WO2022012635A1 - 一种药物组合物及其用途 - Google Patents

一种药物组合物及其用途 Download PDF

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WO2022012635A1
WO2022012635A1 PCT/CN2021/106558 CN2021106558W WO2022012635A1 WO 2022012635 A1 WO2022012635 A1 WO 2022012635A1 CN 2021106558 W CN2021106558 W CN 2021106558W WO 2022012635 A1 WO2022012635 A1 WO 2022012635A1
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ginsenoside
pharmaceutical composition
group
bicyclol
cells
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French (fr)
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WO2022012635A9 (zh
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范代娣
殷诗玉
段志广
严建亚
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陕西巨子生物技术有限公司
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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
    • A61K31/70—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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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 application belongs to the technical field of medicine, and in particular, the application relates to a pharmaceutical composition and use thereof.
  • liver cancer is one of the most common malignant tumors worldwide. The National Cancer Center estimates that in 2015, there will be 466,000 liver cancer incidences and 422,000 liver cancer deaths in my country. Liver cancer is currently the fourth most common malignant tumor and the third leading cause of cancer death in my country, seriously threatening the lives and health of the Chinese people.
  • liver cancer Patients with early-stage liver cancer generally have no obvious symptoms, and most of the patients have middle-stage or late-stage liver cancer when they are diagnosed, while the average survival time of untreated primary liver cancer after diagnosis is less than 4 months.
  • the best treatments for liver cancer are surgical resection and liver transplantation.
  • liver transplantation it is difficult to find a donor; in the case of surgical resection, patients who can undergo surgery are only 20% of all patients, so it is not suitable as a treatment for all patients with liver cancer, and, progress The recurrence rate of stage liver cancer is higher after surgical resection or orthotopic liver transplantation.
  • MDR Multidrug resistance
  • Ginsenoside monomers are the main active components of Chinese medicinal materials such as ginseng, American ginseng, Panax notoginseng, etc. There are more than 30 ginsenoside monomers isolated from ginseng, which have anti-tumor activity, enhance immunity activity, lower blood sugar and improve memory, etc. The role is becoming a hot spot of development.
  • Ginsenoside CK is the metabolite of natural diol-type ginsenoside monomer in human intestinal tract, and it is the entity that ginseng exerts pharmacological activity in vivo. Ginsenoside CK is used for the treatment of rheumatoid arthritis and has entered the clinical trial stage.
  • ginsenoside CK can significantly inhibit the in vitro proliferation of colon cancer HCT-116 cells, SW-480 cells and HT-29 cells, as well as the proliferation and invasion of liver cancer MHCC97-H cells, and can inhibit the colon cancer in vivo in mice.
  • Ginsenoside CK is related to PI3K/Akt signaling pathway, and promotes the apoptosis of breast cancer cell MCF-7; Ginsenoside CK up-regulates p53/p21, FoxO3a-p27/ in HCT-116 cells The expressions of p15 and Smad3, down-regulate the expressions of cdc25A, CDK4/6 and cyclinD1/3, induce apoptosis and G1 phase arrest in HCT-116 cells; Ginsenoside CK affects Annexin A2 protein and NF- ⁇ B p50 isoform in hepatoma HepG2 cells NF- ⁇ B interaction and nuclear colocalization, inhibit the transcriptional activation of NF- ⁇ B and the expression of downstream anti-apoptotic genes X-IAP, c-IAP1, c-IAP2 and Survivin, and promote the activation of Caspase 9 and the
  • Bicyclonol (trade name: Bisino) is the first new anti-hepatitis drug with independent intellectual property rights in my country. The clinical test results show that it can significantly reduce the serum and level of chronic hepatitis B and chronic hepatitis C patients, and can make the hepatitis B virus index of some patients. Turn overcast. Pharmacological studies have shown that bicyclol has the functions of resisting chemical poison-induced liver damage, anti-liver fibrosis, and inhibiting hepatocyte apoptosis caused by immune damage. According to literature reports, bicyclol can significantly inhibit the damage to the cell genome caused by chemical mutagens, and restore the inhibition of hepatic epithelial cell gap communication junction (GJIC) function caused by carcinogens.
  • GJIC hepatic epithelial cell gap communication junction
  • GJIC function and tumorigenesis The relationship between them has been paid more and more attention.
  • Various evidences show that the function of GJIC is closely related to the occurrence and development of tumors, suggesting that it may have a certain blocking effect on the occurrence and development of tumors.
  • Bicyclol can inhibit the in vitro malignant transformation of rat liver epithelial WB-F344 cells induced by 3MC/TPA combination and prevent the occurrence of hepatocellular carcinoma.
  • Bicyclol has a significant inhibitory effect on the adhesion of highly metastatic human hepatoma cell line MHCC97-H to LN and FN, reduces the expression of VEGF mRNA in MHCC97-H cells, and inhibits angiogenesis, thereby inhibiting tumor invasion and metastasis, suggesting that It may have certain potential value in preventing and inhibiting the occurrence and development of liver cancer.
  • Patent Document 1 discloses a liver cancer targeting ginsenoside CK chitosan polymer micelle drug delivery system, which is characterized in that it includes: a targeting head group, a basic carrier, and an anticancer drug; wherein, the targeting The head group is A54 polypeptide, the basic carrier is an amphiphilic polymer, and the anticancer drug is ginsenoside CK; the targeting head group is connected with the hydrophobic end of the basic carrier through a linking group to form a drug delivery carrier; The drug delivery carrier encapsulates ginsenoside CK to form a liver cancer targeting ginsenoside CK-loaded chitosan polymer micelle drug delivery system.
  • Patent Document 2 discloses a composition comprising the following components: ginsenoside Rk1, ginsenoside Rg5, ginsenoside CK, total ginsenoside extract, wherein the composition is an interleukin (IL)-6 modifier.
  • ginsenoside Rk1 ginsenoside Rg5
  • ginsenoside CK total ginsenoside extract
  • Patent Document 3 discloses the use of bicyclic alcohols and pharmaceutically acceptable derivatives thereof for the preparation of medicaments for preventing or treating drug-induced liver injury.
  • ginsenoside monomers or bicyclic alcohols are used, but the effect of preventing and treating tumors is limited, and the prior art does not disclose that ginsenoside monomers can promote the effect of pharmaceutical compositions for preventing and/or treating tumors, There is also no disclosure of the combined use of ginsenoside monomers and bicyclic alcohols to synergize, improve the effect of preventing and/or treating tumors.
  • Patent document 1 CN109793711A publication
  • Patent document 2 CN110090236A publication
  • the present application provides a synergistic antitumor drug composition, which can be safer and more effective. Powerful prevention and treatment of tumors, especially liver cancer.
  • a pharmaceutical composition characterized in that, the pharmaceutical composition comprises: a ginsenoside monomer and a bicyclic alcohol.
  • ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20-(S ,R)-Rg3, ginsenoside 20-(S,R)-Rh2, ginsenoside 20-(S,R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh4;
  • the ginsenoside monomers include ginsenoside CK.
  • ginsenoside monomer and bicyclic alcohol in preparing a pharmaceutical composition for preventing and/or treating tumors.
  • ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20-(S,R )-Rg3, ginsenoside 20-(S,R)-Rh2, ginsenoside 20-(S,R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh4;
  • the ginsenoside monomers include ginsenoside CK.
  • ginsenoside monomer in promoting the effect of a pharmaceutical composition for preventing and/or treating tumors.
  • ginsenoside monomer is selected from one or more of the following: ginsenoside CK, ginsenoside 20-(S, R)-Rg3, ginsenoside 20-(S,R)-Rh2, ginsenoside 20-(S,R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 or ginsenoside Rh4;
  • the ginsenoside monomers include ginsenoside CK.
  • the pharmaceutical composition of the present application comprising ginsenoside monomer and bicyclic alcohol, especially the pharmaceutical composition comprising ginsenoside CK and bicyclic alcohol, can inhibit the proliferation of tumor cells, especially liver cancer cells;
  • the pharmaceutical composition comprising ginsenoside CK and bicyclol has significant synergistic and synergistic effects compared with single drug, and is a kind of high-efficiency and low-toxicity antitumor drug combination.
  • Bicyclol can restore the inhibition of hepatic epithelial cell gap communication junction (GJIC) function caused by chemical carcinogens, protect cellular genomic DNA, and inhibit chemical carcinogen-induced malignant transformation of hepatic epithelial cells and the occurrence and development of liver tumors.
  • GJIC hepatic epithelial cell gap communication junction
  • Ginsenoside CK is related to multiple signaling pathways including PI3K/Akt signaling pathway, NF- ⁇ B signaling pathway and cell cycle regulation signaling pathway, and can inhibit tumor cell growth and metastasis. Ginsenoside CK and bicyclol act on different stages of hepatocarcinogenesis, different targets and signaling pathways, respectively, resulting in stronger curative effects on the prevention and/or treatment of hepatocellular carcinoma.
  • Figure 1(a)-(b) are the morphological diagrams of WB-F344 cells induced by 3MC/TPA in the blank control group and the model group, respectively.
  • Figure 2(a)-(e) are respectively the H&E staining and pathological observation pictures of liver cancer prevention experiment of SD rats in blank control group, model group, ginsenoside CK group, bicyclol group, and ginsenoside CK/bicyclol combination group.
  • the application provides a pharmaceutical composition, characterized in that, the pharmaceutical composition comprises: ginsenoside monomer and bicyclic alcohol.
  • the ginsenoside monomer of the present application can be a commercially available ginsenoside monomer, or a ginsenoside monomer isolated from ginseng cultivated or collected in nature, or another ginsenoside monomer converted from an isolated ginsenoside monomer.
  • the ginsenoside monomer can be selected from ginsenoside CK, ginsenoside 20-(S,R)-Rg3, ginsenoside 20-(S,R)-Rh2, ginsenoside 20-(S,R)-PPD, ginsenoside One or more of the monomers of saponin Rk1, ginsenoside Rg5, ginsenoside Rk3, ginsenoside Rh4, etc., preferably including ginsenoside CK.
  • Ginsenoside CK chemical name is 20-0- ⁇ -D-glucopyranosyl-20-protopanaxadiol (ginsenoside CK), molecular formula: C 36 H 62 O 8 , molecular weight: 622.87, odorless, White powder.
  • ginsenoside CK used in the experiment in this application is the common ginsenoside monomer obtained by extracting ginseng, and the highly active rare ginsenoside CK with a purity of more than 98% is obtained by directional biotransformation technology and separation and purification.
  • the structural formula of ginsenoside CK is:
  • Rh4 "Ginsenoside Rh4", molecular formula: C 36 H 60 O 8 ; molecular weight: 620.4; scientific name: 6-O- ⁇ -D-glucopyranosyl-20(-HO)-trans-protopanaxatriol; odorless, white powder. Soluble in methanol and ethanol, slightly soluble in ethyl acetate, poor in water solubility, insoluble in chloroform and ether; CAS number: 174721-08-5; structural formula:
  • Bicyclic alcohol which has the chemical name 4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2-hydroxymethyl-2'-methoxycarbonyl Biphenyl, with the molecular formula of C 19 H 18 O 9 , can be clinically used to treat elevated aminotransferase caused by chronic hepatitis.
  • Bicycloalcohol is a structural analog of biphenyl diester, and has the functions of anti-hepatitis virus and anti-hepatocyte damage.
  • an effective dose of ginsenoside monomer and bicyclic alcohol are included as active ingredients.
  • the effective dose refers to the dose used when the pharmaceutical composition exerts its pharmaceutical function.
  • ginsenoside monomers and bicyclol are combined according to the proportions defined in this application, in terms of inhibiting the proliferation of liver cancer cells in vitro, inhibiting the malignant transformation of liver cancer precursor cells in vitro, and treating liver cancer in vivo. It showed synergistic effect in the aspect of liver cancer prevention and prevention of rat liver cancer.
  • the inventors have also found that the ginsenoside monomer and bicyclic alcohol are also low in toxicity within the above ratio range.
  • the weight ratio of the ginsenoside monomer and the bicyclic alcohol is preferably 1:0.3-3, specifically 1:0.3, 1:0.4, 1:0.
  • the concentration of ginsenoside monomers may be 3-30 ⁇ M, specifically Available in 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 21 ⁇ M, 22 ⁇ M, 23 ⁇ M, 24 ⁇ M, 25 ⁇ M, 26 ⁇ M, 27 ⁇ M, 28 ⁇ M, 29 ⁇ M, 30 ⁇ M, preferably 5 ⁇ M to 20 ⁇ M; the concentration of bicyclol can be 1 to 20 ⁇ M, specifically 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10
  • the pharmaceutical composition of the present application can be administered orally or parenterally.
  • the dosage varies depending on the degree of symptoms, patient age, sex, body weight, difference in sensitivity, administration method, administration period, administration interval, the nature of the pharmaceutical preparation, the type of active ingredient, etc., and there is no particular limitation, but it is usually an adult (body weight 60 kg) 10mg-1.5g per day, preferably 50mg-900mg, more preferably 100mg-600mg, more preferably 300mg-500mg (based on the total weight of ginsenoside CK), the above-mentioned dosage can usually be administered in 1-3 times a day.
  • the ginsenoside monomer is selected from one or two or three or four or more than five of the following: ginsenoside CK, ginsenoside 20- (S,R)-Rg3, ginsenoside 20-(S,R)-Rh2, ginsenoside 20-(S,R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3, and ginsenoside Rh4.
  • the ginsenoside monomer includes ginsenoside CK.
  • ginsenoside CK it can also contain other ginsenoside monomers selected from one or more of the following: ginsenoside 20-(S,R)-Rg3, ginsenoside 20-(S,R)-Rh2 , ginsenoside 20-(S, R)-PPD, ginsenoside Rk1, ginsenoside Rg5, ginsenoside Rk3 and ginsenoside Rh4, and may not contain other ginsenoside monomers.
  • the total weight of the total ginsenoside monomers included in the pharmaceutical composition is 100
  • the content of the ginsenoside CK is preferably more than 50 parts by weight, more preferably more than 60 parts by weight, more preferably more than 70 parts by weight, more preferably more than 80 parts by weight, more preferably more than 90 parts by weight, More preferably, it is 95 parts by weight or more, more preferably 99 parts by weight or more, and more preferably 100 parts by weight (that is, the ginsenoside monomer contained in the pharmaceutical composition is only ginsenoside CK).
  • the purity of the ginsenoside monomer used in the pharmaceutical composition of the present application may be more than 98%.
  • a pharmaceutically acceptable carrier such as an adjuvant
  • a pharmaceutically acceptable carrier such as an adjuvant
  • the excipients in the pharmaceutical composition of the present application for example, the excipients commonly used in medicines or health products in the technical field can be used.
  • the auxiliary materials are starch, dextrin, lactose, mannitol, sodium hypromellose, xanthan gum, protein sugar and the like.
  • the dosage form of the pharmaceutical composition of the present application is not particularly limited, for example, it can be an oral dosage form or an injection dosage form.
  • the oral dosage form can be a liquid dosage form or a solid dosage form.
  • the oral dosage forms can be, for example, hard capsules, soft capsules, slow-release capsules, compressed tablets, sugar-coated tablets, powders, granules, dropping pills, honeydew pills, syrups or oral liquids;
  • the injection dosage forms can be, for example, solution forms, Suspension type, emulsion type or lyophilized powder.
  • the administration mode of the pharmaceutical composition for improving sleep can be, for example, oral administration, drip or injection.
  • a solid preparation for oral use after adding excipients and optionally binders, disintegrating agents, lubricants, coloring agents, flavoring agents, etc. to the main drug, Tablets, coated tablets, granules, fine granules, powders, capsules and the like are prepared according to conventional methods.
  • excipients for example, lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, silicon dioxide, etc. can be used; cellulose, methylcellulose, acacia, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.; as disintegrants, for example, dry starch, sodium carboxymethyl starch, polyvinylpyrrolidone, carboxymethyl can be used cross-linked polymer of sodium cellulose, etc.; as a lubricant, for example, magnesium stearate, talc, silicon dioxide, etc.
  • a colorant a colorant allowed to be added to a medicine can be used; as a flavoring agent, Cocoa powder, menthol, aromatic acid, peppermint oil, borneol, cinnamon powder can be used.
  • a flavoring agent Cocoa powder, menthol, aromatic acid, peppermint oil, borneol, cinnamon powder
  • the above-mentioned tablets and granules can also be coated with sugar coating, gelatin coating, and other necessary outer coatings.
  • pH adjusters, buffers, suspending aids, solubilizers, stabilizers, isotonic agents, preservatives, etc. can be added to the main drug as required, and then prepared according to conventional methods.
  • examples of suspending aids include methyl cellulose, Tween 80, hydroxyethyl cellulose, acacia, tragacanth powder, sodium carboxymethyl cellulose, and polyoxyethylene sorbitol. Monolaurate, etc.
  • examples of the solubilizer include polyoxyethylene hydrogenated castor oil, Tween 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, and the like.
  • examples of stabilizers include sodium sulfite, sodium metasulfite, and the like; examples of preservatives include methylparaben, ethylparaben, sorbic acid, phenol, cresol, and chlorocresol.
  • Liver cancer can be prevented and/or treated by administering the pharmaceutical composition of the present application to a subject.
  • the subject may be a mammal, such as a human, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, etc., preferably a human.
  • the present application has a significant effect of preventing and/or treating tumors, especially preventing and/or treating liver cancer. Therefore, the present application also provides the use of the pharmaceutical composition of the present application in the preparation of a pharmaceutical composition for preventing and/or treating tumors. Further, the tumor is preferably liver cancer.
  • the present application also provides the use of ginsenoside monomers in promoting the effect of a pharmaceutical composition for preventing and/or treating tumors.
  • the pharmaceutical composition of the present application exhibits synergistic or even significant synergistic effects in inhibiting the proliferation of liver cancer cells in vitro, inhibiting the malignant transformation of liver cancer precursor cells in vitro, treating liver cancer in vivo, and preventing liver cancer.
  • the CI value of the interaction index between ginsenoside monomer and bicyclic alcohol is less than 1, preferably less than or equal to 0.7. Further, by combining ginsenoside monomer and bicyclic alcohol in the ratio defined in this application, a pharmaceutical composition with better effect of preventing and/or treating liver cancer can be obtained.
  • Example 1 Combined use of ginsenoside CK and bicyclol inhibits the proliferation of liver cancer HepG2 cells in vitro
  • the MTT method was used to detect the inhibitory effect of ginsenoside CK on the proliferation of liver cancer HepG2 cells.
  • MTT is chemically named 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide and is a yellow dye.
  • MTT method also known as “MTT colorimetric method” is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, while dead cells have no such function.
  • Dimethyl sulfoxide can dissolve formazan in cells, and its absorbance value is measured at 490nm wavelength with a microplate reader. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. According to the measured absorbance value (OD value), the number of living cells is judged. The larger the OD value, the stronger the cell activity (if the drug toxicity is measured, the lower the drug toxicity).
  • OD value absorbance value
  • This method has been widely used in the activity detection of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity test, and tumor radiosensitivity assay. It is characterized by high sensitivity and economy.
  • T/C value of the combination group, A and B are the T/C values of the drug alone group.
  • the experimental data are expressed as x ⁇ s and analyzed by SPSS17.0 statistical software. Pairwise comparisons between groups were performed using the LSD method. P ⁇ 0.05 means significant difference, P ⁇ 0.01 means extremely significant difference.
  • the inhibitory effects of ginsenoside CK and bicyclol on the in vitro proliferation of liver cancer HepG2 cells measured by MTT method are shown in Table 1.
  • the single drug use of ginsenoside CK with different concentrations and the single drug use of bicyclol with different concentrations have a certain degree of The inhibitory effect of tumor cell growth was in a concentration-dependent manner.
  • the combined use of ginsenoside CK and bicyclol has a stronger inhibitory effect on HepG2.
  • the combination of the two monomers has a significant difference (P ⁇ 0.05), and the CI index also shows that The combination of the two monomers has obvious synergistic effect.
  • Example 2 Combined use of ginsenoside CK and bicyclol inhibits the in vitro malignant transformation of WB-F344 cells to cancer cells induced by 3MC/TPA combination
  • the models for studying tumor chemopreventive drugs mainly include in vitro and in vivo systems.
  • the in vitro system uses various carcinogens to directly or indirectly induce the malignant transformation of normal cells.
  • the effective indicator is that the malignant transformation of cells is inhibited. It can well simulate the multi-stage process of tumorigenesis in vivo and the experimental conditions are easy to control.
  • Rat liver epithelial-like stem cells (WB-F344 cells) are derived from normal adult syngeneic male rats. They are immature liver epithelial cells with the properties of liver stem cells. They are related to the occurrence of liver cancer and are precursor cells of liver cancer. .
  • Model group 1500 WB-F344 cells/well were inoculated in 6-well plates with 2 mL of culture medium per well. 24h after the cells were inoculated, the carcinogen 3-methylcholanthrene (3MC) was added to the culture medium to make the final concentration 2 ⁇ g/mL. After 96h of culture, the 3MC was removed and replaced with fresh medium. After culturing for 3 days, phorbol meat was added.
  • 3MC carcinogen 3-methylcholanthrene
  • TPA Myristyl acetate
  • Blank control group On the basis of the model group, 0.2% DMSO was used to replace 3MC and TPA.
  • Dosing group On the basis of the model group, 24 h after cell inoculation, ginsenoside CK administration group (concentrations of 5 ⁇ M, 7.8 ⁇ M, 2.5 ⁇ M, 2 ⁇ M) and bicyclol administration group (concentration of 2 ⁇ M) were set.
  • the T/C value of the combination group of two monomers, A and B are the T/C values of the two monomers acting alone. When CI ⁇ 1, it means that the two monomers have synergistic effect, and when CI ⁇ 0.7, it means that the synergistic effect is very significant.
  • Figure 1(a) is the cell morphology of the blank control group. It is polygonal, spreads well on the substrate, has abundant cytoplasm and is contact inhibitory.
  • Figure 1(b) The cell morphology of the model group. It can be seen from the figure that the transformed WB-F344 cells are irregular in shape, spindle-shaped or polygonal, with different cell sizes, loss of growth polarity, and loss of contact inhibition. Often overlapping growth, scattered transformation foci can be seen. The transformation foci count results are shown in Table 2 below. At the end of the experiment, the model group formed obvious transformation foci, and the number of transformation foci was 58.6 ⁇ 7.2.
  • Ginsenoside CK group, bicyclol group and ginsenoside CK/bicyclol combination group 1, ginsenoside CK/bicyclol combination group 2, ginsenoside CK/bicyclol combination group 3, ginsenoside CK/bicyclol combination group Group 4 has inhibitory effect on the formation of transformation foci (see Table 2 for specific results), and the CI values of the two monomer interaction indices are both less than 1, which has a synergistic effect on inhibiting the malignant transformation of WB-F344 cells in vitro.
  • a nude mouse xenograft model of human hepatoma was established: 4-5 weeks old female BALB/c nude mice were inoculated with Hep-3B cells subcutaneously in the left armpit, and each nude mouse was injected with 4 ⁇ 10 6 cells. When the tumor volume reached 100-300 mm 3 , the nude mice were randomly divided into 16 groups with 10 animals in each group.
  • the 16 groups were a model group and 15 drug-added groups, respectively, and the 15 drug-added groups were: (1) 5 ginsenoside CK groups, administered by gavage, and the doses were 60 mg/kg and 30 mg/kg, respectively. , 90mg/kg, 100mg/kg, 24mg/kg; (2) 5 bicyclol groups were administered by gavage, and the doses were 60mg/kg, 90mg/kg, 30mg/kg, 20mg/kg, 96mg/kg, respectively.
  • ginsenoside CK/bicyclol combination groups were administered by gavage, and the doses of ginsenoside CK/bicyclol were 60mg/kg/60mg/kg, 30mg/kg/90mg/kg, 90mg respectively /kg/30mg/kg, 100mg/kg/20mg/kg, 24mg/kg/96mg/kg.
  • the administration volume of each drug-added group was 0.2 mL, once a day, for 21 consecutive days. Observations were made once a day, and the clinical status of the animals was recorded, including whether they were dead, dying, appearance, mental status, and activity status.
  • Tumor volume V 0.5 ⁇ a ⁇ b ⁇ b, where a is the length of the tumor, and b is the width of the tumor.
  • Tumor inhibition rate (%) (model group V-medicated group V)/model group V ⁇ 100%.
  • the T/C values of A and B are the T/C values of the two monomers acting alone. When CI ⁇ 1, it means that the two monomers have synergistic effect, and when CI ⁇ 0.7, it means that the synergistic effect is very significant.
  • the results of the tumor volume test are shown in Table 3.
  • the CI index of the combination of the two monomers is 0.68 (CI ⁇ 0.7), which has a significant synergistic effect.
  • the CI index is lower than when the ratio of the two is outside the above range.
  • the ratio is 1:1, the CI index is the smallest and the synergistic effect is the most obvious.
  • Example 4 Combined use of ginsenoside CK and bicyclol to prevent DEN-induced liver cancer in rats
  • Model establishment and administration 100 SD rats were purchased, and after one week of adaptation in the animal room, they were randomly divided into 5 groups, namely blank control group, model group, ginsenoside CK group, bicyclol group, and ginsenoside CK/bicyclol combination Group.
  • the ginsenoside CK group and the bicyclol group were given intragastric administration.
  • the administration dose of the ginsenoside CK group was 60 mg/kg
  • the bicyclol group was 60 mg/kg
  • the ginsenoside CK/bicyclol combination group was administered with a dose of 60 mg/kg.
  • the blank control group and the model group were given equal volume of vehicle, and they were given continuous intragastric administration for 20 weeks.
  • DEN chemical mutagen diethylnitrosamine
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • H&E staining is a commonly used staining method for pathological sections, and it is Hematoxylin-Eosin staining. This method can be used for any fixative-fixed tissue and sections using various embedding methods.
  • Hematoxylin is a basic dye that can dye basophilic substances in tissues blue, such as chromatin in the nucleus, etc.
  • eosin is an acidic dye that can dye eosinophilic substances in tissues red, For example, the cytoplasm and nucleoli of most cells were red in H&E-stained sections.
  • the body weight of the rats in the model group was significantly lower than that in the blank control group, and the liver weight and liver coefficient were significantly higher than those in the blank control group (P ⁇ 0.05).
  • the change of the organ coefficient generally reflects the enlargement, congestion, hyperplasia and hypertrophy of the organ, and is an indicator of liver damage. Intraperitoneal injection of DEN can increase the liver coefficient of the rat, and it is speculated that the rat liver may be damaged.
  • the body weight of the rats in the ginsenoside CK group, the bicyclol group and the ginsenoside CK/bicyclol combination group was significantly higher than that in the model group (P ⁇ 0.05), and the liver coefficient was significantly lower than that in the model group (P ⁇ 0.05).
  • the effect of the bicyclol combination group was significantly better than that of the single administration group.

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Abstract

一种药物组合物，所述药物组合物包括：人参皂苷单体和双环醇。所述药物组合物对肝癌的化学预防和治疗具有显著疗效，相比于与单独使用人参皂苷单体或单独使用双环醇，具有显著的协同和增效作用。人参皂苷单体和双环醇在制备预防和／或治疗肿瘤的药物组合物中的用途，以及人参皂苷单体在促进预防和／或治疗肿瘤的药物组合物的作用中的用途。

Description

一种药物组合物及其用途 技术领域
本申请属于医药技术领域，具体地，本申请涉及一种药物组合物及其用途。
背景技术
原发性肝癌，是世界范围内最常见的恶性肿瘤之一。国家癌症中心预计2015年中国肝癌发病人数46.6万，肝癌死亡人数42.2万，肝癌是目前我国第四位的常见恶性肿瘤及第三位的肿瘤致死病因，严重威胁我国人民的生命和健康。
早期肝癌病人一般无明显症状，多数患者就诊时已经为中期、晚期肝癌，而未经治疗的原发性肝癌确诊后平均生存时间少于4个月。当前，肝癌最好的治疗方法为手术切除和肝移植。然而，在肝移植的情况下，很难找到捐赠者；在手术切除的情况下，可以进行手术的患者在全部患者仅占20％，因此不适合用作所有肝癌患者的治疗方法，而且，进展期肝癌在手术切除或原位肝移植术后复发率都较高。
其他治疗方法有全身抗癌疗法、肝动脉化学注射疗法、肝动脉化疗栓塞和放射疗法等，但这些治疗方法在杀死肝癌细胞的同时，同时还会杀死正常细胞，具有致命的副作用。此外，据报道，作为代表性肝癌靶向药物索拉非尼具有在长期服用时胰腺缩小和耐药问题等副作用。多药耐药性(MDR)是肿瘤治疗的一大障碍，其机制较为复杂。研究发现，肿瘤细胞几乎对所有的单独药物都存在不同程度的耐药性，但多个药物的联合用药能够同时作用于癌细胞的多个靶位点或信号途径，可有效的克服单药耐药性的缺点，为肿瘤的预防和/或治疗提供了新的方法和希望，也将成为了肿瘤预防和/或治疗的未来趋势。
人参皂苷单体是人参、西洋参、三七等中药材主要的活性成分，从人参中分离得到的人参皂苷单体有30余种，其抗肿瘤活性、增强免疫力活性、 降血糖以及提高记忆力等作用正成为开发的热点。人参皂苷CK是天然的二醇型人参皂苷单体在人肠道内的代谢产物，是人参在体内发挥药理活性的实体。人参皂苷CK用于治疗类风湿性关节炎，目前已进入临床试验阶段。抗肿瘤相关研究表明，人参皂苷CK能够明显抑制结肠癌HCT-116细胞，SW-480细胞和HT-29细胞的体外增殖，以及肝癌MHCC97-H细胞的增殖和侵袭，并能够抑制小鼠体内结肠癌和肝癌等肿瘤的生长和肝转移；人参皂苷CK和PI3K/Akt信号通路有关，促进乳腺癌细胞MCF-7的凋亡；人参皂苷CK上调HCT-116细胞中p53/p21,FoxO3a-p27/p15和Smad3的表达，下调cdc25A，CDK4/6和cyclinD1/3的表达，诱导HCT-116细胞的凋亡和G1期阻滞；人参皂苷CK影响肝癌HepG2细胞中Annexin A2蛋白质和NF-κB p50亚基的相互作用和细胞核共定位，抑制NF-κB的转录激活和下游抗细胞凋亡基因X-IAP、c-IAP1、c-IAP2和Survivin的表达，促进Caspase 9的激活和HepG2细胞凋亡的发生，并抑制下游MMP-2/9的表达水平，从而抑制肿瘤细胞转移。
双环醇(商品名：百赛诺)是我国第一个拥有自主知识产权的抗肝炎新药，临床试验结果表明能显著降低慢性乙肝和慢性丙肝病人血清和水平，并能使部分病人的乙肝病毒指标转阴。药理研究结果显示，双环醇具有抗化学毒物引起的肝损伤、抗肝纤维化、抑制免疫损伤引起的肝细胞凋亡等作用。根据文献报道，双环醇能明显抑制化学诱变剂对细胞基因组的损伤，恢复致癌剂所引起的肝上皮细胞细胞间隙通讯连接(GJIC)功能的抑制，细胞间隙连接通讯(GJIC)功能与肿瘤发生之间的关系越来越受到人们的重视，多种证据显示GJIC功能与肿瘤的发生发展有着密切的关系，提示其对肿瘤的发生发展可能有一定的阻断作用。双环醇可抑制3MC/TPA联合诱导的大鼠肝上皮WB-F344细胞的体外恶性转变，预防肝癌的发生。双环醇对高转移人肝癌细胞株MHCC97-H与LN、FN的粘附有明显的抑制作用，降低MHCC97-H细胞的VEGF mRNA的表达，抑制新生血管生成，从而抑制肿瘤的侵袭和转移，提示在预防和抑制肝癌的发生发展方面可能有一定的潜在价值。
专利文献1公开了一种肝癌靶向载人参皂苷CK壳聚糖聚合物胶束递药系统，其特征在于，包括：靶向头基，基础载体，以及抗癌药物；其中，所述靶向头基为A54多肽，所述基础载体为两亲性聚合物，所述抗癌药物为人 参皂苷CK；所述靶向头基通过连接基团与基础载体的疏水端连接形成递药载体；所述递药载体包载人参皂苷CK，形成肝癌靶向载人参皂苷CK壳聚糖聚合物胶束递药系统。
专利文献2公开了一种组合物，其包含下述组分：人参皂苷Rk1、人参皂苷Rg5、人参皂苷CK、人参总皂苷提取物，其中所述组合物为白介素(IL)-6调节剂。
专利文献3公开了双环醇及其可药用衍生物用于制备预防或治疗药物性肝损伤的药物的用途。
现有技术中或者使用人参皂苷单体，或者使用双环醇，然而其预防和治疗肿瘤效果有限，且现有技术没有公开人参皂苷单体可以促进预防和/或治疗肿瘤的药物组合物的作用，也没有公开联合使用人参皂苷单体和双环醇以协同增效、提高预防和/或治疗肿瘤的效果。
现有技术文献
专利文献1 CN109793711A公开文本
专利文献2 CN110090236A公开文本
专利文献3CN109106711A公开文本
发明内容
为了解决现有技术中单独使用人参皂苷单体或单独使用双环醇所带来的预防和治疗肿瘤效果有限的问题，本申请提供一种协同增效的抗肿瘤药物组合物，其可更加安全和强效的预防和治疗肿瘤，尤其是肝癌。
本申请的具体技术方案如下：
1、一种药物组合物，其特征在于，所述药物组合物包括：人参皂苷单体和双环醇。
2、根据项1所述的药物组合物，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:0.3～3。
3、根据项2所述的药物组合物，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:1。
4、根据项1-3中任一项所述的药物组合物，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、 人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3或人参皂苷Rh4；
优选地，所述人参皂苷单体包括人参皂苷CK。
5、人参皂苷单体和双环醇在制备预防和/或治疗肿瘤的药物组合物中的用途。
6、根据项5所述的用途，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:0.3～3。
7、根据项6所述的用途，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:1。
8、根据项5-7中任一项所述的用途，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3和人参皂苷Rh4；
优选地，所述人参皂苷单体包括人参皂苷CK。
9、根据项5-8中任一项所述的用途，其特征在于，所述肿瘤为肝癌。
10、人参皂苷单体在促进预防和/或治疗肿瘤的药物组合物的作用中的用途。
11、根据项10所述的用途，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3或人参皂苷Rh4；
优选地，所述人参皂苷单体包括人参皂苷CK。
12、根据项10或11所述的用途，其特征在于，所述预防和/或治疗肿瘤的药物组合物包括双环醇。
13、根据项10-12中任一项所述的用途，其特征在于，所述作用为协同增效作用。
申请的效果
本申请的包含人参皂苷单体和双环醇的药物组合物，尤其是包含人参皂苷CK和双环醇的药物组合物，能够抑制肿瘤细胞，尤其是肝癌细胞的增殖；抑制化学诱变剂诱导的大鼠肝上皮样干细胞向肝癌细胞的恶性转变；对裸鼠 体内肝癌的生长具有明显的抑制作用；能够预防致癌剂诱导的肝癌。包含人参皂苷CK和双环醇的药物组合物，相比于单一用药，有显著的协同和增效作用，是一类高效低毒的抗肿瘤药物组合。本申请相关的分子生物学研究也提示这一类药物组合具有显著的协同增效的分子机制和理论基础。双环醇可恢复化学致癌剂所引起的肝上皮细胞细胞间隙通讯连接(GJIC)功能的抑制，保护细胞基因组DNA，抑制化学致癌物诱发的肝上皮细胞恶性转化和肝脏肿瘤的发生发展。人参皂苷CK与PI3K/Akt信号通路、NF-κB信号通路和细胞周期调控信号通路等多条信号通路有关，可抑制肿瘤细胞生长和转移。人参皂苷CK和双环醇分别作用于肝癌发生的不同阶段，不同的靶点和信号通路，从而产生更强的预防和/或治疗肝癌的疗效。
附图说明
图1(a)-(b)分别为空白对照组和模型组3MC/TPA联合诱导后的WB-F344的细胞形态图。
图2(a)-(e)分别为空白对照组、模型组、人参皂苷CK组、双环醇组、人参皂苷CK/双环醇联用组的SD大鼠肝癌预防实验肝脏H&E染色病理观察图。
具体实施方式
以下将对本申请做以详细说明。
需要说明的是，在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式，然所述描述乃以说明书的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
本申请提供一种药物组合物，其特征在于，所述药物组合物包括：人参皂苷单体和双环醇。
本申请的人参皂苷单体可以为市售的人参皂苷单体，或从在自然界中栽培或采集的人参分离出的人参皂苷单体，或从分离出的一种人参皂苷单体转换成的另一种人参皂苷单体，或为通过合成方法合成的人参皂苷单体，然而可以使用任何示出本申请的肝癌预防或治疗效果的人参皂苷单体，而没有限制。所述人参皂苷单体可以选自人参皂苷CK、人参皂苷20-(S,R)-Rg3、人 参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3、人参皂苷Rh4等单体中的一种或两种以上，优选包括人参皂苷CK。
“人参皂苷CK”，化学名称为20-0-β-D-吡喃葡萄糖基-20-原人参二醇(人参皂苷CK)，分子式：C 36H 62O 8，分子量：622.87，无臭，白色粉末状。可溶于甲醇、乙醇，微溶与乙酸乙酯，水溶性较差，不溶于三氯甲烷、乙醚，CAS号：39262-14-1，其是作为诸如人参或红参等的参类的主要活性成分的皂苷之一，指的是属于原人参三醇(protopanaxadiol，PPT)类的人参皂苷单体。本申请中实验用的人参皂苷CK，是将人参提取得到的普通人参皂苷单体，通过定向生物转化技术，再经过分离纯化获得的纯度为98％以上的高活性稀有人参皂苷CK。人参皂苷CK的结构式为：
Figure PCTCN2021106558-appb-000001
“人参皂苷20-(S,R)-Rg3”，分子式：C 42H 70O 12，分子量767.0，类白色粉末，溶于甲醇，CAS号：14197-60-5，结构式为：
Figure PCTCN2021106558-appb-000002
“人参皂苷20-(S,R)-Rh2”，分子式：C 36H 62O 8，分子量622.87，无色针晶，易溶于甲醇，CAS号：78214-33-2，结构式为：
Figure PCTCN2021106558-appb-000003
“人参皂苷20-(S,R)-PPD”，分子式：C 30H 52O 3，分子量460.74，白色晶体，可溶于丙酮、DMSO等溶剂。CAS号：30636-90-9，结构式为：
Figure PCTCN2021106558-appb-000004
“人参皂苷Rk1”，分子式：C 42H 70O 12，分子量767.0，无臭，白色粉末状。可溶于甲醇、乙醇，微溶于乙酸乙酯，水溶性较差，不溶于三氯甲烷、乙醚，CAS号：494753-69-4，结构式为：
Figure PCTCN2021106558-appb-000005
“人参皂苷Rg5”，分子式：C 42H 70O 12，分子量：767.0，无臭，白色粉末状。可溶于甲醇、乙醇，微溶于乙酸乙酯，水溶性较差，不溶于三氯甲烷、乙醚，CAS号：74964-14-0，结构式为：
Figure PCTCN2021106558-appb-000006
“人参皂苷Rk3”，分子式：C 36H 60O 8，分子量：620.9，无味，白色粉末状。易溶于水、甲醇、乙醇，CAS号：364779-15-7，结构式为：
Figure PCTCN2021106558-appb-000007
“人参皂苷Rh4”，分子式：C 36H 60O 8；分子量：620.4；学名：6-O-β-D-glucopyranosyl-20(-HO)-trans-protopanaxatriol；无臭，白色粉末状。可溶于甲醇、乙醇，微溶于乙酸乙酯，水溶性较差，不溶于三氯甲烷、乙醚；CAS号：174721-08-5；结构式为：
Figure PCTCN2021106558-appb-000008
“双环醇”，其化学名称为4，4'-二甲氧基-5，6，5'，6'-双(亚甲二氧基)-2-羟甲基-2'-甲氧羰基联苯，分子式为C 19H 18O 9，临床上可用于治疗慢性肝炎所致的氨基转移酶升高。双环醇为联苯双酯结构类似物，具有抗肝炎病毒和抗肝细胞损伤两方面的作用。
本申请的药物组合物中，包含有效剂量的作为活性成分的人参皂苷单体和双环醇。有效剂量是指药物组合物发挥药学功能时的使用剂量。
在一个具体实施方式中，将人参皂苷单体、双环醇按本申请限定的比例范围进行组合，在对肝癌细胞的体外增殖的抑制作用方面、抑制肝癌前体细胞体外恶性转化方面、体内治疗肝癌方面、以及预防大鼠肝癌方面表现出协同增效。发明人还发现，在上述比例范围内，人参皂苷单体和双环醇也是低 毒性的。从协同增效作用显著的角度考虑，所述药物组合物中，人参皂苷单体和所述双环醇的重量比优选为1:0.3～3，具体可为1:0.3，1:0.4，1:0.5，1:0.6，1:0.7，1:0.8，1:0.9，1:1，1:1.1，1:1.2，1:1.3，1:1.4，1:1.5，1:1.6，1:1.7，1:1.8，1:1.9，1:2，1:2.1，1:2.2，1:2.3，1:2.4，1:2.5，1:2.6，1:2.7，1:2.8，1:2.9，1:3，优选为1:1。
在一个具体实施方式中，所述药物组合物在体外抑制癌细胞的增殖过程中，或者是在体外抑制正常细胞向癌细胞的恶性转化过程中，人参皂苷单体浓度可为3～30μM，具体可为3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM、21μM、22μM、23μM、24μM、25μM、26μM、27μM、28μM、29μM、30μM，优选为5μM～20μM；双环醇浓度可为1～20μM，具体可为1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM、11μM、12μM、13μM、14μM、15μM、16μM、17μM、18μM、19μM、20μM，优选为3μM～15μM。
在一个具体实施方式中，本申请的药物组合物，可以口服或非口服施用。施用量因症状程度、患者年龄、性别、体重、敏感性差异、施用方法、施用时期、施用间隔、药物制剂的性质、有效成分的种类等而异，无特殊限制，但通常成人(体重60kg)每日10mg～1.5g、优选50mg～900mg、更优选100mg～600mg、更优选300mg～500mg(以人参皂苷CK的总重量计)，上述施用量通常可每日分1～3次施用。
在一个具体实施方式中，本申请的药物组合物中，所述人参皂苷单体选自下述的一种或两种或三种或四种或五种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3和人参皂苷Rh4。
在一个具体实施方式中，本申请的药物组合物中，所述人参皂苷单体包括人参皂苷CK。除人参皂苷CK之外，还可以包含选自下述一种或两种以上的其它人参皂苷单体：人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3和人参皂苷Rh4，也可以不包含其它人参皂苷单体。在本申请的药物组合物中包含其它人参皂苷单体的情况下，从更好地发挥协同增效作用的角度考虑，以所述药物组合物中包含的总人参皂苷单体的总重量为100重量份计，所述人参皂苷CK的含量优选为50重量份以上、更优选为60重量份以上、更优 选为70重量份以上、更优选为80重量份以上、更优选为90重量份以上、更优选为95重量份以上、更优选为99重量份以上、更优选100重量份(即所述药物组合物中包含的人参皂苷单体仅为人参皂苷CK)。本申请的药物组合物使用的人参皂苷单体纯度可以为98％以上。
在一个具体实施方式中，本申请的药物组合物中可以包含药物可接受的载体，例如辅料。对于本申请的药物组合物中的辅料，没有特殊限制，例如可以使用本技术领域通常用于药品或保健品的辅料。具体地，所述辅料为淀粉、糊精、乳糖、甘露醇、羟丙甲纤维素钠、黄原胶、蛋白糖等。
在一个具体实施方式中，对本申请的药物组合物的剂型没有特殊限制，例如，可以是口服剂型或注射剂型。所述口服剂型可以是液体剂型，也可以是固体剂型。所述口服剂型可以为例如硬胶囊、软胶囊、缓控释放胶囊、压片、糖衣片、粉剂、颗粒剂、滴丸、水蜜丸、糖浆或口服液；所述注射剂型可以为例如溶液型、混悬液型、乳浊液型或冻干粉。所述用于改善睡眠的药物组合物的给药方式可以为例如口服、滴注或注射。
在一个具体实施方式中，制备口服用固体制剂时，可以在向主药中加入赋形剂以及视需要而定的粘合剂、崩解剂、滑润剂、着色剂、矫味剂等后，按照常规方法制成片剂、包衣片剂、颗粒剂、细粒剂、散剂、胶囊剂等。
在上述具体实施方式中，作为赋形剂，可使用例如乳糖、玉米淀粉、白糖、葡萄糖、山梨糖醇、结晶纤维素、二氧化硅等；作为粘合剂，可使用例如聚乙烯醇、乙基纤维素、甲基纤维素、阿拉伯胶、羟基丙基纤维素、羟基丙基甲基纤维素等；作为崩解剂，可使用例如干淀粉、羧甲基淀粉钠、聚乙烯吡咯烷酮、羧甲基纤维素钠的交联聚合物等；作为滑润剂，可使用例如硬脂酸镁、滑石、二氧化硅等；作为着色剂，可使用允许在药品中添加的着色剂；作为矫味剂，可使用可可粉、薄荷脑、芳香酸、薄荷油、龙脑、桂皮粉。当然，也可以在上述片剂、颗粒剂上包覆糖衣、明胶衣、以及其它的必要外衣。
在一个具体实施方式中，制备注射剂时，可以根据需要向主药中添加pH调节剂、缓冲剂、悬助剂、增溶剂、稳定剂、等渗剂、防腐剂等，再按照常规方法制成静脉、皮下、肌肉内注射剂。此时，也可以根据需要，利用常规方法制成冷冻干燥物。
在上述具体实施方式中，作为悬助剂，可列举例如甲基纤维素、吐温80、羟基乙基纤维素、阿拉伯胶、黄蓍树胶粉、羧甲基纤维素钠、聚氧乙烯山梨糖醇单月桂酸盐等。作为增溶剂，可列举例如聚氧化乙烯氢化蓖麻油、吐温80、烟酰胺、聚氧乙烯山梨糖醇单月桂酸盐、聚乙二醇、蓖麻油脂肪酸乙酯等。作为稳定剂，可列举例如亚硫酸钠、偏亚硫酸钠等；作为防腐剂，可列举例如对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、山梨酸、苯酚、甲酚、氯甲酚等。
将本申请的药物组合物施用于对象，可以预防和/或治疗肝癌。所述对象可以是哺乳动物，例如可以是人、大鼠、兔、羊、猪、牛、猫、狗、猴等，优选为人。
另一方面，发明人发现本申请的药物组合物具有显著的预防和/或治疗肿瘤尤其是预防和/或治疗肝癌的效果。因此，本申请也提供本申请的药物组合物在制备预防和/或治疗肿瘤的药物组合物中的应用。进一步地，所述肿瘤优选为肝癌。
本申请还提供人参皂苷单体在促进预防和/或治疗肿瘤的药物组合物的作用中的用途。
本申请的药物组合物，在抑制肝癌细胞的体外增殖方面、抑制肝癌前体细胞体外恶性转化方面、体内治疗肝癌方面、以及预防肝癌方面均表现出协同增效、甚至显著的协同增效作用。具体的，人参皂苷单体与双环醇相互作用指数CI值小于1、优选小于等于0.7。进一步地，以本申请所限定的比例对人参皂苷单体和双环醇进行组合，可以得到预防和/或治疗肝癌效果更为优异的药物组合物。
实施例
以下通过实施例进一步说明本申请的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器。
实施例1人参皂苷CK和双环醇联用抑制肝癌HepG2细胞的体外增殖
本实施例采用MTT法检测人参皂苷CK对肝癌HepG2细胞增殖的抑制作用。“MTT”化学名称为3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐，是一种黄色的染料。“MTT法”又称“MTT比色法”，是一种检测细胞存活和生长的方法。检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT 还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中，而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲臜，用酶标仪在490nm波长处测定其吸光度值，在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。根据测得的吸光度值(OD值)，来判断活细胞数量，OD值越大，细胞活性越强(如果是测药物毒性，则表示药物毒性越小)。该方法已广泛用于一些生物活性因子的活性检测、大规模的抗肿瘤药物筛选、细胞毒性试验以及肿瘤放射敏感性测定等。它的特点是灵敏度高、经济。
首先，取生长状态良好、细胞密度达到80％以上的HepG2细胞，加入胰酶消化，调整细胞密度为1×10 5个/mL，加入到96孔板中，每孔体积100μL，培养于37℃，5％培养箱。24h后，弃掉上清，设置人参皂苷CK组、双环醇组和人参皂苷CK/双环醇联用组，如表1所示向各加药组加入200μL不同浓度的人参皂苷CK、双环醇、人参皂苷CK/双环醇。相应的，空白对照组加入200μL1640培养液，每个浓度设置6个复孔。48h后，每孔加入150μLMTT和细胞培养液的混合液，其对应的体积比为1:2，继续培养4h。小心移除培养基，加入150μL DMSO，缓慢振荡10min，使其溶解生成的蓝紫色结晶Formazan。最后在490nm处检测每个孔对应的吸光度OD值，并利用以下公式来计算细胞抑制率：细胞抑制率(％)＝(1-加药组平均OD值/对照组平均OD值)×100％。
以两种单体相互作用指数CI来判定联用效果，CI＝AB/(A×B)，T为加药组细胞的OD值，C为对照组细胞的OD值，AB为两种单体联用组的T/C值，A、B为药物单独作用组的T/C值。CI＜1时，表示两种单体有协同作用，CI≤0.7时，表示协同作用非常显著。
实验数据以x±s表示，用SPSS17.0统计软件进行分析。组间两两比较用LSD法。P＜0.05表示差异显著，P＜0.01表示差异极显著。
MTT法测得的人参皂苷CK和双环醇对肝癌HepG2细胞的体外增殖的抑制作用结果如表1所示，不同浓度的人参皂苷CK单药使用和不同浓度的双环醇单药使用均有一定程度的抑制肿瘤细胞生长作用，且呈浓度依赖效应。与单用药物相比，人参皂苷CK和双环醇联合使用对HepG2的抑制作用更强，与单用药物相比，两种单体联用具有显著性差异(P＜0.05)，CI指数也显示两种单体联用具有明显的协同效应。并且，在人参皂苷CK与双环醇的总浓度不变的情况下，两者比例在本申请所述的1:0.3-3范围内时，CI指数低，且比例在1:1时，CI指数最小，协同效应最明显。
表1人参皂苷CK和双环醇对肝癌HepG2细胞的体外增殖的抑制作用(x±s)
Figure PCTCN2021106558-appb-000009
实施例2人参皂苷CK和双环醇联用抑制3MC/TPA联合诱导的WB-F344细胞向癌细胞的体外恶性转化
研究肿瘤化学预防药物的模型主要有体外系统和体内系统。体外系统是用各种致癌物直接或间接诱发正常细胞发生恶性转化，其有效指标为细胞恶性转化得到抑制，具有能够很好地模拟体内肿瘤发生的多阶段过程和实验条件易于控制等优点。大鼠肝上皮样干细胞(WB-F344细胞)来自正常成年同基因雄性大鼠，是一种肝脏幼稚上皮细胞，具有肝脏干细胞的性质，它与肝癌的发生有一定的关系，为肝癌前体细胞。
模型组：WB-F344细胞1500个/孔分别接种于6孔板，每孔2mL培养液。细胞接种后24h，培养液中加入致癌物3-甲基胆蒽(3MC)使其终浓度 为2μg/mL，培养96h后，除去3MC更换新鲜培养基，继续培养3天后，加入佛波醇肉豆蔻乙酸酯(TPA)，第8～12天，TPA浓度为50ng/mL进行诱导，随后TPA浓度为80ng/mL进行诱导，第25天，除去TPA，第30天，实验结束，用PBS液冲洗细胞，做Wright-Giemsa染色，镜下观察并计数转化灶数目。
空白对照组：在模型组基础上，用0.2％的DMSO代替3MC和TPA。
加药组：在模型组基础上，于细胞接种后24h加药处理，设置人参皂苷CK单独给药组(浓度为5μM、7.8μM、2.5μM、2μM)、双环醇单独给药组(浓度为5μM、2.2μM、7.5μM、8μM)、人参皂苷CK/双环醇联用组1(浓度为5μM/5μM)、人参皂苷CK/双环醇联用组2(浓度为7.8μM/2.2μM)、人参皂苷CK/双环醇联用组3(浓度为2.5μM/7.5μM)、人参皂苷CK/双环醇联用组4(浓度为2μM/8μM)，直至实验结束，其它处理同模型组。细胞在加入TPA后视细胞状态每1～3天换一次液。
利用以下公式来计算加药组的肝癌细胞抑制率：抑制率(％)＝(1-加药组转化灶数目/空白对照组转化灶数目)×100％。以两种单体相互作用指数CI来判定联用效果，CI＝AB/(A×B)，T为加药组细胞的转化灶数目，C为空白对照组细胞的转化灶数目，AB为两种单体联用组的T/C值，A、B为两种单体单独作用组的T/C值。CI＜1时，表示两种单体有协同作用，CI≤0.7时，表示协同作用非常显著。
实验结束后，空白对照组与模型组的细胞形态观察结果如图1所示，图1(a)为空白对照组的细胞形态，图中可以看出，正常生长的WB-F344细胞形态规则，呈多边形，在基质上铺展良好，胞质丰富，具接触抑制性。图1(b)模型组的细胞形态，图中可以看出，经转化的WB-F344细胞，形态不规则，为梭形或多边形，细胞大小不等，生长的极性消失，失去接触抑制，常有重叠生长，可见散在的转化灶。转化灶计数结果如下表2所示，实验结束时，模型组形成明显的转化灶，转化灶数目为58.6±7.2个，空白对照组细胞培养30天，也有较少的转化灶形成，与模型组相比有极显著性差异(P<0.05)。人参皂苷CK组、双环醇组和人参皂苷CK/双环醇联用组1、人参皂苷CK/双环醇联用组2、人参皂苷CK/双环醇联用组3、人参皂苷CK/双环醇联用组4，对转化灶的形成均有抑制作用(具体结果见表2)，两种单体相互作用 指数CI值均小于1，对于抑制WB-F344细胞的体外恶性转化具有协同增效作用。
表2人参皂苷CK和双环醇对3MC/TPA联合诱导的WB-F344细胞体外恶性转化的影响(x±s)
Figure PCTCN2021106558-appb-000010
与空白组相比， #P<0.05， ##P<0.01；与模型组相比，*P<0.05，**P<0.01，***P<0.01。
实施例3人参皂苷CK和双环醇联用治疗肝癌体内药效评价
建立人肝癌裸鼠异种移植瘤模型：4～5周龄的雌性BALB/c裸鼠，左侧腋窝皮下接种Hep-3B细胞，每只裸鼠注射4×10 6个细胞。肿瘤体积达到100～300mm 3时，将裸鼠随机分为16组，每组10只动物。
16组分别为1个模型组和15个加药组，15个加药组分别为：(1)5个人参皂苷CK组，灌胃给药，给药剂量分别为60mg/kg、30mg/kg、90mg/kg、100mg/kg、24mg/kg；(2)5个双环醇组，灌胃给药，给药剂量分别为60mg/kg、 90mg/kg、30mg/kg、20mg/kg、96mg/kg；(3)5个人参皂苷CK/双环醇联用组，灌胃给药，人参皂苷CK/双环醇给药剂量分别为60mg/kg/60mg/kg、30mg/kg/90mg/kg、90mg/kg/30mg/kg、100mg/kg/20mg/kg、24mg/kg/96mg/kg。各加药组给药体积0.2mL，每天给药一次，连续给药21天。每天观察1次，记录动物的临床状态，观察内容包括是否死亡、濒死、外观、精神状况及活动状况等。每周两次测试瘤长和瘤宽，并按如下公式计算肿瘤体积和抑瘤率(％)。肿瘤体积V＝0.5×a×b×b，式中，a为肿瘤的长，b为肿瘤的宽。抑瘤率(％)＝(模型组V-加药组V)/模型组V×100％。
以两种单体相互作用指数CI来判定联用效果，CI＝AB/(A×B)，T为加药组肿瘤体积，C为模型组的肿瘤体积，AB为两种单体联用组的T/C值，A、B为两种单体单独作用组的T/C值。CI＜1时，表示两种单体有协同作用，CI≤0.7时，表示协同作用非常显著。
连续给药21天，动物临床症状无异常表现。肿瘤体积测试结果如表3所示，两种单体联用的CI指数为0.68(CI＜0.7)，具有显著的协同增效作用。并且，在人参皂苷CK与双环醇的总浓度不变的情况下，两者比例在本申请所述的1:0.3-3范围内时，CI指数低于两者比例在在上述范围之外时的CI指数，且比例在1:1时，CI指数最小，协同效应最明显。
表3人参皂苷CK和双环醇对人肝癌裸鼠异种移植瘤模型裸鼠肿瘤体积的影响
Figure PCTCN2021106558-appb-000011
Figure PCTCN2021106558-appb-000012
与模型组相比，*P<0.05，**P<0.01。
实施例4人参皂苷CK和双环醇联用预防DEN诱导的大鼠肝癌
模型建立及给药：购买SD大鼠100只，动物房适应一周后，随机分成5组，分别是空白对照组、模型组、人参皂苷CK组、双环醇组、人参皂苷CK/双环醇联用组。人参皂苷CK组和双环醇组均为灌胃给药，人参皂苷CK组给药剂量为60mg/kg，双环醇组给药剂量为60mg/kg，人参皂苷CK/双环醇联用组给药剂量为60mg/kg/60mg/kg，空白对照组和模型组给予等体积溶媒，连续灌胃给药20周。灌胃给药一周后，除空白对照组外，各组动物腹腔注射化学诱变剂二乙基亚硝胺(DEN)20mg/kg连续2周，40mg/kg连续2周，60mg/kg连续2周以及80mg/kg连续2周。
指标的检测：灌胃给药20周后，处死动物。动物处死前称量大鼠体重。腹腔注射戊巴比妥钠溶液麻醉大鼠，剖开腹壁，暴露肝脏，观察肝脏的大小、形态、色泽，有无结节等。腹主动脉采血，以供检测谷丙转氨酶(ALT)和谷草转氨酶(AST)。将肝脏完整取出，测量肝重，计算肝脏系数，肝脏系数＝肝重/体重*100％。取部分肝脏样本，以4％甲醛固定，制作H&E染色病理切片。H&E染色是病理切片常用的染色方法，是苏木素-伊红(Hematoxylin-Eosin)染色法。这种方法对任何固定液固定的组织和应用各种包埋法的切片均可使用。苏木素是一种碱性染料,可使组织中的嗜碱性物质染成蓝色，如细胞核中的染色质等；伊红是一种酸性染料，可使组织中的嗜酸性物质染成红色，如多数细胞的胞质、核仁等在H&E染色的切片中均呈红色。
实验结果：
(1)肝癌预防模型大鼠血清ALT、AST和ALP水平
结果如表4所示，与空白对照组比较，模型组大鼠血清ALT、AST和ALP水平显著上升(P<0.05)，说明化学诱变剂DEN在诱导SD大鼠肝癌过程中，伴随着血清肝功指标ALT、AST和ALP水平的变化。与模型组比较，人参皂苷CK组和双环醇组的血清ALT、AST和ALP水平均显著降低(P<0.05)，而人参皂苷CK/双环醇联用组效果明显优于单独用药组，血清ALT、AST和ALP水平接近空白对照组。
表4人参皂苷CK和双环醇对肝癌预防模型大鼠血清ALT、AST和ALP水平的影响(x±s)
Figure PCTCN2021106558-appb-000013
与空白组比较， #P<0.05；与模型组比较，*P<0.05。
(2)肝癌预防模型大鼠体重、肝重及肝脏指数
灌胃给药20周后，如表5所示，模型组大鼠体重显著低于空白对照组，肝重和肝脏系数均显著高于空白对照组(P＜0.05)。脏器系数变化一般反映脏器的肿大、充血、增生肥大等情况，是肝损伤的指标指标，腹腔注射DEN可使大鼠肝脏系数增加，推测大鼠肝脏可能发生损伤。人参皂苷CK组、双环醇组和人参皂苷CK/双环醇联用组大鼠体重显著高于模型组(P＜0.05)，肝脏系数显著低于模型组(P＜0.05)，而且人参皂苷CK/双环醇联用组的效果明显优于单独给药组。
表5人参皂苷CK和双环醇对大鼠体重、肝重和肝脏系数的影响(x±s)
Figure PCTCN2021106558-appb-000014
Figure PCTCN2021106558-appb-000015
与空白对照组比较， #P<0.05；与模型组比较，*P<0.05。
(3)肝脏H&E染色病理切片
灌胃给药20周后处死所有大鼠，如图2(a)-(e)所示，空白对照组大鼠肝脏组织呈深红色，大小正常，柔软脆弱，肝脏表面光滑无异常，H&E染色切片可见规则的肝小叶和肝细胞索结构；而模型组动物肝脏出现严重病变，肝脏质地变硬，各叶散在大小不等的癌结节，有呈巨块状，H&E染色证实为肝细胞癌或中重度肝硬化；人参皂苷CK组及双环醇组亦有部分动物肝脏存在一些灰白色结节，肝癌发生例数及肝脏病变程度较模型组为轻；而人参皂苷CK/双环醇联用组仅有个别只动物肝脏存在少量灰白色结节，肝癌发生例数及肝脏病变程度都明显低于单独给药组。
通过本实施例显示，本申请公开的人参皂苷CK和双环醇两种单体联用在应用于预防致癌剂DEN诱导的肝癌模型时，表现出极强的药效，明显优于单独用药，且未发现明显的不良反应，具有良好的安全性。
以上所述，仅是本申请的较佳实施方式而已，并非是对本申请作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容，依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本申请技术方案的保护范围。

Claims (13)

  1. 一种药物组合物，其特征在于，所述药物组合物包括：人参皂苷单体和双环醇。
  2. 根据权利要求1所述的药物组合物，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:0.3～3。
  3. 根据权利要求2所述的药物组合物，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:1。
  4. 根据权利要求1-3中任一项所述的药物组合物，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3和人参皂苷Rh4；
    优选地，所述人参皂苷单体包括人参皂苷CK。
  5. 人参皂苷单体和双环醇在制备预防和/或治疗肿瘤的药物组合物中的用途。
  6. 根据权利要求5所述的用途，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:0.3～3。
  7. 根据权利要求6所述的用途，其特征在于，所述人参皂苷单体和所述双环醇的重量比为1:1。
  8. 根据权利要求5-7中任一项所述的用途，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、和人参皂苷Rh4；
    优选地，所述人参皂苷单体包括人参皂苷CK。
  9. 根据权利要求5-8中任一项所述的用途，其特征在于，所述肿瘤为肝癌。
  10. 人参皂苷单体在促进预防和/或治疗肿瘤的药物组合物的作用中的用途。
  11. 根据权利要求10所述的用途，其特征在于，所述人参皂苷单体选自下述的一种或两种以上：人参皂苷CK、人参皂苷20-(S,R)-Rg3、人参皂 苷20-(S,R)-Rh2、人参皂苷20-(S,R)-PPD、人参皂苷Rk1、人参皂苷Rg5、人参皂苷Rk3或人参皂苷Rh4；
    优选地，所述人参皂苷单体包括人参皂苷CK。
  12. 根据权利要求10或11所述的用途，其特征在于，所述预防和/或治疗肿瘤的药物组合物包括双环醇。
  13. 根据权利要求10-12中任一项所述的用途，其特征在于，所述作用为协同增效作用。
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