New use of evergreen kounine in the preparation of a medicament for the treatment of prostate cancer

Abstract

The application discloses a new use of evergreen kounine in preparation of a medicine for treating prostate cancer. The evergreen kounine is found to have a significant inhibiting effect on the proliferation of prostate cancer cells through a large number of experiment screening and verification, and the evergreen kounine is first proposed as a new use of a prostate cancer treatment medicine. Therefore, the evergreen kounine is further prepared as an effective component of a medicine for treating and / or preventing prostate cancer, which has an important application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular, the present application relates to a new use of a known compound, and more particularly, the present application relates to a new use of sempervirine in the preparation of a medicament for treating prostate cancer. BACKGROUND

[0002] Prostate cancer is one of the most common malignant tumors in the male urogenital system. The main cause of death of prostate cancer is its high metastasis rate, and the main organs of metastasis are bone (50%), lymph nodes, lung, and liver. Its mortality rate is second only to lung cancer worldwide. Prostate cancer has the characteristics of high individual heterogeneity and exponential growth of incidence and mortality rate with age, and the clinical manifestations are asymptomatic, mostly located in the peripheral zone, and the patient's performance is heterogeneous, with large individual differences. Prostate cancer is divided into two types, namely hormone-sensitive prostate cancer and hormone-resistant prostate cancer, and the main difference is the effect of hormone therapy. Most patients will eventually become hormone-resistant prostate cancer.

[0003] Most patients with advanced prostate cancer often have no choice due to metastasis and drug resistance, and our research on traditional Chinese medicine is expected to fill this gap and give patients new hope. Traditional Chinese medicine has the concept of "treatment based on syndrome differentiation", and has the characteristics of "mild and effective", and has unique advantages in anti-tumor. Traditional Chinese medicine monomer is derived from natural plants, and has the characteristics of multi-target and multi-path comprehensive action, and low toxicity and side effects. By protecting the "vital qi" of the body and improving the patient's disease prevention and resistance, it can effectively control the "toxicity" of tumor formation, and prevent recurrence and metastasis by "clearing the source". In addition, traditional Chinese medicine monomer has unique advantages in reducing side effects of other treatment options, improving patient quality of life, and improving survival rate.

[0004] Gelsemium elegans Benth is a world-famous poisonous plant of the genus Gelsemium, which is distributed in Fujian, Yunnan, Guizhou, Guangxi and other provinces of China. Sempervirine (SPV) is a yohimbine-type alkaloid isolated from Gelsemium elegans Benth. In traditional Chinese medicine, Gelsemium elegans Benth is used to treat some types of skin ulcers and headaches. However, there is still a gap in the research of sempervirine in the field of anti-urogenital tumors. SUMMARY

[0005] The purpose of the present application is to provide a new use of sempervirine in the preparation of a medicament for treating prostate cancer, and to provide a safe and effective drug for the treatment of urogenital tumors, in order to overcome the technical problem that there is an urgent need for effective prostate cancer treatment drugs in the field.

[0006] The above inventive purpose of the present application is realized by the following technical scheme:

[0007] In a first aspect, the present application provides a pharmaceutical composition for treating and / or preventing prostate cancer.

[0008] Further, the pharmaceutical composition comprises a therapeutically and / or prophylactically effective amount of sempervirine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form.

[0009] In the present application, the sempervirine (SPV) is a yohimbine-type alkaloid isolated from strychnos nuxvomica, with a molecular formula of C 19 H 16 N2, a molecular weight of 272.4, and a structural formula as shown in formula (I).

[0010]

[0011] In some embodiments, the pharmaceutical composition further comprises one or more other anti-tumor drugs, and the sempervirine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form and the other anti-tumor drugs are administered simultaneously, sequentially or separately to induce apoptosis of prostate cancer cells.

[0012] In some embodiments, the other anti-tumor drugs include, but are not limited to, cell cycle-specific chemotherapeutic drugs, BCR-ABL tyrosine kinase targeting inhibitors, proteasome inhibitors, and conventional chemotherapeutic drugs other than the above three types.

[0013] In some embodiments, the cell cycle-specific chemotherapeutic drugs refer to a large class of chemotherapeutic drugs known to those skilled in the art that kill proliferating cells in a specific cell cycle phase, including but not limited to cytarabine, 5-fluorouracil, hydroxyurea, 6-mercaptopurine, 6-thioguanine, fludarabine, methotrexate.

[0014] In some embodiments, the BCR-ABL tyrosine kinase refers to the fusion gene BCR-ABL resulting from the chromosomal translocation forming the Philadelphia chromosome, and the corresponding encoded fusion protein has abnormal tyrosine kinase activity. BCR-ABL tyrosine kinase targeting inhibitors can prevent the abnormal increase in activity of this kinase, thereby leading to the death of Bcr-Abl positive cells, and the BCR-ABL tyrosine kinase targeting inhibitors include but are not limited to imatinib, dasatinib, nilotinib.

[0015] In some embodiments, the proteasome inhibitors refer to a class of anti-tumor drugs that block the ubiquitin-proteasome pathway, including but not limited to: Bortezomib, Carfilzomib, Ixazomib, Marizomib, Oprozomib, KZR-616, MG-132.

[0016] In some embodiments, the conventional chemotherapy drugs other than the first three categories refer to a series of chemotherapeutic drugs that kill cells at various cell cycle stages in the conventional sense of the cancer or tumor treatment field, including but not limited to: Desoxycoformycin, Vincristine, Doxorubicin, Daunorubicin, Mitoxantrone, Vinblastine, Vindesine, Huperforine, Etoposide, Teniposide, L-Asparaginase.

[0017] In some embodiments, in addition to the evergreen kounine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form as the main drug ingredient and / or at least one other anti-tumor drug as a combination drug in the pharmaceutical composition provided by the present application, the pharmaceutical composition further optionally comprises a pharmaceutically acceptable excipient and / or excipient.

[0018] In a specific embodiment of the present application, the inventors of the present application first discovered that evergreen kounine can significantly inhibit the proliferation activity of prostate cancer cells, effectively promote the apoptosis of prostate cancer cells, block the G1 / S phase transition of prostate cancer cells, and significantly inhibit the invasion and migration ability of prostate cancer cells. Evergreen kounine can be used as an effective anti-tumor traditional Chinese medicine monomer, and has important application prospects in the treatment of urogenital system tumors.

[0019] In a second aspect, the present application provides a pharmaceutical preparation for treating and / or preventing prostate cancer.

[0020] Further, the pharmaceutical preparation comprises the pharmaceutical composition of the first aspect of the present application.

[0021] Further, the dosage form of the pharmaceutical preparation includes oral administration, parenteral administration and / or topical administration;

[0022] Preferably, the dosage form of the pharmaceutical preparation includes solution, sustained-release, suspension, granules, tablets, capsules, powders, effervescent, emulsion, syrup, drops and / or chewable tablets.

[0023] Further, the pharmaceutical preparation further comprises a pharmaceutically acceptable excipient and / or excipient.

[0024] In some embodiments, the pharmaceutical composition or pharmaceutical preparation according to the present application can be prepared into a dosage form suitable for oral administration, parenteral administration or topical administration by means known per se, for example by conventional mixing, granulating, coating, dissolving or lyophilizing processes into a dosage form, including but not limited to, tablets, capsules, granules, injection solutions, injection powders, transdermal patches, ointments, gels, suppositories, oral solutions, oral suspensions, injection emulsions, oral emulsions, sustained-release tablets, controlled-release tablets and the like.

[0025] In some embodiments, the pharmaceutical composition or pharmaceutical preparation according to the present application can further comprise pharmaceutically acceptable auxiliary agents including but not limited to preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts to adjust osmotic pressure, buffers, masking agents or antioxidants. In addition, the pharmaceutical dosage form can be made to have a prolonged absorption by including an ingredient to retard absorption.

[0026] Those skilled in the art will appreciate that, although the pharmaceutical composition or pharmaceutical preparation according to the present application mentioned above can further comprise pharmaceutically acceptable auxiliary agents and / or excipients, when evergreen kounidine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates or crystalline forms and / or at least one of the other anti-tumor drugs are used as a medicament for humans or animals, they can also be administered as such, i.e. without any of the above-mentioned pharmaceutically acceptable auxiliary agents and / or excipients, to achieve the present application.

[0027] When using the pharmaceutical composition or pharmaceutical preparation according to the present application, the administration and dosage regimen can be selected according to various factors, including the species, age, body weight, sex of the subject, and the type of tumor / cancer to be treated; the severity of the tumor / cancer to be treated; the route of administration; the kidney and liver function of the patient; and the specific compound or its salt used. One administration / dosage regimen can be used, for example, to prevent the disease, inhibit (completely or partially) the disease or stop the development of the disease. In a specific embodiment of the present application, the tumor / cancer refers specifically to prostate cancer.

[0028] Further, the present application also encompasses a kit comprising evergreen kounine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof and / or at least one other antitumor drug, i.e. by formulating evergreen kounine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof and / or at least one other antitumor drug in separate unit formulations, and then combining these separate unit formulations in the form of a kit. In this case, evergreen kounine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof and / or at least one other antitumor drug in the separate unit formulations can be administered to a subject simultaneously, sequentially or separately in time.

[0029] In a third aspect, the present application provides use of evergreen kounine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof for the manufacture of a medicament for treating and / or preventing prostate cancer.

[0030] Further, the evergreen kounine significantly inhibits proliferation and / or promotes apoptosis of prostate cancer cells.

[0031] Further, the evergreen kounine significantly inhibits transition of prostate cancer cells from G1 phase to S phase.

[0032] Further, the evergreen kounine significantly inhibits invasion and migration of prostate cancer cells.

[0033] Preferably, the medicament comprises a therapeutically and / or prophylactically effective amount of evergreen kounine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0034] More preferably, the medicament further comprises a pharmaceutically acceptable adjuvant and / or excipient.

[0035] Preferably, the dosage form of the medicament comprises an oral administration dosage form, a parenteral administration dosage form and / or a topical administration dosage form.

[0036] More preferably, the dosage form of the medicament formulation comprises a solution, a sustained release, a suspension, a granule, a tablet, a capsule, a powder, an effervescent, an emulsion, a syrup, a drop and / or a chewable.

[0037] Preferably, the administration method of the medicament comprises oral, subcutaneous, intravenous, intramuscular, intra-arterial, intranasal, intrathecal, mucosal, intrapulmonary and / or rectal.

[0038] In some embodiments, the pharmaceutical, pharmaceutical composition, or pharmaceutical preparation provided herein can be administered in a variety of administration modes, including but not limited to oral, subcutaneous, intravenous, intra-arterial, intra-coronary, intranasal, intrathecal, transdermal, mucosal, topical (e.g., gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary (e.g., using inhalable technologies or pulmonary delivery systems), vaginal, parenteral, rectal, or intraocular.

[0039] In a fourth aspect, the present application provides a method for inhibiting the proliferation of prostate cancer cells and / or promoting the apoptosis of prostate cancer cells in vitro for non-therapeutic purposes.

[0040] Further, the method comprises treating prostate cancer cells with evergreen secaline or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, or crystalline form thereof.

[0041] In specific embodiments of the present application, the present application has demonstrated through a large number of experiments that the evergreen secaline has a significant effect of inhibiting the growth and proliferation of prostate cancer cells and promoting the apoptosis of prostate cancer cells, and thus the evergreen secaline can be used as an inhibitor for inhibiting the growth and proliferation of prostate cancer cells for non-therapeutic purposes in the field of scientific research, such as more in-depth study of the growth and metabolism mechanism or behavior of prostate cancer cells, screening of potential drugs for treating prostate cancer, etc.

[0042] In addition, the present application also provides a method for treating and / or preventing prostate cancer, which comprises the step of administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of the pharmaceutical, pharmaceutical composition, or pharmaceutical preparation of the present application.

[0043] In some embodiments, the subject includes mammals and non-mammals, and in other embodiments, the subject is preferably a human, including a patient diagnosed with prostate cancer or a subject at high risk of developing prostate cancer.

[0044] In some embodiments, the pharmaceutical, pharmaceutical composition, or pharmaceutical preparation can be administered to the subject by oral administration, injection administration, or topical administration. For example, the method can comprise administering the pharmaceutical, pharmaceutical composition, or pharmaceutical preparation to the subject three times a day, once a day, once every two days, etc. In some embodiments, injection administration can include subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, injection administration can include directly injecting the pharmaceutical, pharmaceutical composition, or pharmaceutical preparation into a lesion or an area close to the lesion of the subject. In some embodiments, a combination of different administration modes can be used.

[0045] In a fifth aspect, the present invention provides an in vitro non-therapeutic method for inhibiting the transition of prostate cancer cells from G1 phase to S phase.

[0046] Furthermore, the method comprises: treating prostate cancer cells with evergreen gelsemine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0047] In a sixth aspect, the present invention provides any of the following applications:

[0048] (1) Use of evergreen gelbine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of a pharmaceutical composition for treating and / or preventing prostate cancer;

[0049] (2) Use of evergreen gelbine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of pharmaceutical preparations for treating and / or preventing prostate cancer;

[0050] (3) Use of evergreen gelbine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates or crystalline forms in the preparation of a medicament for inhibiting prostate cancer proliferation and / or promoting apoptosis of prostate cancer cells;

[0051] (4) Use of evergreen gelbine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form in the preparation of a drug for inhibiting the transition of prostate cancer cells from G1 phase to S phase.

[0052] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood according to its ordinary meaning as understood in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0053] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of evergreen gelseed alkaloids, which means those carboxylates, amino acid addition salts, etc. of the compound (evergreen gelseed alkaloids) described in the present invention, which are suitable for contact with patients within the scope of reliable medical judgment, do not produce undue toxicity, irritation, allergic reactions, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (if possible) the zwitterionic form of the compound (evergreen gelseed alkaloids) described in the present invention.

[0054] In some embodiments, examples of the "pharmaceutically acceptable salt of evergreen gelsemine" include, but are not limited to, salts with (as counter ions) alkali metal ions such as Li + 、Na + or K+ or salts with alkaline earth metal ions such as Mg 2+ or Ca 2+ or salts with any other pharmaceutically acceptable metal ion such as Zn 2+ or Al 3+ ; or pharmaceutically acceptable salts with organic bases such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0055] In some embodiments, the pharmaceutically acceptable base addition salt is formed between metal or an amine, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

[0056] In some embodiments, the base addition salt of an acidic compound can be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid in a conventional manner. Both the free acid and the salt form of the compounds are considered within the scope of the present application. The free acid form differs slightly from its salt form in certain physical properties, such as solubility in polar solvents, but the two forms are otherwise equivalent for the purposes of the present application.

[0057] In some embodiments, salts can be prepared from inorganic acids such as sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include the following: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, gluconate, lactobionate, lauryl sulfate, and isethionate, and the like. Salts can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkyldioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, mesylate, and the like. Pharmaceutically acceptable salts can include cations based on alkali and alkaline earth metals, for example sodium, lithium, potassium, calcium, magnesium, and aluminum, as well as ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids such as arginate, gluconate, galacturonate, and the like, are also encompassed by the term.

[0058] The term "hydrate" refers to a compound of the present application (e.g., retusine) in combination with water. Typically, the ratio of the number of water molecules to the number of molecules of the compound in a hydrate of the compound is defined. Thus, a hydrate of a compound can be represented by, for example, the general formula R x H2O, where R is the compound and x is a number greater than zero. A given compound can form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than zero and less than one, e.g., hemihydrates (R 0.5 H2O), and polyhydrates (x is a number greater than one, e.g., dihydrates (R 2 H2O) and hexahydrates (R 6 H2O).

[0059] The term "solvate" refers to a solvent addition form of a compound which comprises chemically combined or non-chemically combined solvent molecules, including any solvated form of the compounds described herein (picrotoxinine). Conventional solvents include, but are not limited to: water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include chemically combined and non-chemically combined solvates. In some cases, the solvates will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, "solvate" includes both solvated and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0060] The term "pharmaceutically acceptable excipients and / or vehicles" refers to any pharmaceutically acceptable excipient or vehicle well known in the pharmaceutical arts, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each excipient or vehicle should be "acceptable" in the sense of being compatible with the other ingredients and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable excipients include, but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laureate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethylene glycol; phosphate buffer solutions; and other non-toxic compatible substances utilized in pharmaceutical formulations.

[0061] The term "therapeutically effective amount" refers to the amount of a therapeutic agent that is sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of other therapeutic agents. In particular embodiments of the application, the disease, disorder, or condition refers to prostate cancer, a prostate cancer-related disorder, or a condition. In particular embodiments of the application, the therapeutic agent refers to picrotoxinine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, or crystalline form thereof, or a pharmaceutical, pharmaceutical composition, or pharmaceutical formulation provided herein.

[0062] The term "prophylactically effective amount" refers to an amount that is sufficient to prevent a disease, disorder, or condition, or to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent the recurrence of a disease, disorder, or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the process of preventing a disease, disorder, or condition. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis, or enhances the prophylactic effect of other prophylactic agents. In particular embodiments of the application, the disease, disorder, or condition refers to prostate cancer, a prostate cancer-related disorder, or a prostate cancer-related condition.

[0063] The term "subject" includes, but is not limited to, a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) and / or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a bovine, a porcine, a equine, an ovine, a caprine, a rodent, a feline, and / or a canine. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a human. The terms "human," "patient," and "subject" are used interchangeably herein. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 Figure 1: Proliferation activity of evergreen kounidine on prostate cancer cells: A, CCK8 detection of the effect of evergreen kounidine on the proliferation activity of prostate cancer cells PC3 and DU145; B, cell colony formation experiment detection of the effect of evergreen kounidine on the proliferation activity of prostate cancer cells PC3 and DU145; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0065] Figure 2 Figure 2: Apoptosis of prostate cancer cells promoted by evergreen kounidine: A, PC3 cell apoptosis flow cytometry results; B, DU145 cell apoptosis flow cytometry results.

[0066] Figure 3 Figure 3: Cell cycle of prostate cancer cells affected by evergreen kounidine: A, PC3 cycle flow cytometry results; B, DU145 cycle flow cytometry results.

[0067] Figure 4 Figure 4: Cell invasion and migration of prostate cancer cells affected by evergreen kounidine: A, PC3 cell Transwell invasion experiment results; B, DU145 cell Transwell invasion experiment results.

[0068] Figure 5 Figure 1 shows the results of transcriptome sequencing analysis of evergreen kounine, wherein A shows a correlation heat map showing the differences between the drug group and the blank group, B shows a Venn diagram showing 11957 different genes between the two groups, C shows a difference analysis showing that 1857 genes are up-regulated and 833 genes are down-regulated in the different genes, D shows a GO / KEGG enrichment analysis chart showing the top 25 enriched pathways, including the p53 signaling pathway, and E-H show the DO and reaction component enrichment analysis results. DETAILED DESCRIPTION

[0069] The present application will be further described below in conjunction with specific examples, which are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0070] The drugs, reagents and raw materials used in the present application are readily available to those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods not specified in the present application are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer, in particular, the following examples are only used to illustrate the present application and should not limit the scope of the present application in any way.

[0071] Example Application of evergreen kounine in the treatment of prostate cancer

[0072] 1. Experimental materials

[0073] 1.1 Cell lines

[0074] Human prostate cancer cell lines DU145 and PC3 were purchased from ATCC, USA, and were preserved and subcultured in the laboratory.

[0075] 1.2 Drugs and reagents

[0076] Evergreen kounine was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., RPM / MIN 1640 and DMEM culture medium were purchased from Hyclone Company of the United States, penicillin was purchased from Hyclone Company of the United States, high-quality fetal bovine serum was purchased from Biological Industries of Israel, 0.25% trypsin-EDTA digestion solution was purchased from Beijing Solayebio Technology Co., Ltd., DMSO was purchased from Sigma, BCA protein quantification kit was purchased from Beijing Kangwei Century, CCK8, double-antibody sterilization solution, and DAPI were purchased from meilunbio. The concentration of evergreen kounine mother liquor was 10 mM dissolved in dimethyl sulfoxide (DMSO), and stored at -20°C. The final concentration of DMSO was less than 0.1% when acting on cells.

[0077] 1.3 Experimental instruments

[0078] HH-ZK type constant temperature water bath was purchased from Gugyi Yihua Instrument Co., Ltd., FB224 electronic analytical balance was purchased from Shanghai Sunyu Hengping Instrument Co., Ltd., high-speed low-temperature centrifuge and table type normal temperature centrifuge were purchased from Eppendorf Company of the United States, pipettor was purchased from Eppendorf and Gilson Company of the United States, cell culture bottle, culture dish and culture plate were purchased from NEST, ultrapure water device was purchased from Milipore Company of the United States, C6 flow cytometer was purchased from BD Company of the United States, Western Blot instrument was purchased from Beijing Liyi, biological safety cabinet and CO2 incubator were purchased from Thermo Company of the United States, ultralow-temperature refrigerator and ordinary refrigerator were purchased from Thermo Company of the United States, laser confocal microscope was purchased from Germany Leica, CKX 41 type inverted phase contrast microscope was purchased from Japan OLYMPUS Company, and multifunctional enzyme label instrument was purchased from Peridn Elmer Company of the United States.

[0079] 1.4 Required solutions

[0080] (1) PBS solution: 8.0 g of NaCl, 0.2 g of KCl, 3.85 g of Na2HPO4·12H2O, and 0.27 g of KH2PO4 were dissolved in 800 mL of triple distilled water, stirred with a glass rod to fully dissolve, the pH value was adjusted to 7.4, and the volume was adjusted to 1000 mL. After high-pressure sterilization, it was ready for use.

[0081] (2) Cell culture solution: in a clean bench, 50 mL of fetal bovine serum was added to 450 mL of RPM / MINI-1640 or DMEM culture medium, and fully mixed. The prepared complete culture medium was stored in a 4°C refrigerator for standby.

[0082] (3) Cell freezing solution: DMSO, fetal bovine serum, RPM / MINI-1640 or DMEM medium were prepared in a ratio of 1:2:7 in a clean bench. The solution was prepared immediately before use.

[0083] 2. Experimental method

[0084] 2.1 Cell culture

[0085] 2.1.1 Cell recovery

[0086] The cells to be recovered were taken out of liquid nitrogen and immediately placed in a 37°C water bath. The frozen cells were completely thawed in 2 minutes by gently shaking, and the freezing tube was disinfected with 75% alcohol and moved into a biosafety cabinet that had been ultraviolet irradiated for 30 minutes. The cells were suspended by gently blowing them with a pipette and centrifuged at 1000 rpm / min for 5 minutes. The supernatant was discarded, and fresh culture medium containing 10% FBS was added to resuspend the cells. The cells were transferred to a cell culture bottle and incubated in a 5% CO2, 95% humidity, 37°C constant temperature incubator. After 24 hours of incubation, the cell state and adhesion were observed under a microscope. The cell culture medium was usually replaced every other day, or every three days depending on the cell growth.

[0087] 2.1.2 Cell passage

[0088] The cells were observed under a microscope, and when the confluence reached 80-90%, the original culture medium was removed with a pipette, and then the cells were washed 2-3 times with PBS. About 1 mL of 0.25% trypsin-EDTA digestion solution was added to the culture bottle, and the culture bottle was gently shaken to evenly coat the cells with the digestion solution and fully contact the cells. The digestion solution was removed, and the culture bottle was placed in a 37°C constant temperature incubator to digest the cells. The cells were observed under a microscope at any time, and when the cells became round, 2-3 mL of culture medium was added to terminate the digestion. The cells were gently blown with a pipette, and the entire cell suspension was collected and transferred to a centrifuge tube. The tube was centrifuged at 1000 rpm / min for 4 minutes. The supernatant was discarded, and an appropriate amount of fresh culture medium containing 10% FBS was added to the centrifuge tube to gently resuspend the cells into a single cell suspension. The suspension was then inoculated into a culture bottle or plate and gently shaken to evenly distribute the cells.

[0089] 2.1.3 Cell freezing

[0090] When the cells are in the logarithmic growth phase (and the cell confluence is 80%-90%), use a pipette to suck out the original culture medium, then rinse the cells with PBS for 2-3 times. Add about 1 mL of 0.25% trypsin-EDTA digestion solution to the culture bottle, gently shake the culture bottle to make the digestion solution evenly coated on the cell surface and fully contact with the cells, suck out the digestion solution, and place the culture bottle in a 37°C constant temperature incubator to digest the cells, and observe under a microscope at any time. When the cells become round, add 2-3 mL of culture medium to terminate the digestion. Gently blow the cells with a pipette, collect all the cell suspension into a centrifuge tube. Centrifuge at 1000 rpm / min for 4 min. Use a pipette to suck out the supernatant, and pay attention to avoid sucking out the cells to cause cell loss. Add an appropriate amount of prepared freezing solution, and gently blow the cells to form a uniform cell suspension. Move the cell suspension into a freezing tube, and add about 1.5 mL to each freezing tube. Mark the cell name, culture conditions, freezing person, and freezing date on the cell freezing tube, and move it into a freezing box. Place the freezing box in a -80°C refrigerator, and after 24 h, transfer the freezing tube into a liquid nitrogen tank for long-term storage of the cells.

[0091] 2.2 Cell CCK8 survival rate detection

[0092] (1) Take DU145 and PC3 cells, centrifuge after digestion, resuspend the cells, and count the cells. Take an appropriate amount of suspension and dilute it to a concentration of 20,000 cells / mL and 40,000 cells / mL, add 100 μL of the suspension to a 96-well plate, and place it in an incubator for culture;

[0093] (2) When the cells adhere, check the cells under a microscope. If the cells are normal, place 3-6 replicate wells in each group, and sequentially add 50 μL of blank medium containing different concentrations of evergreen gelsemine (DU145 cells (0 μM, 3.5 μM, 7 μM, 14 μM), PC3 cells (0 μM, 2 μM, 4 μM, 8 μM)), and then incubate in a 37°C constant temperature incubator;

[0094] (3) After the above drug-containing cells are cultured for 24 h, discard the supernatant, and add an equal amount of freshly prepared CCK8 working solution (usually diluted 10 times with blank medium) to each well, incubate at 37°C for 1-2 h, and then detect the OD value at 450 nm using a microplate reader;

[0095] (4) Calculate the survival rate of the cells after drug addition and draw a cell survival rate line graph.

[0096] 2.3 In vitro migration experiment

[0097] (1) Take a 6-well cell culture plate, and evenly draw straight lines on the back of the culture plate with a black Maker pen and a ruler. The number of straight lines in each well is 5, and the 5 lines are evenly distributed;

[0098] (2) Take the logarithmic growth phase DU145 and PC3 cells, after trypsin digestion, to make single cell suspension, the cells are inoculated in 6 hole cell culture plate, the density is about 60-70%, in CO2 constant temperature incubator culture;

[0099] (3) Microscope observation cell growth state, when the cell grows to monolayer, discard the original culture medium, and add PBS flush 2-3 times, add serum-free medium, continue to culture in CO2 constant temperature incubator;

[0100] (4) After 24h, 100μL gun head along the ruler on the monolayer cells to draw 5 marks. When marking, the gun head should be vertical, not inclined; Mark should be perpendicular to the back of the black line. Discard the culture medium, and wash with PBS 2-3 times, and wash the suspended cells. Add fresh 1% complete medium containing different drug concentrations (the final concentration of evergreen securinine is PC3 cells (0μM, 20μM, 40μM, 60μM) and DU145 cells (0μM, 10μM, 20μM, 40μM)). Use Leica DM14000B inverted digital microscope, 100x, take photos of the scratch position at 0h of drug treatment. Record the position of the photo according to the scratch and the back black line. Continue to culture in CO2 constant temperature incubator;

[0101] (5) After 24h, end the experiment, under the microscope, according to the last position recorded, take photos of the scratch change; Or observe under the microscope that the untreated group scratch has been "healed", end the experiment.

[0102] 2.4 Cloning experiment

[0103] (1) Take the logarithmic growth phase DU145 and PC3 cells, after trypsin digestion, to make single cell suspension, the cells are inoculated in 6 hole cell culture plate, 800 cells per hole, in CO2 constant temperature incubator culture;

[0104] (2) After 24h, discard the original culture medium, add fresh 10% FBS medium containing different drug concentrations (the final concentration of evergreen securinine is DU145 cells (0μM, 3.5μM, 7μM, 14μM) and PC3 cells (0μM, 2μM, 4μM, 8μM)), continue to culture in CO2 constant temperature incubator;

[0105] (3) After 24h, discard the original culture medium, wash with PBS 2-3 times, add fresh 10% FBS medium, culture in CO2 constant temperature incubator, continue to culture for 14 days, the replacement period is three days;

[0106] (4) After 14 days, discard the original culture medium, wash with room temperature PBS 2-3 times;

[0107] (5) Add 1 mL fixative solution (glacial acetic acid: methanol: deionized water = 1:1:8) to each well, and fix for 10 min on a shaker. Discard the fixative solution, and wash with PBS for 3 times, 10 min each time. Add 0.1% crystal violet staining solution to stain for 10 min, and wash with PBS for 3 times, 5 min each time.

[0108] 2.5 Apoptosis detection

[0109] (1) Take the DU145 and PC3 cells in the logarithmic growth phase, and plant in 6-well plates. Treat the DU145 cells (0 μM, 3.5 μM, 7 μM, 14 μM) and PC3 cells (0 μM, 2 μM, 4 μM, 8 μM) with different concentrations of evergreen gelsemine for 24 h;

[0110] (2) Wash the cells with PBS for 2-3 times, and digest with trypsin without EDTA. Collect the cells by centrifugation (1000 rpm / min, 5 min), and the cell number is not less than 1×10 5 ;

[0111] (3) Wash the cells with PBS for 2 times (1000 rpm / min, 5 min);

[0112] (4) Add 500 μL of Binding Buffer to suspend the cells;

[0113] (5) After mixing 5 μL of Annexin V-FITC, add 5 μL of Propidium Iodide, and mix;

[0114] (6) React for 5-15 min at room temperature in the dark;

[0115] (7) Detect by C6 flow cytometry.

[0116] 2.6 Transwell chamber detection

[0117] 2.6.1 Transwell chamber detection (migration)

[0118] (1) When the cells grow to 90% density, digest the cells, resuspend with serum-free medium, count by a hemocytometer, and adjust the cell density to 2×10 5 cells / mL. Take 100 μL of the cell suspension, and drop to the upper chamber. Add 800 μL of serum-containing medium to the lower chamber of the 24-well plate. Place the 24-well plate in a cell culture incubator to culture overnight;

[0119] (2) After 36 h, gently wipe off the cells in the upper chamber with a cotton swab to prevent scratching the cells on the membrane;

[0120] (3) The chamber was rinsed in PBS liquid for 3 times;

[0121] (4) The chamber was placed in methanol for 15 min;

[0122] (5) The chamber was taken out and dried at room temperature for about 5 min;

[0123] (6) After staining with 1% crystal violet for 15 min, PBS was washed for 3 times;

[0124] (7) After the chamber was dried in air, it was observed and photographed by an inverted microscope.

[0125] 2.6.2 Transwell chamber detection (invasion)

[0126] (1) The matrigel was left at 4°C overnight until it was completely liquefied. The 24-well plate and the sterilized gun tip were pre-cooled at 4°C overnight. The Transwell chamber was sterilized by ultraviolet light overnight in a clean bench;

[0127] (2) The matrigel was diluted with blank medium at a ratio of 1:5. 70 μL of the diluted matrigel was transferred to the upper chamber of the Transwell chamber, which was incubated in a cell incubator for 30 min to allow the matrigel to solidify into a gel;

[0128] (3) The subsequent experimental operation was the same as that of the Transwell migration experiment.

[0129] 2.7 Transcriptomic sequencing

[0130] (1) Preparation of cells. PC3 and DU145 were evenly plated in 8 60 mm cell culture dishes. After adhering overnight, the cells were treated with drugs for 24 h, and then the cell lysate was collected using TriZol lysis;

[0131] (2) Extraction of total RNA. The specific steps are described in the TriZol Reagent reagent instruction;

[0132] (3) Measurement of RNA concentration and purity using NanoDrop2000. The measurement was strictly performed according to the operation procedure. The purity of RNA was determined according to the A260 / 280 ratio. If the value of A260 / 280 was between 1.8 and 2.1, the purity of RNA was qualified, and the subsequent sequencing analysis could be performed;

[0133] (4) RNAseq sample sending. The wall of a 1.5 mL centrifuge tube was labeled with the concentration, and the tube was tightly capped with a sealing film. Three repeated samples were taken from each treatment group and placed in dry ice. The samples were mailed to Novogene Company (Beijing, China) for sequencing;

[0134] (5) Library construction, sequencing and GSEA analysis. According to the suggestions of Novogene Company, the RNAseq data was analyzed using UltraTMRNA Library Prep Kit for Sequencing Libraries were generated by NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, MA, USA). Heatmaps were plotted with individual genes in the ER stress / UPR pathway using the “pheatmap” package. Gene Set Enrichment Analysis (GSEA) was performed by the JAVA program (http: / / software.broadinstitute.org / gsea / index.jsp) using the Molecular Signature Database (MSigDB). 1000 random sample permutations were performed, and the significance threshold was set at p < 0.05 and nominal FDR q < 0.05.

[0135] 3. Experimental results

[0136] 3.1 Evergreen Gelsemine can dose-dependently inhibit the proliferation activity of prostate cancer cells

[0137] Prostate cancer cells DU145 and PC3 were selected to evaluate the effect of evergreen gelsemine on the proliferation activity of prostate cancer. CCK8 experiment was performed by setting concentration gradient on two kinds of cells. Prostate cancer cells DU145 and PC3 cells were inoculated in 96-well plates at 2000 cells / well and 4000 cells / well respectively. After 24 h, the drugs were added according to the preset concentration. After 24 h of drug action, the culture medium was removed from each well, 100 μL of ten-fold diluted CCK8 reagent was added, and the OD value was detected by enzyme-labeled instrument after half an hour of incubation. It was found that evergreen gelsemine could dose-dependently inhibit the proliferation activity of prostate cancer cells, and the 24-hour half-inhibitory concentration (IC50, PC3: 3.374 μM, DU145: 6.602 μM) of evergreen gelsemine on two kinds of prostate cancer cells was calculated. Figure 1 A). According to the half-inhibitory concentration (IC50), the concentration gradient of PC3 cells (0 μM, 2 μM, 4 μM, 8 μM) and DU145 cells (0 μM, 3.5 μM, 7 μM, 14 μM) was set to perform colony formation experiment. Prostate cancer cells DU145 and PC3 were inoculated in 6-well plates at 800 cells / well. After 24 h, the drugs were added according to the preset concentration gradient. After 24 h of drug action, the drugs were removed, and the cells were cultured with 5% fetal bovine complete medium for 2 weeks, then the samples were collected, fixed and stained. It was found that evergreen gelsemine could dose-dependently inhibit the proliferation of prostate cancer cells Figure 1 B).

[0138] 3.2 Evergreen gelsemine can effectively promote the apoptosis of prostate cancer cells

[0139] PC3 and DU145 cells were selected to study the effect of evergreen gelsemine on prostate cancer cell apoptosis. The concentration gradient of 0 μM, 3.5 μM, 7 μM (DU145) and 0 μM, 2.5 μM, 5 μM (PC3) was set to act on prostate cancer cells for 24 h. After 24 h, the cells were collected and detected by Annexin V-EGFP / PI double staining cell apoptosis detection kit and flow cytometry. The results showed that evergreen gelsemine could effectively increase the late apoptosis rate of prostate cancer cells Figure 2 A-B).

[0140] 3.3 Evergreen gelsemine blocks the G1 / S phase transition of prostate cancer

[0141] PC3 and DU145 cells were selected to study the effect of evergreen gelsemine on prostate cancer cell cycle. DU145 cells were treated with 0 μM, 3.5 μM, 7 μM of evergreen gelsemine, and PC3 cells were treated with 0 μM, 2.5 μM, 5 μM of evergreen gelsemine. After 24 h, flow cytometry was performed. The results showed that evergreen gelsemine inhibited the transition of uroepithelial tumor cells from G1 phase to S phase in a dose-dependent manner Figure 3 A-B).

[0142] 3.4 Evergreen gelsemine inhibits the invasion and migration of uroepithelial tumor cells

[0143] PC3 and DU145 cells were selected to study the effect of evergreen gelsemine on prostate cancer cell invasion and migration. The concentration gradient of 0 μM, 3.5 μM, 7 μM (DU145) and 0 μM, 2.5 μM, 5 μM (PC3) was set to perform Transwell invasion Figure 4 A-B) and migration experiments on prostate cancer cells. The results showed that evergreen gelsemine inhibited the invasion and migration of uroepithelial tumor cells.

[0144] 3.5 Evergreen gelsemine transcriptomic sequencing results

[0145] DU145 prostate cancer cells were selected and set to blank group and effective concentration group (7 μM) for transcriptomic sequencing. The correlation heat map showed the differences between each sample and the differences between sample groups. The results were as follows Figure 5As shown in A-H, the results showed that there was a significant difference between the drug group and the blank group. The Venn diagram showed that there were 11957 different genes between the blank group and the drug group. The differential analysis results showed that there were 1857 up-regulated differential genes and 833 down-regulated differential genes. The heat map showed the transcription level difference of each gene between the two groups. The GO / KEGG enrichment analysis results showed that the differential genes were enriched in the p53 signaling pathway.

Claims

1. Use of evergreen gelsemine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing prostate cancer.

2. The use according to claim 1, characterized in that The evergreen gelsemine significantly inhibits the proliferation of prostate cancer cells and / or promotes the apoptosis of prostate cancer cells.

3. The use according to claim 1, characterized in that The evergreen gelsemine significantly inhibits the transition of prostate cancer cells from G1 phase to S phase.

4. The use according to claim 1, characterized in that The evergreen gelsemine significantly inhibits the invasion and migration of prostate cancer cells.

5. The use according to claim 1, characterized in that The medicine comprises a therapeutically and / or preventively effective amount of evergreen gelsemine or a pharmaceutically acceptable salt thereof.

6. The use according to claim 1, characterized in that The drug further comprises pharmaceutically acceptable excipients.

7. The use according to claim 1, characterized in that The dosage form of the drug is an oral dosage form, a parenteral dosage form and / or a topical dosage form.

8. The use according to claim 1, characterized in that The dosage form of the pharmaceutical preparation is solution, sustained-release agent, suspension, granule, tablet, capsule, powder, effervescent agent, emulsion, syrup and / or chewable agent.

9. The use according to claim 1, characterized in that The drug is administered orally, subcutaneously, intravenously, intramuscularly, intraarterially, intranasally, intrathecally, intramucosally, intrapulmonaryly and / or rectally.

10. Use of evergreen gelsemine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating and / or preventing prostate cancer.

11. Use of evergreen gelsemine or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical preparation for treating and / or preventing prostate cancer.

12. Use of evergreen gelsemine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the proliferation of prostate cancer cells and / or promoting the apoptosis of prostate cancer cells.

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