CN111150702B - Gel drug sustained release preparation and preparation method and application thereof - Google Patents
Gel drug sustained release preparation and preparation method and application thereof Download PDFInfo
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- CN111150702B CN111150702B CN201911228063.1A CN201911228063A CN111150702B CN 111150702 B CN111150702 B CN 111150702B CN 201911228063 A CN201911228063 A CN 201911228063A CN 111150702 B CN111150702 B CN 111150702B
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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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
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- A—HUMAN NECESSITIES
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention discloses a gel drug sustained-release preparation, a preparation method and application thereof, belonging to the field of medicines. The gel drug sustained-release preparation disclosed by the invention mainly comprises a gel carrier material, an effective amount of a drug and a solvent, can realize the slow release of a colchicine drug, has the property of thermal gelation, is in a solution state at room temperature and is converted into a gel state at 4-37 ℃, and can be conveniently administered by an injection mode. In addition, after in-situ gelling in vivo, the gel drug sustained-release preparation can release the drug in situ so as to carry out local administration, reduce the toxic and side effects of the whole body, and obtain long-acting anti-inflammation and anti-fibrosis effects through slow release, thereby being suitable for popularization and application in the market.
Description
Technical Field
The invention belongs to the field of medicines, relates to a gel drug sustained-release preparation, a preparation method and application thereof, and particularly relates to a long-acting sustained-release preparation for treating myocardial infarction and a preparation method thereof.
Technical Field
Myocardial infarction is caused by myocardial ischemia and hypoxia necrosis caused by coronary artery blood flow interruption, can cause heart failure, cardiogenic shock or cardiac arrest, and is one of the leading causes of death of diseases in the world.
In addition to myocardial damage caused by ischemia and hypoxia, local excessive inflammatory response of myocardial infarction is an important factor for deterioration of cardiac structure and function. Inflammatory responses can promote apoptosis of cardiomyocytes and accelerate synthesis of extracellular matrix, leading to myocardial fibrosis, myocardial remodeling, and cardiac dysfunction. A new anti-inflammatory treatment scheme is explored to reduce local inflammation after acute myocardial infarction, so that the method is helpful for reducing myocardial damage after myocardial infarction and improving myocardial repair, thereby inhibiting myocardial remodeling and improving patient prognosis.
Colchicine is an alkaloid extracted from colchicine of Liliaceae, and has effects in inhibiting mitosis, destroying spindle body, stopping chromosome in metaphase, and inhibiting fibroblast proliferation; meanwhile, lysosome degranulation can be interfered, the activity of neutrophils is reduced, and the migration of the granulocytes to an inflammation area is inhibited, so that the anti-inflammatory effect is realized. The traditional Chinese medicine composition is usually used for treating gout (anti-inflammatory) and tumor (anti-mitosis) clinically, but the traditional Chinese medicine composition is only used for short-term treatment because the application window is narrow and the toxic and side effects of the whole body are easily caused.
Recently, the colchicine is found to reduce local excessive inflammatory reaction of myocardial infarction and inhibit myocardial fibrosis, thereby playing a role in treating myocardial infarction. However, how to effectively use colchicine to treat myocardial infarction without causing obvious systemic toxic and side effects still remains a great challenge in clinic.
Disclosure of Invention
In order to solve the problems that the colchicine has narrow administration window and is easy to cause systemic toxicity, the invention prepares a long-acting sustained-release preparation for treating myocardial infarction by loading colchicine medicine into PEG/polyester block copolymer thermal hydrogel. Local in situ injection is carried out after myocardial infarction (under ultrasonic guidance) to play the roles of inhibiting local over-inflammatory reaction and resisting myocardial fibrosis. The colchicine medicine can be loaded into the polymer water solution at low temperature, and forms gel in situ after entering into the body, and realizes stable and slow release under the diffusion action of the medicine, thereby realizing the in-situ delivery and local continuous and stable administration of the colchicine medicine.
In order to achieve the purpose, the invention adopts the following technical scheme:
a gel drug sustained release preparation is composed of a gel carrier, a block copolymer, a colchicine and a solvent, wherein the block copolymer is composed of polyethylene glycol as a hydrophilic block and polyester as a hydrophobic block, the colchicine is used as a carried drug, and the solvent is composed of the following components in percentage by weight:
5-40 wt%, preferably 15-30 wt% of polyethylene glycol-polyester block copolymer;
0.25-10mg/mL of colchicine;
the balance being solvent.
Under each concentration, the release kinetics of the colchicine medicine is not influenced by the medicine-loading rate, the toxic and side effects in the release period are only related to the total dose, and the effective medicine-loading rate of the colchicine medicine is 0.5-2.5mg/kg by taking a mouse as a model; according to the equivalent dose ratio between human and animal according to the conversion of body surface area, the colchicine is popularized from 20g of mice to 70kg of adults, and the effective drug-loading rate of the colchicine is 0.055-0.275 mg/kg.
The invention discloses a gel carrier material-amphiphilic block copolymer which comprises the following components:
(1) the polyethylene glycol has an average molecular weight of 600 to 20000 and a content of 10 to 90 wt.%, preferably 25 to 50 wt.%, and is designated as polymer A block;
(2) the polyester content is from 10 to 90% by weight, preferably from 50 to 75% by weight, and is designated as B polymer block;
(3) the B block is one of poly (DL-lactic acid-glycolic acid) PLGA, poly (L-lactic acid-glycolic acid) PLLGA, poly (D-lactic acid-glycolic acid) PDLGA, poly (DL-lactic acid) PLA, poly (L-lactic acid) PLLA and poly (D-lactic acid) PDLA;
(4) the block copolymers may be triblock copolymers of the ABA or BAB type, diblock copolymers of the AB type, graft copolymers of the A-g-B or B-g-A type, radial block copolymers of (A-B) n or (B-A) n, and multiblock copolymers of the A (BA) n or B (AB) n configuration, where n is an integer from 2 to 10.
Preferably, the gel drug sustained-release preparation has injectability, is in a solution state at low temperature, can be transformed into a gel state within 1 minute at 4-37 ℃, and has a preferred gel transformation temperature of 25-37 ℃.
Preferably, the solvent is pure water, physiological saline, buffer solution, tissue culture solution, cell culture solution, body fluid of animals, plants or human bodies, or other aqueous solution or other solvent medium without organic solvent as main body.
Preferably, the gel drug sustained-release preparation can also be added with a regulator, and the weight percentage of the regulator in the sustained-release preparation is 0.01-15 wt%; and the regulator is selected from one or more of sugar, salt, sodium carboxymethylcellulose, (iodine) glycerol, simethicone, propylene glycol, carbomer, mannitol, sorbitol, surfactant, tween 20, tween 40, tween 80, xylitol, oligosaccharide, chondroitin, chitin, chitosan, collagen, gelatin, protein gel, hyaluronic acid and polyethylene glycol.
In addition, the invention also discloses a preparation method for protecting the gel drug sustained-release preparation, and the preparation method of the gel drug sustained-release preparation is selected from one of the following methods:
(1) preparing a block copolymer aqueous solution, adding a medicament, dissolving uniformly to form a gel sustained-release preparation, storing at-20 ℃ or below for later use, and redissolving and injecting in vivo before use;
(2) respectively preparing a block copolymer aqueous solution and a medicine injection, separately subpackaging and storing, and fully and uniformly mixing the block copolymer aqueous solution and the medicine injection before injection to prepare a gel sustained-release preparation;
(3) preparing the medicinal injection, mixing with the block copolymer, dissolving to obtain gel sustained release preparation, storing at-20 deg.C or below, and re-dissolving and injecting in vivo before use;
(4) mixing the block copolymer with the medicine, adding a solvent, and dissolving uniformly to obtain a gel sustained-release preparation; storing at-20 deg.C or below for use, and re-dissolving and injecting in vivo before use.
It should be noted that the four preparation methods have no obvious difference in preparation effect.
Exemplarily, the preparation method of the gel drug sustained-release preparation disclosed by the invention specifically comprises the following steps:
dissolving the gel carrier material in the solvent at low temperature, storing at the temperature below-20 ℃ for later use, re-dissolving the gel carrier material dissolved in the solvent at room temperature before use, adding the colchicine medicine, and uniformly mixing to obtain the gel sustained-release preparation.
Preferably, the low temperature dissolution temperature is not higher than the sol-gel transition temperature of the gel support material.
The invention also discloses an application of the gel drug sustained release preparation or the gel drug sustained release preparation prepared by the preparation method in preparing a drug for treating myocardial infarction.
Preferably, the effective dosage range of the colchicine in the gel drug sustained-release preparation is 0.5-2.5mg/kg by taking a mouse as a model.
Preferably, the effective dose range of the colchicine in the gel drug sustained-release preparation is 0.055 to 0.275mg/kg according to the equivalent dose ratio converted by body surface area between human and animal and is popularized from 20g mice to 70kg adults.
Preferably, the gel drug sustained-release preparation has a thermogelation property, and is in a solution state at room temperature and is transformed into a gel state at 4-37 ℃.
According to the technical scheme, compared with the prior art, the gel drug sustained-release preparation, the preparation method and the application thereof provided by the invention have the following excellent effects:
the gel sustained-release preparation prepared by the invention can realize the slow release of colchicine drugs, has the property of thermal gelation, is in a solution state at room temperature and is converted into a gel state at 4-37 ℃, and can be conveniently administrated in an injection mode; after the preparation is gelatinized in situ in vivo, the preparation can release the medicine in situ so as to carry out local administration, reduce the toxic and side effect of the whole body, and obtain long-acting anti-inflammatory and anti-fibrosis effects by slow release.
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, and 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 phase diagram of copolymer-1 in example 8 of the present invention.
FIG. 2 is a temperature swing dynamic rheology curve of a solution of biopolymer-1 (25 wt%) in example 9 of this invention.
FIG. 3 is a graph of the in vitro release profile of a copolymerer-1 (25 wt%) gel formulation of colchicine drug at a loading concentration of 1mg/mL in example 15 of the present invention.
FIG. 4 is a graph showing the myocardial inflammatory response of mice in each group after 4 weeks from myocardial infarction in example 26 of the present invention, in which FIG. 4(A) is an immunofluorescent stain of inflammatory cells in myocardial sections of each group, and FIG. 4(B) is a statistical analysis of the number of inflammatory cells in myocardial sections of each group.
Fig. 5 shows the heart structure and function of each group of mice after 4 weeks from the myocardial infarction in example 26 of the present invention, wherein fig. 5(a) is a typical image of cardiac ultrasound, and fig. 5(B) is parameter statistics of each group of cardiac ultrasound.
FIG. 6 is the evaluation of toxicity of the colchicine system in example 26 of the present invention, in which FIG. 6(A) is a graph of body weight change and FIG. 6(B) is a graph of liver function status.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying 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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.
Example 1
15g of bishydroxypolyethylene glycol (PEG1500) were weighed into a 250mL three-necked flask, and after sealing the ports with vacuum ester, the water was removed under mechanical stirring at 130 ℃ for 3h under vacuum. Then introducing argon to cool to 80 ℃, adding 37g of D, L-lactide and glycolide with the molar ratio of 1:1 in total, and fully stirring; adding a toluene solution containing 50mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. And then, cooling to 120 ℃, vacuumizing for 3h to remove unreacted monomers, pouring out the product while the product is hot, washing the crude product for 3 times by using deionized water at 80 ℃, and freeze-drying the product to obtain a final product, namely the BAB type triblock polymer PLGA-PEG-PLGA (copolymer-1), wherein the yield is about 85%. The number average and weight average molecular weights (M) of the above-mentioned BAB type triblock polymers were determined by gel permeation chromatography (GPC, polystyrene as standard)n，Mw) 5200 and 6120, respectively, molecular weight distribution coefficient (M)w/Mn，
) 1.30, and the polymer water system has thermal gelation property.
Example 2
10g of bishydroxypolyethylene glycol (PEG1000) was weighed into a 250mL three-necked flask, and after sealing the ports with vacuum ester, the water was removed under mechanical stirring at 130 ℃ for 3h under vacuum. Then introducing argon to cool to 80 ℃, adding 25g of D, L-lactide, and fully stirring; adding a toluene solution containing 50mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. Then cooling to 120 ℃, vacuumizing for 3h to remove unreacted monomers, pouring out the mixture while the mixture is hot, washing the crude product for 3 times by using deionized water at 80 ℃,after freeze drying, the final product is obtained, and the BAB type triblock polymer PLA-PEG-PLA (copolymer-2) is obtained with the yield of about 80 percent. The number average and weight average molecular weights (M) of the above-mentioned BAB type triblock polymers were determined by gel permeation chromatography (GPC, polystyrene as standard)n，Mw) 4750 and 6120, respectively, molecular weight distribution coefficient (M)w/Mn，
) 1.14, the polymer water system has thermal gelation properties.
Example 3
15g of monomethoxypolyethylene glycol (mPEG750) were weighed into a 250mL three-necked flask, and after sealing each port with vacuum ester, water was removed in vacuo at 130 ℃ for 3h with mechanical stirring. Then introducing argon to cool to 80 ℃, adding 30g of D, L-lactide and 10g of glycolide, and fully stirring; adding a toluene solution containing 40mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. And then, cooling to 120 ℃, vacuumizing for 3h to remove unreacted monomers, pouring out the product while the product is hot, washing the crude product for 3 times by using deionized water at 80 ℃, and freeze-drying to obtain a final product, thus obtaining the AB type block polymer mPEG-PLGA (copolymer-3) with the yield of about 79%. The number average and weight average molecular weights (M) of the above AB type block polymers were determined by gel permeation chromatography (GPC, polystyrene as a standard)n，Mw) 2850 and 3300, respectively, molecular weight distribution coefficient (M)w/Mn，
) 1.24, the polymer water system has thermal gelation properties.
Example 4
20g of bishydroxypolyethylene glycol (PEG1000) were weighed into a 250mL three-necked flask, and after sealing the ports with vacuum ester, the water was removed under mechanical stirring at 130 ℃ for 3h under vacuum. Then introducing argon to cool to 80 ℃, adding 30g of D-lactide and 8g of glycolide, and fully stirring; adding a toluene solution containing 60mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. Then cooling to 120 deg.CVacuum for 3h to remove unreacted monomer, pouring out while hot, washing the crude product with deionized water at 80 ℃ for 3 times, and freeze-drying to obtain the final product, namely BAB type triblock polymer PDLGA-PEG-PDLGA (copolymer-4), with the yield of about 83%. The number average and weight average molecular weights (M) of the above-mentioned BAB type triblock polymers were determined by gel permeation chromatography (GPC, polystyrene as standard)n，Mw) 5150 and 5960, respectively, molecular weight distribution coefficient (M)w/Mn，
) 1.17, the polymer water system has thermal gelation properties.
Example 5
20g of bishydroxypolyethylene glycol (PEG1000) were weighed into a 250mL three-necked flask, and after sealing the ports with vacuum ester, the water was removed under mechanical stirring at 130 ℃ for 3h under vacuum. Then introducing argon to cool to 80 ℃, adding 25g of L-lactide and glycolide with a molar ratio of 3:1, and fully stirring; adding a toluene solution containing 40mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. Then cooling to 120 ℃, vacuumizing for 3h to remove unreacted monomers, pouring out the product while the product is hot, washing the crude product for 3 times by using deionized water at 80 ℃, and freeze-drying to obtain a final product, namely BAB type triblock polymer PLLGA-PEG-PLLGA (copolymer-5), wherein the yield is about 80%. The number average and weight average molecular weights (M) of the above-mentioned BAB type triblock polymers were determined by gel permeation chromatography (GPC, polystyrene as standard)n，Mw) 4500 and 5850, respectively, molecular weight distribution coefficient (M)w/Mn，
) 1.25, the polymer water system has thermal gelation properties.
Example 6
11g of monomethoxypolyethylene glycol (mPEG550) were weighed into a 250mL three-necked flask, and after sealing each port with vacuum ester, water was removed in vacuo at 130 ℃ for 3h with mechanical stirring. Then introducing argon to cool to 80 ℃, adding 25g of DL-lactide and glycolide with a molar ratio of 20:1 in total, and fully stirring; adding a toluene solution containing 60mg of stannous octoate, repeatedly vacuumizing for 3 times to remove toluene, heating to 150 ℃, and reacting for 12 hours under the protection of argon. The temperature is then reduced to 120 ℃, vacuum is applied for 3h to remove unreacted monomers, the crude product is dissolved in dichloromethane solution, and cold ether is used for precipitation, and the yield is about 85%.
Dissolving the two-block copolymer in anhydrous toluene, adding equivalent HDMI, carrying out reflux reaction for 8h at 60 ℃, carrying out rotary evaporation and concentration, adding into a large amount of anhydrous ether, freezing in a refrigerator at-20 ℃ overnight for precipitation, filtering to remove impurities, and carrying out vacuum drying for 48h to obtain mPEG-PLGA-mPEG. The number average and weight average molecular weights of the ABA block copolymer (mPEG-PLGA-mPEG, Polymer-17) were 5150 and 6270, respectively, as determined by Gel Permeation Chromatography (GPC) (using polystyrene as a standard and THF as a mobile phase), and the molecular weight distribution coefficient (M)w/Mn，
) Is 1.25. The copolymer (copolymer-6) itself has reversible, thermogelling properties in water.
Example 7
Following the basic procedure given in examples 1 to 6, other block copolymers were synthesized with different molecular weight PEGs or mPEG with different monomers, the properties of which are shown in table 1:
TABLE 1
The block polymers in the above tables all have thermotropic gelling properties. The polymer is prepared into a water solution with a certain concentration, the water solution is in a solution state when the temperature is lower than the gel transition temperature, the water solution is transformed into semisolid gel along with the temperature rise, and the process is reversible.
The block polymers in Table 1 are formulated in proportions to dissolve in water at temperatures below the gel transition temperature and when the temperature is above the gel transition temperature, a solution of the polymer mixture forms a gel.
Example 8
Weighing a proper amount of copolymer-1 in example 1, preparing a series of solutions with the polymer mass percentage content of 10-25 wt% by taking deionized water as a solvent, performing a reverse tube method test, specifically immersing a 2mL sample bottle carrying 0.5mL sample solution in a water bath, immediately inverting for 180 ℃ after balancing for 15min, and if the sample does not flow obviously within 30s, determining that the sample is in a gel state, wherein the temperature is the sol-gel transition temperature. The results of the tube inversion test are shown in FIG. 1, and as shown in FIG. 1, the solution of copolymer-1 can undergo sol-gel phase transition at the temperature range of 0-50 ℃ to be tested, so that it has the property of thermal gelation.
Example 9
A proper amount of 25 wt% copolymer-1 block copolymer solution is taken, and a rotational rheometer is adopted to measure the change of rheological properties such as storage modulus, loss modulus and the like of a polymer water system along with the temperature. The temperature was swept at a shear frequency of 1.59Hz at a heating rate of 1 c/min, and the results are recorded in fig. 2. As shown in FIG. 2, a 25 wt% solution of copolymer-1 polymer has a cross-point between the storage modulus and the loss modulus at 35 ℃ at the test temperature, and can undergo a sol-gel phase transition, thereby exhibiting thermal gelation properties.
Example 10
Weighing a proper amount of block copolymer-7 in the table 1, taking deionized water as a solvent, preparing a series of solutions with the polymer mass percentage content of 10-25 wt%, and performing a pipe inversion method test. The result of the tube inversion method test shows that the obtained copolymer-7 solution can generate sol-gel phase transition along with the temperature rise, and the phase transition temperature can be controlled within the temperature range of 33-39 ℃ according to the difference of concentration, so that the copolymer-7 has the property of thermal gelation.
Example 11
An appropriate amount of the block copolymer-12 of table 1 was weighed, and a 23 wt% aqueous solution of copolymer-12 was prepared using a PBS (pH 7.4) solution as a solvent. The pour tube test shows that 23 wt% solution of the copolymer-12 has sol-gel phase transition with the temperature rise, the phase transition temperature is 32 ℃, and the temperature is just between the room temperature and the physiological temperature, so the copolymer-12 has the property of thermal gelation.
Example 12
With reference to the experimental methods described in examples 8 to 11, the polymers in Table 1 were also dissolved in water at a suitable concentration and at a low temperature, and the sol-gel phase transition occurred with increasing temperature, and thus all had reversible thermal gelation properties.
Example 13
When the aqueous solution of the block copolymer prepared in example 9 was added with 0.025-0.1% by weight of colchicine drug, it was found that it had no effect on both the sol-gel phase transition temperature and injectability.
Example 14
The block copolymer-1 is prepared into a polymer solution with the weight percent of 25 by taking physiological saline as a solvent, and is filtered and sterilized. About 0.1mL of the solution was taken and the solution was injected subcutaneously into the back of a mouse under anesthesia using a C57BL/6 mouse as a model experimental animal. Mice were sacrificed at regular intervals and the degradation of the gel in the mice was followed. The results show that the injectable material remained in the body for four weeks, with no visible gel after the fifth week. Meanwhile, the injection part does not have the phenomena of edema, tissue necrosis and the like in the experimental process.
Example 15
Adding a proper amount of physiological saline into the block copolymer-1, stirring and dissolving the mixture in a refrigerator at 4 ℃ to prepare an aqueous solution with the weight percentage of the polymer of 25 wt%, then adding 0.25,0.5 and 1mg/mL colchicine medicaments, and respectively stirring the mixture uniformly to obtain drug-loaded polymer solutions with three concentrations. The test of a tube inversion method shows that the obtained solution can generate sol-gel transition at physiological temperature, namely, the solution is in a flowable solution state at the temperature lower than the physiological temperature, and when the temperature is raised to the physiological temperature, the sol-gel phase transition is generated to form in-situ gel. In vitro drug release experiments with three drug loading concentrations show that colchicine drug can be slowly released from the gel for more than 8 days, the release kinetics of colchicine in hydrogel is not affected by the drug loading amount, and the results are recorded in fig. 3.
Example 16
The copolymer-7 in the table 1 is added with a proper amount of normal saline, stirred and dissolved in a refrigerator at 4 ℃ to prepare an aqueous solution with the polymer weight percentage of 20 wt%, then 2mg/mL colchicine medicine is added, and the mixture is stirred uniformly to obtain a medicine-carrying polymer solution. The test of a tube inversion method shows that the obtained solution can generate sol-gel transition at physiological temperature, namely, the solution is in a flowable solution state at the temperature lower than the physiological temperature, and when the temperature is raised to the physiological temperature, the sol-gel phase transition is generated to form in-situ gel. In vitro drug release experiments showed that colchicine drug can be slowly released from the gel for more than 3 weeks.
Example 17
The copolymer-8 in the table 1 is added with a proper amount of normal saline, stirred and dissolved in a refrigerator at 4 ℃ to prepare an aqueous solution with the polymer weight percentage of 20 wt%, then 0.25mg/mL colchicine medicine is added, and the mixture is stirred uniformly to obtain a medicine-carrying polymer solution. The test of a tube inversion method shows that the obtained solution can generate sol-gel transition at physiological temperature, namely, the solution is in a flowable solution state at the temperature lower than the physiological temperature, and when the temperature is raised to the physiological temperature, the sol-gel phase transition is generated to form in-situ gel. In vitro drug release experiments showed that colchicine drug can be slowly released from the gel for more than 1 week.
Example 18
The copolymer-9 in Table 1 was dissolved in normal saline with stirring in a refrigerator at 4 ℃ to give an aqueous solution with a polymer weight of 23 wt%, followed by addition of 0.25mg/mL colchicine and stirring to obtain a drug-loaded polymer solution. The test of a tube inversion method shows that the obtained solution can generate sol-gel transition at physiological temperature, namely, the solution is in a flowable solution state at the temperature lower than the physiological temperature, and when the temperature is raised to the physiological temperature, the sol-gel phase transition is generated to form in-situ gel. In vitro drug release experiments showed that colchicine drug can be slowly released from the gel for more than 3 weeks.
Example 19
The copolymer-11 in Table 1 was dissolved in normal saline with stirring in a refrigerator at 4 ℃ to prepare an aqueous solution with a polymer weight percentage of 25 wt%, then 0.25mg/mL colchicine drug was added and stirred uniformly to obtain a drug-loaded polymer solution. The test of a tube inversion method shows that the obtained solution can generate sol-gel transition at physiological temperature, namely, the solution is in a flowable solution state at the temperature lower than the physiological temperature, and when the temperature is raised to the physiological temperature, the sol-gel phase transition is generated to form in-situ gel. In vitro drug release experiments showed that colchicine drug can be slowly released from the gel for more than 1 week.
Example 20
The drug-loaded gel sustained-release preparation in example 16 is added with 1% of sucrose regulator to regulate the drug release rate, so that the in-vitro 2-week release of the drug can be realized.
Example 21
The drug-loaded gel sustained-release preparation in example 18 is added with 5% of sucrose regulator to regulate the drug release rate, so that the in vitro 2-week release of the drug can be realized.
Example 22
The drug-loaded gel sustained-release preparation in example 16 is added with 5% of polyethylene glycol 400 to adjust the drug release rate, so that the in-vitro 2-week release of the drug can be realized.
Example 23
The drug-loaded gel sustained-release preparation in example 18 is added with 10% of polyethylene glycol 200 to adjust the drug release rate, so that the in-vitro 2-week release of the drug can be realized.
Example 24
The drug-loaded gel sustained-release preparation in example 16 is added with 8% xylitol regulator to regulate the drug release rate, so that the in-vitro 1-week release of the drug can be realized.
Example 25
The drug-loaded gel sustained-release preparation in example 18 is added with 5% of mannitol regulator to regulate the drug release rate, so that the in vitro 2-week release of the drug can be realized.
Example 26
Preparing copolymer-1 with normal saline to obtain 25 wt% solution, filtering, sterilizing, adding 1mg/mL colchicine, and stirring at 4 deg.C in refrigerator to obtain injection. A C57BL/6 mouse is used as a model animal, and the proximal end of the left anterior descending branch of the coronary artery is ligated to construct a myocardial infarction mouse model. Then 10 microliters of colchicine aqueous Gel was injected into the peduncle margin region, and the peduncle mice (Col @ Gel group) were injected with the drug-loaded Gel in situ into the peduncle margin region. Meanwhile, Sham-operated mice (Sham group), myocardial infarction mice without hydrogel injection (MI group) were used as blank controls, and myocardial infarction mice injected with an equal dose of colchicine in the abdominal cavity (Col (i.p.) group) were used as positive controls.
The results of the embodiment show that the colchicine gel sustained-release preparation provided by the invention can effectively inhibit the inflammatory reaction of myocardial infarction and improve the structure and function of the heart; the above effect was more pronounced compared to the positive control group without causing systemic toxicity, and the results are recorded in fig. 4-6. As shown in FIG. 4, myocardial infarction caused an inflammatory response, manifested by a marked increase in inflammatory cells (F4/80-positive cells); and Col @ Gel can obviously reduce inflammatory cells. Col (i.p.) at the same dose is less effective than Col @ Gel. As shown in the typical image of cardiac ultrasound in panel 5(A) and panel 5B, EF and FS in myocardial infarction groups were significantly lower than those in sham operation groups (p <0.001), and LVIDs and LVIDd were significantly higher than those in sham operation groups (p <0.001), indicating severe structural and functional damage to the myocardium. All echocardiographic parameters of the Col @ Gel group were significantly improved (p <0.05 or p <0.001) compared to the MI group, whereas the Col (i.p.) group was improved only in EF and FS (p <0.05 or p < 0.001). This indicates that after myocardial infarction, Col @ Gel treatment had a significant improvement in myocardial structure and function, whereas Col (i.p.) treatment was less effective in myocardial structure and function. As shown in FIG. 6(A), the average body weight of the mice in the Col @ Gel group increased 3.42. + -. 0.5g at 4 weeks after myocardial infarction, which was not significantly different from those in the Sham and myocardial infarction groups. The Col (i.p.) group showed significantly less body weight gain than the other three groups (2.95 ± 0.4, p <0.05), indicating that intraperitoneal injection of Col solution caused significant systemic toxicity compared to Col @ Gel. Fig. 6(B) shows that serum AST and ALT levels, Col (i.p.) group, were significantly higher than the other three groups (p <0.001), indicating that Col systemic exposure induced severe liver damage, while local myocardial application of Col @ Gel effectively prevented this side effect. In the figure, represents that two groups of data have significant difference, and p is <0.05, represents that two groups of data have significant difference, and p is < 0.01; indicates that there was a significant difference between the two sets of data and p < 0.001.
Example 27
Preparing copolymer-1 with normal saline to obtain 25 wt% solution, filtering, sterilizing, adding colchicine 10mg/mL, and stirring at 4 deg.C to obtain sustained release preparation 10 mg/mL. About 20g of ICR mice were used as a model, and 5. mu.L of drug-loaded gel was injected subcutaneously into the back, i.e., the actual drug loading was 2.5mg/kg, and the physiological state of the mice was found to be good. The results of this example show that a colchicine concentration dose of 10mg/mL is safe.
Example 28
Preparing copolymer-1 with physiological saline to obtain 25 wt% solution, filtering, sterilizing, adding 0.15,0.25,0.5, 1, 1.5, 2mg/mL colchicine, and stirring at 4 deg.C in refrigerator to obtain various sustained release preparations with different concentrations. Approximately 20g of ICR mice were used as a model, and 0.1mL of drug loaded gel was injected subcutaneously into the back, i.e., the actual drug loading was 0.75,1.25,2.5, 5, 7.5, 10 mg/kg. When the drug loading is less than or equal to 2.5mg/kg, the physiological state of the experimental mouse is good; the drug loading is equal to 5mg/kg, and the experimental mice survive but have poor physiological conditions; when the drug loading is 7.5mg/kg or more, all the patients die within 2 days due to drug toxicity.
The results of this example show that significant systemic toxicity already occurs when the colchicine hydrogel sustained release formulation reaches a dose of 5mg/kg, whereas the colchicine hydrogel sustained release formulation is safe at a dose in the range of 0.5-2.5 mg/kg.
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 method disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the description of the method part.
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 (7)
1. A gel drug sustained release preparation, characterized in that the gel drug sustained release preparation contains 3-10mg/mL colchicine; further comprising: 5-40 wt% of gel carrier material, 0.01-15 wt% of regulator and the balance of solvent; the gel carrier material is an amphiphilic block copolymer which is composed of polyethylene glycol as a hydrophilic block and polyester as a hydrophobic block;
the content of the gel carrier material in the gel drug sustained-release preparation is 15-30 wt%; in the gel carrier material, the average molecular weight of the polyethylene glycol is 600-20000, and the content is 10-90 wt%; the content of the polyester is 10-90 wt%, and the polyester is at least one of poly (DL-lactic acid-glycolic acid) copolymer, poly (L-lactic acid-glycolic acid) copolymer, poly (D-lactic acid-glycolic acid) copolymer, poly (DL-lactic acid) copolymer, poly (L-lactic acid) copolymer and poly (D-lactic acid) copolymer.
2. The sustained-release gel pharmaceutical preparation according to claim 1, wherein the solvent is pure water, physiological saline, a buffer solution, a tissue culture solution, a cell culture solution, animal or plant body fluid or other solvent media not mainly containing an organic solvent; the regulator is selected from one or more of sugar, salt, sodium carboxymethylcellulose, iodine glycerol, simethicone, propylene glycol, carbomer, mannitol, sorbitol, surfactant, Tween 20, Tween 40, Tween 80, xylitol, oligosaccharide, chondroitin, chitin, chitosan, collagen, gelatin, protein gel, hyaluronic acid, and polyethylene glycol.
3. A method for preparing a gel drug delivery formulation according to claim 1 or 2, comprising the steps of:
dissolving the gel carrier material in the solvent at low temperature, storing at the temperature below-20 ℃ for later use, re-dissolving the gel carrier material dissolved in the solvent at room temperature before use, adding the colchicine medicine, and uniformly mixing to obtain the gel sustained-release preparation.
4. The method for preparing a gel drug delivery formulation according to claim 3, wherein the low temperature dissolution temperature is not higher than the sol-gel transition temperature of the gel carrier material.
5. Use of a gel pharmaceutical sustained release formulation according to claim 1 or 2 or a gel pharmaceutical sustained release formulation prepared by the method according to claim 3 in the preparation of a medicament for the treatment of myocardial infarction.
6. The use of a gelated drug depot according to claim 5, wherein the gelated drug depot has a thermogelling property, is in a solution state at room temperature, and is transformed into a gel state at 4-37 ℃; and the effective dosage range of the colchicine in the gel drug sustained-release preparation is 0.5-2.5mg/kg by taking a mouse as a model.
7. The use of a gelated pharmaceutical sustained release formulation according to claim 6 wherein the effective dose of colchicine in the gelated pharmaceutical sustained release formulation ranges from 0.055 to 0.275mg/kg for promotion from 20g mice to 70kg adults according to the equivalent dose ratio between human and animal, reduced by body surface area.
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