CN112891551A

CN112891551A - Nano-drug using irinotecan as carrier and preparation method and application thereof

Info

Publication number: CN112891551A
Application number: CN202110111986.XA
Authority: CN
Inventors: 姜虎林; 邢磊; 宋琪
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-06-04
Anticipated expiration: 2041-01-27
Also published as: CN112891551B

Abstract

The invention discloses a nano-drug taking irinotecan as a carrier and a preparation method thereof, which can reduce the metabolic toxicity of irinotecan and expand the application range of the irinotecan as a small molecular carrier. The nano-drug mainly comprises irinotecan, COX-2 inhibitor or VEGFR inhibitor and dispersant. The COX-2 inhibitor and the VEGFR inhibitor can be prepared into nano-drugs with irinotecan through pi-pi accumulation, hydrophobic interaction, electrostatic interaction, hydrogen bond and the like, and with the assistance of a proper dispersant. Based on physical acting force such as pi-pi accumulation, the COX-2 inhibitor or the VEGFR inhibitor exerts drug effect as a proto-drug. The nano-drug is prepared by a solvent conversion method, has simple method, controllable quality and high drug loading, effectively inhibits the proliferation of metastatic colorectal cancer cells, reduces the generation of new vessels and improves the microenvironment.

Description

Nano-drug using irinotecan as carrier and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a nano-drug taking irinotecan as a carrier, a preparation method thereof and application thereof in tumor treatment.

Background

Cancer is a disease affecting the life span of humans with morbidity and mortality in the first three. In china, clinical treatment conditions are limited and mortality rates are rising year by year. Metastasis occurs in advanced stages of colorectal cancer, and therefore stage IV is referred to as metastatic colorectal cancer. Patients diagnosed with stage I or II disease have 91% and 82% relative survival at 5 years, respectively, while 5-year survival for stage IV disease drops to 15%. Clinically, the effective rate of chemotherapy for the first-line late metastatic colorectal cancer consisting of irinotecan, 5-fluorouracil and folinic acid is less than 40%. The adverse events existing in the irinotecan chemotherapy process are neutropenia, diarrhea, skin toxicity and weakness, many of the toxicity are caused by the toxicity of metabolites on body organs, and after irinotecan enters a human body as a prodrug, two metabolic pathways exist: the main metabolic pathway is the conversion by carboxylesterase (CES, including CES1, CES 2) to produce 7-ethyl-10-hydroxy-camptothecin (SN-38), the active metabolite, which is subsequently inactivated by uridine diphosphate glucuronyl transferase 1a1 (UGT 1a 1), to produce 7-ethyl-10-hydroxy-camptothecin glucuronide (SN-38G) for excretion in vitro; the other metabolic pathway is the metabolism of cytochrome P450 (CYP) 3A4 enzyme and CYP3A5 enzyme into corresponding products to be discharged out of the body. These side effects can reduce the quality of life of the patient. Especially fatal diarrhea during treatment is fundamentally limited in its use.

The lymph node part and the liver metastasis of the patient with the advanced metastatic colorectal cancer have high COX-2 expression higher than that of the in situ tumor. The high dose of the non-steroidal anti-inflammatory drug can inhibit the proliferation of tumor cells with high expression of COX-2. Animal experiments show that the familial adenomatous polyposis can gradually disappear by using the non-steroidal anti-inflammatory drug for a long time. At present, aspirin, sulindac, celecoxib, etodolac, rofecoxib and the like are clinically used for preventing and treating colorectal cancer, and based on clinical reports, the selective treatment effect of COX-2 inhibitors such as celecoxib, etodolac, rofecoxib and the like on colorectal cancer is superior to that of non-steroidal anti-inflammatory drugs such as aspirin, sulindac and the like, and the side effects caused by the inhibition of COX-1 such as gastrointestinal bleeding, nausea, dizziness and the like can be reduced.

At present, in clinical treatment schemes for inhibiting colorectal cancer metastasis, monoclonal antibodies or small molecule inhibitors, such as regorafenib and bevacizumab, which is a targeted drug VEGF receptor, are added to inhibit metastasis, wherein the most common drug types are angiogenesis inhibitors. The VEGFR tyrosine kinase small molecule inhibitor Ruegfenib is approved to be used as a first small molecule targeting preparation for treating metastatic colorectal cancer, inhibits the activity of VEGFR, and achieves the purposes of preventing tumor angiogenesis and resisting tumors, so that the VEGFR tyrosine kinase small molecule targeting preparation is an improvement for treating the metastatic colorectal cancer. Because of individual differences, irinotecan in combination with regorafenib has been shown in certain clinical trials to have no significant differences in the progression-free survival and median survival of patients compared to the placebo group, suggesting that VEGFR inhibitors have limited therapeutic efficacy and that the chemotherapeutic regimen of VEGFR inhibitors also requires adjustment. Currently, other small molecule inhibitors such as sorafenib and furoquintinib are also being clinically tested.

In the traditional clinical first-line treatment scheme of metastatic colorectal cancer, oral administration and intravenous injection are mostly required to be matched with chemotherapy. The design of carriers for oral chemotherapeutic drugs and oral nsaids has led to challenging design of oral carrier systems due to gastrointestinal irritation and the strong acid environment of the stomach. Although most of VEGFR inhibitors are oral preparations, it is difficult to co-localize the two drugs to kill and inhibit tumor tissues.

The traditional polymer carrier has the problems of complex preparation, higher cost, degradation toxicity of monomers, low drug-loading rate and slow effect. Therefore, it is necessary to design a nano-drug using small molecules as carriers, and at the same time, the application range of the small molecule carriers is enlarged, the preparation cost is reduced, the chemical modification to the drug in the preparation process is reduced, the drug loading capacity of the drug is improved, and the problem of metabolic toxicity caused by large dosage is solved.

Disclosure of Invention

The invention discloses a nano-drug taking irinotecan as a carrier, which reduces the metabolic toxicity of the irinotecan and expands the application range of the irinotecan as the carrier. COX-2 and VEGFR are used as two special metastasis targets in metastatic colorectal cancer, their representative drugs can pass pi-pi accumulation effect, electrostatic interaction and hydrogen bond effects with irinotecan, and with the help of appropriate dispersant and control of appropriate drug ratio can be prepared into particle size in 80-150 nm nanometer preparation. Based on physical acting force such as pi-pi accumulation and the like, the COX-2 inhibitor and the VEGFR inhibitor can exert drug effects as proto-drugs.

The first aspect provides a nano-drug taking irinotecan as a carrier, which is a combined drug nano-particle consisting of irinotecan, a hydrophobic drug and a dispersion medium, wherein the hydrophobic drug is one or more of a COX-2 inhibitor and a VEGFR small-molecule inhibitor; wherein, irinotecan and hydrophobic drug are used as main drugs, the dispersion medium is used as a stabilizer, and the mass concentration of the dispersion medium is lower than that of the main drugs.

The mass ratio of the hydrophobic drug to the dispersion medium is 2:1-20:1, and the mass ratio of irinotecan to the hydrophobic drug is 1:1.5-10: 1.

Preferably, the hydrophobic drugs include, but are not limited to: one or more of celecoxib, rofecoxib, etodolac, diclofenac sodium, nabumetone, tolfenamic acid, carprofen, pranoprofen, sorafenib, regorafenib, furoquintinib, ranvatinib and cabozantinib.

Preferably, the dispersion medium is one or more of PEG-2000, F68, D-sorbitol and PVP K-30.

In a second aspect, a method for preparing the nano-drug with irinotecan as a carrier is provided, which comprises the following steps:

a: dissolving irinotecan or its salt and hydrophobic drug in organic solvent to obtain mixed solution;

b: dropping the mixed solution into the aqueous solution containing the dispersion medium, stirring at the speed of 250-1000 r/min for reaction, standing, centrifuging, and taking the supernatant.

In the step A, the organic solvent is one or more of ethanol, methanol, DMSO and DMF;

in the step B, the dropping speed of the mixed solution is 2 s/drop, 5 s/drop or 10 s/drop; the stirring reaction time is 2-10 min.

The aqueous solution includes, but is not limited to, distilled water, glucose, physiological saline.

In some embodiments, the method of preparing a nano-drug comprises:

a: dissolving irinotecan hydrochloride and hydrophobic drug in 0.02-1 ml of organic solvent to obtain a mixed solution;

b: dropping the mixed solution into the aqueous solution containing the dispersion medium according to the ratio of 2 s/drop, 5 s/drop or 10 s/drop, and stirring at the speed of 250-1000 r/min for 2-10 min;

c: b, placing the nanoparticles prepared in the step B for 2-10 min at normal temperature under the condition of ultrasonic treatment or no ultrasonic treatment;

d: centrifuging the nanoparticles at 2000-6000 r/min for 3-20 min;

e: removing insoluble solid to obtain supernatant as target preparation.

The storage condition of the nano-drug prepared by the method comprises but is not limited to 4 ℃ or freezing storage.

In a third aspect, the invention provides an application of the nano-drug taking irinotecan as a carrier in preparing a drug for preventing and/or treating tumor diseases. Further, the tumor diseases are tumors to which irinotecan belongs as an mainly-used medicament, including metastatic colorectal cancer.

Compared with the related art, the invention has the following advantages: the preparation is prepared from irinotecan and hydrophobic drugs (aromatic ring or aromatic heterocyclic hydrophobic COX-2 inhibitor or VEGFR inhibitor) by a solvent conversion method, is simple and environment-friendly, has controllable quality and high drug loading, and is proved by clinical researches of two types of hydrophobic drugs in colorectal cancer. The clinical experiment is combined with the preparation result, so that the purpose of treating the metastatic colorectal cancer can be achieved.

Drawings

The drawings related to the experiment are all obtained by arranging actual data.

Fig. 1 and fig. 2 are respectively a particle size distribution diagram and a Zeta potential diagram of a double-drug nanoparticle obtained by self-assembly of irinotecan and carprofen at a mass ratio of 2: 1.

Fig. 3 is a transmission electron microscope image of double-drug nanoparticles obtained by self-assembly of irinotecan and carprofen at a mass ratio of 2: 1.

Figure 4 is a graph of the time stability of irinotecan and carprofen under nanoparticle storage conditions at a mass ratio of 2: 1.

Fig. 5 shows the particle size change of irinotecan and sorafenib nanoparticles after the PVP K-30 serving as a stabilizer is prolonged.

Fig. 6 shows the changes of the particle size of irinotecan and carprofen nanoparticles with F68 as a stabilizer over time.

Fig. 7 and 8 show that the nanoparticles KINPs and kinnpps obtained when irinotecan and carprofen were added without PVP and with PVP were measured for CAC at 20 ℃ by the conductivity method, respectively.

Figure 9 is the particle size of nanoparticles of irinotecan with carprofen and an aqueous solution containing PVP diluted with glucose for injection.

FIG. 10 is the X-ray diffraction pattern of KINPs, KIPNPs nano-formulations and free drug and stabilizer.

FIGS. 11 and 12 are graphs of data from 24 h and 48 h cytotoxicity experiments on SW620 cells, respectively.

Detailed Description

For better understanding of the technical solutions of the present invention, the following description of the embodiments of the present invention is provided with reference to the accompanying drawings, but the present invention includes, but is not limited to, the following statements.

The hydrochloric acid, the hydrophobic drug containing benzene ring or aromatic heterocycle, the water-soluble organic solvent and the pure water used in the invention

More than 98% of analytically pure commercial chemical reagents are not different from the reagents of different manufacturers.

Example 1 irinotecan in the optimum mass ratio: carprofen is 2:1 weighing 3 groups of 10 mg irinotecan respectively and placing the 3 groups of 10 mg irinotecan in 3 1.5 ml EP tubes, adding 200 mu l DMSO in the 1.5 ml EP tubes and carrying out ultrasonic treatment, weighing 3 groups of 5 mg carprofen respectively and placing the 3 groups of 5 mg carprofen in the solution to dissolve to form mixed liquid. Slowly dropping the mixed solution into 5 ml of 0.5 mg/ml PVP aqueous solution, limiting the dropping speed to be 2 s/drop, stirring at the speed of 500 r/min for 2.5 min, centrifuging at the speed of 2000-6000 r/min for 3-10 min, taking supernatant, measuring the particle size and Zeta potential, and finally obtaining the graph 1 and the graph 2. The grain diameter of the obtained nano-particles is about 78 nm, and the Zeta potential is about 27 mv.

Example 2 according to the preparation method of example 1, irinotecan and carprofen nanoparticles were prepared, the solution was concentrated by gradient centrifugation, 20 μ l was dropped into a copper mesh, phosphotungstic acid was added, drying was performed for 5 min under an infrared lamp, and after moisture evaporation, transmission electron microscopy images were taken, and fig. 3 was obtained. The resulting particle size was about 73 nm.

Example 3 irinotecan in the optimum mass ratio: carprofen is 2:1 weighing 3 groups of 10 mg irinotecan respectively and placing the 3 groups of 10 mg irinotecan in 3 1.5 ml EP tubes, adding 200 mu l DMSO in the 1.5 ml EP tubes and carrying out ultrasonic treatment, weighing 3 groups of 5 mg carprofen respectively and placing the 3 groups of 5 mg carprofen in the solution to dissolve to form mixed liquid. Slowly dropping the mixed solution into 5 ml of 0.5 mg/ml PVP aqueous solution, limiting dropping speed to be 2 s/drop, stirring at 500 r/min for 2.5 min, centrifuging at 2800 r/min for 5 min, taking 3.5 ml of supernate, and measuring the particle size of 1 h. After the particle size was measured, the three groups of nanoparticles were placed in a refrigerator at 4 ℃ and the particle size was measured at the same time every day. Preparing three groups according to the same method, freezing and storing at-20 deg.C after measuring particle size, thawing at normal temperature, measuring the change of particle size at the same time point every day, and obtaining figure 4. The best storage condition for the nanoparticles is refrigerator storage at 4 ℃.

Example 4 irinotecan in mass proportions: sorafenib 3: 1, respectively weighing 5 groups of 15 mg irinotecan and placing the 5 groups of 15 mg irinotecan in a 1.5 ml EP tube, dripping 200 mu l of DMSO into the EP tube to dissolve irinotecan, then respectively weighing 5 groups of 5 mg sorafenib and placing the 5 groups of 5 mg sorafenib in the solution, and ultrasonically dissolving sorafenib. Slowly dripping 200 μ l of the mixed solution into 5 ml of PVP aqueous solution with different concentrations, limiting the dripping speed to 2 s/drop, stirring at 500 r/min for 2.5 min, standing in dark place for 5 min by ultrasonic treatment, and centrifuging at 1500 r/min for 5 min. Taking 3.5 ml of supernatant, and measuring the particle sizes of the supernatant after standing for 1 h, 24 h, 48 h and 72 h. Finally, FIG. 5 is obtained. The experimental result shows that PVP can be used as a stabilizer for irinotecan and sorafenib nanoparticles.

Example 5 irinotecan in a preliminary tented mass ratio: carprofen is 2:1, 5 groups of 2.5 mg irinotecan are weighed and placed in a 1.5 ml EP tube, 200. mu.l DMSO is added in the 1.5 ml EP tube, ultrasonic treatment is carried out, and 5 groups of 1.25 mg carprofen are respectively weighed and placed in the solution to be dissolved to form a mixed solution. Slowly dropping the mixed solution into 5 ml of F68 solution with different concentrations, limiting dropping speed to 2 s/drop, stirring at 500 r/min for 2.5 min, centrifuging at 2800 r/min for 5 min, taking 3.5 ml of supernatant, determining particle size of standing for 1 h and 24 h, and finally obtaining the graph 6. The experimental result shows that F68 can be used as a stabilizer of irinotecan and carprofen nanoparticles.

Example 6 irinotecan and carprofen solids were weighed out in the proportions described in example 1 to give a solution of carprofen concentration 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, 0.03125 mg/ml in a total volume of 100 ml, and a 200 ml solution of KIPNPs 0.0156 mg/ml, 0.0078 mg/ml, 0.0039mg/ml in 2 h to prepare the CAC for assay.

Similarly, by repeating the above steps, KINPs without PVP were prepared by the same concentration treatment, and CAC was prepared within 2 h.

And (3) conductivity measurement: after the solution is prepared, the temperature is kept at 20 ℃, the conductivity instrument is determined to belong to a normal calibration range at the moment, the indoor temperature of a laboratory is 20 ℃, the platinum black electrode is repeatedly washed for three times, and the conductivity of the solution is sequentially measured according to the preparation sequence. When CAC experiment determination and conductivity determination of the same solution are carried out, the measurement needs to be carried out in sequence from low concentration to high concentration, and finally, the results of FIG. 7 and FIG. 8 are obtained. The experimental result is CAC_KIPNPsIs significantly less than CAC_KINPsThe addition of PVP can reduce CAC and improve the stability of the nanoparticles.

Example 7 KIPNPs having a volume of 5 ml were prepared and the particle size was measured according to the method of example 1. Then 2ml of the solution is respectively diluted by one time by glucose solution for injection, and after 1 min of ultrasonic treatment, the gradient dilution is carried out continuously. The concentrations after dilution were 0.500 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, 0.03125 mg/ml, respectively, and the particle size was measured within 0.5 h after dilution, to obtain FIG. 9. The experimental result is that glucose can be used as a diluting medium.

Example 8 according to the proportion of example 1, 100 mg of carprofen and 200 mg of irinotecan are weighed and dissolved in 4 ml of DMSO, and then gradually dropped into 96 ml of distilled water, and stirred at the speed of 500 r/min for 10 min. Finally, the mixture was added to 2 centrifuge tubes of 50 ml and centrifuged at 2800 r/min for 5 min. The precipitate was discarded, the supernatant was retained, and after freeze-drying the KINPs solution, 150 mg of the solid was taken for X-ray diffraction analysis.

Weighing 100 mg of carprofen and 200 mg of irinotecan according to the optimal preparation proportion, dissolving in 4 ml of DMSO, gradually dripping into 96 ml of PVP aqueous solution containing 0.5 mg/ml, and stirring at the speed of 500 r/min for 10 min. Finally, the mixture was added to 2 centrifuge tubes of 50 ml and centrifuged at 2800 r/min for 5 min. The precipitate was discarded, the supernatant was retained, and after freeze-drying the KINPs solution, 150 mg of the solid was taken for X-ray diffraction analysis.

And weighing 150 mg of irinotecan, PVP and carprofen solid respectively, and performing X-ray diffraction analysis respectively to obtain a combined graph 10. The amorphous state of KINPs and KIPNPs shows that the nano-particle taking irinotecan as a carrier actually has physical interaction with carprofen, and PVP is added to assist the stabilization effect.

Example 9 according to 10⁴SW620 cells were plated in L-15 medium containing 10% FBS per well, and after 24 h, KIPNPs were prepared in a volume of 5 ml as in example 1, and 2ml of nanoparticles were serially diluted in 2ml L-15 medium containing no FBS to a carprofen concentration of 0.003 mg/ml. Seven concentration gradients were taken for dosing, two columns as blanks, with six parallel groups per concentration. After 24 h or 48 h of administration, the drug solution was aspirated and 120. mu.l of 0.83 mg/ml MTT was added. After incubation for 4 h at 37 ℃ in the dark, the MTT solution was aspirated, 150. mu.l DMSO was added, shaking in the dark for 15 min, absorbance at 570 nm was measured for each group and blank, and cell survival was calculated.

Press 10⁴SW620 cells were plated per well with L-15 medium containing 10% FBS, and after 24 h, KIPNPs were prepared in a volume of 5 ml as in example 1, 2ml of nanoparticles were first diluted with 1.4 ml of FBS-free L-15 medium and 0.6 ml of DMSO, the solution was mixed, and then sequentially diluted with 2ml of FBS-free L-15 medium in a gradient manner until the Carprofen concentration was 0.003 mg/ml. Seven concentration gradients were taken for dosing, two columns as blanks, with six parallel groups per concentration. After 24 h or 48 h of administration, the drug solution was aspirated and 120. mu.l of 0.83 mg/ml MTT was added. After incubation for 4 h at 37 ℃ in the dark, the MTT solution was aspirated, 150. mu.l DMSO was added, shaking in the dark for 15 min, absorbance at 570 nm was measured for each group and blank, and cell survival was calculated.

Press 10⁴Concentration per well SW620 cells were plated in L-15 medium containing 10% FBS, and 24 hours later, 5 ml of irinotecan solution at a concentration of 1.8 mg/ml was prepared according to the irinotecan content in the method of example 1, and 2ml of the solution was sequentially subjected to gradient dilution with L-15 medium containing no FBS to an irinotecan concentration of 0.006 mg/ml. Seven concentration gradients were taken for dosing, two columns as blanks, with six parallel groups per concentration. After 24 h or 48 h of administration, the drug solution was aspirated and 120. mu.l of 0.83 mg/ml MTT was added. After incubation for 4 h at 37 ℃ in the dark, the MTT solution was aspirated, 150. mu.l DMSO was added, shaking in the dark for 15 min, absorbance at 570 nm was measured for each group and blank, and cell survival was calculated.

Press 10⁴SW620 cells were plated in L-15 medium containing 10% FBS per well, and 24 hours later, 5 ml of a solution of carprofen having a concentration of 0.9 mg/ml was prepared according to the content of carprofen in the method of example 1, and 2ml of the solution was sequentially subjected to gradient dilution with L-15 medium containing no FBS to a concentration of carprofen of 0.003 mg/ml. Seven concentration gradients were taken for dosing, two columns as blanks, with six parallel groups per concentration. After 24 h or 48 h of administration, the drug solution was aspirated and 120. mu.l of 0.83 mg/ml MTT was added. After culturing at 37 ℃ in the dark for 4 h, the MTT solution was aspirated, 150. mu.l DMSO was added, shaking in the dark for 15 min, absorbance at 570 nm was measured for each group and blank, and the fine particle was calculatedCell survival, resulting in fig. 11 and fig. 12. The experimental results show that KIPNPs have better tumor inhibition effect at 48 h than the irinotecan group, the carprofen group and the physical mixing group added with DMSO.

The foregoing are only preferred embodiments of the present invention, and it will be apparent to those skilled in the art that the invention can be carried out with cell type changes, the delivery or carrier assistance of hydrophobic drugs of the same type, surfactant type, etc., without departing from the principles of the present invention, and such modifications are within the scope of the present invention.

Claims

1. A nano-drug taking irinotecan as a carrier is characterized by being a combined drug nano-particle consisting of irinotecan, a hydrophobic drug and a dispersion medium, wherein the hydrophobic drug is one or more of a COX-2 inhibitor and a VEGFR small-molecule inhibitor; wherein, irinotecan and hydrophobic drug are used as main drugs, the dispersion medium is used as a stabilizer, and the mass concentration of the dispersion medium is lower than that of the main drugs.

2. The irinotecan-carried nano-drug according to claim 1, wherein the mass ratio of the hydrophobic drug to the dispersion medium is 2:1 to 20:1, and the mass ratio of the irinotecan to the hydrophobic drug is 1:1.5 to 10: 1.

3. The irinotecan-carried nano-drug according to claim 1, wherein the hydrophobic drug is one or more of celecoxib, rofecoxib, etodolac, diclofenac, nabumetone, tolfenamic acid, carprofen, pranoprofen, sorafenib, regorafenib, furotinib, ranvatinib and cabozantinib.

4. The nano-preparation using irinotecan as a molecular carrier according to claim 1, characterized in that the dispersion medium is one or more of PEG-2000, F68, D-sorbitol, PVPK-30.

5. The method for preparing irinotecan-carried nano-drug according to any one of claims 1 to 4, characterized by comprising:

6. The method for preparing irinotecan-supported nano-drug according to claim 5, characterized in that in the step A, the organic solvent is one or more of ethanol, methanol, DMSO and DMF.

7. The method for preparing irinotecan-supported nano-drugs according to claim 5, wherein in the step B, the mixed solution is dropped at a rate of 2 s/drop, or 5 s/drop, or 10 s/drop; the stirring reaction time is 2-10 min.

8. The method for preparing irinotecan-carried nano-drug according to claim 5, characterized in that the aqueous solution is distilled water, glucose, physiological saline.

9. Use of the irinotecan-carried nanomedicine of any one of claims 1 to 4 in the preparation of a medicament for the prevention and/or treatment of a neoplastic disease.

10. Use according to claim 9, wherein the neoplastic disease is a neoplasm for which irinotecan is indicated as a major agent, including metastatic colorectal cancer.