Drug combination of protocatechuic aldehyde and dacarbazine and its use in the treatment of malignant melanoma

Abstract

The present invention relates to a pharmaceutical combination of protocatechuic aldehyde and dacarbazine and its use in the treatment of malignant melanoma. The pharmaceutical combination includes: protocatechuic aldehyde or a composition containing protocatechuic aldehyde as a first active ingredient; and dacarbazine as a second active ingredient. The pharmaceutical combination according to the embodiments of the present invention can increase the number of DNA double-strand breaks in tumor cells induced by dacarbazine, promote apoptosis of tumor cells, and thus achieve the purpose of treating and controlling cancer, especially treating malignant melanoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular, relates to a drug combination of protocatechuic aldehyde and dacarbazine and use thereof in treating malignant melanoma. Background Art

[0002] Cancer is a disease that seriously threatens human life and health. In recent years, the incidence and mortality of cancer have continued to rise around the world, which is a problem that plagues human life and health. Currently available cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, macromolecular immunotherapy and other treatment methods.

[0003] There are many types of cancer, such as thyroid cancer, nasopharyngeal cancer, adenocarcinoma, liver cancer, skin cancer, etc. Among them, malignant melanoma is the most invasive and deadly skin cancer. The current treatments for malignant melanoma mainly include surgery and adjuvant therapy, radiotherapy, chemotherapy, biological immunotherapy, targeted therapy, etc., but for patients with poor economic conditions, resistance to targeted therapy, and no response to immunosuppressants, chemotherapy is still an important means of treating melanoma. Currently, the commonly used chemotherapy drugs are Dacarbazine (Dacarbazine, DTIC), but the efficacy of DTIC alone is about 8% to 12%, and its use efficiency is relatively low.

[0004] Therefore, there is an urgent need to develop an effective drug for treating cancer. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a drug combination of protocatechuic aldehyde and a DNA alkylating agent, which can promote apoptosis of tumor cells and effectively treat cancers such as melanoma.

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] Dacarbazine was approved by the US FDA for the treatment of malignant melanoma in 1975 and is still the gold standard chemotherapy drug for the clinical treatment of advanced malignant melanoma. Dacarbazine is a DNA alkylating agent, and its mechanism of action is to metabolize into the active compound 5-(3-methyltriazine-1-yl)imidazole-4-amide (MTIC), which further methylates guanine (G) in DNA molecules to form O 6 -methylguanine. 6The formation of O⁶-methylguanine can cause a mismatch in the G-C pairing during DNA replication, resulting in the formation of a G-T pairing. Once the G-T pairing is formed, the intracellular DNA damage repair system will repeatedly cut at the mismatch, thus generating DNA double-strand breaks. The formation of a large number of DNA double-strand breaks will lead to the apoptosis of tumor cells, thereby achieving the purpose of treating and controlling tumors.

[0008] However, the effective rate of using DTIC alone is about 8% - 12%, and its efficiency is not high and tumor patients are prone to drug resistance. To solve this problem, people have tried to use various drugs in combination with DTIC to improve the curative effect and increase the survival benefit of melanoma patients, such as cisplatin, recombinant human endostatin (Endostar), doxorubicin, vincristine, etc. However, the combination of commonly used chemotherapeutic drugs and dacarbazine for chemotherapy cannot improve the response level of melanoma to drugs and the survival of melanoma patients (Chapman PB, Einhorn LH, Meyers ML, et al: Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol 17:2745 - 2751, 1999). Therefore, there is an urgent need to develop a drug with low toxicity and side effects, and can increase the efficacy of DTIC and inhibit or reverse the generation of its drug resistance.

[0009] Protocatechuic aldehyde (PA), with the chemical name of 3,4-dihydroxybenzaldehyde, is a natural phenolic acid compound and is one of the effective active ingredients of traditional Chinese medicines such as Salvia miltiorrhiza, Salvia japonica, Gastrodia elata, Lycium barbarum, Stenoloma chusanum, Ilex chinensis, Tsaoko fruit, Cedrus deodara, Ampelopsis japonica, Acalypha australis, Begonia grandis, etc. According to research reports, it has various pharmacological activities such as antibacterial, anti-inflammatory, anti-tumor, and antioxidant stress. Among them, the structural formula of protocatechuic aldehyde is shown in formula (II):

[0010]

[0011] Based on this, in the first aspect of the present invention, the present invention proposes the use of a compound shown in formula (I) or its salt or its derivative or a composition containing the compound shown in formula (I) or its salt or its derivative in the preparation of a reagent for reducing the expression or activity of MGMT protein,

[0012]

[0013] wherein, R1 is selected from -CHO, -COOH, -CH₂CH₂OH, -CH₂OH or

[0014] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by halogen or hydroxyl, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, Or, it forms a 5- to 10-membered heteroalkyl ring when connected to the C to which it is attached, wherein the 5- to 10-membered heteroalkyl ring is optionally substituted by one or more R7s;

[0015] R4, R5, and R6 are each independently selected from H or -OH;

[0016] R7 is selected from -C 1～6 alkyl optionally substituted by hydroxyl.

[0017] The inventors found through experiments that protocatechuic aldehyde or a drug containing protocatechuic aldehyde can effectively reduce the expression or activity of the MGMT protein. In particular, when a reagent containing protocatechuic aldehyde is contacted with cells, the expression or activity of the MGMT protein in the cells can be reduced, and it can be used for scientific research on in vitro cells.

[0018] It should be noted that in this article, "reducing the expression or activity of the MGMT protein" includes reducing the expression of the MGMT protein and reducing the activity of the MGMT protein.

[0019] Exemplarily, reducing the expression of the MGMT protein includes but is not limited to being achieved in the following ways: reducing the mRNA level of the MGMT protein, inhibiting the translation of the mRNA of the MGMT protein, and promoting the degradation of the MGMT protein.

[0020] Exemplarily, reducing the activity of the MGMT protein may refer to changing the spatial structure of the MGMT protein to decrease the activity of the MGMT protein. According to the embodiments of the present invention, the reduction of the expression of the MGMT protein is achieved by promoting the degradation of the MGMT protein. The inventors found through experiments that contacting the reagent of the present invention with cells can effectively promote the degradation of the MGMT protein.

[0021] According to an embodiment of the present invention, the compound represented by formula (I) or its salt or derivative thereof or the composition containing the compound represented by formula (I) or its salt or derivative thereof includes at least one selected from Salvia miltiorrhiza or its extract, Sage or its extract, Gastrodia elata or its extract, Lycium barbarum or its extract, Pteris crenata or its extract, Ipomoea australis or its extract, Tsaoko or its extract, Cedrus deodara or its extract, Ampelopsis pilosa or its extract, Amaranthus chinensis or its extract, Begonia or its extract, Ranunculus spinulosa or its extract, Cibotium barometz or its extract, Palm and its extract and Persimmon leaf and its extract.

[0022] According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or derivative thereof includes a compound selected from the group consisting of compound danshen tablets, compound danshen dripping pills, compound danshen injection, danshen tablets, xinkeshu tablets, xiaojiantongluo tablets, anshenbuxin pills, tianwangbuxin pills, wenxin granules, shuangdan oral liquid, xintong oral liquid, danhonghuayu oral liquid, danshen water extract, xiakucao oral liquid, fulcong mixture, xiangdan injection, yinaoxin granules, shengpupenyan granules, huoluoxiaotong tablets, osteitisxiaofang, guanxinning injection, danhong injection, dingxiyatong capsules, compound kylin cataplasm, dog spine slices , Shenmai Granules, Shensong Yangxin Capsules, Danshen Chuanxiongqin Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimailing Oral Liquid, Yiganning Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lianshen Tonglin Tablets, Bubai Granules, Kunning Oral Liquid, Shenkangning Tablets, Runing Granules, Rukuaixiao Granules, Jizhi Syrup, Huolisu Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules and Tangmaikang Granules.

[0023] In the second aspect of the present invention, the present invention provides a single-dose MGMT protein inhibitor. According to an embodiment of the present invention, the single-dose MGMT protein degrader comprises 20-200 μM of a compound represented by formula (I) or a salt thereof or a derivative thereof,

[0024]

[0025] Wherein, R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH or

[0026] R2 and R3 are independently selected from H, -C optionally substituted by halogen or hydroxyl 1～6 Alkyl, -C 2～6 Alkenyl, -C(O)R7, -C 1～4 Alkylene -OR7, -Si(CH3)3, or, It forms a 5- to 10-membered heteroalkyl group when connected to the linked C, wherein the 5- to 10-membered heteroalkyl group is optionally substituted by one or more R7;

[0027] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by a hydroxyl group. The single-dose MGMT protein degrader of the present invention can effectively inhibit the expression and activity of MGMT protein in cells, for example, promote the degradation of MGMT protein in cells, and is particularly useful for scientific research on in vitro cells.

[0028] In a third aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises a compound of formula (I) or a salt or derivative thereof, or a composition containing a compound of formula (I) or a salt or derivative thereof,

[0029]

[0030] wherein R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH, or

[0031] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by a halogen or a hydroxyl group, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, or, It forms a 5- to 10-membered heteroalkyl group when connected to the linked C, wherein the 5- to 10-membered heteroalkyl group is optionally substituted by one or more R7;

[0032] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by a hydroxyl group. The kit of the present invention can be used to promote the degradation of MGMT protein in cells.

[0033] Exemplarily, the kit of the present invention is used to control the level of MGMT protein in cells in order to obtain cells with a specific level of MGMT protein.

[0034] According to an embodiment of the present invention, the working concentration of the compound of formula (I) or a salt or derivative thereof is 20 to 200 μM, preferably 40 to 150 μM, and more preferably 40 to 100 μM. Using the above working concentration can further reduce the level of MGMT protein in cells, and particularly can effectively promote the degradation of MGMT protein.

[0035] According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivative includes at least one selected from Salvia miltiorrhiza or its extract, Salvia japonica or its extract, Gastrodia elata or its extract, Lycium barbarum or its extract, Stenoloma chusanum or its extract, Ilex purpurea or its extract, Tsaoko fruit or its extract, Cedrus deodara or its extract, Ampelopsis japonica or its extract, Acalypha australis or its extract, Begonia grandis or its extract, Ranunculus muricatus or its extract, Cibotium barometz or its extract, palm and its extract, and persimmon leaf and its extract.

[0036] In a fourth aspect of the present invention, the present invention provides a method for reducing the expression or activity of MGMT protein. According to an embodiment of the present invention, the method includes: contacting a cell expressing MGMT with a compound represented by formula (I) or its salt or its derivative or a composition containing the compound represented by formula (I) or its salt or its derivative;

[0037]

[0038] wherein, R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH or

[0039] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by halogen or hydroxyl, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, or, together with the C to which it is attached form a 5- to 10-membered heterocycloalkyl, wherein the 5- to 10-membered heterocycloalkyl is optionally substituted by one or more R7;

[0040] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by hydroxyl. The method according to the embodiment of the present invention can effectively promote the degradation of MGMT protein, especially can be used for scientific research on in vitro cells. For example, it can be used to degrade MGMT protein in in vitro cells to obtain cells with a specific level of MGMT protein.

[0041] According to an embodiment of the present invention, the working concentration of the compound represented by formula (I) or its salt or its derivative is 20-200 μM, preferably 40-150 μM, more preferably 40-100 μM. Using the above working concentration can further reduce the level of MGMT protein in cells, especially can effectively promote the degradation of MGMT protein.

[0042] According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivative includes at least one selected from Salvia miltiorrhiza or its extract, Salvia japonica or its extract, Gastrodia elata or its extract, Lycium barbarum or its extract, Stenoloma chusanum or its extract, Ilex purpurea or its extract, Amomum tsaoko or its extract, Cedrus deodara or its extract, Ampelopsis japonica or its extract, Acalypha australis or its extract, Begonia grandis or its extract, Ranunculus muricatus or its extract, Cibotium barometz or its extract, Trachycarpus fortunei and its extract, and Persimmon leaf and its extract.

[0043] In a fifth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: a compound represented by formula (I) or its salt or its derivative, or a composition containing the compound represented by formula (I) or its salt or its derivative; and a DNA alkylating agent;

[0044]

[0045] wherein, R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH or

[0046] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by halogen or hydroxyl, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, or, together with the C to which it is attached, form a 5- to 10-membered heterocycloalkyl group, wherein the 5- to 10-membered heterocycloalkyl group is optionally substituted by one or more R7;

[0047] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by hydroxyl. The pharmaceutical composition according to the embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by the DNA alkylating agent, promote apoptosis of tumor cells, and thus achieve the purpose of treating and controlling cancer.

[0048] The inventors also found through experiments that the pharmaceutical composition can effectively reduce the level of MGMT protein. Further, the inventors detected the mRNA transcription levels of genes related to DNA damage repair in cells and found that, compared with the pharmaceutical composition that can reduce the mRNA level of MGMT protein and inhibit the translation of the mRNA of MGMT protein, the pharmaceutical composition of the present invention reduces the level of MGMT protein mainly by promoting the degradation of MGMT protein.

[0049] According to an embodiment of the present invention, the DNA alkylating agent includes at least one selected from dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lomustine, melphalan, procarbazine hydrochloride, treosulfan, thiotepa, methyl mustard and its derivatives, nitrogen mustard oxide hydrochloride and its derivatives, chlorambucil derivatives, phenylalanine mustard and its derivatives, mechlorethamine benzoate, mono-substituted aryl mustard derivatives, multi-substituted aryl mustard, oligopeptide aryl mustard, macrocyclic polyamine mustard, cyclophosphamide derivatives, linear phosphoramide derivatives, metal complex mustard, uracil mustard and solanesylamine derivatives.

[0050] According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventor found through experiments that the combination of protocatechuic aldehyde and dacarbazine is better compared with other DNA alkylating agents.

[0051] According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

[0052] According to an embodiment of the present invention, the compound represented by formula (I) or its salt or its derivative or the composition containing the compound represented by formula (I) includes at least one of Salvia miltiorrhiza or its extract, Salvia officinalis or its extract, Gastrodia elata or its extract, Lycium barbarum or its extract, Stenoloma chusanum or its extract, Ilex purpurea or its extract, Amomum tsaoko or its extract, Cedrus deodara or its extract, Ampelopsis japonica or its extract, Acalypha australis or its extract, Begonia grandis or its extract, Ranunculus muricatus or its extract, Cibotium barometz or its extract, Trachycarpus fortunei and its extract, and Persimmon leaf and its extract.

[0053] According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivative includes at least one selected from Compound Danshen Tablets, Compound Danshen Dripping Pills, Compound Danshen Injection, Danshen Tablets, XinKeshu Tablets, Xiaojian Tongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella vulgaris Oral Liquid, Fucong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluo Xiaotong Tablets, Guyan Xiaofang, Guanxinning Injection, Danhong Injection, Dingxi Toothache Capsules, Compound Qilin Babuji, Cibotium barometz Decoction Pieces, Shenmai Granules, Shensong Yangxin Capsules, Danshen Ligustrazine Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimai Ling Oral Liquid, Yiganing Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lianshen Tonglin Tablets, Bupai Granules, Kunning Oral Liquid, Shenkangning Tablets, Runing Granules, Rukuai Xiao Granules, Jizhi Syrup, Huolisu Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules, and Tangmaikang Granules.

[0054] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0055] According to an embodiment of the present invention, the molar ratio of the compound of formula (I) or its salt or its derivative to the DNA alkylating agent is (1 to 100):(100 to 1). The inventors found through experiments that using the above ratio can further promote tumor cell apoptosis and improve the therapeutic effect on cancer.

[0056] In the sixth aspect of the present invention, the present invention provides a drug combination or a drug kit. According to an embodiment of the present invention, the drug combination or the drug kit comprises: a compound of formula (I) or its salt or its derivative or a composition containing the compound of formula (I) or its salt or its derivative as a first active ingredient; and a DNA alkylating agent as a second active ingredient;

[0057]

[0058] wherein, R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH or

[0059] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by halogen or hydroxyl, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, or, together with the C to which it is attached form a 5- to 10-membered heterocycloalkyl, wherein the 5- to 10-membered heterocycloalkyl is optionally substituted by one or more R7;

[0060] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by hydroxyl. The drug combination or the drug kit according to an embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by the DNA alkylating agent, promote tumor cell apoptosis, and thus achieve the purpose of treating and controlling cancer.

[0061] The inventors also found through experiments that the drug combination or the drug kit can effectively reduce the level of MGMT protein. Further, the inventors detected the mRNA transcription levels of genes related to DNA damage repair in cells and found that, compared with the drug combination or the drug kit that can reduce the mRNA level of MGMT protein and inhibit the translation of the mRNA of MGMT protein, the drug combination or the drug kit of the present invention reduces the level of MGMT protein mainly by promoting the degradation of MGMT protein.

[0062] According to an embodiment of the present invention, the DNA alkylating agent includes at least one selected from dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lomustine, melphalan, procarbazine hydrochloride, treosulfan, thiotepa, methyl nitrogen mustard and its derivatives, nitrogen mustard oxide hydrochloride and its derivatives, chlorambucil derivatives, phenylalanine mustard and its derivatives, mechlorethamine benzoate, mono-substituted aryl nitrogen mustard derivatives, multi-substituted aryl nitrogen mustard, oligopeptide aryl nitrogen mustard, macrocyclic polyamine nitrogen mustard, cyclophosphamide derivatives, linear phosphoramide derivatives, metal complex nitrogen mustard, uracil mustard, and solanesylamine derivatives.

[0063] According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventors found through experiments that the combination of protocatechuic aldehyde and dacarbazine is better than other DNA alkylating agents.

[0064] According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

[0065] According to an embodiment of the present invention, the compound represented by formula (I) or its salt or its derivative, or the composition containing the compound represented by formula (I) or its salt or its derivative includes at least one of Salvia miltiorrhiza or its extract, Salvia officinalis or its extract, Gastrodia elata or its extract, Lycium barbarum or its extract, Stenoloma chusanum or its extract, Ilex purpurea or its extract, Amomum tsaoko or its extract, Cedrus deodara or its extract, Ampelopsis japonica or its extract, Acalypha australis or its extract, Begonia grandis or its extract, Ranunculus muricatus or its extract, Cibotium barometz or its extract, Trachycarpus fortunei and its extract, and Persimmon leaf and its extract.

[0066] According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivative includes at least one of Compound Danshen Tablets, Compound Danshen Dripping Pills, Compound Danshen Injection, Danshen Tablets, Xin Keshu Tablets, Xiaojian Tongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Water Extract of Salvia miltiorrhiza, Prunella vulgaris Oral Liquid, Fucong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluo Xiaotong Tablets, Guyan Xiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Babuji, Cibotium barometz Decoction Pieces, Shenmai Granules, Shensong Yangxin Capsules, Danshen Chuanxiongqin Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimai Ling Oral Liquid, Yiganing Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lianshen Tonglin Tablets, Pubai Granules, Kunning Oral Liquid, Shenkangning Tablets, Runing Granules, Rukuai Xiao Granules, Jizhi Syrup, Huolisu Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules, and Tangmaikang Granules.

[0067] According to an embodiment of the present invention, the molar ratio of the compound represented by formula (I) or a salt or a derivative thereof to the DNA alkylating agent is (1 to 100):(100 to 1). The inventors found through experiments that using the above ratio can further promote tumor cell apoptosis and improve the therapeutic effect on cancer.

[0068] According to an embodiment of the present invention, the compound represented by formula (I) or a salt or a derivative thereof and the DNA alkylating agent are formulated together or separately.

[0069] According to an embodiment of the present invention, the compound represented by formula (I) or a salt or a derivative thereof and the DNA alkylating agent are used simultaneously or separately.

[0070] In the seventh aspect of the present invention, the present invention provides a single dosage form. According to an embodiment of the present invention, the single dosage form includes: a compound represented by formula (I) or a salt or a derivative thereof, or a composition containing the compound represented by formula (I) or a salt or a derivative thereof as a first active ingredient; and a DNA alkylating agent as a second active ingredient; wherein, the molar ratio of the compound represented by formula (I) or a salt or a derivative thereof to the DNA alkylating agent is (1 to 100):(100 to 1);

[0071]

[0072] wherein, R1 is selected from -CHO, -COOH, -CH2CH2OH, -CH2OH or

[0073] R2 and R3 are each independently selected from H, -C 1～6 alkyl optionally substituted by halogen or hydroxyl, -C 2～6 alkenyl, -C(O)R7, -C 1～4 alkylene-OR7, -Si(CH3)3, or, together with the C to which it is attached forms a 5- to 10-membered heterocycloalkyl, wherein the 5- to 10-membered heterocycloalkyl is optionally substituted by one or more R7;

[0074] R4, R5, and R6 are each independently selected from H or -OH; R7 is selected from -C 1～6 alkyl optionally substituted by hydroxyl. The single dosage form according to an embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by the DNA alkylating agent, promote tumor cell apoptosis, and thus achieve the purpose of treating and controlling cancer.

[0075] According to an embodiment of the present invention, the DNA alkylating agent includes at least one selected from dacarbazine, temozolomide, thiotepa, methyl nitrogen mustard and its derivatives, nitrogen mustard oxide hydrochloride and its derivatives, chlorambucil derivatives, phenylalanine mustard and its derivatives, mechlorethamine benzoate, monosubstituted aryl nitrogen mustard derivatives, polysubstituted aryl nitrogen mustard, oligopeptide aryl nitrogen mustard, macrocyclic polyamine nitrogen mustard, cyclophosphamide derivatives, linear phosphoramide derivatives, metal complex nitrogen mustard, uracil mustard, and solanesylamine derivatives.

[0076] According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventors found through experiments that the combination of protocatechuic aldehyde and dacarbazine is better compared to other DNA alkylating agents.

[0077] According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

[0078] According to an embodiment of the present invention, the compound of formula (I) or its salt or its derivative or the composition containing the compound of formula (I) or its salt or its derivative includes at least one of danshen or its extract, sage or its extract, gastrodia elata or its extract, wolfberry or its extract, stenoloma chusanum or its extract, ilicifolia or its extract, tsaoko fruit or its extract, cedar or its extract, ampelopsis japonica or its extract, acalypha australis or its extract, begonia or its extract, ranunculus muricatus or its extract, cibotium barometz or its extract, palm and its extract, and persimmon leaf and its extract.

[0079] Furthermore, the use described in the first aspect above, the single-dose MGMT protein degrader described in the second aspect, the kit described in the third aspect, the method described in the fourth aspect, the pharmaceutical composition described in the fifth aspect, the drug combination or medicine box described in the sixth aspect, or the single dosage form described in the seventh aspect may further include at least one of the following additional technical features:

[0080] According to an embodiment of the present invention, R2 and R3 are each independently selected from H, -CH3, -CH2(CH3)2, -CH2CH2CH2CH3, -COCH3, -CH2OCH3, -CH2CH2OH, -CH=CH-CH3, -CF3, -CHF2, -Si(CH3)3, Or, it forms a 5-membered heterocycloalkyl group with the C to which it is attached, where the 5-membered heterocycloalkyl group is optionally substituted by two R7s, and R7 is selected from -CH3 or -CH2CH2OH.

[0081] According to an embodiment of the present invention, the compound of formula (I) has the following structure:

[0082]

[0083]

[0084]

[0085] In the eighth aspect of the present invention, there is provided a use of the aforementioned pharmaceutical composition, the aforementioned drug combination or medicine box or the aforementioned single dosage form in the preparation of a drug for preventing and / or treating cancer.

[0086] According to an embodiment of the present invention, the cancer includes at least one of breast cancer, ovarian cancer, mammary cancer, testicular cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, thyroid cancer, lung cancer, prostate cancer, kidney cancer, melanoma, squamous cell carcinoma, digestive tract adenocarcinoma, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, promyelocytic leukemia, meningeal leukemia, multiple myeloma, lymphosarcoma, lymphoma (TLX5 lymphoma, malignant lymphoma, B-cell lymphoma), bladder cancer, head and neck cancer, esophageal cancer, brain cancer, pharyngeal cancer, tongue cancer, synovial cell carcinoma, neuroblastoma, uterine cancer, fibrosarcoma, myxosarcoma, liposarcoma, soft tissue tumor, osteosarcoma (chondrosarcoma, osteosarcoma), chordoma, angiosarcoma, endothelial cell sarcoma, lymphangiosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma,, cervical cancer, cervical carcinoma, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, brain or intrathecal tumor, glioblastoma, and retinoblastoma; preferably, the cancer includes at least one of melanoma, glioma, small cell lung cancer, osteosarcoma and lymphoma.

[0087] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0089] Figure 1 It is a graph showing the results of the survival rate of malignant melanoma cells in each group in Example 1 of the present invention;

[0090] Figure 2Results graph of the survival rates of malignant melanoma and glioma cells in each group in Example 2 of the present invention;

[0091] Figure 3 Results graph of the survival rates of malignant melanoma cells in each group in Example 3 of the present invention;

[0092] Figure 4 Synergy scores of PA and DTIC by the ZIP model and the Bliss model in Example 3 of the present invention;

[0093] Figure 5 Detection results of Western Blotting in Example 4 of the present invention;

[0094] Figure 6 Detection results of Western Blotting in Example 5 of the present invention;

[0095] Figure 7 Results of the alkaline comet assay in Example 6 of the present invention;

[0096] Figure 8 PA content detection standard curve in Example 1 of the present invention. Detailed implementation manners

[0097] The embodiments of the present invention will be described in detail below. The described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0098] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0099] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0100] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0101] In this text, the terms "optionally", "optional", or "option" generally mean that the subsequent described event or condition can but does not necessarily occur, and this description includes the cases where the event or condition occurs and the cases where the event or condition does not occur.

[0102] In this text, the term "substituted" means that a hydrogen atom in a molecule is replaced by other different atoms or groups.

[0103] In this text, the term "optionally substituted" means that "substitution" can occur or not occur, that is, a hydrogen atom in a molecule or group is replaced by other identical or different atoms or groups.

[0104] In this text, the minimum and maximum carbon atom contents in a hydrocarbon group are indicated by a prefix. For example, the prefix C a～b alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C 1～6 alkyl refers to an alkyl group containing 1 to 6 carbon atoms.

[0105] In this text, the term "alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. The alkyl group can be straight-chain or branched-chain. Representative branched-chain alkyl groups have one, two, or three branches. The alkyl group can be optionally substituted by one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl. The alkyl group can also be part of other groups, such as -O(C 1～6 alkyl).

[0106] In this text, the terms "heterocycloalkyl", "heterocycle", and "heteroalkane" all refer to a saturated ring or non-aromatic unsaturated ring containing at least one heteroatom; where the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. In this text, a monovalent saturated or partially unsaturated monocyclic ring usually representing a plurality of ring atoms contains 1, 2, or 3 ring heteroatoms selected from O, and the remaining ring atoms are carbon.

[0107] In this text, the term "5- to 10-membered heterocycloalkyl" refers to a 5-, 6-, 7-, 8-, 9-, or 10-membered saturated or unsaturated heterocycloalkyl, preferably a 5- to 6-membered saturated heterocycloalkyl. The unsaturation described in the present invention means that the group or molecule contains a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-oxygen double bond, a carbon-sulfur double bond, a carbon-nitrogen triple bond, etc.; the unsaturated carbocyclic group of the present invention includes or does not include an aromatic ring group, and the unsaturated heterocyclic group includes or does not include a heteroaromatic group, and those skilled in the art can freely choose.

[0108] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine. Among them, "halogen-substituted alkyl" means that one or more hydrogen atoms in the alkyl are substituted by halogen; for example, trifluoromethyl, difluoromethyl, monofluoromethyl, etc.

[0109] As used herein, the term "-OR" means that the R group is connected to the oxygen atom by a single bond.

[0110] As used herein, in the term "-C(O)R", the oxygen atom is connected to the carbon atom by a double bond.

[0111] As used herein, the term "single dose" refers to a dose for single use; exemplarily, "single dose MGMT protein activity inhibitor" refers to a dose that can inhibit the activity of MGMT protein in cells by adding the MGMT protein activity inhibitor once.

[0112] As used herein, the term "single dosage form" refers to a dosage form that is used up in a single administration of a preparation.

[0113] As used herein, the term "drug combination" refers to the simultaneous or sequential application of two or more drugs for the purpose of treatment, and the result is mainly to increase the efficacy of the drugs or to reduce the toxic and side effects of the drugs.

[0114] As used herein, the term "nitrogen mustard compounds" refers to a class of cytotoxic chemotherapy drugs with a structure similar to mustard gas, such as cyclophosphamide, chlorambucil, melphalan, bendamustine, ifosfamide, and estramustine, etc.

[0115] As used herein, the term "treatment" is used to refer to obtaining the desired pharmacological and / or physiological effects. The effects can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases of mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the disease of the individual, including but not limited to administering a drug containing the compound described herein to an individual in need.

[0116] The compounds mentioned herein include but are not limited to the compound itself and deuterated compounds. The "deuterated compounds" of the present invention refer to compounds in which one or more hydrogen atoms in the molecule or group are substituted by deuterium atoms, and the proportion of deuterium atoms is greater than the natural abundance of deuterium.

[0117] It should be noted that the "compound shown in formula (I)" in this article can be used directly as an active ingredient or can be a prodrug of the active ingredient, without specific restrictions. When taken as a prodrug, it can be pre-treated in vitro before taking, or can be taken directly and then converted into an active ingredient in the body.

[0118] In this article, the term "γH2AX" generally refers to one of the members of the histone H2A family of chromosomes. Under the stimulation of various physical and chemical factors, the double-stranded DNA of cells breaks, and phosphatidylinositol 3-kinases such as ATM and ATR phosphorylate serine at position 139 on H2AX, forming phosphorylated H2AX, that is, γH2AX. γH2AX produced by H2AX phosphorylation can clearly reflect the degree of DNA damage and repair as a biomarker.

[0119] In this article, the term "O 6 -methylguanine-DNA methyltransferase (abbreviated as MGMT)" is a DNA repair protein that can remove alkyl adducts in DNA to eliminate the toxic effects of DNA alkylating agents on cells. Research shows that the expression level of MGMT is significantly increased in many malignant tumors such as melanoma and glioma, which is an important factor for their resistance to chemotherapy drugs (such as DTIC and TMZ).

[0120] In this article, the term "temozolomide (Temozolomide, TMZ)" is a DNA alkylating agent with the same pharmacology as DTIC. The difference is that TMZ can cross the blood-brain barrier and can spontaneously and rapidly degrade in the body to produce the active metabolite MTIC, thereby producing an anti-tumor effect, and is used as a therapeutic drug for adult malignant glioma and malignant melanoma.

[0121] In this article, the term "cycloheximide (Cicloheximide, CHX)" is an organic compound with the chemical formula C 15 H 23 NO4, which can inhibit the protein synthesis of eukaryotes. Among them, the chemical structural formula of cycloheximide is:

[0122]

[0123] In this article, the term "protocatechuic aldehyde (Protocatechuic aldehyde, PA)", with the chemical name of 3,4-dihydroxybenzaldehyde, is a natural phenolic acid compound and is one of the active ingredients of traditional Chinese medicines such as Salvia miltiorrhiza, Salvia japonica, Gastrodia elata, Lycium barbarum, Stenoloma chusanum, Ilex purpurea, Tsaoko fruit, Cedrus deodara, Ampelopsis japonica, Acalypha australis, Begonia grandis, Ranunculus muricatus, Woodwardia japonica, Trachycarpus fortunei, and Persimmon leaves.

[0124] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchases.

[0125] Example 1: Effects of Salvia miltiorrhiza extract and TMZ on the survival rate of malignant melanoma cells

[0126] The inventors investigated the effects of the combined use of Salvia miltiorrhiza extract and TMZ on the survival rate of malignant melanoma cells. The specific experimental steps are as follows:

[0127] 1. Materials and methods

[0128] 1.1 Experimental materials

[0129] 1.1.1 Cell source

[0130] Human melanoma cells A375 are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and A2058 is from ATCC.

[0131] 1.1.2 Preparation of Salvia miltiorrhiza extract and TMZ solutions

[0132] For convenient experimental investigation, the used Salvia miltiorrhiza extract was prepared by dissolving commercially available Compound Danshen Tablets (Shanghai Leyons Pharmaceutical Co., Ltd.) in PBS to form a stock solution with a concentration of 100 mg / mL for standby. TMZ (S1237) was purchased from Selleck and dissolved in DMSO to prepare a stock solution with a concentration of 200 mM for standby.

[0133] 1.1.3 Cell culture reagents and culture conditions

[0134] The culture medium used for cell culture is a complete medium, and its composition is: 89% DMEM high-glucose medium, 10% fetal bovine serum, and 1% penicillin-streptomycin double-antibody solution. The cell culture environment is: in a cell culture incubator at 37°C and 5% CO2.

[0135] 1.1.4 Main reagents

[0136] Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

[0137] 1.1.5 Main instruments and consumables

[0138] Laminar flow hood, cell culture incubator, microplate reader, low-speed centrifuge, freeze dryer, high-performance liquid chromatograph, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube, microporous filter membrane.

[0139] 2. Experimental methods

[0140] 1) CCK8 assay for cell viability

[0141] A3755 and A2058 were cultured using complete medium respectively. Cells with good growth conditions (cell density reaching over 80%) were plated in a sterile environment. After washing the surface of the above cells with sterile PBS, they were digested with trypsin to make the adherent cells detached, and were thoroughly pipetted and mixed to prepare A375 and A2058 melanoma cell suspensions.

[0142] The above A375 and A2058 melanoma cell suspensions were inoculated into 96-well plates at a density of 3000 cells per well and cultured with complete medium. After the cells adhered for 24 hours, a control group, a danshen tablets group, a TMZ group, and a danshen tablets + TMZ group were set up. The original medium was discarded and the medium containing the corresponding danshen extract and / or TMZ was added and cultured in a cell incubator for 48 - 72 h. Among them, the control group was complete medium supplemented with the same concentration of DMSO as the other drug-added groups (without danshen extract and TMZ), the danshen tablets group was added with different concentrations of danshen extract alone, the TMZ group was added with different concentrations of TMZ alone, and the danshen tablets + TMZ group was added with different concentrations of danshen extract and different concentrations of TMZ in combination. For details, see Figure 1 Subsequently, the original medium was discarded. According to the instructions, CCK8 reagent (Shanghai Beyotime Biotechnology Co., Ltd., C0039) was used. After incubating in a 37°C cell incubator for 1 h, the absorbance value of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Each measurement was repeated 3 times.

[0143] The detection results are as Figure 1 shown, where compared with the TMZ group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0144] The results showed that in the A375 and A2058 melanoma cell lines, compared with the use of danshen extract and TMZ alone, the viability of melanoma cells in the danshen tablets + TMZ group was significantly reduced.

[0145] The above experimental results showed that: Danshen extract can significantly enhance the killing effect of TMZ on melanoma cells, and the viability of melanoma cells is reduced.

[0146] 2) Determination of the content of PA in danshen tablets

[0147] Take 10 Danshen tablets (300 mg / tablet), crush them into powder, add 60 ml of water, reflux in a water bath at 100 °C for 1 h, filter, and then freeze-dry and weigh. The weight is 1651 mg. Precisely weigh 1.00 mg of the water extract, add 1 ml of 50% methanol, dissolve it by ultrasonic treatment, and filter it through a 0.22-μm microporous membrane for use as a test sample to be measured. Precisely weigh 1.00 mg of PA, add 50% methanol, dissolve it by ultrasonic treatment, and filter it through a 0.22-μm microporous membrane, and then prepare standard solutions with concentrations of 0.0625, 0.125, 0.20, 0.25, 0.5, and 1 mg / ml respectively. Set the chromatographic conditions: the chromatographic column is Shim-pack GIST (C18, 4.6×250 mm, particle size 5 μm); mobile phase A is 0.1% formic acid aqueous solution, B is acetonitrile, and isocratic elution with 25% acetonitrile; the flow rate is 1 ml / min; the detection wavelength is 280 nm, and the injection volume is 5 μl. After the detection is completed, calculate the chromatographic peak response areas of different concentrations of PA respectively. Take the concentration as the abscissa and the response area as the ordinate to draw a standard curve, perform linear regression on it, and obtain a regression equation. Subsequently, compare the chromatogram of the water extract with the chromatogram of the PA standard product, calculate the corresponding chromatographic peak areas, substitute the calculated chromatographic peak areas into the regression equation to calculate the PA content in the water extract as 2.3%, that is, the PA content in Danshen tablets is 3.8 mg / tablet. The standard curve is as Figure 8 shown.

[0148] Example 2: Effects of PA and TMZ on the survival rates of malignant melanoma cells and glioma cells

[0149] The inventor further analyzed the active ingredients in the Danshen extract, speculated that the main active ingredient in Danshen was protocatechuic aldehyde (PA), and investigated the effects of the combined use of PA and TMZ on the survival rates of malignant melanoma and glioma cells. The experimental steps are referred to Example 1, and the difference is that a control group, a PA group, a TMZ group, and a PA+TMZ group are set respectively. The drug addition amounts in each group are referred to Figure 2 , and the PA (D108405) used in the experiment was purchased from Sigma Company and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200 mM for standby.

[0150] The specific experimental steps are as follows:

[0151] 1. Materials and methods

[0152] 1.1 Experimental materials

[0153] 1.1.1 Cell source

[0154] The human melanoma cells A375 are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and A2058 are from ATCC. The human glioma cells U87MG are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and HS683 are from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0155] 1.1.2 Preparation of PA and TMZ Solutions

[0156] The PA (D108405) used in the experiment was purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200 mM for standby. The TMZ (S1237) was purchased from Selleck and dissolved in DMSO to prepare a stock solution with a concentration of 200 mM for standby.

[0157] 1.1.3 Cell Culture Reagents and Culture Conditions

[0158] Same as Example 1

[0159] 1.1.4 Main Reagents

[0160] Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

[0161] 1.1.5 Main Instruments and Consumables

[0162] Laminar flow hood, cell culture incubator, microplate reader, low-speed centrifuge, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube.

[0163] 2. Experimental Methods

[0164] 1) CCK8 Assay for Cell Viability

[0165] Refer to Example 1, with the difference that the human glioma cell suspensions of U87MG and HS683 are inoculated onto 96-well plates at a quantity of 2000 and 3000 per well respectively, and cultured with complete medium. After the cells adhere for 24 hours, a control group, a PA group, a TMZ group, and a PA + TMZ group are set respectively, and treated for 72 h. The drug addition amounts in each group are referred to Figure 2 .

[0166] The detection results are as Figure 2 shown, where, compared with the TMZ group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

[0167] The experimental results show that the combined use of PA and TMZ can significantly reduce the viability of malignant melanoma cells A375 and A2058, as well as U87MG and HS683 glioma cells.

[0168] Example 3: Effects of PA and DTIC on the survival rate of malignant melanoma cells and their synergistic effect

[0169] 1. Materials and methods

[0170] 1.1 Experimental materials

[0171] 1.1.1 Cell source

[0172] Human melanoma cells A375 were derived from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and SK-MEL-28 was derived from ATCC.

[0173] 1.1.2 Preparation of PA and DTIC solutions

[0174] PA (D108405) used in the experiment was purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200 mM for standby. DTIC (S1221) was purchased from Selleck and dissolved in DMSO to prepare a stock solution with a concentration of 30 mM for standby.

[0175] 1.1.3 Cell culture reagents and culture conditions

[0176] The culture medium used for cell culture was complete medium, and its composition was: 89% high-glucose DMEM medium, 10% fetal bovine serum, and 1% penicillin-streptomycin double-antibody solution. The cell culture environment was: in a cell culture incubator at 37°C and 5% CO2.

[0177] 1.1.4 Main reagents

[0178] Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

[0179] 1.1.5 Main instruments and consumables

[0180] Laminar flow hood, cell culture incubator, microplate reader, low-speed centrifuge, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube.

[0181] 2. Experimental methods

[0182] 1) CCK8 assay for cell survival rate

[0183] A375 and SK-MEL-28 were cultured using complete medium respectively. Cells with good growth conditions (cell density reaching more than 80%) were plated in a sterile environment. After washing the surface of the above cells with sterile PBS, they were digested with trypsin to make the adherent cells detached, and then thoroughly pipetted and mixed to prepare A375 or SK-MEL-28 melanoma cell suspensions.

[0184] The above-mentioned A375 and SK-MEL-28 melanoma cell suspensions were inoculated into 96-well plates at a density of 3000 cells per well and cultured in complete medium. After 24 hours of cell attachment, control group, PA group, DTIC group, and PA+DTIC group were set up. The original medium was discarded and fresh medium containing the corresponding PA and DTIC was added, and the cells were cultured in a cell incubator for 48 - 72 hours. Among them, the control group was complete medium containing the same concentration of DMSO as the other drug-added groups (without PA and DTIC). In the PA group, different concentrations (20, 40, 60, 80, and 100 μM) of PA were added alone. In the DTIC group, different concentrations (20, 40, 60, 80, and 100 μM) of DTIC were added alone. In the PA+DTIC group, different concentrations of PA (20, 40, 60, 80, and 100 μM) and different concentrations of DTIC (20, 40, 60, 80, and 100 μM) were added in combination. Subsequently, the original medium was discarded, and CCK8 reagent (Shanghai Beyotime Biotechnology Co., Ltd., C0039) was used according to the instructions. After incubation in a 37°C cell incubator for 1 hour, the absorbance value of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm, and each measurement was repeated 3 times.

[0185] The detection results are as Figure 3 shown, where compared with the DTIC group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0186] The results showed that in the A375 and SK-MEL-28 melanoma cell lines, the effects of different concentrations of PA and different concentrations of DTIC alone on cell viability were relatively low. Compared with DTIC alone, the viability of melanoma cells in the PA+DTIC group was significantly reduced, indicating that PA could significantly enhance the killing effect of DTIC on melanoma cells and reduce the viability of melanoma cells.

[0187] 2) Determination of synergistic effect

[0188] The experimental method was the same as that for CCK8 to determine cell viability, that is, A375 cells were treated with 20, 40, 80, 160 μM of PA and 5, 10, 20, 40, 80 μM of DTIC separately and in combination for 48 h, and SK-MEL-28 cells were treated with 40, 60, 80, 100 μM of PA and 20, 40, 60, 80, 100 μM of DTIC separately and in combination for 72 h, and the cell viability was measured by the CCK8 method. Then the obtained absorbance was converted to calculate the cell viability, and subsequently, the SynergyFinder software (https: / / synergyfinder.fimm.fi), an online synergy scoring software, was used to evaluate its synergy score. By comparing the observed drug combination response with the expected response and calculating using a reference model assuming no interaction between drugs, the degree of drug combination synergy or antagonism can be quantified. Commonly used reference models include the highest single agent HSA model, Bliss model, Loewe model, and zero interaction potency model ZIP. According to the different assumptions of different models, the inventors selected the Bliss and ZIP models to quantify the synergy degree between PA and DTIC. Among them, the Bliss model assumes a random process in which the two drugs independently exert their effects, and the expected combined effect can be calculated based on the probabilities of independent events. The zero interaction potency ZIP model captures the drug interaction relationship by comparing the potency changes (effects at specific dose levels) of the dose-response curves between individual drugs and their combinations. ZIP assumes that the dose-response curves of two non-interacting drugs change little, and it utilizes the advantages of the additivity of the Loewe model and the independence of the Bliss model, aiming to systematically evaluate various drug interaction patterns that may occur in high-throughput drug combination screening. The results are as Figure 4 shown.

[0189] By definition, when the synergy score is less than -10: the interaction between the two drugs may be antagonistic; from -10 to 10: the interaction between the two drugs may be additive; greater than 10: the interaction between the two drugs may be synergistic.

[0190] The experimental results are as Figure 4 shown: In both cell lines, for the combination of PA and DTIC, regardless of the ZIP model or the Bliss model, the synergy score is greater than 10 (the white dashed box area represents the concentration area with the highest synergy score), indicating that there is a synergistic effect between PA and DTIC.

[0191] Furthermore, the inventors explored the mechanism of the synergistic effect of PA and DTIC, examined the protein levels of genes related to DNA damage repair, and unexpectedly found that the drug combination of PA + DTIC could further increase the protein level of DNA damage protein γH2AX and decrease the protein level of DNA repair protein MGMT. Further, the inventors discovered that the change in the MGMT protein level was caused by PA promoting the degradation of the MGMT protein.

[0192] Example 4: Protein Levels of Genes Related to DNA Damage Repair

[0193] 1. Experimental Materials

[0194] 1.1 Protein Lysis Buffer

[0195] Use RIPA150 lysis buffer (150 indicates that the sodium chloride concentration is 150 mM): protease inhibitor (PI): sodium glycerophosphate: NaF = 90:10:1:1 to prepare the protein lysis buffer.

[0196] 1.2 Preparation of Cycloheximide (CHX) Solution

[0197] CHX (66 - 81 - 9) used in the experiment was purchased from Selleck. After centrifuging the drug powder, it was dissolved in PBS to prepare a stock solution with a final concentration of 15 mg / mL, aliquoted, and frozen at -80 °C for storage and later use.

[0198] 1.3 Cell Source

[0199] Human melanoma cells A375 were derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and SK - MEL - 28 was from ATCC.

[0200] 1.4 Related Buffers and Their Preparation

[0201] 10× Electrophoresis Buffer: 30 g Tris base, 144 g glycine, 10 g SDS, made up to 1 L with ddH2O.

[0202] 1× Transfer Buffer: 28.8 g glycine, 6 g Tris base, 400 ml methanol, made up to 2 L with ddH2O.

[0203] 10× TBS Solution: 88 g sodium chloride, 24 g Tris base, 13 ml concentrated hydrochloric acid, made up to 1 L with ddH2O.

[0204] 1× TBST Solution: Measure 100 ml of 10× TBS solution and mix it with 1 ml of 20% Tween, then make up to 1 L.

[0205] 1.5 Main Instruments and Consumables

[0206] Vertical electrophoresis tank, multi-functional imaging system, 24-well plate, platform scale, analytical balance, shaker, PVDF membrane, filter paper.

[0207] 2 Experimental methods

[0208] 1) Drug treatment

[0209] A375 and SK-MEL-28 cells were cultured with the complete cell culture medium of Example 3 respectively. Cells with good growth conditions (cell density reaching more than 80%) were selected for the experiment. The above two types of cells were respectively set into 4 groups, namely the control group, PA group, DTIC group, and PA+DTIC group. Among them, the control group was the complete culture medium added with DMSO with the same concentration as that in other drug-added groups (without PA and DTIC). PA was added alone in the PA group (the final concentration added to A375 cells was 40 μM, and the final concentration added to SK-MEL-28 cells was 100 μM). DTIC was added alone in the DTIC group (the final concentration added to A375 cells was 40 μM, and the final concentration added to SK-MEL-28 cells was 100 μM). PA and DTIC were added simultaneously in the PA+DTIC group (the final concentration of PA added to A375 cells was 40 μM, the final concentration of PA added to SK-MEL-28 cells was 100 μM, the final concentration of DTIC added to A375 cells was 40 μM, and the final concentration of DTIC added to SK-MEL-28 cells was 100 μM). Then they were put back into the cell incubator at 37 °C and 5% CO2 and continued to be cultured for 48 - 72 h.

[0210] 2) Western blot analysis

[0211] A375 and SK-MEL-28 cells treated and untreated with drugs were lysed with protein lysate, and 1 / 4 of the volume of 5×SDS loading buffer was added to the protein lysate and mixed evenly. The protein samples were heated at 100 °C for 8 minutes to make them completely denatured. Subsequently, after the obtained proteins were separated by SDS-PAGE polyacrylamide gel electrophoresis, they were transferred to a PVDF membrane (O162-0177, Bio-Rad). After incubating with the corresponding primary antibodies (antibodies 1 - 3 in Table 1) and secondary antibodies (antibodies 4 - 5 in Table 1), a chemiluminescence kit (K22030, Abbkine) was used to detect the protein bands. The detection results are shown in Figure 5 .

[0212] Figure 5The results showed that, compared with the control group, the use of PA alone could increase the protein level of the DNA damage protein γH2AX and decrease the protein level of the DNA repair protein MGMT; moreover, the drug combination of PA + DTIC could further increase the protein level of the DNA damage protein γH2AX and decrease the protein level of the DNA repair protein MGMT.

[0213] The above results suggest that the combined use of PA and DTIC has a certain effect on the DNA damage repair ability of tumor cells, and can be used to improve the response levels of malignant tumors such as melanoma, glioma, small cell lung cancer, osteosarcoma, and lymphoma to DNA alkylating agents such as DTIC.

[0214] Table 1: Antibody types

[0215]

[0216] Example 5: Investigation of the effect of PA on the levels of proteins related to DNA damage repair

[0217] Referring to the experimental method of Example 4, the effect of PA on the DNA damage repair-related proteins MGMT protein in the presence of CHX was further investigated. A375 cells and SK-MEL-28 cells were treated with CHX (40 μg / ml) alone and in combination with PA (40 μM, 100 μM) for 0 h, 1 h, 2 h, 4 h, and 8 h, respectively, and then protein samples were collected for Western blot analysis.

[0218] The experimental results are as Figure 6 shown. Treatment of melanoma cells with CHX for different times inhibited protein synthesis in the cells, and a certain amount of DMSO was added as a control. It can be clearly seen from Figures (6A, 6C) that the MGMT protein gradually degraded (i.e., the protein level decreased) with the increase of time, indicating that CHX played a role. In addition, on the basis of adding CHX, a certain drug concentration of PA was added respectively. It can be clearly seen from Figures (6B, 6D) that the protein degradation rate of MGMT was significantly faster than that of the DMSO group (i.e., the protein level was lower), indicating that PA could promote the degradation of the MGMT protein.

[0219] The inventors further verified the synergistic effect of the combined use of PA and DTIC at the cellular level through the comet assay.

[0220] Example 6: Investigation of DNA damage

[0221] 1. Experimental materials

[0222] 1.1 Cell source

[0223] The human melanoma cell line A375 was derived from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and SK-MEL-28 was derived from ATCC.

[0224] 1.2 Main reagents

[0225] Sodium hydroxide (NaOH), absolute ethanol, low melting point agarose gel, Comet Aaasy kit (4250 - 050 - K, Trevigen), Green I (SY1020, Solarbio) staining solution.

[0226] 1.2 Preparation of main buffers

[0227] Alkaline lysis solution (Alkaline Unwinding Solution): Weigh 0.4 g of NaOH, add 250 μL of 0.2 M EDTA solution, and make up to 50 mL with ddH2O.

[0228] Alkaline electrophoresis buffer: Weigh 8 g of NaOH, add 2 mL of 0.5 M EDTA solution with a pH value of 8.0, and make up to 1000 mL with ddH2O.

[0229] 1.5 Main instruments and consumables

[0230] Laminar flow hood, cell counter, horizontal electrophoresis tank, oven, table scale, fluorescence microscope, glass slides, 24 - well plates, low melting point agarose gel.

[0231] 2. Experimental methods

[0232] 1) Drug treatment

[0233] Culture A375 and SK - MEL - 28 cells respectively using the complete cell culture medium of Example 3, and select cells with good growth conditions (cell density reaching more than 80%) for the experiment. Set the above two types of cells into 4 groups, namely the control group, PA group, DTIC group, and PA + DTIC group. Among them, the control group is added with DMSO at the same concentration as the drug - added groups. In the PA group, PA is added alone (the final concentration added to A375 cells is 40 μM, and the final concentration added to SK - MEL - 28 cells is 100 μM). In the DTIC group, DTIC is added alone (the final concentration added to A375 cells is 40 μM, and the final concentration added to SK - MEL - 28 cells is 100 μM). In the PA + DTIC group, PA (the final concentration added to A375 cells is 40 μM, and the final concentration added to SK - MEL - 28 cells is 100 μM) and DTIC (the final concentration added to A375 cells is 40 μM, and the final concentration added to SK - MEL - 28 cells is 100 μM) are added simultaneously. Then place them back into the cell culture incubator at 37°C and 5% CO2 for continued culture, 48 h for A375 cells and 72 h for SK - MEL - 28 cells.

[0234] 2) Alkaline Comet Assay

[0235] The cell samples treated with drugs were digested with trypsin to collect cell suspensions. After washing once with ice-cold PBS, they were resuspended with PBS. They were counted using a cell counter to ensure that the density of the cell suspension was 1×10 5 cell / mL. The cell suspension was mixed with low melting point agarose gel at a volume ratio of 1:10, evenly coated on glass slides, and solidified at 4°C for half an hour. Then it was immediately placed in pre-cooled Lysis Buffer (supplied in the kit) and lysed at 4°C for 2 hours. After the lysis was completed, the excess lysate was wiped dry, and it was immersed in freshly prepared Alkaline Unwinding Solution and incubated at room temperature in the dark for 40 min. The electrophoresis apparatus was installed, about 850 ml of pre-cooled alkaline electrophoresis buffer was poured in, the voltage was set to 21V, and electrophoresis was carried out for 45 min. Subsequently, the glass slides were taken out, immersed in ddH2O twice, 5 min each time, and immersed in 70% ethanol twice, 5 min each time. After completing the above steps, the glass slides were placed in an oven at 37°C to dry the slides. After staining the slides with Green I, the samples were observed using a fluorescence microscope. The results are shown in Figure 7 .

[0236] 3. Experimental Results

[0237] As Figure 7 shown, the cell samples treated with drugs were subjected to the alkaline comet assay, and the tailing of cells in different groups was observed. The results showed that the comet tails in the PA + DTIC combination group were significantly longer and thicker, indicating that the DNA double-strand breakage was more severe.

[0238] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0239] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, Comprising: Protocatechuic aldehyde or a composition containing protocatechuic aldehyde as the first active ingredient; And A DNA alkylating agent as the second active ingredient; Wherein, the DNA alkylating agent is dacarbazine and / or temozolomide; The molar ratio of the protocatechuic aldehyde to the DNA alkylating agent is (1 - 100):(100 - 1).

2. The pharmaceutical composition according to claim 1, wherein The composition containing protocatechuic aldehyde is selected from at least one of Compound Danshen Tablets, Compound Danshen Dripping Pills, and Compound Danshen Injection.

3. The pharmaceutical composition according to claim 1, wherein Further comprising pharmaceutically acceptable excipients.

4. A drug combination or medicine box, characterized in that, Comprising: Protocatechuic aldehyde or a composition containing protocatechuic aldehyde as the first active ingredient; And A DNA alkylating agent as the second active ingredient; Wherein, the DNA alkylating agent is dacarbazine and / or temozolomide; The molar ratio of the protocatechuic aldehyde to the DNA alkylating agent is (1 - 100):(100 - 1).

5. The pharmaceutical combination or kit according to claim 4, characterized in that, The composition containing protocatechuic aldehyde is selected from at least one of Compound Danshen Tablets, Compound Danshen Dripping Pills, and Compound Danshen Injection.

6. The pharmaceutical combination or kit according to claim 4, wherein The protocatechuic aldehyde or the composition containing protocatechuic aldehyde and the DNA alkylating agent are formulated together or separately.

7. Use of the pharmaceutical composition according to any one of claims 1 - 3, the pharmaceutical combination or the medicine box according to any one of claims 4 - 6 in the preparation of a medicine for preventing and / or treating cancer; The cancer is at least one of melanoma, glioma, small cell lung cancer, osteosarcoma, and lymphoma.

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