WO2014079377A1

WO2014079377A1 - Antitumor prodrugs with function of p-glycoprotein inhibition

Info

Publication number: WO2014079377A1
Application number: PCT/CN2013/087617
Authority: WO
Inventors: 张志平; 谭松巍
Original assignee: 武汉平华生物医药科技有限公司
Priority date: 2012-11-21
Filing date: 2013-11-21
Publication date: 2014-05-30
Also published as: CN102935236B; CN102935236A

Abstract

Antitumor prodrugs with function of P-glycoprotein inhibition are disclosed. The prodrugs are amphiphilic substances composing of antitumor drug covalently linked with polyethylene glycol succinate with function of P-glycoprotein inhibition via a linker. The linker has a sensitive bond and two reactive groups separately covalently linked with antitumor drug and polyethylene glycol succinate. The sensitive bond is a scissile chemical bond in the intracelluar reducing environment or acidic environment of tumor cells.

Description

Anti-tumor prodrug with P-glycoprotein inhibitory function

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.

Background technique

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. However, 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. The emergence of this multidrug resistance (MDR) has greatly reduced the efficacy of the drug, leading to chemotherapy failure. In addition, 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 tumor site, thereby reducing its toxic side effects. However, in clinical studies, such prodrugs have not shown an enhanced therapeutic effect, which may be surrounded by the polymer chain, which is not easily reduced to the original structure by the enzyme and can only rely on the slow hydrolysis of the linkage to cause drug release. Slow speed is related. For drug-resistant tumors, this type of prodrug has no special effects. Summary of the invention

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.

In order to solve the above technical problems, 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. Covalently linked to the amphiphilic substance, 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.

According to the present invention, 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.

Represents the part of the anti-tumor drug; represents the part of the connector.

According to a preferred aspect of the present invention, 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.

Further preferably, 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.

According to a preferred aspect, the structural formula of the anti-tumor prodrug having glycoprotein inhibitory function according to the present invention is as follows:

Style

Representing antineoplastic agents

V7T ; m, P are independently integers between 1 and 12 _; M represents S or Se.

Further preferably, m, 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.

Further, m is preferably the same as P, and when m is the same as P, the structure formed is relatively symmetrical, and thus relatively more stable, the polyethylene glycol succinate is preferably selected from the group consisting of 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. Preferably, the polyethylene glycol in the polyethylene glycol succinate has a number average molecular weight of from 500 to 6,000. Specifically, the polyethylene glycol in the polyethylene glycol succinate is PEG500, PEG 1000, PEG2000, PEG3350 or PEG5000.

According to the present invention, 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.

According to the present invention, 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. Taking the connector as 3,3'-dithiodipropionic acid as an example, the anti-tumor prodrug can be synthesized by the following steps;

(1), refluxing 3,3'-dithiodipropionic acid in acetyl chloride to obtain 3,3'-dithiodipropionic anhydride;

(2) Dissolving polyethylene glycol succinate (such as TPGS) and 3,3'-dithiodipropionic anhydride having P-glycoprotein inhibitory function in anhydrous DMSO, and adding appropriate amount of triethylamine. After adding DMAP, the reaction was stirred at room temperature to obtain TPGS--S-S-COOH;

(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;

(4), using an appropriate amount of anhydrous dichloromethane or DMSO to dissolve the anti-tumor drug (such as sputum), mixed with the activated product obtained in step (3), stirring at room temperature, can be obtained with Ρ-glycoprotein inhibition Functional anti-tumor prodrug

(TPGS-S-S-PTX) o

The above synthetic route is represented by a chemical reaction formula as follows:

T Y {1 )DCC/NHS

TPGS ” TPGS each S-COOH * TPGS S S PTX.

(2) PTX

DMAP '

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). 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. At the same time, 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.

Due to the use of the above technical solutions, 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. Moreover, 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. On the one hand, drug derivatives have less steric hindrance and are easily reduced to effective drug tracts. Structure, can achieve rapid release of drugs. On the other hand, the free polyethylene glycol succinate binds to P-gp, inhibits its pumping of the drug, and increases the intracellular concentration of the drug.

^Illustration

Figure 1 is a nuclear magnetic resonance spectrum of TPGS in the implementation of Figure 1;

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 14 is an in vitro release profile of TPGS-CO-NH-NH=C-DOX prodrug (Example 7);

Figure 15 shows the results of a 24-hour cytotoxicity test for DOX, mPEG-DOX prodrugs, and TPGS-CO-NH-N:»:::C-DOX prodrugs.

Figure 16 shows the tumor growth inhibition results of TPGS-CO-NH-NH=C-DOX prodrug and clinical preparation doxorubicin (DOX) against H22 liver cancer mice. detailed description

Example 1

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.

(2), 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.

(3) 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 dialysis, the ethanol was evaporated.

Figure 1 to Figure 3 are hydrogen nuclear magnetic resonance spectra of TPGS, TPGS-S-S-COOH and paclitaxel prodrugs, respectively.

:. Successful synthesis of the structure of paclitaxel prodrugs.

Example 2

The present invention provides a doxorubicin prodrug having a P-glycoprotein inhibitory function, which is synthesized by the following steps;

(1), activated TPGS-S-S-COOH was obtained in the same manner as in Example 1 before: hydrazine.

(2), connect doxorubicin: 0.6g 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;

Hydrogen nuclear magnetic resonance imaging was performed on the doxorubicin prodrug. The spectrum is shown in Figure 4 to confirm the successful synthesis of the above-described structure of doxorubicin prodrug.

Example 3

This embodiment provides a paclitaxel prodrug which has a P-glycoprotein inhibitory function, which is synthesized by the following steps -

(1) Synthesis of diselenomalonic acid: Weigh i.5g sodium borohydride in iOOmL round bottom flask, add 20ml deionized water, and then add 3.2g selenium powder; after stirring for 10 minutes, heat the solution to 70Ό The reaction was completed until the selenium powder completely disappeared. Under a nitrogen atmosphere, a solution of 3-chloropropionic acid in tetrahydrofuran (4, 36 g of Z50 ml) was added, and the reaction was carried out for 12 h under 50 Torr. The tetrahydrofuran was removed by rotary evaporation, and then washed with diethyl ether to obtain diselenyl. Diacid.

(2) Synthesis of TPGS-Se-Se-COOH: Weigh i.5g TPGS into a 100 mL round bottom flask, dry for 3 to 5 hours under vacuum conditions in a vacuum drying oven, and then add 0.306 g of selenium. Diacetic acid, 0,183 g of 4-dimethylaminopyridine, (U5 mL. Triethylamine, dissolved in 5-1 mL of DMSO; the reaction was stirred at room temperature for 24 hours in an anhydrous environment.

(3) Activation of TPGS-Se-Se-COOH: The above product was dialyzed against water in a dialysis bag with a molecular weight of 1000 for 24 to 48 hours, and then lyophilized to obtain TPGS-Se-Se-COOH; using 5 iOmL two Chloroform (DCM) redissolves the lyophilizate, adding N-hydroxysuccinimide (NHS) and hydrazine, Ν'-dicyclohexylcarbodiimide (DCC), stirring in a dry environment for 24 h at room temperature .

(4), connecting paclitaxel: in a 100 mL round bottom flask: L02g of paclitaxel (ΡΤΧλ is dissolved with 5 15mL DCM; the product obtained in step (3) is filtered, and the filtrate is added to the paclitaxel solution; in an anhydrous environment, 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 the dialysis was completed, the paclitaxel prodrug (TPGS-Se-Se-COPTX) was obtained by the following method, and the structural formula was as follows -

Example 4

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.

Example 5

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

Example 6

This example provides a P-glycoprotein inhibitory function of epirubicin prodrug, which is synthesized by the following steps:

(1), TPGS p-carboxybenzaldehyde ester synthesis: Weigh 1.5g TPGS into a lOOmL round bottom flask, dry in a vacuum drying oven under 60 Ό 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.

(2), connect epirubicin: Add 0.3g of doxorubicin to a 50mL round bottom flask, add 2- 10mL of chloroform / methanol mixed solvent (1/1, v / v) and 0.06mi ethylamine dissolved And do light treatment; 0.8g TPGS p-carboxybenzaldehyde ester dissolved in l-8mi of the same chloroform / methanol mixed solvent, added to doxorubicin solution; in an anhydrous environment, stirring at room temperature for 24h; The dialysis bag with a molecular weight of 2000 was dialyzed in a mixed solvent of absolute ethanol and DMSO for 2448 h. After the dialysis was completed, the doxorubicin pH-sensitive prodrug containing the connector of Schiff base was obtained by boiling the ethanol.

(TPGS-CN-DOX), the structure is as follows;

Example 7

This example provides a glycoprotein inhibitory function of doxorubicin prodrug which is synthesized by the following steps:

(1), Ding? Synthesis of 08 succinate (TPGS-COOH): Weigh ί5g TPGS into OOmL round bottom flask, dry in a vacuum oven at 60 °C for 3-5 hours, and then add 0.15g succinic anhydride. , 0,] 83g DMAP and 0.15mL of triethylamine, dissolved in 5-20mL anhydrous dichloromethane (DCM); in a water-free environment, stirring at room temperature for 24 hours, rotary evaporation to remove most of DCM After precipitation and washing in diethyl ether, TPGS succinate was obtained.

(2) Preparation of 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. .

(3), connect doxorubicin: Add (), 6g doxorubicin and 0.8g TPGS succinic hydrazide in a lOOmL round bottom flask, add 5-20raL methanol and 0.12mi triethylamine to dissolve, drop Add one drop: trifluoroacetic acid, and avoid it in the dark; in an anhydrous environment, stir under 60'overnight; the product obtained is dialyzed in a mixture of absolute ethanol and DMSO for 24-48h in a dialysis bag with a molecular weight of 2000. After the completion of the evaporation of ethanol, the doxorubicin pH-sensitive prodrug (TPGS-CO-NH- = C- DOX) can be used as a connector. The structure is as follows:

A hydrogen nuclear magnetic resonance test was performed on the doxorubicin prodrug. The spectrum is shown in Figure 5 to confirm the successful synthesis of the above-described structure of doxorubicin prodrug. Characterization and performance testing of paclitaxel prodrugs with P-glycoprotein inhibitory function (Example 1)

(- P-glycoprotein inhibitory function of paclitaxel prodrug stability

1, the preparation of the solution

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.

2. Evaluation indicators

(1) Apparent characteristics

According to the above method for preparing a solution, 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.

Referring to Figure 6, it has been observed that the prodrugs of each concentration are stable and evenly dispersed, and remain unchanged for one month at room temperature.

(2) Stability of prodrugs in different environments

According to the preparation method of the solution described in the above (1), 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. Referring to Figure 7, 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.

(B), in vitro release of paclitaxel prodrugs with P-glycoprotein inhibitory function

1. Preparation of standard curve

Weigh 12.2 mg of paclitaxel in 10 ml of methanol, and then dilute to the following concentrations: 3.05, 6, 10, 9.15, 15.25, 2, .35, 27.45-ug/mL., using ultraviolet spectrophotometry ^ at a wavelength of 227 nm The absorbance of the solution was measured and plotted as a standard curve (as shown in Figure 8).

2. In vitro release of paclitaxel prodrugs

To investigate the release behavior of the traditional prodrug mPEG-PTX and the novel prodrug TPGS-S-S-PTX in different environments: Add or not add OmM glutathione in PBS pH 7.4; dissolve paclitaxel prodrug In each medium, add to the dialysis bag with a molecular weight of 1000, as the inner liquid of the bag; place 5 (00mL of the corresponding medium as the external liquid, shake in the shaker, constant temperature 37Ό; respectively at 0,5h, lh , 2, 4h, 8h, 12h, 24h, 48h, take a certain amount of liquid, and add the same amount of liquid. Use the ultraviolet spectrophotometer to measure the absorbance at 227nm, substitute the standard curve drawn in step 1 above, calculate The corresponding paclitaxel concentration was obtained, and the in vitro release profile of paclitaxel was plotted (as shown in Figure 9). It can be seen that the release rate of the mPEG-PTX prodrug with the ester bond as the linkage was added to the reducing agent glutathione (GSH). The TPGS-SS-PTX prodrug linked by disulfide bond showed obvious reduction responsiveness, and the drug release was significantly accelerated in the GSH environment.

(≡), cytotoxicity of prodrugs on cells of non-resistant neoplastic cells and peony tumors

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 μΐ/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 μΜ). The culture solution was aspirated, and 100 μM 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 μM 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.

Referring to Figure iO, it can be seen that the lethality of 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.

(iv) Pharmacokinetics of prodrugs

SD rats were randomly divided into 2 groups, 3 in each group, respectively, in the Taxol group and TPGS-S-S-PTX group. Dosage: PTX 10 mg/kgo After tail vein injection, 30 min, 1 h After 2 h, 4 h, 8 h, 12 h, 24 h, 48 h and 72 h, blood samples were taken and the PTX content was determined by HPLC. The pharmacokinetic parameters were calculated by DAS statistical software (version 2, 1.1, Mathematical Phamiacology Professional Committee, Chii: ia). TPGS-S-S-PTX prodrug and clinical preparation Taxol (Taxd) pharmacokinetic parameters calculation results parameter unit Taxoi TPGS-SS- PTX

AUCo- _t iiig/L/h 23.452 soil i.929 4CL985 soil 6.478

AUC mg/L/h 4 i. i 84.士 6.599

Inch

h 1.747^0.134 9, U 3士 0, i70

MRT h 1 .809 0.440 h 2,221i0.20 I 9.429 soil 0'396

ί:,,

ο

CL L/lv'kg 0.426^0.033 0,247 soil 0.036

H- : Γ,

VL/kg L372 soil 0.227 3.343 soil 0J36 τ ^E max h 0.005±0.000 0,500士0 00

, mg/L ! 1382.4:0.624

Referring to Figure 11 and Table 1 above, it can be seen that the rate of clearance of Taxol in the blood is high, while the blood circulation time of the TPGS-S-S ΡΤΧ prodrug is significantly prolonged, compared with Taxi, half-life t _{i / 2} The prolongation was 4,25 times, the M:RT increased to 5,22 times, the area under the curve (AUC) ± was 1.75 times larger, and the corresponding clearance rate (CL value) was greatly reduced, only 0.58 times that of Taxol. In terms of toxicity, Taxol was in the toxic concentration region within 45 minutes after injection (higher than 8.54μ ₈ /ηΛλ and the maximum concentration of TPGS-SS-PTX prodrug was 7.03 g/mL, respectively. Bu, this greatly reduces the side effects of PTX.

(5) Tissue distribution of prodrugs

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. Referring to Figure 12, it can be seen that compared with Taxol, the metabolic rate of TPGS-S-S- sputum prodrugs in all tissues is reduced, heart, lung and kidney enrichment are significantly reduced, and liver and spleen concentrations are slightly There is an increase, the enrichment ability of the tumor site is significantly enhanced and can maintain a high concentration for a long time, indicating that TPGS-SS-ΡΤΧ prodrug can more effectively target the tumor tissue in the sputum and reduce the systemic toxicity of sputum.

(6) Antitumor activity of prodrugs

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. On the 9th day, the mice were sacrificed and the tumors were excised and weighed. The tumor inhibition rates of Taxol and TPGS-SS-PTX were 45.31% and 52.43%, respectively. This indicates that the TPGS-S S-PTX prodrug has better tumor inhibition ability than Taxol. Characterization and performance testing of doxorubicin prodrugs with P-glycoprotein inhibitory function (Example 7)

(a), P-glycoprotein inhibition function of the doxorubicin prodrug

In vitro release of paclitaxel prodrug

The release behavior of the doxorubicin prodrug of Example 7 at pH was examined (test method with the in vitro release of paclitaxel prodrug) (shown in Figure 9). It can be seen that the release rate of the prodrug under neutral conditions pH=:7.4) is slower; while under weakly acidic conditions (pH=5.0), the release rate of DOX is significantly accelerated, showing a significant pH response.

(B), the cytotoxicity of prodrugs on non-resistant tumor cells and drug-resistant tumor cells

Breast cancer sensitive cells MCF-7 and drug resistant cells MCF-7/ADR were selected as models, and cell culture and cytotoxicity assays were combined with paclitaxel prodrugs. As can be seen in Figure 10, the doxorubicin prodrug is more potent against the sensitive cells MCF-7 than the DOX and mPEG-DOX prodrugs; whereas for the resistant cells MCF 7/ADR, DOX has almost no killing effect, mPEG - DOX prodrugs exhibit a certain killing ability, while doxorubicin prodrugs still exhibit strong cytotoxicity.

(≡), the ability of prodrugs to inhibit tumor growth in tumor-bearing mice

Kunming mice bearing H22 liver cancer were selected as tumor models, and the tumor volume changes of each group were recorded after group administration, as shown in Fig. 16. It can be seen that both doxorubicin and doxorubicin prodrugs show better tumor inhibition, but the efficacy of doxorubicin prodrugs is significantly better than the clinical spike doxorubicin.

The above described embodiments are merely illustrative of the technical concept and the features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the present invention and to implement the present invention, and the scope of the present invention is not limited thereto. Equivalent variations or modifications made in accordance with the spirit of the invention are intended to be included within the scope of the invention.

Claims

Rights request:

An anti-tumor prodrug having a Ρ-glycoprotein inhibitory function, characterized in that: the anti-tumor prodrug is an anti-tumor drug and a polyethylene glycol succinate having a Ρ-glycoprotein inhibitory function through a connector Valence linking the amphiphilic substance, the linker contains a sensitive bond and contains at least two reactive functional groups for covalently linking the antitumor drug and the polyethylene glycol succinate, the sensitivity A bond is a chemical bond that is easily broken in a reducing or acidic environment within a tumor cell.

2. The anti-tumor prodrug having glycoprotein stagnation function according to claim 1, wherein: the sensitive bond is a disulfide bond, a diselenide bond, a hydrazone bond, a sulphur base bond, a hydrazone bond, an orthoester Bond, acetal or ketal bond.

The anti-tumor prodrug having a scorpion-glycoprotein inhibitory function according to claim 1 or 2, which is characterized in that: two reactions of covalently linking an antitumor drug and polyethylene glycol succinate The functional groups are the same or different and are each selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a substituted amino group, an amide group, a cyano group and an isocyanate group, an aldehyde group, and a carbonyl group.

4. The anti-tumor prodrug having P-glycoprotein inhibitory function according to claim 3, wherein: the connector is composed of a sensitive bond, two reactive functional groups, and is respectively connected between the sensitive bond and the two reactive functional groups. The number is the methylene composition of Bu 12 .

The antitumor prodrug having the Ρ-glycoprotein inhibitory function according to claim 4, wherein the connector is 3,3 '-dithiodipropionic acid, 4,4'-dithiodi Butyric acid, 3,3'-diselenodipropionic acid, p-carboxybenzaldehyde, 3-carboxybenzaldehyde, glyoxylic acid, pyruvic acid, succinic hydrazide or adipic hydrazide.

The anti-tumor prodrug having a Ρ-glycoprotein inhibitory function according to claim 4, which has the structural formula:

In the formula I] _:

Representative of the anti-tumor drug; m, P are independently integers between 1 and 12; M stands for S or Se.

The anti-tumor prodrug having glycoprotein inhibitory function according to claim 1 or 6, wherein the polyethylene glycol succinate is selected from the group consisting of polyethylene glycol vitamin E succinate and polyethylene glycol. Cholesterol succinate and polyethylene glycol One of the fatty alcohol succinates.

The antitumor prodrug having a P-glycoprotein inhibitory function according to claim 1 or 6, wherein the polyethylene glycol in the polyethylene glycol succinate has a number average molecular weight of 500 to 6,000.

9. The anti-tumor prodrug having glycoprotein stagnation function according to claim 8, wherein: the polyethylene glycol in the polyethylene glycol succinate is PEG500, PEG 1000, PEG2000, PEG3350 or: PEG5 ()00.

The anti-tumor prodrug having P-glycoprotein inhibitory function according to claim 1 or 6, wherein the antitumor drug is selected from the group consisting of paclitaxel, docetaxel, doxorubicin, epirubicin, One of vinblastine, vincristine, sorghum, and camptothecin.