Adriamycin prodrug, preparation method and application thereof

文档序号：657726 发布日期：2021-04-27 浏览：67次中文

阅读说明：本技术 一种阿霉素前药及其制备方法与应用 (Adriamycin prodrug, preparation method and application thereof ) 是由彭媛媛张安林许慧邓泽平成佳于 2020-12-29 设计创作，主要内容包括：本发明属于生物医用高分子材料领域,具体涉及一种阿霉素前药及其制备方法与其作为前药的应用。目的在于提供一种阿霉素前药及其制备方法与应用,该阿霉素前药具有良好的生物相容性,低毒副作用和抑制肿瘤细胞增殖的能力,该制备方法合成条件不苛刻,提纯方便,适合工业化生产。(The invention belongs to the field of biomedical high polymer materials, and particularly relates to an adriamycin prodrug, a preparation method thereof and application thereof as the prodrug. The adriamycin prodrug has good biocompatibility, low toxic and side effects and the capacity of inhibiting tumor cell proliferation, and the preparation method has the advantages of mild synthesis conditions, convenience in purification and suitability for industrial production.)

1. An doxorubicin prodrug having the structural formula shown in formula (I):

wherein n is 4.

2. The process for the preparation of doxorubicin prodrug according to claim 1, characterized by comprising the steps of:

(1) taking anhydrous tetrahydrofuran as a solvent, taking polyethylene glycol and tert-butyl acrylate as raw materials in the presence of Na or tetramethyl ammonium hydroxide as a catalyst, and performing nucleophilic substitution reaction to prepare polyethylene glycol with a tert-butyl ester group at the tail end, wherein the molar ratio of the polyethylene glycol to the tert-butyl acrylate is 1: 1.5-3;

(2) using dichloromethane as a solvent, and performing hydrolysis reaction on the polyethylene glycol with the terminal of tert-butyl ester group prepared in the step (1) and trifluoroacetic acid to prepare polyethylene glycol with the terminal of carboxyl, wherein the concentration of the trifluoroacetic acid is 20-50%;

(3) in the presence of triethylamine, using dichloromethane as a solvent, and carrying out condensation reaction on the polyethylene glycol with the terminal carboxyl prepared in the step (2) and N, N '-disuccinimidyl carbonate to prepare polyethylene glycol with the terminal succinimide ester group, wherein the molar ratio of the polyethylene glycol with the terminal carboxyl to the N, N' -disuccinimidyl carbonate is 1: 2-3;

(4) and (3) carrying out amidation reaction on the polyethylene glycol with the terminal N-ester group succinimide obtained in the step (3), RGD and adriamycin in the presence of phosphate buffer solution to obtain the compound shown in the formula (I), wherein the molar ratio of the polyethylene glycol with the terminal succinimide ester group, the RGD and the adriamycin is 1: 0.5-0.8:1.2-1.5.

3. The method of preparing an doxorubicin prodrug according to claim 2, wherein in the step (1), polyethylene glycol is added to anhydrous tetrahydrofuran, Na is added under the protection of inert gas, after stirring and reacting until no bubble is generated, tert-butyl acrylate dissolved in anhydrous tetrahydrofuran is added to the reaction solution, after completion of the reaction of polyethylene glycol is detected, a dilute hydrochloric acid solution is added dropwise, an organic solvent is concentrated, the mixture is washed with water and a saturated aqueous sodium chloride solution, then drying and concentrating are performed, the crude product is mixed with silica gel and subjected to column chromatography, and polyethylene glycol with a tert-butyl ester group at a terminal is prepared, wherein the molar ratio of polyethylene glycol to Na is 1-3: 0.1.

4. The method of preparing an doxorubicin prodrug according to claim 2, wherein in the step (1), polyethylene glycol is added to anhydrous tetrahydrofuran, tetramethylammonium hydroxide is added, tert-butyl acrylate dissolved in anhydrous tetrahydrofuran is added to the reaction solution, after completion of the reaction of polyethylene glycol is detected, a dilute hydrochloric acid solution is added dropwise, the organic solvent is concentrated, the mixture is washed with water and a saturated aqueous solution of sodium chloride, and then dried and concentrated, and the crude product is subjected to sample mixing with silica gel and column chromatography to prepare polyethylene glycol having a tert-butyl ester group at a terminal, wherein the molar ratio of polyethylene glycol to tetramethylammonium hydroxide is 1: 0.1-0.3.

5. The process for preparing an adriamycin prodrug according to claim 2, wherein the step (2) comprises dissolving the polyethylene glycol with the terminal of tert-butyl ester group prepared in the step (1) in dichloromethane, adding trifluoroacetic acid, refluxing for 3-4 days to complete the reaction, concentrating the mixture to remove trifluoroacetic acid, slowly dropping the concentrated solution into glacial ethyl ether to precipitate a solid, filtering the precipitated solid, adding dichloromethane to dissolve the solid, dropping the solution into glacial ethyl ether, repeating the process for 4-5 times, performing suction filtration, and removing the residual solvent from the filtered solid by rotary evaporation to obtain polyethylene glycol with the terminal of carboxyl group.

6. The method for preparing the doxorubicin prodrug according to claim 2, wherein in the step (3), N' -disuccinimidyl carbonate and triethylamine are sequentially added into dichloromethane, the mixture is reacted and stirred at a certain temperature until the reaction is finished, the mixture is subjected to reduced pressure rotary evaporation, oily liquid obtained by the rotary evaporation is dripped into ice petroleum ether under stirring to generate white granular solid, the solid is filtered, the obtained white solid is transferred into a single-mouth bottle, and the residual solvent is removed by the reduced pressure rotary evaporation at normal temperature, so that the polyethylene glycol with the terminal succinimide ester group is finally obtained.

7. The method for preparing an adriamycin prodrug according to claim 2, wherein the step (4) is that RGD and polyethylene glycol with a terminal succinimide ester group are weighed and added into phosphate buffer solution, adriamycin is added into the phosphate buffer solution after stirring for 2 hours, and the mixture is reacted at room temperature in a dark place for 48 hours until the reaction is finished and then is post-treated to obtain the compound of formula (I).

8. The process for the preparation of doxorubicin prodrug according to claim 6, characterized in that the pH of the phosphate buffered saline solution in step (4) is 7.2-8.

9. An injectable doxorubicin prodrug composition comprises a doxorubicin prodrug and an acceptable pharmaceutical adjuvant, wherein the structural formula of the doxorubicin prodrug is shown in formula (I):

wherein n is 4.

10. Use of the doxorubicin prodrug according to claim 1 or 9 for the preparation of an antitumor medicament.

Technical Field

The invention belongs to the field of biomedical high polymer materials, and particularly relates to an adriamycin prodrug, a preparation method thereof and application thereof as the prodrug.

Background

Adriamycin is a broad-spectrum anthracycline antitumor drug with nonspecific cell cycle. Since the discovery in the sixties of the last century till now, the drug has been widely applied to the treatment of various tumors in clinic, such as prostate cancer, ovarian cancer, breast cancer and other tumors of various tissue origins. However, similar to most common small molecule chemotherapy drugs, doxorubicin has the disadvantages of low tissue specificity and selectivity, and is very easy to form serious toxic and side effects on normal human tissues, especially cardiotoxicity, so that the application dose of doxorubicin in clinical chemotherapy is greatly limited. In addition, long-term use of adriamycin for chemotherapy can induce tumor cells to show multidrug resistance (MDR), so that the tumor cells can resist adriamycin and other chemotherapeutic drugs, and the treatment effect is obviously reduced.

The prodrug is a compound which is obtained by modifying a chemical structure of a drug, has no or little activity in vitro, and releases an active drug by enzymatic or non-enzymatic conversion in vivo to exert a drug effect. The prodrug can improve the utilization rate of the medicament, improve the targeting property of the medicament and reduce the toxic and side effects of the medicament.

There is a need in the art to provide an adriamycin drug that improves biocompatibility, drug toxicity and side effects, and targeting.

Disclosure of Invention

The invention aims to provide an adriamycin prodrug, a preparation method and application thereof, wherein the adriamycin prodrug has good biocompatibility, low toxic and side effects and the capability of inhibiting tumor cell proliferation, and the preparation method has the advantages of non-harsh synthesis conditions, convenience in purification and suitability for industrial production.

The technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides an doxorubicin prodrug, which has a structural formula shown in formula (I):

wherein n is 4.

In a second aspect, the present invention provides a method for preparing an doxorubicin prodrug, which is characterized by comprising the following steps:

Preferably, in the step (1), polyethylene glycol is added into anhydrous tetrahydrofuran, Na is added under the protection of inert gas, stirring is carried out until no bubbles are generated, tert-butyl acrylate dissolved in the anhydrous tetrahydrofuran is added into a reaction solution, after the polyethylene glycol is detected to be completely reacted, a dilute hydrochloric acid solution is dropped, an organic solvent is concentrated, a mixture is washed by water and a saturated sodium chloride aqueous solution, then drying and concentrating are carried out, a crude product is mixed with silica gel and subjected to column chromatography, and the polyethylene glycol with the terminal tert-butyl ester group is prepared, wherein the molar ratio of the polyethylene glycol to the Na is 1-3: 0.1.

Preferably, in the step (1), polyethylene glycol is added into anhydrous tetrahydrofuran, then tetramethylammonium hydroxide is added, tert-butyl acrylate dissolved in the anhydrous tetrahydrofuran is added into a reaction solution, after the polyethylene glycol is detected to react completely, a dilute hydrochloric acid solution is dropped, an organic solvent is concentrated, a mixture is washed by water and a saturated sodium chloride aqueous solution, then drying and concentrating are performed, a crude product is mixed with silica gel and subjected to column chromatography, and the polyethylene glycol with a tert-butyl ester group at the end is prepared, wherein the molar ratio of the polyethylene glycol to the tetramethylammonium hydroxide is 1: 0.1-0.3.

Preferably, in the step (2), the polyethylene glycol with the terminal of tert-butyl ester group obtained in the step (1) is dissolved in dichloromethane, trifluoroacetic acid is added, the reflux reaction is carried out for 3-4 days, the reaction is completed, the mixture is concentrated to remove the trifluoroacetic acid, the concentrated solution is slowly dripped into the ethyl glacial ether to precipitate solids, the precipitated solids are filtered, the dichloromethane is added to dissolve the precipitated solids, the solution is dripped into the ethyl glacial ether, the process is repeated for 4-5 times, the suction filtration is carried out, and the residual solvent is removed by rotary evaporation of the leached solids to obtain the polyethylene glycol with the terminal of carboxyl group.

Preferably, in the step (3), N' -disuccinimidyl carbonate and triethylamine are sequentially added into dichloromethane, the mixture is reacted and stirred at a certain temperature until the reaction is finished, the mixture is subjected to reduced pressure rotary evaporation, oily liquid obtained by the rotary evaporation is dripped into stirred ice petroleum ether to generate white granular solid, the solid is filtered, the obtained white solid is transferred into a single-neck bottle, the residual solvent is removed by the reduced pressure rotary evaporation at normal temperature, and finally the polyethylene glycol with the terminal of the succinimide ester group is obtained.

Preferably, in the step (4), RGD and polyethylene glycol with a terminal succinimide ester group are weighed and added into a phosphate buffer solution, doxorubicin is added after stirring for 2 hours, the reaction is carried out for 48 hours at room temperature in a dark place until the reaction is finished, and the compound of the formula (I) is obtained after post-treatment.

Preferably, the pH value of the phosphate buffered salt solution in the step (4) is 7.2-8.

The synthetic route of the preparation method of the adriamycin prodrug provided by the invention can be expressed as follows:

the third aspect of the present invention provides an injectable doxorubicin prodrug composition, which comprises a doxorubicin prodrug and an acceptable pharmaceutical adjuvant, wherein the structural formula of the doxorubicin prodrug is shown in formula (I):

wherein n is 4.

The fourth aspect of the invention provides the application of the adriamycin prodrug in preparing antitumor drugs.

Drawings

FIG. 1 is a standard curve of doxorubicin

FIG. 2 is prodrug DOX-PEG₄MTT profile of RGD and DOX prodrugs on Bcap37 cells

FIG. 3 is prodrug DOX-PEG₄MTT profile of RGD and DOX prodrugs versus SKOV3 cells

FIG. 4 is prodrug DOX-PEG₄MTT map of-RGD and DOX prodrugs on A549 cells

FIG. 5 is PEG prodrug DOX-PEG₄MTT profiles 48h and 72h after RGD treatment of Bcap37 cells

FIG. 6 is PEG prodrug DOX-PEG₄MTT profiles 48h and 72h after RGD treatment of A549 cells

FIG. 7 shows the morphology of control Bcap37 cells

FIG. 8 is a graph of l 0. mu.g/mL DOX prodrug treated Bcap37 cells for 72h

FIG. 9 shows the morphology of prodrug DOX-PEG4-RGD after treating Bcap37 cells for 72h

FIG. 10 shows the BALB/c nude mouse lotus Bcap37 model

FIG. 11 shows the state of dissolution of DOX observed under an optical microscope

FIG. 12 shows the DOX and DOX-PEG model of nude mouse with Bcap37 human breast cancer transplantable tumor₄Photographs of the tumors of each group at the end of the experiment under RGD

FIG. 13 shows the nude mouse model of human breast cancer transplantable tumor with Bcap37 loaded on doxorubicin and DOX-PEG₄Tumor volume change curve under effect of RGD (arginine-glycine-aspartic acid) drug

FIG. 14 shows the nude mouse model of human breast cancer transplantable tumor with Bcap37 loaded on doxorubicin and DOX-PEG₄Body weight profile under the action of RGD drugs

FIG. 15 shows PBS, DOX prodrugs and PEG prodrug DOX-PEG₄Pathological section map of-RGD group animal heart tissue

FIG. 16 shows PBS, DOX prodrugs and PEG prodrug DOX-PEG₄Pathological section map of tumor tissue of RGD group animal

FIG. 17 is DOX-PEG₄HPLC profile of RGD

FIG. 18 is DOX-PEG₄LC-MS spectra of RGD

Detailed Description

To facilitate understanding, the present application will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the application are shown. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

EXAMPLE 1 Synthesis of Compound of formula IV

400g of compound II are dissolved in 0.5L of anhydrous THF, N₂While protecting, 1.58g of Na was carefully added in portions, and after stirring the reaction until the sodium cake completely disappeared and no air bubbles were generated, 511.43g of III was sufficiently dissolved in 800mL of anhydrous THF and added in portions to the reaction system, and the reaction was stirred overnight.

After the completion of the reaction of II was detected by LC-MS and TLC, a dilute hydrochloric acid solution was added dropwise. The organic solvent was concentrated to the usual volume size and the mixture was washed twice with 500mL X2 water and once with 500mL saturated aqueous sodium chloride solution. The mixture was dried over anhydrous sodium sulfate and concentrated, and the crude product was stirred with silica gel and purified by column chromatography. Eluting with PE/EA as 5:1 to obtain intermediate product IV.

EXAMPLE 2 Synthesis of Compound of formula IV

EXAMPLE 3 Synthesis of Compound of formula IV

400g of compound II are dissolved in 0.5L of anhydrous THF, N₂56.22g of tetramethylammonium hydroxide was carefully added in portions under protection, and after stirring the reaction until the tetramethylammonium hydroxide was completely disappeared, 791.86g of III was sufficiently dissolved in 800mL of anhydrous THF and added in portions to the reaction system, and the reaction was stirred overnight.

EXAMPLE 4 Synthesis of Compound of formula IV

400g of compound II are dissolved in 0.5L of anhydrous THF, N₂Under protection, 18.76g of tetramethylammonium hydroxide was carefully added in portions, and after stirring the reaction until the tetramethylammonium hydroxide was completely disappeared, 395.93g of III was sufficiently dissolved in 800mL of anhydrous THF and added in portions to the reaction system, and the reaction was stirred overnight.

EXAMPLE 5 Synthesis of Compound of formula V

230g of product IV are dissolved in 230mL of EDCM, 350mL of 20% strength trifluoroacetic acid are added and the mixture is refluxed at 45 ℃ for 3 to 4 days. After completion of the assay IV reaction, the mixture was concentrated to remove trifluoroacetic acid. Slowly dropping the concentrated solution into glacial ethyl ether at-40 deg.C to precipitate solid. The solid was filtered and dissolved in DCM and added dropwise to-40 ℃ diethyl ether and repeated 4-5 times. And (4) removing residual solvent by rotary evaporation of the pumped solid to obtain a product V.

EXAMPLE 6 Synthesis of Compound of formula V

230g of product IV are dissolved in 230mL of EDCM, 350mL of 50% strength trifluoroacetic acid are added and the mixture is refluxed at 45 ℃ for 3 to 4 days. After completion of the assay IV reaction, the mixture was concentrated to remove trifluoroacetic acid. Slowly dropping the concentrated solution into glacial ethyl ether at-40 deg.C to precipitate solid. The solid was filtered and dissolved in DCM and added dropwise to-40 ℃ diethyl ether and repeated 4-5 times. And (4) removing residual solvent by rotary evaporation of the pumped solid to obtain a product V.

EXAMPLE 7 Synthesis of Compound of formula V

230g of product IV are dissolved in 230mL of CCM, 350mL of 35% strength trifluoroacetic acid are added and the mixture is refluxed at 45 ℃ for 3 to 4 days. After completion of the assay IV reaction, the mixture was concentrated to remove trifluoroacetic acid. Slowly dropping the concentrated solution into glacial ethyl ether at-40 deg.C to precipitate solid. The solid was filtered and dissolved in DCM and added dropwise to-40 ℃ diethyl ether and repeated 4-5 times. And (4) removing residual solvent by rotary evaporation of the pumped solid to obtain a product V.

EXAMPLE 8 Synthesis of a Compound of formula VI

To a 100ml single-neck flask was added 50ml of dichloromethane, followed by addition of Compound V (8.45g, 2.5mmol), DSC (1.29g,5mmol), Et₃N (0.09g, 0.9mmol), the reaction was stirred at 30 ℃ for 12 h. After the reaction was complete (monitored by TLC), the reaction was reduced pressure rotary evaporated to a small volume and the oily liquid was dropped into stirred ice petroleum ether to yield a white granular solid. And (3) filtering the solid, dissolving the obtained filter cake into a small amount of dichloromethane, removing isopropanol by rotary evaporation, continuously settling the obtained product in petroleum ether, and filtering the obtained solid. The white solid was transferred to a 500ml single-neck flask, oftenThe residual solvent such as petroleum ether was distilled off at room temperature using a reduced pressure rotary evaporator to obtain intermediate product VI (11.04g, white powdery solid). Yield: 83 percent.

EXAMPLE 9 Synthesis of a Compound of formula VI

To a 100ml single-neck flask was added 50ml of dichloromethane, followed by addition of Compound V (8.45g, 2.5mmol), DSC (1.61g,6.25mmol), Et₃N (0.09g, 0.9mmol), the reaction was stirred at 30 ℃ for 12 h. After the reaction was complete (monitored by TLC), the reaction was reduced pressure rotary evaporated to a small volume and the oily liquid was dropped into stirred ice petroleum ether to yield a white granular solid. And (3) filtering the solid, dissolving the obtained filter cake into a small amount of dichloromethane, removing isopropanol by rotary evaporation, continuously settling the obtained product in petroleum ether, and filtering the obtained solid. The white solid was transferred to a 500ml single-neck flask, and the residual solvent such as petroleum ether was distilled off at normal temperature using a reduced pressure rotary evaporator to obtain intermediate product VI (11.7g, white powdery solid). Yield: 88 percent.

EXAMPLE 10 Synthesis of a Compound of formula VI

To a 100ml single-neck flask was added 50ml of dichloromethane, followed by the addition of Compound VI (8.45g, 2.5mmol), DSC (1.94g,7.5mmol), Et₃N (0.09g, 0.9mmol), the reaction was stirred at 30 ℃ for 12 h. After the reaction was complete (monitored by TLC), the reaction was reduced pressure rotary evaporated to a small volume and the oily liquid was dropped into stirred ice petroleum ether to yield a white granular solid. And (3) filtering the solid, dissolving the obtained filter cake into a small amount of dichloromethane, removing isopropanol by rotary evaporation, continuously settling the obtained product in petroleum ether, and filtering the obtained solid. The white solid was transferred to a 500ml single-neck flask, and the residual solvent such as petroleum ether was distilled off at normal temperature using a reduced pressure rotary evaporator to obtain an intermediate product VI (12.2g, white powdery solid). Yield: 91.76 percent.

EXAMPLE 11 Synthesis of Compound of formula I

RGD (1.73g,5mmol) and SPA-PEG were accurately weighed₄-SPA (5.32g,10mmol) was added to 100mL PBS buffer (PH 7.2), after stirring for 2 hours DOX (7.2g,12mmol) was added thereto, the reaction was left to react at room temperature for 48 hours until the reaction was completed, and 200mL of dichloromethane/methanol (V/V5: 1) extraction water was addedDrying the organic phase, concentrating under reduced pressure, and purifying the crude product by column chromatography to obtain 2.58g of pure product I with the yield of 43.15%.

EXAMPLE 12 Synthesis of Compound of formula I

RGD (2.42g,7mmol) and SPA-PEG were accurately weighed₄-SPA (5.32g,10mmol) was added to 100mL PBS buffer (PH 7.6), after stirring for 2 hours DOX (8.37g,14mmol) was added thereto, the reaction was carried out at room temperature for 48 hours in the dark until the reaction was completed, 200mL dichloromethane/methanol (V/V5: 1) was added to extract the aqueous phase, the organic phase was dried and concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain 2.81g pure product I with a yield of 47%.

EXAMPLE 13 Synthesis of Compound of formula I

RGD (2.77g,8mmol) and SPA-PEG were accurately weighed₄-SPA (5.32g,10mmol) was added to 100mL PBS buffer (PH 8), after stirring for 2 hours DOX (9g,15mmol) was added thereto, the reaction was carried out at room temperature in the dark for 48h until the reaction was completed, 200mL dichloromethane/methanol (V/V5: 1) was added to extract the aqueous phase, the organic phase was dried and concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain 2.72g pure I with a yield of 45.5%.

Example 14DOX-PEG₄MTT assay of-RGD

In the embodiment, human breast cancer cells Bcap37, ovarian adenocarcinoma cells SKOV3 and lung cancer cells A549 are selected as in-vitro experimental models, technical DOX is taken as reference, and an MTT method is adopted to determine PEG prodrug DOX-PEG₄-in vitro anti-tumor activity of RGD.

Because the uptake behavior of the tumor cells to the drugs also has very important influence on the activity of the drugs, in order to observe the PEG prodrug DOX-PEG of the tumor cells more deeply and intuitively₄RGD uptake, we observed our synthetic prodrug DOX-PEG with Bcap37 line as in vitro experimental model and bulk DOX as reference by laser confocal microscopy (CLSM)₄-the route of RGD entry into non-drug resistant cancer cells.

(1) The following experimental instruments were used for this experiment:

(2) the following drugs and reagents were used for this experiment:

name of reagent	Company name	Purity of
			Adriamycin hydrochloride	Sigma-Aldrich	99％
MTT	Shanghai Kanglang Biotech Co Ltd	99％
			DMEM culture solution (1640)	THERMO FISHER SCIENTIFIC (CHINA) Co.,Ltd.
GNM31800RPMI 1640	THERMO FISHER SCIENTIFIC (CHINA) Co.,Ltd.
			Calf serum (10%)	Beijing Lvyuan Berd Biotech Ltd
Green streptomycin solution (1%)	Beijing Lvyuan Berd Biotech Ltd
			Dimethyl sulfoxide (DMSO)	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Sodium chloride	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
			Potassium chloride	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Disodium hydrogen phosphate	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
			Potassium dihydrogen phosphate	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Hematoxylin Eosin (HE) staining kit	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.

(3) Determination of the Adriamycin Standard Curve

As shown in FIG. 1, the concentration of doxorubicin standard solution is plotted on the abscissa, and the corresponding OD value is plotted on the ordinate, thereby drawing a standard curve of the UV absorption. It can be seen that the signal is 5.52 × 10^-7-6.44×10^-7The linear relation is good in the mol/mL range, and the linear equation is that Y is 7.15 xX, X represents OD value, X represents the concentration of the standard solution of DOX, and the linear correlation coefficient R is obtained by carrying out linear regression analysis on the linear relation¹Is 0.998.

(4) Cell culture

BCap37、SKOV₃And A549 tumor cells in 35mm cell culture bottle, selecting RPMI 1640 culture solution containing 10% calf serum (FCS) and 1% streptomycin solution, and culturing in 5% CO₂Relative humidity 90%), and at 37 deg.C.

(5) In vitro cytotoxicity evaluation of PEG prodrugs (MTT assay)

The PEG prodrug DOX-PEG synthesized by us₄And dissolving RGD and DOX reference raw drug in PBS buffer solution to prepare standard liquid medicine, and quantifying by using the previous DOX standard curve for later use. The cultured cell line was digested with 0.25% trypsin and seeded in a 96-well plate at a density of 5X 10⁷Wells, after overnight incubation; adding medicine according to the set concentration (adriamycin equivalent concentration of 0.001,0.01,0.1,0.5,1,5,10,100 mu g/mL) and continuing to culture for 48h, centrifuging to remove the culture solution, washing the pore plate with sterile PBS buffer, repeating for three times, adding fresh culture solution without medicine into each pore and continuing to culture for 24 h; adding sterile MTT solution into each well, continuously culturing for 4h, centrifuging to remove the culture solution containing MTT, re-dissolving the purple crystal with 150 μ l/well DMSO, shaking for 10min, and reading the OD value of the crystal at 562nm by using an enzyme-labeling instrument. Finally, the cell survival rate and the cell growth inhibition rate were calculated according to the following formulas, and a graph of the cell survival rate was plotted using IC₅₀The calculator calculates the corresponding IC₅₀. The specific calculation formula is as follows:

cell viability (%). ratio (test OD/blank OD) × 100%

Cell growth inhibition (%) was [1- (test OD value/blank OD value) ] × 100%

Prodrug DOX-PEG₄MTT maps of RGD and DOX prodrugs on Bcap37 cells, SKOV3 cells and A549 cells are shown in FIG. 2, FIG. 3 and FIG. 4, respectively.

As can be seen from FIGS. 2-4, the PEG prodrug DOX-PEG₄RGD retains the high cytotoxicity of DOX drug per se and exhibits good dose dependence, i.e., the survival rate of various cancer cells gradually decreases with increasing dose. The experimental results shown in FIGS. 2-4 also indicate that the PEG prodrug DOX-PEG₄The cytotoxicity of RGD on tumor cells is weaker than that of DOX technical, and specific differences are shown in IC₅₀Comparison of values (table 1).

TABLE 1

Group of	Bcap37 cell line	SKOV3 cell line	A549 cell line
				IC₅₀ of DOX-PEG₄-RGD(μg/mL)	0.255	0.204	0.295
IC₅₀of DOX(ug/mL)	0.160	0.0169	0.0245

Except that PEG prodrug DOX-PEG was investigated₄Dose-dependence of the cytotoxicity of RGD, we also examined its toxicity as a function of time. As shown in FIGS. 5 and 6, PEG prodrug DOX-PEG₄After the RGD is used for treating Bcap37 and A549 tumor cell strains, the cytotoxicity of 72h is obviously enhanced compared with 48h, which indicates that the longer the action time of the medicine is, the in vitro antitumor activity of the medicine is also gradually enhanced, namely, the time dependence exists.

(6) In vitro cytotoxicity evaluation of PEG prodrugs (cell morphology observation)

In the experiment, human breast cancer cells Bcap37 are used as an observation object, after the drug is added according to a set concentration (doxorubicin equivalent concentration of 0.1,1,10, l00 mug/mL), the cells are cultured for 48 hours, then washed with PBS buffer solution for three times, then the culture solution is added for continuous culture for 24 hours, and then the culture dish is placed under an optical microscope for observation. As a result, as shown in FIG. 7, it was found that the cell morphology of the control group of the cell line was relatively uniform and the growth was rapid.

As shown in fig. 8 and 9, after treating the Bcap37 cells with l0ug/mL of DOX prodrug and DOX prodrug DOX for 72h, it was observed that the integrity of most cancer cells in both experimental groups was lost, and a large amount of cell debris was seen in the visual field, indicating that a large amount of apoptosis or necrosis of the Bcap37 occurred after the drug treatment. The PEG prodrug DOX-PEG4-RGD is shown to retain the characteristic of high cytotoxicity of DOX technical material and has strong in-vitro cytotoxicity.

Example 15 in vivo study of DOX-PEG4-RGD

After good in vitro experimental results were obtained, we further performed DOX-PEG, a PEG prodrug₄RGD has been studied in vivo. The embodiment mainly comprises two parts: firstly, mice are used as experimental animals, and DOX-PEG serving as adriamycin prodrug is investigated₄-pharmacokinetic properties of RGD; secondly, establishing and utilizing a nude mouse experimental model of the ovarian cancer-bearing transplantation tumor, taking the change of the tumor volume and the change of the weight of the nude mouse as the observation standards, combining the histopathology technology, and observing the PEG prodrug DOX-PEG₄-in vivo biosafety and antitumor activity of RGD.

(1) Laboratory apparatus

Name of instrument	Company name
		Enzyme-linked immunosorbent assay (ELISA) instrument	Shanghai Woyuan science and technology Co
96-well culture plate	Costa, USA Inc
		Electronic balance	Shanghai precision instruments & meters Co Ltd
Ultrapure water instrument	NANJING OKHJ TECHNOLOGY Co.,Ltd.
		Biological safety cabinet	SUZHOU ANTAI AIRTECH Co.,Ltd.
Centrifugal machine	Zhang hong City Ruiteng centrifuge manufacturing Co Ltd
		High Performance Liquid Chromatography (HPLC)	Waters
Constant temperature water bath box	Shanghai Jinghong experiment equipment Co Ltd

(2) Medicine and reagent

Name of reagent	Company name	Purity of
			Adriamycin hydrochloride	Sigma-Aldrich	99％
Heparin (Heparin)	Shanghai Kanglang Biotech Co Ltd	99％
			Sodium chloride	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Potassium chloride	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
			Tetrahydrofuran (THF)	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Dimethyl sulfoxide (DMSO)	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
			Ethanol	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Disodium hydrogen phosphate	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
			Potassium dihydrogen phosphate	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR
Acetonitrile	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	AR

(3) Cell culture and experimental animal culture

Inoculating human breast cancer cell Bcap37 into cell culture bottle, selecting 1640 complete culture solution containing 5% CO₂The culturing and passage process was carried out in a cell culture chamber at 37 ℃.

(4) Establishment of tumor-bearing nude mouse model

Experimental nude mice were randomly divided into PBS group, DOX group and DOX-PEG group₄RGD groups of 4 mice each. Culturing breast cancer cells according to the formula of 1x10⁶The cells/mouse were inoculated subcutaneously into the right back of the 6-8 week BALB/C nude mouse, and the experiment was started when the tumor diameter was 3-4mm, and the BALB/C nude mouse model is shown in FIG. 10.

(5) Preparation of experimental medicine

PBS (pH 7.4) buffer was used as a blank control in this experiment.

The preparation method of the DOX group of medicines comprises the following steps: the adriamycin hydrochloride medicine is accurately weighed, dissolved by a small amount of purified water, diluted by a pre-prepared PBS buffer solution (the PBS buffer solution and the sterilized purified water are uniformly mixed according to the volume ratio of 1: 1) to prepare a DOX injection solution of 1mg/mL, and the DOX injection solution is reserved for standby.

DOX-PEG₄The preparation method of the-RGD group medicament comprises the following steps: firstly, weighing a certain amount of PEG prodrug DOX-PEG₄RGD, which is fully dissolved in about 0.5mL of acetonitrile, and then the acetonitrile solution is slowly dripped into the PBS buffer solution (ensuring that the final concentration of DOX-PEG4-RGD is about 1 mg/mL) under the conditions of keeping out of light and vigorous stirring, and the whole dripping process is controlled to be completed within 2 minutes. After the dropwise addition is finished, continuously and violently stirring for 10 minutes under the condition of keeping out of the sun, completely removing the solvent acetonitrile under the vacuum reduced pressure state to obtain a clear and transparent mauve solution, accurately measuring the accurate drug concentration through the calibrated standard curve of the adriamycin, and storing for later use.

On the other hand, in order to compare the solubility differences of DOX, 10mg each of DOX was weighed and dissolved in 1ml of PBS solution sufficiently, and the dissolution of the sample was observed by an optical microscope, as shown in FIG. 11, it was clearly found that DOX-PEG was dissolved in the same concentration₄Only a small fraction of undissolved solids in the PBS solution of RGD, but a large amount of undissolved solids in the PBS solution of DOX, is sufficient to show a significant improvement in the solubility of PEG-modified DOX.

(6) PEG prodrug DOX-PEG₄In vivo safety and anticancer Activity experiment of-RGD

When the tumor diameter grows to 3-4mm, the PBS injection solution and the DOX injection solution are respectively used as negative and positive controls to investigate DOX-PEG₄In vivo safety and antitumor activity of RGD injection solutions. We chose 5mg/kg DOX equivalent dose, administered once every 4 days via tail vein, the first administration was recorded as day 1, administered 7 times in total, and at the same time, the body weight and tumor volume of the animals were recorded, and the reaction and activity status of nude mice were observed. Plotting tumor suppression curves and body weight change curves from the measured tumor volume and body weight data, and determining the weight of each group of animals at the end of the experimentTumor volume the tumor Inhibition Rate of Treatment (IRT) was calculated, tumor volume V ═ a × b²(a, tumor major diameter; b, tumor minor diameter). Tumor inhibition rate IRT (%). 100% × (m)_c-m_e)/m_c。m_cAnd m_eMean tumor weights for experimental and control groups).

As is evident from FIG. 12, PEG prodrug DOX-PEG in comparison to the PBS negative control group₄The appearance of the tumor of the experimental mice of the group of-RGD and DOX is obviously smaller, which shows that the DOX shows a very obvious tumor inhibition effect, the tumor inhibition rate of the DOX reaches 43 percent after the experiment is finished, and the DOX-PEG₄The tumor inhibition rate of RGD reaches 40%. Although the tumor inhibition effect of the DOX technical positive control group is better than that of DOX-PEG₄The RGD is obviously improved, but the weight of the nude mice of the DOX technical positive control group is extremely obviously and rapidly reduced, as shown in figure 14, the weight is reduced by nearly 40 percent, while the DOX-PEG₄The weight change of the nude mice in the RGD experimental group showed only a small decrease compared to the PBS negative control group. In fact, the nude mice of the DOX control group had to terminate the experiment after 5 times of administration due to the high lean/failure status and 90% of the nude mice died. These results indicate that the PEG prodrug DOX-PEG₄Compared with DOX technical product, the RGD has obviously reduced toxic and side effects in vivo, obviously improved safety, and retained ideal tumor inhibiting activity. As a novel prodrug, PEG prodrug DOX-PEG₄The in vivo safe dosage range of the RGD is obviously improved compared with that of DOX original drug, namely, the DOX-PEG of the adriamycin prodrug₄RGD has the advantages of high in-vivo anti-tumor effect and low toxic and side effects.

(7) Histopathological study of transplantable tumors and hearts

To compare and evaluate DOX technical material and PEG prodrug DOX-PEG more deeply₄In vivo safety and tumor-inhibiting effect of RGD, we fixed the heart and tumor tissues of each group of experimental nude mice and histopathological observation of their sections was performed. As shown in FIG. 15, the PBS group did not have obvious lesions in the heart, the arrangement of the myocardial fibers was relatively regular, no rupture of the myocardial fibers was found, no hypertrophy or atrophy was observed, the cytoplasm was uniform and abundant, no edema was observed, no inflammatory cell infiltration was observed, and the cell membrane was not thickened.Comparative PBS, DOX and DOX-PEG₄The heart tissue section of the RGD group shows that compared with the PBS group, the heart muscle tissue of the mice in the DOX group shows obvious myocardial cell hypertrophy, disorganized muscle cell arrangement and obvious inflammatory cell infiltration, has serious interstitial fibrosis and has obvious histopathological characteristics of heart failure. DOX-PEG₄Compared with the DOX group, the RGD treatment group mice have no obvious reconstruction expression of myocardial structures, the arrangement of myocardial cells is relatively regular, and more obvious myocardial cell hypertrophy or inflammatory infiltration is not seen. PBS and DOX-PEG₄The cardiac tissue sections of both groups of the-RGD group were very similar, indicating that even 7 consecutive treatments of animals gave up to 5mg/kg DOX equivalent of PEG prodrug DOX-PEG per treatment₄RGD, which still did not show strong cardiotoxicity, consistent with the previous good status and no abnormal body weight changes in this group of experimental animals.

Tumor tissue sections were subjected to H&E staining was observed, and as shown in FIG. 16, DOX prodrug and Dox-PEG, doxorubicin prodrug₄The cancer cells of the RGD experimental group tumor tissues mostly show typical apoptosis forms, such as obvious reduction of cell density, cytoplasm disappearance, vacuolation of cell plasma, shrinkage or fragmentation of nuclear chromatin, and the like, and the characteristic changes are shown in DOX-PEG₄The RGD group was more pronounced than the DOX group. These experimental results further indicate that PEG prodrug DOX-PEG₄RGD not only has obviously improved in-vivo safety compared with DOX technical, but also has greatly improved safe dosage compared with DOX technical, and has extremely ideal tumor inhibition effect.

The above-mentioned embodiments only express the embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Adriamycin prodrug, preparation method and application thereof

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