In-situ phase change gel sustained release preparation and preparation method and application thereof

Abstract

The invention provides an in-situ injection phase change gel sustained-release preparation as well as a preparation method and application thereof, and particularly relates to a stable injectable gel sustained-release preparation prepared from phospholipid, oil for injection and active pharmaceutical ingredients, and the stable injectable gel sustained-release preparation has the characteristics of good biocompatibility, remarkable sustained-release effect, wide application range, simplicity in preparation and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to an in-situ injectable phase change gel sustained-release formulation, specifically to a stable injectable gel sustained-release formulation prepared from phospholipids and injectable oils, which falls within the field of pharmaceutical technology. Background Technology

[0002] Proteins exhibit good selectivity for human-specific targets. Their larger molecular size and diverse structures enhance their interaction with specific protein pockets, resulting in protein drugs with better bioactivity and lower toxicity compared to smaller molecules. Currently, protein drugs are widely used to treat major diseases such as cancer, cardiovascular and cerebrovascular diseases, immune diseases, neurodegenerative diseases, and metabolic diseases. However, protein drugs are still difficult to administer non-injectable routes because they are extremely unstable in the environment of pepsin and gastric acid and have difficulty crossing the complex gastrointestinal barrier. This results in very low bioavailability when administered orally. Subcutaneous, intramuscular, or intravascular administration is also problematic because the in vivo half-life of proteins is usually very short, requiring frequent dosing to maintain therapeutic concentrations. This not only leads to low compliance and high costs but also causes other adverse reactions. For example, when using anti-vascular endothelial growth factor (VEGF) drugs to treat neovascular fundus diseases, frequent intravitreal injections are required, which can easily induce complications such as cataracts, retinal hemorrhage, and detachment. These problems limit the bioactivity and efficacy of protein drugs. Therefore, developing injectable, long-acting, sustained-release formulations for protein drugs is an important means to improve bioavailability, facilitate medication, stabilize blood drug concentrations, reduce toxic side effects, and enhance patient compliance.

[0003] Currently, various injectable sustained-release systems have been designed and manufactured to release drugs. Among them, microspheres are the most researched and successfully marketed injectable long-acting sustained-release formulations, with PLGA (polylactic acid-glycolic acid copolymer) microspheres being the most widely used. When microspheres are injected subcutaneously or intramuscularly, the drug is slowly released (over several weeks to months) as the matrix material hydrolyzes and dissolves, exerting its therapeutic effect in the body for a long time, thereby reducing the frequency of dosing, stabilizing drug release to reduce drug exposure-related side effects, and improving patient compliance. Protein drugs have complex chemical structures and spatial conformations, poor stability, and are prone to structural changes, resulting in reduced activity and increased immunogenicity. Currently, there are no long-acting protein-loaded microspheres on the market. On the one hand, the preparation of microspheres often requires ultrasonic treatment and the use of organic solvents, which can affect the protein structure, causing physicochemical changes such as aggregation, adsorption, precipitation, oxidation, and hydrolysis. On the other hand, the acidic environment generated by microsphere degradation may cause local inflammatory reactions and reduce protein stability. These disadvantages limit the application of PLGA microspheres.

[0004] Multivesicular liposomes (MVLs) are a type of sustained-release formulation first discovered and studied by Kim's team at the University of Washington in 1983. Using phospholipids, cholesterol, and neutral lipids as encapsulation materials, they are spherical formulations composed of multiple non-concentric aqueous chambers, or vesicles. As the vesicles rupture one by one, the drug is released, achieving sustained release. Compared to traditional liposomes, MVLs have advantages such as high encapsulation efficiency and low drug leakage, making them suitable for encapsulating water-soluble small molecule drugs and bioactive macromolecules. One relatively successful and commercially available MVL is the bupivacaine liposome injection suspension (trade name: Exparal) prepared by Pacira using DepoFoam multivesicular liposome technology, which can continuously release the drug for up to 72 hours. Patent CN102274183B reports a method for preparing and applying multivesicular liposomes, which encapsulates exenatide and provides sustained in vitro release for 160 hours. Patent CN110339166B reports a liraglutide multi-capsule liposome that exhibits a hypoglycemic effect for up to 312 hours in vivo. Although multi-capsule liposomes achieve a good sustained-release effect, they still have several shortcomings: First, the preparation of MVLs requires at least amphiphilic lipids (such as phospholipids), neutral lipids (such as triglycerides), emulsifiers, and osmotic pressure regulators, making the formulation and preparation process complex and leaving residual organic solvents. Second, the drug concentration difference between the internal and external aqueous phases of MVLs persists, leading to drug permeation issues. Furthermore, MVLs are prone to sedimentation and aggregation during storage, affecting formulation stability and making them inconvenient for transportation and preservation. These drawbacks limit the development of MVLs.

[0005] Vesicular phospholipid gels (VPGs) are also under investigation. VPGs are semi-solid phospholipid dispersions, morphologically similar to vesicles, but distinct from traditional liposome gels and ordinary liposome compartment structures. Due to their unique three-dimensional network structure, VPGs can serve as drug reservoirs, enabling sustained drug release. Patents CN102626381A, CN102697741A, and CN102716095A have reported vesicular phospholipid gel injections of cefoperazone sodium, oxaliplatin, and paclitaxel, respectively, but the sustained-release time of these formulations does not exceed 24 hours, indicating a relatively short release period.

[0006] Our laboratory has developed an in-situ phase change gel sustained-release system (CN102526753A) using high-concentration phospholipids as the main matrix and ethanol as the solvent. It has advantages such as good biocompatibility, significant sustained-release effect, and good in vivo degradation. It has good sustained-release effect when applied to protein and peptide drugs. For example, octreotide acetate can be stably sustained-released for about a month in rats, rabbits and dogs with very little drug burst release, which is better than commercially available octreotide acetate microspheres (MX Wang, et al. Pharmacokinetic and pharmacodynamic study of a phospholipid-based phaseseparation gel for once a month administration of octreotide, Journal of Controlled Release 230 (2016) 45–56); exenatide acetate phospholipid gel has almost no burst release and can be sustained-released for up to a month in rats, maintaining a stable hypoglycemic effect for more than 20 days (M Hu, et al. Long-Acting Phospholipid Gel of Exenatide for Long-Term Therapy of Type II Diabetes, Pharmaceutical research 33 (2016): 1318–1326). In existing technologies, including CN102526753A, common solvents used in phospholipid gel systems are organic solvents such as ethanol and NMP. The reason for using these organic solvents is not only because of their good solubility for various drugs and excipients, but also because of their good biocompatibility and safety. However, in our research, we also found that, on the one hand, these phospholipid gel formulations using ethanol and NMP as solvents still inevitably cause irritation to a certain extent, leading to excessive inflammation at the injection site. On the other hand, as those skilled in the art know, organic solvents such as ethanol are common chemical factors that cause protein degradation and inactivation. The presence of organic solvents such as ethanol makes it easy for protein drugs to be adsorbed and aggregated, thereby reducing stability and efficacy, and is also not conducive to the transportation and storage of the formulation.

[0007] Therefore, for the development of long-acting formulations of protein drugs, ensuring the stability of protein drugs in the formulation, improving the preparation efficiency of protein drug sustained-release systems, reducing local irritation, and achieving excellent sustained-release effects are urgent technical problems to be solved in terms of preparation process and formulation. Summary of the Invention

[0008] The purpose of this invention is to overcome the defects of the existing technology and provide an in-situ injection phase change gel sustained-release formulation, its preparation method and application.

[0009] The research unexpectedly revealed that phospholipids, when mixed with injectable ethyl oleate and / or ethyl linoleate under certain temperature conditions, can form a clear, transparent, and homogeneous system. This system remains clear even after cooling to room temperature and exhibits good fluidity, making it a suitable replacement for organic solvents in existing technologies. The resulting gel system has a suitable viscosity for injection and good biocompatibility. Furthermore, through extensive experimentation, the inventors discovered that for protein drugs, ethyl oleate and / or ethyl linoleate, as solvents in phospholipid gel systems, achieve significantly superior technical effects compared to other similar injectable oils. This significantly improves the stability of protein drugs and can replace commonly used organic solvents such as ethanol and NMP in existing gel-type drugs, achieving unexpected technical benefits. When this system encounters a small amount of bodily fluid, its fluidity decreases significantly, and it solidifies. Dissolving or dispersing the drug in this system and injecting it into the body allows the bodily fluid to penetrate, reducing the viscosity of the formulation at the injection site and forming a semi-solid gel. This gel acts as a carrier and barrier for sustained drug release, effectively controlling drug release.

[0010] Based on the aforementioned innovative findings, using recombinant human coagulation factor VIII (a protein-based drug) as a model drug, we prepared a formulation with a phospholipid to ethyl oleate ratio of 1:1 (w:w). After subcutaneous injection of 0.94 mL into rats, the drug was released for over 384 hours. Compared to intravenous injection of recombinant human coagulation factor VIII, the half-life was significantly extended. Under the same administration method, the in-situ injection phase change gel sustained-release formulation showed significantly improved bioavailability, almost no irritation to the injection site, and significantly increased stability.

[0011] One objective of this invention is to provide an in-situ injection phase change gel sustained-release system prepared from phospholipids, injectable oils, and pharmaceutical active ingredients. This system does not use organic solvents, has extremely low local irritation, and greatly improves drug stability. The pharmaceutical active ingredient is a protein.

[0012] One of the objectives of this invention is to provide a phospholipid gel sustained-release formulation containing protein-based active ingredients that has a wide range of applications, undergoes a rapid phase transition after injection, achieves long-lasting sustained release, and is easy to inject.

[0013] The phospholipid gel sustained-release formulation of the present invention comprises, by weight, 10-60 parts phospholipid, 45-90 parts injectable oil, and 0.0025-20 parts of active pharmaceutical ingredient. The active pharmaceutical ingredient is a protein.

[0014] In the phospholipid gel sustained-release formulation system, the ratio of phospholipid to injectable oil in the carrier is 2:8 to 6:4 (w:w), preferably 3:7 to 5.5:4.5 (w:w), and more preferably 3:7 to 5:5 (w:w).

[0015] In the phospholipid gel sustained-release formulation system, the content of injectable oil, by weight percentage, is 40% to 90%, preferably 45% to 80%, more preferably 50% to 70%, and even more preferably 50% to 60%.

[0016] The phospholipids suitable for the phospholipid gel sustained-release formulation of the present invention include, but are not limited to, one or more combinations of natural phospholipids, semi-synthetic phospholipids, and synthetic phospholipids.

[0017] The natural phospholipids are selected from egg yolk lecithin, soybean phospholipids, or combinations thereof; the semi-synthetic phospholipids are selected from hydrogenated egg yolk lecithin, hydrogenated soybean phospholipids, or combinations thereof; the synthetic phospholipids are selected from one or more combinations of dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, myristoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylglycerol, and dipalmitoyl phosphate.

[0018] In a specific implementation, the present invention preferably uses soybean lecithin S100 and / or egg yolk lecithin E80.

[0019] The injectable oil suitable for the phospholipid gel sustained-release formulation of the present invention includes one or a combination of ethyl oleate and ethyl linoleate. Preferably, the injectable oil is ethyl oleate.

[0020] Proteins suitable for preparing the phospholipid gel sustained-release formulation of the present invention include, but are not limited to, insulin, glucagon, α-interferon, β-interferon, pegylated interferon, interleukin, erythropoietin (EPO), colony-stimulating factor (GSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), adenosine deaminase, β-glucocerebrosidase, α-galactosidase, tissue plasminogen activator, streptokinase, urokinase, antithrombin, asparaginase, and uric acid. Enzymes, trypsin, afotazone α, cell growth factors, coagulation factors, prostaglandins, botulinum toxin, rituximab, reperatumab, omalizumab, denosumab, trastuzumab, inetuzumab, tocilizumab, bevacizumab, infliximab, adalimumab, etanercept, CD25 antibody, adebenonemab, sugelizumab, envorimab, putelizumab, slulizumab, cepalimumab, penaprip, tislelizumab, camrelizumab, sintilimab, toripalimab, candunizumab, vedipicatumab, telitacicept, zabetoumab, oxotimib, ambavirin, roximatemab Misvir / Mexicoside, Conbercept, Nimotuzumab, Iodine-131 Tumor Cell Nuclear Antibody, CD3 Antibody, IL-8 Antibody, Iodine-131 Metuximab, Tirecizumab, Detrastuzumab, Verporutuzumab, Pesolizumab, Nacetuzumab, Moglizumab, Goxatozumab, Ramosinumab, Inellizumab, Imalimumab, Ofatuzumab, Oga-Itozumab, Sesticumab, Meppolizumab, Datoximab, Ipilimumab, Oxetuzumab, Sattriumab, Brosoxuromumab, Lanalimumab, Belintoumab, Dupremumab, Bropremumab, Brentuximab Verdelizumab, atezolizumab, trastuzumab emtansine, abatacept, alixirozumab, gusetizumab, belimumab, durvalumab, ichzumab, pecelizumab, daratumumab, secukinumab, pertuzumab, emecizumab, ikuzumab, evolotumumab, pembrolizumab, nivolumab, edasizumab, golimumab, baliximab, cetuximab, ustekinumab, ranibizumab, dalizumab, aflibercept, gemtozumab, polotumumab, trastuzumab, enrofloxacin, rontoximab, tesoximab, and somituzumab.

[0021] Preferably, the protein is selected from one or more of insulin, coagulation factors, pembrolizumab, adalimumab, dupilumab, and aflibercept.

[0022] Preferably, the protein is selected from recombinant human coagulation factor VIII.

[0023] Furthermore, the in-situ phase change gel of the present invention may also contain pharmaceutically acceptable excipients.

[0024] Furthermore, the pharmaceutically acceptable excipients are selected from one or more of the following: preservatives, surfactants, antioxidants, and metal ion chelating agents.

[0025] Furthermore, the in-situ injection phase change gel sustained-release system of the present invention does not contain organic solvents.

[0026] Furthermore, the organic solvent is selected from one or more of methanol, ethanol, propylene glycol, tert-butanol, dimethyl sulfoxide, and N,N-dimethylpyrrolidone.

[0027] Furthermore, the in-situ injection phase change gel sustained-release system of the present invention is preferably used in the preparation of drugs for treating metabolic diseases, tumors, and cardiovascular and cerebrovascular diseases.

[0028] The in-situ injection phase change gel sustained-release system of the present invention is a novel dosage form in which the active pharmaceutical ingredient can be dissolved or dispersed in a composition of phospholipids and injectable oil to become a free-flowing injectable formulation. After injection into the body, body fluids permeate into the formulation, causing the formulation at the injection site to immediately form a semi-solid gel encapsulating the active pharmaceutical ingredient, thus exhibiting a good sustained-release effect.

[0029] The in-situ injection phase change gel sustained-release system of the present invention can also preserve the active pharmaceutical ingredient and the blank phospholipid injection oil matrix separately, and dissolve or disperse them evenly before administration.

[0030] In a specific implementation scheme, the preparation method of the present invention includes the following steps:

[0031] (1) Take phospholipids and injectable oil and mix them at 0 to 120°C to completely dissolve the phospholipids. The mixing method is selected from one or more combinations of stirring, vortexing and ultrasound. The mixing temperature is preferably 30°C to 60°C to obtain a clear and transparent phospholipid gel.

[0032] (2) Take the active pharmaceutical ingredient and prepare drug microparticles by common pharmaceutical crystallization or pulverization methods;

[0033] (3) Mix the phospholipid gel from step (1) and the drug particles from (2) evenly, let stand for a while to remove air bubbles in the preparation, dispense, seal, and obtain the final product.

[0034] The in-situ injectable phase change gel sustained-release formulation of the present invention can be administered by the following methods: subcutaneous injection, intramuscular injection, intracavitary injection through tissue, and intracavitary injection into an open body cavity without penetrating the tissue.

[0035] This invention, through inventive research, uses phospholipids and injectable oils as main raw materials, and recombinant human coagulation factor VIII (a protein drug) as a model drug. Through simple mixing, a free-flowing, easily injectable, in-situ injectable phase change gel sustained-release formulation is obtained. In vivo animal experiments have demonstrated its good sustained-release effect. Its irritation to the injection site was investigated, and the results show that the formulation has good biocompatibility and does not cause irritation to the administration site.

[0036] This invention provides an in-situ gel formulation that can universally achieve sustained release of various drugs. It can effectively solve the problems of local irritation and decreased drug stability caused by existing in-situ drug gel formulations, and has good application prospects.

[0037] The in-situ phase change gel sustained-release formulation provided by this invention has the following significant advantages:

[0038] (1) It has good fluidity and is easy to inject. After being injected into the body, the body fluid seeps in and causes the preparation at the injection site to solidify rapidly and undergo a phase change, forming a semi-solid gel, which becomes a reservoir for drug release and slowly releases the drug.

[0039] (2) The raw and auxiliary materials used have good biocompatibility and biodegradability, do not contain organic solvents, and will hardly cause irritation to the administration site.

[0040] (3) It has a wide range of applications and can significantly improve the stability of active drugs. Drugs of different properties are not easily degraded.

[0041] (4) The preparation method is simple and easy to industrialize. Attached Figure Description

[0042] Figure 1 The images show the appearance of the in-situ phase change gel sustained-release formulation before and after the phase change.

[0043] Figure 2 Photograph of in-situ phase change gel injected into water using a 26G injection needle.

[0044] Figure 3 The elastic modulus and viscous modulus are those of Example 1 and Example 1 15 minutes after subcutaneous injection.

[0045] Figure 4 HE staining images of the skin at the injection site 7 and 14 days after subcutaneous injection of the in-situ phase change gel sustained-release formulation of this invention and the phospholipid gel of patent CN102526753A (×5).

[0046] Figure 5 HE staining images of skin tissue at the injection site 7 and 14 days after subcutaneous injection of the in-situ phase change gel sustained-release formulation of this invention and the phospholipid gel of patent CN102526753A (×200).

[0047] Figure 6 HE staining images (×200) of subcutaneous tissue at the injection site 7 and 14 days after subcutaneous injection of the in-situ phase change gel sustained-release formulation of this invention and the phospholipid gel of patent CN102526753A.

[0048] Figure 7 The pharmacokinetic curves for Example 1 and the recombinant human coagulation factor VIII solution are shown. Detailed Implementation

[0049] The following embodiments are further illustrations of the present invention, but are by no means limitations on the scope of the invention. The present invention is further described in detail below with reference to the embodiments; however, those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Furthermore, those skilled in the art can make equivalent substitutions, combinations, improvements, or modifications to the present invention based on the description thereof, but all such substitutions and modifications will be included within the scope of the present invention.

[0050] Example 1

[0051] Take 5g of soybean lecithin S100 and 5g of ethyl oleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel with good flowability, such as... Figure 2 Add 7500 IU of recombinant human coagulation factor VIII to a blank phospholipid gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the recombinant human coagulation factor VIII phospholipid gel sustained-release formulation.

[0052] Example 2

[0053] Take 4g of soybean lecithin S100 and 6g of ethyl oleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 100mg of insulin powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the insulin lecithin gel sustained-release formulation.

[0054] Example 3

[0055] Take 3g of soybean lecithin S100 and 7g of ethyl oleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 500mg of pembrolizumab fine powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will have the pembrolizumab lecithin gel sustained-release formulation.

[0056] Example 4

[0057] Take 2g of soybean lecithin S100 and 8g of ethyl oleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 250mg of etanercept fine powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the etanercept lecithin gel sustained-release formulation.

[0058] Example 5

[0059] Take 5g of soybean lecithin S100 and 5g of ethyl linoleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 30mg of interferon powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and the interferon lecithin gel sustained-release preparation is obtained.

[0060] Example 6

[0061] Take 4g of soybean lecithin S100 and 6g of ethyl linoleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 30mg of erythropoietin, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will have the erythropoietin gel sustained-release preparation.

[0062] Example 7

[0063] Take 4.5g of egg yolk phospholipid E80 and 5.5g of ethyl oleate, stir at 60℃ until the phospholipids are completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank phospholipid gel. Take 10000IU of asparaginase fine powder, add it to the blank phospholipid gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the asparaginase phospholipid gel sustained-release preparation.

[0064] Example 8

[0065] Take 3g of soybean lecithin S100 and 7g of ethyl linoleate, stir at 50℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 16mg of interleukin powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the interleukin lecithin gel sustained-release preparation.

[0066] Example 9

[0067] Take 2g of egg yolk phospholipid E80 and 8g of ethyl linoleate, stir at 50℃ until the phospholipids are completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank phospholipid gel. Take 500mg of trastuzumab emtansine fine powder, add it to the blank phospholipid gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will have the sustained-release formulation of trastuzumab emtansine phospholipid gel.

[0068] Example 10

[0069] Take 5.5g of soybean lecithin S100 and 4.5g of ethyl oleate, stir at 40℃ until the lecithin is completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank lecithin gel. Take 360mg of afotazyme α fine powder, add it to the blank lecithin gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will get the afotazyme α lecithin gel sustained-release preparation.

[0070] Example 11

[0071] Take 5g of hydrogenated egg yolk lecithin, 3g of ethyl oleate, 2g of ethyl linoleate, and 1mg of tert-butylhydroxyanisole. Stir at 45℃ until the phospholipids are completely dissolved. After cooling to room temperature (25℃), a clear and transparent blank phospholipid gel is obtained. Take 5000IU of recombinant human coagulation factor IX fine powder, add it to the blank phospholipid gel, stir thoroughly, let stand until the bubbles completely disappear, and seal to obtain the recombinant human coagulation factor IX phospholipid gel sustained-release formulation.

[0072] Example 12

[0073] Take 5.5g of egg yolk phospholipid E80 and 4.5g of ethyl linoleate, stir at 40℃ until the phospholipids are completely dissolved, and cool to room temperature (25℃) to obtain a clear and transparent blank phospholipid gel. Take 3mg of conbercept fine powder, add it to the blank phospholipid gel, stir thoroughly, let stand until the bubbles completely disappear, seal, and you will have the conbercept phospholipid gel sustained-release formulation.

[0074] Experimental Example 1: Selection of Injection Oil

[0075] Weigh out 4g of egg yolk lecithin E80 per portion, add 6g of each of the 9 injectable oils listed in the table below, stir, observe the appearance, and measure the viscosity using a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria) (measurement parameters: temperature 25℃, shear rate 25s). -1 The results are shown in the table below:

[0076] Table 1: Solubility properties of injectable oil and egg yolk phospholipid E80

[0077]

[0078] As shown in the table above, both ethyl oleate and ethyl linoleate have good solubility for E80.

[0079] Weigh out 4g of soybean lecithin S100, add 6g of each of the 9 injectable oils listed in the table below, stir, observe the appearance, and measure the viscosity using a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria) (measurement parameters: temperature 25℃, shear rate 25s). -1 The results are shown in the table below:

[0080] Table 2: Solubility properties of oil for injection and soybean lecithin S100

[0081]

[0082]

[0083] As shown in the table above, both ethyl oleate and ethyl linoleate have good solubility for S100.

[0084] Experimental Example 2: Selection of Injection Oil Ratio

[0085] Weigh out 10g of phospholipids and injection oil in different proportions according to the table below, stir, observe the appearance, and measure the viscosity (temperature 25℃, shear rate 25s) using a modular intelligent advanced rotational rheometer (model: MCR302, manufacturer: Anton Paar GmbH, Austria). -1 The results are shown in the table below:

[0086] Table 3: Screening of Phospholipid to Injection Oil Ratio

[0087]

[0088]

[0089] As shown in the table above, when the ratio of phospholipid to injection oil reaches 6:4 (w:w), the viscosity of the system increases significantly, making it difficult to inject smoothly through a syringe. Therefore, the preferred ratio of phospholipid to injection oil is 2:8 to 5.5:4.5 (w:w), and more preferably, the ratio is 2:8 to 5:5 (w:w). Figure 1 It can be seen that when the ratio of phospholipids to injectable oil is 2:8 to 5.5:4.5, both can change from a liquid with good fluidity before phase change to a semi-solid state after adding a small amount of water.

[0090] Experimental Example 3: Rheological Properties

[0091] The product of Example 1 was injected subcutaneously into SD rats, and removed 15 minutes later. Its elastic modulus (storage modulus) G' and viscous modulus (loss modulus) G" were measured using a modular intelligent advanced rotational rheometer. The measurement parameters were: strain 1%, angular frequency 0.1–100 rad / s, and temperature 25°C. The results are as follows: Figure 3As shown, initially, both the elastic modulus G' and the viscous modulus G" are very small, with G' being less than G" indicating that the formulation is in a liquid state; 15 minutes after injection into the subcutaneous tissue of rats, G' and G" increase, and G' becomes greater than G" indicating that the formulation has transformed into a solid state.

[0092] Experiment Example 4: Investigation of Local Irritation

[0093] Preparation of the in-situ phase change gel sustained-release formulation of the present invention: Take 5g of soybean phospholipid S100 and 5g of ethyl oleate, stir at 50°C to completely dissolve the phospholipid, and cool to room temperature (25°C) to obtain the final product.

[0094] Preparation of phospholipid gel according to patent CN102526753A: Take 7g of soybean phospholipid S100, 1.5g of medium-chain fatty acid glycerides and 1.5g of anhydrous ethanol, stir to completely dissolve the phospholipids, and you will get the gel.

[0095] The in-situ phase change gel sustained-release formulation of this invention and the phospholipid gel of patent CN102526753A were injected subcutaneously into SD rats. Adverse reactions such as skin ulceration, edema, and erythema at the injection site were observed. Skin tissue from the injection site was collected on days 7 and 14 for histopathological examination. The pathological results are shown in the table below:

[0096] Table 4: Adverse Reactions

[0097]

[0098] Following the injection, all experimental animals exhibited normal behavior and diet, and no obvious erythema, ulceration, or edema was observed at the injection site. Figures 4-6 As shown, on day 7, both the in-situ phase change gel sustained-release formulation of this invention and the phospholipid gel of patent CN102526753A exhibited varying degrees of inflammatory reactions in the subcutaneous tissue after injection. The former showed normal skin tissue, while the latter was accompanied by mild local necrosis of the skin tissue. These pathological changes recovered somewhat by day 14. The subcutaneous inflammatory reaction in the in-situ phase change gel sustained-release formulation group changed from moderate to mild, and the changes in the skin at day 14 were almost within the normal range. The inflammatory reaction in the phospholipid gel group of patent CN102526753A changed from mild to moderate.

[0099] Example 5: In vivo pharmacokinetic study

[0100] Recombinant human coagulation factor VIII solution: The recombinant human coagulation factor VIII fine powder was prepared into a solution with a concentration of 250 IU / mL using 0.9% sodium chloride injection.

[0101] Fifteen male SD rats (weighing 200±20g) were randomly divided into three groups: an intravenous injection group, a subcutaneous injection group, and a subcutaneous injection group (Example 1), with five rats in each group. The intravenous injection group received a single tail vein injection of recombinant human coagulation factor VIII solution at a dose of 250 IU / kg. The subcutaneous injection groups and the Example 1 group received single subcutaneous injections of recombinant human coagulation factor VIII solution and Example 1 solution, respectively, at a dose of 2500 IU / kg. At 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240, 312, and 384 hours post-administration, approximately 300 μL of blood was collected from the orbital sinus. The supernatant plasma was collected by centrifugation, and the concentration of recombinant human coagulation factor VIII in the SD rat plasma was determined using a validated chromogenic substrate method. The results are shown in the table below.

[0102] Table 7: Pharmacokinetic Results of Recombinant Human Coagulation Factor VIII

[0103]

[0104] Based on the pharmacokinetic data results and pharmacokinetic curves in the table above... Figure 7 As can be seen, compared with the recombinant human coagulation factor VIII solution, Example 1 significantly prolonged the half-life and mean residence time of recombinant human coagulation factor VIII. Under the same administration method, Example 1 improved the bioavailability of recombinant human coagulation factor VIII.

Claims

1. An in situ injectable phase transition gel sustained release formulation characterized in that, The composition comprises phospholipid 10-60 parts by weight, injection oil 45-90 parts by weight, and pharmaceutical active ingredient 0.0025-20 parts by weight; the pharmaceutical active ingredient is a protein; the injection oil is selected from one or a combination of ethyl oleate and ethyl linoleate.

2. The in situ injectable phase transition gel sustained release formulation according to claim 1, wherein, The phospholipid is selected from one or a combination of natural phospholipid, semi-synthetic phospholipid, and synthetic phospholipid; the natural phospholipid is selected from one or a combination of egg yolk lecithin, soybean phospholipid; the semi-synthetic phospholipid is selected from one or a combination of hydrogenated egg yolk lecithin and hydrogenated soybean phospholipid; the synthetic phospholipid is selected from one or a combination of dipalmitoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, dioleoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl glycerol, and dipalmitoyl phosphatidic acid.

3. The in situ injectable phase transition gel sustained release formulation according to claim 1, wherein The phospholipid is selected from one or a combination of soybean phospholipid S100 and egg yolk phospholipid E80.

4. The in situ injectable phase transition gel sustained release formulation according to claim 1, wherein, The protein is selected from one or more of insulin, glucagon, alpha-interferon, beta-interferon, pegylated interferon, interleukin, erythropoietin (EPO), colony stimulating factor (GSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), adenosine deaminase, beta-glucocerebrosidase, alpha-galactosidase, tissue plasminogen activator, streptokinase, urokinase, antithrombin, asparaginase, uricase, trypsin, alfa-afutase, cell growth factor, coagulation factor, prostaglandin, botulinum toxin, rituximab, ranibizumab, omalizumab, denosumab, trastuzumab, inotuzumab, tocilizumab, bevacizumab, infliximab, adalimumab, etanercept, CD25 antibody, adalimumab, sucralfate, enavlituzumab, pateclizumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, sarilumab, 5. The in situ injectable phase-transition gel sustained release preparation according to claim 1, wherein ​ 6. The in situ injectable phase-transition gel depot preparation according to claim 1, wherein, ​ 7. The in situ injectable phase-transition gel depot preparation according to claim 1, wherein ​ 8. The in situ injectable phase-transition gel depot preparation according to claim 1, wherein, ​ 9. A process for the preparation of the in situ injectable phase- transition gel sustained release formulation as claimed in any one of claims 1 to 8, characterized by ​ (1) Take phospholipid and injectable oil to mix, mix at 0-120℃ to make phospholipid completely dissolved, obtain clear transparent phospholipid gel; (2) Take the active ingredient of medicine to prepare medicine micro-particle through crystallization or crushing method; (3) Mix the phospholipid gel of step (1) and the medicine micro-particle of (2) uniformly, stand to remove the air bubble in preparation, dispense, seal, obtain.

10. The use of the in-situ injectable phase-transition gel sustained-release preparation of any one of claims 1-8 in the preparation of medicine for treating metabolic disease, tumor, cardiovascular and cerebrovascular disease.

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