CN114209847A

CN114209847A - Secondary nanocrystallized albumin paclitaxel medicament and application thereof

Info

Publication number: CN114209847A
Application number: CN202111665880.0A
Authority: CN
Inventors: 王刚; 郑骏年; 韩露露; 陈玉鑫; 李慧忠; 柴大飞; 邱小燕
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-03-22
Anticipated expiration: 2041-12-31
Also published as: CN114209847B

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a secondary nanocrystallized albumin paclitaxel preparation and application thereof. Specifically, the invention provides a preparation method of a medicine with low toxic and side effects, which comprises the steps of carrying out twice nano treatment on an active ingredient or carrying out nano treatment on a nano medicine again.

Description

Secondary nanocrystallized albumin paclitaxel medicament and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a secondary nanocrystallized albumin paclitaxel medicament and application thereof.

Background

In the last 60 s, american scientists found anticancer active ingredients in the bark of the pacific yew tree: paclitaxel. Paclitaxel was approved for ovarian cancer therapy in 1992. However, the extraction of paclitaxel from yew tree alone is far from sufficient to satisfy the needs of many clinical patients, and scientists continue to develop research and find abundant semi-finished products from european yew tree to semi-synthesize paclitaxel, solving the problem of paclitaxel supply. However, paclitaxel can only be dissolved by means of polyoxyethylated castor oil to ensure a certain solubility and stability, but in 20-40% of patients, injection of this solvent can cause severe allergic reactions.

The new generation of taxoids is nanoparticle albumin-bound paclitaxel Nab-paclitaxel, and the latest albumin-bound nanoparticle technology is adopted, so that toxic solvents are removed, the problem of allergy is solved, and the treatment effect is improved. In vitro studies showed that albumin-bound paclitaxel was 1.33 times as concentrated in tumor tissues as conventional solvent-based paclitaxel, while in normal tissues the drug concentration was 1/2, which is common. The existing guidelines recommend that albumin-bound paclitaxel is used alone for metastatic breast cancer, pancreatic cancer and other solid tumors.

In view of the good solubility of albumin-bound paclitaxel, it is common practice in clinical treatment to increase the dosage of the drug to achieve better therapeutic effect. However, high doses of albumin-bound paclitaxel also induce significant toxic side effects, such as myelosuppression, alopecia, and peripheral neurotoxicity. Therefore, aiming at the problems of the prior albumin-bound paclitaxel in the clinical application process, the reduction of the toxic and side effects while maintaining the curative effect is a very worthy research direction.

Disclosure of Invention

In order to reduce the toxic and side effects of albumin paclitaxel in treatment, the albumin paclitaxel nanoparticles are subjected to secondary nano-packaging, the obtained secondary nano-albumin paclitaxel avoids the problems of bone marrow suppression and limited hematopoiesis, and the albumin paclitaxel nanoparticles show a very good anti-tumor effect by combining radiotherapy, and the lung cancer experiment shows that the effect is multiplied.

Preparation method

In one aspect, the invention provides a method for preparing a low toxic side effect drug, the method comprising twice nanometering an active ingredient or again nanometering a nanometering drug.

Preferably, the nanocrystallization process includes any method of nanoparticle preparation. Specifically, for example: antisolvent method, emulsification method, thermal gel method, albumin nanometer combination technology, self-assembly technology, and nanometer spray drying technology.

Preferably, the two nano-treatments can adopt the same or different nano-particle preparation methods.

Preferably, the nanocrystallization treatment is performed by an anti-solvent method, and the anti-solvent method comprises the following steps: mixing serum albumin (HSA) with active component or nano-drug, slowly dripping ethanol or acetone, and finally adding cross-linking agent to obtain nano-particles.

Preferably, the step of the antisolvent process comprises the steps of:

1) mixing HSA + active ingredient or nano-drug; preferably, the mixed solution is stirred at a high rotation speed; preferably, a high rotational speed refers to 1000 rpm;

2) adding ethanol or acetone into the solution of the step 1), and continuously stirring; preferably, stirring is for at least 5 minutes (specifically including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes); more preferably, stirring for 10 minutes;

3) adding a cross-linking agent to the solution of 2); preferably, after stirring uniformly, incubating in an environment above 25 ℃ for at least 1 hour (specifically including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours); more preferably, the incubation is for 4 hours.

Preferably, the cross-linking agent comprises formaldehyde, glutaraldehyde;

preferably, the cross-linking agent is glutaraldehyde; more preferably, it is 8% glutaraldehyde.

Preferably, the ethanol or acetone is slowly dropped; more preferably, the dropwise addition rate of the ethanol or acetone is 1 ml/min;

preferably, the operation step further comprises the step of taking the nano solution for centrifugal resuspension. Specifically, the step of centrifugation resuspension is: after centrifugation at 12000rpm, the supernatant was discarded, air-dried, and resuspended in sterile water. Preferably, filtration through a 0.22nm filtration membrane is also required.

Secondary nano medicine

On the other hand, the invention provides a secondary nano-drug, which is prepared by performing nano-treatment on an active ingredient twice or performing nano-treatment on a nano-drug again. That is, the present invention provides the product prepared by the aforementioned preparation method.

Suitable "active ingredients" in the present invention illustratively include 5-aminosalicylic acid (salicilic acid), abacavir (abacayir), abarelix (abarelix), abavacp (abatacept), acamprosate (acamprosate), acarbose (acarbose), aceclofenac (aceclofenac), acetylsalicylic acid, abamectin (acitretin), aclarubicin (aclarubicin), actinomycin (actinomycin), acyclovir (acyclovir), adalimumab (adalimumab), adefovir (adefovir), adefovir dipivoxil (adefovir dipivoxil), adenosine, ademetionine (adenosionine), adrenaline (adriacin).

Preferably, the active ingredient is an anticancer active ingredient.

Preferably, the anticancer active ingredient includes, but is not limited to, any plant-derived ingredient having anticancer activity, including plant-derived active ingredients, specifically, Sulphoraphane, Paclitaxel (Paclitaxel), Epipodophyllotoxin, Vincristine (Vincristine), Vinblastine (Vinblastine), Vinorelbine (Vinorelbine), Vindesine (Vindesine), Vinblastine (Vinflunine), allicin (Pomiferin), Epigallothechin-3-gallate, Combrestatin A-4, sphastatin, Roscovitine (Roscovitine), Flavopirimidyl (Flavopiridol), Noscapine (Noscapine), Formononectin, Cabazitaxel, Colchicine (Colocine), Combrestatin, docetaxel (docetaxel), Duratoxin (Isotaxinol), Potentillin (Potentillin-2, Verticine.

Preferably, the anticancer active ingredient is paclitaxel.

Preferably, the nano-drug includes, but is not limited to, albumin-bound paclitaxel (b

For example as described in patents WO2014105644 and WO 2008057562) and liposomal daunorubicin (e.g., (r) ((r))

For example as described in patents EP0004467 and US 20070286897).

Preferably, the nano-drug is albumin-bound paclitaxel (also referred to herein simply as "albumin paclitaxel").

Preferably, the particle size of the secondary nano-drug provided by the invention is in the range of 50-150 nm; preferably about 100 nm.

Cells

In another aspect, the present invention provides a cell comprising the drug produced by the above-described preparation method or the above-described secondary nanometerized drug.

Preferably, the cells comprise animal cells;

preferably, the cell is a human cell;

more preferably, the cell is a neutrophil.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition comprising the drug prepared by the above preparation method or the above secondary nanometerized drug.

Preferably, the pharmaceutical composition further comprises any active pharmaceutical substance capable of being an ingredient of a multiparticulate dosage form, a tablet comprising pellets, a mini-tablet, a capsule, a sachet, an effervescent tablet or a dry powder for oral suspension;

pharmaceutical compositions of the compounds of the present invention may be administered by any of the following means: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracardiac, intrasternal or intravenous administration.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

Preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the national food and drug administration for use in humans or livestock.

Preferably, the pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drip, transdermal preparations, ointments, lotions, adhesive preparations, suppositories, pellets, nasal preparations, pulmonary preparations, eye drops and the like, oral or parenteral preparations.

System for controlling a power supply

In another aspect, the invention provides a cancer treatment system with combined chemoradiotherapy, the system comprising at least the following two modules:

1) a radiotherapy module to perform radiotherapy, in particular, the radiotherapy module comprises an instrument or reagent to perform radiotherapy;

2) the chemotherapy module for administering the medicine prepared by the preparation method or the secondary nanocrystallization medicine to a patient preferably can also comprise other instrument devices required for administering the medicine.

Preferably, the radiotherapy module and the chemotherapy module are repeatedly administered alternately;

preferably, the chemotherapy module is administered 1-3 days prior to administration of the radiotherapy module; one radiotherapy and one chemotherapy can be called as one course of treatment, and a patient can be treated by a plurality of courses of treatment; specifically, it may be: a first-use radiotherapy module (abbreviated as "first radiotherapy") -a first-use chemotherapy module (abbreviated as "first chemotherapy") -a second-use radiotherapy module (abbreviated as "second radiotherapy") -a second-use chemotherapy module (abbreviated as "second chemotherapy");

the radiotherapy and the radiotherapy have the same meanings and can be replaced mutually. Radiation therapy is one of three cancer core therapies. Researchers have found that tumor-targeted radiation therapy can act as a "cancer vaccine" to induce neutrophil-mediated tumor cell death, stimulating the immune system against other metastatic cancer cells.

The chemotherapy and the chemical drug treatment are the same in meaning and can be replaced with each other. Specifically, the chemical drug used in the present invention is a drug prepared by the above-mentioned preparation method or the above-mentioned secondary nanocrystallized drug.

Applications of

In another aspect, the invention provides a product prepared by the preparation method, a secondary nanocrystallized medicament, a cell, a pharmaceutical composition and application in preparing a medicament for treating cancer.

Preferably, the medicament for treating cancer comprises a medicament for use in conjunction with radiotherapy.

Preferably, the radiation comprises ionizing radiation, particle beam radiation.

Preferably, the particle beam comprises electrons, protons, neutrons, heavy ions such as carbon ions or mesons.

Preferably, the ionizing radiation comprises x-ray radiation, ultraviolet radiation, infrared radiation, gamma ray radiation or microwave radiation.

In another aspect, the invention provides the use of the aforementioned cancer treatment system in the manufacture of a product for the treatment of cancer.

Preferably, the cancer comprises: cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional epithelial cancer of the bladder, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumors, mast cell tumors, multiple myeloma, melanoma, glioma, and sarcoma.

Preferably, the cancer comprises any cancer that can be treated with paclitaxel. Specifically, ovarian cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, non-Hodgkin's lymphoma, carcinoma of large intestine, melanoma, head and neck cancer, lymphoma, and cerebroma have certain curative effects.

Preferably, the cancer is lung cancer.

Preferably, the cancer treatment is a model organism constructed against LLC lung cancer cells.

Preferably, the model organism is a mouse.

Method of treatment

In another aspect, the present invention provides a method for treating cancer, which comprises administering any one of the secondary nanocrystallization drugs, the products, the cells and the pharmaceutical compositions prepared by the preparation method to a patient.

Preferably, the method further comprises subjecting the patient to radiation therapy.

Drawings

Fig. 1 is a graph showing the results of particle size measurement of double nanocrystallized albumin paclitaxel, fig. 1A is a graph showing the results of detection, and fig. 1B is a data statistical graph.

Fig. 2 is a graph showing the results of potential measurement of double nanocrystallized albumin paclitaxel, fig. 2A is a graph showing the results of detection, and fig. 2B is a data statistical graph.

FIG. 3 shows the stability test results of two-time nanocrystallized albumin paclitaxel.

FIG. 4 shows the results of particle size stability test of the double nanocrystallized albumin paclitaxel.

FIG. 5 is a morphology of double nanocrystallized albumin paclitaxel under electron microscope.

FIG. 6 is a graph showing the result of the measurement of the drug loading of neutrophils under the stimulation of radiotherapy.

FIG. 7 is a graph showing the results of measurement of drug loading of neutrophils under PMA stimulation, FIG. 7A is a graph showing the results of flow cytometry, and FIG. 1B is a graph showing the results under a microscope.

FIG. 8 is a graph showing the results of a chemotaxis experiment, FIG. 8A is a graph showing the results of detection, and FIG. 8B is a data statistics graph.

FIG. 9 is a graph showing the results of ROS detection, FIG. 9A is a graph showing the results of detection, and FIG. 9B is a data statistics graph.

FIG. 10 is a graph showing the results of measurement of the expression levels of il-1. beta. and il-6. beta. in FIG. 10A, il-6. beta. and il-6 in FIG. 10B.

FIG. 11 is a flow chart of a mouse experiment.

Fig. 12 is a tumor solid map.

Fig. 13 is a statistical graph of tumor volume and tumor weight change, fig. 13A is volume, and fig. 13B is weight.

FIG. 14 is a statistical graph of the change in body weight of mice.

Fig. 15 is a graph of the percent of drug-loaded neutrophils within the tumor, fig. 15A is a flow chart, and fig. 15B is a statistical chart.

Fig. 16 is a graph of HE staining results for heart, liver, spleen, lung and kidney, fig. 16A: heart, fig. 16B: lung, fig. 16C: the liver of a human being is treated with a drug,

FIG. 16D: spleen, fig. 16E: kidneys.

FIG. 17 is a graph showing the results of blood routine and liver renal function tests.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 preparation of Secondary Nanometrized Albumin paclitaxel

The preparation method comprises the following steps:

1. 1ml of an albumin-binding paclitaxel solution (trade name: Aiyue; Hengsui pharmaceutical Co., Ltd.) having a concentration of 20mg/ml was taken and put into a 50ml conical flask containing a stirring rotor, followed by stirring while slowly adjusting the rotation speed (1000 rpm);

2. slowly dripping 3.45ml of absolute ethyl alcohol into the conical flask at the speed of 1ml/min, sealing the conical flask opening by using a preservative film, and stirring for 10min at room temperature;

3. finally, 20 mul of 8% glutaraldehyde is added, the solution is transferred to a 15ml centrifuge tube after being stirred uniformly, and the solution is incubated for 4 hours at room temperature (>25 ℃);

4. taking 500 mu L of the nano solution, loading the nano solution into 1.5ml Ep tubes, centrifuging the tubes at 12000rpm for 15min, removing supernatant, and then airing the tubes at room temperature;

5. adding 1ml of sterilized water to resuspend the precipitate, filtering by a 0.22nm filter membrane, and storing at 4 ℃ in the dark.

Feature verification

And scanning the prepared product under an electron microscope, and measuring the particle size, potential, stability and particle size stability of the product. The scanograms and the results of the measurements are shown in FIGS. 1-5.

Example 2 Effect of Secondary Nanometrized Albumin paclitaxel on neutrophils

Considering that the therapeutic effect of the modified nano-drug is mediated by neutrophils, the influence of the modified nano-drug on neutrophils is judged.

The nano-drug prepared as shown in fig. 6 can be phagocytized by neutrophils normally in vivo and in vitro (detected by flow and electron microscope), and is more obvious under inflammatory stimulation (fig. 7), and the maximum drug loading can reach 99.1%.

In order to determine the influence of the modified nano-drug on the migration characteristic, oxygen burst and inflammatory cytokine expression of the nano-drug, the migration characteristic of the drug-loaded neutrophilic granulocyte is evaluated through a Transwell experiment; secondly, evaluating the change of the intracellular ROS level of the neutrophilic granulocyte after drug loading through flow cytometry and fluorescence intensity detection; and the expression of neutrophil associated inflammatory cytokines, in particular il-1 β, il-6, after loading was assessed by RT-qPCR.

The chemotaxis experiment result is shown in FIG. 8 (in the figure, fMLP is fMLP, which is called N-formamyl-methionyl-leucyl-phenylalanine, Chinese name: N-formyl-L-methionyl-L-leucyl-L-phenylalanine tripeptide; a leukocyte chemotactic peptide found in bacteria, has strong chemotaxis and inflammation-causing activity, and can rapidly activate leukocytes). The ROS detection results are shown in fig. 9 (H2DCFDA, a probe conventionally used for detecting Reactive Oxygen Species (ROS) in cells). FIG. 10 shows the results of measurement of the expression levels of inflammatory cytokines il-1. beta. and il-6.

The above results indicate that drug loading does not affect neutrophil migration itself, ROS production and expression of related inflammatory cytokines.

Example 3, in vivo experiments: lung cancer model

Constructing a mouse LLC lung cancer subcutaneous tumor model at the thigh part on the right side of the mouse; radiotherapy is carried out one day before the medicine treatment; mice body weight and tumor volume were monitored periodically. The specific flow is shown in fig. 11.

The tumor mass is shown in fig. 12, and the change in the statistical volume and weight of the data is shown in fig. 13. The mice were also monitored for weight change and the statistics are shown in figure 14.

Whether the drug loading of the neutrophils in the tumor is detected is shown in fig. 15, and the result shows that about 89% of the neutrophils are loaded with the drug.

Example 4 judgment of drug safety

After the nano-drug can obviously inhibit tumors after being definitely modified, the toxic and side effects of the nano-drug after being definitely modified are definitely determined by the HE staining of the heart, the liver, the spleen, the lung and the kidney of a mouse, and the result is shown in figure 16; simultaneously, the liver and kidney functions and the peripheral blood cell changes of the mice were detected by a mouse biochemical analyzer and a blood conventional analyzer, respectively (fig. 17).

The results show that the modified nano-drug has no obvious toxic or side effect and can obviously reduce the bone marrow suppression effect of the albumin-bound paclitaxel.

Claims

1. A process for preparing the medicine with low toxic by-effect includes such steps as nano treating the active component twice or nano treating the nano-class medicine again.

2. The method of claim 1, wherein the active ingredient is an anticancer active ingredient, and the nano-drugs include, but are not limited to, albumin-bound paclitaxel and liposomal daunorubicin;

preferably, the anticancer active ingredient comprises Sulphoraphane, paclitaxel, vinblastine, Epipodophyllotoxin, vincristine, vinblastine, vinorelbine, vindesine, allicin, Epigallothecin-3-white, Combretastatin A-4phosphate, Roscovitine, Flapiridol, Nocardia, Formononetin, Cabazitaxel, Colchicine, Combretastatin, docetaxel, isoliquiritigenin, Larotaxel, Podophyllotoxin, Resveratrotrol, 2S-abyssine II, Verubulin.

3. The method of claim 2, wherein the anticancer active ingredient is paclitaxel and the nano-drug is albumin-bound paclitaxel.

4. The production method according to claim 1, wherein the nanocrystallization treatment comprises using any of the following methods: anti-solvent method, emulsification method, thermal gel method, albumin nanometer combination technology, self-assembly technology, nanometer spray drying technology;

preferably, the nanocrystallization treatment adopts an anti-solvent method.

5. The method of claim 4, wherein the step of antisolvent comprises: mixing serum albumin with active ingredients or nano-drugs, slowly dripping ethanol or acetone, and finally adding a cross-linking agent to obtain nano-particles;

preferably, the step of the antisolvent process comprises the steps of:

1) mixing HSA + active components, or taking nanometer medicine;

2) adding ethanol or acetone into the solution of 1);

3) adding a cross-linking agent to the solution of 2);

preferably, the cross-linking agent comprises formaldehyde, glutaraldehyde;

preferably, the cross-linking agent is glutaraldehyde; more preferably, 8% glutaraldehyde;

preferably, the ethanol or acetone is slowly dropped;

more preferably, the dropwise addition rate of ethanol or acetone is 1 ml/min.

6. A medicament obtainable by the process of any one of claims 1 to 5.

7. A cell comprising the agent of claim 6;

preferably, the cells comprise animal cells;

preferably, the cell is a human cell;

more preferably, the cell is a neutrophil.

8. A pharmaceutical composition comprising the medicament of claim 6;

preferably, the pharmaceutical composition is administered by any of the following means: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracardiac, intrasternal or intravenous administration;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient;

preferably, the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration or the national food and drug administration for use in humans or livestock;

preferably, the dosage form of the pharmaceutical composition includes tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release drugs, aerosols, films, injections, intravenous drip, transdermal absorption drugs, ointments, lotions, adherent drugs, suppositories, pellets, nasal drugs, pulmonary drugs, eye drops.

9. A chemoradiotherapy-combined cancer treatment system, comprising at least the following two modules:

1) a radiotherapy module for an instrument or reagent for performing radiotherapy;

2) a chemotherapy module for administering the drug of claim 6 to a patient.

10. Use of the medicament of claim 6, the cell of claim 7, the pharmaceutical composition of claim 8, the cancer therapy system of claim 9 in the manufacture of a product for the treatment of cancer;

preferably, the cancer comprises: cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, transitional epithelial cancer of the bladder, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, cancer of the female reproductive tract, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumors, mast cell tumors, multiple myeloma, melanoma, glioma, and sarcoma;

preferably, the cancer is lung cancer.