Nothing Special   »   [go: up one dir, main page]

WO2014079377A1 - 一种具有p-糖蛋白抑制功能的抗肿瘤前药 - Google Patents

一种具有p-糖蛋白抑制功能的抗肿瘤前药 Download PDF

Info

Publication number
WO2014079377A1
WO2014079377A1 PCT/CN2013/087617 CN2013087617W WO2014079377A1 WO 2014079377 A1 WO2014079377 A1 WO 2014079377A1 CN 2013087617 W CN2013087617 W CN 2013087617W WO 2014079377 A1 WO2014079377 A1 WO 2014079377A1
Authority
WO
WIPO (PCT)
Prior art keywords
prodrug
tumor
polyethylene glycol
bond
group
Prior art date
Application number
PCT/CN2013/087617
Other languages
English (en)
French (fr)
Inventor
张志平
谭松巍
Original Assignee
武汉平华生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉平华生物医药科技有限公司 filed Critical 武汉平华生物医药科技有限公司
Publication of WO2014079377A1 publication Critical patent/WO2014079377A1/zh

Links

Classifications

    • 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
    • A61K31/337—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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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 invention belongs to the technical field of chemical medicines, and particularly relates to an anti-tumor prodrug which can realize the treatment of tumors, especially drug-resistant tumors.
  • Cancer is one of the leading causes of death in modern society.
  • the results of the national death survey released by the Ministry of Health in 2008 showed that cancer has become the first cause of death in China's cities and the second in rural areas.
  • An important means of treating cancer is chemotherapy.
  • tumor patients often develop resistance to chemotherapeutic drugs during treatment, and also cross-resistance to other drugs with different chemical structures and different mechanisms of action.
  • MDR multidrug resistance
  • most anti-tumor drugs have low water solubility, and the modification of hydrophilic polymer materials can greatly increase the solubility of drugs, and can be passively targeted by high permeability and retention effect (EPR effect).
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and to provide an anti-tumor prodrug having a P-glycoprotein inhibitory function, which is expected to more effectively achieve treatment of tumors and drug-resistant tumors.
  • the present invention adopts the following technical scheme - an anti-tumor prodrug having a glycoprotein inhibitory function, which is linked by an antitumor drug to a polyethylene glycol succinate having a P-glycoprotein inhibitory function.
  • the linker containing the sensitive bond contains at least two reactive functional groups for covalent attachment of the antitumor drug and polyethylene glycol succinate, the sensitive bond being in the tumor cell A chemical bond that is easily broken in a reducing environment or an acidic environment.
  • the sensitive key is characterized in that two substances connected together are dissociated under a reducing environment or acidic condition in the tumor cells, and therefore, the sensitive key can be further divided into a reduction sensitive key and pH sensitive key.
  • Commonly used reduction-sensitive bonds are disulfide bonds, di-selenium bonds, etc.; common pH-sensitive bonds are oxime bonds, sulphur base bonds, oxime bonds, orthoester bonds, acetals or ketal bonds.
  • the antitumor prodrug of the present invention having the inhibitory function of the I S protein can be represented by the formula I.
  • the two energy groups for covalently linking the antitumor drug and the polyethylene glycol succinate may be the same or different, and are selected from the group consisting of a hydroxyl group, a carboxyl group (including an acid anhydride form), an amino group, a substituted amino group. , amide group, cyano group and isocyanate group, aldehyde group and carbonyl group.
  • the connector is composed of a sensitive bond, two reactive functional groups, and a methylene group of 1 to 12, respectively, which is bonded between the sensitive bond and the two reactive functional groups.
  • Specific connectors are such as 3,3'-dithiodipropionic acid, 4,4'-dithiodibutyric acid, 3,3'-diselenodipropionic acid, p-carboxybenzaldehyde, 3- Carboxybenzaldehyde, glyoxylic acid, pyruvic acid, succinic hydrazide, adipic hydrazide, and the like.
  • the structural formula of the anti-tumor prodrug having glycoprotein inhibitory function according to the present invention is as follows:
  • n, P are independently integers between ⁇ 6, such as m, :P are both, 2 or 3; or for example, m is 2 and P is 3.
  • the polyethylene glycol succinate is preferably selected from the group consisting of polyethylene glycol vitamin E amber.
  • polyethylene glycol vitamin E amber One of an acid ester, a polyethylene glycol cholesterol succinate, and a polyethylene glycol fatty alcohol succinate, of which polyethylene glycol vitamin E succinate (TPGS) is more preferred.
  • TPGS polyethylene glycol vitamin E succinate
  • the polyethylene glycol in the polyethylene glycol succinate has a number average molecular weight of from 500 to 6,000.
  • the polyethylene glycol in the polyethylene glycol succinate is PEG500, PEG 1000, PEG2000, PEG3350 or PEG5000.
  • the antitumor drug may be any known antitumor drug, preferably a drug required for the P gp substrate, including but not limited to paclitaxel (PTX), docetaxel, doxorubicin (DOX). , epirubicin, vinblastine, vincristine, sorghum: cedar ester and camptothecin.
  • PTX paclitaxel
  • DOX doxorubicin
  • epirubicin vinblastine
  • vincristine vincristine
  • sorghum cedar ester and camptothecin.
  • the above-mentioned antitumor prodrug having a P-glycoprotein inhibitory function can be prepared by a conventional organic synthesis method starting from an existing raw material.
  • the anti-tumor prodrug can be synthesized by the following steps;
  • step (3) Dissolving the product obtained in the step (2) in anhydrous dichloromethane, stirring at room temperature in the presence of N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC) The reaction is activated;
  • NHS N-hydroxysuccinimide
  • DCC dicyclohexylcarbodiimide
  • TPGS TPGS each S-COOH * TPGS S S PTX.
  • the inventive idea of the present invention is as follows; It is known that overexpression of a P-glycoprotein (P-gp) encoded by the H3 mdrl gene is an important mechanism for MDR production. In recent years, the P-gp inhibition of PEG derivatives has been extensively studied and confirmed, and the most effective inhibitory effect is polyethylene glycol vitamin E succinate (TPGS).
  • TPGS polyethylene glycol vitamin E succinate
  • the invention utilizes TPGS and a similar function of polyethylene glycol succinate and a disulfide bond, a selenium bond and the like to design a response point in a tumor cell intracellular reducing environment or an acidic environment, having P-gp A sensitive prodrug that inhibits function.
  • the prodrug can self-assemble to form a micellar structure, which is aggregated in tumor tissue by EPR effect and enters into tumor cells by endocytosis. Dissociation of intracellular sensitive bonds forms free TPGS polymers and derivatives of antitumor drugs.
  • the derivative does not have a steric hindrance of a high molecular chain, and thus is easily reduced to a drug substance structure having high antitumor activity.
  • TPGS binds to P-gp, inhibits P-gp activity, reduces anti-tumor drug efflux, and reverses multidrug resistance of drug-resistant cells, greatly improving the therapeutic effect of anti-tumor drugs.
  • the present invention has the following advantages over the prior art:
  • the prodrug of the invention can self-assemble to form a polymer micelle, which greatly improves the solubility and stability of the hydrophobic drug, realizes a long circulation of the drug in the body, and is enriched in the tumor site by the EPR effect.
  • the prodrug can be rapidly dissociated into a polyethylene glycol succinate having a P-glycoprotein inhibitory function and a derivative of a drug under reducing conditions or acidic conditions in a fat tumor cell.
  • drug derivatives have less steric hindrance and are easily reduced to effective drug tracts. Structure, can achieve rapid release of drugs.
  • the free polyethylene glycol succinate binds to P-gp, inhibits its pumping of the drug, and increases the intracellular concentration of the drug.
  • Figure 1 is a nuclear magnetic resonance spectrum of TPGS in the implementation of Figure 1;
  • Example 2 is a nuclear magnetic resonance spectrum of TPGS-S-S-COOH in Example 1;
  • Figure 3 is a nuclear magnetic resonance spectrum of the paclitaxel prodrug of Example 1;
  • Figure 4 is a nuclear magnetic resonance spectrum of the doxorubicin prodrug of the invention.
  • Figure 5 is a nuclear magnetic resonance spectrum of the doxorubicin prodrug of Example 7.
  • Figure 6 is a photograph of different concentrations of TPGS-S-S-PTX solution
  • Figure 7 shows the particle size change curve of TPGS-S-S-PTX under different pH and GSH conditions
  • Figure 8 is a PTX standard curve
  • Figure 9 shows the release profile of the raPEG-PTX prodrug and the TPGS-S-S-PTX prodrug
  • Figure 10 shows the results of 24, 48, and 72-hour cytotoxicity tests of PTX, mPEG-PTX prodrug and TPGS-S-S-PTX prodrug.
  • Figure 11 shows the pharmacokinetic profile of the TPGS-S-S-PTX prodrug and the clinical preparation Taxoi.
  • Figure 12 shows the results of tissue distribution of TPGS-S-S-PTX prodrug and Taxol in S180 sarcoma-bearing mice.
  • Figure i3 shows the tumor growth inhibition of TPGS-S-S-PTX prodrug and Taxoi against S180 sarcoma mice.
  • Figure 15 shows the results of a 24-hour cytotoxicity test for DOX, mPEG-DOX prodrugs, and TPGS-CO-NH-N:»:::C-DOX prodrugs.
  • This example provides a paclitaxel prodrug having a P-glycoprotein inhibitory function, which is synthesized by, for example, a T step: synthesis of (1), 3,3'-dithiodipropionic anhydride: weighing 2 g 3, 3' - Dithiodipropionic acid was placed in a 50 ml round bottom flask, 20 ml of acetyl chloride was added, and the reaction was refluxed for 3 hours at 65 ° C. The impurities such as acetic acid and acetyl chloride were distilled off under reduced pressure; the mixture was washed three times with diethyl ether This gave 3,3'-dithiodipropionic anhydride.
  • TPGS-SS-COOH synthesis Weigh 1.5g TPGS into a lOOmL round bottom flask, dry in a vacuum drying oven at 60 ° C for 3-5 hours, and then put 0.276g 3,3' - Dithiodipropionic anhydride, 0.183 g of 4-dimethylaminopyridine, 0.15 laL: triethylamine, dissolved in 5 - 10 mL of dimethyl sulfoxide; in a dry environment, stir the reaction at room temperature for 24 hours! Inch.
  • TPGS-SS-COOH Activation of TPGS-SS-COOH: The above product was dialyzed against water in a dialysis bag with a molecular weight of 1000, taken out after 24 to 48 hours, and frozen to obtain TPGS-S-S-COOH; with 5- lOniL dichloromethane (DCM) Re-dissolve the frozen material, add N-hydroxysuccinimide and N'-dicyclohexylcarbodiimide, stir in an anhydrous environment at room temperature (4), connecting paclitaxel: in a 100 mL round bottom flask, add 1.02 g of paclitaxel (PTX), dissolved with 5 - 15 mL of DCM; filter the product obtained by the step (3), the filtrate is added to the paclitaxel solution; Stir at room temperature for 48 h ; the obtained product was dialyzed against absolute ethanol in a dialysis bag with a molecular weight of 2000 for 24-48 h. After di
  • Figure 1 to Figure 3 are hydrogen nuclear magnetic resonance spectra of TPGS, TPGS-S-S-COOH and paclitaxel prodrugs, respectively.
  • the present invention provides a doxorubicin prodrug having a P-glycoprotein inhibitory function, which is synthesized by the following steps;
  • step (1) doxorubicin is added to the lOOraL round bottom flask, 5-1 OmL DMSO and 0.12mi: triethylamine is dissolved, and is protected from light; step (1) The product was filtered, and the filtrate was added to the doxorubicin solution; the mixture was stirred at room temperature for 48 hours in an anhydrous environment; the obtained product was dialyzed against a dialysis bag of molecular weight 2000 in a mixed solvent of absolute ethanol and DMSO for 24-48 hours, and dried after dialysis.
  • Ethanol is a doxorubicin prodrug (TPGS-S-S-DOX), and its structure is as follows;
  • This embodiment provides a paclitaxel prodrug which has a P-glycoprotein inhibitory function, which is synthesized by the following steps -
  • This embodiment provides a camptothecin prodrug of P-glycoprotein inhibitory function, which is synthesized by the following steps: (U5g camptothecin is added to a lOOmL round bottom flask, dissolved in 5-1000 mL of DMSO, and avoided. Light treatment; added activated TPGS-S-S-COOH to camptothecin solution; stirred in room temperature for 48 h at room temperature ; obtained product was dialyzed against absolute ethanol in dialysis bag with molecular weight of 2000 24-48bu After the dialysis is completed, the ethanol is obtained as a camptothecin prodrug.
  • This embodiment provides a scutellarin prodrug of a P-glycoprotein inhibitory function, which is synthesized by the following procedure - 0,55 g of homoharringtonine is added to a 100 mL round bottom flask, and 5-- 10m: L DMSO is dissolved and treated as a dark; the activated TPGS-S-S-COOH is added to the homoharringtonine solution; in an anhydrous environment, room temperature
  • This example provides a P-glycoprotein inhibitory function of epirubicin prodrug, which is synthesized by the following steps:
  • TPGS p-carboxybenzaldehyde ester synthesis Weigh 1.5g TPGS into a lOOmL round bottom flask, dry in a vacuum drying oven under 60 ⁇ conditions for 3-5 hours, and then put 0.60g p-carboxybenzaldehyde, 0.825 g DCC and 0,243 g DMAP' 0.15 mL: triethylamine, dissolved in 50-100 mL of anhydrous tetrahydrofuran (THF): In an anhydrous environment, the reaction was stirred at room temperature for 48 hours, then the precipitate was filtered off and evaporated to remove large Part of the THF was precipitated and washed in diethyl ether to give TPGS p-carboxybenzaldehyde.
  • THF tetrahydrofuran
  • This example provides a glycoprotein inhibitory function of doxorubicin prodrug which is synthesized by the following steps:
  • TPGS succinylhydrazide Add 0,075 g of hydrazine hydrochloride to a 50 mL round bottom flask, add 2-10 mL of anhydrous DCM: and 0,18 ml. Dissolve in triethylamine and protect from light. 1.2g TPGS-COOH was activated in the same manner as in Example 3, step 3, added dropwise to hydrazine DCM solution, reacted at room temperature for 48 hours, then evaporated to remove most of the DCM, and then precipitated and washed in diethyl ether to obtain TPGS succinic hydrazide. .
  • the paclitaxel prodrug of Example 1 was dissolved in ethanol, slowly dropped into PBS of pH 7.4, stirred while dripping, and the ethanol was stirred and stirred overnight.
  • a solution having a concentration of a prodrug of 1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL, 7 mg/mL, and 10 mg/mL was separately prepared, and sedimentation was observed at room temperature.
  • three groups of solutions having a concentration of 1 mg/m:L of the prodrug are respectively prepared, one group is a PBS solution of pH 5.5, and the second group is a PBS solution of pH 5.5, and Add iOmM: glutathione (GSH), the third group is pH 7, 4 PBS solution, the fourth group is pH 7.4 PBS solution, add iOmM GS1-L at 37 ° C constant temperature, The particle size of the micelles in the solution was measured at lh, 2h, 3h, 4h, 6h, 8h, 24h, 48h.
  • the results show that the prodrugs exhibit good stability at pH 7, 4 and 5.5, and the particle size remains unchanged: while the addition of GSH, the stability of the prodrug is reduced, due to the dissociation of disulfide bonds, The prodrug micelles have a reduced particle size.
  • Cell culture ovarian cancer sensitive cell A2780 and drug-resistant cell A2780/T]3 ⁇ 4 RPM containing 16 ml/L inactivated fetal bovine serum, lOOU/ml penicillin and 100 U/nil streptomycin] 1640 medium, placed at 5% C0 2 cell incubator, 37 V constant temperature and humidity culture.
  • Cytotoxicity assay A2780 and A2780/T cells in logarithmic growth phase were washed with PBS, 0,25% chymotrypsin, and centrifuged to a cell suspension at a concentration of IxlO 5 /ml. The suspension was added to a 96-well cell culture plate at 100 ⁇ /well for 24 h to allow the cells to completely adhere. Paclitaxel was used as a model drug. According to the anti-drug group (TPGS-SS-PTX prodrug), the common group (mPEG prodrug, ester bond as the linkage), PTX monotherapy group and blank group (physiological saline) were configured in a series of different Concentration of micelle/reference solution (0.001-100 ⁇ ).
  • the culture solution was aspirated, and 100 ⁇ M of different concentrations of micelle/reference solution were added to each well, and cultured for 24, 48, and 72 h. Each well was then replaced with 200 MTT solution (0.5 mg/mi) and culture continued for 4 h. Finally, 150 ⁇ M DMSO was added to each well and shaken for 10 min. The absorbance of each well at 570 nm was measured with a microplate reader and the corresponding: C50 was calculated.
  • TPGS-SS-PTX prodrug on sensitive cell A2780 is comparable to that of PTX, much higher than that of mPEG PTX prodrug; and for drug-resistant cell A2780/T, PTX has almost no killing effect.
  • the mPEG PTX prodrug showed weaker killing ability, while the TPGS-S-S-PTX prodrug still showed strong cytotoxicity.
  • a transplanted tumor model of Kunming mice bearing S180 sarcoma was established. When the tumor grew to 50-100 mm 3 , it was randomly divided into two groups (Taisu group and TPGS-S-S-PTX group). The dose was administered; PTX 10 mg/kg, and some mice were sacrificed at 6, 12, and 24 h, respectively, and the organs were taken, washed and homogenized, and the sputum content was determined by HPLC.
  • mice S180 sarcoma mice were randomly divided into three groups (saline group, Taxol group and TPGS-S-S PTX group) when the tumor grew to 50 100 mm 3 , and the drug was started (recorded as day 1).
  • the dose is 10 mg/kg of PTX and the injection time is 1, 3, 5, and 7 days.
  • Tumor size was measured daily and the tumor volume was calculated. See Figure 13, all Salme The tumor volume of the mice in the group increased almost linearly, while the prodrugs of Taxol and TPGS-S-S-PTX significantly inhibited the growth of the tumor, and the growth inhibition ability of the prodrug was the strongest.
  • the mice were sacrificed and the tumors were excised and weighed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种具有P-糖蛋白抑制功能的抗肿瘤前药，其是通过连接器共价连接抗肿瘤药物与具有P-糖蛋白抑制功能的聚乙二醇琥珀酸酯构成的两亲性物质，所述连接器含有敏感键以及两个分别用于共价连接抗肿瘤药物和聚乙二醇琥珀酸酯的反应官能团，所述敏感键是在肿瘤细胞内的还原性环境或酸性环境下易断裂的化学键。

Description

一种具有 P-糖蛋白抑制功能的抗肿瘤前药 技术领域
本发明属于化学药物技术领域， 具体涉及一种可实现肿瘤特别是耐药性肿瘤的治疗 的抗肿瘤前药。
背景技术
癌症是现代社会的主要致死疾病之一。 2008年卫生部发布的全国死因调查结果显示： 癌症已成为我国城市首位、 农村第二位的死因。 治疗癌症的一种重要手段就是化疗。 但 是， 治疗过程中肿瘤患者往往会对化疗药产生耐药性， 同 还对其他不同化学结构、 不 同作用机制的药物产生交叉耐药性。 这种多药耐药性 (multidrug resistance, MDR) 的出 现极大程度上降低了药物的疗效， 导致化疗失效。 此外， 大多数抗肿瘤药物的水溶性较 低， 通过亲水性高分子材料的改性可以很大程度上增加药物的溶解度， 并能通过高通透 和滞留效应（EPR效应） 被动靶向到肿瘤部位， 从而降低其毒副作用。 但在临床研究中， 这类前药并未表现出增强的治疗效果， 这可能与药物被高分子链所包围， 不易被酶还原 成原始结构而只能依靠连接键的缓慢水解从而导致药物释放速度慢有关。对耐药性肿瘤, 这类前药也未见特殊效果。 发明内容
本发明所要解决的技术问题是克服现有技术的不足， 提供一种具有 P-糖蛋白抑制功 能的抗肿瘤前药， 其可望更高效地实现对肿瘤及耐药性肿瘤的治疗。
为解决以上技术问题， 本发明采取如下技术方案- 一种具有 糖蛋白抑制功能的抗肿瘤前药， 其是由抗肿瘤药物与具有 P-糖蛋白抑制 功能的聚乙二醇琥珀酸酯通过连接器共价连接构成的两亲性物质， 连接器含有敏感键 至少含有两个分别用于共价连接抗肿瘤药物和聚乙二醇琥珀酸酯的反应官能团， 所述的 敏感键是在肿瘤细胞内的还原性环境或酸性环境下易断裂的化学键。
根据本发明， 敏感键的特点是， 通过其连接在一起的两个物质在舯瘤细胞内的还原 性环境或酸性条件下即会解离， 因此， 敏感键可迸一步分为还原敏感键和 pH敏感键。 常 见的还原敏感键有二硫键、 二硒键等； 常见的 pH敏感键有腙键、 西弗碱键、 肟键、 原酸 酯键、 缩醛或缩酮键等。
本发明的具有 I S蛋白抑制功能的抗肿瘤前药可用式 I表示。
Figure imgf000002_0001
Figure imgf000003_0001
代表的是抗肿瘤药物的部分； 代表的是连接器的部分。
根据本发明的一个优选方面， 用于共价连接抗肿瘤药物和聚乙二醇琥珀酸酯的两个 能团可以相同或不同， 旦选自羟基、 羧基 （包括酸酐形式）、 氨基、 取代氨基、 酰 胺基、 氰基及异氰酸基、 醛基和羰基。
进一步优选地， 连接器由敏感键、 两个反应官能团以及分别连接在敏感键与两个反 应官能团之间的个数为 1〜12的亚甲基组成。具体的连接器有^如 3，3'-二硫代二丙酸 , 4,4'- 二硫代二丁酸， 3,3'-二硒代二丙酸， 对羧基苯甲醛、 3-羧基苯甲醛、 乙醛酸、 丙酮酸、 琥 珀酰胼、 己二酰胼等。
根据一个优选方面， 根据本发明的具有 糖蛋白抑制功能的抗肿瘤前药的结构通式 如式： Π:
式
代表的是抗肿瘤药
Figure imgf000003_0002
V7T ; m,P独立地为 1〜12之间的整数 ; M代表 S或 Se。
进一步优选地， m，P独立地为〗〜6之间的整数， 例如 m,:P均为】， 2或 3 ; 或者例如 m为 2， P为 3。
进一歩优选地， m与 P相同， 当 m与 P相同时， 形成的结构较对称， 因而相对更稳 进 ·步地， 聚乙二醇琥珀酸酯优选为选自聚乙二醇维生素 E琥珀酸酯、 聚乙二醇胆 固醇琥珀酸酯及聚乙二醇脂肪醇琥珀酸酯中的一种， 其中， 更优选聚乙二醇维生素 E琥 珀酸酯 （TPGS )。 优选地， 聚乙二醇琥珀酸酯中的聚乙二醇的数均分子量为 500〜6000。 具体地说， 聚 乙二醇琥珀酸酯中的聚乙二醇为 PEG500、 PEG 1000, PEG2000、 PEG3350或 PEG5000。
根据本发明， 抗肿瘤药物可以是己知的各种抗肿瘤药物， 优选是需要用于 P gp底物 的药物， 包括但不限于紫杉醇 （PTX)、 多西紫杉醇、 多柔比星 （DOX)、 表柔比星、 长 春碱、 长春新碱、 高 Ξ:尖杉酯碱及喜树碱等。
根据本发明， 上述的具有 P-糖蛋白抑制功能的抗肿瘤前药可以丛已有的原料出发， 经过常规的有机合成方法制备得到。 以连接器为 3,3'-二硫代二丙酸为例， 抗肿瘤前药可 通过如下歩骤合成；
( 1 )、 使 3,3'-二硫代二丙酸在乙酰氯中回流， 得到 3,3'-二硫代二丙酸酐；
(2)、 将具有 P-糖蛋白抑制功能的聚乙二醇琥珀酸酯 （如 TPGS)、 3,3'-二硫代二丙 酸酐溶解于无水 DMSO中， 加入适量三乙胺， 在加入 DMAP条件下， 室温下搅拌反应， 得到 TPGS- - S- S- COOH;
( 3 )、将步骤 (2)中所得的产物溶解在无水二氯甲烷中，在 N-羟基琥珀酰亚胺 (NHS)、 二环己基碳二亚胺 （DCC) 存在下， 室温下搅拌反应对其进行活化；
(4 )、 用适量无水二氯甲烷或 DMSO溶解抗 )ΐ中瘤药物 （如 ΡΤΧ)， 与步骤 （3 ) 中所 得活化产物混合， 室温下搅拌反应， 即可得具有 Ρ-糖蛋白抑制功能的抗肿瘤前药
(TPGS-S-S-PTX) o
上述合成路线用化学反应式来表示如下：
T Y {1 )DCC/NHS
TPGS 」 一 TPGS各 S-COOH * TPGS S S PTX。
(2)PTX
DMAP '
本发明的发明思路如下； 已知， H3 mdrl 基因编码的 P-糖蛋白 (P- glycoprotein, P-gp) 的过表达是 MDR.产生的一个重要机制。 近年来， PEG衍生物的 P-gp抑制作用得到了广 泛的研究和证实， 其中抑制效果最佳的就是聚乙二醇维生素 E琥珀酸酯 （TPGS)。 本发 明利用 TPGS及具有类似功能的聚乙二醇琥珀酸酯和二硫键、 二硒键等敏感键， 设计出 一种以肿瘤细胞内还原性环境或酸性环境为响应点， 具有 P- gp抑制功能的敏感前药， 该 前药可自组装形成胶束结构， 通过 EPR效应在肿瘤组织聚集， 经胞吞进入肿瘤细胞。 在 胞内敏感键解离， 形成游离的 TPGS聚合物和抗肿瘤药物的衍生物。 该衍生物不存在高 分子链的位阻， 因而较易被还原成具有高抗肿瘤活性的原药结构。 同时， TPGS 与 P- gp 结合， 抑制 P- gp活性， 降低抗肿瘤药物的外排， 而逆转耐药细胞的多药耐药性， 大幅 度提高抗肿瘤药物的的治疗效果。
由于上述技术方案的运用， 本发明与现有技术相比具有如下优势：
本发明的前药可自组装形成高分子胶束， 迸而极大地改善疏水药物的溶解度和稳定 性， 实现药物在体内的长循环， 通过 EPR效应富集于肿瘤部位。 并旦本前药在胖瘤细胞 内还原条件或酸性条件下能迅速解离成具有 P-糖蛋白抑制功能的聚乙二醇琥珀酸酯和和 药物的衍生物。 一方面， 药物衍生物具有较小的空间位阻， 很容易被还原成有效药物结 构， 能实现药物的快速释放。 另一方面， 游离的聚乙二醇琥 ¾酸酯与 P -gp结合， 抑制其 对药物的泵出， 提高药物的胞内浓度。
^图说明
图 1为实施^ 1中 TPGS的核磁共振氢谱谱图；
图 2为实施例 1中 TPGS- S-S-COOH的核磁共振氢谱谱图；
图 3为实施例 1的紫杉醇前药的核磁共振氢谱谱图；
图 4为实施^ 2的多柔比星前药的核磁共振氢谱谱图；
图 5为实施例 7的多柔比星前药的核磁共振氢谱谱图；
图 6为不同浓度的 TPGS- S- S- PTX溶液照片；
图 7为 TPGS- S-S- PTX在不同 pH值和 GSH条件下的粒径变化曲线；
图 8为 PTX标准曲线；
图 9为 raPEG- PTX前药和 TPGS- S- S- PTX前药的体夕卜释放曲线；
图 10为 PTX, mPEG-PTX前药和 TPGS-S-S-PTX前药的 24、 48、 72小时细胞毒性 实验结果。
图 11为 TPGS- S-S-PTX前药和临床制剂泰素 （Taxoi) 的药代动力学曲线。
图 12为 TPGS- S- S- PTX前药和泰素（Taxol)在荷 S180肉瘤小鼠上的组织分布结果。 图 i3为 TPGS- S- S- PTX前药和泰素（Taxoi)对荷 S180肉瘤小鼠肿瘤生长抑制结果。 图 14为 TPGS-CO-NH-NH=C-DOX前药 （实施例 7) 的体外释放曲线;
图 15为 DOX、 mPEG-DOX前药和 TPGS- CO- NH- N:»:::C- DOX前药的 24小时细胞毒 性实验结果。
图 16为 TPGS- CO- NH- NH=C- DOX前药和临床制剂多柔比星（DOX)对荷 H22肝癌 小鼠肿瘤生长抑制结果。 具体实施方式
实施例 1
本实施例提供一种具有 P-糖蛋白抑制功能的紫杉醇前药， 其通过如 T步骤合成： ( 1 )、3,3'-二硫代二丙酸酐的合成：称取 2g 3,3'-二硫代二丙酸放入 50ml圆底烧瓶中 , 加入 20ml乙酰氯， 65'Ό回流反应 3小时， 减压蒸馏去除乙酸、 乙酰氯等杂质； 在乙醚中 洗涤 3次， 减压挥发乙醚后得 3,3'-二硫代二丙酸酐。
(2)、 TPGS-S-S-COOH的合成： 称取 1.5g TPGS放入 lOOmL圆底烧瓶中， 在真空 千燥箱中 60°C条件下干燥 3-5小时， 再投入 0.276g 3,3'-二硫代二丙酸酐， 0.183g 4-二甲 氨基吡啶， 0.15raL：三乙胺， 溶在 5- lOmL二甲亚砜中； 在无水的环境中， 室温下搅拌反 应 24小! ΐ寸。
( 3 )、 TPGS-S-S-COOH的活化： 将上述产物用分子量 1000的透析袋在水中透析， 24〜48h后取出， 冻千即得 TPGS- S- S- COOH; 用 5- lOniL二氯甲烷 (DCM) 重新溶解冻 千物， 加入 N-羟基琥珀酰亚胺和 N'-二环己基碳二亚胺， 在无水环境中， 室温下搅拌反 (4)、连接紫杉醇：在 lOOmL的圆底烧瓶中加入 1.02g紫杉醇（PTX),用 5- 15mL DCM 溶解； 将歩骤 （3 ) 所得产物过滤， 滤液加至紫杉醇溶液中； 在无水环境中， 室温下搅拌 48h; 所得产物用分子量 2000的透析袋在无水乙醇中透析 24- 48h, 透析完毕后挥干乙醇 即
Figure imgf000006_0001
图 1〜图 3分别是 TPGS、 TPGS-S-S-COOH以及紫杉醇前药的氢核磁共振谱图， 确证
：.述结构的紫杉醇前药的成功合成。
实施例 2
本实施 ^提供一种 P-糖蛋白抑制功能的多柔比星前药， 其通过如下步骤合成；
( 1 ) , 按照与实施例 1前 Ξ:歩相同的方法获得活化的 TPGS- S- S- COOH。
(2)、连接多柔比星：将 0.6g多柔比星加在 lOOraL圆底烧瓶里， 5-1 OmL DMSO 和 0.12mi :三乙胺溶解， 并做避光处理； 将步骤（1 ) 的产物过滤， 滤液加至多柔比星溶液 中；在无水环境中，室温下搅拌 48h;所得产物用分子量 2000的透析袋在无水乙醇和 DMSO 混合溶剂中透析 24- 48h, 透析完毕后挥干乙醇即得多柔比星前药 （TPGS- S- S- DOX)， 其 结构如下；
Figure imgf000007_0001
对多柔比星前药进行了氢核磁共振测试， 谱图参见图 4， 确证上述结构的多柔比星前 药的成功合成。
实施例 3
本实施飼提供 ·种 P-糖蛋白抑制功能的紫杉醇前药， 其通过如下歩骤合成-
( 1 )、 二硒代丙二酸的合成： 称取 i.5g硼氢化钠于 iOOmL圆底烧瓶中， 加入 20ml 去离子水， 再投入 3.2g硒粉； 搅拌 10分钟后， 加热溶液至 70Ό反应， 直至硒粉完全消 失； 在氮气保护下加入 3-氯丙酸的四氢呋喃溶液 （4,36gZ50ml), 50Ό下反应 12h, 旋蒸 去掉四氢呋喃， 冻千后用乙醚洗涤， 即得二硒代丙二酸。
(2)、 TPGS-Se-Se-COOH的合成： 称取 i.5g TPGS放入 IOOmL圆底烧瓶中， 在真空 千燥箱中 60Ό条件下千燥 3〜5小时， 再投入 0.306g 二硒代二乙酸， 0,183g 4 -二甲氨基吡 啶， (U5mL .三乙胺,溶在 5- lOmL DMSO中；在无水的环境中， 室温下搅拌反应 24小时。
(3 )、 TPGS-Se-Se-COOH的活化： 将上述产物用分子量为 1000的透析袋在水中透 析' 24〜48h后取出， 冻干即得 TPGS- Se- Se- COOH; 用 5 iOmL二氯甲烷 (DCM)重新溶 解冻干物， 加入 N-羟基琥珀酰亚胺 (NHS)和 Ν,Ν'-二环己基碳二亚胺 (DCC), 在无水环境 中， 室温下搅拌反应 24h。
(4)、连接紫杉醇： 在 IOOmL的圆底烧瓶中加入 :L02g的紫杉醇（ΡΤΧλ 用 5 15mL DCM 溶解； 将步骤 （3 ) 得到的产物过滤， 滤液加至紫杉醇溶液中； 在无水环境中， 室 温下搅拌 48h; 所得产物用分子量为 2000的透析袋在无水乙醇中透析 24- 48h, 透析完毕 后挥千乙醇即得紫杉醇前药 （TPGS- Se-Se- COPTX), 其结构式如下-
Figure imgf000008_0001
实施例 4
本实施飼提供一种 P-糖蛋白抑制功能的喜树碱前药， 其通过如下步骤合成： 将 (U5g 喜树碱加在 lOOmL圆底烧瓶里， 加入 5- lOmL的 DMSO溶解， 并做避光处理； 将活化的 TPGS- S- S-COOH加至喜树碱溶液中； 在无水环境中， 室温下搅拌 48h; 所得产物用分子 量为 2000的透析袋在无水乙醇中透析 24- 48bu 透析完毕后挥千乙醇即得喜树碱前药， 其 结
Figure imgf000008_0002
实施例 5
本实施飼提供 ·种 P-糖蛋白抑制功能的高≡尖杉酯碱前药， 其通过如下步骤合成- 将 0,55g的高三尖杉酯减加在 lOOmL的圆底烧瓶里， 加入 5- 10m:L的 DMSO溶解， 并做 避光处理； 将将活化的 TPGS- S- S- COOH加至高三尖杉酯碱溶液中； 在无水环境中， 室温
Figure imgf000009_0001
实施例 6
本实施例提供一种 P-糖蛋白抑制功能的表柔比星前药， 其通过如下歩骤合成：
( 1 )、 TPGS对羧基苯甲醛酯的合成： 称取 1.5g TPGS放入 lOOmL圆底烧瓶中， 在 真空干燥箱中 60Ό条件下千燥 3-5小时，再投入 0.60g对羧基苯甲醛，0.825g DCC和 0,243g DMAP' 0.15mL :三乙胺， 溶在 50- lOOmL无水四氢呋喃 (THF) 中： 在无水的环境中， 室 温下搅拌反应 48小时后滤去沉淀，旋蒸去掉大部分 THF后在乙醚中沉淀、洗涤，得 TPGS 对羧基苯甲醛酯。
(2)、连接表柔比星： 将 0.3g多柔比星加在 50mL的圆底烧瓶里， 加入 2- 10mL的氯 仿 /甲醇混合溶剂 （1/1 , v/v) 和 0.06mi 乙胺溶解， 并做避光处理； 将 0.8g TPGS对羧 基苯甲醛酯溶于 l-8mi相同的氯仿 /甲醇混合溶剂， 加至多柔比星溶液中； 在无水环境中， 室温下搅拌 24h;所得产物 分子量为 2000的透析袋在无水乙醇和 DMSO混合溶剂中透 析 2448h, 透析完毕后挥千乙醇即得含有西弗碱的连接器的多柔比星 pH 敏感前药
(TPGS-C-N-DOX), 其结构如下；
Figure imgf000010_0001
实施例 7
本实施例提供一种 糖蛋白抑制功能的多柔比星前药， 其通过如下步骤合成：
( 1 )、 丁？08琥¾酸酯 （TPGS- COOH) 的合成： 称取 ί .5g TPGS放入】 OOmL圆底烧 瓶中，在真空干燥箱中 60 °C条件下干燥 3-5小时，再投入 0.15g琥珀酸酐， 0,】83g DMAP 和 0.15mL三乙胺， 溶在 5- 20mL无水二氯甲垸 （DCM ) 中； 在无水的环境中， 室温下搅 摔反应 24小时， 旋蒸去掉大部分 DCM后在乙醚中沉淀、 洗涤， 得 TPGS琥珀酸酯。
( 2) , TPGS琥珀酰胼的制备： 将 0,075g盐酸联氨加在 50mL的圆底烧瓶里， 加入 2-10mL的无水 DCM:和 0,18ml .三乙胺溶解， 并做避光处理； 1.2g TPGS- COOH采用与实 例 1歩骤 3相同的方法活化， 滴加进联氨 DCM溶液， 室温反应 48小 后旋蒸去掉大部 分 DCM后在乙醚中沉淀、 洗涤， 得 TPGS琥珀酰肼。
(3 )、 连接多柔比星： 将（),6g多柔比星和 0.8g TPGS琥珀酰胼加在 lOOmL的圆底烧 瓶里， 加入 5- 20raL的甲醇和 0.12mi三乙胺溶解， 滴加一滴:三氟乙酸， 并做避光处理； 在无水环境中， 60'Ό下搅拌过夜;所得产物用分子量为 2000的透析袋在无水乙醇和 DMSO 混合溶剂中透析 24-48h,透析完毕后挥干乙醇即得以腙键为连接器的多柔比星 pH敏感前 药 （TPGS- CO- NH- =C- DOX ), 其结构如下：
Figure imgf000011_0001
对多柔比星前药进行了氢核磁共振测试， 谱图参见图 5 , 确证上述结构的多柔比星前 药的成功合成。 具有： P—糖蛋白抑制功能的紫杉醇前药 （实施例 1 ) 的表征以及性能测试
(― P—糖蛋白抑制功能的紫杉醇前药的稳定性
1、 溶液的制备
将实施例 1的紫杉醇前药溶解在乙醇中， 缓慢滴至 pH7.4的 PBS中， 边滴边搅拌， 过夜搅拌挥干其中的乙醇。
2、 评份指标
( 1 ) 表观特征
按照上述溶液制备的方法， 分别配成前药浓度为 lmg/mL、 2mg/mL、 3mg/mL、 5mg/mL, 7mg/mL、 lOmg/mL的溶液， 置室温下观察是否发生沉降。
参见图 6， 经观察， 各个浓度前药都很稳定， 分散均匀， 室温下放置一个月性状无变 化。
( 2 ) 前药在不同环境下的稳定性
按照上述 ( 1 ) 中所述溶液的制备方法， 分别配成三组前药浓度为 lmg/m:L的溶液， 一组为 pH 5.5的 PBS溶液， 第二组为 pH 5.5的 PBS溶液， 且加入 iOmM:的谷胱甘肽 ( GSH)， 第三组为 pH 7,4的 PBS溶液， 第四组为 pH 7。4的 PBS溶液， 加入 iOmM的 GS1-L 在 37°C恒温条件下， 分别在 lh、 2h、 3h、 4h、 6h、 8h、 24h、 48h时间点测定溶液 中胶束的粒径大小。 参见图 7, 结果表明前药在 pH 7,4和 5.5下均表现出良好的稳定性， 粒径保存不变： 而在加入 GSH后， 前药稳定性降低， 由于二硫键的解离， 前药胶束粒径减小。
(二）、 P—糖蛋白抑制功能的紫杉醇前药的体外释放
1、 标准曲线的制备
称取 12.2mg的紫杉醇溶于 10ml甲醇中, 再依次稀释成以下浓度： 3.05、 6,10, 9.15、 15.25、 2〗.35、 27.45-ug/mL., 利用紫外分光光度^在波长为 227nm处测定溶液的吸光度， 绘成标准曲线 （如图 8所示）。
2、 紫杉醇前药的体外释放
考察传统前药 mPEG- PTX和新型前药 TPGS-S- S-PTX在不同环境下的释放行为： 在 pH 7.4的 PBS中， 分别加入或不加入 OmM的谷胱甘肽； 将紫杉醇前药溶解在各自的介 质中， 加至分子量为 1000的透析袋中， 作为袋内液； 袋外放置 5( 00mL的相应介质作 为外液， 在摇床中震荡， 恒温 37Ό ; 分别在 0,5h、 lh、 2、 4h、 8h、 12h、 24h、 48h取出 一定量的夕卜液， 并补充等量的液体。 利用紫外分光光度计在波长 227nm处测定吸光度， 代入上述步骤 1 中所绘标准曲线， 算得到对应的紫杉醇浓度， 绘制出紫杉醇体外释放 曲线（如图 9所示）。可以看到， 以酯键为连接键的 mPEG- PTX前药在加入还原剂谷胱 肽 （GSH) 时， 释放速度与不加几乎一致； 而以二硫键连接的 TPGS- S-S- PTX前药则表 现出明显的还原响应性， 在 GSH环境中药物释放明显加快。
(≡)、 前药对非耐药舯瘤细胞和酎药肿瘤的细胞的细胞毒性
细胞培养：卵巢癌敏感细胞 A2780和耐药细胞 A2780/T ]¾含 100 ml/L灭活胎牛血清、 lOOU/ml青霉素和 100U/nil链霉素的 RPM】1640培养液， 置于 5% C02细胞培养箱， 37 V 恒温恒湿培养。
细胞毒性实验： 取对数生长期的 A2780和 A2780/T细胞 ¾ PBS洗涤， 0,25%姨蛋白 酶消化， 离心后再配置成浓度为 IxlO5个 /ml的细胞悬浮液。 将该悬浮液按 100 μΐ/孔加入 96 孔细胞培养板中培养 24 h, 让细胞完全贴壁。 以紫杉醇为模型药物， 按照抗耐药组 (TPGS-S-S-PTX前药），普通组（mPEG前药，酯键为连接键）、 PTX单药组及空白组（生 理盐水） 配置一系列不同浓度的胶束 /参比溶液 （0.001- 100μΜ)。 吸除培养液， 每孔依次 加入 100 μΐ不同浓度的胶束 /参比溶液， 培养 24、 48、 72 h。 之后每孔再用 200 MTT溶 液 （0.5 mg/mi)进行更换， 继续培养 4 h。 最后每孔加入 150 μΐ DMSO, 振荡 10 min。 用 酶标仪检测各孔于 570 nm处的吸收值， 并计算相应的 :C50。
参见图 iO,可以看到， TPGS-S-S-PTX前药对敏感细胞 A2780的杀伤力与 PTX相当， 远高于 mPEG PTX前药；而对耐药细胞 A2780/T而言， PTX几乎无杀伤作用， mPEG PTX 前药表现出较弱的杀伤能力， 而 TPGS- S- S-PTX前药仍表现出很强的细胞毒性。
(四） 前药的药代动力学
SD大鼠随机分为 2组， 每组 3只， 分别为泰素组和 TPGS-S- S- PTX组， 给药剂量： PTX 10 mg/kgo 尾静脉注射给药后， 30 min, 1 h， 2 h， 4 h， 8 h, 12 h, 24 h, 48 h和 72h之后 分别取血样处理， HPLC检测 PTX含量。药代动力学参数通过 DAS统计软件 （version 2,1.1, Mathematical Phamiacology Professional Committee, Chii:ia)i十算得 - --到。 . TPGS- S- S- PTX前药和临床制剂泰素 （Taxd) 的药代动力学参数计算结果 参数 单位 Taxoi TPGS-S-S- PTX
AUCo-t iiig/L/h 23.452土 i.929 4CL985土 6.478
AUC mg/L/h 4 i. i 84.士 6.599
寸
h 1.747^0.134 9, U 3士 0, i70
MRT h 1 .809 0.440 h 2,221i0.20 I 9.429土 0'396
ί：、、
ο
CL L/lv'kg 0.426^0.033 0,247土 0.036
H- ： Γ、
V L/kg L372土 0.227 3.343土 0J36 τ E max h 0.005±0.000 0,500士 0 00
、 mg/L ! 1382.4:0.624
参见图 11和上方的表 1，可以看到泰素在血液中的清除速率很高，而 TPGS-S- S ΡΤΧ 前药的血液循环时间显著延长，与泰素相比，半衰期 ti / 2延长为 4,25倍、 M:RT增加为 5,22 倍、 曲线下面积 （AUC) ±曾大 1.75倍， 相应的清除率（CL值）极大地降低， 仅为泰素的 0.58倍。在毒性方面，泰素在注射后 45分钟内其浓度处于毒性浓度区域（高于 8.54μ8/ηΛλ 而 TPGS- S-S- PTX前药的最大浓度分别为 7.03 g/mL, 都在毒性区域以夕卜， 这大大减轻了 PTX的毒副作用。
(五） 前药的组织分布
建立荷 S180肉瘤昆明小鼠移植瘤模型， 在当肿瘤长至 50-100 mm3 时， 随机分为 2 组 （泰素组和 TPGS- S- S- PTX组）， 尾静詠注射给药， 给药剂量； PTX 10 mg/kg, 分别于 6、 12 , 24h时处死部分小鼠， 取各脏器， 洗干净后均浆， HPLC检测 ΡΤΧ含量。 参见图 12, 可以看到， 与泰素相比， TPGS- S- S- ΡΤΧ前药的在各组织的代谢速率都有所 降低， 心、 肺、 肾富集明显降低， 肝、 脾浓度略有提高， 舯瘤部位的富集能力显著增强 且能长时间保持较高浓度，说明 TPGS- S-S- ΡΤΧ前药能更有效地靶向 )ΐ中瘤组织、降低 ΡΤΧ 的系统毒性。
(六） 前药的抗肿瘤活性
取荷 S180肉瘤小鼠， 在当肿瘤长至 50 100 mm3 时, 随机分为三组 （生理盐水组、 泰素组和 TPGS- S- S PTX组）, 幵始用药 (记为第 1天）, 剂量为 PTX浓度 10 mg/kg, 注 射时间为第 1、 3、 5、 7天。 每天测量肿瘤尺寸、 计算胖瘤体积。 参见图 13, 所有 Salme 组的小鼠肿瘤体积几乎呈直线上升， 而泰素和 TPGS- S- S- PTX前药都能明显抑制肿瘤的 生长， 前药的生长抑制能力最强。 在第 9天处死小鼠， 剥离胂瘤称重， ^算可得泰素和 TPGS-S-S-PTX的抑瘤率分别为 45.31%和 52.43%。 这说明了 TPGS- S S- PTX前药的舯瘤 抑制能力要优于泰素。 具有 P—糖蛋白抑制功能的多柔比星前药 （实施例 7) 的表征以及性能测试
(一）、 P—糖蛋白抑制功能的多柔比星前药的体夕卜释放
紫杉醇前药的体外释放
考察实施例 7的多柔比星前药在 pH值下的释放行为（测试方法同紫杉醇前药体外释 放部分） （如图 9所示）。 可以看到， 前药在中性条件下 pH=:7.4) 释放速度较为缓慢； 而在弱酸性条件下 （pH=5.0) DOX的释放速度明显加快， 表现出明显的 pH响应性。
(二）、 前药对非耐药肿瘤细胞和耐药肿瘤的细胞的细胞毒性
选取乳腺癌敏感细胞 MCF- 7和耐药细胞 MCF- 7/ADR为模型， 细胞培养和细胞毒性 实验方法同紫杉醇前药部分。 图 10可以看到， 多柔比星前药对敏感细胞 MCF- 7的杀 伤力要强于 DOX和 mPEG-DOX前药； 而对耐药细胞 MCF 7/ADR而言， DOX几乎无杀 伤作用， mPEG-DOX前药表现出一定的杀伤能力， 而多柔比星前药仍表现出很强的细胞 毒性。
(≡)、 前药对荷瘤小鼠的肿瘤生长抑制能力
选用荷 H22肝癌昆明小鼠作为肿瘤模型， 分组给药后记录各组的肿瘤体积变化， 如 图 16所示。 可以看到， 多柔比星和多柔比星前药都表现出了较好的肿瘤抑制效果， 但多 柔比星前药的疗效明显优于临床刺剂多柔比星。
上述实施飼只为说明本发明的技术构思及特点， 其目的在于让熟悉此项技术的人士能够 了解本发明的内容并据以实施， 并不能以此限制本发明的保护范围。 凡根据本发明精神 实质所作的等效变化或修 ¾i , 都应涵盖在本发明的保护范围之内。

Claims

权利要求：
一种具有 Ρ-糖蛋白抑制功能的抗肿瘤前药， 其特征在于： 该抗舯瘤前药是由抗肿瘤药 物与具有 Ρ-糖蛋白抑制功能的聚乙二醇琥珀酸酯通过连接器共价连接构成的两亲性物 质， 所述连接器含有敏感键且至少含有两个分别用于共价连接所述抗肿瘤药物和所述聚 乙二醇琥珀酸酯的反应官能团， 所述的敏感键是在肿瘤细胞内的还原性环境或酸性环境 下易断裂的化学键。
2、 根据权利要求 1所述的具有 糖蛋白抑刺功能的抗肿瘤前药， 其特征在于： 敏感键为 二硫键、 二硒键、 腙键、 西弗碱键、 肟键、 原酸酯键、 缩醛或缩酮键。
3、 根据权利要求 1或 2所述的具有 Ρ-糖蛋白抑制功能的抗肿瘤前药， 其特征在干： ]¾于 共价连接抗肿瘤药物和聚乙二醇琥珀酸酯的两个反应官能团相同或不同， 且分别选自羟 基、 羧基、 氨基、 取代氨基、 酰胺基、 氰基及异氰酸基、 醛基、 羰基。
4、 根据权利要求 3所述的具有 P-糖蛋白抑制功能的抗肿瘤前药， 其特征在于： 连接器由 敏感键、两个反应官能团以及分别连接在敏感键与两个反应官能团之间的个数为卜12的 亚甲基组成。
5、 根据权利要求 4所述的具有 Ρ -糖蛋白抑制功能的抗肿瘤前药， 其特征在于； 连接器为 3,3 '-二硫代二丙酸， 4,4'-二硫代二丁酸， 3,3'-二硒代二丙酸， 对羧基苯甲醛， 3-羧基苯甲 醛， 乙醛酸， 丙酮酸， 琥珀酰胼或己二酰肼。
6、 根据权利要求 4所述的具有 Ρ-糖蛋白抑制功能的抗肿瘤前药， 其结构式为式 Π所示：
式 I】中:
Figure imgf000015_0001
代表的是抗 肿瘤药物的部分； m, P独立地分别为 1〜12之间的整数； M代表 S或 Se。
7、 根据权利要求 1或 6所述的具有 糖蛋白抑制功能的抗肿瘤前药， 其特征在于： 聚乙 二醇琥珀酸酯为选自聚乙二醇维生素 E琥珀酸酯、 聚乙二醇胆固醇琥珀酸酯及聚乙二醇 脂肪醇琥珀酸酯中的一种。
8、 根据权利要求 i或 6所述的具有 P-糖蛋白抑制功能的抗肿瘤前药， 其特征在于； 聚乙 二醇琥珀酸酯中的聚乙二醇的数均分子量为 500~6000。
9、 根据权利要求 8所述的具有 糖蛋白抑刺功能的抗肿瘤前药， 其特征在于： 聚乙二醇 琥珀酸酯中的聚乙二醇为 PEG500, PEG 1000, PEG2000, PEG3350或: PEG5()00。
10、 根据权利要求 1或 6所述的具有 P-糖蛋白抑制功能的抗肿瘤前药， 其特征在于： 抗 肿瘤药物为选自紫杉醇、 多西紫杉醇、 多柔比星、 表柔比星、 长春碱、 长春新碱、 高≡ 尖杉酯碱及喜树碱中的一种。
PCT/CN2013/087617 2012-11-21 2013-11-21 一种具有p-糖蛋白抑制功能的抗肿瘤前药 WO2014079377A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201210475046.XA CN102935236B (zh) 2012-11-21 2012-11-21 一种具有p-糖蛋白抑制功能的抗肿瘤前药
CN201210475046.X 2012-11-21

Publications (1)

Publication Number Publication Date
WO2014079377A1 true WO2014079377A1 (zh) 2014-05-30

Family

ID=47694181

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/087617 WO2014079377A1 (zh) 2012-11-21 2013-11-21 一种具有p-糖蛋白抑制功能的抗肿瘤前药

Country Status (2)

Country Link
CN (1) CN102935236B (zh)
WO (1) WO2014079377A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333749A (zh) * 2020-03-27 2020-06-26 广州古泉生物科技有限公司 一种聚环氧丙烷-海藻酸钠水凝胶的制备和应用

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105853357A (zh) * 2016-04-26 2016-08-17 中国药科大学 一种多功能型协同混合胶束递药系统及其制备方法
CN106632272B (zh) * 2016-11-29 2019-07-26 沈阳药科大学 白蛋白结合型5-氟尿嘧啶前体药物的合成及应用
CN106620717B (zh) * 2016-12-13 2020-11-24 上海交通大学 一种具有逆转肿瘤多药耐药性功能的两亲性缀合物抗肿瘤纳米药及其制备方法和应用
CN106727284B (zh) * 2017-01-09 2020-06-02 武汉工程大学 一种磁性还原增强型药物敏感释放纳米凝胶及其制备和保存方法
CN108245683B (zh) * 2018-01-26 2020-02-14 华中科技大学 一种具有p-糖蛋白抑制功能的抗肿瘤前药及制备方法
CN109485748A (zh) * 2018-09-04 2019-03-19 澳门大学 一类苄泽修饰壳聚糖嫁接物及其制备方法、应用
CN109350748B (zh) * 2018-10-24 2021-06-11 沈阳药科大学 氧化还原双敏感键桥连小分子前药及其自组装纳米粒
CN109824884B (zh) * 2019-02-19 2021-04-06 安徽大学 一种pH敏感和活性氧增敏的普兰尼克聚合物及其制备方法和应用
CN112494460B (zh) * 2020-12-10 2022-03-11 浙江大飞龙动物保健品股份有限公司 替米考星粉剂及其制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1226176A (zh) * 1996-08-02 1999-08-18 奥索·麦克尼尔药品公司 含有与n－末端共价结合的单个水溶性聚合物的多肽类
CN102114246A (zh) * 2011-03-01 2011-07-06 中国药科大学 生物体病灶部位特异性释药的两亲性多糖衍生物载体及其药学组合物的制备和应用
CN102735809A (zh) * 2012-06-29 2012-10-17 成都欧林生物科技股份有限公司 测定Hib结合疫苗中高分子结合物含量的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1226176A (zh) * 1996-08-02 1999-08-18 奥索·麦克尼尔药品公司 含有与n－末端共价结合的单个水溶性聚合物的多肽类
CN102114246A (zh) * 2011-03-01 2011-07-06 中国药科大学 生物体病灶部位特异性释药的两亲性多糖衍生物载体及其药学组合物的制备和应用
CN102735809A (zh) * 2012-06-29 2012-10-17 成都欧林生物科技股份有限公司 测定Hib结合疫苗中高分子结合物含量的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU, XIANG ET AL.: "Preparation and Characterization of Camptothecin-hyaluronic Aicd Conjugate.", JOURNAL OF XINYANG NORMAL UNIVERSITY (NATURAL SCIENCE EDITION), vol. 24, no. 4, October 2011 (2011-10-01), pages 541 - 517 *
NA CAO ET AL.: "Doxorubicin conjugated to D- a -tocopheryl polyethylene glycol 1000 succinate (TPGS): Conjugation chemistry, characterization, in vitro and in vivo evaluation.", BIOMATERIALS, vol. 29, no. 28, 7 July 2008 (2008-07-07), pages 3856 - 3865 *
VANANGAMUDI ANBHARASI ET AL.: "Doxorubicin conjugated to D- a -tocopheryl polyethylene glycol succinate and folic acid as a prodrug for targeted chemotherapy.", J. BIOMED. MATER. RES. PART A, vol. 94A, no. 3, 1 September 2010 (2010-09-01), pages 730 - 743 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333749A (zh) * 2020-03-27 2020-06-26 广州古泉生物科技有限公司 一种聚环氧丙烷-海藻酸钠水凝胶的制备和应用

Also Published As

Publication number Publication date
CN102935236A (zh) 2013-02-20
CN102935236B (zh) 2015-08-26

Similar Documents

Publication Publication Date Title
WO2014079377A1 (zh) 一种具有p-糖蛋白抑制功能的抗肿瘤前药
Wang et al. Cancer nanomedicines stabilized by π-π stacking between heterodimeric prodrugs enable exceptionally high drug loading capacity and safer delivery of drug combinations
Mu et al. Acid-sensitive PEGylated paclitaxel prodrug nanoparticles for cancer therapy: Effect of PEG length on antitumor efficacy
Shan et al. A paclitaxel prodrug with bifunctional folate and albumin binding moieties for both passive and active targeted cancer therapy
CN108904447B (zh) 一种肝肿瘤靶向载体材料、胶束制剂及其制备方法
KR20180120220A (ko) 난소암을 특이적으로 표적하는 생분해성 양친성 폴리머, 이로부터 제조된 폴리머 배시클 및 용도
CN108670954B (zh) 一种共载化疗药物的甘草次酸前药胶束及其制备方法
Liu et al. A multi-stimuli responsive nanoparticulate SN38 prodrug for cancer chemotherapy
Liang et al. Carboplatin-loaded SMNDs to reduce GSH-mediated platinum resistance for prostate cancer therapy
JP2024508128A (ja) 注射用の新規アビラテロン誘導体の調製方法及び用途
CN108245683B (zh) 一种具有p-糖蛋白抑制功能的抗肿瘤前药及制备方法
Li et al. Design and synthesis of polymer prodrugs for improving water-solubility, pharmacokinetic behavior and antitumor efficacy of TXA9
CN114230634B (zh) 一种酸响应性抗癌肽及其制备方法
CN104262614B (zh) 一种具有抗癌活性的化合物及抗癌组合物
CN107090082B (zh) 一种具有肿瘤组织还原敏感性的茄尼醇衍生物、其制备方法和应用
Duan et al. Highly water-soluble methotrexate-polyethyleneglycol-rhodamine prodrug micelle for high tumor inhibition activity
Guan et al. Design, synthesis, and characterization of glycyrrhetinic acid-mediated multifunctional liver-targeting polymeric carrier materials
CN108524951A (zh) 一种具有肝肿瘤靶向作用的索拉菲尼纳米制剂
CN112641728A (zh) 一种色胺酮纳米脂质体及其制备方法
CN111363062B (zh) 一种tpgs修饰的羧甲基壳聚糖-大黄酸偶联物及其合成工艺和用途
CN107714641A (zh) 一种用于联合用药的喜树碱前药负载双药物超分子水凝胶的制备方法
CN107441043A (zh) 一种pH敏感性混合胶束及其制备方法与应用
KR20120126356A (ko) 양친성 저분자량 히알루론산 복합체를 포함하는 나노 입자 및 그의 제조 방법
CN102558546B (zh) 聚乙二醇甘草次酸酯、复合载药胶束及其制备方法和用途
Liu et al. A novel dextran-based dual drug conjugate targeted tumors with high biodistribution ratio of tumors to Normal tissues

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13856628

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13856628

Country of ref document: EP

Kind code of ref document: A1