CN115634294A - Polymer prodrug released in redox environment response and preparation and application thereof - Google Patents
Polymer prodrug released in redox environment response and preparation and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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Abstract
The invention provides a polymer prodrug released in response to a redox environment, and preparation and application thereof, wherein the polymer prodrug is obtained by connecting telmisartan with heparin sodium through esterification reaction of 2,2' -dithioethanol, and responds to release a telmisartan prototype drug under a redox condition. The nanoparticles formed by the polymer prodrug have stable structure, can form micelles by self-aggregation in an aqueous medium, and have strong passive targeting tumor effect and high in-vivo stability. The polymer prodrug releases telmisartan proto-type medicine in a human body by responding to an oxidation-reduction environment so as to enhance permeation and retention effects, down-regulate alpha-smooth muscle actin, reverse tumor fibroblast phenotype, inhibit tumor matrix barrier, improve the medicine distribution capacity in tumors, increase the medicine concentration of intracellular targets and improve the treatment efficacy of patients.
Description
Technical Field
The invention relates to the field of pharmacy, in particular to a polymer prodrug released by redox environment response and preparation and application thereof.
Background
Tumors are one of the most serious diseases that threaten the quality of life of humans. More than 90% of tumor patients die from metastasis, and the main reason is the low efficiency of conventional treatment means such as surgery, radiotherapy, chemotherapy and the like. Wherein, the tumor tissue hypoxia microenvironment is the key to cause tumor metastasis and low survival rate of patients, and 50-60% of solid tumors have hypoxia areas. Therefore, alleviating tumor hypoxic microenvironments and inhibiting tumor metastasis are becoming the focus of interest to researchers. The clinical efficacy of tumor therapy is still not ideal due to the lack of extensive research on the interaction of various pathological barriers and related mechanisms during tumor development by researchers.
Tumor fibroblasts are a major source of collagen and other matrix secretion in the tumor microenvironment. Telmisartan is an angiotensin receptor inhibitor, and related researches report that Telmisartan has an anti-tumor effect. However, the therapeutic effect of single treatment of telmisartan is not ideal due to the fact that telmisartan is slightly soluble, and telmisartan released by a traditional drug delivery system contains sulfydryl, and the sulfydryl is also a prodrug to a certain extent, so that the spatial combination of telmisartan and AT1R receptors is influenced, and the therapeutic effect of the telmisartan is reduced or even lost. Therefore, there is a need for a polymeric prodrug based on a drug delivery system that responds to release in a redox environment.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a polymer prodrug released in response to a redox environment, and a preparation method and an application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a polymeric prodrug that releases in response to a redox environment, the polymeric prodrug having the following structural formula:
wherein n ranges from 16 to 52.
Further, the polymer prodrug is a polymer prodrug obtained by connecting telmisartan through esterification reaction of 2,2' -dithioethanol with heparin sodium, and the polymer prodrug responds to release of telmisartan proto-drug under redox conditions.
In another aspect, the present invention provides a method for preparing a polymer prodrug released in response to a redox environment, which specifically comprises the following steps:
(1) Telmisartan coupled 2,2' -dithioethanol
Dissolving telmisartan in a mixed solution of dimethyl sulfoxide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine, stirring for reaction, adding bis (2-hydroxyethyl) disulfide, and stirring for 12 hours to obtain a coupling substance of telmisartan and 2,2' -dithioethanol;
(2) Activation of heparin sodium
Dissolving heparin sodium with molecular weight of 8000-25000 in a mixed solution of dimethyl sulfoxide and water, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine into the mixed solution, and stirring for reaction to obtain activated heparin sodium;
(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug
And (2) mixing a coupling compound of telmisartan and 2,2' -dithioethanol with activated heparin sodium to react for 24 hours to obtain a reaction liquid, dialyzing the reaction liquid in pure water for 3 days, and freeze-drying to obtain the polymer prodrug.
Further, in the step (1), the molar ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the 4-dimethylaminopyridine to the telmisartan is as follows: 5-8.
Further, in step (1), the molar ratio of bis (2-hydroxyethyl) disulfide to telmisartan is 1.
Further, in the step (2), the volume ratio of dimethyl sulfoxide to water is 8-9.
Further, in the step (2), the molar ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide to the 4-dimethylaminopyridine to the heparin sodium is as follows: 5-8.
Further, in the steps (1) and (2), the reaction conditions of the stirring reaction are stirring for 1 hour under a nitrogen atmosphere at 60 ℃.
In another aspect, the polymer prodrug released by redox response prepared by the method is used for preparing a medicament for treating tumors, and the polymer prodrug realizes redox response release of telmisartan interfering with tumor-related fibroblasts in a tumor microenvironment, so that a tumor matrix barrier is damaged, and intratumor permeability is increased.
The invention has the beneficial effects that:
the invention provides a polymer prodrug which responds to a redox environment in cytoplasm to intervene in a tumor pathological barrier in a tumor microenvironment. The invention integrates two functions of reversing the phenotype of tumor fibroblasts and anticoagulation on nanoparticles formed by the same polymer prodrug in a chemical synthesis mode. The polymer prodrug is composed of three parts, wherein the hydrophobic part is a telmisartan pharmacological action structure, the hydrophilic part is a heparin sodium pharmacological action structure, and the hydrophilic part and the hydrophobic part are connected through a chemical bond responding to a redox environment.
The nanoparticles formed by the polymer prodrug have stable structure, can form micelles by self-aggregation in an aqueous medium, and have strong passive targeting tumor effect and high in-vivo stability. The polymer prodrug releases telmisartan proto-type medicine in a human body by responding to an oxidation-reduction environment so as to enhance permeation and retention effects, down-regulate alpha-smooth muscle actin, reverse tumor fibroblast phenotype, inhibit tumor matrix barrier, improve the medicine distribution capacity in tumors, increase the medicine concentration of intracellular targets and improve the treatment efficacy of patients.
Drawings
FIG. 1 is a synthetic process scheme of the present invention;
figure 2 shows the survival of activated mouse fibroblasts following 48 hours treatment with different concentrations of telmisartan and polymeric prodrug;
figure 3 shows the expression levels of α -smooth muscle actin by activated mouse fibroblasts following treatment with different concentrations of telmisartan and polymeric prodrug;
FIG. 4 is a graph showing the response of telmisartan released from a polymer prodrug to glutathione as measured by high performance liquid chromatography.
Detailed Description
Example 1
A polymeric prodrug released in response to a redox environment, as shown in figure 1, prepared by the following method:
(1) Telmisartan coupled 2,2' -dithioethanol
Dissolving 80mg of telmisartan, 148mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 8mg of 4-dimethylaminopyridine in 3mL of dimethyl sulfoxide, stirring for 1 hour at 60 ℃ under a nitrogen atmosphere, adding 57mg of bis (2-hydroxyethyl) disulfide, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2' -dithioethanol;
(2) Activation of heparin sodium
Dissolving 160mg of heparin sodium in 10mL of a mixed solution of dimethyl sulfoxide and water (volume ratio is 8;
(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug
And (2) mixing a coupling compound of telmisartan and 2,2' -dithioethanol with activated heparin sodium to react for 24 hours to obtain a reaction liquid, dialyzing the reaction liquid in pure water for 3 days, and freeze-drying to obtain the polymer prodrug.
The invention evaluates the micelle cell survival of the polymer prodrug by the tumor-related fibroblast inhibition rateAnd (4) rate. The test is measured by tetrazolium salt colorimetry. Using mouse breast cancer 4T1 cell activated mouse fibroblast NIH/3T3 cell as model, adding 200 μ L of 2 × 10-containing solution into each well of 96-well cell culture plate 3 Culture medium of activated NIH/3T3 cells, at 37 ℃ and 5% CO 2 Incubating in an incubator for 12 hours, after the cells are completely attached to the wall, respectively adding telmisartan and polymer prodrug (experimental group) with different concentrations into the cell holes of a 96-hole cell culture plate, and setting a plurality of holes in each hole by taking untreated blank cells as a reference; after 48 hours of incubation, 20. Mu.L of thiazolium blue solution with the concentration of 5mg/mL is added into each hole, the incubation is continued for 4 hours, then the supernatant is discarded, 200. Mu.L of dimethyl sulfoxide is added into each hole, the absorbance at 570nm is measured by an enzyme-linked detector, and the cell survival rate is calculated according to the following formula:
cell survival rate (%) = absorbance in experimental group/absorbance in control group × 100%
Toxicity of telmisartan and the polymeric prodrug on activated mouse fibroblasts is shown in fig. 2, and it can be seen from the figure that the survival rate of the fibroblasts treated with the polymeric prodrug is lower than that of the fibroblasts treated with telmisartan, indicating that the polymeric prodrug has stronger drug effect on the mouse fibroblasts than telmisartan.
Example 2
A polymeric prodrug released in response to a redox environment, as shown in figure 1, prepared by the following method:
(1) Telmisartan coupled 2,2' -dithioethanol
Dissolving 100mg of telmisartan, 190mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 12mg of 4-dimethylaminopyridine in 4mL of dimethyl sulfoxide, stirring for 1 hour at 60 ℃ under a nitrogen atmosphere, adding 67mg of bis (2-hydroxyethyl) disulfide, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2' -dithioethanol;
(2) Activation of heparin sodium
Dissolving 200mg of heparin sodium in 16mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8;
(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug
The coupling compound of telmisartan and 2,2' -dithioethanol and activated heparin sodium are mixed and reacted for 24 hours to obtain reaction liquid, the reaction liquid is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze drying.
The invention evaluates the phenotypic ability of the polymer prodrug micelle to reverse tumor fibroblasts according to the expression level of alpha-smooth muscle actin in the tumor-associated fibroblasts. The test adopts Western blotting method, uses mouse fibroblast NIH/3T3 cell activated by mouse breast cancer 4T1 cell as model, adds 2.5mL of cell culture plate containing 2X 10 5 Culture medium of activated NIH/3T3 cells, at 37 ℃ and 5% CO 2 Incubating for 12 hours, after the cells are completely attached to the wall, adding the polymer pro-drugs with different concentrations into the cell holes of a 6-hole cell culture plate, incubating for 48 hours, removing the supernatant, washing for 2 times by using phosphate buffer solution, putting the 6-hole cell culture plate on ice, adding 100 microliter of cell complete lysate into each hole to fully lyse the cells, centrifuging and precipitating at 4 ℃, 12000rpm for 20 minutes, measuring the protein concentration and discarding the quantitative calculation of the sample addition amount, performing high-temperature water bath for 10 minutes (100 ℃) on the protein, performing electrophoresis, sealing and incubating a primary antibody and exposing, and the result is shown in figure 3. The results in fig. 3 indicate that the polymeric prodrugs affect the expression level of alpha-smooth muscle actin in tumor-associated fibroblasts, and that the expression level correlates with drug concentration.
Example 3
A polymeric prodrug released in response to a redox environment, as shown in figure 1, prepared by the following method:
(1) Telmisartan coupled 2,2' -dithioethanol
Dissolving 160mg of telmisartan, 296mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 16mg of 4-dimethylaminopyridine in 6mL of dimethyl sulfoxide, stirring at 60 ℃ for 1 hour under a nitrogen atmosphere, adding 114mg of bis (2-hydroxyethyl) disulfide, and stirring for 12 hours to obtain a coupling product of telmisartan and 2,2' -dithioethanol;
(2) Activation of heparin sodium
Dissolving 320mg of heparin sodium in 20mL of a mixed solution of dimethyl sulfoxide and water (volume ratio 8;
(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug
The coupling compound of telmisartan and 2,2' -dithioethanol and activated heparin sodium are mixed and reacted for 24 hours to obtain reaction liquid, the reaction liquid is dialyzed in pure water for 3 days, and the polymer prodrug is obtained after freeze drying.
The release profile of telmisartan from the polymeric prodrug was studied using dialysis. Equal amounts of telmisartan and the polymer prodrug are filled into a dialysis bag, then dialyzed with 25mL of phosphate buffered saline or 25mL of phosphate buffered saline and glutathione with different concentrations for 72 hours, dialysate is extracted at a predetermined time and new phosphate buffered saline is added, and the reaction spectrum of the telmisartan released from the polymer prodrug on the glutathione is determined by high performance liquid chromatography, and the result is shown in FIG. 4. The results in fig. 4 show that the polymer prodrug has the same peak value in a glutathione environment as telmisartan at 28-30, and the polymer prodrug can release a telmisartan prototype drug in the glutathione environment.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention may be apparent to those skilled in the relevant art and are intended to be within the scope of the present invention.
Claims (9)
1. A polymeric prodrug releasing in response to a redox environment, wherein the polymeric prodrug has the following structural formula:
wherein n ranges from 16 to 52.
2. A redox environment-responsive release polymer prodrug according to claim 1, wherein the polymer prodrug is a polymer prodrug obtained by linking telmisartan to heparin sodium by esterification with 2,2' -dithioethanol, which polymer prodrug is responsive to release of telmisartan proto-drug under redox conditions.
3. A method for preparing a polymeric prodrug for redox environment responsive release comprising the steps of:
(1) Telmisartan coupled 2,2' -dithioethanol
Dissolving telmisartan in a mixed solution of dimethyl sulfoxide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine, stirring for reaction, adding bis (2-hydroxyethyl) disulfide, and stirring for 12 hours to obtain a coupling substance of telmisartan and 2,2' -dithioethanol;
(2) Activation of heparin sodium
Dissolving heparin sodium in a mixed solution of dimethyl sulfoxide and water, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine into the mixed solution, and stirring for reaction to obtain activated heparin sodium;
(3) Synthesis of heparin sodium coupled telmisartan polymer prodrug
And (2) mixing a coupling compound of telmisartan and 2,2' -dithioethanol with activated heparin sodium to react for 24 hours to obtain a reaction liquid, dialyzing the reaction liquid in pure water for 3 days, and freeze-drying to obtain the polymer prodrug.
4. The method of claim 3, wherein the polymeric prodrug is released in response to a redox environment,
in the step (1), the molar ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide to 4-dimethylaminopyridine to telmisartan is as follows: 5-8.
5. The method of claim 3, wherein the polymeric prodrug is released in response to a redox environment,
in the step (1), the molar ratio of bis (2-hydroxyethyl) disulfide to telmisartan is 1.
6. A method of preparing a redox environment responsive release polymer prodrug of claim 3,
in the step (2), the volume ratio of dimethyl sulfoxide to water is 8-9.
7. A method of preparing a redox environment responsive release polymer prodrug of claim 3,
in the step (2), the molar ratio of 1-ethyl- (3-dimethylaminopropyl) carbodiimide to 4-dimethylaminopyridine to heparin sodium is as follows: 5-8.
8. A method of preparing a redox environment responsive release polymer prodrug of claim 3,
in the steps (1) and (2), the reaction condition of the stirring reaction is that the stirring is carried out for 1 hour under the nitrogen atmosphere at the temperature of 60 ℃.
9. Use of a redox environment responsive release polymeric prodrug as claimed in any one of claims 1 to 2 in the manufacture of a medicament for the treatment of tumours.
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| CN202211333409.6A CN115634294A (en) | 2022-10-28 | 2022-10-28 | Polymer prodrug released in redox environment response and preparation and application thereof |
| PCT/CN2022/133438 WO2024087273A1 (en) | 2022-10-28 | 2022-11-22 | Polymer prodrug releasable in response to redox environment, and preparation and use thereof |
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