined in claim 5;
preferably, the concentration of the benzisoselenazole derivative and the platinum anticancer drug is 1-100 mu M,
preferably, the action time is 10-120 h.
8. A pharmaceutical composition comprises a benzisoselenazole derivative and a platinum-based anti-cancer drug;
preferably, the benzisoselenazole derivative has the meaning of claim 2 or 3, the platinum-based anticancer drug has the meaning of claim 4, and the ratio of the benzisoselenazole derivative to the platinum-based anticancer drug has the meaning of claim 5;
preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient.
9. The benzisoselenazole derivative and the platinum anticancer drug are combined to prepare the drug for inhibiting the postoperative recurrence of the tumor;
preferably, the tumor has the meaning as claimed in claim 5;
preferably, the benzisoselenazole derivative has the meaning as defined in claim 2 or 3, and the platinum-based anticancer drug has the meaning as defined in claim 4;
preferably, the ratio of the benzisoselenazole derivative to the platinum-based anticancer drug has the meaning of claim 5.
10. A pharmaceutical composition for inhibiting postoperative recurrence of a tumor, comprising a benzisoselenazole derivative and a platinum-based anticancer agent;
preferably, the benzisoselenazole derivative has the meaning of claim 2 or 3, the platinum-based anticancer drug has the meaning of claim 4, and the ratio of the benzisoselenazole derivative to the platinum-based anticancer drug has the meaning of claim 5;
preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient.
Technical Field
The invention belongs to the technical field of tumor treatment, and particularly relates to an application of a benzisoselenazole derivative and a platinum drug in preparation of a drug for treating tumor and a drug for treating postoperative tumor recurrence.
Background
Tumors, especially malignant tumors, are global human health killers and are one of the leading causes of high mortality from disease. Clinical treatment modalities for tumors include mainly surgery, radiation therapy and chemotherapy. However, even with the above treatments, the probability of rapid proliferation and recurrence of tumors remains high, making anticancer therapy one of the most challenging problems worldwide.
The remaining of cancer cells in the body caused by the incomplete cancer treatment is a main cause of recurrence of cancer cells. The main treatment means of cancer is surgery, radiotherapy and chemotherapy, the surgery is a mechanical means, the local treatment is thorough, but the cancer cell is dropped after the cancer is attacked due to the metastasis of the cancer cell, and the cancer cell is proliferated or metastasized to the periphery and far away through the ways of local diffusion, blood vessels, lymphatic vessels and the like. According to the monitoring data of the cancer condition of the disease pre-control center in China, the recurrence and metastasis rate of cancer patients in China after operation for 3 months is 50%, the recurrence and metastasis rate of cancer patients in China after operation for 6 months is as high as 69%, and the recurrence or metastasis rate of cancer patients after operation for five years is as high as more than 90%.
Overall, recurrent cancer is more difficult than initial cancer treatment, mainly because: 1. the initial local treatment such as surgery or radiotherapy has removed cancer cells as much as possible, but some very small cancer cell population may remain, and the cancer cells have migrated out of the scope of surgery and radiotherapy very early. These cancer cell populations tend to be very aggressive (rapid growth and rapid spread), i.e., recurring tumors are composed of a very high malignancy subpopulation, and treatment difficulties are greatly increased for tumors of further increased malignancy. 2. Cancer is resistant to the initial chemoradiotherapy treatment and the side effects of the initial treatment can also lead to difficulties in the re-treatment of the recurrent tumor.
The platinum anticancer drug is used as a first-line anticancer drug of various cancers, is suitable for ovarian cancer, lung cancer, liver cancer and the like, and can play a broad-spectrum anticancer role through various mechanisms. However, when the platinum anticancer drug is clinically used as an anticancer drug alone or a postoperative recurrence drug, a large gap time is required for eliminating toxicity in use due to its large toxicity, which affects anticancer effects and has a poor effect of suppressing cancer recurrence for postoperative patients.
Therefore, the combination of different target and low-toxicity medicaments with platinum medicaments is an important way for effective treatment, and the finding of medicaments which can coordinate platinum medicaments to play a better anti-tumor effect and a better effect of inhibiting tumor growth, can show low toxicity, inhibit or eliminate the growth of cancer cells fundamentally and effectively inhibit initial tumors and postoperative recurrent tumors becomes an urgent technical problem to be solved.
Disclosure of Invention
The invention provides an application of a benzisoselenazole derivative and a platinum anti-cancer drug in preparation of a drug for treating tumors.
According to the invention, the benzisoselenazole derivative has a structure of a compound shown as a formula A, and is selected from at least one of the compound shown as the formula A, a precursor, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug and a solvate thereof,
wherein R is1、R2Identical or different, independently of one another, from H or the following radicals: c1-12Alkyl radical, C3-20A cycloalkyl group; preferably, it is selected from H, or the following groups: c1-6Alkyl radical, C3-10A cycloalkyl group; illustratively, R1、R2Is selected from H.
Wherein R is selected from C1-12Alkylene, phenylene, biphenylene, triphenylene, or
Preferably, in the compound shown in the formula A, R1、R2Identical or different, independently of one another, from H, -CH3、-CH2CH3、-CH(CH3)2、-C(CH3)3、-CH(CH2)4or-CH (CH)2)5(ii) a R is selected from-CH2-、-C2H4-、-C4H8-, phenylene-C6H4-。
According to an exemplary embodiment of the present invention, the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -butane, the structure of which is shown below:
according to the present invention, the platinum-based anticancer drug is selected from at least one of Cisplatin (CDDP), Carboplatin (CBP), oxaliplatin (1-OHP), nedaplatin (CDGP), etc.; illustratively, the platinum-based anti-cancer drug is selected from Cisplatin (CDDP).
According to the invention, the molar ratio of the benzisoselenazole derivative to the platinum anticancer drug is (1-99) to (1-99); for example, the molar ratio is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6); illustratively, the molar ratio is 1:1, 2:1, 60:1, 70:1, 80: 1.
According to the invention, the tumors include solid tumors and non-solid tumors, and may be benign and malignant. For example, the tumors include, but are not limited to: liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, gastric cancer, skin cancer, bladder cancer, ovarian cancer, prostate cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, lymph cancer, oral epithelial cancer, epithelial cervical cancer or chronic myelogenous leukemia. Preferably, the tumor is a drug-resistant tumor, which means resistance to an antitumor drug acting on tumor cells in a dividing and proliferating state. Illustratively, the tumor is selected from liver cancer, ovarian cancer, lung cancer, rectal cancer or epithelial cervical cancer.
Furthermore, the invention provides the application of the benzisoselenazole derivative and a platinum anti-cancer drug in combination for inhibiting the proliferation of tumor cells. For example, the use of benzisoselenazole derivatives in combination with platinum-based anti-cancer drugs for inhibiting the proliferation of tumor cells in vitro or in vivo; preferably, for use in inhibiting the proliferation of tumor cells in vitro.
For example, the tumor cells include, but are not limited to: human liver cancer cell HepG2, human liver cancer cell Bel-7402, human liver cancer cell Huh7721, human colorectal cancer cell LoVo, human colorectal cancer cell RKO, human colorectal cancer cell SW480, human lung cancer cell A549, human lung cancer cell H1299, human lung cancer cell SPCA-1, human epithelial cervical cancer cell HeLa, human breast cancer cell MCF-7, human chronic myelogenous leukemia cell k562, human esophageal cancer cell KYSE150, human esophageal cancer cell KYSE450 or human esophageal cancer cell KYSE 510. According to the technical scheme, the tumor cell is selected from human liver cancer cell Bel-7402, human colorectal cancer cell LoVo, human epithelial cervical cancer cell HeLa and human lung cancer cell A549.
According to an exemplary technical scheme of the invention, the benzisoselenazole derivative is combined with a platinum anticancer drug to inhibit the proliferation of human hepatoma cell Bel-7402. For example, the compound can be used for inhibiting the proliferation of human hepatoma cell Bel-7402 in vitro. Preferably, the protein is used for inducing Bel-7402 apoptosis, for example, inducing Bel-7402 apoptosis by reducing Bcl-2/Bax protein expression ratio. For another example, the combination is also used for inhibiting the activity of TrxR (thioredoxin oxidoreductase) in Bel-7402 cells.
Furthermore, the invention provides a method for inhibiting tumor cell proliferation (including in vitro proliferation and in vivo proliferation) by combining the benzisoselenazole derivative and the platinum anticancer drug, wherein the benzisoselenazole derivative and the platinum anticancer drug act on tumor cells according to a certain proportion.
Preferably, the certain mixture ratio is as follows: the molar ratio of the benzisoselenazole derivative to the platinum anti-cancer drug is (1-99) to (1-99); for example, the molar ratio is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6); illustratively, the molar ratio is 1:1, 2:1, 60:1, 70:1, 80: 1. Specifically, when the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is not very different, for example, the molar ratio is (1-10): (1-10), (1-6): 1-6), and exemplarily, the molar ratio is 1:1 and 2:1, the combination of the benzisoselenazole derivative and the platinum-based anticancer drug can preferably act to inhibit the in vitro proliferation of tumor cells. When the benzisoselenazole derivative is used in vivo, because the in vivo metabolism of the drug to reach the concentration of a tumor part is greatly related to the in vivo property, the dosage of the benzisoselenazole derivative orally taken needs to be far larger than that of a platinum anti-cancer drug (conventionally injected) for administration, for example, the molar ratio of the benzisoselenazole derivative to the platinum anti-cancer drug is (50-90): 1-30), (60-70): 1-10, and exemplarily, the molar ratio is 60:1, 70:1 and 80:1, the combination of the benzisoselenazole derivative and the platinum anti-cancer drug can preferably act on inhibiting the in vivo proliferation of tumor cells.
Preferably, the concentrations of the benzisoselenazole derivative and the platinum anticancer drug are both 1-100 mu M, such as 5-60 mu M and 10-50 mu M; illustratively, the concentration is 15, 20, 30, 40 μ M.
Preferably, the action time is 10-120 h, such as 15-105 h and 24-96 h; the action times are, for example, 24h, 48h, 72 h.
Preferably, the mode of action is incubation.
Furthermore, the invention also provides a pharmaceutical composition, which comprises the benzisoselenazole derivative and a platinum anti-cancer drug. Preferably, the benzisoselenazole derivative and the platinum-based anti-cancer drug have the meanings and the proportion as described above.
Preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient.
Preferably, the pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, and the like.
For example, the diluent is selected from lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like. The binder is, for example: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. For example, the antioxidant is selected from vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, and the like. For example, the pH adjusting agent is selected from hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like. For example, the preservative is selected from methyl paraben, ethyl paraben, m-cresol, benzalkonium chloride, and the like. For example, the lubricant is selected from magnesium stearate, aerosil, talc, and the like. For example, the disintegrant is selected from starch, methylcellulose, xanthan gum, croscarmellose sodium, and the like.
The dosage form of the pharmaceutical composition may be in the form of oral preparations such as tablets, capsules, pills, powders, granules, suspensions, syrups, and the like; it can also be administered by injection, such as injection solution, powder for injection, etc., by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.
Further, the present invention also provides a method for treating tumors by using the above pharmaceutical composition, and a therapeutically effective amount of the pharmaceutical composition is administered to an individual in need thereof.
Preferably, the subject may be a mammal, such as a human.
Preferably, the tumor has the meaning as described above, preferably liver cancer.
Furthermore, the invention also provides application of the benzisoselenazole derivative and a platinum anti-cancer drug in preparation of a drug for inhibiting postoperative recurrence of tumors.
Preferably, the molar ratio of the benzisoselenazole derivative to the platinum anti-cancer drug is (1-99) to (1-99); for example, the molar ratio is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6); illustratively, the molar ratio is 1:1, 2:1, 60: 1.
Preferably, the dosage of the benzisoselenazole derivative is 10-600 mg/kg based on the weight of a subject (such as a mouse); for example, 50 to 500mg/kg, 100 to 400 mg/kg; illustratively, the amount administered is 180 mg/kg.
Preferably, the dosage of the platinum anticancer drug is 0.05-5 mg/kg based on the weight of an administration object (such as a mouse); for example, 0.1 to 4mg/kg, 0.5 to 3 mg/kg; illustratively, the amount administered is 2 mg/kg.
Preferably, the tumor has the meaning as described above, preferably lung cancer, e.g. mouse Lewis lung cancer.
Preferably, the benzisoselenazole derivative is used in combination with a platinum-based anticancer drug to increase the number of white blood cells, red blood cells, and platelets) in a blood routine.
Preferably, the benzisoselenazole derivative is combined with a platinum-based anti-cancer drug to synergistically inhibit the expression of TrxR in tumor tissues.
Furthermore, the invention also provides a pharmaceutical composition for inhibiting postoperative recurrence of tumors, which comprises the benzisoselenazole derivative and a platinum anti-cancer drug. Preferably, the benzisoselenazole derivative and the platinum-based anti-cancer drug have the meanings and the proportion as described above.
Preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient. Preferably, the adjuvant has the meaning as described above.
Further, the present invention also provides the above pharmaceutical composition in combination with a method of inhibiting postoperative tumor recurrence by administering a therapeutically effective amount of the pharmaceutical composition to an individual in need thereof.
Preferably, the subject may be a mammal, such as a human, a mouse.
Preferably, the tumor has the meaning as described above, preferably lung cancer, e.g. mouse Lewis lung cancer.
The invention has the beneficial effects that:
the invention unexpectedly discovers that the combined application of the BS and the CDDP has excellent effect on inhibiting the proliferation of various tumor cells and has great application value on the treatment of various tumors. Particularly, the two are combined, so that the CDDP administration dosage with high toxicity can be effectively reduced, and the safety of anticancer drugs is improved; the apoptosis of the Bel-7402 cell can be induced by reducing the expression ratio of Bcl-2/Bax protein, and the proliferation rate of tumor cells is obviously inhibited.
The invention also unexpectedly discovers that the combined application of the BS and the CDDP has excellent effect on inhibiting the postoperative recurrence of the tumor. The concrete expression is as follows: the combination of the two can synergistically inhibit the expression of TrxR in tumor tissues, obviously reduce the proliferation rate of postoperative tumor cells and improve the growth inhibition rate of the postoperative tumor cells. Furthermore, the combination of BS and CDDP can also relieve the toxicity of CDDP and improve the immunity.
The combination of BS and platinum anticancer drugs as a mechanism for treating tumors and postoperative recurrence:
platinum anti-cancer drugs, particularly cisplatin, as an anti-tumor chemotherapeutic drug in the first line of clinical practice have definite anti-tumor effects, but have obvious cytotoxicity correspondingly, and cisplatin is not suitable for postoperative tumor patients in theory, but is clinically still selected for postoperative recurrence medication in order to kill recurrent tumors immediately and control tumor recurrence and growth. The BS is used as thioredoxin reductase inhibiting medicine and has the characteristic of inhibiting tumor cell proliferation and recurrent growth. The invention unexpectedly discovers that when the BS and the cisplatin are combined, the synergistic anti-tumor effect generated by the superposition of different action mechanisms of the BS and the cisplatin can be exerted, and the dosage of the cisplatin and the dependence of an organism on the cisplatin are reduced, so that the toxic and side effect bearing capacity of a postoperative patient is reduced, and the recovery of the patient is further assisted.
Definition and description of terms
Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present specification.
The term "C1-12Alkyl is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C1-6An alkyl group. "C1-6Alkyl "is understood to mean preferablyStraight or branched chain saturated monovalent hydrocarbon radicals having 1,2, 3, 4, 5, 6 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group or a 1, 2-dimethylbutyl group. In particular, the radicals have 1,2, 3, 4, 5, 6 carbon atoms ("C)1-6Alkyl groups) such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly groups having 1,2, 3 or 4 carbon atoms ("C)1-4Alkyl groups) such as methyl, ethyl, n-propyl, isopropyl or butyl.
The term "C3-20Cycloalkyl is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C3-10Cycloalkyl groups ". The term "C3-10Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Said C is3-10Cycloalkyl groups may be monocyclic hydrocarbon groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic hydrocarbon groups such as decalin rings.
The term "C1-12Alkylene is understood to mean preferably a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C, with the loss of two hydrogen atoms1-4An alkylene group. "C1-4Alkylene "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4 carbon atoms which has lost two hydrogen atoms. The alkylene group is, for example, methylene, ethylene, propylene, butylene.
The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present invention sufficient to effect the intended use, including but not limited to the treatment of a disease as defined below. The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the particular compound selected, the dosage regimen to be followed, whether to administer it in combination with other compounds, the timing of administration, the tissue to be administered and the physical delivery system carried.
Drawings
FIG. 1 is a plot of the growth inhibition rate of BS, CDDP and their combination for 24 h;
a: BEL-7402 cells; b: LoVo cells; c: HeLa cells; d: a549 cells. Data are presented as mean. + -. standard deviation, n.gtoreq.3.
FIG. 2 is a graph showing the growth inhibition rate of BS, CDDP and their combination for 48 h;
a: BEL-7402 cells; b: LoVo cells; c: HeLa cells; d: a549 cells. Data are presented as mean. + -. standard deviation, n.gtoreq.3.
FIG. 3 is a plot of the growth inhibition rate of BS, CDDP and their combination for 72 h;
a: BEL-7402 cells; b: LoVo cells; c: HeLa cells; d: a549 cells. Data are presented as mean. + -. standard deviation, n.gtoreq.3.
FIG. 4 is a graph of DRI values of BEL-7402, LoVo and HeLa cells when BS and CDDP were administered in combination in examples 1 and 2;
a: example 1 DRI value analysis profile of BEL-7402 cells on 48 h;
b: example 2 DRI value analysis profile of BEL-7402 cells on 48 h;
c: example 1 DRI value analysis plot of the effect on BEL-7402 cells for 72 h;
d: example 2 DRI value analysis plot of the effect on BEL-7402 cells for 72 h;
example 1 DRI value analysis of HeLa cells for 48 h;
f: example 2 DRI value analysis profile of HeLa cells on 48 h;
g: example 1 DRI value analysis profile of HeLa cells on 72 h;
h: example 2 DRI value analysis profile of HeLa cells on 72 h;
i: example 1 plot of DRI value analysis on LoVo cells for 48 h;
j: example 2 plot of DRI value analysis on LoVo cells for 48 h;
k: example 1 DRI value analysis profile of 72h on LoVo cells;
l: example 2 graph analysis of DRI values on LoVo cells for 72 h.
FIG. 5 is a graph showing the expression levels of TrxR1, Trx1, β -actin proteins and TrxR activity 48 hours after the action of BS or CDDP;
a is Westernblot analysis of the levels of TrxR1, Trx1 and β -actin in BEL-7402 cells after 48 hours of BS action, B is Westernblot analysis of the levels of TrxR1, Trx1 and β -actin in BEL-7402 cells after 48 hours of CDDP action, and C is the effect of BS on the relative levels of TrxR activity (left), TrxR1 (middle) and Trx1 (right) proteins in BEL-7402 cells, wherein the data are expressed as mean value + -standard deviation, and n is more than or equal to 3.*P<0.05, which indicates a statistical difference from the control group;#P<0.05, representing a statistical difference compared to the
FIG. 6 is a graph of BS or CDDP induction of apoptosis in BEL-7402 cells after 48 hours of action;
a: pro-apoptotic effect on BEL-7402 cells after 48 hours of action with 0 (control), 20, 30, 40. mu.M BS or CDDP. Upper left quadrant: dead cells(ii) a Upper right quadrant: late apoptotic cells; lower left quadrant: a living cell; right lower quadrant: early apoptotic cells; b: total apoptosis rate of BEL-7402 cells after 48 hours of BS action. Data are presented as mean. + -. standard deviation, n.gtoreq.3. P<0.01, which indicates a statistical difference from the control group;#P<0.01, representing a statistical difference compared to the
FIG. 7 is a graph showing that the group administered with BS and CDDP enhanced the apoptosis-inducing effect of BEL-7402 cells;
effect on apoptosis 48 hours after exposure to 30 μ M BS (B), 30 μ M (c) and 30 μ M combination (BS: CDDP ═ 1:1) in BEL-7402 cells.
FIG. 8 is a graph showing that the combination of BS and CDDP induces apoptosis by decreasing the expression ratio of Bcl-2/Bax protein in BEL-7402 cells;
western blot analysis of the levels of Bax, Bcl-2, β -actin in BEL-7402 cells 48 hours after the combined (BS: CDDP 1:1) 30. mu.M, 30. mu.M and 30. mu.M groups A, B, total apoptosis rate of BEL-7402 cells 48 hours after the combined (BS: CDDP 1:1) and B, the individual and combined (BS: CDDP) groups, data are expressed as mean. + -. standard deviation, n.gtoreq.3<0.01, which indicates a statistical difference from the control group;#P<0.05, representing a statistical difference compared to the
FIG. 9 shows tumor growth in groups of mice. Mouse tumor size was measured every two days using a vernier caliper according to the formula: tumor length x tumor width 2 x 0.5236 tumor volume was calculated. (a) The method comprises the following steps Photographs of each group of tumors at the end of the experiment; (b) the method comprises the following steps Tumor volume changes in mice of each group. Data are presented as mean ± standard deviation (n ═ 6). P <0.05, indicating a statistical difference compared to the control group.
FIG. 10 shows the body weight change of mice. (a) The method comprises the following steps The weight change of the mice in the administration process; (b) the method comprises the following steps Weight gain during administration in each group of mice. P <0.05, indicating a statistical difference compared to the control group.
FIG. 11 shows the expression level of TrxR protein in mouse tumor tissue. (a) The method comprises the following steps Mouse tumor tissue TrxR expression levels; (b) the method comprises the following steps Relative expression level of TrxR. Data are presented as mean ± standard deviation, n ═ 3. P<0.05, which indicates a statistical difference from the control group;#P<0.05, which indicates a statistical difference from the CDDP group.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The experimental method comprises the following steps:
1. test drugs, cells and animals:
BS: the chemical name of the product is 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -ketone) ] -butane, which is referred to Chinese patent No. 02158917.8.
Cisplatin (cissplatin, CDDP): purchased from Hai Wei, China (Beijing) Gene science and technology, Inc.
Human liver cancer cell Bel-7402, human colorectal cancer cell LoVo, human epithelial cervical cancer cell HeLa and human lung cancer cell A549: all purchased from cell center of basic medical college of the university of medical science.
Balb/c mice: 4 weeks old, weight 16g-18g, male, purchased from the center of laboratory animals of the department of medicine of Beijing university, and production license number SYXK (Jing) 2012-0036. The breeding environment is clean, the breeding temperature is 25 +/-2 ℃, the illumination is 12h for 12h, the breeding environment is dark, and the food and water supply is sufficient.
2. The test method comprises the following steps:
2.1 assay of inhibition of cell proliferation (SRB method):
(1) digesting the wall-attached cultured cells into a single-cell suspension, and adjusting the cell concentration to be 2 multiplied by 104one/mL, and was inoculated uniformly in a 96-well plate (Corning), 180. mu.L per well;
(2) placing the 96-well plate in an incubator for 24h, and adding 20 mu L of compound per well according to the specified concentration after the cells adhere to the wall;
(3) at the end of the drug action, 100 μ L of 10% (v/v) trichloroacetic acid precooled at 4 ℃ is added into each well, and a 96-well plate is fixed for 1h at 4 ℃;
(4) carefully discarding the fixative, slowly washing with deionized water from one side of the 96-well plate for 4 times, blow-drying with electric heating blower or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);
(5) adding 100 μ L of 0.057% (w/v) SRB staining solution into each well, and staining for 30min at room temperature;
(6) carefully discarding the dye solution, washing each well with 150 μ L of 1% (v/v) acetic acid for 4 times, drying by blowing with electric heat or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);
(7) accurately adding 200 mu L of 10mM Tris destaining solution into each hole, standing at room temperature to dissolve the Tris destaining solution naturally or placing the Tris destaining solution on a shaking table to dissolve the Tris destaining solution;
(8) the absorbance of the sample at 492nm was measured using a microplate reader (Thermo Fisher Scientific). The cell proliferation inhibition rate was calculated according to the following formula:
wherein, the control well is inoculated with 180 μ L of cell suspension, and the compound is replaced by medium (20 μ L) of the same volume; for the inoculation of the blank wells, 180. mu.L of medium was added, and for the administration, 20. mu.L of medium was added. Three duplicate wells were set for each drug concentration.
2.2Western Blot:
extracting total protein of tissue, determining protein concentration (30-50 μ g), adding 5 × loading buffer solution, adding RIPA lysate to make up volume, mixing, and performing denaturation treatment at 95 deg.C for 10 min. After fully and uniformly mixing, adding a clean 1.5mm rubber plate to a proper position, and sealing with n-butanol. Adding 1mL of 1 XSEPARATION GEL buffer solution, and standing at room temperature overnight; inserting a 1.5mm sample adding comb, and standing at room temperature for 2h to wait for gelation.
Pulling out the sample adding comb, and loading the sample by a conventional method; and (4) performing constant voltage electrophoresis at 80V until the front edge of the bromophenol blue enters the separation gel, increasing the voltage to 160V, continuing the constant voltage electrophoresis, and stopping the electrophoresis until the bromophenol blue migrates to the end of the separation gel.
Accurately shearing 6 pieces of filter paper with the same size as the separation gel and a 0.2 mu m PVDF membrane, soaking the PVDF membrane in methanol for 10s, soaking in deionized water for 3min, and soaking in an electrotransformation buffer solution for 3 min;
the electricity changes sandwich and lays by the order of positive pole to negative pole in proper order: one sponge, three pieces of filter paper, a PVDF membrane, separation glue, three pieces of filter paper and one sponge (all soaked in an electric transfer buffer solution in advance), air bubbles are expelled from the layers by a glass rod, and an electric transfer clamp and an ice box are clamped and put into an electric transfer tank;
and (5) placing the electric rotating tank in an ice-water bath, and rotating for 2 hours at a constant current of 250 mA.
After the electrotransfer is finished, taking out the PVDF membrane, and placing the PVDF membrane in a 5% closed state; washing the membrane with TBST for 3 times, sealing the PVDF membrane and a diluted secondary antibody solution with a proper proportion in a hybridization bag, incubating for 1h at room temperature, washing the membrane with TBST for 3 times, and developing;
and opening a gel imaging system, uniformly mixing the solution A and the solution B of the ECL luminous liquid in equal volume, uniformly distributing the mixture on the PVDF membrane, and carrying out exposure analysis.
2.3 flow cytometry-based apoptosis assay:
1) cells from logarithmic growth phase were seeded in six-well plates (Corning),
2) after 24h of drug action, the recovery medium is centrifuged at 1200rpm for 2min in a 5mL centrifuge tube, and the supernatant is discarded. Rinsing adherent cells in the culture dish once by using PBS precooled at 4 ℃, adding 300 mu L of pancreatin, and placing in an incubator for digestion for 3 min;
3) and (3) blowing the cells into single cells by using 1mL of culture medium, re-suspending the precipitated cells in a centrifuge tube by using 1mL of PBS, combining the precipitated cells with the adherent cells in a culture dish of the corresponding group, centrifuging at 400rpm for 2min, and discarding the supernatant. Resuspend the cells with 1mL PBS, screen through 300 mesh cell screen;
4) resuspending the precipitated cells with 190. mu.L of binding solution, adding 10. mu.l of Annexin V-FITC, incubating at room temperature in the dark for 10min, centrifuging at 4000rpm for 2min, and discarding the supernatant;
5) the cells were resuspended in 195. mu.L of binding solution, 5. mu.L of PI solution was added and immediately detected on a flow cytometer (BDFACSCalibur).
2.4 establishment of tumor recurrence model after mouse Lewis lung cancer surgery:
balb/c mice subcutaneously transplanted with murine lung carcinoma cells Lewis were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm3In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm3Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:
a blank control group (5% CMC-Na, i.g., q.d.); drug low, medium and high dose groups: 90 mg/kg-1,i.g.,q.d.；180mg·kg-1,i.g.,q.d；360mg·kg-1,i.g.,q.d.。
Mice body weight, tumor volume and activity were recorded at intervals during the experiment. The mice were sacrificed eight days after administration, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed.
2.5 preparation of the liquid medicine
BS medicine mother liquor: BS was weighed and dissolved in DMSO to prepare a 10mM stock solution, which was stored at-20 ℃ until use.
CDDP mother liquor: CDDP was weighed and dissolved in PBS to prepare a 10mM stock solution, which was stored at room temperature for further use.
The stock solution was diluted to the desired concentration (15, 20, 30, 40 μ M) in serum-free medium as required before use. The latter experiments were diluted as required for the experiment.
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