CN114767868B - Application of COX-2 inhibitor and chemotherapeutic drug in preparation of antitumor drug - Google Patents
Application of COX-2 inhibitor and chemotherapeutic drug in preparation of antitumor drug Download PDFInfo
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Abstract
The invention discloses an application of a COX-2 inhibitor and a chemotherapeutic drug in combination in preparation of an antitumor drug, and also protects an application of a small molecular double prodrug consisting of the COX-2 inhibitor and the chemotherapeutic drug in preparation of a drug delivery system, and also protects an application of small molecular double prodrug nanoparticles consisting of the COX-2 inhibitor and the chemotherapeutic drug in preparation of the drug delivery system, wherein the drug is an antitumor drug, in particular an anti-breast cancer drug. Meanwhile, the preparation methods of the micromolecular double prodrug and the micromolecular double prodrug nanoparticle are also described. According to the invention, the COX-2 specific inhibitor is selected to be combined with chemotherapeutic drugs, and the synergistic regulation and control of immunosuppressive tumor microenvironment by the double-targeting tumor cells and MDSCs cells is realized through the amphiphilic micromolecule double-prodrug nanoparticles.
Description
Technical Field
The invention relates to the technical field of antitumor drugs, in particular to application of a COX-2 inhibitor and a chemotherapeutic drug in preparation of antitumor drugs.
Background
Breast cancer is one of the most common malignant tumors in women, seriously threatens the life health of women, is difficult to treat clinically due to easy invasion, easy drug resistance and high lethality rate, and currently, chemotherapy and radiotherapy are generally used as main clinical treatments. Studies have found that dynamic interactions between breast cancer cells and immune or stromal cells in the Tumor Microenvironment (TME) play an important role in chemotherapy resistance, promoting tumor development, invasion and metastasis. This complex immunosuppressive tumor microenvironment is a major obstacle to the success of breast cancer therapy.
In recent years, anti-tumor immunotherapy based on remodeling of the Immunosuppressive Tumor Microenvironment (ITM) has received widespread attention. Wherein highly expressed myeloid-derived suppressor cells (MDSCs) play an extremely important role in ITM. It causes cytotoxic T lymphocyte tolerance by relying on the production of Reactive Oxygen Species (ROS); and indirectly inhibits the function of T lymphocytes by over-expressing arginase 1, TGF and IL-10 and promoting the development and accumulation of immunosuppressive regulatory T cells (Tregs), and differentiates into immunosuppressive Tumor Associated Macrophages (TAMs) to block antitumor immune response. Therefore, MDSCs are considered as important targets of tumor immunotherapy, inhibit the proliferation and intratumoral accumulation of MDSCs, and can effectively regulate ITM to improve the anti-tumor efficacy. Interestingly, it was found that Gemcitabine (GEM), a chemotherapeutic drug, in addition to killing tumor cells directly by chemotherapy, has been shown to selectively deplete MDSCs from tumor-bearing mice, abrogate the immunosuppressive function of MDSCs, and increase CD8 + T cell antitumor activity.
It is also noteworthy that the process by which MDSCs produce large amounts of ROS to induce immune tolerance is highly dependent on the activity of cyclooxygenase-2 (COX-2). COX-2 is a catalyst for the synthesis of prostaglandin 2 (PGE) from arachidonic acid 2 ) The rate-limiting enzyme of (2), PGE 2 Tumor progression is supported by the control of cell proliferation, angiogenesis and immunosuppression by modulating downstream targets. In addition, high expression of COX-2 blocks Dendritic Cell (DCs) migration, recruits immunosuppressive cells, and increases the barrier between T cells and tumor cells by upregulating C-X-C chemokine ligand 12 (CXCL 12). This suggests that COX-2 is also a favorable target for the modulation of ITM, inhibiting COX-2/PGE 2 The pathway may significantly improve ITM, sweeping the barrier for further effective killing of tumor cells.
Based on the research background and the defects of poor physicochemical property, low bioavailability, poor curative effect and the like of single-drug treatment, if the chemotherapeutic drug can be combined with the specificity inhibition of COX-2, the tumor effect can be improved, and based on the complementary synergy of the two drugs in an anti-tumor action mechanism, the ITM is effectively reversed through double-targeting tumor cells and MDSCs cells, so that the chemotherapeutic-immune synergy can enhance the anti-tumor effect. However, how to realize that two small molecule drugs with distinct physicochemical properties can be efficiently and accurately co-delivered to tumor cells and MDSCs cells in vivo is a prerequisite and key for successful treatment of breast cancer by chemotherapy in combination with immunization.
In recent years, a carrier-free small molecule double-prodrug self-assembly nano drug delivery system makes a great contribution in the fields of realizing high-efficiency co-delivery and accurate cooperative treatment of combined drugs. Compared with carrier-assisted nanoparticles, the carrier-free nanoparticles have the remarkable advantages of simple preparation process, strong drug-loading capacity, high drug bioavailability, avoidance of immeasurable potential toxicity of the carrier and the like.
Based on the above advantages, the combined medicine can be accurately co-delivered to tumor cells. However, it is desirable to achieve the desired combined anti-tumor efficacy, and how to ensure effective drug release and tumor accumulation is another key issue that needs to be addressed.
Therefore, how to combine the chemotherapeutic drug and the COX-2 inhibitor into an anti-tumor drug and ensure the effective release of the drug and tumor accumulation is a technical problem that needs to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the invention selects COX-2 specific inhibitor combined with chemotherapeutic drugs, and realizes the synergistic regulation and control of immunosuppressive tumor microenvironment by the double-targeting tumor cells and MDSCs cells through the amphiphilic small molecule double prodrug nanoparticles.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the combination of the COX-2 inhibitor and the chemotherapeutic drug in preparing the anti-tumor drug comprises the following components: breast, lung and ovarian cancer.
The invention also provides application of a small molecule double prodrug consisting of a COX-2 inhibitor and a chemotherapeutic drug in preparation of a drug delivery system as an inventive concept same as the technical scheme, wherein the drug is an anti-tumor drug, and the tumor comprises: breast, lung and ovarian cancer.
As the same inventive concept as the above technical solution, the present invention also claims the application of small molecule double prodrug nanoparticles composed of COX-2 inhibitor and chemotherapeutic drugs in the preparation of drug delivery systems, wherein the drugs are anti-tumor drugs, and the tumors comprise: breast, lung and ovarian cancer. .
The invention also provides a small molecular double prodrug which is used for ensuring the effective release and tumor accumulation of the antitumor drug, and the invention is the same as the technical proposal and comprises the following raw materials: COX-2 inhibitors, anhydrides, and chemotherapeutic agents.
As a preferable embodiment of the above technical means, the COX-2 inhibitor comprises Celecoxib (CXB) or Rofecoxib (RXB); the chemotherapeutic drug comprises irinotecan hydrochloride (IR) Gemcitabine (GEM), paclitaxel (PTX) or other antitumor drugs containing active amino or hydroxyl, wherein the acid anhydride is any one of oxalic anhydride, adipic anhydride and succinic anhydride;
as the same inventive concept as the above technical solution, the present invention also claims a preparation method of a small molecule dual prodrug for ensuring effective release and tumor accumulation of an antitumor drug, comprising the following processes: firstly, a COX-2 inhibitor and anhydride are subjected to chemical catalysis to obtain an intermediate, and then chemotherapeutic drugs and the intermediate are further reacted to obtain the amphiphilic micromolecular double prodrug.
As the preferable technical scheme of the technical scheme, the process for obtaining the intermediate by chemically catalyzing the COX-2 inhibitor and the anhydride comprises the following steps: COX-2 inhibitor, acid anhydride, and DMAP are dissolved in an organic solvent 1 in a molar ratio of 1 2 Placing the mixture in an oil bath at 40-90 ℃ under the protection condition, magnetically stirring the mixture for reaction for 12-30 h to obtain reaction liquid, precipitating the reaction liquid into a hydrochloric acid aqueous solution, continuously stirring the mixture, extracting the reaction liquid by using an organic solvent 1, combining organic layers, washing the organic layers by using a sodium chloride solution, drying, concentrating, separating and purifying to obtain an intermediate;
the process of further reacting the chemotherapeutic drug with the intermediate to obtain the amphiphilic micromolecular double prodrug comprises the following steps: dissolving the intermediate, DMAP and chemotherapeutic drug in an organic solvent 1, and slowly adding the mixture under the ice bath conditionSlow addition of EDCI at N 2 Magnetic stirring reaction is carried out for 12 to 36 hours at a temperature of between 4 and 30 ℃ under protection; after the reaction is finished, extracting by using an organic solvent 1, washing by using a sodium chloride solution, collecting an organic layer, concentrating, separating, purifying and drying in vacuum to prepare a product CXB-GEM amphiphilic micromolecule double prodrug; wherein the molar ratio of the intermediate, DMAP, chemotherapeutic drug and EDCI is 1.05-0.2;
the organic solvent 1 is any one of anhydrous pyridine, anhydrous tetrahydrofuran, anhydrous N, N-dimethylformamide, anhydrous dioxane, anhydrous dichloromethane and ethyl acetate.
The invention also provides a micromolecular double prodrug nanoparticle which ensures the effective release and tumor accumulation of the antitumor drug, and the micromolecular double prodrug nanoparticle is prepared by self-assembling the micromolecular double prodrug under the low-speed magnetic stirring.
As a preferable technical solution of the above technical solution, the process includes:
dissolving the amphiphilic micromolecule double prodrug in an organic solvent 2 to obtain a solution, dropwise adding the solution into the solution A, volatilizing the organic solvent 2 under low-speed magnetic stirring, and self-assembling the amphiphilic micromolecule double prodrug to form nanoparticles with uniform size.
As a preferable embodiment of the above technical solution, the organic solvent 2 is any one of methanol, ethanol, acetone, tetrahydrofuran, dioxane, and pyridine; the solution A is any one of phosphate buffer solution, hepes solution and water with the pH value of 7.4.
According to the technical scheme, the invention discovers that the chemotherapeutic drug and the COX-2 inhibitor have synergistic effect in the aspect of treating tumors, the two solutions can be mixed to effectively inhibit the proliferation activity of breast cancer 4T1 cells, and the effect of the physical mixture of the two drugs is stronger than that of any single free solution; moreover, the invention discovers for the first time that amphiphilic small molecule double prodrugs consisting of chemotherapeutic drugs and COX-2 inhibitors can be self-assembled to form uniform nanoparticles.
The invention has the following specific effects:
1) The CXB-GEM amphiphilic micromolecule double prodrug is designed and synthesized, and the synthesis method is simple and easy to operate;
2) CXB-GEM nanoparticles with uniform particle size are prepared, and the preparation process is simple and easy to implement;
3) The CXB-GEM nanoparticle prepared by using the CXB solution and the GEM solution as a control has remarkable capacity of inhibiting tumor cell proliferation and inducing apoptosis;
4) The prepared CXB-GEM nanoparticles have the advantages of good anti-tumor effect and tumor lung metastasis inhibition.
In conclusion, the small-molecule double-prodrug nanoparticle overcomes the defects of complex carrier structure, high carrier toxicity, poor biocompatibility and the like, researches two small-molecule drugs with different physicochemical properties to tumor cells and MDSCs cells in a highly-efficient, synchronous, accurate and co-delivery manner by investigating the pharmaceutical characteristics and in-vivo and in-vitro anti-tumor effects of the small-molecule double-prodrug nanoparticle, and realizes the advantage of chemotherapy-immune combined treatment on breast cancer by cracking micelles to release the drugs under the response of intracellular hydrolase and a low pH environment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram of the particle size and transmission electron microscopy of CXB-GEM nanoparticles according to the present invention;
FIG. 2 is a graph of the in vitro release of CXB-GEM nanoparticles according to the invention;
FIG. 3 is a graph of the toxicity results of formulations of CXB-GEM nanoparticles of the present invention;
FIG. 4 is a diagram of the induction of apoptosis of 4T1 cells by CXB-GEM nanoparticles according to the present invention;
FIG. 5 is a diagram showing the in vivo antitumor drug efficacy of CXB-GEM nanoparticles of the present invention.
FIG. 6 is a diagram showing the results of CXB-GEM nanoparticles inhibiting tumor lung metastasis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1: synthesis of CXB intermediate by reacting CXB with SA to form amide
CXB (0.025 mmol), SA (0.075 mmol) and DMAP (0.002 mmol) were dissolved in ethyl acetate under N 2 Placing the mixture in an oil bath at 40-90 ℃ under the protection condition, magnetically stirring the mixture for reaction for 12-30 h, stopping the reaction, precipitating the reaction solution into a hydrochloric acid aqueous solution, continuously stirring the mixture, extracting the reaction solution by using ethyl acetate, combining organic layers, washing the organic layers by using a sodium chloride solution, drying the organic layers on magnesium sulfate, and separating and purifying the organic layers by using a silica gel column chromatography to obtain a CXB intermediate as shown in a formula I;
example 2 Synthesis of CXB-GEM amphiphilic Small molecule Dual prodrugs
CXB intermediate (4.157 mmol), DMAP (0.425 mmol) and GEM (5.56 mmol) were dissolved in ethyl acetate and 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI, 5.37 mmol) was added slowly under ice-bath conditions over N 2 Magnetically stirring and reacting for 12-36 h at 4-30 ℃ in a protective atmosphere. After the reaction is finished, ethyl acetate is adopted for extraction, the mixture is washed by sodium chloride solution, an organic layer is collected and concentrated, the separation and purification are further carried out by silica gel column chromatography, and the product CXB-GEM amphiphilic micromolecule double prodrug is finally prepared after vacuum drying, wherein the product is shown as a formula II;
example 3 Synthesis of RXB-SA intermediates by reaction of RXB with SA to amides
RXB (0.035 mmol), SA (0.055 mmol) and DMAP (0.008 mmol) were dissolved in ethyl acetate and the mixture was dissolved in N 2 And (2) placing the mixture in an oil bath at 40-90 ℃ under the protection condition, magnetically stirring the mixture for reaction for 12-36 h, stopping the reaction, precipitating the reaction liquid into a hydrochloric acid aqueous solution, continuously stirring the mixture, extracting the reaction liquid by using ethyl acetate, combining organic layers, washing the combined organic layers by using a sodium chloride solution, drying the combined organic layers on magnesium sulfate, concentrating the organic layers, and separating and purifying the organic layers by using a silica gel column chromatography to obtain the RXB-SA intermediate.
Example 4 Synthesis of RXB-GEM amphiphilic Small molecule Biprodrugs
RXB intermediate (4.162 mmol), DMAP (0.434 mmol) and GEM (5.54 mmol) are dissolved in ethyl acetate, and 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5.51 mmol) is slowly added under ice-bath conditions, and the mixture is stirred under N 2 Magnetically stirring and reacting for 12-36 h at 4-30 ℃ in a protective atmosphere. And after the reaction is finished, extracting by using ethyl acetate, washing by using a sodium chloride solution, collecting an organic layer, concentrating, further separating and purifying by using a silica gel column chromatography, and drying in vacuum to finally prepare the product RXB-GEM amphiphilic micromolecular double prodrug.
Example 5 Synthesis of CXB-IR amphiphilic Small molecule Dual prodrugs
CXB intermediate (4.145 mmol), DMAP (0.436 mmol) and IR (5.52 mmol) were dissolved in ethyl acetate and 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (5.38 mmol) was added slowly under ice-bath conditions over N 2 Magnetic stirring reaction is carried out for 12 to 36 hours at a temperature of between 4 and 30 ℃ under the protective atmosphere. And after the reaction is finished, extracting by using ethyl acetate, washing by using a sodium chloride solution, collecting an organic layer, concentrating, further separating and purifying by using a silica gel column chromatography, and finally preparing the product CXB-IR amphiphilic micromolecule double prodrug after vacuum drying.
Example 6 preparation of CXB-GEM nanoparticles
Weighing 5mgCXB-GEM amphiphilic micromolecule double prodrug, dissolving in 0.5mL methanol, dropwise adding the mixture into 5mL deionized water under the condition of stirring, stirring at low speed overnight to volatilize the solvent, and carrying out self-assembly to form the nano-particles with uniform size.
The particle size and appearance of the CXB-GEM nanoparticle prepared in example 3 were measured by a dynamic light scattering particle size analyzer and a transmission electron microscope, and the results are shown in fig. 1, which indicates that the CXB-GEM nanoparticle prepared was a spherical particle with a small particle size and a uniform size.
Example 7 preparation of RXB-GEM nanoparticles
Weighing 5mgRXB-GEM amphiphilic micromolecule double prodrug, dissolving in 0.5mL of methanol, dropwise adding the mixture into 5mL of deionized water under the condition of stirring, and stirring at low speed overnight to volatilize the solvent, so that the solvent is self-assembled to form nanoparticles with uniform size.
Example 8 preparation of CXB-IR nanoparticles
Weighing 5mgCXB-IR amphiphilic micromolecule double prodrug, dissolving the amphiphilic micromolecule double prodrug in 0.5mL of methanol, dropwise adding the amphiphilic micromolecule double prodrug into 5mL of deionized water under the stirring condition, stirring at a low speed overnight to volatilize the solvent, and enabling the amphiphilic micromolecule double prodrug to be self-assembled to form the nano-particles with uniform size.
Example 9 in vitro release experiments of CXB-GEM nanoparticles.
The tumor cell lysosomes have a pH near 5.0 and are rich in various hydrolases, which promote the cleavage of ester or amide bonds. Therefore, phosphate buffer solutions with pH7.4 and pH5.0 are used as release media to simulate physiological environment and tumor microenvironment in vitro, and the release condition of the drug in the CXB-GEM nanoparticles is investigated. Placing 2mL of nanoparticle solution in a dialysis bag, placing in 50mLpH7.4 and pH5.0 release media, setting GEM solution and CXB solution as free drug control groups, shaking in a shaker at 37 ℃, sampling at set time points and adding an equal amount of fresh media, and determining the concentration of GEM and CXB by high performance liquid chromatography.
As a result, as shown in fig. 2, in both release media, the free CXB and GEM solutions released the drug rapidly in a shorter time, and less than 10% of the drug was released by the CXB-GEM nanoparticles in the ph7.4 medium compared to the free solution group. The release of the drug in the medium with pH5.0 is improved by 4 times, which shows that the CXB-GEM nanoparticle in vitro release drug has pH correspondence. This is because ester and amide linkages in CXB-GEM nanoparticles are more easily cleaved under acidic conditions to trigger drug release. Compared with free solution for quickly releasing the medicine, the nano-particle has the effect of slow release, and can obviously prolong the action time of the medicine.
Example 10 cytologic toxicity study of CXB-GEM nanoparticles
The toxicity of the CXB-GEM nanoparticles on breast cancer 4T1 cells is examined by adopting an MTT method. Inoculating the breast cancer 4T1 cells into a 96-well cell culture plate, placing the cell culture plate in a cell culture box for overnight incubation, removing old culture solution, and then administering CXB solution, GEM solution, physical mixture solution of CXB and GEM and CXB-GEM nanoparticle solution which are diluted by fresh culture medium and have different concentrations. After 72h incubation, 50. Mu.L of MTT solution at a concentration of 2mg/mL was added to each well for 4h incubation. Old culture medium was aspirated off by pipette gun and 100. Mu.L of LDMSO was added to each well. And measuring the OD value at 490nm by using an enzyme-labeling instrument, and calculating the cell activity.
The results are shown in fig. 3, the CXB solution and the GEM solution both have a certain activity of inhibiting the proliferation of breast cancer 4T1 cells, and the effect of the physical mixture of the two drugs is stronger than that of any single free solution group, which indicates that the combination of the two drugs can kill tumor cells synergistically, and provides a basis for the combination of the two drugs. In addition, compared with a physical mixture, the CXB-GEM nanoparticle has the strongest cell proliferation inhibition capability and presents obvious antitumor advantages.
Example 11 study of CXB-GEM nanoparticles inducing MDSCs apoptosis
MDSCs were sorted from the spleen of 4T1 tumor-bearing mice by flow cytometry sorting as follows. Firstly, 4T1 cells with good growth state are inoculated subcutaneously to BALB/c mice until the tumor volume is 500mm 3 The mice are sacrificed in an ultra-clean bench and the spleen is dissected out, ground and filtered, added with erythrocyte lysate for cracking, centrifuged, cleaned, added with APC-CD11b and PE-Gr-1 antibody for staining, washed by precooled PBS, added with PBS for resuspension, and immediately subjected to cell sorting by a flow cytometer to obtain the MDSCs. MDSCs were seeded into six-well cell culture plates, placed in a cell culture incubator for overnight incubation, the old culture medium was discarded, and CXB solution (3.75 μ g/mL), GEM solution (2.5 μ g/mL), a solution of a physical mixture of CXB and GEM, CXB-GEM nanoparticle solution diluted with fresh medium were administered. After further incubation for 24h, useAnnexin V-FITC/PI apoptosis detection kit staining, and detecting the MDSCs apoptosis induction capability of different drugs by a flow cytometer.
The results are shown in fig. 4, the CXB solution and the GEM solution induce apoptosis of 6.9% and 9.9% of MDSCs cells respectively, compared with the two solutions, the physical mixture of the CXB solution and the GEM solution has stronger apoptosis inducing ability, but the CXB-GEM nanoparticle can induce apoptosis of 44%, and presents the strongest ability of inducing apoptosis of MDSCs cells in vitro, which is beneficial for the CXB-GEM nanoparticle to effectively co-deliver CXB and GEM to MDSCs cells in vivo, and enhances the micro environment of tumor immunosuppression by the combined regulation of the CXB solution and the GEM solution to play a role of synergistic antitumor.
Example 12 in vivo antitumor drug efficacy studies of CXB-GEM nanoparticles.
Female BABL/c mice (18-22 g) were taken and injected subcutaneously with 4T1 cells to establish tumor models. The tumor volume is up to 50mm 3 The method is divided into five groups at random, namely a physiological saline group, a CXB solution, a GEM solution, a physical mixture solution of CXB and GEM, and a CXB-GEM nanoparticle solution. Dosing was performed on days 0, 3, 6, and mouse tumor volume and body weight were recorded. Each group of 2 mice was sacrificed 21 days after drug administration, tumors were removed and photographed, and at the same time, the tumor volume, weight, and survival days of the remaining mice were recorded, giving in vivo antitumor effects of the nanoparticles. When the tumor volume grows to 1000mm 3 Left and right, i.e. considered dead, mice were sacrificed.
The results are shown in fig. 5, and it is seen visually that compared with the normal saline group, both the free drug solution groups can inhibit tumor volume growth, and the inhibition effect of the physical mixture is better, but compared with the physical mixture group, the CXB-GEM nanoparticle has the strongest inhibition capability on tumor volume, the tumor of the treated mice is minimal, and the survival period of the mice is significantly prolonged. The CXB-GEM nanoparticles can efficiently co-deliver two medicines to the tumor part, release the medicines in tumor cells and MDSCs, regulate and control the tumor immunosuppression microenvironment through chemotherapy-immunity combination, and play the best anti-tumor effect. In addition, the weight of the mouse monitored after the drug treatment is not obviously reduced, which shows that the CXB-GEM nano-particle has good in-vivo biological safety and is a nano delivery system capable of effectively treating the breast cancer.
Example 13 study of in vivo inhibition of lung metastasis by CXB-GEM nanoparticles.
Each group of tumor-bearing mice 21 days after administration in effect example 4 was sacrificed 1, lung tissues were taken out and washed with physiological saline, fixed with Bouin's fixative, and photographed after decoloration with absolute ethanol, and the number of metastatic nodules in the lung was observed.
The results are shown in fig. 6, and the lung of the mice after the saline group treatment showed a large number of tumor metastasis nodules, which indicates that the breast cancer has the characteristic of lung metastasis. After different medicines are adopted for treatment, pulmonary nodules are gradually reduced, wherein pulmonary metastasis nodules of mice treated by the CXB-GEM nanoparticles are the least, and the mice show obvious capacity of inhibiting tumor pulmonary metastasis, which is consistent with pharmacodynamic results.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. A micromolecular double prodrug nanoparticle for ensuring effective release and tumor accumulation of antitumor drugs is characterized by comprising the following raw materials: COX-2 inhibitors, anhydrides, and chemotherapeutic drugs; the COX-2 inhibitor is celecoxib and the chemotherapeutic agent is gemcitabine;
the structural formula of the double prodrug is shown as the formula II:
II。
2. the preparation method of the small molecule double prodrug nanoparticles of claim 1, comprising the following steps:
1) Firstly, chemically catalyzing a COX-2 inhibitor and anhydride to obtain an intermediate, and then further reacting a chemotherapeutic drug with the intermediate to obtain an amphiphilic micromolecular double prodrug; wherein, the process of obtaining the intermediate by chemically catalyzing the COX-2 inhibitor and the anhydride comprises the following steps: a COX-2 inhibitor, an acid anhydride, and DMAP are dissolved in an organic solvent 1 at a molar ratio of 1 to 4 2 Placing the mixture in an oil bath at the temperature of 40-90 ℃ under the protection condition, magnetically stirring the mixture for reaction for 12-30 hours to obtain a reaction solution, precipitating the reaction solution into a hydrochloric acid aqueous solution, continuously stirring the mixture, extracting the reaction solution by using an organic solvent 1, combining organic layers, washing the organic layers by using a sodium chloride solution, drying, concentrating, separating and purifying to obtain an intermediate;
2) And further reacting the chemotherapeutic drug with the intermediate to obtain the amphiphilic micromolecular double prodrug: dissolving the intermediate, DMAP and chemotherapeutic drug in an organic solvent 1, slowly adding EDCI under the ice bath condition, and adding N 2 Magnetically stirring and reacting for 12 to 36 hours at the temperature of 4-30 ℃ under protection; after the reaction is finished, extracting by using an organic solvent 1, washing by using a sodium chloride solution, collecting an organic layer, concentrating, separating, purifying and drying in vacuum to prepare a product CXB-GEM amphiphilic micromolecule double prodrug; wherein the molar ratio of the intermediate, DMAP, chemotherapeutic agent and EDCI is 1.05 to 0.2; the organic solvent 1 is any one of anhydrous pyridine, anhydrous tetrahydrofuran, anhydrous N, N-dimethylformamide, anhydrous dioxane, anhydrous dichloromethane and ethyl acetate;
3) The amphiphilic micromolecular double prodrug is self-assembled into nanoparticles under the low-speed magnetic stirring.
3. The preparation method of the small molecule double prodrug nanoparticles for ensuring the effective release and tumor accumulation of the antitumor drug according to claim 2, wherein the specific process of the step 3) is as follows: dissolving the amphiphilic micromolecule double prodrug in an organic solvent 2 to obtain a solution, dropwise adding the solution into the solution A, volatilizing the organic solvent 2 under low-speed magnetic stirring, and self-assembling the amphiphilic micromolecule double prodrug to form CXB-GEM nanoparticles with uniform size; wherein the organic solvent 2 is any one of methanol, ethanol, acetone, tetrahydrofuran, dioxane and pyridine; the solution A is any one of phosphate buffer solution with pH of 7.4, hepes solution and water.
4. The use of small molecule dual prodrug nanoparticles of claim 1 for the preparation of a drug delivery system, wherein the drug is an anti-tumor drug, the tumor comprising: breast, lung and ovarian cancer.
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| CN1346282A (en) * | 1998-12-23 | 2002-04-24 | G.D.西尔公司 | Use of a cyclooxygenase-2 inhibitor and one or more antineoplastic agents as combination therapy method in treating neoplasia |
| CN107441492A (en) * | 2016-05-30 | 2017-12-08 | 复旦大学 | The medical composition and its use of Cyclooxygenase-2 Inhibitor and Nano medication delivery system |
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| CN1346282A (en) * | 1998-12-23 | 2002-04-24 | G.D.西尔公司 | Use of a cyclooxygenase-2 inhibitor and one or more antineoplastic agents as combination therapy method in treating neoplasia |
| CN107441492A (en) * | 2016-05-30 | 2017-12-08 | 复旦大学 | The medical composition and its use of Cyclooxygenase-2 Inhibitor and Nano medication delivery system |
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