WO2014131360A1 - 普罗布考及其衍生物抗肿瘤转移的用途 - Google Patents

普罗布考及其衍生物抗肿瘤转移的用途 Download PDF

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WO2014131360A1
WO2014131360A1 PCT/CN2014/072632 CN2014072632W WO2014131360A1 WO 2014131360 A1 WO2014131360 A1 WO 2014131360A1 CN 2014072632 W CN2014072632 W CN 2014072632W WO 2014131360 A1 WO2014131360 A1 WO 2014131360A1
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probucol
metastasis
pharmaceutical composition
tumor
derivative
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French (fr)
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李亚平
张志文
刘泽莹
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中国科学院上海药物研究所
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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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention relates to a novel use of probucol or a derivative thereof, in particular to the preparation of anti-tumor metastasis, reducing the incidence of malignant tumor metastasis, reducing the number of metastases, and improving the survival rate of tumor-bearing animals. Use in medicine. Background technique
  • Cancer metastasi s refers to the process in which tumor cells are freed from the primary tumor, infiltrated into the surrounding tissues, invaded into the circulatory system and subsequently metastasized in the body, and adhered to endothelial cells to form metastatic tumors.
  • Tumor metastasis is one of the main causes of tumor failure and recurrence, and it is also a key factor leading to the death of tumor patients.
  • Clinical diagnosis shows that more than 60% of newly diagnosed tumor patients have metastasized, and their 5-year survival rate is less than 20%. At present, only a very small number of patients with metastasis can be treated effectively by surgery, while other clinical treatments are very limited. Therefore, the prevention and treatment of tumor metastasis faces very serious challenges.
  • Tumor metastasis pathways mainly include lymphatic metastasis, hematogenous metastasis, implant metastasis, and local diffusion and metastasis. Lymphatic metastasis and blood metastasis are the most important pathways. Therefore, it is one of the prevention and treatment of tumor metastasis by effectively inhibiting various related factors in the process of invasion, migration and implantation of tumor cells, and improving the lymphatic targeting of drugs and the selective distribution of tumor metastasis sites.
  • probucol and its compositions have anti-tumor metastasis effects, can reduce the incidence of malignant tumor metastasis, reduce the number of metastases, and improve the survival rate of tumor-bearing animals.
  • a use of probucol or a derivative thereof for the preparation of a medicament (pharmaceutical composition) for inhibiting tumor metastasis ⁇ 90 ⁇ % ⁇
  • the content of the drug (pharmaceutical composition) of the probucol or its derivative is 0. 01- 99wt%, preferably, 0. 1- 90wt%.
  • the medicament (pharmaceutical composition) is also used to inhibit the MMP-9 gene or its protein, or the VCAM-1 factor.
  • the effective concentration of the probucol or its derivative is
  • O.OlnM/L-lmM/L preferably 0.1 nM/L-50 ( ⁇ M/L, optimally 4 nM/L-40 ( ⁇ M/L.
  • the tumor comprises breast cancer.
  • the metastasis comprises a lung metastasis.
  • the probucol derivative comprises a monosuccinate of probucol, i.e., amber or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt of amber cloth comprises a salt formed by reacting with a base: a potassium salt, a sodium salt, a calcium salt or a magnesium salt.
  • the probucol succinate is amber cloth (AGI-1067).
  • a use of a probucol pharmaceutical composition for the preparation of a medicament for inhibiting tumor metastasis comprising a therapeutically effective amount of one or more selected from the group consisting of Pro Compounds in Bucai or derivatives thereof and pharmaceutical excipients.
  • the tumor comprises breast cancer.
  • the metastasis comprises a lung metastasis.
  • the probucol pharmaceutical composition is a self-microemulsion, a nanosuspension, a nanoparticle or a nanoemulsion.
  • the probucol pharmaceutical composition is a capsule, a tablet, a solid dispersion or a granule.
  • the probucol derivative is probucol succinate AGI-1067.
  • a method for non-therapeutic inhibition of tumor cell metastasis in vitro comprising the steps of: culturing said tumor cells in a culture system comprising an effective amount of probucol or a derivative thereof.
  • the tumor cells are derived from breast cancer.
  • a pharmaceutical composition for treating or inhibiting tumor metastasis which comprises probucol or a derivative thereof as an active ingredient, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a chemotherapeutic agent that inhibits tumor cell growth or proliferation.
  • a method of treating or inhibiting metastasis wherein a safe and effective amount of probucol or a derivative thereof, or a pharmaceutical composition comprising probucol or a derivative thereof, is administered to a desired subject.
  • the present invention provides an inhibitor of the MMP-9 gene or a protein thereof, or a VCAM-1 gene or a protein thereof, wherein the inhibitor is an effective amount of probucol or a derivative thereof Or a pharmaceutical composition containing probucol or a derivative thereof.
  • the probucol derivative described herein may be probucol succinate AGI-1067.
  • the probucol pharmaceutical composition comprises a therapeutically effective amount of one or more compounds selected from probucol or a derivative thereof, and a pharmaceutical excipient.
  • the probucol pharmaceutical composition may be a self-microemulsion, a nanosuspension, a nanoparticle or a nanoemulsion.
  • the probucol pharmaceutical composition can be a capsule, a tablet, a solid dispersion or a granule.
  • Figure 1 shows in vitro experiments with probucol or its derivatives to inhibit lung metastasis in breast cancer.
  • Figure 2 shows that probucol or its derivatives inhibit the onset of lung metastasis in breast cancer.
  • Figure 3 shows a comparison of the effects of probucol and its nanoformulations in cell scratch assays, where TX100 represents Triton X-100 (Triton X-100) as a negative control and DNP represents the nanoformulation prepared in Example 11. .
  • Figure 4 shows a comparison of the effects of probucol and its nanoformulations in cell migration experiments.
  • Figure 5 shows a comparison of the effects of probucol and its nanoformulations in cell invasion experiments.
  • Figure 6 shows a comparison of the effects of amber cloth and its nanoformulation in a cell scratch test, wherein P188 represents Poloxamer P188 as a negative control and PSN represents the nanoformulation prepared in Example 12.
  • Figure 7 shows a comparison of the effects of amber cloth and its nanoformulations in cell migration experiments.
  • Figure 8 shows a comparison of the effects of amber cloth and its nanoformulations in cell invasion experiments.
  • Figure 9 shows a comparison of the efficacy of probucol and its nanoformulations in survival experiments in breast cancer model mice.
  • Figure 10A shows the effect of the intervention of probucol and its nanoformulations on lung metastasis in breast cancer.
  • Figure 10B shows the effect of intervention on the inhibition of lung metastasis in amber cloth and its nanoformulations.
  • Figure 11A shows the effect of probucol and its nanoformulations on lung cancer metastasis in vivo. Comparison of pathological specimens.
  • Figure 11B shows a comparison of the effect of amber cloth and its nanoformulation on the inhibition of lung metastasis in vivo.
  • Figure 12 shows that probucol and its nanoformulations have no effect on cancer cell activity over a range of concentrations.
  • Figure 13 shows that probucol and its nanoformulations have no significant effect on cancer cell apoptosis over a range of concentrations.
  • Figure 14 shows that probucol and its nanoformulations have no significant effect on tumor cell growth in vivo.
  • Figure 15 shows that amber cloth and its nanoformulation have no effect on cancer cell activity over a range of concentrations.
  • Figure 16 shows that amber cloth and its nanoformulation have no significant effect on cancer cell apoptosis over a range of concentrations.
  • Figure 17 shows that probucol and its nanoformulations are capable of inhibiting the expression of ⁇ P-9.
  • FIG. 18 shows that amber cloth and its nanoformulation inhibit the expression of VCAM-1.
  • the inventors have conducted long-term and in-depth research and screened a large number of active compounds, and for the first time unexpectedly found that probucol or its derivatives for the treatment of cardiovascular diseases (such as atherosclerosis) can be used for malignant solid tumors (especially It is a metastasis of breast cancer (such as lung metastasis) with good inhibitory activity.
  • cardiovascular diseases such as atherosclerosis
  • breast cancer such as lung metastasis
  • the inventors have also found through experiments that probucol or its derivatives do not inhibit the growth or proliferation of tumor cells themselves, and their inhibition of tumor metastasis may be closely related to the inhibition of MMP-9 or VCAM-1 expression. Relationship. On the basis of this, the present invention has been completed. the term
  • the term "probucol derivatives” refers to the production of probucol after reaction with an acid.
  • the ester and the pharmaceutically acceptable salt further formed from the ester and base.
  • the acid includes hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; organic acids include: formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1, 5), asiatic acid, oxalic acid, tartaric acid , lactic acid, salicylic acid, benzoic acid, valeric acid, diethyl acetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, styrene Acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluen
  • the terms "amber cloth test”, “probucol succinate”, “probucol monosuccinate”, “AGI-1067” are used interchangeably and refer to both probucol and succinic acid.
  • the amber cloth of the present invention can be prepared by conventional preparation techniques in the field.
  • the pharmaceutically acceptable salt of the probucol monoester comprises a potassium salt, a sodium salt, a calcium salt or a magnesium salt.
  • Probucol also known as probucol, was the first lipid-lowering drug marketed in the United States in 1977. It fades out of the market because it lowers cholesterol while lowering high-density lipoprotein cholesterol (HDL).
  • the main pharmacological effects of probucol include lowering cholesterol synthesis and promoting cholesterol breakdown, altering the nature and function of high-density lipoprotein subtypes, anti-oxidation, inhibiting the formation of foam cells, delaying the formation of atherosclerotic plaques, and regressing Formation of atherosclerotic lesions.
  • the invention also provides a pharmaceutical composition having significant anti-tumor efficacy, comprising a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable salts Carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of anagrelide hydrochloride, and one or more pharmaceutically acceptable carriers (or pharmaceutical excipients).
  • the compound itself or a mixture of a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, diluent or the like may be administered orally in the form of a tablet, capsule, granule, powder or syrup or may be administered orally in the form of an injection. Dosing.
  • the pharmaceutical composition preferably contains, as an active ingredient, a compound of the formula I of the present invention or a pharmaceutically acceptable salt thereof in an amount of from 0.01% to 99% by weight, more preferably from 0.1% to 90% by weight of the active ingredient.
  • compositions can be prepared by conventional pharmaceutical methods.
  • useful pharmaceutical adjuvants include excipients (e.g., saccharide derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, Potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose; gum arabic; dextran Silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc., binders (eg gelatin, polyvinylpyrrole) Anthrone and polyethylene glycol), disintegrants (such as cellulose derivatives such as sodium carboxymethylcellulose, polyvinylpyrrolidone), lubricants (such as talc, calcium stea
  • the dose of the compound of the present invention, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof varies depending on the age, sex, race, condition, and the like of the patient.
  • a dose of 200 mg/kg was used, and those skilled in the art can screen a safe and effective amount of the human drug according to the amount of the in vivo experiment.
  • the daily dose for an adult is about 10 mg to 2000 mg, preferably 50 mg to 1000 mg.
  • Probucol or a derivative thereof of the present invention can be prepared by a conventional means in the art, and for example, can be formulated into tablets, capsules, emulsions, suspensions.
  • probucol or a derivative thereof can be prepared as a nano preparation.
  • the present invention provides a method of inhibiting metastasis of tumor cells, the method comprising administering to a subject cell in need thereof an effective amount of probucol or a derivative thereof or a pharmaceutical composition comprising probucol or a derivative thereof.
  • the pharmaceutical composition comprises: (a) a therapeutically effective amount of probucol or a derivative thereof; and (b) a pharmaceutically acceptable carrier.
  • the tumor comprises: breast cancer, lung cancer.
  • the compound of formula I has an effective concentration of 0.01 nM/L-lmM/L, preferably 0.1 nM/L-50 ( ⁇ M/L, most preferably lnM/L-10). ( ⁇ M/L.
  • the invention also provides a method for preparing a drug or pharmaceutical composition for anti-tumor metastasis, the method comprising: mixing a therapeutically effective amount of probucol or a derivative thereof with a pharmaceutically acceptable carrier to form a pharmaceutical combination Things.
  • the pharmaceutical composition further comprises a component selected from the group consisting of: a chemotherapeutic agent for inhibiting tumor growth or proliferation.
  • the invention opens up a new use of probucol or its derivatives for inhibiting tumor metastasis, especially for breast cancer lung metastasis, which can effectively inhibit the occurrence of breast cancer lung metastasis, improve the survival rate, and reduce the incidence of metastasis, indicating that Compounds and their compositions have potential applications in inhibiting metastasis of malignant tumors.
  • the probucol is prepared into a drug-containing granule by wet granulation, dry granulation or direct mixing by using starch, pregelatinized P powder, microcrystalline cellulose, lactose, povidone, and the like.
  • Probucol capsules Use probucol with starch, pregelatinized starch, microcrystalline cellulose, lactose, povidone, etc.
  • the granules, dry granulation or direct mixing are prepared into drug-containing granules, and then the probucol tablet is compressed by a tableting machine.
  • Example 5 Probucol capsules
  • Probucol and starch, pregelatinized starch, microcrystalline cellulose, lactose, povidone, etc. are prepared by wet granulation, dry granulation or direct mixing to prepare drug-containing granules, which are then loaded into a capsule shell to prepare Probucol capsules.
  • Example 6 Probucol porous carrier complex
  • the probucol is mixed with a porous material containing pores such as porous starch and crospovidone, and is dispersed in ethanol, ultrasonically mixed, and then evaporated to dryness to remove an organic solvent.
  • Example 7 Probucol Derivative (Amber Cloth, AGI-1067) Porous Carrier Complex The probucol derivative (AGI-1067) was mixed with mesoporous silica, diatomaceous earth, etc., and dispersed together in ethanol. , ultrasonically mixed, and then evaporated to remove the organic solvent, that is, obtained.
  • Example 8 Probucol Nanoemulsion
  • Probucol is mixed with Tween 80, zhonglian triglyceride and polyethylene glycol stearate HS-15 (1:2:1:1, w/w), dissolved in ethanol, evaporated to dryness to remove organic solvents Add water to mix.
  • Example 9 Probucol Derivative (Amber Cloth, AGI-1067) Self-microemulsion
  • the probucol derivative (AGI-1067) is mixed with Cremphol EL, zhonglian triglyceride and Transcutol HP: 10:3:7, w/w), sonicated, and mixed with water when administered.
  • Example 10 Probucol Solid Dispersion
  • Probucol is mixed with phospholipids, sodium cholate (1: 2:1, w/w), dissolved in methanol, and evaporated to remove the organic solvent.
  • Example 11 Probucol Nanoparticles
  • the probucol was mixed with the surfactant Triton X-100 in a ratio of 1:2 (w/w), dissolved in ethanol, and evaporated to dryness at 37 ° C to remove the organic solvent, and shaken with water to obtain DNP.
  • Example 12 Probucol Derivative (Amber Cloth, AGI-1067) Nanoparticles
  • the probucol derivative (amber cloth test, AGI-1067) and the surfactant Poloxamer P188 were mixed in a ratio of 1:3, dissolved in ethanol, and evaporated to dryness at 37 ° C to remove the organic solvent, and shaken with water to obtain PSN.
  • AGI-1067 amber cloth test
  • Poloxamer P188 surfactant Poloxamer P188
  • Probucol was mixed with povidone PVPkl2 and sodium dodecyl sulfate and ground in a planetary mill for 30 minutes to obtain a probucol nanosuspension.
  • Test Example 1 Probucol and its nano preparations inhibit cancer cell metastasis in vitro
  • 4T1 cells purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences
  • the cells were inoculated into a 6-well plate at a ratio of 5 ⁇ 10 6 cells/well.
  • the cells were formed into a single layer thick, the culture solution was aspirated, and the tip was evenly scratched and washed with PBS. 3 times, replaced with fresh medium, photographed under the microscope, added Probucol (40 ng / mL, 400 ng / mL), preparation excipients (equivalent to 40 ng / mL, 400 ng / mL Probucol)
  • the nano-preparation group (40 ng/mL, 400 ng/mL) was further cultured for 36 hours, and the healing of the scratch was photographed.
  • the results showed that the probucol and nanoformulation groups were able to effectively inhibit the healing of cell scratches compared to the control (Fig. 3).
  • FBS-free medium is added to the upper layer of the Transwell chamber, 600 ⁇ of FBS-containing medium is added to the lower layer, and Probucol (40 ng/mL, 400 ng/mL) is added to the upper and lower chambers.
  • Probucol 40 ng/mL, 400 ng/mL
  • the ng/mL, 400 ng/mL probucol) and nano-preparation groups were continued for 24 hours.
  • the Transwell chamber was removed, fixed in 90% ethanol for 10 min, dried and stained with crystal violet solution for 30 min, and the cells in the upper layer of the chamber were wiped clean, and then photographed under a microscope. The results showed that the probucol and nanoformulation groups were able to effectively inhibit the cell migration ability of 4T1 cells compared with the control (Fig. 4).
  • Transwell method was used to study the invasive properties of cells.
  • the upper layer of Tanswell chamber was added with ⁇ matrigel solution (containing 10% Matrigel) overnight, and 2 ⁇ 10 5 4T1 cells were suspended in ⁇ FBS-free medium and added to Transwell.
  • the upper layer of the chamber the lower layer is added with 600 ⁇ FBS-containing culture.
  • the Transwell l chamber was removed, fixed in 90% ethanol for 10 min, dried and stained with crystal violet solution for 30 min.
  • the unmigrated cells in the upper chamber were wiped clean and photographed under a microscope.
  • the results showed that the probucol and nano-formulation groups were able to effectively inhibit the cell invasion of 4T1 cells compared with the control, and the ability of the nano-preparation group to reduce cell invasion at 400 ng/mL was significantly better than that of the probucol group (Fig. 5).
  • DNP is the probucol nano preparation group (Example 11) 1. 4 Probucol nano preparations comparison
  • the transwel l chamber was used to test the inhibitory effect of probucol on the metastasis of tumor cells cultured in vitro.
  • the nanocomposites prepared in Examples 8 and 11 and the cells were co-cultured in a chamber with a medium containing no FBS, and the drug probucol or AGI 1067 was administered at a concentration of 100 g ⁇ mL - blank cells without any drug were
  • the cells were stained with crystal violet, and the unmigrated cells in the chamber were removed with a cotton swab, and the cell migration was observed under a microscope.
  • the results are shown in Fig. 1, indicating that the probucol nano drug composition can significantly inhibit the migration of breast cancer cell 4T1 cells.
  • Mouse mammary fat pad inoculation 4T 1-LUC cells, 4T1 established orthotopic tumor model, n 8, oral administration of the composition prepared in Example 1, Test Example 11, at a dose of 200 ⁇ 3 ⁇ 4 ⁇ kg- 1, is administered daily. Once, continuous administration for 30-40 days, the lung metastasis of breast cancer was examined, and the results are shown in Figure 2 and Table 1.
  • Example 11 1/8 75% 1. 0 ⁇ 1. 7
  • the death of the animals was recorded, and the results showed that the nano-formulation group was able to effectively prolong the survival rate of the tumor model (Fig. 9).
  • Example 12 1/8 75% 0. 6 ⁇ 0 ⁇ 5 Test Example 5 Detection of inhibition of tumor cells by probucol
  • the cytotoxicity of probucol and its nano-preparation in 4T1 cells was measured by sputum method, and 4T1 cells were seeded in a 96-well plate at 8000 cells/well, overnight, and sequentially added 0. 004, 0. 04, 0.4, 4 and 4 (1 g/mL of probucol, TX100 (equivalent to probucol concentration) and nano-preparation group, continued to culture for 48 h and then detected cytotoxicity by MTT method, the results showed that 4 ng / mL to 4 g / mL Within the concentration range, there was almost no change in cell viability, and probucol and nanoformulations were not cytotoxic (Fig. 12).
  • Flow cytometry was used to detect apoptosis of probucol and its nano preparations in 4T1 cells.
  • 4T1 cells were seeded in 6-well plates at 1.5 ⁇ 10 5 cells/well overnight, and 40 and 400 ng were added respectively.
  • /mL of probucol, TX100 (equivalent to Probucol concentration) and nano-preparation group continue to culture for 48h After staining with the Annexin V-FITC/PI test kit, the apoptosis characteristics of the cells were detected by flow cytometry. The results showed that the apoptosis characteristics were almost unchanged at 40 ng/mL and 400 ng/mL compared with the control (Fig. 13).
  • a 4T1 orthotopic tumor model was established, and the probucol and nano-preparation groups were administered daily to detect tumor growth.
  • the results showed that the probucol and nano-prepared groups could not inhibit tumor growth compared with the saline group.
  • the tumor growth curve is shown in Figure 14.
  • probucol has no cytotoxic effect on cancer cells, and thus cannot inhibit the growth or proliferation of tumor cells, nor induce apoptosis of tumor cells.
  • Test Example 6 Inhibition of tumor cells by amber cloth test
  • Example 7 Probucol, amber cloth and its nano-preparation mechanism of tumor metastasis inhibiting gene expression studies on probucol, amber cloth and its nano-preparation in tumor cell metastasis, the results are shown in Figure 17. 18.
  • Figure 17 shows the immunofluorescence expression of MMP-9 expression in healthy mice and 4T1-induced in situ breast cancer metastasis model under the treatment of saline, probucol and DNP, respectively. Green (bright gray) fluorescent marker. It can be seen that in the model group treated with probucol, the expression of ⁇ P-9 was significantly lower than that of the control group, and the effect of the nano preparation was better.
  • Figure 18 shows the immunofluorescence of VCAM-1 expression in healthy mice and 4T1-induced lung metastasis models under normal saline, amber cloth and PSN treatments, which can be seen after amber cloth test.
  • the expression of VCAM-1 was significantly lower than that of the control group, and the effect of the nano-formulation was better.

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Abstract

本发明涉及一种普罗布考或其衍生物在制备用于抑制肿瘤转移，优选乳腺癌转移，更优选乳腺癌肺转移的药物中的用途。本发明还涉及一种普罗布考药物组合物在制备用于抑制肿瘤转移优选乳腺癌转移，更优选乳腺癌肺转移的药物中的用途，其中，所述普罗布考药物组合物包含治疗有效量的一种或多种选自普罗布考或其衍生物中的化合物以及药物辅料。

Description

普罗布考及其衍生物抗肿瘤转移的用途
技术领域
本发明涉及普罗布考或其衍生物的新用途， 具体涉及普罗布考或其衍生物 在制备具有抗肿瘤转移， 降低恶性肿瘤转移发生率， 减少转移灶的数目， 提高 荷瘤动物生存率的药物中的用途。 背景技术
肿瘤转移 （Cancer metastasi s ) 是指肿瘤细胞从原发瘤游离， 向周围组 织浸润， 侵袭进入循环系统并随之在体内转移， 并且与内皮细胞粘附浸润形成 转移瘤等过程。 肿瘤转移是肿瘤难以治愈和复发的主要原因之一， 也是导致肿 瘤患者死亡的关键因素。 临床诊断结果显示， 约 60%以上的初诊肿瘤患者已经 发生转移， 其 5年生存率不到 20%。 目前， 只有极少数的肿瘤转移患者能够通 过手术进行有效治疗， 而其他临床治疗方式效果非常有限。 因此， 肿瘤转移的 防治面临着非常严峻的挑战。
肿瘤转移的过程非常复杂， 主要包括各种基质金属蛋白酶如匪 P-2、匪 P-9 等， 炎症因子如 IL-6、 TNF- α 等， 信号转导通路以及各种粘附分子如 VCAM- 整合素和选择素等多种转移相关因子的共同参与。 肿瘤转移途径主要有淋巴道 转移、 血道转移、 种植转移和局部扩散转移等， 其中淋巴转移和血液转移是最 主要的途径。 因此， 有效抑制肿瘤细胞的侵袭、 迁移和着床等转移过程中的多 种相关因子， 提高药物的淋巴靶向性和肿瘤转移病灶部位的选择性分布， 是防 治肿瘤转移的之一。
因此， 本领域迫切需要开发抑制肿瘤转移的新型药物制剂， 从而延长肿瘤 患者的生存时间， 并改善生存质量。 发明内容
本发明人发现普罗布考及其组合物具有抗肿瘤转移的作用， 能降低恶性肿 瘤转移发生率， 减少转移灶的数目， 提高荷瘤动物生存率。 本发明第一方面， 提供了一种普罗布考或其衍生物在制备用于抑制肿瘤转 移的药物（药物组合物）中的用途。 在另一优选例中， 所述的药物（药物组合物）中普罗布考或其衍生物的含量 为 0. 01- 99wt%， 较佳地， 为 0. 1- 90wt%。 在另一优选例中， 所述的药物（药物组合物）还用于抑制 MMP-9基因或其蛋 白、 或 VCAM-1因子。
在另一优选例中， 所述普罗布考或其衍生物的有效浓度为
O.OlnM/L-lmM/L, 较佳地为 0.1nM/L-50(^M/L， 最佳地为 4nM/L-40(^M/L。
在另一优选例中， 所述肿瘤包括乳腺癌。
在另一优选例中， 所述转移包括肺转移。
在另一优选例中， 所述普罗布考衍生物包括普罗布考的单琥珀酸酯即琥珀 布考或其药学上可接受的盐。
在另一优选例中， 所述的琥珀布考药学上可接受的盐包括与碱反应形成的 盐： 钾盐、 钠盐、 钙盐或镁盐。
在另一优选例中， 所述的普罗布考琥珀酸酯为琥珀布考 (AGI-1067)。 本发明第二方面， 提供了一种普罗布考药物组合物在制备用于抑制肿瘤转 移的药物中的用途， 所述普罗布考药物组合物包含治疗有效量的一种或多种选 自普罗布考或其衍生物中的化合物以及药物辅料。
在另一优选例中， 所述肿瘤包括乳腺癌。
在另一优选例中， 所述转移包括肺转移。
在另一优选例中， 所述普罗布考药物组合物为自微乳、 纳米混悬液、 纳米 粒或纳米乳。
在另一优选例中， 所述普罗布考药物组合物为胶囊、 片剂、 固体分散体或 颗粒剂。
在另一优选例中， 所述普罗布考衍生物为普罗布考琥珀酸酯 AGI-1067。 本发明第三方面， 提供了一种体外非治疗性的抑制肿瘤细胞转移的方法， 包括步骤： 在含有有效量的普罗布考或其衍生物的存在的培养体系下， 培养所 述肿瘤细胞。
在另一优选例中， 所述的肿瘤细胞来自乳腺癌。
本发明第四方面， 提供了一种用于治疗或抑制肿瘤转移的药物组合物， 所 述的药物组合物含有普罗布考或其衍生物作为活性成分， 以及药学上可接受的 载体。 在另一优选例中， 所述的药物组合物还包括抑制肿瘤细胞生长或增殖的化 疗剂。
本发明第五方面， 提供了一种治疗或抑制肿瘤转移的方法， 向所需要的对 象施用安全有效量的普罗布考或其衍生物， 或含有普罗布考或其衍生物的药物组 合物。
在另一优选例中， 本发明还提供了一种 MMP-9基因或其蛋白、 或 VCAM-1 基因或其蛋白的抑制剂，所述的抑制剂为有效量的普罗布考或其衍生物或含有普罗布 考或其衍生物的药物组合物。
本发明的目的是提供普罗布考或其衍生物在制备用于抑制肿瘤转移， 优选 乳腺癌转移， 更优选乳腺癌肺转移的药物中的用途。
本发明的目的是提供一种普罗布考药物组合物在制备用于抑制肿瘤转移， 优选乳腺癌转移， 更优选乳腺癌肺转移的药物中的用途。
本申请所述普罗布考衍生物可以为普罗布考琥珀酸酯 AGI-1067。
所述普罗布考药物组合物包含治疗有效量的一种或多种选自普罗布考或 其衍生物中的化合物以及药物辅料。
所述普罗布考药物组合物可以为自微乳、 纳米混悬液、 纳米粒或纳米乳。 所述普罗布考药物组合物可以为胶囊、 片剂、 固体分散体或颗粒剂。 附图说明
图 1显示了普罗布考或其衍生物抑制乳腺癌肺转移体外实验。
图 2显示了普罗布考或其衍生物在体抑制乳腺癌肺转移的发生。
图 3 显示了普罗布考及其纳米制剂在细胞划痕实验中的效果对比， 其中 TX100代表曲拉通 X-100 ( Triton X-100 ) 作为阴性对照， DNP代表实施例 11 中制备的纳米制剂。
图 4显示了普罗布考及其纳米制剂在细胞迁移实验中的效果对比。
图 5显示了普罗布考及其纳米制剂在细胞侵袭实验中的效果对比。
图 6显示了琥珀布考及其纳米制剂在细胞划痕实验中的效果对比， 其中， P188代表泊洛沙姆 P188 ( Poloxamer P188 ) 作为阴性对照， PSN代表实施例 12中制备的纳米制剂。
图 7显示了琥珀布考及其纳米制剂在细胞迁移实验中的效果对比。
图 8显示了琥珀布考及其纳米制剂在细胞侵袭实验中的效果对比。 图 9显示了普罗布考及其纳米制剂在乳腺癌模型小鼠生存率实验中的效果 对比。
图 10A显示了普罗布考及其纳米制剂在体内对乳腺癌肺部转移的干预效果 对比统计， 图 10B显示了琥珀布考及其纳米制剂体内抑制肺部转移的干预效果 干预效果对比统计。
图 11A显示了普罗布考及其纳米制剂在体内对乳腺癌肺部转移的干预效果 病理标本对比， 图 11B显示了琥珀布考及其纳米制剂体内抑制肺部转移的干预 效果病理标本对比。
图 12 显示了普罗布考及其纳米制剂在一定浓度范围内对癌细胞活性无影 响。
图 13 显示了普罗布考及其纳米制剂在一定浓度范围内对癌细胞的凋亡无 显著影响。
图 14 显示了普罗布考及其纳米制剂在体内， 对肿瘤细胞的生长也无显著 影响。
图 15 显示了琥珀布考及其纳米制剂在一定浓度范围内对癌细胞活性无影 响。
图 16 显示了琥珀布考及其纳米制剂在一定浓度范围内对癌细胞的凋亡无 显著影响。
图 17显示了普罗布考及其纳米制剂能够抑制匪 P-9的表达。
图 18显示了琥珀布考及其纳米制剂能够抑制 VCAM-1的表达。 具体实施方式
本发明人经过长期而深入的研究， 并筛选了大量活性化合物， 首次意外地 发现用于治疗心血管疾病（如动脉粥样硬化）的药物普罗布考或其衍生物可对 恶性实体肿瘤（尤其是乳腺癌）的转移（如肺转移）具有良好的抑制活性。 此外， 发明人还通过实验发现， 普罗布考或其衍生物对肿瘤细胞的生长或增殖本身并 不具有抑制作用， 其抑制肿瘤转移的作用可能与抑制 MMP-9或 VCAM-1 的表达 有密切的关系。 在此基础上， 完成了本发明。 术语
如本文所用， 术语 "普罗布考的衍生物"指普罗布考经与酸的反应后产生 的酯以及所述的酯与碱进一步形成的药学上可接受的盐。 通常， 所述的酸包括 盐酸、 氢溴酸、 磷酸、 硝酸、 硫酸； 有机酸包括： 甲酸、 乙酸、 丙酸、 丁二酸、 萘二磺酸 (1，5)、 亚细亚酸、 草酸、 酒石酸、 乳酸、 水杨酸、 苯甲酸、 戊酸、 二 乙基乙酸、 丙二酸、 琥珀酸、 富马酸、 庚二酸、 己二酸、 马来酸、 苹果酸、 氨 基磺酸、 苯丙酸、 葡糖酸、 抗坏血酸、 烟酸、 异烟酸、 甲磺酸、 对甲苯磺酸、 柠檬酸， 以及氨基酸。 优选地， 所述的酸包括琥珀酸。
如本文所用， 术语 "琥珀布考" 、 "普罗布考的琥珀酸酯" 、 "普罗布考 的单琥珀酸酯" 、 "AGI-1067 " 可互换使用， 均指普罗布考与琥珀酸反应后产 生的普罗布考单酯或其药学上可接受的盐的形式。 本发明琥珀布考可采用本领 域常规制备技术制得。 优选地， 所述普罗布考单酯的药学上可接受的盐包括钾 盐、 钠盐、 钙盐或镁盐。 普罗布考
普罗布考 （probucol ) 又名丙丁酚， 是 1977年首先在美国上市的降血脂 药。 由于其在降胆固醇的同时降低了高密度脂蛋白胆固醇 （HDL) 而淡出市场。 普罗布考的主要药理作用包括降低胆固醇合成和促进胆固醇分解， 改变高密度 脂蛋白亚型的性质和功能， 抗氧化作用， 抑制泡沫细胞的形成， 延缓动脉粥样 硬化斑块的形成， 消退已形成的动脉粥样硬化病变。 药物组合物
本发明还提供了一种药物组合物， 其具有显著的抗肿瘤功效， 其中含有治疗 有效量的所述式 I化合物或其药学上可接受的盐， 以及一种或多种药学上可接受的 载体。在本发明的另一优选例中，所述的药物组合物中含有治疗有效量的阿那格雷 盐酸盐， 以及一种或多种药学上可接受的载体 (或药物辅料)。
可将化合物本身或其药学上可接受的盐与可药用赋形剂、 稀释剂等的混合物 以片剂、胶囊、颗粒剂、散剂或糖浆剂的形式口服给药或以注射剂的形式非口服给 药。该药物组合物优选含有重量比为 0.01%-99%的本发明的式 I化合物或其药学上 可接受的盐作为活性成分， 更优选含有重量比为 0.1%-90%的活性成分。
上述制剂可通过常规制药方法制备。 可用的药用辅剂的例子包括赋形剂 (例如 糖类衍生物如乳糖、蔗糖、葡萄糖、甘露糖醇和山梨糖醇； 淀粉衍生物如玉米淀粉、 土豆淀粉、 糊精和羧甲基淀粉； 纤维素衍生物如结晶纤维素、 羟丙基纤维素、 羧甲 基纤维素、 羧甲基纤维素钙、 羧甲基纤维素钠； 阿拉伯胶； 右旋糖酐； 硅酸盐衍生 物如偏硅酸镁铝； 磷酸盐衍生物如磷酸钙； 碳酸盐衍生物如碳酸钙； 硫酸盐衍生物 如硫酸钙等)、 粘合剂 (例如明胶、 聚乙烯吡咯垸酮和聚乙二醇)、 崩解剂 (例如纤维 素衍生物如羧甲基纤维素钠、 聚乙烯吡咯垸酮)、 润滑剂 (例如滑石、 硬脂酸钙、 硬 脂酸镁、 鲸蜡、 硼酸、 苯甲酸钠、 亮氨酸)、 稳定剂 (对羟基苯甲酸甲酯、 对羟基苯 甲酸丙酯等)、矫味剂 (例如常用的甜味剂、酸味剂和香料等)、稀释剂和注射液用溶 剂 (例如水、 乙醇和甘油等)。
本发明的化合物、 其药学上可接受的盐， 或其药物组合物的给药量随患者的 年龄、 性别、 种族、 病情等的不同而不同。 在小鼠实验中， 采用了 200mg/kg的给 药量， 本领域技术人员可以根据该体内实验用量筛选安全有效量的人类药物用量。 通常， 成人的日给药量为大约 10mg-2000mg， 优选 50mg-1000mg。
本发明普罗布考或其衍生物的制剂可采用本领域常规手段制备获得， 例如 可以制成片剂、 胶囊剂、 乳剂、 混悬剂。 优选地， 可将普罗布考或其衍生物制 备成纳米制剂。 抗肿瘤转移的药物（药物组合物）及其制备
本发明提供了一种抑制肿瘤细胞转移的方法， 所述方法包括， 向需要的对象 细胞施用有效量的普罗布考或其衍生物或包含普罗布考或其衍生物的药物组合物。
在本发明的一个优选例中， 所述的药物组合物包括： （a)治疗有效量的普罗布 考或其衍生物； 和 (b)药学上可接受的载体。
在另一优选例中， 所述的肿瘤包括： 乳腺癌、 肺癌。
在另一优选例中， 所述式 I化合物的有效浓度为 0.01nM/L-lmM/L， 较佳地 为 0.1nM/L-50(^M/L， 最佳地为 lnM/L-10(^M/L。
本发明还提供了一种抗肿瘤转移的药物或药物组合物的制备方法， 所述方法 包括：将治疗有效量的普罗布考或其衍生物与药学上可接受的载体混合，从而形成 药物组合物。
在另一优选例中， 所述药物组合物还包括选自下组的组分： 用于抑制肿瘤生 长或增殖的化疗剂。
对被治疗对象施用治疗有效量的普罗布考或其衍生物或包括普罗布考或其衍 生物的药物组合物， 可以治疗或抑制肿瘤的转移。 本发明的有益效果：
本发明开拓了普罗布考或其衍生物抑制肿瘤转移， 尤其是抗乳腺癌肺转移 的新用途， 它能有效抑制乳腺癌肺转移的发生， 提高了生存率， 降低了转移发 生率， 显示该化合物及其组合物在抑制恶性肿瘤转移方面具有开发应用前景。
此外， 本发明采用的化合物作为曾经上市的药物， 其毒副作用已经得以验 证， 在进一步的临床应用开发中相对安全性更高。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法，通常按照常规条件，例如 Sambrook等人， 分子克隆：实验室手册（New York： Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂 商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。 实施例 1 普罗布考混悬液
将普罗布考原料药 （河北武邑慈航药业有限公司， 纯度为 99. 7%) 分散于 水、 缓冲液、 含有 0. 5%羧甲基纤维素钠或者 0. 1%羟丙基纤维素的溶液中。 实施例 2 普罗布考衍生物 AGI-1067混悬液
将普罗布考衍生物 AGI-1067 (上海皓元化学科技有限公司， 纯度 98. 0%) 分散于水、 缓冲液、 含有 0. 5%羧甲基纤维素钠或者 0. 1%羟丙基纤维素的溶液 中。 实施例 3 普罗布考颗粒
将普罗布考与淀粉、 预胶化 P粉、 微晶纤维素、 乳糖、 聚维酮等采用湿法 制粒、 干法制粒或者直接混合的方法制备成含药物颗粒。 实施例 4普罗布考片剂
将普罗布考与淀粉、 预胶化淀粉、 微晶纤维素、 乳糖、 聚维酮等采用湿法 制粒、 干法制粒或者直接混合的方法制备成含药物颗粒， 然后用压片机压制普 罗布考片剂。 实施例 5 普罗布考胶囊
将普罗布考与淀粉、 预胶化淀粉、 微晶纤维素、 乳糖、 聚维酮等采用湿法 制粒、干法制粒或者直接混合的方法制备成含药物颗粒，进而装载于胶囊壳中， 制备普罗布考胶囊。 实施例 6 普罗布考多孔载体复合物
将普罗布考与含有孔洞的多孔材料如多孔淀粉、 交联聚维酮混合， 一起分 散于乙醇中， 超声混合， 然后蒸干除去有机溶剂， 即得。 实施例 7 普罗布考衍生物 （琥珀布考， AGI-1067) 多孔载体复合物 将普罗布考衍生物 （AGI-1067) 与介孔二氧化硅、 硅藻土等混合， 一起分 散于乙醇中， 超声混合， 然后蒸干除去有机溶剂， 即得。 实施例 8 普罗布考纳米乳
将普罗布考与 Tween 80， 中联甘油三酯和聚乙二醇硬脂酸酯 HS-15 (1:2:1:1, w/w) 混合， 在乙醇中溶解， 蒸干除去有机溶剂， 加水混合即得。 实施例 9普罗布考衍生物 （琥珀布考， AGI-1067) 自微乳
将普罗布考衍生物 （AGI-1067 ) 与 Cremphol EL, 中联甘油三酯和 Transcutol HP: 10: 3:7， w/w) 混合， 超声溶解， 给药时加水混合即可。 实施例 10 普罗布考固体分散体
将普罗布考与磷脂、 胆酸钠 （1: 2:1, w/w) 混合， 于甲醇中溶解， 蒸干 除去有机溶剂， 即得。 实施例 11 普罗布考纳米粒
将普罗布考与表面活性剂曲拉通 X-100按照 1:2 比例混合（w/w)， 溶于乙 醇， 37°C蒸干除去有机溶剂， 加水振摇， 即得 DNP。 实施例 12普罗布考衍生物 （琥珀布考， AGI-1067) 纳米粒
将普罗布考衍生物 （琥珀布考， AGI-1067) 与表面活性剂 PoloxamerP188 按照 1:3比例混合， 溶于乙醇， 37°C蒸干除去有机溶剂， 加水振摇， 即得 PSN。 实施例 13 普罗布考纳米混悬剂
将普罗布考与聚维酮 PVPkl2 和十二垸基硫酸钠混合， 于行星式研磨仪中 研磨 30分钟， 即得普罗布考纳米混悬剂。 测试例 1: 普罗布考及其纳米制剂体外抑制癌细胞转移实验
1.1 划痕实验， Wound Healing assay
将 4T1细胞（购自中国科学院上海细胞资源中心）以 5X106个 /孔的比例接 种于 6孔板中， 待细胞形成单层厚， 吸弃培养液， 用枪头均匀划痕， 用 PBS洗 涤 3次，更换为新鲜的培养液，在显微镜下拍照，分别加入普罗布考 （40 ng/mL、 400 ng/mL) 、 制剂辅料 （相当于 40 ng/mL 、 400 ng/mL普罗布考） 和纳米制 剂组 （40 ng/mL 、 400 ng/mL) ， 继续培养 36 小时， 拍照观察在划痕的愈合 情况。 结果表明与对照相比， 普罗布考和纳米制剂组能够有效抑制细胞划痕的 愈合（图 3)。
1.2 细胞迁移 ， Transwell migration assay
采用 Transwell 方法研究细胞的迁移特性， 将 1X105个 4T1 细胞混悬于
ΙΟΟμί不含 FBS的培养液， 加入到 Transwell小室的上层， 下层加入 600μί含 FBS的培养液， 上下两室同时加入普罗布考 （40 ng/mL 、 400 ng/mL) 、 制剂 辅料 （相当于 40 ng/mL 、 400 ng/mL普罗布考） 和纳米制剂组 （40 ng/mL 、 400 ng/mL)， 继续培养 24小时。取出 Transwell小室， 用 90%乙醇固定 lOmin, 挥干后用结晶紫溶液染色 30min， 将小室上层未迁移的细胞擦干净， 之后于显 微镜下拍照。 结果表明与对照相比， 普罗布考和纳米制剂组能够有效抑制 4T1 细胞的细胞迁移能力（图 4)。
1.3 细胞侵袭 Transwell invasion assay
采用 Transwell方法研究细胞的侵袭特性， Tanswell小室上层加入 ΙΟΟμΙ^ 基质胶溶液 （含 10%的 Matrigel) 过夜， 将 2 X 105个 4T1细胞混悬于 ΙΟΟμΙ^不 含 FBS的培养液， 加入到 Transwell小室的上层， 下层加入 600μί含 FBS的培 养液， 上下两室同时加入普罗布考 （40 ng/mL、 400 ng/mL ) 、 制剂辅料 （相 当于 40 ng/mL、 400 ng/mL普罗布考）和纳米制剂组（40 ng/mL、 400 ng/mL ) ， 继续培养 24小时。 取出 Transwel l小室， 用 90%乙醇固定 10min， 挥干后用结 晶紫溶液染色 30min， 将小室上层未迁移的细胞擦干净， 之后于显微镜下拍照。 结果表明与对照相比， 普罗布考和纳米制剂组能够有效抑制 4T1细胞的细胞侵 袭， 400ng/mL时纳米制剂组降低细胞侵袭的能力显著优于普罗布考组（图 5)。
其中 DNP为普罗布考纳米制剂组 （实施例 11 ) 1. 4普罗布考纳米制剂的比 较
1. 4利用 transwel l小室来检验普罗布考对体外培养的肿瘤细胞转移作用 的抑制作用。 将实施例 8、 11制备的纳米组合物和细胞在小室内用不含 FBS的 培养基共培养， 药物普罗布考或者 AGI 1067的药物浓度为 lOOr^ mL— 其中未 加任何药物的空白细胞为对照组， 共培养 24h后， 用结晶紫对细胞进行染色， 用棉签除去小室内未迁移细胞后， 用显微镜观察细胞迁移情况。 结果如图 1所 示，表明含有普罗布考纳米药物组合物能显著抑制乳腺癌细胞 4T1细胞的迁移。
以上结果表明， 普罗布考普通制剂及其纳米制剂在体外的癌细胞迁移、 侵 袭实验中， 能够有效地抑制癌细胞的转移。 且在高浓度下， 纳米制剂在体外的 抑制作用强于普通制剂。 测试例 2 琥珀布考及其纳米制剂体外抑制癌细胞转移实验
按测试例 1. 1、 1. 2以及 1. 3的方法， 对琥珀布考或其纳米制剂（测试例 12 中制备的纳米制剂)在体外抑制 4T1 癌细胞的迁移和侵袭进行了研究。 结果如 图 1 D和图 6-图 8可见： 其中 PSN为琥珀布考纳米制剂组 （实施例 12 )
与对照相比，琥珀布考及其纳米制剂能够有效抑制细胞划痕的愈合（图 6)、 有效抑制 4T1细胞的细胞迁移能力（图 7、 图 1)， 还能够有效抑制 4T1细胞的 细胞侵袭（图 8)。 测试例 3普罗布考及其纳米制剂体内抑制癌细胞转移实验
在小鼠乳腺脂肪垫接种 4T 1-LUC细胞， 建立 4T1原位瘤模型， 每组 8只， 口服给予测试例 1、 11制备的组合物，给药剂量为 200π¾· kg—1 ,每天给药一次， 连续给药 30-40天， 考察乳腺癌肺转移情况， 结果如图 2， 表 1所示。
表 1 普罗布考及其自组装纳米粒在体抑制乳腺癌肺转移 转移率 生存率 平均转移灶数 生理盐水 6/8 37. 5% 11. 7 ± 3. 1
实施例 1 3/8 50% 9. 3 ± 1. 2
实施例 11 1/8 75% 1. 0 ± 1. 7 当连续给药 38 天， 记录动物死亡情况， 结果表明纳米制剂组能够有效延 长肿瘤模型的存活率（图 9)。
将动物处死， 取肺部， 记录肉眼可见的肿瘤转移灶， 结果表明纳米制剂组 存活动物中只有一半发生转移， 而生理盐水和普罗布考组均发生转移， 纳米制 剂能够有效降低肿瘤的肺部转移， 其转移灶的数目不到普罗布考的 10% (图 10、 11)。
结果表明普罗布考或其纳米制剂组合物相比生理盐水组发生肺转移的概 率降低， 小鼠生存率提高， 同时肺部转移灶数目也显著降低（图 2)。 测试例 4琥珀布考及其纳米制剂体内抑制癌细胞转移实验
按照测试例 3的方法， 观察琥珀布考及其纳米制剂在体内抑制癌细胞转移 的效果（图 2)。
转移率 生存率 平均转移灶数
实施例 12 1/8 75% 0. 6 ± 0· 5 测试例 5 普罗布考对肿瘤细胞的抑制作用检测
5. 1 细胞活性检测
采用 ΜΤΤ法检测普罗布考及其纳米制剂在 4T1细胞中的细胞毒性，以 8000 个 /孔将 4T1细胞接种于 96孔板中， 过夜， 依次加入 0. 004、 0. 04、 0. 4、 4和 4(^g/mL的普罗布考， TX100 (相当于普罗布考的浓度） 和纳米制剂组， 继续培 养 48h后采用 MTT法检测细胞毒性， 结果表明在 4ng/mL到 4 g/mL的浓度范围 内， 细胞活性几乎没有变化， 普罗布考和纳米制剂没有细胞毒性（图 12)。
5. 2 细胞凋亡检测
采用流式细胞仪检测普罗布考及其纳米制剂在 4T1细胞中的细胞凋亡， 以 1. 5 X 105个 /孔将 4T1细胞接种于 6孔板中， 过夜， 分别加入 40和 400 ng/mL 的的普罗布考， TX100 (相当于普罗布考的浓度） 和纳米制剂组， 继续培养 48h 后， Annexin V-FITC/PI检测试剂盒染色后， 流式细胞仪检测细胞的凋亡特性， 结果表明在 40ng/mL和 400ng/mL 时， 细胞凋亡特性与对照相比几乎没有变化 (图 13)。
5. 3 普罗布考及其纳米制剂抑制乳腺癌细胞 4T1转移的体内评价
建立 4T1原位瘤模型， 每天给予普罗布考和纳米制剂组， 检测肿瘤生长的 情况， 结果表明与生理盐水组相比， 普罗布考和纳米制剂组均不能抑制肿瘤的 生长。 肿瘤生长曲线如图 14所示。 由此可见， 普罗布考对癌细胞并不存在细胞毒性作用， 因此无法抑制肿瘤 细胞的生长或增殖， 也无法诱导肿瘤细胞的凋亡。 测试例 6 琥珀布考对肿瘤细胞的抑制作用检测
方法同实施例 5， 结构可见琥珀布考及其纳米制剂在 4 μ g/ml 内无法抑制 肿瘤细胞的生长或增殖， 400ng/ml也无法诱导肿瘤细胞的凋亡（图 15、 16)。 测试例 7普罗布考、琥珀布考及其纳米制剂抑制肿瘤转移的作用机制研究 对普罗布考、 琥珀布考及其纳米制剂进行了在肿瘤细胞转移中的基因表达 研究， 结果见图 17、 18。
其中， 图 17显示了在健康小鼠和 4T1诱导的原位乳腺癌转移模型中， 在 生理盐水、 普罗布考以及 DNP分别治疗下， MMP-9表达的免疫荧光表现， 其中 匪 P-9以绿色（亮灰色）荧光标记。 可见在经过普罗布考处理的模型组中， 匪 P-9 的表达显著低于对照组， 且纳米制剂的效果更佳。
同样， 图 18显示了在健康小鼠和 4T1诱导的肺转移癌模型中， 在生理盐 水、 琥珀布考以及 PSN的分别治疗下， VCAM-1表达的免疫荧光表现， 可见在经 过琥珀布考处理的模型组中， VCAM-1的表达显著低于对照组， 且纳米制剂的效 果更佳。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims

权 利 要 求
1、 一种普罗布考或其衍生物在制备用于抑制肿瘤转移的药物中的用途。
2、 根据权利要求 1所述的用途， 其中， 所述肿瘤包括乳腺癌。
3、 根据权利要求 2所述的用途， 其中， 所述转移包括肺转移。
4、 根据权利要求 1-3 中任意一项所述的用途， 其中， 所述普罗布考衍生 物包括普罗布考的单琥珀酸酯即琥珀布考或其药学上可接受的盐。
5、 一种普罗布考药物组合物在制备用于抑制肿瘤转移的药物中的用途， 其中， 所述普罗布考药物组合物包含治疗有效量的一种或多种选自普罗布考或 其衍生物中的化合物以及药物辅料。
6、 根据权利要求 5所述的用途， 其中， 所述肿瘤包括乳腺癌。
7、 根据权利要求 6所述的用途， 其中， 所述转移包括肺转移。
8、 根据权利要求 5 所述的用途， 其中， 所述普罗布考药物组合物为自微 乳、 纳米混悬液、 纳米粒或纳米乳。
9、 根据权利要求 5所述的用途， 其中， 所述普罗布考药物组合物为胶囊、 片剂、 固体分散体或颗粒剂。
10、 根据权利要求 5-9中任意一项所述的用途， 其中， 所述普罗布考衍生 物为普罗布考琥珀酸酯。
11、 一种体外非治疗性的抑制肿瘤细胞转移的方法， 其特征在于， 包括步 骤： 在普罗布考或其衍生物的存在下， 培养所述肿瘤细胞。
12、 一种用于治疗或抑制肿瘤转移的药物组合物， 其中， 所述的药物组合 物含有有效量的普罗布考或其衍生物作为活性成分， 以及药学上可接受的载 体。
13. 一种治疗或抑制肿瘤转移的方法， 其特征在于， 向所需要的对象施用 安全有效量的普罗布考或其衍生物， 或含有普罗布考或其衍生物的药物组合 物。
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