WO2017133599A1 - 肿瘤基因甲基化调节剂的新用途及抗肿瘤药物 - Google Patents

肿瘤基因甲基化调节剂的新用途及抗肿瘤药物 Download PDF

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WO2017133599A1
WO2017133599A1 PCT/CN2017/072505 CN2017072505W WO2017133599A1 WO 2017133599 A1 WO2017133599 A1 WO 2017133599A1 CN 2017072505 W CN2017072505 W CN 2017072505W WO 2017133599 A1 WO2017133599 A1 WO 2017133599A1
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tumor
antitumor
gene methylation
drug
active ingredients
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French (fr)
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覃扬
董庆
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覃扬
董庆
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Priority to US16/075,248 priority Critical patent/US11179349B2/en
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Publication of WO2017133599A1 publication Critical patent/WO2017133599A1/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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the invention belongs to the field of tumor treatment, and particularly relates to a novel use of a tumor gene methylation regulator and an antitumor drug.
  • DNA methylation When it is methylated, it often leads to gene silencing, which causes important genes such as tumor suppressor genes and DNA repair genes to lose their functions, resulting in normal Cell growth and differentiation regulation disorders and DNA damage cannot be repaired in time, which is related to the formation of a variety of tumors. DNA methylation is reversible. If DNA methylation regulators are used to demethylate some important genes and restore their normal functions, inhibition of DNA methyltransferase activity has become a new research and development strategy.
  • DNA methyltransferase inhibitors are 5-azacytidine nucleoside (Azacitidine, 5-Aza-CR) and 5-aza-2-deoxycytidine (5-aza-2-deoxycytidine, 5-Aza). -CdR). Their mechanism of action is thought to inhibit DNA methylation by replacing cytosine during DNA replication and inhibiting the activity of DNMT after forming a covalent bond with DNMT. They are widely used to study the biological processes of DNA methylation and to treat acute myeloid leukemia and myelodysplast syndrome (MDS). However, these two drugs limit their clinical application due to side effects such as mutagenicity, cytotoxicity and myelosuppression.
  • SAM S-adenosyl methionine
  • SAM S-adenosyl methionine
  • SAM is known to be involved in more than 40 metabolic reactions in vivo involving the transfer of methyl groups of S-adenosylmethionine to substrates such as nucleic acids, proteins and fats. In the United States, it has been sold under the name of SAM as a nutritional supplement, which has the effect of improving mood, maintaining the liver and comfortable joints.
  • Cordyceps is a traditional Chinese medicine produced in Cambodia, Qinghai, Sichuan, Yunnan and Gansu. The research on Cordyceps has been carried out for several years and has achieved a lot of results. It has been found that the main component of the grass contains 3'-deoxyadenosine (Cordycepin, CAS: 73-03-0).
  • 3'-deoxyadenosine is a novel broad-spectrum antibiotic. At present, some studies have been carried out on the pharmacology of 3'-deoxyadenosine in antibacterial, anti-inflammatory, anti-HIV-I viruses, selective inhibition of Clostridium, and immunomodulation. 3'-deoxyadenosine anti-tumor research Previously focused on anti-leukemia research, in the United States in 1997, 3'-deoxyadenosine was used in the treatment of acute pre-B and pre-T lymphocytic leukemia phase I clinical trial, but no Can carry out subsequent work.
  • the technical problem to be solved by the present invention is to overcome the defects of the existing tumor treatment technical solutions and provide a new and effective choice for tumor treatment.
  • the technical solution of the present invention to solve the technical problem is to provide a use of a tumor gene methylation regulator in the preparation of an antitumor drug.
  • tumor gene methylation regulators are shellac red pigment, 3'-deoxyadenosine, 5-azacytidine, genistein and [1-( ⁇ -D-furan) At least one of riboside-1,2-dihydropyrimidin-2-one] (zebularine).
  • 5-azacytidine 5-Azacytidin Molecular formula: C 8 H 12 N 4 O 5; molecular weight: 244.21 structural formula is as follows:
  • the above tumor is a hematoma or a solid tumor.
  • the hematoma is at least one of leukemia, multiple myeloma, glioma or malignant lymphoma.
  • the solid tumor described above is at least one of breast cancer, lung cancer, stomach cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, uterine cancer, kidney cancer, bladder cancer or skin cancer.
  • the antitumor drug described in the above application contains at least one other antitumor active ingredient in addition to the tumor gene methylation regulator.
  • the other antitumor active ingredients described in the above uses are 5-fluorouracil, capecitabine, gemcitabine, potassium oxonate (octacilin potassium), oltipraz, tegafur, paclitaxel, and more.
  • the other antitumor active ingredients described in the above uses are bevacizumab, aflibercept, pertuzumab, trastuzumab, At least one of a monoclonal antibody such as Cetuximab, Rituximab, Alemtuzumab or Panitumumab.
  • the other anti-tumor active ingredients described in the above uses and the tumor gene methylation regulator may be separately packaged separately.
  • the present invention provides an antitumor drug.
  • the anti-tumor drug is made of one of the main active ingredients of a tumor gene methylation regulator.
  • the tumor gene methylation regulator described in the above antitumor drug is a shellac red pigment, a class A anti-cancer gene methylation compound, genistein or [1-( ⁇ -D) At least one of - sulphur riboside-1,2-dihydropyrimidin-2-one] (zebularine).
  • the tumor described in the above antitumor drug is a hematoma or a solid tumor.
  • the hematoma described in the antitumor drug is at least one of leukemia, multiple myeloma, glioma or malignant lymphoma.
  • the solid tumor described in the antitumor drug is at least one of breast cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, colorectal cancer, liver cancer, prostate cancer, skin cancer, uterine cancer, renal cancer, and bladder cancer.
  • the antitumor drug further contains at least one other antitumor active ingredient.
  • the other anti-tumor active ingredients described in the above technical solutions are platinum anticancer drugs, anthracycline anticancer drugs, antimetabolite anticancer drugs, alkaloid anticancer drugs or targeted antibody anticancer drugs. At least one of them.
  • anthracycline anticancer drug is at least one of daunorubicin, doxorubicin, epirubicin, mitoxantrone or various pharmaceutically acceptable salts thereof.
  • the above-mentioned antimetabolite anticancer drug is at least one of pemetrexed, tegafur, capecitabine, gemcitabine, 5-fluorouracil or various pharmaceutically acceptable salts thereof.
  • the above platinum anticancer drug is at least one of cisplatin, carboplatin, oxaliplatin, nedaplatin or various pharmaceutically acceptable salts thereof.
  • the alkaloid anticancer drug described above is a taxane anticancer drug. Further, the taxane anticancer drug is at least one of paclitaxel, docetaxel, cephalosporin, 10-deacetylpaclitaxel, baccatin III or various pharmaceutically acceptable salts thereof.
  • the other antitumor active ingredients described in the above antitumor drugs are bevacizumab, aflibercept, pertuzumab, trastuzumab (Trastuzumab). And at least one of a monoclonal antibody such as Cetuximab, Rituximab, Alemtuzumab or Panitumumab.
  • the relative proportions of each type of tumor methylation regulator and other anti-tumor active ingredients and their respective specificities can be adjusted according to specific conditions and purposes. Dosage and method of administration.
  • the antitumor drug is prepared by adding a pharmaceutically acceptable excipient or an auxiliary component to the tumor gene methylation regulator as one of the main active ingredients.
  • the above-mentioned antitumor drug dosage form is an oral preparation, an injection preparation, a spray preparation or an intravenous drip preparation.
  • the auxiliary or auxiliary component described in the above antitumor drug includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, a protective agent, and adsorption.
  • a diluent an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, a protective agent, and adsorption.
  • a combination of one or more of a carrier or a lubricant includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, a protective agent, and adsorption.
  • the other antitumor active ingredient and the tumor gene methylation regulator described in the above antitumor drug may be a combination preparation.
  • the other antitumor active ingredients described in the above antitumor drugs and the tumor gene methylation regulator may be separately packaged separately.
  • the above-mentioned antitumor drug is in the form of an oral preparation or an injection.
  • the injection described in the above antitumor drug is an injection or a powder injection.
  • the present invention provides a method of treating at least one condition or symptom associated with tumor growth in a human.
  • the party comprises administering to a human a pharmaceutical composition comprising a tumor gene methylation modulator and administering at least one effective amount of an additional anti-tumor active ingredient.
  • the tumor gene methylation regulator described in the above method and other anti-tumor active ingredients may be administered to the patient together, or may be administered to the patient separately, as long as any one of the patients has a higher than normal physiological amount in the patient.
  • the administration of the blood concentration to the other can be considered as a combined use.
  • the dosage of the tumor gene methylation modulator administered by the above method is a dose effective to treat the condition or symptom associated with tumor growth in combination with at least one other anti-tumor active ingredient.
  • the tumor gene methylation regulator according to the above method is the tumor gene methylation regulator: shellac red pigment, genistein, 3'-deoxyadenosine, 5-aza cell At least one of glycosides or [1-( ⁇ -D-ribofuranoside)-1,2-dihydropyrimidin-2-one].
  • the other antitumor active ingredients described in the above method are 5-fluorouracil, gemcitabine, tegafur, capecitabine, potassium oxonate, oltipraz, paclitaxel, docetaxel, epirubicin At least one of a vegetarian, doxorubicin, arubicin, mitoxantrone, irinotecan, cisplatin, carboplatin, oxaliplatin, nedaplatin, and various salts thereof.
  • the other antitumor active ingredients described in the above methods are bevacizumab, aboxicept, pertuzumab, trastuzumab, cetuximab, rituximab, At least one of alemtuzumab or panitumumab.
  • the tumor described in the above method is a hematoma or a solid tumor.
  • the tumor gene methylation regulator described in the above method when administered simultaneously with other anti-tumor active ingredients, the patient is administered by the same or different administration routes.
  • the tumor gene methylation regulator described above is in the same preparation or in a different preparation when the same antibiotic active ingredient is administered to the patient simultaneously with the same administration route.
  • the present invention also provides a method for improving the efficacy of a tumor prevention and treatment drug or reducing the onset of a tumor prevention and treatment drug.
  • the tumor gene methylation regulator is used in combination with at least one effective amount of other anti-tumor active ingredients to increase the sensitivity of the tumor to other anti-tumor active ingredients, thereby improving the therapeutic effect or reducing the on-effect dosage.
  • the tumor gene methylation regulator described in the above method and other anti-tumor active ingredients may be administered to the patient together, or may be administered to the patient separately, as long as any one of the patients has a higher than normal physiological amount in the patient.
  • the administration of the blood concentration to the other can be considered as a combined use.
  • the dose of the tumor gene methylation regulator administered in the above method is a dose for increasing the effect of other antitumor active ingredients or reducing the effective amount of other antitumor active ingredients.
  • the tumor gene methylation regulator according to the above method is the tumor gene methylation regulator: shellac red pigment, genistein, 3'-deoxyadenosine, 5-aza cell Glycosides or [1-( ⁇ -D-ribofuranoside)-1,2-dihydrogen At least one of pyrimidin-2-one].
  • the other antitumor active ingredients described in the above method are 5-fluorouracil, gemcitabine, tegafur, capecitabine, potassium oxonate, oltipraz, paclitaxel, docetaxel, epirubicin At least one of a vegetarian, doxorubicin, arubicin, mitoxantrone, irinotecan, cisplatin, carboplatin, oxaliplatin, nedaplatin, and various salts thereof.
  • the other antitumor active ingredients described in the above methods are bevacizumab, aboxicept, pertuzumab, trastuzumab, cetuximab, rituximab, At least one of alemtuzumab or panitumumab.
  • the tumor described in the above method is a hematoma or a solid tumor.
  • the tumor gene methylation regulator described in the above method when administered simultaneously with other anti-tumor active ingredients, the patient is administered by the same or different administration routes.
  • the tumor gene methylation regulator described above is in the same preparation or in a different preparation when the same antibiotic active ingredient is administered to the patient simultaneously with the same administration route.
  • the present invention also provides a method for preparing the aforementioned antitumor drug.
  • the invention finds that the tumor gene methylation regulator is used as a sensitizer in combination with a tumor therapeutic drug, and can be used for preparing an anti-tumor drug.
  • the anti-tumor drug of the invention can significantly reduce the dosage of the tumor therapeutic drug while increasing the curative effect. Therefore, the side effects of the tumor treatment drug on the body can be significantly reduced.
  • the invention can reduce the pain of the patient and improve the quality of life, and can obviously reduce the treatment cost, and has a good application prospect.
  • Extract A in each of the drawings refers to 3'-deoxyadenosine.
  • the inventors noticed the similarity between the structure and S-adenosylmethionine (SAM, methyl donor) when studying natural 3'-deoxyadenosine, suggesting that 3'-deoxyadenosine may also act as a non-A Base donors involved in competing with SAM for DNMTs
  • SAM S-adenosylmethionine
  • the binding process changes the normal ratio of intracellular SAM/SAH (S-adenosyl-L-homocyteine) and reverses the methylation status of the tumor suppressor promoter region, which may inhibit the growth of tumor cells.
  • the present inventors used in vitro human solid tumor cells (including liver cancer cell line HuH-7, breast cancer cell line T47D, MCF-7) and treated with 3'-deoxyadenosine alone.
  • a series of experiments were designed to confirm that 3'-deoxyadenosine alone can inhibit the proliferation of the above multiple cell lines, and at the DNA level, the inventors proposed to inhibit the methylation of tumor suppressor genes and restore the expression of tumor suppressor genes. mechanism. However, the effect is still not satisfactory.
  • a tumor gene methylation regulator represented by 3'-deoxyadenosine such as shellac red pigment, 3'-deoxyadenosine, 5-azacytidine, genistein Or zebularine, etc.
  • the present invention refers to such tumor gene methylation regulators as sensitizers for tumor therapeutic drugs.
  • the use of a sensitizer in combination with a tumor therapeutic agent can achieve synergistic effects in a variety of tumor models, and can significantly reduce the amount of anti-tumor drugs, thereby reducing the side effects of anti-tumor drugs.
  • tumor gene methylation regulators and other anti-tumor drugs can be prepared as new anti-tumor drugs.
  • the present invention also develops a new tumor treatment method, that is, a combination of a tumor gene methylation regulator and other anti-tumor drugs, thereby increasing the therapeutic effect or reducing the amount of other anti-tumor drugs to reduce toxic side effects.
  • test compound combination was used in combination with 3'-deoxyadenosine-paclitaxel, 3'-deoxyadenosyl-doxorubicin, and 3'-deoxyadenosine-cisplatin.
  • Control compound group 3'-deoxyadenosine, paclitaxel, doxorubicin, cisplatin.
  • FBS fetal bovine serum (Invitrogen-10099141);
  • the above-mentioned tumor cell lines derived from ATCC were cultured in an incubator at 37 ° C, 5% CO 2 . Cell lines were passaged twice a week by trypsinization. Cells in the logarithmic growth phase are used for plating.
  • the plate was incubated overnight in an incubator at 37 ° C, 5% CO 2 , and 100% relative humidity.
  • the compound stock solution was taken out from the -80 ° C refrigerator and thawed at room temperature.
  • the 96-well cell plate was returned to the incubator for 48 hours.
  • a CellTiter-Glo working solution was prepared by adding 10 ml of CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.
  • the cell culture plate was taken out and allowed to stand for room temperature for 30 minutes.
  • the plate was shaken on an orbital shaker for 2 minutes to induce cell lysis.
  • Table 2 shows that for human breast cancer cells, 3'-deoxyadenosine 10uM or 5uM- is combined with paclitaxel at the same IC 50 , ie, 50% inhibition, and the dose is less than 5 times or 10 times.
  • the dosage of paclitaxel is 1/189 or 1/37 of the drug alone, that is, the total dose of the two drugs is 0.004 uM or 0.02 uM when the same inhibition rate is reached, which is much smaller than 1/1 of each of the two drugs alone.
  • Table 3 shows that for human breast cancer, 3'-deoxyadenosine 10uM or 5uM is combined with paclitaxel at the same IC 50 , ie, the same 50% inhibition rate, when the dose is 5 times or 10 times less than the single dose,
  • the dosage of doxorubicin is 1/4 or 1/1.4 of the drug alone, that is, when the same inhibition rate is reached, the total dose of the two drugs is 0.116 uM or 0,388 uM, which is much smaller than that of the two drugs alone.
  • the animals were started after 7 days of feeding in the experimental environment. Animals were housed in an IVC (independent air supply system) cage at the SPF level animal house (5 per cage). Each cage animal information card indicates the number of animals in the cage, gender, strain, date of receipt, dosing schedule, experiment number, group, and start date of the experiment. All cages, litter and drinking water are sterilized prior to use. Cage, feed and drinking water are changed twice a week.
  • IVC independent air supply system
  • Human leukemia HL60, human gastric cancer cell line SGC-7901, human breast cancer cell MCF-7, human colon cancer cell line HT-29, human lung cancer A-549, human liver cancer Huh-7 were cultured in vitro, digested and centrifuged to prepare concentration A cell suspension of about 1 x 10 7 cells/mL, 0.2 mL / was inoculated subcutaneously into the right axilla of the mouse.
  • the drug was stopped 14 days after the administration, and the animals were sacrificed on the 7th day, and the animals were sacrificed by cervical dislocation.
  • the solid tumors were weighed.
  • the data were analyzed by SPSS statistical analysis software.
  • Tumor growth inhibition rate (average tumor weight of vehicle control group - mean tumor weight of administration control group) / mean tumor weight of vehicle control group ⁇ 100%.
  • nude mice of each group in the following experiments were 7 in each group.
  • Experimental group drug (capecitabine 400mg/Kg+3'-deoxyadenosine 20mg/Kg (3'-deoxyadenosine is represented by extract A in the figure, the following figures are the same)) oral administration of human breast cancer
  • the tumor-injected mice showed a tumor inhibition rate of 85% on the 11th day of administration compared with the negative control group (salt group), while the positive control group (capecitabine 400 mg/Kg/day) showed A similar tumor inhibition rate was 86.1%; in the positive group, 5 experimental animals died on the 11th day of the experiment. It was shown that capecitabine combined with control A not only had a good tumor suppressing effect, but no experimental animals died at 11 days, indicating that the toxicity of the combination drug was significantly reduced.
  • the experimental results are shown in Figure 2.
  • the results showed that the experimental group was given oral mice with human leukemia xenografts (adriamycin 25 mg/week + 3-deoxyadenosine 20 mg/kg/week; administration method: doxorubicin 5 mg/kg/+3'
  • the tumor growth rate was 16.7% compared with the negative control group (salt group), while the positive control group (60 mg/kg/) Week; 20 mg/kg, 3 times/week each inhibited tumor growth rate of 21.4%, but on the 14th day of the experiment, one experimental animal died in this group.
  • the dose of the experimental group was 41.7% of the positive control group (60 mg/kg/week), although the inhibition rate of tumor inhibition in animals decreased slightly (21.4% vs. 16.7%), but it was safe. Increased sex, no experimental animal death occurred. It is worth exploring further research in patients with cancer.
  • the experimental group (Tiggio 2.5mg/kg/day +3'-deoxyadenosine 20mg/kg/day) was orally administered to mice with human breast cancer xenografts on the 18th day of administration, and negative In the control group (salt group), the inhibition rate against tumor was 66.9%; it was equivalent to the positive control group (Tiggio 5 mg/kg/day) inhibiting tumor effect (66.2%); but the positive control group had one animal died. .
  • the transplanted tumor model was successful for 14 days and then treated with drugs.
  • the experimental results are shown in Figure 4.
  • the results showed that the experimental group was administered to mice with human lung cancer xenografts (cisplatin 2.5mg/kg/day+3'-deoxyadenosine 20mg/kg/day), and the experiment continued until the 28th day.
  • the tumor growth inhibition rate was 25%; it is worth noting that none of the experimental animals died at the end of the experiment.
  • the positive control group (cisplatin 5mg/kg/day; on day 7 nude mice died 2, on the 10th day a total of 4 deaths; on the 15th day cisplatin dose was reduced to 2.5mg/kg; on the 18th day, a total of 5 deaths Only.
  • high dose of cisplatin (5mg/kg) has a good inhibitory effect on tumor growth, due to the high toxicity, the experimental animals died more (5/7 animals died) and the experimental work could only be terminated early.
  • the transplanted tumor model was successful for 23 days and then treated with drugs. See Figure 5 for the experimental results.
  • the results showed that: the experimental group was administered intravenously to mice bearing human liver cancer xenografts (adriamycin 1.67 mg/Kg/+3'-deoxyadenosine, 3'-deoxyadenosine 12.5 mg/Kg, Daily administration ⁇ 6 times); on the 37th day, compared with the negative control group (salt group), the tumor inhibition rate was 33.8%; and the positive control group (2.5 mg/kg) inhibited the tumor growth rate of 25.7%.
  • the experimental results show that although the dose of doxorubicin (1.67mg/kg) in the experimental group is 66.8% of the amount of doxorubicin (2.5mg/kg) in the positive control group, the tumor suppressing effect of 3'-deoxyadenosine on doxorubicin There is a synergistic effect and still have a good inhibition rate (31%).
  • the experimental results are shown in Figure 7.
  • the experimental results showed that the experimental group was given oral mice with colon cancer xenografts for 14 consecutive days (capecitabine 250mg/Kg+3'-deoxyadenosine 20mg/Kg). On the 31st day, the tumor inhibition rate was 72.6% compared with the negative control group (salt group), and the inhibition rate of the positive control group (capecitabine 400 mg/Kg) was 80.1%.
  • the experimental results are shown in Figure 8.
  • the results showed that the experimental group was administered orally for 14 days (Tiogio 2.5mg/kg + 3'-deoxyadenosine 20mg/Kg) to the mice with human colon cancer xenografts.
  • the tumor inhibition rate was 76.7% as compared with the negative control group (salt group), and the inhibition rate of the positive control group (Tiggio: 5 mg/kg) was 66.1%.
  • Each group was administered once daily (volume 0.2 mL, intravenously) and measured twice a week.
  • the experimental group (5-fluorouracil 3mg/kg+3'-deoxyadenosine 20mg/Kg) was intravenously injected into mice with human gastric cancer (SGC 7901).
  • SGC 7901 human gastric cancer
  • the experimental results are shown in Figure 9 (the CDP in the figure refers to 3) '-deoxyadenosine.
  • the tumor inhibition rate was 81.3% as compared with the blank control group (salt group), and the inhibition rate of the positive control group (5-fluorouracil 6 mg/kg) was 67.5%.

Abstract

肿瘤基因甲基化调节剂在制备抗肿瘤药物中的用途。以及，肿瘤基因甲基化调节剂可作为增敏剂与肿瘤治疗药物联合用于制备抗肿瘤药物。

Description

肿瘤基因甲基化调节剂的新用途及抗肿瘤药物 技术领域
本发明属于肿瘤治疗领域，具体涉及肿瘤基因甲基化调节剂的新用途以及抗肿瘤药物。
背景技术
肿瘤发生的过程复杂,肿瘤发生机理研究认为人体遗传学(genetics)以及基因表观遗传学(epigenetic)等的变化也可能是引起恶性肿瘤的原因之一。遗传学改变如基因的突变、缺失会导致编码区结构和功能破坏。而基因表观遗传学改变不引起DNA序列的改变，但通过对DNA自身的化学修饰导致转录水平的改变可能影响基因的表达，从而调控DNA功能。DNA甲基化是哺乳动物DNA最常见的表观遗传学修饰方式之一，也是目前肿瘤生物学关注的热点。基因启动子区的CpG岛在正常状态下一般是非甲基化的,当其发生甲基化时,常导致基因转录沉寂,使重要基因如抑癌基因、DNA修复基因等丧失功能,从而导致正常细胞的生长分化调控失常以及DNA损伤不能被及时修复,这与多种肿瘤的形成相关。DNA甲基化是可逆,如应用DNA甲基化调节剂可以使一些重要基因发生去甲基化,而恢复其正常功能，因此抑制DNA甲基转移酶活性已成为新的防治肿瘤的研究思路。
已有大量文献报道，在人乳腺癌中参与抑制细胞增殖、DNA损伤修复和抑制肿瘤转移的抑癌基因的启动子被高甲基化所沉默，如p16，INK4A，p14，ARF，p15，CCDN2，DAPK，DNA repair，MGMT，hMLH1，GSTP1，RARβ2，APC，ERβ，CDH1，CDH13。导致抑癌基因甲基化的重要机制是乳腺癌细胞内DNA甲基转移酶高表达。
已知的DNA甲基转移酶抑制剂有5-氮杂胞嘧啶核苷(Azacitidine,5-Aza-CR)和5-氮杂脱氧胞嘧啶核苷(5-aza-2-deoxycytidine，5-Aza-CdR)。它们的作用机理被认为是通过在DNA复制过程中取代胞嘧啶及与DNMT形成共价键后抑制DNMT的活性两种途径来抑制DNA甲基化。它们被广泛应用于研究DNA甲基化的生物过程和治疗急性髓细胞性白血病及骨髓增生异常综合征(myelodysp lastic syndrome,MDS)。但这两种药物由于其致突变、细胞毒性和骨髓抑制等副作用而限制了它们的临床应用。
DNA甲基修饰过程除了需要DNA甲基转移酶外，还需要一种重要的辅酶，即S-腺苷基甲硫氨酸(英文：S-adenosyl methionine，缩写：SAM)参与甲基转移反应。它因带 有“活泼甲基”而在甲基转移作用中扮演重要的角色。现已知SAM参与了40多种体内的代谢反应，涉及将S-腺苷基蛋氨酸的甲基转移到核酸、蛋白质和脂肪等底物上去。在美国已有用SAM的名字按营养补品销售，有改善情绪、保养肝脏和舒适关节的功效。
虫草是我国特产的一种中药，分布在西藏、青海、四川、云南和甘肃。有关虫草的研究开展了若干年,取得了大量的成果，其中发现了种草的主要成分包含3’-脱氧腺苷(Cordycepin，CAS：73-03-0)等。
3’-脱氧腺苷是一种新型的广谱抗菌素。目前对3’-脱氧腺苷在抗菌、抗炎、抗HIV-Ⅰ型病毒、选择性抑制梭菌以及免疫调节等药理学方面开展了一些研究。3’-脱氧腺苷抗肿瘤方面以往的研究集中在抗白血病的研究上，1997年美国已将3’-脱氧腺苷用于治疗急性前B和前T淋巴细胞白血病一期临床实验，但没能进行后继工作。目前也有一些报导3’-脱氧腺苷治疗实体瘤的试验研究，但多是鼠系细胞株中进行的实验，且单独使用时其抗肿瘤活性偏低，至今也没有作为治疗药物用于肿瘤的治疗。本领域有研究发现3’-脱氧腺苷对人肿瘤DNA的甲基化有调节作用。
发明内容
本发明要解决的技术问题是克服现有肿瘤治疗技术方案的缺陷，为肿瘤治疗提供一种新的有效选择。
本发明解决技术问题的技术方案为提供了肿瘤基因甲基化调节剂在制备抗肿瘤药物中的用途。
其中，所述的肿瘤基因甲基化调节剂为虫胶红色素、3’-脱氧腺苷、5-氮杂胞苷、金雀异黄素(genistein)和[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮](zebularine)中的至少一种。
所述5-氮杂胞苷的英文名称：5-Azacytidin分子式：C8H12N4O5；分子量：244.21结构如下式所示：
Figure PCTCN2017072505-appb-000001
其中，上述的肿瘤为血液瘤或者实体瘤。
其中，上述的血液瘤为白血病、多发性骨髓瘤、胶质瘤或恶性淋巴瘤中的至少一种。
其中，上述的实体瘤为乳腺癌、肺癌、胃癌、胰腺癌、食道癌、结直肠癌、肝癌、前列腺癌、子宫癌、肾癌、膀胱癌或皮肤癌中的至少一种。
其中，上述用途中所述的抗肿瘤药物中除肿瘤基因甲基化调节剂还含有至少一种其他的抗肿瘤活性成分。
其中，上述用途中所述的其他抗肿瘤活性成分为5-氟尿嘧啶、卡培他滨、吉美嘧啶、氧嗪酸钾(奥替拉西钾)、奥替拉西、替加氟、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂或它们各自的可药用盐中至少一种。
其中，上述用途中所述的其他的抗肿瘤活性成分为贝伐珠单抗(bevacizumab)、阿柏西普(aflibercept)，帕妥珠单抗(Pertuzumab)，曲妥珠单抗(Trastuzumab)、西妥昔单抗(Cetuximab)、利妥昔单抗(Rituximab)，阿仑单抗(Alemtuzumab)或帕尼单抗(Panitumumab)等单抗类抗肿瘤药物中的至少一种。
进一步的，上述用途中所述的其他抗肿瘤活性成分与肿瘤基因甲基化调节剂可是分别独立包装的。
同时，本发明提供了一种抗肿瘤药物。该抗肿瘤药物是由肿瘤基因甲基化调节剂为主要活性成分之一制成。
其中，上述抗肿瘤药物中所述的肿瘤基因甲基化调节剂为虫胶红色素、A类抗抑癌基因甲基化化合物、金雀异黄素(genistein)或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮](zebularine)中的至少一种。
其中，上述抗肿瘤药物中所述的肿瘤为血液瘤或者实体瘤。
其中，上述抗肿瘤药物中所述的血液瘤为白血病、多发性骨髓瘤、胶质瘤或恶性淋巴瘤中的至少一种。
其中，上述抗肿瘤药物中所述的实体瘤为乳腺癌、肺癌、胃癌、胰腺癌、食道癌、结直肠癌、肝癌、前列腺癌、皮肤癌、子宫癌、肾癌、膀胱癌中的至少一种。
进一步的，上述抗肿瘤药物中除肿瘤基因甲基化调节剂还含有至少一种其他的抗肿瘤活性成分。
其中，上述技术方案中所述的其他的抗肿瘤活性成分为铂类抗癌药、蒽环类抗癌药、抗代谢类抗癌药、生物碱类抗癌药或靶向抗体类抗癌药中的至少一种。
其中，上述的蒽环类抗癌药物为柔红霉素，阿霉素、表阿霉素、米托蒽醌或他们各自的各种可药用盐中的至少一种。
其中，上述的抗代谢类抗癌药为培美曲赛、替吉奥、卡培他滨、吉西他滨、5-氟尿嘧啶或他们各自的各种可药用盐中的至少一种。
其中，上述的铂类抗癌药为顺铂、卡铂、奥沙利铂、奈达铂或他们各自的各种可药用盐中的至少一种。
其中，上述的所述生物碱类抗癌药为紫杉烷类抗癌药。进一步的所述紫杉烷类抗癌药为紫杉醇、多烯紫杉醇、三尖杉宁碱、10-去乙酰紫杉醇、巴卡亭III或他们各自的各种可药用盐中的至少一种。
5-氟尿嘧啶、卡培他滨、吉美嘧啶、氧嗪酸钾(奥替拉西钾)、奥替拉西、替加氟、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂或它们各自的可药用盐中至少一种。
其中，上述抗肿瘤药物中所述的其他的抗肿瘤活性成分为贝伐珠单抗(bevacizumab)、阿柏西普(aflibercept)，帕妥珠单抗(Pertuzumab),曲妥珠单抗(Trastuzumab)、西妥昔单抗(Cetuximab)、利妥昔单抗(Rituximab)，阿仑单抗(Alemtuzumab)或帕尼单抗(Panitumumab)等单抗类抗肿瘤药物中的至少一种。
显然，在针对不同的肿瘤类型，以及使用的不同上述其他抗肿瘤活性成分时，可以根据具体情况和目的，调整各类肿瘤甲基化调节剂和其他抗肿瘤活性成分的相对比例和各自的具体用量以及施用方式。
其中，上述抗肿瘤药物是由肿瘤基因甲基化调节剂为主要活性成分之一添加药剂学上的辅料或辅助性成分制备而成的药剂。
其中，上述的抗肿瘤药物的剂型为口服制剂、注射制剂、喷雾制剂或静脉滴注制剂。
其中，上述的抗肿瘤药物中所述的辅料或辅助性成分包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、保护剂、吸附载体或润滑剂中的一种或几种的组合。
其中，上述的抗肿瘤药物中所述的其他的抗肿瘤活性成分与肿瘤基因甲基化调节剂可为复方制剂。
其中，上述的抗肿瘤药物中所述的其他的抗肿瘤活性成分与肿瘤基因甲基化调节剂可是分别独立包装的联用制剂。
其中，上述的抗肿瘤药物的剂型为口服制剂或者注射剂。
其中，上述的抗肿瘤药物的中所述的注射剂为注射液或粉针剂。
同时，本发明还提供治疗人的至少一种和肿瘤生长有关的病症或症状的方法。该方 法包括向人施予药物组合物，该药物组合物包括肿瘤基因甲基化调节剂，并配合施予至少一种有效量其他的抗肿瘤活性成分。
其中，上述方法中所述的肿瘤基因甲基化调节剂和其他的抗肿瘤活性成分可以一起施予患者，也可分别施予患者，只要在任意一者在患者体内有高于正常生理量的血药浓度时施予另一者均可被视为配合使用。
其中，上述方法施予的肿瘤基因甲基化调节剂的剂量为能与至少一种其他的抗肿瘤活性成分配合而有效治疗该与肿瘤生长有关的病症或症状的剂量。
其中，上述方法中所述的肿瘤基因甲基化调节剂为所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮]中的至少一种。
其中，上述方法中所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂和它们的各种盐的至少一种。
其中，上述方法中所述的其他的抗肿瘤活性成分为贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
其中，上述方法中所述的肿瘤为血液瘤或者实体瘤。
其中，上述方法中所述的肿瘤基因甲基化调节剂与其他抗肿瘤活性成分同时施用时，用相同或者不同的给药途径施与患者。
其中，上述所述的肿瘤基因甲基化调节剂与其他的抗肿瘤活性成分用相同的给药途径同时施与患者时，处于同一制剂中或者处于不同的制剂中。
与此同时，本发明还提供了提高肿瘤防治药物疗效或降低肿瘤防治药物起效用量的方法。将肿瘤基因甲基化调节剂与至少一种有效量其他的抗肿瘤活性成分配合使用，达到提高肿瘤对其他的抗肿瘤活性成分敏感性，从而提高疗效或降低起效用量的目的。
其中，上述方法中所述的肿瘤基因甲基化调节剂和其他的抗肿瘤活性成分可以一起施予患者，也可分别施予患者，只要在任意一者在患者体内有高于正常生理量的血药浓度时施予另一者均可被视为配合使用。
其中，上述方法中所述施予的肿瘤基因甲基化调节剂的剂量为提高其他的抗肿瘤活性成分效或降低其他的抗肿瘤活性成分起效用量的剂量。
其中，上述方法中所述的肿瘤基因甲基化调节剂为所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢 嘧啶-2-酮]中的至少一种。
其中，上述方法中所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂和它们的各种盐的至少一种。
其中，上述方法中所述的其他的抗肿瘤活性成分为贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
其中，上述方法中所述的肿瘤为血液瘤或者实体瘤。
其中，上述方法中所述的肿瘤基因甲基化调节剂与其他抗肿瘤活性成分同时施用时，用相同或者不同的给药途径施与患者。
其中，上述所述的肿瘤基因甲基化调节剂与其他的抗肿瘤活性成分用相同的给药途径同时施与患者时，处于同一制剂中或者处于不同的制剂中。
最后，本发明还提供了前述的抗肿瘤药物的制备方法。
本发明创造性发现将肿瘤基因甲基化调节剂作为增敏剂与肿瘤治疗药物联合，可用于制备抗肿瘤药物，本发明抗肿瘤药物在增加疗效的同时，还可以显著的降低肿瘤治疗药物的用量，从而能够明显地降低肿瘤治疗药物对机体的副作用。本发明在减轻患者的痛苦，提高其生活质量的同时还能明显地降低治疗费用，具有很好的应用前景。
附图说明
各附图中的提取物A均指3’-脱氧腺苷。
图1、卡培他滨和3’-脱氧腺苷抗乳腺癌(MCF-7)的试验结果。
图2、阿霉素和3’-脱氧腺抗白血病(HL60)的实验结果。
图3、替吉奥和3’-脱氧腺抗乳腺癌(MCF-7)的实验结果。
图4、顺铂和3’-脱氧腺抗肺癌(A-549)的实验结果。
图5、阿霉素和3’-脱氧腺苷抗肺癌(huh-7)的实验结果。
图6、卡培他滨和3’-脱氧腺苷抗胃癌(SGC-7901)的实验结果。
图7、卡培他滨和3’-脱氧腺苷抗结肠癌(HT-29)的实验结果。
图8、替吉奥和3’-脱氧腺苷抗结肠癌(HT-29)的实验结果。
图9、5-氟尿嘧啶与3'-脱氧腺抗胃癌(SGC-7901)的实验结果。
具体实施方式
发明人在研究天然3’-脱氧腺苷时注意到其结构与S-腺苷甲硫氨酸(SAM，甲基供体)的相似性，从而推测3’-脱氧腺苷也可能作为非甲基供体涉及与SAM共同竞争DNMTs的 结合过程，改变细胞内SAM/SAH(S-adenosyl-L-homocyteine)的正常比例，逆转抑癌基因启动子区的甲基化状态，而有可能抑制肿瘤细胞的生长。
基于此，本发明人在前期的工作中，运用体外培养的人实体瘤细胞(包括肝癌细胞系HuH-7，乳腺癌细胞系T47D，MCF-7)，用3’-脱氧腺苷单独进行处理，设计了一系列试验，证实3’-脱氧腺苷单独施用可以抑制上述多个细胞系增殖，并在DNA水平验证了发明人提出的抑制抑癌基因甲基化作用，恢复抑癌基因表达的机制。但是，其效果仍不理想。
在后继研究中，本发明创造性地发现，以3’-脱氧腺苷为代表的肿瘤基因甲基化调节剂，如虫胶红色素、3’-脱氧腺苷、5-氮杂胞苷、genistein或zebularine等，具有能增强肿瘤细胞对现有的肿瘤治疗药物的敏感性的功效，本发明将这一类肿瘤基因甲基化调节剂称之为肿瘤治疗药物的增敏剂。本发明使用增敏剂与肿瘤治疗药物联合使用，能在多种肿瘤模型中获得协同作用，以及可以显著降低抗肿瘤药物的用量，从而降低抗肿瘤药物的毒副作用。在上述创造性发现的基础上，肿瘤基因甲基化调节剂和其他抗肿瘤药物可以制备成新的抗肿瘤药物。本发明还开发出了新的肿瘤治疗方法，即肿瘤基因甲基化调节剂和其他抗肿瘤药物联合使用，从而增加疗效，或者减少其他抗肿瘤药物的用量以减少毒副作用。
以下通过具体实施方式的介绍对本发明进行详细的说明
实施例一、抗肿瘤细胞实验结果
1、参试药物
受试化合物组合：3’-脱氧腺苷-紫杉醇联用、3’-脱氧腺苷-阿霉素联用、3’-脱氧腺苷-顺铂联用。
对照化合物组：3’-脱氧腺苷、紫杉醇、阿霉素、顺铂。
2.实验设计
表1.在不同细胞中检测的化合物及其浓度
Figure PCTCN2017072505-appb-000002
Figure PCTCN2017072505-appb-000003
3.材料
3.1.细胞系
人乳腺癌MCF-7，人肝癌Huh-7；肿瘤细胞生长特点：MCF-7，Huh-7均为贴壁生长
3.2.培养基
RPMI 1640(Invitrogen-22400089)；DMEM(Invitrogen-11960077)
FBS胎牛血清(Invitrogen-10099141)；
双抗(青霉素、链霉素)(Invitrogen-15140122)
3.3.多孔板
Greiner
Figure PCTCN2017072505-appb-000004
96-孔板，平底黑板(带盖及透明底),#655090.
3.4.细胞活性实验所用试剂及仪器
(1).Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7571).
(2).2104
Figure PCTCN2017072505-appb-000005
读板器，PerkinElmer.
4.实验方法及步骤
4.1.细胞培养
将ATCC来源的上述肿瘤细胞系在37℃，5％CO2的培养箱中进行培养。每周将细胞系用胰酶消化传代2次。将处于对数生长期的细胞用于铺板。
4.2.铺板
(1).用台盼兰进行细胞染色并计数活细胞。
(2).将细胞浓度调整至2×104个/ml(约2.0×103个/孔)。
(3).在培养板中每孔加入100μl细胞悬液，在空白对照孔中加入不含细胞的培养液。
(4).将培养板在37℃,5％CO2,及100％相对湿度的培养箱中培养过夜。
4.3.20X化合物工作液的配制及化合物处理细胞
(1).将化合物储存液从-80℃冰箱中取出，室温解冻。
(2).配制20X化合物工作液
(3).加药：取5μl的20X化合物工作液加入到细胞培养板中。在溶媒对照和空白对照中加入5μl DMSO-细胞培养液混合液。
(4).将96孔细胞板放回培养箱中培养48小时。
4.4.CellTiter-Glo发光法细胞活性检测
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7571)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入10ml CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置30分钟使其平衡至室温。
(6).在每孔中加入80μl的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在2104EnVision读板器上检测发光信号。
4.5数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(RLU溶媒对照–RLU化合物)/(RLU溶媒对照–RLU空白对照)*100％.在Excel中计算不同浓度化合物的抑制率，然后用XLFit软件(方程式205)作抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率，绝对IC50及相对IC50。
5.结果
5.1.细胞活性实验中化合物抗增殖参数结果总结
表2.人乳腺癌细胞活性实验抗细胞活性参数
Figure PCTCN2017072505-appb-000006
Figure PCTCN2017072505-appb-000007
表3.抗人乳腺癌细胞活性实验参数
Figure PCTCN2017072505-appb-000008
表2显示，对于人乳腺癌细胞，在相同IC50，即同等达到50％抑制率情况下，3’-脱氧腺苷10uM或5uM-与紫杉醇联合，即用药剂量低于单独用药5倍或10倍时，紫杉醇的用量分别是单独用药的1/189或1/37，即达到同等抑制率时这两种药的总剂量为0.004uM或0.02uM，其远小于两种药单独用药各1/2剂量的总和(0.85/2uM+0.75/2＝0.8uM)。
表3显示，对于人乳腺癌，在相同IC50，即同等50％抑制率情况下，3’-脱氧腺苷10uM或5uM与紫杉醇联合，即用药剂量低于单独用药5倍或10倍时，阿霉素的用量分别是单独用药的1/4或1/1.4，即达到同等抑制率时，这两种药的总剂量为0.116uM或0,388uM，其远小于两种药单独用药各1/2剂量的总和(0.85/2+0.51/2＝0.68uM)。表明为3’-脱氧腺苷在10uM或5uM时与阿霉素具有协同作用。
表4、人肝癌细胞活性实验抗细胞活性参数。
Figure PCTCN2017072505-appb-000009
表4显示，对于人肝癌，在相同IC50，即同等50％抑制率情况下，3’-脱氧腺苷与阿霉素联合，这两种药的总剂量为0.28uM，其远小于两种药单独用药各1/2剂量的总和(1.346/2uM+1.2/2uM＝1.27uM)，表明3’-脱氧腺苷与阿霉素具有协同作用；3’-脱氧腺苷与顺铂联合，这两种药的总剂量为0.995uM，其远小于两种药单独用药各1/2剂量的总和(1.346/2uM+5/2＝3.17uM),表明3’-脱氧腺苷与阿霉素具有协同作用。
结论：肿瘤细胞的重要特征之一是无限性生长，因此研究抗肿瘤药物首先是观察其抗肿瘤细胞生长的作用。研究结果表明；对于人乳腺癌细胞，在3’-脱氧腺苷10uM或5uM时，与紫杉醇联合具有协同作用，尤其在10uM时，紫杉醇的用量仅为单独用药剂量的1/189；在3’-脱氧腺苷10uM或5uM时，与阿霉素有协同作用；对于人肝癌细胞，3’-脱氧腺苷50uM与阿霉素具有协同作，3’-脱氧腺苷50uM与顺铂具有协同作用。
实施例二抗肿瘤活性动物实验
1、实验动物
种属：小鼠
品系：BALB/c裸小鼠
性别及数量：雌性，每组7只
开始给药时周龄及体重：6～8周龄，体重18～22克。
2、实验动物饲养条件
动物到达后在实验环境饲养7天后方开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼5只)。每笼动物信息卡注明笼内动物数目，性别，品系，接收日期，给药方案，实验编号，组别以及实验开始日期。所有笼具、垫料及饮水在使用前均灭菌。笼具、饲料及饮水每周更换两次。
3、参试模型制备及药物处理说明
人白血病HL60、人胃癌细胞株SGC-7901、人乳腺癌细胞MCF-7、人结肠癌细胞HT-29、、人肺癌A-549、人肝癌Huh-7体外常规培养，消化离心后制成浓度约1×107个细胞/mL的细胞混悬液，0.2mL/只接种于小鼠右侧腋窝皮下。
取出上述已接种好的生长良好的皮下移植瘤，切成约2×2×2mm大小的肿瘤组织块备用。在麻醉后取小鼠左侧卧位，至腋窝处，放入肿瘤组织块，缝合皮肤，消毒后，放回笼内正常饲养。待肿瘤直径长至7～8mm左右时分组。一般分组后连续灌胃给药或空白(溶媒：0.9％(g/mL)氯化钠注射液)14天，1次/天，给药体积均为20mL/kg，特殊情况在相应实例中单独加以说明。给药前、后观察动物的一般症状。首次给药后每周测 量2次实体瘤的长(a)短(b)径，按公式体积(V)＝1/2×a×b2计算肿瘤体积，并对动物进行称重。给药14天后停药，第7天时对动物进行称重后颈椎脱臼处死动物，取出实体瘤称重。用SPSS统计分析软件对所得数据进行分析。
疗效评价公式：
肿瘤生长抑制率＝(溶媒对照组平均瘤重-给药对照组平均瘤重)/溶媒对照组平均瘤重×100％。
4、各个模型具体用药及实验结果
如无特别说明，以下各实验中各组的裸鼠均为每组7只。
实验1  卡培他宾和3'-脱氧腺苷抗乳腺癌(MCF-7)实验及结果
实验结果参见图1。实验组药(卡培他宾400mg/Kg+3'-脱氧腺苷20mg/Kg(3'-脱氧腺苷在图中用提取物A表示，以下各图同))经口腔给带人体乳腺癌移植瘤的小鼠，在给药的第11天，与阴性对照组(生理盐水组)比较，其肿瘤抑制率为85％；而阳性对照组(卡培他宾400mg/Kg/天)则显示出类似的肿瘤抑制率为86.1％；，阳性组在实验的第11天有5只实验动物死亡。表明卡培他宾联合对照物A不但有良好的抑制肿瘤效果，其在11天时无实验动物死亡，也说明联合用药的毒性明显减少。
实验2、阿霉素和3’-脱氧腺抗白血病(HL60)实验及结果
实验结果参见图2。结果表明：实验组药经口腔给带人体白血病移植瘤的小鼠(阿霉素25mg/周+3-脱氧腺苷20mg/kg/周；给药方式：阿霉素5mg/kg/+3’-脱氧腺苷4mg/kg/天，连续给药5天)的第14天，与阴性对照组(生理盐水组)比较，其抑制肿瘤生长率为16.7％；而阳性对照组(60mg/kg/周；每次20mg/kg,3次/周)抑制肿瘤生长率为21.4％，但在实验的第14天，本组有1只实验动物死亡。实验组的用药量(25mg/kg/周)为阳性对照组(60mg/kg/周)的41.7％，虽然在动物抑制肿瘤的抑制率有小幅度下降(21.4％对16.7％)，但其安全性提高，无实验动物死亡发生。值得探索在肿瘤病患者做进一步的研究。
实验3、替吉奥和3’-脱氧腺抗乳腺癌(MCF-7)实验及结果
实验结果参见图3。实验组药(替吉奥2.5mg/kg/天+3’-脱氧腺苷20mg/kg/天)经口腔给带有人体乳腺癌移植瘤的小鼠，在给药的第18天，与阴性对照组(生理盐水组)比较，其对肿瘤的抑制率为66.9％；与阳性对照组(替吉奥5mg/kg/天)抑制肿瘤效果(66.2％)相当；但阳性对照组有一只动物死亡。说明替吉奥用量减少(2.5mg/kg)，但联合3’-脱氧腺苷3’-脱氧腺苷可达到倍量替吉奥(5mg/kg)的抑制肿瘤效果，而且毒性减少。
实验4、顺铂和3’-脱氧腺抗肺癌(A-549)实验及结果
接种A-549细胞后，14天移植瘤模型成功，然后进行药物处理。实验结果参见图4。结果表明：实验组药经注射给带人体肺癌移植瘤的小鼠给药(顺铂2.5mg/kg/天+3’-脱氧腺苷20mg/kg/天)，实验一直持续到了第28天，与阴性对照组(生理盐水组)比较，其抑制肿瘤生长率为25％；值得注意到是这组实验动物在实验结束时无一死亡。
而阳性对照组(顺铂5mg/kg/天；第7天裸鼠死亡2只，第10天共死亡4只；第15天顺铂剂量减为2.5mg/kg；第18天，共死亡5只。虽然高剂量的顺铂(5mg/kg)对肿瘤生长有较好的抑制作用，但因毒性大，实验动物死亡多(5/7只动物死亡)而只能提前终止实验工作。
实验5、阿霉素和3’-脱氧腺苷抗肺癌(huh-7)实验及结果
接种人肝癌Huh7细胞后，23天移植瘤模型成功，然后进行药物处理。实验结果参见图5。结果表明：实验组药经静脉注射给带有人体肝癌移植瘤的小鼠给药(阿霉素1.67mg/Kg/+3’-脱氧腺苷，3’-脱氧腺苷12.5mg/Kg，间日给药×6次)；在第37天，与阴性对照组(生理盐水组)比较，其肿瘤抑制率为33.8％；而阳性对照组(2.5mg/kg)抑制肿瘤生长率为25.7％。
实验结果说明虽然实验药组阿霉素用量(1.67mg/kg)是阳性对照组阿霉素用量(2.5mg/kg)的66.8％，由于3’-脱氧腺苷对阿霉素的抑制肿瘤作用有协同效应，仍对肿瘤有较好的抑制率(31％)。
实验6、卡培他宾和3’-脱氧腺苷抗胃癌(SGC-7901)实验及结果
结果参见图6。结果表明：实验组药在造模成功后连续给药14天(卡培他宾400mg/Kg+3’-脱氧腺苷20mg/Kg)经口腔给带有人体胃癌移植瘤的小鼠；在第27天，与阴性对照组(生理盐水组)比较，其肿瘤抑制率为89.2％；而阳性对照组(卡培他宾400mg/Kg)的抑制率为57.8％。
实验结果说明3’-脱氧腺苷3’-脱氧腺苷对卡培他滨的抑制胃肿瘤作用有协同效应，使其对肿瘤的抑制率提高了54％。
实验7、卡培他宾和3’-脱氧腺苷抗结肠癌(HT-29)实验及结果
实验结果参见图7。实验结果表明：实验组药在造模成功后连续14天(卡培他宾250mg/Kg+3’-脱氧腺苷20mg/Kg)经口腔给带有人体结肠癌移植瘤的小鼠，在第31天，与阴性对照组(生理盐水组)比较，其肿瘤抑制率为72.6％；而阳性对照组(卡培他宾400mg/Kg)的抑制率为80.1％。
实验结果说明3’-脱氧腺苷3’-脱氧腺苷对阿霉素的抑制结肠肿瘤作用有协同效应，虽然实验组卡培他宾的用量(250mg/Kg)为阳性对照组用量(卡培他宾400mg/Kg)的2/3，但由于3’-脱氧腺苷3’-脱氧腺苷的协同作用，仍对结肠肿瘤表现出较好抑制率作用(72.6％比80.1％)。因为减少卡培他滨的用量，对癌症病人的毒性作用也将相应的减少。
实验8、替吉奥和3’-脱氧腺苷抗结肠癌(HT-29)实验及结果
实验结果见图8。结果表明：实验组药在造模成功后连续给药14天(替吉奥2.5mg/kg+3’-脱氧腺苷20mg/Kg)经口腔给带有人体结肠癌移植瘤的小鼠，在第31天，与阴性对照组(生理盐水组)比较，其肿瘤抑制率为76.7％；而阳性对照组(替吉奥：5mg/kg)的抑制率为66.1％。实验结果说明3’-脱氧腺苷3’-脱氧腺苷3’-脱氧腺苷对替吉奥抑制结肠肿瘤作用有协同效应，虽然实验组替吉奥的用量(2.5mg/Kg)比较阳性对照组用量(替吉奥5mg/kg)的少，但由于3’-脱氧腺苷3’-脱氧腺苷3’-脱氧腺苷的协同作用，仍对结肠肿瘤表现出较好抑制率作用(76.7％比66.1％)。
实验9、5-氟尿嘧啶与3'-脱氧腺苷抗胃癌(SGC 7901)的实验结果
各组每日给药一次(体积0.2mL,静脉注射)，每周测量2次。实验组(5-氟尿嘧啶3mg/kg+3'-脱氧腺苷20mg/Kg)药经静脉注射给带有人体胃癌(SGC 7901)的小鼠，实验结果参见图9(图中的CDP即指3'-脱氧腺苷)。在给药的第28天，与空白对照组(生理盐水组)比较，其肿瘤抑制率为81.3％；而阳性对照组(5-氟尿嘧啶6mg/kg)的抑制率为67.5％。
实验结果说明3'-脱氧腺苷对5-氟尿嘧啶抗胃癌作用有协同效应。实验组中虽然5-氟尿嘧啶的用量(3mg/Kg)比阳性对照组5-氟尿嘧啶用量(6mg/kg)减少了50％，但由于3’-脱氧腺苷的协同作用，仍对胃癌表现出更好的抑制作用。

Claims (36)

  1. 肿瘤基因甲基化调节剂在制备抗肿瘤药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于：所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮]中的至少一种。
  3. 根据权利要求1所述的用途，其特征在于：所述的肿瘤为血液瘤或实体瘤。
  4. 根据权利要求1～3任一项所述的用途，其特征在于：所述的抗肿瘤药物除肿瘤基因甲基化调节剂还含有至少一种其他的抗肿瘤活性成分。
  5. 根据权利要求1～4任一项所述的用途，其特征在于：所述的其他的抗肿瘤活性成分为铂类抗癌药、蒽环类抗癌药、抗代谢类抗癌药、生物碱类抗癌药或靶向抗体类抗癌药中的至少一种。
  6. 根据权利要求1～5任一项所述的用途，其特征在于：所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂及它们的各种盐、或者贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
  7. 根据权利要求1～6任一项所述的用途，其特征在于：所述的其他的抗肿瘤活性成分与肿瘤基因甲基化调节剂是分别独立包装的制剂。
  8. 抗肿瘤药物，其特征在于是由肿瘤基因甲基化调节剂为主要活性成分之一制成；还含有至少一种其他的抗肿瘤药物。
  9. 根据权利要求8所述的抗肿瘤药物，其特征在于：所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮]中的至少一种。
  10. 根据权利要求8或9所述的抗肿瘤药物，其特征在于：所述的肿瘤为血液瘤或者实体瘤。
  11. 根据权利要求8～10任一项所述的抗肿瘤药物，其特征在于：所述的其他的抗肿瘤活性成分为铂类抗癌药、蒽环类抗癌药、抗代谢类抗癌药、生物碱类抗癌药或靶向抗体类抗癌药中的至少一种。
  12. 根据权利要求8～11任一项所述的抗肿瘤药物，其特征在于：所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂和它们的各种盐的至少一种。
  13. 根据权利要求8～12任一项所述的抗肿瘤药物，其特征在于：所述的其他的抗肿瘤活性成分为贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
  14. 根据权利要求8～13任一项所述的抗肿瘤药物，其特征在于：所述的其他的抗肿瘤活性成分与肿瘤基因甲基化调节剂是复方制剂。
  15. 根据权利要求8～14任一项所述的抗肿瘤药物，其特征在于：所述的其他的抗肿瘤活性成分与肿瘤基因甲基化调节剂是分别独立包装的。
  16. 根据权利要求8～15任一项所述的抗肿瘤药物，其特征在于：所述的抗肿瘤药物的剂型为口服制剂、注射制剂或喷雾制剂。
  17. 治疗人的至少一种和肿瘤生长有关的病症或症状的方法，包括向人施予包含肿瘤基因甲基化调节剂的药物的步骤，以及与肿瘤基因甲基化调节剂配合使用至少一种有效量的其他的抗肿瘤活性成分。
  18. 根据权利要求17所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂和其他的抗肿瘤活性成分可以一起施予患者，也可分别施予患者，只要在任意一者在患者体内有高于正常生理量的血药浓度时施予另一者均可被视为配合使用。
  19. 根据权利要求17或18所述的方法，其特征在于：所述施予的肿瘤基因甲基化调节剂的剂量为能与至少一种其他的抗肿瘤活性成分配合而有效治疗该与肿瘤生长有关的病症或症状的剂量。
  20. 根据权利要求17～19任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂为所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮]中的至少一种。
  21. 根据权利要求17～20任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为铂类抗癌药、蒽环类抗癌药、抗代谢类抗癌药、生物碱类抗癌药或靶向抗体类抗癌药中的至少一种。
  22. 根据权利要求17～21任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂和它们的各种盐的至少一种。
  23. 根据权利要求17～22任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
  24. 根据权利要求17～23任一项所述的方法，其特征在于：所述的肿瘤为血液瘤或者实体瘤。
  25. 根据权利要求17～24任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂与其他抗肿瘤活性成分同时施用时，用相同或者不同的给药途径施与患者。
  26. 根据权利要求17～25任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂与其他的抗肿瘤活性成分用相同的给药途径同时施与患者时，处于同一制剂中或者处于不同的制剂中。
  27. 提高肿瘤防治药物疗效或降低肿瘤防治药物起效用量的方法；其特征在于：将肿瘤基因甲基化调节剂与至少一种有效量其他的抗肿瘤活性成分配合使用，达到提高肿瘤对其他的抗肿瘤活性成分敏感性，从而提高疗效或降低起效用量的目的。
  28. 根据权利要求27所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂和其他的抗肿瘤活性成分可以一起施予患者，也可分别施予患者，只要在任意一者在患者体内有高于正常生理量的血药浓度时施予另一者均可被视为配合使用。
  29. 根据权利要求27或28所述的方法，其特征在于：所述施予的肿瘤基因甲基化调节剂的剂量为提高其他的抗肿瘤活性成分效或降低其他的抗肿瘤活性成分起效用量的剂量。
  30. 根据权利要求27～29任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂为所述的肿瘤基因甲基化调节剂为虫胶红色素、金雀异黄素、3’-脱氧腺苷、5-氮杂胞苷或[1-(β-D-呋喃核糖苷)-1,2-二氢嘧啶-2-酮]中的至少一种。
  31. 根据权利要求27～30任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为铂类抗癌药、蒽环类抗癌药、抗代谢类抗癌药、生物碱类抗癌药或靶向抗体类抗癌药中的至少一种。
  32. 根据权利要求27～31任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为5-氟尿嘧啶、吉美嘧啶、替加氟、卡培他滨、氧嗪酸钾、奥替拉西、紫杉醇、多烯紫杉醇、表阿霉素、阿霉素、阿柔比星、米托蒽醌、伊立替康、顺铂、卡铂、奥沙利铂、奈达铂和它们的各种盐的至少一种。
  33. 根据权利要求27～32任一项所述的方法，其特征在于：所述的其他的抗肿瘤活性成分为贝伐珠单抗、阿柏西普、帕妥珠单抗、曲妥珠单抗、西妥昔单抗、利妥昔单抗、阿仑单抗或帕尼单抗中的至少一种。
  34. 根据权利要求27～33任一项所述的方法，其特征在于：所述的肿瘤为血液瘤或者实体瘤。
  35. 根据权利要求27～34任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂与其他抗肿瘤活性成分同时施用时，用相同或者不同的给药途径施与患者。
  36. 根据权利要求27～35任一项所述的方法，其特征在于：所述的肿瘤基因甲基化调节剂与其他的抗肿瘤活性成分用相同的给药途径同时施与患者时，处于同一制剂中或者处于不同的制剂中。
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