CN110759928B

CN110759928B - Preparation of camptothecin drug nanocrystals by reversible decomposition method

Info

Publication number: CN110759928B
Application number: CN201810840039.2A
Authority: CN
Inventors: 龚涛; 杨琴; 宋旭; 张志荣; 孙逊; 谭田田; 赵娟; 周楚楚; 刘兴
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2022-04-01
Anticipated expiration: 2038-07-27
Also published as: CN110759928A

Abstract

The invention provides a method for preparing camptothecin drug nanocrystals by a reversible decomposition method. The nano crystal provided by the invention is needle-shaped or rod-shaped, can not contain any auxiliary material, retains the anti-tumor activity of the medicament, enhances the tumor penetration effect of the medicament, and reduces toxic and side effects. The preparation process is simple, is suitable for industrial production, and has wide application prospect.

Description

Preparation of camptothecin drug nanocrystals by reversible decomposition method

Technical Field

The invention specifically relates to a nano-crystal of a camptothecin medicament, and a preparation method and application thereof, and belongs to the technical field of medicines.

Background

Antitumor therapy mainly relies on small molecule antitumor drugs, which generally have more clinical application defects and limitations, such as: the drug has low solubility and is difficult to dissolve in clinically available solvents; the half-life of the drug in plasma is short, and the drug must be frequently administered to maintain effective blood concentration; the antitumor drug has side effects on organisms, and the toxic and side effects are aggravated by frequent administration. Some antitumor drugs with higher activity, such as camptothecin, paclitaxel, etc., are limited by low solubility of the drugs in water, and can not be directly administered intravenously, and the drug solubility must be improved by means of chemical modification, solubilizer or new formulation technology.

The camptothecin medicine is a quinoline alkaloid with cytotoxicity, is a specific inhibitor of topoisomerase I, and has a certain inhibiting effect on various tumor cells. Currently, camptothecin drugs approved by the FDA for marketing are irinotecan (CPT-11) and topotecan, two prodrugs obtained by chemically modifying 7-ethyl-10-hydroxycamptothecin (SN 38). The in vitro anti-tumor activity of CPT-11 is one percent or even one thousandth of that of SN38, and the CPT-11 can play the drug effect only after being metabolized to the parent drug SN38 under the action of carboxylesterase II and liver microsomes in vivo. However, the carboxylesterase activity in humans is not as strong as that in murine carboxylesterase, and thus the pharmacological activity of CPT-11 in humans is not as significant as that in animal experiments. Research shows that the proportion of CPT-11 converted into SN38 in 24h of a human body is only about 2-8%. In addition, under physiological conditions, camptothecin medicaments have an E-ring opening/closing dynamic process, and the open-ring carboxylic acid form camptothecin medicaments lose pharmacological activity and can be quickly cleared out of a body. Therefore, there is a need to find a camptothecin pharmaceutical preparation with strong anti-tumor activity and suitable for clinical application.

CN 106963731A provides a precursor compound 7-ethyl-10 tert-butyloxycarbonylcatothecin obtained by derivatization modification of SN38, and forms pH sensitive micelle. The prodrug micelle group has obvious antitumor activity relative to a physiological group. CN 105777770A provides a long-circulating liposome which is prepared into a prodrug of SN38 modified by saturated long-chain fatty acid through chemical modification of SN 38. In vitro experiments show that the prototype ring-opening speed of the prodrug long-circulating liposome is greatly reduced, and the concentration of the active drug is effectively improved. However, the preparation methods have the potential safety hazards of low drug loading, introduction of novel auxiliary materials, organic solvent residues (such as chloroform and dichloromethane), preparation stability and the like.

In recent years, nanocrystal drugs (NCs) have been studied for increasing the efficiency of drug entry into cells, maximizing drug efficacy, and reducing toxic side effects due to their drug loading of 100% and have received much attention (Muller RH, Gohla S, Keck cm. state of the art of nanocrystals-specialty targets, production, nanotopography enzymes and intracellular delivery. european Journal of pharmaceuticals and biopharmaceuticals.2011; 78: 1-9.). As a preparation intermediate, NCs can be further prepared into preparations suitable for various administration routes such as oral administration, injection, spraying, transdermal administration and the like. Currently, 6 nanocrystal intermediate drug varieties are approved by FDA for marketing. Therefore, the camptothecin drug nanocrystal which is safe, low in toxicity and high in drug loading capacity is expected to be prepared into a camptothecin drug preparation which is high in anti-tumor activity and suitable for clinical application.

The preparation theory of the nano-crystal is mainly as follows: from top to bottom, from bottom to top and by comprehensive utilization of two preparation methods. The crystals formed under supersaturation conditions are usually needle-shaped or tendril-shaped and highly targeted to the mononuclear macrophagy system (Wang YC, Zheng Y, Zhang L, Wang QW, Zhang DR.Stablility of nanosuspensions in drug delivery.journal of Controlled Release.2013; 172: 1126-41.). CN 106466296A and literature (Yang dao Feng, Guo Rui Qi, Su Wen Jing, etc. hydroxycamptothecin nanocrystalline prescription process screening and industrial first exploration, volume 12 of the 2016 month 12, 35 of the pharmaceutical guidance) provide a camptothecin drug spherical nanocrystal containing stabilizer prepared by 'alkali dissolution-twice acid precipitation'. The provided hydroxycamptothecin nanocrystal is spherical, and has poor stability at 4 ℃ when no stabilizer is contained. Literature studies indicate that acidic species can accelerate the Ostwald ripening process of nanocrystals, affecting the stability of nanocrystals (M.P. Hendricks, B.M. Cossairt, J.S. Owen. the immunity of nanocrystalline precursors conversion kinetics: mechanism of the interaction between the reaction between carbon carboxylate and cadmium bis (diphenyl thiophosphinate). Acs Nano 2012; 6(11): 10054-. Nanocrystals prepared according to conventional theories generally have crystalline or amorphous structural characteristics. Research reports show that the passive transport efficiency and the tumor infiltration capacity of the nanorods with sharp edges or special shapes are obviously superior to those of spherical nano preparations with regular shapes. Although the theory of nanocrystal preparation has become relatively mature, few nanocrystals have a particle size below 220 nm. Moreover, the nanocrystals are affected by Ostwald ripening, have the defect of rapid growth of particle size, and need to be added with a stabilizer to maintain the relative stability of the particle size. Many factors limit the clinical utility of nanocrystal formulations (Al-Kassas R, Bansal M, Shaw J. Nanosing technologies for improving bioavailability of drugs. Journal of Controlled Release: office Journal of the Controlled Release facility 2017; 260: 202-12.).

Through a large number of experiments, the inventor discovers that the camptothecin drug nanocrystal which is needle-shaped or rod-shaped and has the particle size of about 150nm, good stability and no auxiliary materials can be prepared by a simple preparation process based on the structural characteristic that the camptothecin drug 'E' ring has 'closed loop/open loop' dynamic conversion under physiological conditions through a reversible decomposition method, can promote the penetration of the drug in tumor parts, reduces toxic and side effects, and has good clinical application prospect.

Disclosure of Invention

The invention provides a needle-shaped or rod-shaped nanometer crystal preparation of camptothecin medicaments with the particle size of 100-250 nm, and the nanometer crystal preparation only consists of the camptothecin medicaments.

One of the purposes of the invention is to provide a method for preparing camptothecin drug nanocrystals.

One of the purposes of the invention is to provide a camptothecin drug nanocrystal which is mainly prepared by a reversible decomposition method.

Wherein, the reversible decomposition method is as follows: under certain pH conditions, the camptothecin drug E ring is dynamically converted into prototypes/carboxylic acid types (in the dynamic conversion example of SN38, as shown in the following, the dynamic equilibrium of ring opening/ring closing exists under physiological conditions), and based on the characteristic, a prototype/carboxylic acid type camptothecin mixture can be prepared. The open-ring carboxylic acid type camptothecin drug has a hydrophobic condensed ring structure and a hydrophilic 'E ring' open-ring structure, and has the characteristics similar to a surfactant. When the mixture is dispersed in water, strong intermolecular force is generated between the hydrophobic end of the carboxylic acid type camptothecin and the proto-type drug to form a stable hydrophobic inner core; the carboxylic acid structure of the ring-opened E ring has stronger hydrophilic capacity, can form an outer ring hydrophilic end to solubilize the prototype drug, and can obtain a highly dispersed and self-assembled mixed drug nanocrystal. Finally, the volatile alkaline reagent is removed by means of preparation, the carboxylic acid type camptothecin drug without pharmacological activity is reversed to be the prototype drug, the solubilized mixed type drug nanocrystal is reversed to be the prototype camptothecin drug nanocrystal, and the pharmacological activity of the drug is retained.

The camptothecin drug nanocrystal provided by the invention is characterized in that the nanocrystal consists of 100% of drugs. The form of the nano crystal is needle-shaped or rod-shaped. The grain size of the nano crystal is 100-250 nm. The drug constituting the nanocrystal exists mainly in the form of proto-drug, and the proportion of the proto-drug in the camptothecin drug is more than 95%.

The camptothecin drug nanocrystal provided by the invention is characterized by consisting of a camptothecin drug and a freeze-drying protective agent. The grain size of the nano crystal is 100-250 nm.

The camptothecin drug is selected from one or more of irinotecan (CPT-11), 7-ethyl-10-hydroxycamptothecin (SN38), 10-Hydroxycamptothecin (HCPT), belotecan (CDK-602) and topotecan (TPT), and the camptothecin drug can also be selected from the acid salt forms of the raw materials, preferably hydrochloride. Wherein, the structural formula of the camptothecin medicament is shown as follows:

wherein the freeze-drying protective agent is selected from one or more of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin, preferably trehalose or human serum albumin; the mass ratio of the freeze-drying protective agent to the camptothecin medicaments is (0:1) - (100:1), and the preferable mass ratio of the added freeze-drying protective agent is (15:2) - (50: 1).

One of the purposes of the invention is to provide a method for preparing camptothecin drug nanocrystals by a reversible decomposition method, which comprises the following steps:

(1) dispersing camptothecin drug in a reaction system, stirring at room temperature in a dark place until the solution is clear, and stopping the reaction; the reaction system comprises a reaction reagent and a reaction solvent;

(2) removing part of the reaction reagent and the reaction solvent in the reaction (1) to obtain solid powder or suspension solution. Wherein the mass ratio of the carboxylic acid type medicament to the prototype medicament is 1: 5-10: 1;

(3) dispersing the solid powder obtained in the step (2) in water, and uniformly dispersing to obtain a solution I;

(4) filtering the solution I, and freeze-drying to obtain camptothecin drug nanocrystal freeze-dried powder;

(5) before use, adding an injection solvent to redissolve the camptothecin drug nanocrystal freeze-dried powder obtained in the step (4), and uniformly dispersing to obtain the camptothecin drug nanocrystal freeze-dried powder for use.

In the invention, the reaction reagent in step (1) is a low-boiling point and basic compound, including but not limited to triethylamine, diethylamine, ethylamine, trimethylamine, dimethylamine, monomethylamine or ammonia, preferably one or a mixture of monomethylamine and ammonia; the volume/mass ratio of the reaction reagent to the camptothecin drug is (2:1) - (20:1) (mL/g), and preferably, the volume/mass ratio is (5:1) - (10:1) (mL/g).

Wherein the reaction solvent is characterized by: the solvent has strong polarity and can be mutually dissolved with the alkaline reaction reagent, such as methanol, ethanol, tetrahydrofuran or water, and is preferably one of methanol, ethanol and water.

The method of removing the reaction solvent in the step (2) is freeze-drying, vacuum-drying, or reduced-pressure rotary evaporation, and preferably reduced-pressure rotary evaporation. The rotary evaporation temperature is 30-60 ℃, and preferably 37-50 ℃.

The dispersion mode in the step (3) includes but is not limited to one or more of rapid stirring, water bath ultrasound, probe ultrasound or high pressure homogenization, and preferably probe ultrasound and/or high pressure homogenization.

The filtration mode in the step (4) is selected from 0.22 mu m microporous membrane sterile filtration.

The solvent for injection in step (5) includes, but is not limited to, one or more of 5% glucose solution, 0.9% sodium chloride solution or water for injection, and preferably one of 5% glucose solution and water for injection.

And (3) a freeze-drying protective agent can be added, specifically, the solid powder obtained in the step (2) is dispersed in an aqueous solution containing the freeze-drying protective agent. The freeze-drying protective agent is selected from one or more of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin. The prepared nanocrystal solution containing the freeze-drying protective agent can be stored at the temperature of minus 20 ℃. The mass ratio of the freeze-drying protective agent to the camptothecin medicaments is (0:1) - (100: 1). When the lyoprotectant is added, the mass ratio is preferably (15:2) - (50: 1).

Advantages of the invention

(1) The needle-shaped or rod-shaped camptothecin drug nanocrystal provided by the invention not only retains the anti-tumor activity of the drug, reduces the toxic and side effects, and promotes the penetration of the drug at the tumor part, but also has the advantages of simple preparation process, lower requirements on preparation instruments, suitability for industrial production, and wide application prospect.

(2) The camptothecin drug nanocrystal preparation provided by the invention can be added with no auxiliary materials, has high drug loading and good safety, and effectively avoids the safety risk caused by the auxiliary materials.

(3) The method for preparing camptothecin drug nanocrystals by the reversible decomposition method provided by the invention is simple to operate, has lower requirements on instruments and equipment, obtains the carboxylic acid type compound with increased polarity based on the reversible decomposition property of a camptothecin drug prototype, utilizes the amphipathy of the carboxylic acid type compound to solubilize the insoluble prototype drug, and then reversibly decomposes the insoluble prototype drug into the prototype drug, avoids the use of auxiliary materials such as a surfactant and the like, avoids the introduction of a precursor compound or a derivative, avoids the introduction of an acidic reagent, is favorable for improving the stability of a nanocrystal preparation, is favorable for improving the safety of the preparation, retains the antitumor activity of the drug, and reduces toxic and side effects.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: thermogram of SN38 prototype/carboxylic acid type mixture. (A) SN38 differential scanning calorimetry of crude drug. (B) SN38 prototype/carboxylic acid type mixtures differential scanning calorimetry thermograms. (C) Thermogravimetric analysis of SN38 bulk drug. (D) Thermogravimetric analysis of a prototype SN 38/carboxylic acid type mixture.

FIG. 2: crystal diffraction analysis pattern of SN38 nanocrystal powder.

FIG. 3: HPLC schematic before and after lyophilization of SN38 prototype/carboxylic acid type mixtures. (A) HPLC schematic of a prototype SN 38/carboxylic acid type mixture. (B) HPLC schematic of SN38 nanocrystal solutions.

FIG. 4: formulation characterization of nanocrystals. (A) Particle size distribution of SN38 nanocrystal solution. (B) Zeta potential distribution of SN38 nanocrystal solutions. (C) Particle size distribution of SN 38/Albumin nanocrystal solution. (D) Zeta potential distribution of SN 38/albumin nanocrystal solution. (E) Transmission electron microscopy analysis chart of SN38 nanocrystal solution. (F) Transmission electron microscopy analysis chart of SN 38/albumin nanocrystal solution. (G) Transmission electron microscopy analysis of HCPT nanocrystal solutions. (H) Scanning electron microscope analysis picture of SN38 nanocrystal freeze-dried sample. (I) Scanning electron microscope analysis chart of SN38 nanocrystal solution.

FIG. 5: drug concentration-time profiles of SN38 nanocrystal formulations with irinotecan hydrochloride in healthy SD rats (n ═ 5).

FIG. 6: the organ distribution map of the SN38 nanocrystal preparation and irinotecan hydrochloride in tumor-bearing mice at different time points (n ═ 5). And (A-D) are SN38 drug concentration statistical graphs in each main organ of 1h,4h,8h and 24 h. (E) Statistics were made for SN38 concentrations at various time points in the tumor.

FIG. 7: pharmacodynamic experiments of the SN38 nanocrystal preparation and irinotecan hydrochloride in tumor-bearing mice (n ═ 5) are carried out. (A) Tumor volume-time curve. (B) Body weight-time curve of tumor-bearing mice. (C) Tumor display plots for each dosing group. (D) Tumor weight for each group administered. (E) Tumor inhibition rate of each administration group.

Detailed Description

The following examples are further illustrative of the present invention and are in no way intended to limit the scope of the invention. The present invention is further illustrated in detail below with reference to examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Also, equivalent substitutions, combinations, improvements or modifications of the invention may be made by those skilled in the art based on the description of the invention, but these are included in the scope of the invention.

Example 1

(1) Preparation of a prototype SN 38/Carboxylic acid type mixture

Dissolving 1mL of ammonia water in 5mL of water, weighing SN38(0.2g) and dispersing in the ammonia water solution, reacting at room temperature in a dark place until the solution is clear, stopping the reaction, and removing the reaction solvent by rotary evaporation under reduced pressure at 60 ℃ to obtain an SN38 prototype/carboxylic acid type mixture (light yellow flaky solid), wherein the mass percent of the prototype drug is 82.1%.

(2) Preparation of SN38 nanocrystal formulations

Dispersing the mixture (20mg) obtained in the step (1) in 20mL of purified water, and performing ultrasonic treatment on the mixture for 10min (100W) by using a probe to obtain a solution of blue-green opalescence. The formulation solution was then filtered through a 0.22 μm filter and freeze dried to produce a lyophilized powder of SN38 nanocrystals. And (3) re-dissolving the freeze-dried powder in 20mL of 5% glucose solution for injection, and shaking and dispersing to obtain an SN38 nanocrystal solution.

Example 2

(1) Preparation of camptothecin drug proto/carboxylic acid type mixture

Dissolving 2mL of ammonia water in 5mL of water, weighing camptothecin medicaments (0.2g, including CPT-11, HCPT, CDK-602 or TPT) to be dispersed in the ammonia water solution, reacting at room temperature in a dark place until the solution is clear, stopping the reaction, and removing the reaction solvent by reduced pressure rotary evaporation at 60 ℃ to obtain camptothecin medicament prototype/carboxylic acid type mixture solid powder or suspension (light yellow flaky solid or light yellow solution).

(2) Preparation of nano crystal preparation of camptothecin medicine

And (2) dispersing the mixture (20mg) obtained in the step (1) in 20mL of purified water (or metering the suspension to 250mL and 25mL), and performing ultrasonic treatment on the probe for 10min (100W) to obtain a blue-green opalescent solution. The formulation solution was then filtered through a 0.22 μm filter and freeze dried to produce a nanocrystal lyophilized powder. 5mL of 5% glucose solution for injection is taken to redissolve the freeze-dried powder, and the corresponding medicine nanocrystal solution is obtained by ultrasonic dispersion.

TABLE 1 particle size and Zeta distribution of solutions of nano-crystalline camptothecin drugs prepared in EXAMPLE 2

Example 3

(1) Preparation of a prototype SN 38/Carboxylic acid type mixture

Dissolving 4mL of triethylamine in 4mL of water, weighing SN38(0.2g) and dispersing in the triethylamine aqueous solution, stirring at room temperature in a dark place until the solution is clear, stopping reaction, and drying in vacuum to remove most of alkaline reagents to obtain SN38 prototype/carboxylic acid type mixture suspension (light yellow liquid), wherein the mass percent of the prototype medicament is 18.3%.

(2) Preparation of SN38 nanocrystal formulations

And (2) metering the volume of the mixture suspension prepared in the step (1) to 50mL, taking (25mL of aqueous solution (50 mg of human serum albumin is added), quickly stirring to obtain a blue-green opalescent preparation solution, then freeze-drying the preparation solution to obtain SN 38/albumin nanocrystal freeze-dried powder, re-dissolving the freeze-dried powder in 30mL of 0.9% physiological saline, shaking and dispersing to obtain an SN38 nanocrystal solution with the particle size of 135.8nm and the PDI of 0.102, and placing the nanocrystal solution prepared in the step at the temperature of-20 ℃ for freezing and placing according to requirements.

Example 4

(1) Preparation of a prototype SN 38/Carboxylic acid type mixture

The same as in example 1.

(2) Preparation of SN38 nanocrystal formulations

The SN38 mixture (10mg) obtained in step (1) was weighed, dispersed in 200mL of an aqueous solution (containing 500mg of trehalose), and homogenized under high pressure (pressure 1000bar, 5 times) to obtain a blue-green opalescent formulation solution. The formulation solution was then filtered through a 0.22 μm filter and freeze dried to produce SN 38/trehalose nanocrystal lyophilized powder. 10mL of water for injection is taken to re-dissolve the freeze-dried powder, and the solution is ultrasonically dispersed to obtain SN38 nanocrystal solution with the particle size of 165.1nm and the PDI of 0.183. The nanocrystal solution prepared by the steps can be frozen and stored at the temperature of minus 20 ℃ as required.

Example 5

(1) Preparation of a prototype SN 38/Carboxylic acid type mixture

Dissolving 4mL of triethylamine in 5mL of methanol, weighing SN38(0.2g) and dispersing in the triethylamine/methanol solution, stirring and reacting at room temperature in a dark place until the solution is clear, stopping the reaction, and freeze-drying to obtain an SN38 prototype/carboxylic acid type mixture (light yellow flaky solid), wherein the prototype SN38 accounts for 52.6 percent of the theoretical mass percent of the mixture

The alkaline reactant can be selected from diethylamine, ethylamine, trimethylamine, dimethylamine or methylamine, and the reaction solvent can be one or more of water, tetrahydrofuran, methanol or ethanol. The reaction time is adjusted according to the actual situation, and the HPLC monitoring shows that the reaction can be stopped when the mass percent of the proto-drug is less than 10%. The proto/carboxylic acid type mixture solid or suspension is obtained by rotary evaporation, and after the proto/carboxylic acid type mixture solid or suspension is prepared into the nano crystal by a double decomposition method, the mass fraction of the carboxylic acid type medicament is less than 5 percent (shown in a table 2).

TABLE 2 percent preparation of SN38 nanocrystals AC-SN38 at different reaction conditions

Note: R-AC is the mass percent of AC-SN38 in the reaction system when the reaction is stopped; M-AC is the mass percent of AC-SN38 in the mixture or suspension after rotary evaporation; NCs-AC is the mass percent of AC-SN38 in the freeze-dried nanocrystal powder or the reconstituted nanocrystal solution.

(2) Preparation of SN38 nanocrystal formulations

The resulting mixture (10mg) of (1) was weighed out and dispersed in 10mL of an aqueous solution (containing 1g of sucrose), and stirred rapidly for 20min to obtain a blue-green opalescent formulation solution. The formulation solution was then filtered through a 0.22 μm filter and freeze dried to produce a SN 38/sucrose nanocrystal lyophilized powder. 10mL of 5% glucose solution is taken to redissolve the freeze-dried powder, and the SN38 nanocrystal solution is obtained by ultrasonic dispersion.

The above lyoprotectant can be selected from mannitol, lactose, maltose, glucose or no lyoprotectant, and the rest operations are the same as above. After re-dissolving, the nano-crystal with the particle size of 150-250 nm (as shown in table 3) can be prepared, and the nano-crystal solution can be frozen and stored at the temperature of-20 ℃ by adding the freeze-drying protective agent.

TABLE 3 average particle size of SN38 nanocrystals prepared with different classes of lyoprotectants

Comparative example

About 17mg of irinotecan hydrochloride (CPT-11) was weighed and dissolved in 10mL of a glucose solution having a pH of 6.05% to prepare a solution having a molar concentration equivalent to that of SN38(1 mg/mL).

Test example 1

The preparation of a prototype SN 38/carboxylic acid mixture (M-SN38) from example 1 was carried out¹H-NMR detection analysis, (DMSO-d)₆400MHz) 10.29(brs,1H),8.02(d, J ═ 6.0Hz,1H),7.41-7.39(m,2H),7.24(s,1H),6.50(s,1H),5.42(s,2H),5.72(s,2H),3.09-3.07(m,2H),1.88-1.85(m,2H),1.28(t, J ═ 7.2Hz,3H),0.88(t, J ═ 7.2Hz, 3H). Comparative results show that the parent structure of the SN38 prototype/carboxylic acid type mixture, SN38, has no other structural changes.

Test example 2

After vacuum drying the SN38 prototype/carboxylic acid type mixture (M-SN38) prepared in example 1 and the SN38 bulk drug, weighing 10mg of powder, placing the powder in an aluminum tray, scanning the powder in the range of 0-350 ℃, heating at the speed of 10 ℃/min in a nitrogen atmosphere, and carrying out differential scanning calorimetry and thermogravimetric analysis. As shown in figure 1, the SN38 bulk drug (A, C) loses about 4.7 percent of weight loss at 145 ℃ of 115-cost and loses one molecule of crystal water (4.39 percent of theoretical weight loss), is in a molten state at 209 ℃ of 206-cost and is subjected to decarboxylation degradation under high temperature. FIG. 1(B, D) shows that the SN38 prototype/carboxylic acid type mixture exothermed at 60-85 ℃ with a weight loss of about 4.2%, presumably a small amount of volatile alkaline reagent remained in the mixture, which is also a prerequisite for the presence of the SN38 carboxylic acid type in the mixture; loss of crystal water is about 0.72 percent of weight loss at the temperature of 101-127 ℃; 237-290 ℃ in a molten state accompanied by an endotherm, followed by decarboxylation degradation to release heat.

Test example 3

A small amount of the SN38 nanocrystal lyophilized powder prepared in example 1 and the SN38 drug substance were coated on a substrate, compressed, and placed on an X-ray diffractometer to perform a small angle X-ray diffraction experiment with NCs-0% as shown in FIG. 2. The prepared NCs-0% has Bragg parameters completely consistent with those of a SN38 bulk drug reference product. Based on the high performance liquid chromatogram analysis of fig. 3, it is presumed that, in the presence of both the SN38 prototype and the carboxylic acid type mixture, carboxylic acid type SN38 having a good hydrophilicity has a reversible hydrophilic end and a hydrophobic end of SN38 fused ring itself, and can function as a surfactant to obtain a highly dispersed mixed nanocrystal. Removing volatile alkaline substances by freeze drying, and reversing carboxylic acid type SN38 to prototype SN38 to obtain SN38 nanocrystal with particle size of about 150 nm.

Test example 4

The particle size and distribution of the nanosuspension were measured by dynamic light scattering, and SN38 nanocrystal lyophilized solid powders and solutions, SN 38/albumin nanocrystal solutions, and HCPT nanocrystal solutions prepared in examples 1 and 3, and example 2 were formulated for characterization, and SN38 and HCPT nanocrystal solutions prepared in examples 1 and 2 were placed at 4 ℃ and the nanocrystal solutions were monitored for particle size and PDI changes within 15 days, as shown in table 4. The fine particles obtained in example 1 were found to have an average particle diameter of 150.9nm, PDI of 0.086 and Zeta potential of-16.5 mV in aqueous solution, as shown in FIG. 4(A, B), transmission electron microscopy of FIG. 4(E), and scanning electron microscopy of the nanocrystal solid powder and the nanocrystal solution of FIG. 4(H, I). The average particle diameter of the SN38 albumin nanocrystal particles prepared in example 3 in an aqueous solution was 169.4nm, PDI was 0.193, Zeta potential was-19.4 mV, as shown in FIG. 4(C, D), and as shown in FIG. 4(F) by transmission electron microscopy. The HCPT nanocrystal solution of example 2 was subjected to transmission electron microscopy characterization as shown in fig. 4 (G). The prepared camptothecin medicament nanocrystal powder is in a loose porous structure by utilizing the reversible open-loop/closed-loop characteristic of the camptothecin medicament and introducing a volatile basic reagent, and the prepared camptothecin medicament nanocrystal particles can form needle-shaped or rod-shaped nanocrystals with better stability in an aqueous solution.

Table 4 stability of particle size of NCs in the dark at 4 ℃ (n ═ 3)

Test example 5

The SN38 nanocrystal solution prepared in example 1 and the control were used for drug plasma clearance experiments. Injecting equal molar quantity of SN38(6.5mg/kg) into tail vein of healthy SD rat, collecting blood 100 microliter through orbit vein of rat at 10min,20min,30min,1h,2h,4h,8h,12h,24h and 36h, adding 2 times volume of organic solvent (acetonitrile: methanol is 1:1, 0.5% acetic acid, v/v), mixing, standing in dark for 1h, centrifuging at 13500rpm, 10min, collecting supernatant, and performing LC-MS/MS quantitative analysis, wherein the experimental result is shown in FIG. 5.

As can be seen from FIG. 5, irinotecan can be rapidly cleared in vivo, and the nanocrystals prepared by the invention can improve the retention time of SN38 in blood. As can be seen from table 5, the blood clearance half-life of the nanocrystal formulation drug of the present invention is about 2 times that of irinotecan hydrochloride, and the area under the drug concentration-time curve (AUC) is more than 5 times that of irinotecan hydrochloride.

Table 5 pharmacokinetic parameters for SN38 nanocrystal formulations and irinotecan hydrochloride (n ═ 5)

AUC_0→t: area under drug concentration-time curve

t_1/2: half life of blood concentration

Test example 6

The SN38 nanocrystal preparations prepared in example 1 and the control example were used to examine the distribution of drugs in the major organs of the body. Selecting Balb/c female mice with good physical condition, and inoculating 4T to the position of the left side of the mouse close to the underarm mammary fat pad₁Tumor cells (approximately 5 x 10 cells per mouse inoculation)⁵Individual tumor cell), the tumor volume is up to 200-300mm³The mice were sacrificed at 1h,4h,8h and 24h after the administration by postcaudal intravenous injection (the equivalent administration dose of SN38 is 10mg/kg), blood, heart, liver, spleen, lung, kidney and tumor were collected for subsequent LC/MS/MS quantitative detection and analysis, and the results of the distribution experiment were examined and shown in FIG. 6.

As can be seen from fig. 6(a to D), in the comparative example, irinotecan hydrochloride rapidly distributed and cleared in the organs, the SN38 nanocrystal preparation prepared by the present invention mainly distributed in the liver, spleen and lung, and the retention time in the organs was long. As shown in FIG. 6(E), the SN38 nanocrystal preparation prepared by the invention has longer accumulation time in tumors and has obvious advantage in accumulation concentration compared with irinotecan hydrochloride.

Test example 7

CPT-11, HCPT, CDK-602 and TPT nanocrystal preparations prepared in example 2 and a control example are taken to carry out in vivo experimental investigation of the antitumor effect of tumor-bearing mice. Selecting Balb/c female mice with good physical condition, and inoculating 4T to the position of the left side of the mouse close to the underarm mammary fat pad₁Tumor cells (approximately 5 x 10 cells per mouse inoculation)⁵Tumor cells) until the tumor volume is 50-100 mm³The later beginning and the end of the body quietThe injection is injected in pulse (equivalent dosage is converted into SN38 to be 5mg/kg), the injection is administered every two days for 3 times, mice are killed on the 20 th day after the tumor is inoculated, the tumor is separated, the tumor weight is recorded, and the tumor inhibition rate is calculated according to the formula: TGI% ((m))_S-m)/m _s100% (TGI is tumor inhibition rate, m)_sTumor weight in physiological group and tumor weight in administration group), the tumor inhibition rate was shown in the table below.

TABLE 6 statistics of in vivo pharmacodynamic tumor inhibition of several camptothecin drug nanocrystals (n ═ 5)

Test example 8

The SN38 nanocrystal preparation prepared in example 1 and the control example are used for carrying out in vivo experimental investigation of the antitumor effect of tumor-bearing mice. Selecting Balb/c female mice with good physical condition, and inoculating 4T to the position of the left side of the mouse close to the underarm mammary fat pad₁Tumor cells (approximately 5 x 10 cells per mouse inoculation)⁵Tumor cells) until the tumor volume is 50-100 mm³After that, tail vein injection administration (SN38 equivalent dose is 5mg/kg) is started, 5 times are given in total every other day, the size of the tumor and the body weight of the mouse are recorded every other day, and the tumor volume of the mouse is calculated according to the formula: v ═ D (L × D)²) (V is tumor volume, L is tumor major diameter, D is tumor minor diameter), tumor volume-time curve and body weight-time curve were plotted as shown in FIG. 7(A, B). The mice are killed at the 29 th day after tumor inoculation, tumors are separated, the tumor weight is recorded, and the tumor inhibition rate is calculated according to the following formula: TGI% ((m))_S-m)/m _s100% (TGI is tumor inhibition rate, m)_sTumor weight in physiological group and tumor weight in administration group), as shown in FIG. 7(E, C, D).

As can be seen from fig. 7, at the set administration concentration, the size of the irinotecan hydrochloride tumor is not significantly different from that of the normal saline group, no significant tumor inhibition effect is generated, and the tumor growth rate is high, whereas the SN38 nanocrystal preparation provided by the invention has slow tumor growth during administration, slow tumor growth rate after administration is stopped, and significant tumor inhibition effect is different from that of the physiological saline and the irinotecan hydrochloride group. The SN38 nanocrystal provided by the invention has excellent antitumor activity.

Claims

1. A method for preparing a nanocrystal preparation comprises the following steps: (1) dispersing camptothecin drugs in a reaction system consisting of a reaction reagent and a reaction solvent, and carrying out a light-resistant reaction at room temperature;

(2) removing part of the reaction reagent and the solvent in the step (1) to obtain solid powder or suspension, wherein the mass ratio of the carboxylic acid type drug to the prototype drug in the solid powder or suspension is 1: 5-10: 1;

(3) fully dispersing the mixture powder or suspension obtained in the step (2) in water;

(4) freeze drying to obtain nanometer crystalline preparation of camptothecin medicine;

the reaction reagent is selected from one or a mixture of triethylamine, diethylamine, ethylamine, trimethylamine, dimethylamine, monomethylamine or ammonia water;

the nano-crystal preparation only consists of camptothecin drugs and is in a needle shape or a rod shape.

2. The method for preparing a nanocrystal formulation according to claim 1, wherein the particle size of the nanocrystal formulation is 100 to 250 nm.

3. The method for preparing a nanocrystal formulation according to claim 1, wherein the camptothecin drug is selected from one or more of irinotecan, 7-ethyl-10 hydroxycamptothecin, 10-hydroxycamptothecin, belotecan, topotecan or salt form of the above drugs.

4. The method for preparing a nanocrystal formulation according to claim 1, further comprising a step of adding a lyoprotectant, wherein the lyoprotectant is selected from one or more of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin, and the mass ratio of the lyoprotectant to the drug is (0:1) - (100: 1).

5. The method of claim 4, wherein the lyoprotectant is selected from the group consisting of trehalose and human serum albumin.

6. The method for preparing a nanocrystal formulation according to claim 4, wherein the mass ratio of the lyoprotectant to the drug is (15:2) - (50: 1).

7. The method for preparing the nanocrystal preparation of claim 1, wherein the camptothecin drug nanocrystal preparation obtained in step (4) is redissolved by adding an injection solvent, and the dispersion is uniform, so that the camptothecin drug nanocrystal preparation can be used, wherein the injection solvent is one or more selected from a 5% glucose solution, a 0.9% sodium chloride solution or water for injection.

8. The method for preparing a nanocrystal formulation according to claim 7, wherein the solvent for injection is selected from a 5% glucose solution or water for injection.

9. The method of claim 1, wherein the reagent is selected from the group consisting of monomethylamine and ammonia.

10. The method of claim 1, wherein the volume/mass ratio of the reactive agent to the camptothecin drug is (2:1) - (20:1) (mL/g).

11. The method for preparing a nanocrystal formulation according to claim 1, wherein the reaction solvent is selected from one or more of methanol, ethanol, tetrahydrofuran or water.

12. The method of claim 1, wherein the reaction solvent is selected from one or more of methanol, ethanol, and water.

13. The method of claim 1, wherein the volume/mass ratio of the reactive agent to the camptothecin drug is (5:1) - (10:1) (mL/g).

14. The method for preparing the nanocrystal preparation according to claim 1, wherein the removing of the partial reaction reagent and solvent in the reaction (1) in the step (2) is freeze drying, vacuum drying or reduced pressure rotary evaporation, wherein the rotary evaporation temperature is 30-60 ℃; the dispersion mode is selected from one or more of rapid stirring, water bath ultrasound, probe ultrasound or high pressure homogenization.

15. The method for preparing the nanocrystal preparation according to claim 13, wherein the removing of the partial reaction reagent and the solvent in the reaction (1) in the step (2) is performed by reduced pressure rotary evaporation at a temperature of 37-50 ℃; the dispersion mode is selected from probe ultrasonic or high pressure homogenization.

16. Use of the nanocrystal formulation of any one of claims 1 to 15 for the preparation of a medicament for the treatment of colorectal cancer, bladder cancer, gastric cancer, esophageal cancer, tonsil cancer, nasopharyngeal cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, chronic myelogenous leukemia, lymphoma, skin cancer.