WO2017177947A1 - 局部注射双硫仑制剂治疗胸腹膜癌症的方法 - Google Patents

局部注射双硫仑制剂治疗胸腹膜癌症的方法 Download PDF

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WO2017177947A1
WO2017177947A1 PCT/CN2017/080463 CN2017080463W WO2017177947A1 WO 2017177947 A1 WO2017177947 A1 WO 2017177947A1 CN 2017080463 W CN2017080463 W CN 2017080463W WO 2017177947 A1 WO2017177947 A1 WO 2017177947A1
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disulfiram
thoracic
cancer
group
copper
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PCT/CN2017/080463
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English (en)
French (fr)
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王伟光
王志鹏
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诺马（北京）科技有限公司
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Priority to EP17781923.2A priority Critical patent/EP3449919B1/en
Priority to US16/093,187 priority patent/US10828271B2/en
Publication of WO2017177947A1 publication Critical patent/WO2017177947A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/30—Copper compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of cancer treatment, and in particular to a method for treating thoracic peritoneal in situ and metastasizing cancer by locally injecting a disulfiram formulation.
  • Disulfiram has been used in alcohol treatment for more than 60 years (Johansson, 1992). Disulfiram is sensitive to ethanol, and patients can develop an uncomfortable response even if they ingest a small amount of ethanol during treatment. Disulfiram inhibits the activity of Aldehyde Dehydrogenase (ALDH) and blocks the oxidation of ethanol in the acetaldehyde stage. Thus, after inhalation of disulfiram, the concentration of acetaldehyde in human blood is 5 to 10 times higher than that of a subject who consumes the same amount of ethanol but does not take disulfiram.
  • ALDH Aldehyde Dehydrogenase
  • disulfiram-ethanol-like reaction which is proportional to the amount of disulfiram and ethanol ingested. This reaction continues until the ethanol is completely metabolism.
  • Disulfiram even with a small amount of ethanol, can cause facial flushing, severe pulsation of the head and neck, pulsating headache, difficulty breathing, nausea, vomiting, sweating, dry mouth, chest pain, palpitations, hyperventilation , tachycardia, hypotension, syncope, restlessness, fatigue, dizziness, blurred vision and paralysis.
  • disulfiram is highly cytotoxic to a variety of cancer cells (Wang et al., 2003, Cen et al., 2004, Liu et al., 2012, Yip et al., 2011, Liu et. Al., 2014).
  • the effective concentration of disulfiram on tumor cells is at a nanomolar level, which is better than many clinically used anticancer drugs (such as daunorubicin, 5-fluorouracil, gemcitabine, paclitaxel, platinum drugs, vinblastine, etc.). More significant anti-cancer effects in vitro.
  • the results of the in vitro experiments are very encouraging, few studies have reported the anticancer activity of disulfiram in animal experiments.
  • disulfiram has not been used as an anticancer drug in humans because the potential clinical application of disulfiram in cancer treatment is limited by the existing oral preparation of disulfiram.
  • disulfiram is extremely unstable in gastric acid, and most oral disulfiram is rapidly degraded into carbon disulfide (CS 2 ) and diethylamine (DEA) (Johansson, 1992).
  • CS 2 carbon disulfide
  • DEA diethylamine
  • the absorbed disulfiram reacts with the thiol group of serum albumin in the bloodstream and is rapidly reduced by the glutathione reductase system in the blood red blood cells (half-life: 4 minutes), which is rapidly converted to diethyldithiocarbamate.
  • DDC diethyldithiocarbamic acid
  • Most of the disulfiram absorbed by oral administration and its derivatives can be enriched by the liver.
  • the sulfhydryl groups in DDC and disulfiram molecules are rapidly methylated or glucuronylated in the liver to form methyl DDC and glucuronic acid DDC (Agarwal et al., 1983, Agarwal et al., 1986, Gessner and Jakubowski, 1972, Kaslander, 1963, Prickett and Johnston, 1953).
  • Our unpublished data confirm that both have completely lost anticancer activity. Therefore, an oral dose of 500 mg of disulfiram has a blood concentration below the detection limit. The above reaction does not affect the alcohol withdrawal effect of disulfiram.
  • disulfiram is a divalent metal ion chelating agent that produces a large amount of reactive oxygen species (ROS) when chelated with copper ions and other divalent metal ions.
  • ROS reactive oxygen species
  • ROS has a strong killing effect on cancer cells.
  • ROS has a very short life (in nanoseconds) in body fluids. Therefore, disulfiram and copper ions must produce a chelation reaction in cancer tissues to kill cancer cells (Tawari, 2015).
  • the final product, diethyldithio copper, formed by disulfiram and copper ions also has a killing effect on cancer cells. Whether it is the chelation reaction itself or the formation of the final product, the thiol group (-SH) in the disulfiram molecule is indispensable. In the methylation and glucuronidation process, the sulfhydryl group of disulfiram is destroyed, thus losing the ability to produce active oxygen and its diethyldithio copper. Our unpublished experimental data demonstrate that methylated disulfiram completely loses its anticancer activity.
  • disulfiram for local application of thoracic cancer by topical administration. Treatment, thus overcoming the bottleneck of short half-life in disulfiram.
  • the present invention provides a method of treating primary and metastatic cancer of the thoracic peritoneum comprising administering to the subject in need of treatment a therapeutically effective amount of disulfiram or a derivative thereof intraperitoneally.
  • the present invention also provides the use of disulfiram or a derivative thereof for the preparation of a medicament for treating a chest and peritoneal cancer in a subject by intrathoracic administration.
  • the present invention also provides an effective administration preparation of disulfiram or a derivative thereof for treating a chest and peritoneal cancer in a subject by intrathoracic intraperitoneal administration.
  • the present invention also provides disulfiram or a derivative thereof for treating a thoracic and peritoneal cancer in a subject by intrathoracic intraperitoneal administration.
  • the invention also provides disulfiram or a derivative thereof as an active ingredient (eg DDC) combined and non-combined methods of copper, zinc, iron, gold and other divalent transition metal elements.
  • disulfiram or a derivative thereof such as DDC
  • the invention also provides a combination and non-combination of disulfiram or a derivative thereof (such as DDC) with copper, zinc, iron, gold and other divalent transition metal elements and is used in combination with existing anticancer drugs for chest Peritoneal cancer treatment, thereby increasing the anticancer activity of existing anticancer drugs, and/or reducing the toxic side effects of existing anticancer drugs.
  • disulfiram or a derivative thereof such as DDC
  • the invention also provides a kit comprising: (1) disulfiram as a therapeutic agent or a derivative thereof; and (2) instructions for use, wherein the instructions for use of disulfiram or a derivative thereof for passage through the thoracic and peritoneum Intracavitary administration treats patients with thoracic and peritoneal cancer.
  • Figure 1 shows the inhibition of mesothelioma cells. Survival of MSTO, Ju77, 2591, E58 cells after 3 days of incubation with pemetrexed (PMTX), DS/Cu, and both, respectively. In the case of DS/Cu and the two, the inhibition of mesothelioma cells was significantly higher than that of pemetrexed, which was also higher than DS/Cu.
  • Figure 2 shows the inhibition of mesothelioma cells.
  • the inhibition of mesothelioma cells was significantly higher than that of cisplatin and also higher than DS/Cu.
  • Figure 3 shows the inhibition of ovarian cancer cells. Survival of A2780 and SKOV-3 cells after incubation with cisplatin, DS/Cu, and both for 3 days. In the case of DS/Cu and the two, the inhibition of ovarian cancer cells was significantly higher than that of cisplatin and also higher than DS/Cu.
  • Figure 4 shows the overall condition of the mice: After intraperitoneal injection of S-180 ascites tumor cells in BalB/C mice, the mice were generally in good condition during the test. From the third day after the injection, the abdominal cavity of the control group gradually became larger, and it was a boat on the 7th and a spherical shape on the 11th. The control group was sacrificed at 11 days. The treatment group was sacrificed after 21 consecutive days of observation. The control group showed abdominal distension and the treatment group was normal.
  • Figure 5 shows a peritoneal cancer nodule. After intraperitoneal injection of ascites tumor cells in BalB/C mice, multiple cancer nodules were seen in the abdominal cavity on the 11th day of the control group. The masses were clearly seen in the abdominal wall and mesentery, 5-7 per case, and the diameter was 0.2-1 cm. The drug-administered group was sacrificed on the 21st day, and the mouse's abdominal cavity was clean and no cancerous nodules were visible.
  • FIG. 6 After intraperitoneal injection of ascites tumor cells in BalB/C mice, on the 11th day, the control group of mice was sacrificed, and a large amount of bloody ascites was observed in the laparotomy, about 10 ml/head. The diarrhea of the control group is shown. The abdominal cavity of the drug-administered group was completely normal and there was no ascites.
  • Figure 7 Mouse body weight change curve. After intraperitoneal injection of ascites tumor cells in BalB/C mice, the control group formed a rapid increase in body weight due to the formation of cancer ascites. The treatment group had a slight increase in body weight at the initial stage, which was associated with intra-abdominal growth of cancer. After 4 days of treatment, the weight gain of the treatment group was flat, suggesting that intraperitoneal cancer proliferation was controlled by drugs. After 14 days due to the drug effect, the cancer cells were completely killed, the mice lost weight and returned to normal weight in 21 days.
  • Figure 8 Comparison of the number of cancer nodules. After intraperitoneal injection of ascites tumor cells in BalB/C mice, the average number of cancer nodules per mouse in the control group (day 11) was significantly higher (p ⁇ 0.01) than in the treatment group (21 days, 0/mouse).
  • Figure 9 Comparison of ascites volume. After intraperitoneal injection of ascites tumor cells in BalB/C mice, the mean ascites volume (10 ml) per pawn of the control group (day 11) was significantly greater (p ⁇ 0.01) than in the treatment group (21 days, 0 ml/mouse).
  • Figure 10 Comparison of intra-abdominal invasion of cancer cells (100x magnification). After intraperitoneal injection of ascites tumor cells in BalB/C mice, the control group (Day 11) cancer cells invaded the subserosal muscle layer. Blue arrow: cancer tissue; red arrow: muscle tissue. The mean ascites volume (10 ml) per paw cavity was significantly greater (p ⁇ 0.01) than in the treatment group (21 days, 0 ml/mouse). The muscle tissue of the treatment group was completely normal and there were no cancer cells.
  • Figure 11 Microscopic morphology of important organs. After intraperitoneal injection of ascites tumor cells in BalB/C mice, no toxic damage (100-fold magnification) was observed in the main organs (lung, kidney, liver) of the control (11 days) and the treatment group (21 days).
  • Figure 13 Comparison of ascites volume. After intraperitoneal injection of MSTO in CD1 nude mice, the ascites of the control group was obvious on the 60th day, but no obvious ascites (90 days) was observed in the DS/Cu treatment group.
  • Figure 14 Comparison of the number of cancer nodules. After intraperitoneal injection of mesothelioma cells with MSTO in CD1 nude mice, the number of cancer nodules in the control group was significantly higher than that in the DS/Cu treatment group (90 days) by day 60. p ⁇ 0.01.
  • Figure 15 shows a peritoneal cancer nodule. After CD1 nude mice were injected intraperitoneally with mesothelioma cells by MSTO, by the 60th day, a large number of cancer nodules were observed in the control group. Multiple masses (indicated by arrows) are clearly visible in the abdominal wall, liver and mesentery. The mice in the DS/Cu treatment group had a clean abdominal cavity with almost no cancerous nodules.
  • Figure 16 Pathological photograph of cancer nodules. A. Mesenteric invasion. B. Intra-abdominal nodules. C. Liver invasion. D. High power field of view.
  • Figure 18 Comparison of ascites volume. After intraperitoneal injection of ovarian cancer cells into CD1 nude mice, the ascites of the control group was obvious on the 30th day, but no obvious ascites was observed in the DS/Cu treatment group (60 days).
  • Figure 20 shows a peritoneal cancer nodule. After CD1 nude mice were intraperitoneally injected with ovarian cancer cells, by the 30th day, a large number of cancer nodules were observed in the control group. Multiple masses were clearly visible in the abdominal wall and mesentery. The mice in the DS/Cu treatment group had a clean abdominal cavity and fewer nodules (60 days).
  • the inventors found through experiments that disulfiram has a strong killing effect on human malignant mesothelioma and ovarian cancer cell lines in vitro, and that disulfiram can synergistically enhance pemetrexed and cisplatin in humans in vitro.
  • the killing effect of mesothelioma and ovarian cancer cell lines Considering that the topical application of thoracic and peritoneal cavity can overcome the bottleneck of short half-life of disulfiram blood, the inventors performed intraperitoneal injection of disulfiram (5mg/kg) on S180 sarcoma tumor-bearing mice, and oral administration of copper gluconate. A thorough elimination effect on peritoneal metastases was achieved.
  • This exciting result prompted the inventors to further implement treatment experiments for human peritoneal mesothelial cancer and ovarian cancer.
  • the inventors used only 1/20 (2 mg/kg) of the dose of disulfiram anti-alcohol, allowing 60% and 90% of peritoneal mesothelioma and ovarian cancer mice to survive for a long time (90 And 60 days).
  • disulfiram may be due to the direct delivery of disulfiram to the vicinity of the tumor tissue when intraperitoneal injection of disulfiram, thereby avoiding other modes of administration such as oral and intravenous injection.
  • the present invention provides a method of treating a thoracic and peritoneal cancer comprising administering to a subject in need of treatment a therapeutically effective amount of disulfiram or a derivative thereof, intrapleurally and/or intraperitoneally.
  • intrapleural administration also referred to as “pleural administration” or “intrathoracic administration” refers to the delivery of a drug through the pleura into the pleural cavity.
  • intraperitoneal administration refers to the delivery of a drug through the peritoneum to the peritoneal cavity. Inside.
  • thoracic intraperitoneal administration refers to the delivery of a drug through the pleura or peritoneum into the pleural cavity or peritoneal cavity. It will be understood by those skilled in the art that intrapleural and intraperitoneal administration can be performed simultaneously in some cases, such as when there is a cancer to be treated on both the pleura and the peritoneum. The term “thoracic intraperitoneal administration” also encompasses the simultaneous administration to the pleural and peritoneal cavities.
  • Thoraco-intraperitoneal administration has the same meaning as "intrapleural and/or intraperitoneal administration”.
  • disulfiram or its derivatives After being delivered into the chest or abdominal cavity, disulfiram or its derivatives can rapidly spread to the tumor tissue through pleural effusion or peritoneal effusion, thereby exerting an anticancer effect.
  • Thoracic intraperitoneal administration can be conveniently performed by syringe, peristaltic pump or thoracic and abdominal catheter.
  • cancer includes any suitable cancer, such as malignant mesothelioma, gastric cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, endometrial cancer, pancreatic cancer, lymphoma, thyroid cancer. , bone cancer, central nervous system cancer, leukemia, liver cancer, prostate cancer, lung cancer, colon cancer, rectal cancer, brain cancer, melanoma, and human and animal malignant tumors that have been or are not currently reported.
  • pleural and/or peritoneal carcinoma refers to a malignant tumor that grows on the pleura and/or peritoneum of a patient, including primary thoracic and peritoneal cancer and thoracic peritoneal metastases.
  • the primary thoracic and peritoneal cancers mentioned herein include malignant mesothelioma and any other malignant tumor that originates in the thoracic and peritoneum.
  • the method of the invention is particularly useful for the treatment of malignant mesothelioma.
  • Malignant mesothelioma is a rare tumor that occurs in the pleura and peritoneum. Its incidence is closely related to asbestos.
  • Malignant pleural mesothelioma is the most common type of pleural primary tumor. Clinical manifestations are associated with invasive behavior, which usually locally invades the pleural cavity and surrounding structures. If left untreated, the median survival is 4 to 12 months.
  • the thoracic and peritoneal metastatic cancer referred to herein refers to a cancer that is transferred to the pleura and/or peritoneum from other parts of the body. It mainly includes gastric cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, liver cancer, lung cancer, colorectal cancer, etc. and any other metastatic cancer.
  • the method of the invention is particularly suitable for the treatment of peritoneal metastatic cancer.
  • peritoneal metastatic cancers include ovarian cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, and the like, and any other cancer that metastasizes to the peritoneum.
  • Peritoneal metastases have previously been considered the terminal stage of cancer. Supportive care, systemic chemotherapy, and palliative surgery are difficult to improve patient survival.
  • peritoneal metastasis rates of gastric cancer, ovarian cancer, pancreatic cancer, and colon cancer were 99%, 90%, 50%, and 32%, respectively, due to the location of the diseased organ.
  • cancer of other organs can also occur in distant metastasis and spread to the peritoneum, such as breast cancer, lung cancer and lymphoma.
  • the pharmaceutical preparation of the present invention can be used to prevent and/or inhibit the proliferation of tumor cells and/or tumor stem cells.
  • the ineffectiveness of cancer treatments on cancer stem cells is thought to be the cause of many cancer treatment failures.
  • treatment refers to the management and care of a patient to combat a disease or disorder.
  • the term includes broad-spectrum treatments for a particular condition, such as administration of an active compound to alleviate symptoms or complications, delaying the progression of the disease, alleviating or alleviating symptoms and complications, curing or eliminating the disease or condition, and including preventing the said Disease or bad condition.
  • the subject to be treated is preferably a mammal, especially a human, but also includes animals such as dogs, cats, horses, cows, sheep and pigs.
  • R 1 , R 2 , R 3 and R 4 in the thiuram disulfide may be the same or different.
  • R 1 , R 2 , R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl and heteroaryl.
  • the alkyl group may be a linear, branched or cyclic C 1 -C 10 alkyl group, such as a C 1 -C 8 alkyl group, such as a C 1 -C 6 alkyl group, such as C 1 -C 4 alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutane Base, cyclopentyl, cyclohexyl, cycloheptyl.
  • a linear, branched or cyclic C 1 -C 10 alkyl group such as a C 1 -C 8 alkyl group, such as a C 1 -C 6 alkyl group, such as C 1 -C 4 alkyl, such as methyl, ethyl, propyl, is
  • the alkyl group may include a cycloalkyl group and a heterocycloalkyl group such as a cyclopropyl group, a cyclobutyl group and the like.
  • the alkenyl group may be a C 2 -C 10 alkenyl group, such as a C 2 -C 8 alkenyl group, such as a C 2 -C 6 alkenyl group, such as a C 2 -C 4 alkenyl group, such as a vinyl group. , propylene or butenyl.
  • the alkynyl group may be a C 2 -C 10 alkynyl group, for example a C 2 -C 8 alkynyl group, for example a C 2 -C 6 alkynyl group, for example a C 2 -C 4 alkynyl group, for example an ethynyl group , propynyl or butynyl.
  • the alkoxy group may be a C 2 -C 10 alkoxy group, such as a C 2 -C 8 alkoxy group, such as a C 2 -C 6 alkoxy group, such as a C 2 -C 4 alkoxy group.
  • the aryl group may be a C 6 -C 14 monocyclic or bicyclic aromatic group such as a phenyl group, a fluorenyl group or the like, which may optionally further have a substituent such as a benzyl group or the like.
  • the heteroaryl group is an aromatic group containing one or two heteroatoms independently selected from nitrogen, oxygen or sulfur, such as furyl, thienyl, pyrrolyl, thiazolyl, imidazolyl , pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl.
  • R 1 , R 2 and N and R 3 , R 4 and N may form a nitrogen-containing heterocycloalkane or a heterocyclic aromatic hydrocarbon.
  • the R 1 , R 2 , R 3 and R 4 are each independently selected from the group consisting of imidazolinyl, dimethylidene, bisphenol, cyclopropylmethyl, pentamethylene, hydroxyethyl Base or methylglucosamine. In disulfiram, R 1 , R 2 , R 3 and R 4 are all ethyl.
  • disulfiram includes compounds of the above formula and metabolites thereof, and salts thereof formed with sodium ions, potassium ions, ammonium ions, etc., or with copper, zinc, iron, gold, and other divalent transitions.
  • a chelate product of a metal element such as DDC-Cu, DDC-Zn, or the like.
  • the "derivative of disulfiram” includes the dithiol metabolite diethyl disulfide, such as diethyldithiocarbamic acid (DDC), and its combination with sodium ions and potassium. a salt formed by ions, ammonium ions, or the like, or a chelate product of copper, zinc, iron, gold, and other divalent transition metal elements, such as DDC-Cu, DDC-Zn, and the like.
  • DDC diethyldithiocarbamic acid
  • Typical examples of disulfiram derivatives include: diethyldithiocarbamate or diethyldithiocarbamic acid (DDC), sodium diethyldithiocarbamate (DDC-Na), diethyldisulfide Ammonium diethyldithiocarbamate (DDC-NH 3 ), copper diethyldithiocarbamate, zinc diethyldithiocarbamate, and diethyldithiocarbamic acid formed with other metal ions Salt or chelate.
  • DDC diethyldithiocarbamate or diethyldithiocarbamic acid
  • DDC-Na sodium diethyldithiocarbamate
  • DDC-NH 3 diethyldisulfide Ammonium diethyldithiocarbamate
  • copper diethyldithiocarbamate zinc diethyldithiocarbamate
  • disulfiram or a derivative thereof may be administered alone to treat a thoracic and peritoneal cancer, or may be administered in combination with a divalent transition metal preparation.
  • the pharmaceutical preparation of the present invention comprising disulfiram or a derivative thereof (also referred to as "the pharmaceutical preparation of the present invention") can be used in combination with a divalent transition metal preparation such as a copper-containing preparation, thereby Further enhance the anti-cancer effect.
  • a divalent transition metal preparation such as a copper-containing preparation
  • Both formulations may be administered separately, simultaneously or sequentially.
  • disulfiram can be administered intraperitoneally and a copper containing formulation such as copper gluconate can be administered orally.
  • Disulfiram and copper-containing formulations can also be administered simultaneously or sequentially by intraperitoneal injection.
  • the clinician can determine the dosage and mode of administration of the disulfiram and copper-containing formulation based on factors such as the patient's condition, physical condition, and response to the drug.
  • divalent transition metal preparation herein refers to a substance or preparation containing a divalent transition metal element.
  • divalent transition metal includes copper, zinc, iron, gold, and other divalent transition metal elements.
  • Typical examples of the divalent transition metal preparation are a copper-containing preparation and a zinc-containing preparation.
  • particularly suitable copper-containing formulations include copper gluconate, copper citrate, copper chloride, and other organic and inorganic copper formulations that are available for oral administration to patients.
  • particularly suitable zinc-containing formulations include zinc gluconate, zinc chloride, zinc citrate, and other organic and inorganic zinc formulations that are available for oral administration to patients.
  • the invention also encompasses the use of a pharmaceutical formulation of the invention in combination with other active agents conventionally used in the treatment of cancer for the treatment of cancer.
  • the formulations of the invention may be used in combination with surgery or radiation therapy, and/or in combination with one or more drugs, such as cumene, 5-fluorouracil, doxorubicin, paclitaxel, and gemcitabine.
  • drugs such as cumene, 5-fluorouracil, doxorubicin, paclitaxel, and gemcitabine.
  • the present invention provides a pharmaceutical composition or pharmaceutical preparation comprising disulfiram or a derivative thereof as an active ingredient, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be used for treating thoracic and peritoneal cancer in a subject by intrathoracic intraperitoneal administration.
  • the pharmaceutical composition of the present invention may utilize a conventional pharmaceutically acceptable carrier or filler as well as other excipients such as cyclodextrin, sucrose, gelatin, magnesium stearate, olive oil, phospholipids and the like. See, eg, Remington: The Science and Practice of Pharmacy , 19 th Edition, Gennaro, Ed, Mack Publishing Co., Easton, PA, 1995..
  • the compounds of the present invention can be prepared in a variety of suitable dosage forms, such as liquid injections, powders for injection, tablets for injection, or encapsulated in liposomes or other nano-excipients.
  • the formulation will comprise from about 0.005% to about 95%, preferably from about 0.5% to about 50% by weight of the active ingredient, depending on the mode of administration employed.
  • the percentage of active compound in the formulation will depend on the particular nature of the formulation and on the activity of the compound and the needs of the patient.
  • the ratio of the active ingredient in the solution is usually from 0.01% to 90%, and if the preparation is obtained by solid dilution, the content can be higher.
  • the liquid formulation contains from 1% to 50% active compound.
  • terapéuticaally effective amount refers to an amount of active compound that is sufficient to cause a biological or medical response desired by a clinician in a subject.
  • a “therapeutically effective amount” of disulfiram or a derivative thereof can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, condition, and the like.
  • the formulation will take the form of a unit dosage.
  • the formulation can be placed in a vial or other container.
  • the vial or other container may contain a liquid, a solid to be diluted into a suspension, a dry powder, a lyophilizate, or any other suitable form of formulation.
  • the disulfiram or derivative thereof can be administered at a dose of from about 250 to about 500 mg/day (the disulfiram tolerated dose of the alcoholic patient is from 250 to 500 mg/day).
  • the patient preferably takes 2 mg of copper simultaneously, such as copper gluconate. Copper gluconate is preferably used orally.
  • the dosage of disulfiram or a derivative thereof can range from about 1 mg/kg to 10 mg/kg, preferably from about 1 mg/kg to 20 mg/kg.
  • the disulfiram dose can also be calculated based on the amount of ascites in 10 micromoles (2960 micrograms per liter) of disulfiram per liter of ascites. If the patient has 10 liters of ascites, the amount of disulfiram is about 30 mg.
  • kits comprising: (1) disulfiram as a therapeutic agent or a derivative thereof; and (2) instructions for use, wherein the use of the disulfiram or a derivative thereof is specified
  • the thoracic and peritoneal cancer is treated in the subject by intrathoracic intraperitoneal administration.
  • the kit of the invention is particularly suitable for the treatment of thoracic and peritoneal metastases.
  • disulfiram or a derivative thereof may be present in unit dosage form, and the unit dose may be 10 mg/piece (e.g., 10 ml).
  • the dose of disulfiram or a derivative thereof may be from 1 to 10 mg/kg/day.
  • Kits of the invention may also include an optional divalent transition metal formulation.
  • optional means “may or may not have” or “non-essential”.
  • optional copper formulation means that the copper formulation may or may not be included, which may be selected by those skilled in the art based on actual conditions.
  • MSTO, Ju77, 2591, E58 mesothelioma cells were donated by Professor Peter Szlosarek of Queen Mary College of London, cultured in 10% fetal calf serum supplemented with 10 mg/ml of streptomycin and 2 mmol/L L-glutamine.
  • the DMEM medium was cultured at 37 ° C under 5% CO 2 . They were incubated with pemetrexed, DS/Cu (disulfiram/copper gluconate), and cisplatin for 3 days. Cell survival was determined by MTT assay.
  • S180 ascites cells were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences and cultured in DMEM containing 10% fetal calf serum supplemented with 10 mg/ml of streptomycin and 2 mmol/L L-glutamine at 37 ° C, 5%. Culture under CO 2 conditions.
  • mice After S-180 cells were injected 2.5 x 10 6 / into the abdominal cavity of male Kunming mice, the animals were randomly divided into 2 groups (8/group). From the next day after injection, the control mice were orally and intraperitoneally injected with 0.1 ml of PBS. The mice in the drug-administered group were orally administered with 5 mg/kg of gluconate (CuGlu), and 4 hours later, intraperitoneal injection of disulfiram 5 mg/kg. Three times a week. The mice were weighed three times a week. After the end of the test, the animals were sacrificed by necking. All mice were dissected open to observe the formation of ascites and abdominal wall tumors. The liver, spleen, kidney, lung and other important organs were removed, embedded in paraffin, and sectioned for HE staining.
  • CuGlu gluconate
  • mice were generally in good condition. From the third day after the injection, the abdominal cavity of the control group gradually became larger, and it was a boat on the 7th and a spherical shape on the 11th (Fig. 4). One week after the injection of the cells, the body weight of the mice in the administration group was significantly lower than that of the control group (Fig. 7). On the 11th day, the control group of mice was sacrificed. A large amount of bloody ascites was observed in the laparotomy, about 10 ml/only. ( Figure 6). The masses were clearly visible in the abdominal wall and mesentery, 5-7 per case, and the diameter was 0.2-1 cm (Figs. 5 and 8). HE staining confirmed these tumors as tumor tissue (Fig. 10).
  • mice in the administration group were sacrificed, and ascites and mass formation were not observed in the laparotomy (Fig. 9). No obvious lesions were found in vital organs such as liver, spleen, kidney and lung (Fig. 11).
  • MSTO mesothelioma cells and SKVO-3 ovarian cancer cells were cultured in DMEM containing 10% fetal calf serum supplemented with 10 mg/ml of streptomycin and 2 mmol/L L-glutamine at 37 ° C, 5 Culture under % CO 2 conditions.
  • mice in the control group were intraperitoneally injected with 0.1 ml of PBS.
  • the mice in the drug-administered group were orally administered with 5 mg/kg of gluconate (CuGlu), and 4 hours later, 40 ⁇ g/dithione was injected intraperitoneally. Three times a week. The mice were weighed three times a week. After the end of the test, the animals were sacrificed by necking. All mice were dissected open to observe the formation of ascites and abdominal wall tumors.
  • mice After injection of cell mesothelioma and ovarian cancer cells, the control mice died on days 60 and 30, respectively (Fig. 12, 17), and a large amount of ascites was seen in the laparotomy (Fig. 13, 18). A large number of masses were clearly visible in the abdominal wall, mesentery and liver (Figs. 14, 15, 19, 20). The survival rates of mice in the mesothelioma and ovarian cancer treatment groups on days 90 and 60 were 60% and 90%, respectively (Fig. 12, 17). Ascites was not seen in the laparotomy, and only a small amount of mass was formed ( Figures 13-15, 18-20).
  • Malignant thoracic peritoneal mesothelioma is the most common type of pleural primary tumor. Clinical manifestations are associated with invasive behavior, which usually locally invades the thoracic cavity and surrounding structures. If left untreated, the median survival is 4 to 12 months. At present, there is no effective treatment for malignant thoracic and peritoneal mesothelioma in the world. Chest and peritoneal metastases have previously been considered the terminal stage of cancer. The most common thoracic and peritoneal metastases include lung cancer, breast cancer, ovarian cancer, pancreatic cancer, liver cancer, stomach cancer, colorectal cancer, and any other cancer that metastasizes to the thoracic and peritoneum. Supportive care, systemic chemotherapy, and palliative surgery are difficult to improve patient survival.
  • Disulfiram thoracic intraperitoneal treatment is very likely to be a curative means of thoracic peritoneal mesothelioma.
  • intraperitoneal application of disulfiram can inhibit abdominal metastasis and ascites formation in cancer. It can effectively relieve symptoms and prolong survival.
  • the currently internationally recognized animal model of abdominal metastatic cancer is the injection of S180 into the ascites tumor cell with extremely high malignancy. The abdominal cavity of the mouse causes the mice to produce ascites and tumor nodules.
  • mice in the drug-administered group received no ascites and tumor nodules in the intraperitoneal administration group, and no toxic effects were observed in the main organs. This exciting result prompted the inventors to further implement treatment experiments for intraperitoneal mesothelioma and ovarian cancer.
  • Example 3 the inventors used only a dose of 1/20 (2 mg/kg) of the disulfiram anti-alcohol dose, allowing 60% and 90% of the peritoneal mesothelioma and ovarian cancer mice to survive for a long time (90). And 60 days).
  • the inventors administered the mice 3 weeks after inoculation of mesothelial cancer cells in the peritoneal cavity of the mouse, and the tumor-bearing mice had produced ascites.
  • Disulfiram was discontinued after only 6 weeks (12 times) and continued for 4 weeks. 60% of mice survived for a long time (90 days). Therefore, it can be reasoned that the intraperitoneal administration of disulfiram may be a curative means of malignant thoracic and mesenteric carcinoma.
  • disulfiram has no systemic toxicity to humans and animals, it can be used for a long time. It can be concluded that the intraperitoneal injection of disulfiram can significantly inhibit the formation of metastatic carcinoma of the thoracic and peritoneal cavity, prolong the survival of animals and cancer patients and improve the quality of life. Because copper, zinc and other divalent transition metal ions and disulfiram can form DDC copper, zinc and other divalent transition metal ion derivatives (DDC-Cu, DDC-Zn, etc.) in the thoracic and peritoneal cavity, DDC-Cu is A very stable compound that can be absorbed into the bloodstream through the peritoneum and has a therapeutic effect on the primary cancer. Therefore, disulfiram is a potential drug candidate for anti-thoracic peritoneal metastases.
  • DDC-Cu zinc and other divalent transition metal ion derivatives
  • Synergy index CI 0.9-1.1: additive effect; 0.8-0.9: light; 0.6-0.8: medium; 0.4-0.6: synergistic; 0.2-0.4: strong.
  • Synergy index CI 0.9-1.1: additive effect; 0.8-0.9: light; 0.6-0.8: medium; 0.4-0.6: synergistic; 0.2-0.4: strong.
  • Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease. Cancer Ther, 3, 1049-60.
  • Disulfiram a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res, 66, 10425-33.
  • Liposome encapsulated Disulfiram inhibits NF ⁇ B pathway and targets breast cancer stem cells in vitro and in vivo. Oncotarget, 5, 7471-85.

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Abstract

本发明提供了治疗胸腹腔癌症的方法，包括对需要治疗的受试者胸膜内和/或腹膜内给予治疗有效量的双硫仑或其衍生物。本发明还提供了包含双硫仑或其衍生物的药物组合物和试剂盒。

Description

局部注射双硫仑制剂治疗胸腹膜癌症的方法 技术领域
本发明涉及癌症治疗领域，具体而言，涉及通过局部注射双硫仑制剂治疗胸腹膜原位及转移癌症的方法。
背景技术
双硫仑(disulfiram)用于戒酒的治疗超过60年(Johansson,1992)。双硫仑对乙醇敏感，病人在治疗期间即使摄取很少量的乙醇，也会产生不适的反应。双硫仑抑制乙醛脱氢酶[Aldehyde Dehydrogenase(ALDH)]活性，阻断了乙醇在乙醛阶段的氧化。因而摄入双硫仑后，人体血液中乙醛的浓度比饮用等量乙醇但未服用双硫仑的受试者乙醛的浓度高5至10倍。乙醛在血液中的蓄积会产生极度不适症状，称为双硫仑-乙醇样反应，其程度与摄入的双硫仑和乙醇的量均成正比，这种反应会一直持续到乙醇被完全代谢。双硫仑即使与很少量的乙醇同服，也会产生面部潮红、头颈部血管剧烈搏动、搏动性头痛、呼吸困难、恶心、呕吐、出汗、口干、胸痛、心悸、过度换气、心动过速、低血压、晕厥、躁动不安、乏力、眩晕、视力模糊和恍惚等症状。
体外实验证明，双硫仑对多种癌症细胞都具有极强的细胞毒性(Wang et al.,2003,Cen et al.,2004,Liu et al.,2012,Yip et al.,2011,Liu et al.,2014)。双硫仑对肿瘤细胞的细胞毒性作用有效浓度处于纳摩尔水平，比许多临床使用的抗癌药物(如柔红霉素，5-氟尿嘧啶，吉西他滨，紫杉醇，铂类药物，长春碱类等)具有更加显著的体外抗癌效应。尽管体外实验结果很令人振奋，但是鲜有研究报道双硫仑在动物实验中的抗癌活性。况且，目前所有原型双硫仑药物动物抗癌实验均需给予小鼠极高剂量的双硫仑后方能取得有限的抗癌效应(Iljin et al.,2009,Brar et al.,2004,Chen et al.,2006)。所用剂量对重要脏器(肺，肝，肾等)均会造成损伤。故临床癌症患者是不可耐受的。目前有几项已经完成或正在进行的使用双硫仑治疗癌症的临床试验[(Stewart et al.,1987,Verma et al.,1990)ClinicalTrials.gov:NCT00256230,NCT00742911,NCT01118741]，迄今尚未有积极成果的报道。由体外、体内和临床数据之间的差异导出下述结论：口服双硫仑制剂不适合用于人类癌症的治疗。
因此，截至目前，双硫仑未作为抗癌药物用于人体，这是因为双硫仑在癌症治疗方面的潜在临床应用受到现有双硫仑口服制剂的制约。口服给药后，双硫仑在胃酸中极不稳定，并且大部分口服的双硫仑快速降解成二硫化碳(CS2)和二乙胺(DEA)(Johansson,1992)。吸收的双硫仑在血流中与血清白蛋白的巯基反应，并被血液红细胞中谷胱甘肽还原酶系统迅速还原(半衰期：4分钟)，迅速转化为二乙基二硫代氨基甲酸(diethyldithiocarbamate或diethyldithiocarbamic acid，DDC)。绝大部分经口 服吸收的双硫仑及其衍生物可被肝脏富集。DDC及双硫仑分子中的巯基在肝脏中被迅速的甲基化或葡萄糖醛酸化形成甲基DDC及葡萄糖醛酸DDC(Agarwal et al.,1983,Agarwal et al.,1986,Gessner and Jakubowski,1972,Kaslander,1963,Prickett and Johnston,1953)。我们尚未发表的数据证实二者均完全丧失抗癌活性。因此，口服剂量为500mg的双硫仑，其血药浓度低于检测极限。上述反应并不影响双硫仑的戒酒效应。因为肝脏为酒精代谢器官，口服的双硫仑迅速甲基化并聚集于肝脏，甲基化双硫仑对ALDH具有极强的抑制效应。我们的实验证明双硫仑作为二价金属离子螯合剂，在与铜离子及其它二价金属离子螯合时产生大量的活性氧类(Reactive Oxygen Species,ROS)。ROS对癌细胞具强大的杀伤作用。然而ROS在体液中寿命极短(以纳秒计)。所以双硫仑与铜离子必须在癌组织内产生螯合反应，方能对癌细胞起杀伤作用(Tawari,2015)。双硫仑与铜离子形成的终产物二乙基二硫代铜亦对癌细胞具有杀伤作用。无论是螯合反应本身或终产物的形成，双硫仑分子中的巯基(-SH)均是不可或缺的。而在甲基化及葡萄糖醛酸化过程中，双硫仑的巯基均被破坏，因而丧失产生活性氧及其二乙基二硫代铜的能力。我们尚未发表的实验数据证明甲基化双硫仑完全丧失其抗癌活性。双硫仑在血液中迅速代谢及降解对双硫仑的临床抗癌应用提出了严峻的挑战，同时解释了临床实验未获理想结果的原因(http://www.clinicaltrials.gov/)。体外细胞毒性实验和患者抗癌疗效的差异表明由胃肠道系统给药的双硫仑由于在胃肠道及血液中快速转化降解、经肝首过效应和肝内转化降解，阻止了治疗量的药物到达癌症部位，因此成为临床使用双硫仑进行癌症治疗的主要限制因素。
发明内容
鉴于双硫仑体外实验具有极强的抗癌活性，而其超短的血内半衰期严重限制其临床抗癌应用，在本发明中我们通过局部给药将双硫仑用于胸腹膜癌症的局部治疗，从而克服了双硫仑体内半衰期短的瓶颈。
本发明提供了治疗胸腹膜原发及转移癌症的方法，包括对需要治疗的受试者胸腹膜腔内给予治疗有效量的双硫仑或其衍生物。
本发明还提供了双硫仑或其衍生物在制备用于通过胸腹膜内给药治疗受试者胸腹膜癌症的药物中的用途。
本发明还提供了双硫仑或其衍生物的有效给药制剂，用于通过胸腹膜内给药治疗受试者胸腹膜癌症。
本发明还提供了用于通过胸腹膜内给药治疗受试者胸腹膜癌症的双硫仑或其衍生物。
在特别优选的实施方案中，本发明还提供了作为活性成分的双硫仑或其衍生物 (如DDC)与铜、锌、铁、金及其它二价过渡金属元素的联合及非联合用药方法。例如，在本发明的具体实施方案中，双硫仑或其衍生物(如DDC)可以为胸腹膜腔给药；铜、锌、铁、金及其它二价过渡金属元素可以为口服或胸腹腔错时或同时给药，用于治疗受试者胸腹膜癌症。
本发明还提供了双硫仑或其衍生物(如DDC)与铜、锌、铁、金及其它二价过渡金属元素的联合及非联合用药并与现有抗癌药物组合应用，用于胸腹膜癌症治疗，藉此提高现有抗癌药物的抗癌活性，和/或减轻现有抗癌药物的毒副作用。
本发明还提供了试剂盒，包含：(1)作为治疗剂的双硫仑或其衍生物；以及(2)使用说明，所述使用说明中指明双硫仑或其衍生物用于通过胸腹膜腔内给药治疗受试者胸腹膜癌症。
附图说明
图1显示间皮瘤细胞被抑制情况。MSTO、Ju77、2591、E58细胞分别与培美曲塞(PMTX)、DS/Cu、以及两者共同孵育3天后的存活情况。DS/Cu和两者共同孵育情况下，对间皮瘤细胞抑制作用显著高于培美曲塞,亦高于DS/Cu。
图2显示间皮瘤细胞被抑制情况。MSTO、Ju77、2591、E58细胞分别与顺铂、DS/Cu、以及两者共同孵育3天后的存活情况。DS/Cu和两者共同孵育情况下，对间皮瘤细胞抑制作用显著高于顺铂,亦高于DS/Cu。
图3显示卵巢癌细胞被抑制情况。A2780、SKOV-3细胞分别与顺铂、DS/Cu、以及两者共同孵育3天后的存活情况。DS/Cu和两者共同孵育情况下，对卵巢癌细胞抑制作用显著高于顺铂,亦高于DS/Cu。
图4显示小鼠总体情况：BalB/C小鼠腹腔注射S-180腹水瘤细胞后，试验过程中，小鼠一般状态良好。从注射后第3日起，对照组小鼠腹腔逐渐变大，7日时呈舟状、11日时呈球状。对照组于11天处死。治疗组连续观察21天后处死。图示对照组腹部膨隆，治疗组体态正常。
图5显示腹膜癌结节。BalB/C小鼠腹腔注射腹水瘤细胞后，对照组第11天腹腔内可见多发癌结节。腹腔内腹壁和肠系膜上清晰可见肿块5-7个/只,直径为0.2-1厘米。给药组于第21天处死，小鼠腹腔干净，无癌结节可见。
图6：BalB/C小鼠腹腔注射腹水瘤细胞后，第11日，处死对照组小鼠,剖腹可见大量血性腹水，约10毫升/只。图示对照组腹水性状。给药组腹腔完全正常，无腹水。
图7：小鼠体重变化曲线。BalB/C小鼠腹腔注射腹水瘤细胞后，对照组因癌症腹水形成，小鼠体重迅速增加。治疗组在初始阶段体重稍有增加，与癌症腹腔内种植生长有关。治疗4天后治疗组体重增长趋坪，提示腹腔内癌症增殖受到药物控制。14天由于药物效应，癌症细胞被彻底杀灭，小鼠体重回落，21天恢复正常体重。
图8：癌结节数目比较。BalB/C小鼠腹腔注射腹水瘤细胞后，对照组(第11天)每鼠腹腔平均癌结节数目显著(p<0.01)多于治疗组(21天，0/鼠)。
图9：腹水量比较。BalB/C小鼠腹腔注射腹水瘤细胞后，对照组(第11天)每鼠腹腔平均腹水量(10毫升)显著(p<0.01)多于治疗组(21天，0毫升/鼠)。
图10：癌细胞腹腔内侵犯比较(100倍放大)。BalB/C小鼠腹腔注射腹水瘤细胞后，对照组(第11天)癌细胞侵犯浆膜下肌层。蓝色箭头：癌症组织；红色箭头：肌肉组织。每鼠腹腔平均腹水量(10ml)显著(p<0.01)多于治疗组(21天，0ml/鼠)。治疗组肌肉组织完全正常，无癌细胞。
图11：重要脏器显微形态学表现。BalB/C小鼠腹腔注射腹水瘤细胞后，对照(11天)及治疗组(21天)主要脏器(肺，肾，肝)未见毒性损伤(100倍放大)。
图12：显示小鼠生存情况。CD1裸鼠腹腔注射间皮瘤细胞MSTO后，至第60天，对照组小鼠全部死亡。至第90天，DS/Cu治疗组小鼠仍然存活60％。差异显著(p＝0.016)。
图13：腹水量比较。CD1裸鼠腹腔注射间皮瘤细胞MSTO后，至第60天，对照组小鼠腹水明显，而DS/Cu治疗组小鼠未见明显腹水(90天)。
图14：癌结节数目比较。CD1裸鼠腹腔注射间皮瘤细胞MSTO后，至第60天，对照组平均癌结节数目显著多于DS/Cu治疗组(90天)。p<0.01。
图15：显示腹膜癌结节。CD1裸鼠腹腔注射间皮瘤细胞MSTO后，至第60天，对照组可见大量癌结节。腹腔内腹壁、肝脏和肠系膜上清晰可见多发肿块(箭头所指)。DS/Cu治疗组小鼠腹腔干净，几乎无癌结节可见。
图16:癌结节病理照片。A.肠系膜侵犯。B.腹腔内结节。C.肝脏侵犯。D.高倍视野。
图17：显示小鼠生存情况。CD1裸鼠腹腔注射卵巢癌细胞后，至第30天，对照组小鼠全部死亡。至第60天，DS/Cu治疗组小鼠仍然存活90％。P＝0.0001,n＝10
图18：腹水量比较。CD1裸鼠腹腔注射卵巢癌细胞后，至第30天，对照组小鼠腹水明显，而DS/Cu治疗组(60天)小鼠未见明显腹水。
图19：癌结节数目比较。CD1裸鼠腹腔注射卵巢癌细胞后，至第30天，对照组可见多数癌结节。至第60天，DS/Cu治疗组小鼠腹腔内结节明显少于对照组。P<0.01,n＝10
图20：显示腹膜癌结节。CD1裸鼠腹腔注射卵巢癌细胞后，至第30天，对照组可见大量癌结节。腹腔内腹壁和肠系膜上清晰可见多发肿块。DS/Cu治疗组小鼠腹腔干净，癌结节较少(60天)。
具体实施方式
发明人通过实验发现，双硫仑在体外对人恶性间皮癌及卵巢癌细胞系有极强的杀伤效应，而且双硫仑在体外可协同性地增强培美曲塞及顺铂对人恶性间皮癌及卵巢癌细胞系的杀伤效应。考虑到胸腹膜腔局部应用可克服双硫仑血内半衰期短的瓶颈，发明人对S180肉瘤腹腔荷瘤小鼠实施了双硫仑(5mg/kg)腹膜腔内注射，同时口服葡萄糖酸铜，取得了对腹膜转移癌的彻底清除效应。此剂量仅约为双硫仑抗酗酒剂量(250mg/人/天；人:鼠＝1:9)的1/9。此令人振奋的结果促使发明人进一步实施了人腹膜腔内间皮癌及卵巢癌的治疗实验。在进一步的实验中，发明人仅用了双硫仑抗酗酒剂量的1/20(2mg/kg)的剂量，使60％及90％的腹膜间皮癌及卵巢癌小鼠得以长期存活(90及60天)。
考虑到过往研究的结果，这一发现是极其令人振奋的。
不受任何特定理论的束缚，发明人认为，这可能是由于腹腔内注射双硫仑时，可以将双硫仑直接递送到肿瘤组织附近，从而避免了采用其他给药方式例如口服及静脉注射时，双硫仑在胃肠道系统和血液循环过程中的快速代谢及降解的弊端，而且这也有利于双硫仑与口服后通过血循环渗入腹腔液中的二价铜离子在癌细胞附近迅速发生作用，对腹腔内癌细胞产生杀伤效应。
因此，本发明提供了治疗胸腹膜癌症的方法，包括对需要治疗的受试者胸膜内和/或腹膜内给予治疗有效量的双硫仑或其衍生物。
此处所用的术语“胸膜内给药”(intrapleural administration)，也称“胸膜腔内给药”或“胸腔内给药”，是指将药物穿过胸膜递送至胸膜腔内。
此处所用的术语“腹膜内给药”(intraperitoneal administration)，也称“腹膜腔内给药”、“腹腔内给药”或“腹腔给药”，是指将药物穿过腹膜递送至腹膜腔内。
此处所用的术语“胸腹膜内给药”是指将药物穿过胸膜或腹膜递送至胸膜腔或腹膜腔内。本领域技术人员可以理解，在某些情况下，例如当胸膜和腹膜上均存在待治疗的癌症时，可以同时进行胸膜内和腹膜内给药。术语“胸腹膜内给药”也涵盖同时给药至胸膜腔和腹膜腔的情形。
“胸腹膜内给药”与“胸膜内和/或腹膜内给药”具有相同的含义。
在被递送至胸腔或腹腔内之后，双硫仑或其衍生物可以通过胸腔积液或腹腔积液迅速扩散至肿瘤组织，从而发挥抗癌作用。
可以通过注射器、蠕动泵或胸腹腔导管方便地进行胸腹膜内给药。
此处所用的术语“癌症”(cancer)包括任何适合的癌症，例如恶性间皮瘤、胃癌、肾癌、膀胱癌、卵巢癌、乳腺癌、子宫内膜癌、胰腺癌、淋巴癌、甲状腺癌、骨癌、中枢神经系统癌症、白血病、肝癌、前列腺癌、肺癌、结肠癌、直肠癌、脑癌、黑色素瘤及目前已发现或未报道的人类及动物恶性肿瘤。
此处所用的术语“胸腹膜癌症”(pleural and/or peritoneal carcinoma)是指生长于患者胸膜和/或腹膜上的恶性肿瘤，包括原发性胸腹膜癌症以及胸腹膜转移癌。
此处所提到的原发性胸腹膜癌症包括恶性间皮瘤及其它任何原发于胸腹膜的恶性肿瘤。
本发明的方法特别适用于恶性间皮瘤的治疗。
恶性间皮瘤(malignant mesothelioma)是罕见肿瘤，多发生于胸膜和腹膜，其发病多与石棉密切相关。
恶性胸膜间皮瘤是胸膜原发肿瘤中最多见的类型。临床表现与侵袭行为有关，它通常局部侵袭胸膜腔及周围结构。如果不治疗，中位生存期4～12个月。
此处所提到的胸腹膜转移癌是指由机体其它部位转移至胸膜和/或腹膜的癌症。主要包括胃癌、卵巢癌、胰腺癌、淋巴癌、白血病、肝癌、肺癌、结直肠癌等及其它任何转移性癌症。
本发明的方法特别适合于腹膜转移癌的治疗。最常见的腹膜转移癌包括卵巢癌、胰腺癌、肝癌、胃癌、结直肠癌等及其它任何转移于腹膜的癌症。
腹膜转移癌以往被视为癌症的终末阶段。支持性护理、全身化疗以及姑息性手术均难以改善患者的生存期。
由于发病器官位置的原因，胃癌、卵巢癌、胰腺癌、结肠癌的腹膜转移率分别为99％、90％、50％和32％。此外，其它器官的癌症也可发生远处转移，播散到腹膜，如乳腺癌、肺癌和淋巴瘤等。
本发明的药物制剂可以用于阻止和/或抑制肿瘤细胞和/或肿瘤干细胞的增殖。癌症治疗方法对肿瘤干细胞无效被认为是导致很多癌症治疗失败的原因。
本文所用术语“治疗”指为抵御疾病或紊乱状态而对患者进行的管理和照料。本术语包括针对特定状况而进行的广谱治疗，例如给予活性化合物以减轻症状或并发症，延缓疾病的进程，缓和或减轻症状和并发症，治愈或消除疾病或不良状态，也包括预防所述疾病或不良状态。被治疗的受试者优选为哺乳动物，尤其是人类，但也包括动物，如狗、猫、马、牛、羊和猪。
双硫仑和其衍生物
本文所用术语“双硫仑”(disulfiram，简称DS)的化学结构式为：
Figure PCTCN2017080463-appb-000001
双硫仑的衍生物可以用以下化学结构式表示：
Figure PCTCN2017080463-appb-000002
其基本结构为秋兰姆二硫化物。秋兰姆二硫化物中的R1、R2、R3和R4可以相同或不同。在上述通式中R1、R2、R3和R4各自独立地选自氢、烷基、烯基、炔基、烷氧基、芳基和杂芳基。在具体实施方式中，所述烷基可以是直链、支链或环状的C1-C10烷基，例如C1-C8烷基，例如C1-C6烷基，例如C1-C4烷基，例如甲基、乙基、丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、环丙基、环丁基、环戊基、环己基、环庚基。烷基可包括环烷基及杂环烷基，例如环丙基、环丁基等。在具体实施方式中，所述烯基可以是C2-C10烯基，例如C2-C8烯基，例如C2-C6烯基，例如C2-C4烯基，例如乙烯基、丙烯基或丁烯基。在具体实施方式中，所述炔基可以是C2-C10炔基，例如C2-C8炔基，例如C2-C6炔基，例如C2-C4炔基，例如乙炔基、丙炔基或丁炔基。在具体实施方式中，所述烷氧基可以是C2-C10烷氧基，例如C2-C8烷氧基，例如C2-C6烷氧基，例如C2-C4烷氧基，例如甲氧基、乙氧基、丙氧基、正丁氧基、异丁氧基或叔丁氧基。在具体实施方式中芳基可以是C6-C14单环或双环芳香性基团，例如苯基、蒽基等，其还可以任选地地进一步具有取代基，例如苄基等。在具体实施方式中，所述杂芳基是含有一个或两个独立地选自氮、氧或硫的杂原子的芳香性基团，例如呋喃基、噻吩基、吡咯基、噻唑基、咪唑基、吡啶基、吡嗪基、嘧啶基或哒嗪基。在具体实施方式中，R1、R2与N及R3、R4与N可形成含氮杂环烷烃或杂环芳香烃。在一些实施方式中，所述R1、R2、R3和R4各自独立地选自咪唑啉基、二甲亚基、双酚基、环丙基甲基、五亚甲基、羟乙基或甲基葡萄糖胺基。在双硫仑中，R1、R2、R3和R4都是乙基。
术语“双硫仑的衍生物”包括以上通式的化合物及其代谢产物，及其与钠离子、钾离子以及铵离子等形成的盐，或者与铜、锌、铁、金及其它二价过渡金属元素的螯合产物，如DDC-Cu、DDC-Zn等。
在本发明的具体实施方案中，“双硫仑的衍生物”包括双硫仑代谢产物二乙基二硫代物，例如二乙基二硫代氨基甲酸(DDC)，及其与钠离子、钾离子以及铵离子等形成的盐，或者与铜、锌、铁、金及其它二价过渡金属元素的螯合产物，如DDC-Cu、DDC-Zn等。
双硫仑衍生物的典型例子包括：二乙基二硫代氨基甲酸(diethyldithiocarbamate或diethyldithiocarbamic acid，DDC)、二乙基二硫代氨基甲酸钠盐(sodium  diethyldithiocarbamate，DDC-Na)、二乙基二硫代氨基甲酸铵盐(ammonium diethyldithiocarbamate，DDC-NH3)、二乙基二硫代氨基甲酸铜、二乙基二硫代氨基甲酸锌，以及二乙基二硫代氨基甲酸与其它金属离子形成的盐类或者螯合物。
与二价过渡金属制剂联合给药
出于本发明的目的，双硫仑或其衍生物可以单独给药以治疗胸腹膜癌症，也可以与二价过渡金属制剂联合给药。
根据本发明的优选实施方案，本发明的包含双硫仑或其衍生物的药物制剂(也可称为“本发明的药物制剂”)可以与二价过渡金属制剂例如含铜制剂联合使用，从而进一步增强抗癌作用。两制剂可以是单独、同时或循序给药。例如，可以腹膜内给予双硫仑，并口服含铜制剂，例如葡萄糖酸铜。也可以通过腹膜内注射同时或先后给予双硫仑和含铜制剂。临床医师可以根据患者的病情、身体状况、对药物的反应等因素确定双硫仑和含铜制剂的剂量和给药方式。
此处的“二价过渡金属制剂”指的是含有二价过渡金属元素的物质或制剂。此处所述的“二价过渡金属”包括铜、锌、铁、金及其它二价过渡金属元素。二价过渡金属制剂的典型例子是含铜制剂和含锌制剂。特别适合的含铜制剂的例子包括葡萄糖酸铜、柠檬酸铜、氯化铜以及其他可供病人口服的有机及无机铜制剂。特别适合的含锌制剂的例子包括葡萄糖酸锌、氯化锌、柠檬酸锌以及其他可供病人口服的有机及无机锌制剂。
联合治疗
许多疾病可以使用多于一种药物联合治疗，或同时使用，或循序使用。因此，本发明也包括利用本发明的药物制剂与其他常规用于癌症治疗的活性物质联合使用用于癌症治疗。
例如，本发明的制剂可与手术或放射治疗联合使用，和/或与一种或多种药物联合治疗，例如陪美曲塞、5-氟尿嘧啶、阿霉素、紫杉醇和吉西他滨。
药物组合物
本发明提供了药物组合物或药物制剂，包括作为活性成分的双硫仑或其衍生物，以及可药用载体。本发明的药物组合物可以用于通过胸腹膜内给药治疗受试者胸腹膜癌症。
本发明的药物组合物可以利用常规的可药用载体或者填充剂以及其他辅料，例如环糊精、蔗糖、明胶、硬脂酸镁、橄榄油、磷脂等。参见例如Remington:The Science and Practice of Pharmacy,19th Edition,Gennaro,Ed.,Mack Publishing Co.， Easton,PA,1995。
本发明化合物可制备成各种合适的剂型，例如液体注射剂、注射用粉剂、注射用片剂，或者包裹于脂质体或其它纳米赋形剂中。
一般情况下，根据所采用的给药方式，制剂包含的活性成分质量分数约0.005％至95％，优选约0.5％至50％。制剂中的活性化合物的百分比取决于制剂的具体性质，也取决于化合物的活性和患者的需求。溶液中活性成分的比例通常采用是0.01％至90％，同时如果该制剂是经固体稀释得到的，其含量可以更高。在一些实施例中，该液体制剂中含有1％到50％活性化合物。
治疗有效量
本文所用的术语“治疗有效量”指的是足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。双硫仑或其衍生物的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。
在一个优选的实施例中，该制剂将采取单位剂量形式。例如，该制剂可放置在小瓶或其它容器中。小瓶或其它容器可以含有液体，将被稀释成混悬液的固体，干燥粉末，冻干物，或任何其它合适形式的制剂。
在一些实施方案中，双硫仑或其衍生物的给药剂量可以为大约250-大约500毫克/天(酗酒患者的双硫仑耐受剂量为250至500毫克/天)。患者优选同时服用2mg铜，如葡萄糖酸铜。葡萄糖酸铜优选通过口服使用。
在一些实施方案中，双硫仑或其衍生物给药剂量范围可以是大约1mg/kg～10mg/kg，优选大约1mg/kg～20mg/kg。双硫仑剂量亦可根据腹水量按每升腹水10微摩尔(2960微克/升)双硫仑计算。如病人腹水量为10升，双硫仑用量则约为30毫克。
试剂盒
本发明另一方面提供了试剂盒，包含：(1)作为治疗剂的双硫仑或其衍生物；以及(2)使用说明，所述使用说明中指明所述双硫仑或其衍生物用于通过胸腹膜内给药治疗受试者胸腹膜癌症。
本发明的试剂盒特别适合于胸腹膜转移癌的治疗。
在试剂盒中，双硫仑或其衍生物可以以单位剂型的形式存在，其单位剂量可以是10毫克/支(例如10毫升)。双硫仑或其衍生物的剂量可以为1至10mg/kg/天。
本发明的试剂盒还可以包括任选的二价过渡金属制剂。
本文所用的术语“任选”(optional)是指“可以有也可以没有”或“非必需”的含义。例如，“任选的铜制剂”是指可以包含铜制剂，也可以不包含，这可以由本领域技术人员根据实际情况进行选择。
下面将参照附图和实施例对本发明进行进一步描述，附图和实施例只是出于解释本发明的目的，不应当被理解为是对本发明的限制。
实施例1
材料和方法
MSTO、Ju77、2591、E58间皮瘤细胞由伦敦Queen Mary医学院Peter Szlosarek教授惠赠，培养于含10％胎牛血清并添加10mg/ml的青链霉素和2mmol/L L-谷氨酰胺的DMEM培养液中，在37℃，5％CO2条件下培养。分别与培美曲塞、DS/Cu(双硫仑/葡萄糖酸铜)、顺铂共同孵育3天。MTT法测定细胞存活情况。
结果
DS/Cu对间皮瘤细胞的抑制情况明显强于培美曲塞和顺铂，与两者联用效果更佳(图1、2，表1、2)。
DS/Cu对人卵巢癌细胞的抑制情况亦明显强于顺铂，与顺铂联用有显著的协同效应(图3，表3、4)。
实施例2
材料和方法
S180腹水瘤细胞购于中科院上海细胞库，培养于含10％胎牛血清并添加10mg/ml的青链霉素和2mmol/L L-谷氨酰胺的DMEM培养液中，在37℃，5％CO2条件下培养。
动物实验
S-180细胞2.5x 106/只注射入雄性昆明小鼠腹腔后，动物被随机分为2组(8只/组)。注射次日起，对照组小鼠分别口服和腹腔注射PBS 0.1、1毫升，给药组小鼠口服给予葡萄糖酸铜(CuGlu)5mg/kg，4小时后腹腔注射双硫仑5mg/kg。每周给药三次。每周称量小鼠体重三次。试验结束后脱颈处死动物。所有小鼠均剖开腹腔观察腹水形成、腹壁肿瘤情况。摘取肝、脾、肾、肺等重要脏器，固定后石蜡包埋，切片做HE染色。
结果
试验过程中，小鼠一般状态良好。从注射后第3日起，对照组小鼠腹腔逐渐变大，7日时呈舟状、11日时呈球状(图4)。注射细胞1周后，给药组小鼠体重显著低于对照组(图7)。第11日，处死对照组小鼠，剖腹可见大量血性腹水，约10毫升/只 (图6)。腹腔内腹壁和肠系膜上清晰可见肿块5-7个/只,直径为0.2-1cm(图5、8)。HE染色证明这些肿块为肿瘤组织(图10)。注射细胞3周后，处死给药组小鼠，剖腹未见腹水和肿块形成(图9)。肝、脾、肾、肺等重要脏器未见明显病变(图11)。
实施例3
材料和方法
MSTO间皮瘤细胞和SKVO-3卵巢癌细胞培养于含10％胎牛血清并添加10mg/ml的青链霉素和2mmol/L L-谷氨酰胺的DMEM培养液中，在37℃，5％CO2条件下培养。
动物实验
MSTO间皮瘤细胞、SKVO-3卵巢癌细胞3x106/只注射入雌性CD1裸鼠腹腔后，动物被随机分为2组(10只/组)。注射次日起，对照组小鼠腹腔注射PBS 0.1毫升，给药组小鼠口服给予葡萄糖酸铜(CuGlu)5mg/kg，4小时后腹腔注射双硫仑40μg/只。每周给药三次。每周称量小鼠体重三次。试验结束后脱颈处死动物。所有小鼠均剖开腹腔观察腹水形成、腹壁肿瘤情况。
结果
注射细胞间皮瘤和卵巢癌细胞后，对照组小鼠分别于第60天和第30天全部死亡(图12、17)，剖腹可见大量腹水(图13、18)。腹腔内腹壁、肠系膜和肝脏上清晰可见大量肿块(图14、15、19、20)。间皮瘤和卵巢癌治疗组小鼠于第90天和第60天生存率分别为60％和90％(图12、17)。剖腹未见腹水，仅有少量肿块形成(图13-15、18-20)。
讨论
恶性胸腹膜间皮癌是胸膜原发肿瘤中最多见的类型。临床表现与侵袭行为有关，它通常局部侵袭胸腹膜腔及周围结构。如果不治疗，中位生存期4～12个月。目前国际上对恶性胸腹膜间皮癌尚无有效治疗手段。胸腹膜转移癌以往被视为癌症的终末阶段。最常见的胸腹膜转移癌包括肺癌、乳腺癌、卵巢癌、胰腺癌、肝癌、胃癌、结直肠癌等及其它任何转移于胸腹膜的癌症。支持性护理、全身化疗以及姑息性手术均难以改善患者的生存期。
由于恶性胸腹膜间皮癌以局部侵袭性生长为主，较少远处转移。双硫仑胸腹膜腔内治疗极有可能成为胸腹膜间皮癌的治愈性手段。对于其它胸腹膜腔转移癌，双硫仑胸腹膜腔内应用可抑制癌症的腹腔转移和腹水形成。可有效缓解症状延长生存期。目前国际公认的腹腔转移癌动物模型是将恶性程度极高的腹水瘤细胞S180注射入 小鼠腹腔，使小鼠产生腹水和肿瘤结节。在本研究中，我们应用此模型评价DS对腹腔转移癌的治疗作用。未给药的对照组在注射肿瘤细胞2天后可见腹部逐渐增大，伴随体重快速增长，癌细胞接种后第11天处死动物剖腹观察可见明显肿瘤结节。给药组动物接受连续给药3周后，DS腹腔注射给药组小鼠无腹水和肿瘤结节形成，且对主要脏器未见毒性作用。此令人振奋的结果促使发明人进一步实施了腹膜腔内间皮癌及卵巢癌的治疗实验。在实施例3中，发明人仅用了双硫仑抗酗酒剂量的1/20(2mg/kg)的剂量，使60％及90％的腹膜间皮癌及卵巢癌小鼠得以长期存活(90及60天)。在皮癌实验中，发明人在小鼠腹膜腔内接种间皮癌细胞后3周给小鼠用药，此时荷瘤小鼠已产生腹水。双硫仑仅用了6周(12次)后终止用药，继续观察4周。60％小鼠获长期存活(90天)。由此可有理由推断双硫仑胸腹膜腔内用药可能成为恶性胸腹膜间皮癌的治愈性手段。
由于双硫仑对人畜不具系统性毒性，可长期用药。由此可以判定双硫仑胸腹膜腔注射可显著抑制胸腹膜腔转移癌形成，延长动物及癌症患者的生存期并改善生存质量。由于铜、锌及其它二价过渡金属离子与双硫仑可于胸腹膜腔内形成DDC铜、锌及其它二价过渡金属离子衍生物(DDC-Cu、DDC-Zn等)，DDC-Cu乃非常稳定的化合物，可经腹膜吸收入血并对原发癌起治疗作用。因此，双硫仑是极具开发潜力的抗胸腹膜腔转移癌的候选药物。
表1 顺铂(CDDP)、培美曲塞(PMTX)、以及联合应用DS+Cu抑制间皮瘤的IC50s(nM)
Figure PCTCN2017080463-appb-000003
表2 顺铂(CDDP)、培美曲塞(PMTX)与DS+Cu抑制间皮瘤的协同作用
Figure PCTCN2017080463-appb-000004
协同指数CI:0.9-1.1:相加效应；0.8-0.9:轻；0.6-0.8:中；0.4-0.6:协同；0.2-0.4:强。
表3 顺铂(CDDP)单独应用以及联合应用DS+Cu对卵巢癌细胞抑制作用的IC50s(nM)
Figure PCTCN2017080463-appb-000005
表4 顺铂(CDDP)与DS+Cu对卵巢癌细胞抑制的协同作用
Figure PCTCN2017080463-appb-000006
协同指数CI:0.9-1.1:相加效应；0.8-0.9:轻；0.6-0.8:中；0.4-0.6:协同；0.2-0.4:强。 参考文献
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Claims (26)

  1. 治疗胸腹膜癌症的方法，包括对需要治疗的受试者胸膜内和/或腹膜内给予治疗有效量的双硫仑或其衍生物。
  2. 权利要求1的方法，其中所述胸腹膜癌症为原发性胸腹膜癌症。
  3. 权利要求2的方法，其中所述原发性胸腹膜癌症为胸腔内和/或腹腔内的恶性间皮瘤。
  4. 权利要求1的方法，其中所述胸腹膜癌症为胸腹膜转移癌。
  5. 权利要求1-4之任一项的方法，其中所述双硫仑的衍生物选自由二乙基二硫代氨基甲酸、二乙基二硫代氨基甲酸钠、二乙基二硫代氨基甲酸铵、二乙基二硫代氨基甲酸铜、二乙基二硫代氨基甲酸锌构成的组。
  6. 权利要求1-5之任一项的方法，还包括同时或循序给予二价过渡金属制剂。
  7. 权利要求6的方法，其中所述二价过渡金属制剂为含铜制剂或含锌制剂。
  8. 权利要求7的方法，其中所述含铜制剂选自由葡萄糖酸铜、柠檬酸铜和氯化铜构成的组。
  9. 权利要求7的方法，其中所述含锌制剂选自由葡萄糖酸锌、氯化锌和柠檬酸锌构成的组。
  10. 权利要求1-9之任一项的方法，其中所述双硫仑或其衍生物的给药剂量为1mg/kg～大约10mg/kg。
  11. 权利要求1-10之任一项的方法，其中所述双硫仑或其衍生物与另外的抗癌药物联用以治疗所述受试者。
  12. 双硫仑或其衍生物在制备用于通过胸腹膜内给药治疗受试者胸腹膜癌症的药物中的用途。
  13. 权利要求12的用途，其中所述胸腹膜癌症为原发性胸腹膜癌症。
  14. 权利要求13的用途，其中所述原发性胸腹膜癌症为胸腔内和/或腹腔内的恶性间皮瘤。
  15. 权利要求12的用途，其中所述胸腹膜癌症为胸腹膜转移癌。
  16. 权利要求12-15之任一项的用途，其中所述双硫仑的衍生物选自由二乙基二硫代氨基甲酸、二乙基二硫代氨基甲酸钠、二乙基二硫代氨基甲酸铵、二乙基二硫代氨基甲酸铜、二乙基二硫代氨基甲酸锌构成的组。
  17. 试剂盒，包含：
    (1)作为治疗剂的双硫仑或其衍生物；以及
    (2)使用说明，所述使用说明中指明双硫仑或其衍生物用于通过胸腹膜内给药治疗受试者胸腹膜癌症。
  18. 权利要求17的试剂盒，其中所述胸腹膜癌症为原发性胸腹膜癌症。
  19. 权利要求18的试剂盒，其中所述原发性胸腹膜癌症为胸腔内和/或腹腔内的恶性间皮瘤。
  20. 权利要求17的试剂盒，其中所述胸腹膜癌症为胸腹膜转移癌。
  21. 权利要求17-20之任一项的试剂盒，其中所述双硫仑的衍生物选自由二乙基二硫代氨基甲酸、二乙基二硫代氨基甲酸钠、二乙基二硫代氨基甲酸铵、二乙基二硫代氨基甲酸铜、二乙基二硫代氨基甲酸锌构成的组。
  22. 权利要求17-21之任一项的试剂盒，其中所述试剂盒中还包括任选的二价过渡金属制剂。
  23. 权利要求22的试剂盒，其中所述二价过渡金属制剂为含铜制剂或含锌制剂。
  24. 权利要求23的试剂盒，其中所述含铜制剂选自由葡萄糖酸铜、柠檬酸铜和氯化铜构成的组。
  25. 权利要求23的试剂盒，其中所述含锌制剂选自由葡萄糖酸锌、氯化锌和柠檬酸锌构成的组。
  26. 权利要求17-25之任一项的试剂盒，其中所述双硫仑或其衍生物的给药剂量为1mg/kg～大约10mg/kg。
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