CN111297799B - Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof - Google Patents
Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a sustained-release composition preparation of polypeptide protein medicines and a preparation method thereof. The sustained-release composition preparation is prepared from polypeptide protein medicines, a composite reagent and a temperature-sensitive polymer aqueous solution; the compound reagent is protamine, a composition of protamine and metal salt, a composition of protamine, sodium phosphate salt and calcium chloride, a composition of protamine, sodium carbonate and calcium chloride or lauroyl arginine ethyl ester; the sustained-release preparation composition can realize accurate and controllable sustained release of the polypeptide protein medicines on the premise of not influencing the drug effect of the polypeptide protein medicines, and effectively reduce the injection and administration times of the medicines, thereby improving the compliance of patients and ensuring stable release rate. All steps of the preparation method of the sustained-release composition preparation are carried out in the water phase, so that the structure and the biological activity of the polypeptide protein medicine are effectively maintained.
Description
Technical Field
The invention belongs to the field of polypeptide protein medicine preparations, and particularly relates to a sustained-release composition preparation of a polypeptide protein medicine and a preparation method thereof.
Background
Compared with the traditional small molecule drugs which always dominate the whole drug market, the polypeptide protein drugs have higher specificity, higher activity and lower toxicity, and can be used for treating tumors, chronic metabolic diseases, central nervous system, immune system, cardiovascular diseases and other diseases. However, most polypeptide protein drugs require multiple or even long-term administration, and are poorly compliant for patients treated with common injections. Therefore, the key of the research and development of polypeptide protein drugs is to reduce the administration times and improve the administration compliance by prolonging the drug release time through a drug sustained release technology. The polypeptide protein sustained release preparations on the market at present all adopt microspheres as carriers, so that the medicines are slowly released for 1-4 weeks, and the medication compliance of the medicines is obviously improved, but the microsphere preparations have the problems of complex preparation process, influence on the medicine activity, difficulty in controlling the medicine release and the like. In recent years, in-situ temperature-sensitive gel injection gradually becomes a novel polypeptide protein drug sustained-release formulation.
The in-situ temperature-sensitive gel injection is prepared by taking a temperature-sensitive polymer as a carrier and dispersing or dissolving a drug in a temperature-sensitive polymer aqueous solution, and the injection is converted from a liquid state to a semisolid hydrogel by utilizing the temperature-responsive phase transition of the temperature-sensitive polymer aqueous solution at the administration part, so that the drug is slowly released. The in-situ temperature-sensitive gel injection has the advantages of simple drug loading process, drug stability maintenance, good histocompatibility and the like, has long retention time at the drug delivery part, plays a role of drug storage and can delay the release of the drug. There are several types of temperature-sensitive polymers reported at present for sustained release of polypeptide and protein drugs, including natural polymer systems (such as Hydroxypropyl Methylcellulose (HMPC), chitosan and other polysaccharides and their combination with salts) and synthetic polymer systems (including poly (N-isopropylacrylamide) (PNIPAM) and other non-degradable polymers and polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA), polylactic acid-glycolic acid copolymer-polyethylene glycol-polylactic acid-glycolic acid copolymer (PLGA-PEG-PLGA) and other degradable polymers).
Although the polysaccharide/salt system has relatively simple preparation process and low cost, the problems are mainly that the hydrophilicity of a skeleton polymer is poor, the concentration of the polymer in a solution is low, and therefore, the formed gel skeleton has low strength, water is easy to lose, the degradation is fast, and the requirement of slow release and long acting of a medicament cannot be met. In the prior art, for example, the carboxymethyl chitosan quaternary ammonium salt temperature-sensitive gel and the preparation method disclosed in the Chinese patent application CN108653197A, the gelation temperature of the obtained temperature-sensitive gel is the same as the temperature of a human body, the gelation time is extremely short, the antibacterial performance is obvious, but the drug slow-release performance is poor, so that the application range is limited to skin wound surfaces and other parts.
Block copolymers are one of the most widely studied temperature sensitive gel materials. More than 30 block copolymer temperature-sensitive gel systems are published at present, wherein ABA type triblock copolymers are taken as the main components. The method has the advantages that the concentration of the polymer solution is high, the structural strength of the gel framework is high, and the temperature sensitivity, the degradability, the minimum critical solution temperature (LCST) and the like of the material can be regulated and controlled according to requirements.
The gel is a temperature-sensitive in-situ gel product developed by MacroMed company, and the temperature-sensitive gel material used by the gel product is PLGA-PEG-PLGA with low molecular weight; the gel system can realize the sustained-release function for 1-6 weeks and has the basis of developing sustained-release injection. However, it has been found that,
there are also problems with burst release and limited gel regulation. Chinese patent ZL200910049664.6 discloses that a mixed gel composed of two or more PLGA-PEG-PLGA block copolymers has temperature-sensitive characteristics which are not possessed by a single polymer gel, and can more flexibly adjust the properties of LCST, degradation speed and the like of the gel. However, due to the aqueous environment of the temperature-sensitive gel, water-soluble drugs such as polypeptide protein and the like are easy to diffuse through the internal channel, thereby causing burst release and limiting the application of the drugs. Chinese patent publication No. CN103622902B discloses a temperature-sensitive gel pharmaceutical preparation, which is prepared from metal salt, saccharide and hydrophilic material for injectionOne or more auxiliary materials in the polymer are used to reduce the burst release of the water-soluble drug, thereby achieving the purpose of slow release. The fine adjustment of gel release is realized by blending at least two temperature-sensitive polymers and adding various auxiliary materials, and the design of the preparation is relatively complex.
Disclosure of Invention
The invention aims to provide a sustained-release composition preparation of polypeptide protein medicines and a preparation method thereof, and the sustained-release composition preparation can effectively reduce the burst release of the polypeptide protein medicines and realize the accurate and controllable sustained release of the polypeptide protein medicines, thereby reducing the injection administration times and improving the medication compliance of patients.
The invention finds that the temperature-sensitive polymer and various composite reagents have synergistic effect, can obviously improve the slow release effect of the polypeptide protein drug slow release preparation, has stable slow release rate, and maintains effective treatment concentration for a long time, thereby realizing accurate controllable slow release of the polypeptide protein drug.
A sustained-release composition preparation of polypeptide protein medicines is prepared from polypeptide protein medicines, a composite reagent and a temperature-sensitive polymer aqueous solution.
The compound reagent is used for compounding with polypeptide protein medicines in advance, and one of five compound reagents, namely a composition of protamine and metal salt, a composition of protamine, sodium phosphate salt and calcium chloride, a composition of protamine, sodium carbonate and calcium chloride, protamine and lauroyl arginine ethyl ester is selected. The selected composite reagent and the temperature-sensitive polymer have a synergistic effect, the sustained-release effect of the polypeptide protein drug sustained-release preparation can be obviously improved, the sustained-release rate is stable, and the effective treatment concentration is maintained for a long time, so that the accurate controllable sustained release of the polypeptide protein drug is realized.
The metal salt is pharmaceutically acceptable and is dissociated into divalent metal cations in aqueous solution; the divalent metal cation is preferably zinc ion.
The temperature-sensitive polymer is one or more of PLA-PEG-PLA and PLGA-PEG-PLGA.
In the sustained-release composition preparation, the dosage of the polypeptide protein drug can be designed according to the therapeutic dosage when the drug is used; the dosage of the compound reagent is selected to be proper for realizing the effective compound with the polypeptide protein medicine; the temperature-sensitive polymer is automatically converted into hydrogel under the condition of body temperature by mainly utilizing the temperature-responsive phase transition of the temperature-sensitive polymer, and is synergistic with the composite reagent to slowly release the medicine, reduce the administration times and improve the administration compliance of patients, and the dosage of the temperature-sensitive polymer is selected by the dosage of the polypeptide protein medicine and the dosage of the composite reagent.
In order to achieve a better effect of the invention, the following preferable steps are performed:
the dosage of the protamine is 0.1-5% of the weight of the sustained-release composition preparation, and is further preferably 0.1-1%, and the protamine has more remarkable synergistic effect with the temperature-sensitive polymer.
The dosage of the metal salt is 0.1-10% of the weight of the sustained-release composition preparation, and the metal salt has more remarkable synergistic effect with the temperature-sensitive polymer. The metal salt is zinc acetate; the dosage of the zinc acetate is 0.1-3% of the weight of the sustained-release composition preparation, and the more preferable dosage is 0.1-0.5%. The dosage range can better ensure the complete combination of the zinc acetate and the polypeptide protein medicines, and the synergistic effect is stronger.
The total dosage of the sodium phosphate and the calcium chloride is 0.5 to 7.5 percent of the weight of the sustained-release composition preparation; wherein the weight ratio of the sodium phosphate to the calcium chloride is 1: 2-4. The dosage range can better ensure sodium phosphate, calcium chloride and formed calcium phosphate (Ca)3(PO4)2) The compound is effectively compounded with polypeptide protein medicines, and the synergistic effect is stronger. Further preferably: the dosage of the sodium phosphate is 1 to 2.2 percent of the weight of the sustained-release composition preparation; the dosage of the calcium chloride is 4-6% of the weight of the sustained-release composition preparation. The sodium phosphate salt is preferably sodium phosphate, disodium hydrogen phosphate or sodium dihydrogen phosphate.
The total dosage of the sodium carbonate and the calcium chloride is 0.5 to 7.5 percent of the weight of the sustained-release composition preparation; wherein the weight ratio of the sodium carbonate to the calcium chloride is 1: 2-4. The dosage range can better ensure the effective composition of the sodium carbonate, the calcium chloride and the formed calcium carbonate and the polypeptide protein medicine, and the synergistic effect is stronger. Further preferably: the dosage of the sodium carbonate is 1 to 2 percent of the weight of the sustained-release composition preparation; the dosage of the calcium chloride is 4-6% of the weight of the sustained-release composition preparation.
The dosage of the lauroyl arginine ethyl ester is 0.5 to 5 percent of the weight of the sustained-release composition preparation, and the preferable dosage is 1 to 3 percent. The dosage range can better ensure the effective combination of the lauroyl arginine ethyl ester and the polypeptide protein medicine, and the synergistic effect is stronger.
The dosage of the polypeptide protein medicine is 0.1-5% of the weight of the sustained-release composition preparation, and the preferable dosage is 0.1-0.5%. Further ensuring the blood concentration of the polypeptide protein drugs to be maintained within the range of a treatment window when the sustained-release composition preparation is applied, and simultaneously reducing the possibility of side effects of high-concentration drugs on organisms.
The polypeptide protein medicine is one or more of the existing polypeptide protein medicines, including insulin (insulin), insulin analogues, interferon (interferon), Ovalbumin (OVA), recombinant human growth hormone (rhGH), glucagon-like peptide (GLP), glucagon-like peptide analogues, immunoglobulin (IgG), programmed death receptor 1(PD-1), and cell type death-ligand (PD-L1); can be prepared by adopting a commercial product or according to the existing preparation method.
Preferably, the number average molecular weight of the PEG block in the temperature-sensitive polymer is 1000-2000, the weight percentage is 20-40%, the number average molecular weight of the PLA or PLGA block is 1000-2000, the weight percentage is 60-80%, and the molar ratio of the lactic acid unit (LA) to the glycolic acid unit (GA) in the PLGA block is 1-3: 1. The lowest critical solution temperature of the aqueous solution of the temperature-sensitive polymer with the selected molecular weight range and the block proportion is 20-35 ℃, and the normal-temperature preparation of the temperature-sensitive hydrogel and the rapid gelation under the body temperature condition can be realized. The temperature-responsive phase transition of the temperature-sensitive polymer is automatically converted into hydrogel under the condition of body temperature, and the temperature-sensitive polymer has more remarkable synergy with a composite reagent, slowly releases the medicine, reduces the administration times and improves the medication compliance of patients. Further preferably, the temperature-sensitive polymer is PLGA-PEG-PLGA, wherein the number average molecular weight of the PEG block is 1000-1450, the weight percentage is 29.4-32.6%, the number average molecular weight of the PLGA block is 1200-1500, the weight percentage is 67.4-70.6%, and the molar ratio of LA to GA in the PLGA block is 3: 1.
The weight percentage concentration of the temperature-sensitive polymer aqueous solution is 10-30%, and the preferred weight percentage concentration is 20%.
The preparation method of the sustained-release composition preparation of the polypeptide protein medicine comprises the following steps:
(1) dripping the composite reagent aqueous solution into the polypeptide protein medicine aqueous solution, uniformly mixing to obtain polypeptide protein medicine compound suspension, and centrifuging to remove supernatant or freeze-drying to obtain the polypeptide protein medicine compound;
(2) and (2) uniformly mixing the temperature-sensitive polymer aqueous solution with the polypeptide protein drug compound in the step (1) to obtain a sustained-release composition preparation of the polypeptide protein drug.
The concentrations of the polypeptide protein medicine aqueous solution and the compound reagent aqueous solution are not particularly limited, and a small amount or a proper amount of water is required mainly for providing an aqueous solution environment.
The temperature-sensitive slow-release composition preparation entrapping the polypeptide protein medicine can be used as an in-situ temperature-sensitive gel injection.
The raw materials used in the invention are all the existing raw materials, can adopt products sold in the market, can also be prepared according to the existing preparation method, and only need to meet the medicinal quality standard.
Compared with the prior art, the invention has the advantages that:
(1) the invention adopts the temperature-sensitive polymer aqueous solution with proper LCST and polypeptide protein medicine combination, can realize local injection administration, automatically converts into hydrogel under the condition of body temperature through the temperature-responsive phase transition of the temperature-sensitive polymer, slowly releases the medicine, reduces the administration times and improves the medication compliance of patients.
(2) The invention adopts protamine, a composition of protamine and metal salt, a composition of protamine and sodium phosphate and calcium chloride, a composition of protamine and sodium carbonate and calcium chloride or lauroyl arginine ethyl ester as a composite reagent to compound the polypeptide protein medicine, and the temperature-sensitive polymer and the composite reagent have synergistic interaction, so that the burst release of the polypeptide protein medicine can be effectively reduced, the slow release effect of the medicine is further obviously improved, the stability of the polypeptide protein medicine is kept, the slow release rate is stable, the effective treatment concentration is maintained for a long time, and the accurate controllable slow release of the polypeptide protein medicine is realized.
(3) The preparation method adopted by the invention is simple, has higher drug content, and all the steps are carried out in the water phase, thus effectively maintaining the structure and the biological activity of the polypeptide protein drug and being suitable for industrial popularization and application.
(4) The raw materials adopted by the invention have no immunogenicity and good biocompatibility.
Drawings
FIG. 1 is a graph of the in vitro release profile of rhGH from the rhGH complexes of comparative example 1 and comparative example 2 of the present invention.
Fig. 2 is a graph of the in vitro release profile of rhGH in the sustained release compositions of comparative example 3, comparative example 5, and example 1, example 3, example 4, example 5, and example 7 of the present invention.
Fig. 3 is an in vitro release profile of rhGH in sustained release compositions of comparative example 4, comparative example 6 and example 2, example 6 of the present invention.
Fig. 4 is an in vitro release profile of rhGH in sustained release compositions of examples 11, 12, 13, 14 and 3 of the present invention.
FIG. 5 is a time-course curve of rhGH in the sustained-release compositions of examples 1, 3 and comparative example 5 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making creative efforts based on the embodiments of the present invention, belong to the protection scope of the present invention.
Example 1
Precisely weighing 200mg of PLGA1500-PEG1450-PLGA1500 (the number average molecular weight, the weight percentage of the PEG block is 32.6%, the weight percentage of the PLGA block is 67.4%, and the molar ratio of LA/GA is 3:1), adding 0.80ml of deionized water, and dissolving under magnetic stirring at room temperature to obtain a temperature-sensitive polymer aqueous solution with the concentration of 20% (w/w), wherein the LCST is 32 ℃. Precisely weighing 1mg of recombinant human growth hormone (rhGH), and adding 0.3ml of water to dissolve to obtain an rhGH aqueous solution; precisely weighing 1mg protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.3ml water to obtain protamine water solution; under the condition of magnetic stirring, dropwise adding the protamine aqueous solution into the rhGH aqueous solution, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH sustained-release composition, wherein the rhGH and the protamine account for 0.1 percent and 0.1 percent of the rhGH sustained-release composition respectively by weight.
Example 2
200mg of PLGA1200-PEG1000-PLGA1200 (the number average molecular weight, the weight percentage of the PEG block is 29.4%, the weight percentage of the PLGA block is 70.6%, and the LA/GA molar ratio is 3:1) is precisely weighed, 0.80ml of deionized water is added, the mixture is dissolved under magnetic stirring at 15 ℃, and the temperature-sensitive polymer aqueous solution with the concentration of 20% (w/w) is obtained, and the LCST is 20 ℃. Precisely weighing 1mg of recombinant human growth hormone, and adding 0.3ml of water to dissolve to obtain rhGH aqueous solution; precisely weighing 1mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.3ml of water to obtain protamine water solution; dropwise adding the protamine aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH sustained-release composition, wherein the rhGH and the protamine account for 0.1 percent and 0.1 percent of the rhGH sustained-release composition respectively by weight.
Example 3
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Precisely weighing 1mg of recombinant human growth hormone, and adding 0.3ml of water to dissolve to obtain rhGH aqueous solution; accurately weighing 1mg of zinc acetate, and dissolving in 0.3ml of water to obtain a zinc acetate aqueous solution; precisely weighing 1mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.3ml of water to obtain protamine water solution; respectively dropwise adding the zinc acetate aqueous solution and the protamine aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH sustained-release composition, wherein the rhGH, the protamine and the zinc acetate account for 0.1%, 0.1% and 0.1% of the rhGH sustained-release composition respectively by weight.
Example 4
The same procedure as in example 1 was repeated except that the amount of protamine was 5mg, to obtain a rhGH sustained-release composition, wherein the rhGH and protamine were 0.1% and 0.5% by weight, respectively, of the rhGH sustained-release composition.
Example 5
The same procedure as in example 1 was repeated except that the amount of protamine was 10mg, to obtain a rhGH sustained-release composition, wherein the rhGH and protamine were 0.1% and 1.0% by weight, respectively, of the rhGH sustained-release composition.
Example 6
The same procedure as in example 3 was repeated except that 200mg of PLGA1500-PEG1450-PLGA1500 (all having a number average molecular weight, 32.6% by weight of PEG block, 67.4% by weight of PLGA block and 3:1 by weight of LA/GA) was replaced with 200mg of PLGA1200-PEG1000-PLGA1200 (all having a number average molecular weight, 29.4% by weight of PEG block and 70.6% by weight of PLGA block) and 3:1 by weight of LA/GA), to obtain a rhGH sustained-release composition, wherein the weight ratios of rhGH, protamine and zinc acetate to the rhGH sustained-release composition were 0.1%, 0.1% and 0.1%, respectively.
Example 7
The same procedure as in example 3 was repeated except that rhGH was used in an amount of 5mg, protamine was used in an amount of 5mg, and zinc acetate was used in an amount of 5mg, to obtain a rhGH sustained-release composition, wherein rhGH, protamine, and zinc acetate were 0.5%, and 0.5% by weight, respectively, of the rhGH sustained-release composition.
Example 8
The same procedure as in example 3 was repeated except that rhGH was changed to insulin, to obtain an insulin sustained-release composition, wherein the weight ratios of insulin, protamine and zinc acetate to the insulin sustained-release composition were 0.1%, 0.1% and 0.1%, respectively.
Example 9
The procedure of example 3 was followed except that rhGH was replaced with OVA to obtain an OVA sustained-release composition, wherein OVA, protamine and zinc acetate were 0.1%, 0.1% and 0.1% by weight, respectively, of the OVA sustained-release composition.
Example 10
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Precisely weighing 5mg of recombinant human growth hormone, and adding 0.30ml of water to dissolve to obtain rhGH aqueous solution; precisely weighing 1mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.30ml of water to obtain protamine water solution; dropwise adding the protamine aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and (3) freeze-drying the rhGH compound suspension to obtain the rhGH compound freeze-dried powder. And uniformly mixing the rhGH compound freeze-dried powder with a temperature-sensitive polymer aqueous solution to obtain the rhGH sustained-release composition, wherein the rhGH and the protamine account for 0.5 percent and 0.1 percent of the rhGH sustained-release composition respectively by weight.
Example 11
200mg of PLGA1500-PEG1450-PLGA1500 (number average molecular weight, weight percentage of PEG block) are precisely weighed32.6 percent of PLGA block, 67.4 percent of PLGA block and 3:1 mole ratio of LA/GA), adding 0.80ml of deionized water, and dissolving under magnetic stirring at 15 ℃ to obtain a temperature-sensitive polymer aqueous solution with the concentration of 20 percent (w/w), wherein the LCST is 32 ℃. Precisely weighing 3mg of recombinant human growth hormone, and dissolving in 8mL of water to obtain rhGH aqueous solution; 3mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) was precisely weighed and dissolved in 8mL of water to obtain an aqueous protamine solution. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, and vigorously stirred for 5 minutes, followed by slow stirring for 1.5 hours to obtain a protamine-rhGH complex solution. To the protamine-rhGH Complex solution was added 2mL of 70mM NaH2PO4(16.8mg) in water, and 2mL of 250mM CaCl was added dropwise2(55.5mg) of the aqueous solution, vigorously stirred for 10 minutes, then slowly stirred for 1 hour, and finally centrifuged to obtain rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH slow-release composition. Wherein rhGH, protamine and NaH2PO4And CaCl2The weight ratio of the rhGH slow release composition is 0.28%, 1.56% and 5.15%.
Example 12
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Precisely weighing 3mg of recombinant human growth hormone, and dissolving in 8mL of water to obtain rhGH aqueous solution; 3mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) was precisely weighed and dissolved in 8mL of water to obtain an aqueous protamine solution. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, and vigorously stirred for 5 minutes, followed by slow stirring for 1.5 hours to obtain a protamine-rhGH complex solution. To the protamine-rhGH Complex solution was added 2mL of 70mM Na2HPO4(19.9mg) aqueous solution, and 2mL of 250mM CaCl was added dropwise2(55.5mg) of the aqueous solution, vigorously stirred for 10 minutes, then slowly stirred for 1 hour, and finally centrifuged to obtain rhGH complex. Polymerizing rhGH complex with temperature-sensitive polymerThe water solution is mixed evenly to obtain the rhGH slow-release composition. Wherein rhGH, protamine and Na2HPO4And CaCl2The weight ratio of the rhGH sustained-release composition is 0.28%: 0.28%: 1.84 percent and 5.13 percent.
Example 13
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Precisely weighing 3mg of recombinant human growth hormone, and dissolving in 8mL of water to obtain rhGH aqueous solution; 3mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) was precisely weighed and dissolved in 8mL of water to obtain an aqueous protamine solution. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, and vigorously stirred for 5 minutes, followed by slow stirring for 1.5 hours to obtain a protamine-rhGH complex solution. To the protamine-rhGH complex solution was added 2mL of 70mM (mmol/L) Na2CO3(14.8mg) aqueous solution, 2mL of 250mM CaCl was added dropwise2(55.5mg) of the aqueous solution, vigorously stirred for 10 minutes, then slowly stirred for 1 hour, and finally centrifuged to obtain rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH slow-release composition. Wherein rhGH, protamine and Na2CO3And CaCl2The weight ratio of the rhGH slow release composition is 0.28%, 1.38% and 5.16%.
Example 14
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Precisely weighing 1mg of recombinant human growth hormone, and adding 0.30ml of water to dissolve to obtain rhGH aqueous solution; accurately weighing 10mg of lauroyl arginine ethyl ester, and adding 0.30ml of water to dissolve to obtain lauroyl arginine ethyl ester water solution; dropwise adding the lauroyl arginine ethyl ester aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension to obtain the rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the rhGH slow-release composition. Wherein the rhGH and the lauroyl arginine ethyl ester respectively account for 0.1 percent and 1 percent of the weight of the rhGH sustained-release composition.
Example 15
Except that "2 mL of 70mM Na2HPO4(19.9mg) aqueous solution "replace" 2mL70mM Na3PO4(23.0mg) aqueous solution ", the other procedures were the same as in example 12 to obtain a rhGH sustained-release composition. Wherein rhGH, protamine and Na3PO4And CaCl2The weight ratio of the rhGH sustained-release composition is 0.28%: 0.28%: 2.12 percent and 5.12 percent.
Comparative example 1
Precisely weighing 5mg of recombinant human growth hormone, and adding 0.50ml of water to dissolve to obtain rhGH aqueous solution; precisely weighing 1mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.50ml of water to obtain protamine water solution; dropwise adding the protamine aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. The rhGH and protamine account for 0.5 percent and 0.1 percent of the suspension of the rhGH complex by weight.
Comparative example 2
Precisely weighing 5mg of recombinant human growth hormone, and adding 0.33ml of water to dissolve to obtain rhGH aqueous solution; accurately weighing 1mg of zinc acetate, and dissolving in 0.33ml of water to obtain a zinc acetate aqueous solution; precisely weighing 1mg of protamine (Tokyo chemical industry Co., Ltd. (TCI)) and dissolving in 0.33ml of water to obtain protamine aqueous solution; respectively dropwise adding the zinc acetate aqueous solution and the protamine aqueous solution into the rhGH aqueous solution under stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. The rhGH, the zinc acetate and the protamine account for 0.5 percent, 0.1 percent and 0.1 percent of the suspension of the rhGH complex by weight.
Comparative example 3
200mg of PLGA1500-PEG1450-PLGA1500 (all with number average molecular weight, the weight percentage of PEG block being 32.6%, the weight percentage of PLGA block being 67.4%, the molar ratio of LA/GA being 3:1) was precisely weighed, 0.80ml of deionized water was added and dissolved under magnetic stirring at 15 ℃ to obtain a temperature sensitive polymer aqueous solution with a concentration of 20% (w/w) and an LCST of 32 ℃. Accurately weighing 5mg of recombinant human growth hormone, and uniformly mixing with the temperature-sensitive polymer aqueous solution to prepare the slow-release composition with the rhGH accounting for 0.5 percent of the weight of the slow-release composition.
Comparative example 4
200mg of PLGA1200-PEG1000-PLGA1200 (the number average molecular weight, the weight percentage of the PEG block is 29.4%, the weight percentage of the PLGA block is 70.6%, and the LA/GA molar ratio is 3:1) is precisely weighed, 0.80ml of deionized water is added, the mixture is dissolved under magnetic stirring at 15 ℃, and the temperature-sensitive polymer aqueous solution with the concentration of 20% (w/w) is obtained, and the LCST is 20 ℃. Accurately weighing 5mg of recombinant human growth hormone, and uniformly mixing with the temperature-sensitive polymer aqueous solution to prepare the slow-release composition with the rhGH accounting for 0.5 percent of the weight of the slow-release composition.
Comparative example 5
Precisely weighing 200mg of PLGA1500-PEG1450-PLGA1500 (the number average molecular weight, the weight percentage of the PEG block is 32.6%, the weight percentage of the PLGA block is 67.4%, and the molar ratio of LA/GA is 3:1), adding 0.80ml of deionized water, and dissolving under magnetic stirring at room temperature to obtain a temperature-sensitive polymer aqueous solution with the concentration of 20% (w/w), wherein the LCST is 32 ℃. Precisely weighing 1mg of recombinant human growth hormone, and adding 0.3ml of water to dissolve to obtain rhGH aqueous solution; accurately weighing 1mg of zinc acetate, and adding 0.3ml of water for dissolving to obtain a zinc acetate water solution; dropwise adding a zinc acetate aqueous solution into an rhGH aqueous solution under the condition of magnetic stirring, and uniformly mixing to obtain a rhGH compound suspension; and centrifuging the rhGH complex suspension, and removing the supernatant to obtain the rhGH complex. And uniformly mixing the rhGH compound with a temperature-sensitive polymer aqueous solution to obtain the slow-release composition, wherein the rhGH and the zinc acetate respectively account for 0.1 percent and 0.1 percent of the weight of the slow-release composition.
Comparative example 6
The sustained release composition was obtained by following the same procedure as in comparative example 5 except that 200mg of PLGA1200-PEG1000-PLGA1200 (number average molecular weight, 29.4% by weight of PEG block, 70.6% by weight of PLGA block, and 3:1 molar ratio of LA/GA) was used instead of 200mg of PLGA1500-PEG1450-PLGA1500 (number average molecular weight, 32.6% by weight of PEG block, 67.4% by weight of PLGA block, and 3:1 molar ratio of LA/GA), wherein rhGH and zinc acetate were 0.1% and 0.1% by weight of the sustained release composition, respectively.
TABLE 1 summary of the compositions of the examples and comparative formulations
Drug release test: the drug release behavior of each of the samples prepared in examples 1 to 15 and comparative examples 1 to 6 was examined by dialysis, as follows: weighing a proper amount of sample in a Spectra/Por dialysis tube (MWCO100KDa), wherein a drug release medium is 10mL of pH 7.4 phosphate buffer solution, placing the dialysis tube in a constant temperature shaking table, and setting parameters of the constant temperature shaking table: the temperature was 37 ℃ and the rotation speed was 100 rpm. Accurately aspirating 2ml of phosphate buffer from the drug delivery system at different time points, respectively, and then adding the same volume of phosphate buffer release medium to the delivery system. The concentration of the polypeptide protein drug in the sample is determined by high performance liquid chromatography (e.g. the chinese pharmacopoeia 2015 edition), and the experimental results are shown in fig. 1-fig. 4 and table 2.
TABLE 2 summary of drug release data for examples and comparative examples
In the table, # denotes that P < 0.01; 0.01< P <0.05 indicates significant variability; p <0.01 indicates that the variability is very significant.
Compared with the release curves of the polypeptide protein compound and the polypeptide protein temperature-sensitive gel mixture in the comparative examples, the sustained-release composition preparation of the embodiment of the invention has no burst release phenomenon, excellent sustained-release effect and stable sustained-release rate, and the cumulative release amount of 6 days and 20 days is obviously lower than that of the corresponding comparative example preparation; compared with comparative examples 3 and 5, the sustained release rate of the example preparation is stable and is obviously better than that of the comparative example preparation, the cumulative release amount of the example preparation in 6 days is obviously lower than that of the comparative example preparation (P <0.05), and the cumulative release amount of the example preparation in 20 days (long-acting sustained release) is obviously lower than that of the comparative example preparation (P < 0.05); example 11, example 12, example 13, example 14 and example 15, respectively, compared to comparative example 3, the release from the example formulation was significantly slower than the release from the comparative example formulation, the release rate from the example formulation was significantly smoother than the release rate from the comparative example formulation, the cumulative release from the example formulation over 6 days was significantly lower than the comparative example formulation (P <0.05), the cumulative release from the example formulation over 20 days (long-lasting sustained release) was significantly lower than the comparative example formulation (P < 0.05); compared with comparative examples 4 and 6, examples 2 and 6 with the same temperature-sensitive polymer material have the advantages that the sustained release rate of the example preparation is stable and is obviously better than that of the comparative example preparation, the cumulative release amount of the example preparation in 6 days is obviously lower than that of the comparative example preparation (P <0.05), and the cumulative release amount of the example preparation in 20 days is obviously lower than that of the comparative example preparation (P < 0.05). Wherein the cumulative release amount of comparative example 1 and comparative example 2 in 1 day is more than 80%, and comparative example 3, comparative example 4, comparative example 5 and comparative example 6 are capable of prolonging the release of protein due to the formation of temperature sensitive gel, but the sustained release rate is not stable enough. Compared with a comparative preparation, the sustained-release composition preparations of examples 1 to 15 prepared by adding the composite reagent for compounding have more excellent sustained-release effect, can obviously improve the sustained-release effect of the polypeptide protein drug sustained-release preparation, have stable sustained-release rate, are mainly based on the synergistic interaction of the temperature-sensitive polymer and the composite reagent, so that the accurate controllable sustained release of the polypeptide protein drug is realized, and the burst release can be effectively reduced.
Animal experiments: female SD rats of 200g to 250g are used as experimental animals, 300 mul of the sustained-release composition prepared in the example is injected subcutaneously, orbital bleeding is carried out at a certain time point, and blood samples are centrifuged to obtain serum. The concentration of the drug in serum was tested using ab190811 human growth hormone ELISA kit to obtain a drug timing curve. The control group was the sustained-release composition prepared in comparative example 5, and the subcutaneous injection dose was 300. mu.l. The results of the experiment are shown in FIG. 5.
In the case of growth hormone, literature and clinical data indicate that blood levels of growth hormone (Cp) are maintained at 5ng/ml to achieve effective clinical therapeutic effect (Cleland, Jeffrey L., et al. "biocompatible human growth hormone poly (lactic-co-glycolic acid) (PLGA) microspheres precursor a long-acting effect." Journal of Controlled release49.2-3(1997): 193-. Experimental results show that the composition provided by the embodiment of the invention can realize controllable slow release of growth hormone in animal bodies, has better slow release effects of the embodiment 3 and the embodiment 4, can maintain the blood concentration above 5ng/ml for 14 days, can maintain the effective treatment concentration for a long time, and can achieve the effective clinical treatment effect. Example 1 can maintain the plasma concentration at 5ng/ml level for 14 days, and examples 2, 5, 6, 7, 10, 11, 12, 13, 14 and 15 can maintain the plasma concentration at 5ng/ml level for 7 to 10 days, and can also maintain the effective treatment concentration for a long time, thereby achieving the effective clinical treatment effect. As a control, comparative example 5 (combination of temperature sensitive gel and growth hormone/zinc complex) decreased to below therapeutic concentration after 2-3 days of sustained release of effective concentration in rats, failing to maintain effective therapeutic concentration for a long period of time. The release of the polypeptide protein medicine in the animal body is influenced by the aperture of the gel and the stability of the compound, and the dissociation behavior of the growth hormone can be changed by adopting the combination of the compound reagent and the growth hormone, so that the slow release effect of the human growth hormone in the animal body can be obviously prolonged.
Examples 8 and 9 can maintain effective therapeutic concentrations for more than one week and can achieve effective clinical therapeutic effects.
According to the invention, the combination of the composite reagent and polypeptide protein medicines such as growth hormone, insulin and OVA can change the dissociation behavior of the polypeptide protein medicines, and because the interaction between proteins is stronger than the composite action of zinc ions and the polypeptide protein medicines and the synergistic interaction of the temperature-sensitive polymer and the composite reagent, the slow release effect of the polypeptide protein medicines in an animal body can be obviously prolonged, and the slow release rate is stable. The accurate controllable slow release of the polypeptide protein medicines is realized on the premise of not influencing the drug effect of the polypeptide protein medicines, the injection administration times of the medicines are effectively reduced, the compliance of patients is improved, and the effective treatment concentration is maintained for a long time. The change of the parameters in the preparation method does not influence the preparation of the sustained-release composition preparation of the polypeptide protein drugs, so that the combination of any parameter in the preparation method can realize the preparation of the sustained-release composition preparation of the polypeptide protein drugs. And will not be described in detail herein.
Claims (10)
1. A sustained-release composition preparation of polypeptide protein medicines is characterized by being prepared from polypeptide protein medicines, a composite reagent for being compounded with the polypeptide protein medicines in advance and a temperature-sensitive polymer aqueous solution;
the compound reagent is a composition of protamine and metal salt, a composition of protamine, sodium phosphate salt and calcium chloride, a composition of protamine, sodium carbonate and calcium chloride, protamine or lauroyl arginine ethyl ester;
the metal salt is pharmaceutically acceptable and is dissociated into divalent metal cations in aqueous solution; the divalent metal cation is zinc ion;
the temperature-sensitive polymer is one or more than two of PLA-PEG-PLA and PLGA-PEG-PLGA.
2. The sustained-release composition preparation of polypeptide protein drugs as claimed in claim 1, wherein the amount of protamine is 0.1-5% of the weight of the sustained-release composition preparation;
and/or the dosage of the metal salt is 0.1-10% of the weight of the sustained-release composition preparation;
or the total dosage of the sodium phosphate and the calcium chloride is 0.5 to 7.5 percent of the weight of the sustained-release composition preparation; wherein the weight ratio of the sodium phosphate to the calcium chloride is 1: 2-4;
or the total dosage of the sodium carbonate and the calcium chloride is 0.5 to 7.5 percent of the weight of the sustained-release composition preparation; wherein the weight ratio of the sodium carbonate to the calcium chloride is 1: 2-4;
or the dosage of the lauroyl arginine ethyl ester is 0.5 to 5 percent of the weight of the sustained-release composition preparation.
3. The sustained-release composition preparation of polypeptide protein drugs according to claim 1 or 2, wherein the metal salt is zinc acetate; the dosage of the zinc acetate is 0.1 to 3 percent of the weight of the sustained-release composition preparation.
4. The sustained-release composition preparation of a polypeptide protein drug as claimed in claim 1 or 2, wherein the sodium phosphate salt is sodium phosphate, disodium hydrogen phosphate or sodium dihydrogen phosphate.
5. The sustained-release composition preparation of a polypeptide protein drug according to claim 1, wherein the dosage of the polypeptide protein drug is 0.1-5% by weight of the sustained-release composition preparation.
6. The sustained-release composition preparation of polypeptide protein drug according to claim 1 or 5, wherein the polypeptide protein drug comprises one or more of insulin, interferon, ovalbumin, recombinant human growth hormone, glucagon-like peptide, immunoglobulin, programmed death receptor 1, and apoptosis-ligand.
7. The sustained-release composition preparation of a polypeptide protein drug as claimed in claim 1, wherein the lowest critical solution temperature of the temperature-sensitive polymer aqueous solution is 20 ℃ to 35 ℃.
8. The sustained-release composition preparation of polypeptide protein drugs as claimed in claim 1, wherein the weight percentage concentration of the temperature-sensitive polymer aqueous solution is 10-30%.
9. The sustained-release composition preparation of polypeptide protein drugs as claimed in claim 1, wherein the number average molecular weight of the PEG block in the temperature-sensitive polymer is 1000-2000, the weight percentage is 20-40%, the number average molecular weight of the PLA or PLGA block is 1000-2000, the weight percentage is 60-80%, and the molar ratio of LA to GA in the PLGA block is 1-3: 1.
10. The method for preparing the sustained-release composition preparation of the polypeptide protein drug according to any one of claims 1 to 9, comprising the following steps:
(1) dripping the composite reagent aqueous solution into the polypeptide protein medicine aqueous solution, uniformly mixing to obtain polypeptide protein medicine compound suspension, and centrifuging to remove supernatant or freeze-drying to obtain the polypeptide protein medicine compound;
(2) and (2) uniformly mixing the temperature-sensitive polymer aqueous solution with the polypeptide protein drug compound in the step (1) to obtain a sustained-release composition preparation of the polypeptide protein drug.
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