CN113952465A

CN113952465A - Camptothecin prodrug and preparation method and application thereof

Info

Publication number: CN113952465A
Application number: CN202111220732.8A
Authority: CN
Inventors: 柯学; 乔颖玉
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2022-01-21

Abstract

The invention discloses a camptothecin prodrug, a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The invention utilizes a prodrug strategy to chemically bond camptothecin to a Low Molecular Weight Heparin (LMWH) molecular chain to form a prodrug, and synthesized LMWH-CPT is self-assembled to form nanoparticles by virtue of the hydrophobicity of the camptothecin and the hydrophilicity of the low molecular weight heparin. The camptothecin prodrug LMWH-CPT not only has the advantages of a high-molecular prodrug, but also ensures that camptothecin molecules in a nanoparticle core obviously increase the equilibrium constant pKa by means of the unique hydrophobic interaction of the camptothecin molecules, and ensures that the camptothecin molecules have higher ring-closing rate after being stored and injected in a neutral environment.

Description

Camptothecin prodrug and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a self-assembled nano delivery system of a redox-sensitive camptothecin-low molecular weight heparin prodrug.

Background

Tumor metastasis has been a major problem for researchers, and solid tumors can "release" millions of tumor cells into the blood circulation every day, and even if tumors are removed by early surgery, a large number of tumor cells can enter the blood. To avoid cancer recurrence caused by tumor metastasis, there are two major current solutions: firstly, postoperative treatment and secondly, independent increase of the dose of the chemotherapeutic drugs. Increasing the dose of chemotherapeutic drugs can significantly reduce the number of circulating tumor cells and even kill residual tumor cells, but is often accompanied by serious toxic and side effects. Therefore, it is of some significance to design an effective method with low toxicity.

Camptothecin (CPT) is a kind of tryptophan-terpene alkaloids which are extracted and separated from the specific plant camptotheca acuminata in China in the last 60 th century, is a plant anticancer drug which has clinical application value after paclitaxel, and is mainly used for treating diseases such as liver cancer, leukemia and the like. Camptothecin can inhibit topoisomerase I to exert anticancer effect. However, camptothecin drugs face problems of poor water solubility in clinical application and low ring-closing rate caused by unstable structure of active ester under physiological conditions. In recent years, researchers have proposed two strategies to address the problem of unstable camptothecin structures: namely, the medicament is treated by an acidic medium to enable the medicament to have high ring-closing rate in the initial period of administration, or the hydrolysis rate of the medicament under physiological conditions is reduced by utilizing the barrier effect of a macromolecular carrier, however, the strategies do not fundamentally solve the problem of unstable structure of the camptothecin medicament. Therefore, there is a interest in delivering camptothecin in a more safe and effective manner through dosage form design.

Of the two structural forms (closed-loop form and open-loop form) of camptothecin antineoplastic drugs, only the closed-loop form has drug effect, the open-loop form only has toxic and side effects, the half-life of the conversion is about 20min under physiological conditions, and the equilibrium closed-loop rate is only 10% -20%. Camptothecin drugs can generate specific hydrophobic interaction with certain polymers, improve the equilibrium constant in the ring opening process of camptothecin, move the equilibrium towards the closed-loop direction and realize the closed-loop rateAnd (4) rising. The reaction essentially changes the hydrolysis process of the camptothecin drugs and changes the chemical properties of the drugs. For example, the pK of 10-hydroxycamptothecin in phosphate buffer_aIs 6.6 (the equilibrium ring closure ratio is about 15%), and the pK of the 10 wt% P123 micelle solution_aIt was 7.5 (the equilibrium closed-loop rate was about 45%).

Prodrug is a drug that is obtained by modifying the chemical structure of a drug to obtain a compound that is inactive or less active in vitro and is metabolized in vivo to release the parent biologically active substance. The polymer prodrug system obtained by modifying the drug by using the polymer carrier has the dual advantages of prodrug modification and micro-nano drug loading, can enhance the stability of the drug in vivo and in vitro, increase the targeting property of the drug, improve the bioavailability of the drug, reduce adverse drug reactions and improve the treatment effect. The preparation of camptothecin drugs into polymeric prodrugs can improve the solubility of the camptothecin drugs and the drug loading capacity, and at present, much work is done in this direction, however, few reports exist on the research of preparing camptothecin drugs into polymeric prodrugs to improve the self-ring-closing rate and increase the stability of the camptothecin drugs in vivo.

Disclosure of Invention

The invention aims to provide a camptothecin prodrug for resisting tumor metastasis, which is characterized in that camptothecin is chemically bonded to a molecular chain of Low Molecular Weight Heparin (LMWH), and synthesized molecules LMWH-CPT are self-assembled to form nanoparticles by virtue of the hydrophobicity of the camptothecin and the hydrophilicity of the low molecular weight heparin. The camptothecin prodrug LMWH-CPT not only has the advantages of a high-molecular prodrug, but also ensures that CPT molecules in nanoparticle cores obviously increase the equilibrium constant pKa by means of the unique hydrophobic interaction of the CPT molecules, and ensures that the CPT molecules have higher closed-loop rate after being stored and injected in a neutral environment.

In order to achieve the purpose, the invention adopts the following technical scheme:

a camptothecin prodrug is formed by connecting camptothecin and low molecular weight heparin through amide bond.

The preparation method of the camptothecin prodrug comprises the following steps:

the method comprises the following steps: adding adipic dihydrazide ADH, carbodiimide hydrochloride EDCI, hydroxyl benzotriazole HOBT and low molecular weight heparin LMWH into phosphate buffer solution with the pH value of 6.8, and reacting at room temperature to obtain an intermediate product LMWH-ADH;

step two: dissolving camptothecin CPT, 4-dimethylaminopyridine DMAP, carbodiimide hydrochloride EDCI and oleic acid OA in anhydrous dichloromethane for reaction, and separating and purifying to obtain an intermediate product CPT-OA;

step three: reacting CPT-OA, carbodiimide hydrochloride EDCI and hydroxyl phenylpropyl triazole HOBT in formamide solution, filtering, collecting precipitate, adding into formamide solution of LMWH-ADH, and reacting to obtain final product LMWH-CPT.

The LMWH-CPT has the following structural formula:

wherein: r₁Is H or SO₃Na、R₂Is SO₃Na or COCH₃(ii) a When the CPT-OA feeding ratio is 20 percent of LWMH-ADH, the n-m/n is 3 percent.

A nanometer medicinal preparation is prepared by self-assembling the above camptothecin prodrug in solvent to form nanometer granule.

Further, the solvent is pure water, a buffer solution, a body fluid, a tissue culture solution or other solvent media without organic solvent as a main body.

The invention utilizes a prodrug strategy to chemically bond camptothecin to a Low Molecular Weight Heparin (LMWH) molecular chain to form a prodrug, and then synthesized LMWH-CPT is self-assembled to form nanoparticles by virtue of the hydrophobicity of the camptothecin and the hydrophilicity of the low molecular weight heparin. The synthesized LMWH-CPT not only has the advantages of a high-molecular prodrug, but also ensures that the equilibrium constant pKa is obviously increased by virtue of the unique hydrophobic interaction of CPT molecules in the nanoparticle core, and ensures that the CPT molecules have higher ring-closing rate after being stored and injected in a neutral environment. The LMWH-CPT mediates the adhered P-selectin on the surface of the tumor cells through the LMWH, has high targeting property on circulating tumor cells, simultaneously, the LMWH-CPT nanoparticle realizes the rapid release of the CPT in the tumor cells and kills the tumor cells by utilizing reductive sensitive bonds such as disulfide bonds, has high ring-closing rate, and has better toxic and side effect reducing effect compared with the common camptothecin prodrug.

Drawings

FIG. 1 is an electron microscope image of LMWH-CPT nanoparticles of the present invention.

FIG. 2 shows the results of the storage stability of LMWH-CPT nanoparticles of the present invention.

FIG. 3 shows the results of the serum stability of LMWH-CPT nanoparticles of the present invention.

FIG. 4 shows the in vitro cumulative release results of LMWH-CPT nanoparticles of the present invention.

FIG. 5 is a comparison of the survival rate of each group of cells in the cytotoxicity assay of the present invention.

Detailed Description

The invention is further illustrated by the following examples.

Example 1

Synthesis of camptothecin prodrug LMWH-CP

1. Synthesis of intermediate LMWH-ADH

30mL (4mg/mL) of LMWH (MW 4500Da, Nanjing Nanda pharmaceutical industry responsibility Co., Ltd.) is precisely weighed, added into a proper amount of phosphate buffer solution with pH 6.8, added with 141.10mg of ADH, 155.28mg of EDCI and 109.59mg of HOBT after the LMWH is fully dissolved, stirred at room temperature, and the reaction system is adjusted by 0.1M hydrochloric acid to ensure that the pH value is about 6.7 and the reaction lasts for at least 24 hours. In order to remove the remaining reactants and small molecule by-products, the crude product obtained was dialyzed against distilled water. The dialyzed filtrate was filtered and lyophilized to give an intermediate LMWH-ADH.

2. Synthesis of intermediate CPT-OA

100mg (0.3mmol) of camptothecin, 73.30mg (0.6mmol) of DMAP, 93.17mg (0.6mmol) of EDCI and 104.52mg (0.6mmol) of OA were weighed out precisely and dissolved in a dry round-bottomed flask by adding anhydrous dichloromethane, and heated under reflux for 36 hours under nitrogen atmosphere (three times with suction) and allowed to drop dropwise. The reaction process is dynamically monitored, and the reaction end point is determined. Washing the obtained product with 0.01M dilute hydrochloric acid for 3 times, then washing with 0.9% sodium chloride solution for three times, adding magnesium sulfate, drying overnight, filtering, removing solvent from the filtrate with a rotary evaporator, and redissolving. Separating and purifying the solution by using a silica gel column to obtain an intermediate product CPT-OA.

3. Synthesis of end product LMWH-CPT

Dissolving 30mg of CPT-OA in formamide solution, adding EDCI 575mg and HOBT 405.39mg, stirring at room temperature for 24h in the dark, carrying out ice bath, adding 15 times of ethyl glacial ether, stirring for 30min, filtering, collecting precipitate, adding the precipitate into formamide solution containing LWMH-ADH100mg, and heating to 38 ℃ for reaction for 48 h. The unreacted CPT-OA was removed by dialysis with DMF for more than 12h, during which time the DMF was replaced. The color of the product solution in the dialysis bag was observed or it was placed under a 254nm fluorescent lamp to observe the phenomenon.

Example 2

Preparation of LMWH-CPT nano-particle

Precisely weighing 10mg of camptothecin prodrug LMWH-CPT, dissolving the camptothecin prodrug LMWH-CPT in 2mL of tetrahydrofuran solution, dropwise adding the camptothecin prodrug LMWH-CPT into 10mL of ultrapure water while stirring by using a magnetic stirrer, stirring until the tetrahydrofuran is completely volatilized, carrying out ultrasonic treatment for 30min (power is 200W, work is 1s, and pause is 2s) by using a probe under ice bath, and then passing the camptothecin prodrug LMWH-CPT through a 0.45-micrometer filter membrane to obtain a faint yellow LMWH-CPT nanoparticle solution with a Tyndall effect.

Test examples

1. Measurement of particle diameter, potential and drug-loading

An appropriate amount of LMWH-CPT nanoparticle solution is measured, and the particle size and distribution of the drug-loaded nanoparticles are measured by a dynamic light scattering particle size analyzer.

An appropriate amount of LMWH-CPT nanoparticle solution is measured, diluted by distilled water and dripped on a copper mesh, and a sample is negatively dyed by phosphotungstic acid. After drying, the morphology was observed by TEM and photographed.

Precisely measuring a proper amount of nanoparticle solution, adding DMSO to destroy the structure of the nanoparticles to release CPT, and adding DMSO to dilute to a proper concentration. Measuring the absorbance of the solution at the maximum absorption wavelength of camptothecin by using a UV-vis spectrophotometer, calculating the mass of CPT in the polymer, and calculating the drug loading according to the following formula:

wherein, W_loadedIs the amount of CPT in the nanoparticles, W_totalIs the total mass of the nanoparticles.

TABLE 1 measurement results of particle diameter, potential and drug-loading rate of nanoparticles

As can be seen from the above table, the drug loading of the nanoparticles provided by the invention is 8.2%, and the particle size is about 148.08 nm. The PDI is 0.152 and less than 0.2, which indicates that the LMWH-CPT nano-particle is relatively stable. The zeta potential of-21.72 mV, with an absolute value of less than 25, further indicates that the system is relatively stable.

The morphology and appearance of the LMWH-CPT nanoparticles are shown in figure 1, and from the aspect of morphology, the nanoparticles are in a regular spherical shape, and the particle size distribution is uniform.

2. Storage stability of nanoparticles

The LMWH-CPT nanoparticles are placed at room temperature and under a certain humidity environment, and the particle sizes of the nanoparticles at 0d, 1d, 2d, 4d and 9d and the change of PDI are monitored to evaluate the storage stability of the nanoparticles.

The results are shown in fig. 2, the particle size of the LMWH-CPT nanoparticles is not substantially changed within 9 days, indicating that they have good storage stability as a formulation.

3. Serum stability of nanoparticles

Mixing the LMWH-CPT nanoparticle solution with fetal calf serum solution according to a volume ratio of 9:1, incubating for 24h at 37 ℃, and monitoring the particle size change of the nanoparticles at 0h, 2h, 6h, 12h and 24h to evaluate the serum stability of the nanoparticles.

The result is shown in fig. 3, the particle size of the LMWH-CPT nanoparticles is not substantially changed within 24 hours; the defects of volatile activity and the like of the camptothecin are improved to a certain extent, and the camptothecin can be used as a nano-drug for later-stage follow-up researches on cytotoxicity and the like.

4. Release Rate investigation

Accurately weighing 5mg of CPT, dissolving the CPT in PBS (pH7.5) containing 0.05% (w/v) Tween 80, diluting to obtain CPT mother liquor, diluting the CPT mother liquor step by step to 0.02, 0.05, 0.1, 0.25, 0.5 and 1 mu g/mL, sequentially measuring the fluorescence absorption I of each concentration, performing linear regression on the concentration C of the CPT by using the fluorescence intensity I, and drawing a standard curve.

The in vitro release of LMWH-CPT nanoparticles in phosphate buffer solution (100mmol/l, pH7.4) medium was studied by dynamic dialysis study. 1mL of the nanoparticle solution was added as a sample solution to a dialysis bag, which was then placed in 30mL of the above release medium. 5mL samples were taken at the set time points and immediately supplemented with an equal volume of release medium. The amount of CPT released at each time point was measured and plotted using a fluorescence spectrophotometer with the free CPT solution as a control, and the cumulative amount of drug released over 24h was calculated.

The results are shown in fig. 4, and compared with the free CPT solution, the LMWH-CPT nanoparticles show better sustained-release characteristics.

5. Cytotoxicity test

Digesting the CT-26 cells in the logarithmic growth phase by pancreatin to prepare cell suspension, inoculating the cell suspension into a 96-hole cell culture plate, adding 200 mu L of the cell suspension into each hole, and absorbing and removing the upper culture solution after the cells adhere to the wall; a series of CPT, LMWH-CPT nanoparticle solutions with different concentrations diluted by culture medium were added to make the final concentration of the sample 0.01, 0.05, 0.1, 0.5, 1, 5, 10. mu.g/mL, the culture medium without drug was used as a blank control, and 6 duplicate wells were set for each group. After 24h of incubation, 20. mu.L of MTT solution (5mg/mL) was added to each well, incubation was continued for 4h and the medium was aspirated; mu.L of dimethyl sulfoxide was added to each well, and the solution was shaken with a micro-shaker for 10min to dissolve formazan sufficiently. Then, the absorbance A of each well at a wavelength of 570nm was measured by a microplate reader, and the cell survival rate was calculated using a blank medium as a control.

The results are shown in fig. 5, the toxicity of the CPT and LMWH-CPT nanoparticle solution to colon cancer cells CT-26 is significantly increased with the increase of the CPT concentration, indicating that the LMWH-CPT nanoparticles have significant killing ability to colon cancer cells.

Claims

1. A camptothecin prodrug, characterized in that: is formed by connecting camptothecin and low molecular weight heparin through amido bond.

2. A process for the preparation of the camptothecin prodrug of claim 1, wherein: the method comprises the following steps:

3. A nano-formulation prepared by self-assembling the camptothecin prodrug of claim 1 in a solvent to form nanoparticles.

4. The nano-formulation of claim 3, wherein: the solvent is pure water, buffer solution, body fluid, tissue culture solution or other solvent media which does not take organic solvent as a main body.