CN111297799A - Sustained-release composition preparation of polypeptide protein medicine and preparation method thereof - Google Patents

Abstract

The invention discloses a sustained-release composition preparation of polypeptide and protein drugs and a preparation method thereof. The sustained-release composition preparation is made of polypeptide protein drugs, composite reagents and a temperature-sensitive polymer aqueous solution; the composite reagents are protamine, the composition of protamine and metal salt, protamine and sodium phosphate salt and the composition of calcium chloride, the composition of protamine, sodium carbonate and calcium chloride or lauroyl arginine ethyl ester; the sustained-release preparation composition can be realized under the premise of not affecting the efficacy of polypeptide protein drugs The precise and controllable sustained release of polypeptide and protein drugs can effectively reduce the number of drug injections, thereby improving patient compliance, and the release rate is stable. All steps of the preparation method of the sustained-release composition preparation are carried out in the aqueous phase, which effectively maintains the structure and biological activity of the polypeptide and protein drugs.

Description

Sustained-release composition preparation of polypeptide and protein drugs and preparation method thereof

technical field

The invention belongs to the field of polypeptide protein drug preparations, in particular to a sustained-release composition preparation of polypeptide protein drugs and a preparation method thereof.

Background technique

Compared with traditional small molecule drugs that have dominated the entire drug market, polypeptide and protein drugs have higher specificity, higher activity and lower toxicity, and can be used in tumors, chronic metabolic diseases, central nervous system, and immune system. , cardiovascular and other diseases treatment. However, most polypeptide and protein drugs require multiple or even long-term administration, and are treated with ordinary injections, resulting in poor patient compliance. Therefore, prolonging the drug release time through drug sustained-release technology to reduce the number of administrations and improve drug compliance has always been the key to the development of polypeptide protein drugs. At present, the sustained-release preparations of polypeptide and protein on the market all use microspheres as carriers, so that the drug can be slowly released for 1 to 4 weeks, which significantly improves the drug compliance of such drugs. Release control difficulties and other issues. In recent years, in situ thermosensitive gel injection has gradually become a new type of sustained-release formulation of polypeptide and protein drugs.

In situ thermosensitive gel injection is an injection prepared by dispersing or dissolving a drug in a thermosensitive polymer aqueous solution by using thermosensitive polymer as a carrier. Transition from a liquid state to a semi-solid hydrogel, allowing slow release of the drug. In situ thermosensitive gel injection has the advantages of simple drug loading process, maintaining drug stability, good histocompatibility, etc., and has a long residence time at the administration site, which acts as a drug reservoir and can delay drug release. There are several types of thermosensitive polymers reported for the sustained release of polypeptide and protein drugs, including natural polymer systems (such as hydroxypropyl methylcellulose (HMPC) and chitosan and other polysaccharides and their combinations with salts) And synthetic polymer systems (including non-degradable polymers such as poly(N-isopropylacrylamide) (PNIPAM) and polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA), polylactic acid-glycolic acid copolymerization Degradable polymers such as poly(ethylene glycol)-polylactic acid-glycolic acid copolymer (PLGA-PEG-PLGA)).

Although the preparation process of the polysaccharide/salt system is relatively simple and the cost is low, the main problems are that the skeleton polymer has poor hydrophilicity and the polymer concentration in the solution is low, so the formed gel skeleton has low strength, easy water loss and degradation. Faster, can not meet the needs of sustained release and long-acting drugs. In the prior art, such as a kind of carboxymethyl chitosan quaternary ammonium salt thermosensitive gel and preparation method disclosed in Chinese patent application CN108653197A, the gelation temperature of the obtained thermosensitive gel is the same as the human body temperature, and the gelation time is extremely short and long. It has remarkable antibacterial properties, but its drug sustained release performance is poor, so its application scope is limited to skin wounds and other parts.

是MacroMed公司开发的温敏原位凝胶产品，其所用的温敏凝胶材料为低分子量的PLGA-PEG-PLGA；该凝胶系统可以实现1周～6周的缓释功能，具备开发成缓释注射剂的基础。然而研究发现，

也存在突释和凝胶调节受限等问题。中国专利ZL200910049664.6公开了由两种或两种以上PLGA-PEG-PLGA嵌段共聚物组成的混合凝胶具有单一聚合物凝胶所不具有的温敏特性，可以更加灵活地调节凝胶的LCST、降解速度等性质。但由于温敏凝胶的水性环境，多肽蛋白等水溶性药物很容易通过内部通道扩散，从而造成突释，限制了其应用。公告号CN103622902B的中国专利公开了一种温敏凝胶药物制剂，其采用由金属盐、糖类物质以及注射药用的亲水性聚合物中的一种或多种组成的辅料来降低水溶性药物的突释，达到缓释目的。其采用至少两种温敏聚合物共混以及通过多种辅料的添加实现对凝胶释放的微调，制剂的设计相对复杂。Block copolymers are one of the most widely studied thermosensitive gel materials. There are more than 30 kinds of block copolymer thermosensitive gel systems that have been published, among which ABA type triblock copolymers are the main ones. The advantages are that the polymer solution concentration is higher, the gel skeleton structure strength is better, and the temperature sensitivity, degradability and minimum critical solution temperature (LCST) of the material can be adjusted as needed.

It is a thermosensitive in situ gel product developed by MacroMed Company. The thermosensitive gel material used is low molecular weight PLGA-PEG-PLGA; the gel system can achieve a sustained-release function for 1 to 6 weeks, and has the ability to develop into a sustained-release injection. Base. However, the study found that

There are also issues such as burst release and limited gel regulation. Chinese patent ZL200910049664.6 discloses that the mixed gel composed of two or more PLGA-PEG-PLGA block copolymers has temperature-sensitive properties that a single polymer gel does not have, and can flexibly adjust the gel LCST, degradation rate and other properties. However, due to the aqueous environment of thermosensitive gels, water-soluble drugs such as polypeptide proteins are easily diffused through internal channels, resulting in burst release, which limits its application. The Chinese patent of Bulletin No. CN103622902B discloses a thermosensitive gel pharmaceutical preparation, which adopts an adjuvant composed of one or more of metal salts, carbohydrate substances and hydrophilic polymers for injection to reduce the effect of water-soluble drugs. Sudden release to achieve the purpose of sustained release. It adopts the blending of at least two temperature-sensitive polymers and the fine-tuning of the gel release through the addition of various excipients, and the design of the formulation is relatively complicated.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a sustained-release composition preparation of polypeptide and protein drugs and a preparation method thereof. The sustained-release composition preparation can effectively reduce the burst release of polypeptide and protein drugs and realize precise and controllable sustained release of polypeptide and protein drugs. , thereby reducing the number of injections and improving the patient's medication compliance.

The invention finds that the temperature-sensitive polymer and various composite reagents have a synergistic effect, which can significantly improve the sustained-release effect of the sustained-release preparation of polypeptide and protein drugs, the sustained-release rate is stable, and the effective therapeutic concentration is maintained for a long time, so as to achieve the realization of the polypeptide protein drug. Accurate and controlled release of drug-like drugs.

The invention relates to a sustained-release composition preparation of polypeptide and protein drugs, which is prepared from polypeptide protein drugs, composite reagents and a temperature-sensitive polymer aqueous solution.

The compound reagent is used for compounding with polypeptide and protein drugs in advance, and the composition of protamine and metal salt, the composition of protamine and sodium phosphate and calcium chloride, the composition of protamine and sodium carbonate and chloride are selected. One of the five complex reagents of calcium composition, protamine, and lauroyl arginine ethyl ester. The selected compound reagent and temperature-sensitive polymer have a synergistic effect, which can significantly improve the sustained-release effect of the sustained-release preparation of polypeptide and protein drugs. The sustained-release rate is stable and the effective therapeutic concentration is maintained for a long time. precise controllable sustained release.

The metal salt is a pharmaceutically acceptable metal salt that dissociates into a divalent metal cation in an aqueous solution; the divalent metal cation is preferably a zinc ion.

The temperature-sensitive polymer is selected from one or more of PLA-PEG-PLA and PLGA-PEG-PLGA.

In the sustained-release composition preparation, the dosage of the polypeptide and protein drugs can be designed according to the therapeutic dose when the drug is used; the dosage of the compound reagent is to select an appropriate dosage for the main purpose of achieving effective compounding with the polypeptide and protein drugs; The sensitive polymer mainly uses its temperature-responsive phase transition to automatically transform into a hydrogel under the condition of body temperature, and synergizes with the compound reagent to release the drug slowly, reduce the number of administrations, and improve the patient's medication compliance. The dosage of polypeptide and protein drugs and the dosage of compound reagents are selected.

In order to achieve better invention effect, the following optimizations are carried out:

The dosage of the protamine is 0.1%-5% by weight of the sustained-release composition preparation, more preferably 0.1%-1%, and the synergistic effect with the temperature-sensitive polymer is more significant.

The dosage of the metal salt is 0.1% to 10% of the weight of the sustained-release composition preparation, and the synergistic effect with the temperature-sensitive polymer is more significant. The metal salt is selected from zinc acetate; the dosage of the zinc acetate is 0.1%-3% by weight of the sustained-release composition preparation, more preferably 0.1%-0.5%. The dosage range can better ensure the complete compounding of zinc acetate and polypeptide protein drugs, and the synergistic effect is stronger.

The total dosage of the sodium phosphate and calcium chloride is 0.5%-7.5% by weight of the sustained-release composition preparation; wherein, the weight ratio of sodium phosphate and calcium chloride is 1:2-4. The dosage range can better ensure the effective compounding of sodium phosphate, calcium chloride and the formed calcium phosphate (Ca ₃ (PO ₄ ) ₂ ) with polypeptide and protein drugs, and the synergistic effect is stronger. Further preferably: the dosage of the sodium phosphate is 1%-2.2% 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 calcium chloride is 0.5%-7.5% by weight of the sustained-release composition preparation; wherein, the weight ratio of sodium carbonate and calcium chloride is 1:2-4. The dosage range can better ensure the effective compounding of sodium carbonate, calcium chloride and the formed calcium carbonate with polypeptide protein drugs, and the synergistic effect is stronger. Further preferably: the dosage of the sodium carbonate is 1%-2% 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%-5% by weight of the sustained-release composition preparation, more preferably 1%-3%. The dosage range can better ensure the effective compounding of lauroyl arginine ethyl ester and polypeptide protein drugs, and the synergistic effect is stronger.

The dosage of the polypeptide and protein drugs is 0.1%-5% by weight of the sustained-release composition preparation, more preferably 0.1%-0.5%. It further ensures the maintenance of the blood drug concentration of the polypeptide and protein drugs within the therapeutic window range when the sustained-release composition preparation is applied, and at the same time reduces the possibility of side reactions caused by the high concentration drug to the body.

The polypeptide protein drugs are existing polypeptide protein drugs, including insulin (insulin), insulin analogs, interferon (interferon), ovalbumin (OVA), recombinant human growth hormone (rhGH), glucagon One of the like peptide (GLP), glucagon-like peptide analog, immunoglobulin (IgG), programmed death receptor 1 (PD-1), programmed cell death-ligand (PD-L1), etc. Or two or more; can be prepared by using commercially available products or according to existing preparation methods.

Preferably, the number-average molecular weight of the PEG block in the temperature-sensitive polymer is 1000-2000, and the weight percentage is 20%-40%, and the number-average molecular weight of the PLA or PLGA block is 1000-2000, and the weight percentage is 60%. ~80%, the molar ratio of lactic acid units (LA) to glycolic acid units (GA) in the PLGA block is 1-3:1. The minimum critical dissolution temperature of the aqueous solution of the temperature-sensitive polymer with the molecular weight range and block ratio selected in the present invention is 20°C to 35°C, which can realize the normal temperature preparation of the temperature-sensitive hydrogel and the rapid gelation under the condition of body temperature. The temperature-responsive phase transition of this type of thermosensitive polymer automatically transforms into a hydrogel under the condition of body temperature, and the synergy with the composite reagent is more significant, releasing the drug slowly, reducing the number of administrations, and improving the patient's medication compliance. 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%, and 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% to 30%, more preferably 20%.

The preparation method of the sustained-release composition preparation of the polypeptide and protein drugs comprises the following steps:

(1) drop the compound reagent aqueous solution into the polypeptide and protein drug aqueous solution, and mix evenly to obtain the polypeptide protein drug complex suspension, and centrifuge to remove the supernatant or freeze-dry to obtain the polypeptide protein drug complex;

(2) Mixing the temperature-sensitive polymer aqueous solution with the polypeptide-protein drug complex in step (1) uniformly to obtain a sustained-release composition preparation of the polypeptide-protein drug.

The concentrations of the polypeptide protein drug aqueous solution and the composite reagent aqueous solution are not particularly limited, mainly to provide an aqueous environment, a small amount or an appropriate amount of water is sufficient.

The thermosensitive sustained-release composition preparation encapsulating the polypeptide and protein drugs of the present invention can be used as an in situ thermosensitive gel injection.

The raw materials used in the present invention are all existing raw materials, and can be commercially available products or can be prepared according to the existing preparation methods, as long as they meet the pharmaceutical quality standards.

Compared with the prior art, the advantages of the present invention are:

(1) The present invention adopts the combination of a temperature-sensitive polymer aqueous solution with a suitable LCST and a polypeptide protein drug, which can realize local injection and administration, and is automatically converted into water under the condition of body temperature through the temperature-responsive phase transition of the temperature-sensitive polymer. Gel, slow release of drugs, reduce the number of doses, and improve the patient's medication compliance.

(2) the present invention adopts the composition of protamine, protamine and metal salt, the composition of protamine and sodium phosphate salt and calcium chloride, the composition of protamine and sodium carbonate and calcium chloride or the laurel Acylarginine ethyl ester is used as a compound reagent to compound polypeptide and protein drugs. The temperature-sensitive polymer and compound reagent have a synergistic effect, which can effectively reduce the burst release of polypeptide and protein drugs, further significantly improve the sustained release effect of drugs, and maintain polypeptide protein drugs. The stability of the drug, the sustained release rate is stable, and the effective therapeutic concentration is maintained for a long time, so as to realize the precise and controllable sustained release of the polypeptide and protein drugs.

(3) The preparation method adopted in the present invention is simple, has a high drug content, and all steps are carried out in the aqueous phase, which effectively maintains the structure and biological activity of the polypeptide and protein drugs, and is suitable for industrialization and application.

(4) The raw materials used in the present invention have no immunogenicity and good biocompatibility.

Description of drawings

Figure 1 shows the in vitro release curves of rhGH in the rhGH complexes of Comparative Example 1 and Comparative Example 2 of the present invention.

2 is the in vitro release curve 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.

3 is the in vitro release curve of rhGH in the sustained-release compositions of Comparative Example 4, Comparative Example 6, Example 2, and Example 6 of the present invention.

4 is the in vitro release curve of rhGH in the sustained-release compositions of Example 11, Example 12, Example 13, Example 14 and Comparative Example 3 of the present invention.

5 is the drug-time curve of rhGH in the sustained-release compositions of Example 1, Example 3 and Comparative Example 5 of the present invention.

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the embodiments of the present invention, and the described embodiments are a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative labor conditions fall within the protection scope of the present invention.

Example 1

Precisely weigh 200mg PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80ml to Ionized water was dissolved under magnetic stirring at room temperature to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 1mg of recombinant human growth hormone (rhGH), add 0.3ml of water to dissolve to obtain rhGH aqueous solution; accurately weigh 1mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)), add 0.3ml of water to dissolve, to obtain protamine Aqueous solution; under the condition of magnetic stirring, add the protamine aqueous solution dropwise to the rhGH aqueous solution, and mix evenly to obtain the suspension of rhGH complex; centrifuge the suspension of rhGH complex, remove the supernatant, namely The rhGH complex was obtained. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition, wherein the weight ratio of rhGH and protamine in the rhGH sustained-release composition is 0.1% and 0.1%, respectively.

Example 2

Accurately weigh 200 mg of PLGA1200-PEG1000-PLGA1200 (both number average molecular weight, 29.4% by weight of PEG block, 70.6% by weight of PLGA block, LA/GA molar ratio of 3:1), add 0.80ml of Deionized water was dissolved under magnetic stirring at 15°C to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 20°C. Accurately weigh 1mg of recombinant human growth hormone, add 0.3ml of water to dissolve to obtain rhGH aqueous solution; accurately weigh 1mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)), add 0.3ml of water to dissolve, to obtain protamine Aqueous solution; add the protamine aqueous solution dropwise to the rhGH aqueous solution under stirring, and mix evenly to obtain a suspension of rhGH complex; centrifuge the suspension of rhGH complex and remove the supernatant to obtain rhGH complex thing. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition, wherein the weight ratio of rhGH and protamine in the rhGH sustained-release composition is 0.1% and 0.1%, respectively.

Example 3

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 1mg of recombinant human growth hormone, add 0.3ml of water to dissolve, to obtain rhGH aqueous solution; accurately weigh 1mg of zinc acetate, add 0.3ml of water to dissolve to obtain zinc acetate aqueous solution; accurately weigh 1mg of protamine (Tokyo) Chemical Industry Co., Ltd. (TCI)), add 0.3ml of water to dissolve to obtain the protamine aqueous solution; add the zinc acetate aqueous solution and the protamine aqueous solution dropwise to the rhGH aqueous solution under stirring, respectively, and mix well to obtain the rhGH complex The suspension of rhGH complex was centrifuged, and the supernatant was removed to obtain rhGH complex. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition, wherein the weight ratios of rhGH, protamine and zinc acetate in the rhGH sustained-release composition are 0.1%, 0.1%, and 0.1%, respectively.

Example 4

Except that the amount of protamine was 5 mg, other operations were the same as in Example 1, to obtain an rhGH sustained-release composition, wherein the weight ratios of rhGH and protamine in the rhGH sustained-release composition were 0.1% and 0.5%, respectively.

Example 5

Except that the amount of protamine was 10 mg, other operations were the same as those in Example 1, to obtain an rhGH sustained-release composition, wherein the weight ratios of rhGH and protamine in the rhGH sustained-release composition were 0.1% and 1.0%, respectively.

Example 6

In addition to replacing 200 mg of PLGA1500- Except PEG1450-PLGA1500 (both are number average molecular weight, the weight percent of PEG block is 32.6%, the weight percent of PLGA block is 67.4%, and the mol ratio of LA/GA is 3:1), other operations are the same as in Example 3, The rhGH sustained-release composition is obtained, wherein the weight ratios of rhGH, protamine and zinc acetate in the rhGH sustained-release composition are 0.1%, 0.1% and 0.1% respectively.

Example 7

Except that the consumption of rhGH is 5mg, the consumption of protamine is 5mg, and the consumption of zinc acetate is 5mg, other operations are the same as in Example 3, to obtain rhGH sustained-release composition, wherein rhGH, protamine and zinc acetate account for rhGH slow-release composition. The weight ratios of the release compositions were 0.5%, 0.5%, and 0.5%, respectively.

Example 8

Except for replacing rhGH with insulin, the rest of the operations are the same as in Example 3 to obtain an insulin sustained-release composition, wherein the insulin, protamine and zinc acetate account for 0.1%, 0.1%, and 0.1% by weight of the insulin sustained-release composition, respectively. .

Example 9

Except for replacing rhGH with OVA, other operations are the same as in Example 3, to obtain an OVA sustained-release composition, wherein the weight ratios of OVA, protamine and zinc acetate in the OVA sustained-release composition are 0.1%, 0.1%, and 0.1%, respectively. .

Example 10

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 5mg of recombinant human growth hormone, add 0.30ml of water to dissolve, obtain rhGH aqueous solution; accurately weigh 1mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)), add 0.30ml of water to dissolve, to obtain protamine The aqueous solution of protamine is added dropwise to the rhGH aqueous solution under stirring, and the mixture is evenly mixed to obtain a suspension of the rhGH complex; the suspension of the rhGH complex is freeze-dried to obtain a freeze-dried powder of the rhGH complex. The rhGH complex freeze-dried powder and the temperature-sensitive polymer aqueous solution are mixed uniformly to obtain the rhGH sustained-release composition, wherein the weight ratio of rhGH and protamine in the rhGH sustained-release composition is 0.5% and 0.1%, respectively.

Example 11

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 3 mg of recombinant human growth hormone and dissolve it in 8 mL of water to obtain an aqueous solution of rhGH; accurately weigh 3 mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)) and dissolve it in 8 mL of water to obtain an aqueous solution of protamine. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, vigorously stirred for 5 minutes, and then slowly stirred for 1.5 hours to obtain a protamine-rhGH complex solution. To the protamine-rhGH complex solution, add 2 mL of 70 mM NaH ₂ PO ₄ (16.8 mg) aqueous solution, then add 2 mL of 250 mM CaCl ₂ (55.5 mg) aqueous solution dropwise, stir vigorously for 10 minutes, then slowly stir for 1 hour, and finally centrifuge to obtain rhGH Complex. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition. The weight ratios of rhGH, protamine, NaH ₂ PO ₄ and CaCl ₂ in the rhGH sustained-release composition were 0.28%, 0.28%, 1.56%, and 5.15%, respectively.

Example 12

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 3 mg of recombinant human growth hormone and dissolve it in 8 mL of water to obtain an aqueous solution of rhGH; accurately weigh 3 mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)) and dissolve it in 8 mL of water to obtain an aqueous solution of protamine. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, vigorously stirred for 5 minutes, and then slowly stirred for 1.5 hours to obtain a protamine-rhGH complex solution. Add 2mL 70mM Na ₂ HPO ₄ (19.9mg) aqueous solution to the protamine-rhGH complex solution, then add 2mL 250mM CaCl ₂ (55.5mg) aqueous solution dropwise, stir vigorously for 10 minutes, then slowly stir for 1 hour, and finally centrifuge to obtain rhGH complex thing. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition. The weight ratios of rhGH, protamine, Na ₂ HPO ₄ and CaCl ₂ in the rhGH sustained-release composition were 0.28%: 0.28%: 1.84% and 5.13%, respectively.

Example 13

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 3 mg of recombinant human growth hormone and dissolve it in 8 mL of water to obtain an aqueous solution of rhGH; accurately weigh 3 mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)) and dissolve it in 8 mL of water to obtain an aqueous solution of protamine. The protamine aqueous solution was added dropwise to the rhGH aqueous solution, vigorously stirred for 5 minutes, and then slowly stirred for 1.5 hours to obtain a protamine-rhGH complex solution. To the protamine-rhGH complex solution, 2 mL of 70 mM (mmol/L) Na ₂ CO ₃ (14.8 mg) aqueous solution was added, and then 2 mL of 250 mM CaCl ₂ (55.5 mg) aqueous solution was added dropwise, followed by vigorous stirring for 10 minutes, followed by slow stirring for 1 hour , and finally centrifuged to obtain the rhGH complex. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition. The weight ratios of rhGH, protamine, Na ₂ CO ₃ and CaCl ₂ in the rhGH sustained-release composition were 0.28%, 0.28%, 1.38% and 5.16%, respectively.

Example 14

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Accurately weigh 1 mg of recombinant human growth hormone, add 0.30 ml of water to dissolve, and obtain an aqueous solution of rhGH; accurately weigh 10 mg of ethyl lauroyl arginine, add 0.30 ml of water to dissolve, and obtain an aqueous solution of ethyl lauroyl arginine; The aqueous solution of ethyl lauroyl arginine is added dropwise to the rhGH aqueous solution under stirring, and mixed evenly to obtain a suspension of rhGH complex; the suspension of rhGH complex is centrifuged to obtain rhGH complex. The rhGH complex and the temperature-sensitive polymer aqueous solution are uniformly mixed to obtain the rhGH sustained-release composition. The weight ratios of rhGH and lauroyl arginine ethyl ester in the rhGH sustained-release composition were 0.1% and 1%, respectively.

Example 15

Except for replacing "2mL 70mM Na ₂ HPO ₄ (19.9 mg) aqueous solution" with "2mL 70 mM Na ₃ PO ₄ (23.0 mg) aqueous solution", other operations were the same as in Example 12 to obtain the rhGH sustained-release composition. The weight ratios of rhGH, protamine, Na ₃ PO ₄ and CaCl ₂ in the rhGH sustained-release composition were 0.28%: 0.28%: 2.12% and 5.12%, respectively.

Comparative Example 1

Accurately weigh 5mg of recombinant human growth hormone, add 0.50ml of water to dissolve to obtain rhGH aqueous solution; accurately weigh 1mg of protamine (Tokyo Chemical Industry Co., Ltd. (TCI)), add 0.50ml of water to dissolve, to obtain protamine Aqueous solution; add the protamine aqueous solution dropwise to the rhGH aqueous solution under stirring, and mix evenly to obtain a suspension of rhGH complex; centrifuge the suspension of rhGH complex and remove the supernatant to obtain rhGH complex thing. The weight ratios of rhGH and protamine in the suspension of the rhGH complex were 0.5% and 0.1%.

Comparative Example 2

Accurately weigh 5mg of recombinant human growth hormone, add 0.33ml of water to dissolve, to obtain rhGH aqueous solution; accurately weigh 1mg of zinc acetate, add 0.33ml of water to dissolve, obtain zinc acetate aqueous solution; accurately weigh 1mg of protamine (Tokyo) Chemical Industry Co., Ltd. (TCI)), add 0.33ml of water to dissolve to obtain an aqueous solution of protamine; add the aqueous solution of zinc acetate and the aqueous solution of protamine dropwise to the rhGH aqueous solution under stirring, and mix well to obtain the rhGH complex The suspension of rhGH complex was centrifuged, and the supernatant was removed to obtain rhGH complex. The weight ratios of rhGH, zinc acetate and protamine in the suspension of the rhGH complex are 0.5%, 0.1% and 0.1%.

Comparative Example 3

Precisely weigh 200 mg of PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80 ml of deionized water, dissolved under magnetic stirring at 15°C, to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Precisely weigh 5 mg of recombinant human growth hormone, mix it with the temperature-sensitive polymer aqueous solution uniformly, and prepare a sustained-release composition in which the weight ratio of rhGH to the sustained-release composition is 0.5%.

Comparative Example 4

Accurately weigh 200 mg of PLGA1200-PEG1000-PLGA1200 (both number average molecular weight, 29.4% by weight of PEG block, 70.6% by weight of PLGA block, LA/GA molar ratio of 3:1), add 0.80ml of Deionized water was dissolved under magnetic stirring at 15°C to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 20°C. Precisely weigh 5 mg of recombinant human growth hormone, mix it with the temperature-sensitive polymer aqueous solution uniformly, and prepare a sustained-release composition in which the weight ratio of rhGH to the sustained-release composition is 0.5%.

Comparative Example 5

Precisely weigh 200mg PLGA1500-PEG1450-PLGA1500 (all are number average molecular weight, the weight percentage of PEG block is 32.6%, the weight percentage of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), add 0.80ml to Ionized water was dissolved under magnetic stirring at room temperature to obtain a temperature-sensitive polymer aqueous solution with a concentration of 20% (w/w), and the LCST was 32°C. Precisely weigh 1 mg of recombinant human growth hormone, add 0.3 ml of water to dissolve, to obtain rhGH aqueous solution; accurately weigh 1 mg of zinc acetate, add 0.3 ml of water to dissolve to obtain zinc acetate aqueous solution; under the condition of magnetic stirring, add zinc acetate aqueous solution dropwise into the rhGH aqueous solution, and mix evenly to obtain a suspension of the rhGH complex; centrifuge the suspension of the rhGH complex, and remove the supernatant to obtain the rhGH complex. The sustained-release composition is obtained by uniformly mixing the rhGH complex and the temperature-sensitive polymer aqueous solution, wherein the weight ratios of rhGH and zinc acetate in the sustained-release composition are 0.1% and 0.1% respectively.

Comparative Example 6

In addition to replacing 200 mg of PLGA1500- Except for PEG1450-PLGA1500 (both number-average molecular weight, the weight percent of PEG block is 32.6%, the weight percent of PLGA block is 67.4%, the molar ratio of LA/GA is 3:1), other operations are the same as Comparative Example 5, A sustained-release composition was obtained, wherein the weight ratios of rhGH and zinc acetate in the sustained-release composition were 0.1% and 0.1%, respectively.

Table 1 embodiment and comparative example preparation composition summary

Drug release test: The dialysis method was used to investigate the drug release behavior of each sample prepared in Examples 1-15 and Comparative Examples 1-6. The steps were as follows: Weigh an appropriate amount of the sample into a Spectra/Por dialysis tube (MWCO100KDa), and the drug release medium was 10 mL of pH 7.4 phosphate buffer, the dialysis tube was placed in a constant temperature shaker, and the parameters of the constant temperature shaker were set: the temperature was 37°C, and the rotation speed was 100rpm. Accurately draw 2 ml of phosphate buffer from the drug release system at different time points, and then add the same volume of phosphate buffer release medium to the release system. The drug concentration of polypeptide and protein in the sample is detected by high performance liquid phase method (for example, Chinese Pharmacopoeia 2015 edition). The experimental results are shown in Figures 1-4 and Table 2.

Table 2 embodiment and comparative example drug release data summary

In the table, * means 0.01<P<0.05, # means P<0.01; 0.01<P<0.05 means significant difference; P<0.01 means extremely significant difference.

By comparing the release curves of the polypeptide-protein drug sustained-release composition preparation of the present invention with the polypeptide-protein complex and the polypeptide-protein thermosensitive gel mixture in the comparative example, it can be seen that the sustained-release composition preparation of the embodiment of the present invention has no burst release phenomenon, The sustained-release effect is excellent, the sustained-release rate is stable, and the cumulative release amount at 6 days and 20 days is significantly lower than that of the corresponding comparative formulations; Example 5, Example 7, Example 8, Example 9 and Example 10 were compared with Comparative Example 3 and Comparative Example 5, respectively. The sustained release rate of the example preparation was stable, which was obviously better than that of the comparative example preparation, and the example preparation was 6 days. The cumulative release of the preparations was significantly lower than that of the comparative preparations (P<0.05), and the cumulative release (long-acting sustained release) of the example preparations for 20 days was significantly lower than that of the comparative preparations (P<0.05); Example 12, Example 13, Example 14 and Example 15 were compared with Comparative Example 3, respectively. The release of the Example formulation was significantly slower than that of the Comparative Example formulation, and the release rate of the Example formulation was significantly higher than that of the Comparative Example formulation. More stable, the cumulative release amount of the example preparation for 6 days was significantly lower than that of the comparative example preparation (P<0.05), and the cumulative release amount (long-acting sustained release) of the example preparation for 20 days was significantly lower than that of the comparative example preparation (P<0.05). ); Comparing Example 2 and Example 6 with the same temperature-sensitive polymer material with Comparative Example 4 and Comparative Example 6, the sustained-release rate of the example preparation is stable, which is obviously better than that of the comparative example preparation, and the cumulative release of the example preparation for 6 days The doses of the preparations were significantly lower than those of the comparative preparations (P<0.05), and the cumulative release amount of the example preparations in 20 days was significantly lower than that of the comparative preparations (P<0.05). Among them, the cumulative release amount of Comparative Example 1 and Comparative Example 2 in one day exceeded 80%. Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 can prolong the release of protein due to the formation of thermosensitive gel, but slow release The speed is not stable enough. Compared with the preparation of the comparative example, the sustained-release composition preparation of Examples 1-15 obtained by adding the composite reagent for composite preparation in the present invention has better sustained-release effect, and can significantly improve the sustained-release effect of the sustained-release preparation of polypeptide and protein drugs. The stable release rate is mainly based on the synergistic effect of the temperature-sensitive polymer and the composite reagent, so as to realize the precise and controllable sustained release of polypeptide and protein drugs, and can effectively reduce the burst release.

Animal experiment: 200g-250g female SD rats were used as experimental animals, 300μl of the sustained-release composition prepared in the example was injected subcutaneously, blood was collected from the orbit at a certain time point, and the blood samples were centrifuged to obtain serum. The drug concentration in serum was tested with ab190811 human growth hormone ELISA kit, and the drug-time curve was obtained. The control group is the sustained-release composition prepared in Comparative Example 5, and the subcutaneous injection dose is 300 μl. The experimental results are shown in Figure 5.

Taking growth hormone as an example, literature and clinical data show that the plasma concentration (Cp) of growth hormone can be maintained at the level of 5ng/ml to achieve an effective clinical therapeutic effect (Cleland, Jeffrey L., et al. "Recombinant humangrowth hormone poly( lactic-co-glycolic acid) (PLGA) microspheres provide a longlasting effect." Journal of Controlled Release 49.2-3(1997):193-205.). The experimental results show that the composition of the embodiment of the present invention can realize the controllable sustained release of growth hormone in animals, and the better sustained release effect is in Example 3 and Example 4, which can maintain the blood drug concentration above 5ng/ml level for as long as possible. For 14 days, the effective therapeutic concentration can be maintained for a long time, and an effective clinical therapeutic effect can be achieved. Example 1 can maintain the blood concentration above the 5ng/ml level for 14 days, and Examples 2, 5, 6, 7, 10, 11, 12, 13, 14 and 15 can maintain the blood concentration at 5ng/ml The number of days above the level is 7 days to 10 days, and the effective therapeutic concentration can also be maintained for a long time, and an effective clinical therapeutic effect can be achieved. As a control, the effective concentration of Comparative Example 5 (the combination of thermosensitive gel and growth hormone/zinc complex) in rats decreased to below the therapeutic concentration after 2-3 days of release, and the effective therapeutic concentration could not be maintained for a long time. The release of polypeptide and protein drugs in animals is affected by the pore size of the gel and the stability of the complex. The combination of the complex reagent and the growth hormone can change the dissociation behavior of the growth hormone, so it can significantly prolong the human growth hormone in the animal body. slow release effect.

Examples 8 and 9 can maintain the effective therapeutic concentration for more than one week, and can achieve effective clinical therapeutic effect.

In the present invention, the combination of the composite reagent and the polypeptide and protein drugs such as growth hormone, insulin and OVA can change the dissociation behavior of the polypeptide and protein drugs. At the same time, the synergistic effect of the temperature-sensitive polymer and the composite reagent can significantly prolong the sustained-release effect of the polypeptide and protein drugs in animals, and the sustained-release rate is stable. Under the premise of not affecting the efficacy of polypeptide and protein drugs, the precise and controllable sustained release of polypeptide and protein drugs can be achieved, the number of drug injections can be effectively reduced, the patient's compliance can be improved, and an effective therapeutic concentration can be maintained for a long time. The change of the parameters in the preparation method of the present invention does not affect the preparation of the sustained-release composition preparation of polypeptide protein drugs, so any combination of parameters in the preparation method of the present invention can realize the preparation of the sustained release composition preparation of polypeptide protein drugs. It is not repeated here.

Claims (10)

1. the sustained-release composition preparation of a polypeptide protein drug, is characterized in that, is made by polypeptide protein drug, composite reagent and temperature-sensitive polymer aqueous solution; The composite reagent is the composition of protamine and metal salt, the composition of protamine and sodium phosphate and calcium chloride, the composition of protamine and sodium carbonate and calcium chloride, protamine or lauroyl Arginine ethyl ester; The metal salt is a pharmaceutically acceptable metal salt that dissociates into a divalent metal cation in an aqueous solution; the divalent metal cation is a zinc ion; The temperature-sensitive polymer is one or more of PLA-PEG-PLA and PLGA-PEG-PLGA. 2. The sustained-release composition preparation of polypeptide and protein drugs according to claim 1, wherein the consumption of the protamine is 0.1%-5% by weight of the sustained-release composition preparation; And/or, the amount of the metal salt is 0.1% to 10% by weight of the sustained-release composition preparation; Alternatively, the total dosage of the sodium phosphate and calcium chloride is 0.5% to 7.5% by weight of the sustained-release composition preparation; wherein, the weight ratio of the sodium phosphate to the calcium chloride is 1:2 to 4; Or, the total dosage of the sodium carbonate and calcium chloride is 0.5% to 7.5% by weight of the sustained-release composition preparation; wherein, the weight ratio of sodium carbonate to calcium chloride is 1:2 to 4; Alternatively, the amount of the lauroyl arginine ethyl ester is 0.5% to 5% by weight of the sustained-release composition preparation. 3. The sustained-release composition preparation of polypeptide and protein drugs according to claim 1 or 2, wherein the metal salt is zinc acetate; the consumption of the zinc acetate is 0.1% by weight of the sustained-release composition preparation ~3%. 4. The sustained-release composition preparation of polypeptide and protein drugs according to 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 polypeptide and protein drugs according to claim 1 , wherein the dosage of the polypeptide and protein drugs is 0.1% to 5% by weight of the sustained-release composition preparation. 6 . 6. The sustained-release composition preparation of polypeptide and protein drugs according to claim 1 or 5, wherein the polypeptide and protein drugs comprise insulin, insulin analogs, interferon, ovalbumin, recombinant human growth hormone , one or more of glucagon-like peptides, glucagon-like peptide analogs, immunoglobulins, programmed death receptor 1, and programmed cell death-ligands. 7 . The sustained-release composition preparation of polypeptide and protein drugs according to claim 1 , wherein the minimum critical dissolution temperature of the temperature-sensitive polymer aqueous solution is 20° C.˜35° C. 8 . 8 . The sustained-release composition preparation of polypeptide and protein drugs according to claim 1 , wherein the temperature-sensitive polymer aqueous solution has a weight percentage concentration of 10% to 30%. 9 . 9 . The sustained-release composition preparation of polypeptide and protein drugs according to claim 1 , wherein the number-average molecular weight of the PEG block in the temperature-sensitive polymer is 1000-2000, and 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 preparation method of the sustained-release composition preparation of polypeptide and protein drugs according to any one of claims 1 to 9, characterized in that, comprising the following steps: (1) drop the compound reagent aqueous solution into the polypeptide and protein drug aqueous solution, and mix evenly to obtain the polypeptide protein drug complex suspension, and centrifuge to remove the supernatant or freeze-dry to obtain the polypeptide protein drug complex; (2) Mixing the temperature-sensitive polymer aqueous solution with the polypeptide-protein drug complex in step (1) uniformly to obtain a sustained-release composition preparation of the polypeptide-protein drug.

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