Human growth hormone fc fusion protein injection preparation, preparation method and application thereof in preparation of burn and fracture drugs

Abstract

The application belongs to the technical field of biological pharmacy, and provides a human growth hormone Fc fusion protein injection preparation, a preparation method and application in preparation of burn and fracture drugs. The preparation comprises human growth hormone-Fc fusion protein, polysorbate 80, disodium edetate and a buffer system. By using polysorbate 80 as a surfactant, the aggregation of the protein is effectively prevented, and the stability of the protein is maintained; disodium edetate is used as a metal chelating agent to reduce the degradation of the protein by metal ions and prolong the effective period of the protein. The buffer system avoids the influence of pH fluctuation on the protein, so that the activity of the protein is maintained. Through the previous in-vivo pharmacodynamic experiment verification, the application of the injection in the treatment of fractures and burns has a remarkable repair effect, promotes fracture healing and accelerates burn wound repair.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and particularly relates to a human growth hormone Fc fusion protein injection formulation, its preparation method, and its application in the preparation of burn and fracture drugs. Background Technology

[0002] Human growth hormone is an important drug used clinically to treat growth hormone deficiency. However, traditional recombinant human growth hormone has a short half-life in the body and usually requires daily subcutaneous injection. This frequent administration places a significant treatment burden and psychological stress on patients, especially children, severely affecting medication adherence. To overcome this shortcoming, the development of long-acting growth hormone has become a research hotspot in this field. Among them, fusing human growth hormone with antibody Fc fragments through gene recombination technology, i.e., human growth hormone-Fc fusion protein, and utilizing the recycling mechanism of Fc fragments to extend the drug's half-life, is a very promising long-acting strategy.

[0003] However, human growth hormone-Fc fusion protein, as a large protein molecule, is extremely unstable in aqueous solution, readily undergoing physical aggregation (such as the formation of polymers) and chemical degradation (such as oxidation and deamidation). This makes it difficult to prepare it into a liquid formulation that can be stored for a long time. Therefore, some existing drugs containing this type of fusion protein have to be in the form of freeze-dried powder for injection, requiring patients to reconstitute before use. This process is cumbersome and prone to introducing risks of microbial contamination and dosage errors, causing inconvenience for clinical application.

[0004] Furthermore, while various drugs and therapies exist in the field of fracture treatment, limitations remain. For example, long-term use of some chemical drugs poses safety risks; some biological agents, such as bone morphogenetic proteins, are expensive and may induce heterotopic ossification; and teriparatide, as one of the few drugs that can effectively promote bone formation, is not only expensive but also requires daily injections, leading to poor patient compliance and potential safety risks. Therefore, developing a new fracture treatment drug with better efficacy, higher safety, and more convenient administration is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0005] In view of the aforementioned technical deficiencies in the prior art, the technical problem to be solved by this application is to provide a human growth hormone-Fc fusion protein injection formulation to overcome the technical difficulty of its easy oxidation, polymerization and other degradation reactions, making it difficult to prepare into a liquid formulation that can be stably stored for a long time. Another objective of this application is to provide the application of the aforementioned human growth hormone-Fc fusion protein injection in the preparation of drugs for treating fractures, so as to overcome the defects of existing fracture treatment drugs such as limited efficacy, inconvenient administration or safety risks.

[0006] Specifically, the present invention is achieved through the following scheme: This invention provides a human growth hormone-Fc fusion protein injection formulation, the injection formulation comprising: human growth hormone-Fc fusion protein, polysorbate 80, disodium edetate, and a buffer system.

[0007] Optionally, the concentration of the human growth hormone-Fc fusion protein is 10-30 mg / mL.

[0008] Optionally, the concentration of polysorbate 80 is 1-5 mg / mL, and the concentration of disodium edetate is 0.1-0.3 mg / mL.

[0009] Optionally, the buffer system is a 5-20 mM citrate buffer or phosphate buffer, and the buffer system maintains the pH of the injection solution at 6.5-7.5.

[0010] Optionally, sodium chloride can be used to adjust the osmotic pressure to 280-320 mOsm / kg.

[0011] Further optionally, the injection formulation comprises: 10-30 mg / mL human growth hormone-Fc fusion protein, 1-5 mg / mL polysorbate 80, 0.1-0.3 mg / mL disodium edetate, and sodium chloride to adjust the osmotic pressure to 280-320 mOsm / kg. The buffer system is a 5-20 mM citrate buffer or phosphate buffer, and the buffer system maintains the pH of the injection at 6.5-7.5.

[0012] Optionally, the stable injection solution has a high-performance liquid chromatography purity of ≥98% after being stored at 25°C for 28 days.

[0013] Optionally, the stable injection solution has an electrophoretic purity of ≥85% after being stored at 25°C for 28 days.

[0014] Optionally, the injectable formulation further comprises a small molecule antioxidant, wherein the small molecule antioxidant is at least one selected from reduced glutathione, thiourea, L-cysteine, and sodium thiogluconate. The concentration of the small molecule antioxidant is 0.05-0.15 mg / mL.

[0015] Further optionally, the small molecule antioxidant is a compound of reduced glutathione and sodium thiogluconate, with a mass ratio of 1-4:1-4.

[0016] Another aspect of the application provides a method for preparing a human growth hormone-Fc fusion protein injection formulation, comprising: adding human growth hormone-Fc fusion protein, polysorbate 80 and disodium edetate to a buffer system.

[0017] Optionally, sodium chloride can be used to adjust the osmotic pressure.

[0018] Alternatively, human growth hormone-Fc fusion protein, polysorbate 80, and disodium edetate can be added to the buffer system, and the osmotic pressure can be adjusted to 280-320 mOsm / kg using sodium chloride, with the pH value maintained at 6.5-7.5.

[0019] Optionally, the human growth hormone-Fc fusion protein is a fully humanized fusion protein of human growth hormone and antibody Fc. This protein is synthesized by fusing the Fc segment gene of human immunoglobulin IgG4 with the C-terminus of human growth hormone via a linker gene using genetic engineering technology and recombining the fusion into the expression vector p327.7. The constructed plasmid is stably transfected into CHO-K1 cells and expressed extracellularly. hGH-Fc not only retains the biological activity of natural GH, but also eliminates ADCC, CDC, and ADCP effects through Fc segment molecular design. Simultaneously, hGH-Fc retains the FcRn-mediated in vivo recycling effect, thereby significantly prolonging the half-life of growth hormone in the human body. This characteristic enables hGH-Fc to achieve the goal of extending the dosing interval.

[0020] Another aspect of this application provides the use of a human growth hormone Fc fusion protein injection formulation in the preparation of medicaments for fractures and / or burns.

[0021] This invention improves the stability and purity of proteins through rational formulation design. Using polysorbate 80 as a surfactant effectively prevents protein aggregation and precipitation, ensuring high protein stability in solution. Test results show that even after long-term storage, the electrophoretic purity of the protein remains at a high level, demonstrating the protective effect of polysorbate 80 on protein stability and structural integrity, reducing the risk of protein degradation and aggregation. Furthermore, disodium edetate, as a metal chelating agent, effectively chelates metal ions in solution, preventing metal-catalyzed protein degradation reactions, thereby extending the protein's shelf life. Test results show that under accelerated conditions, the formulation containing disodium edetate exhibits minimal protein degradation, further enhancing the long-term stability of the preparation. This stability ensures the biological activity and efficacy of the protein in the injection solution, making it suitable for long-term storage and clinical application. This invention also uses sodium chloride to adjust the osmotic pressure to match the physiological osmotic pressure of the human body, ensuring the physiological compatibility of the injection solution when used in vivo. This osmotic pressure adjustment effectively avoids cell damage and discomfort caused by osmotic pressure imbalance, while the use of a buffer solution ensures a stable pH of approximately 7.0, further preventing protein denaturation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 3D modeling of tibial CT scans of rats in each group at 28 days; Figure 2 Images of safranin-fast green staining of tibial pathological sections from 28-day-old rats in each group.

[0024] Figure 3 Photographs show the appearance of skin lesions in rats.

[0025] Figure 4 The image shows the scoring results for MVSS.

[0026] Figure 5 Examples of pathological sections of rats in each of the 7D groups.

[0027] Figure 6 Examples of pathological sections of rats in each of the 21D groups.

[0028] Figure 7 The expression diagrams of IGF-1 and IGFBP-3 in rats of each group are shown.

[0029] Figure 8 The graph shows the expression of TGF-β1 in rats of each group.

[0030] Figure 9 This is a diagram showing the expression of VEGF-A in rats.

[0031] Figure 10 The graph shows the expression of TNF-α in rats of each group. Detailed Implementation

[0032] For ease of understanding and implementation, and unless otherwise expressly defined or the context otherwise requires, the numerical values, parameters, and intervals mentioned in this specification and claims shall be interpreted as closed intervals including the endpoints; within a reasonable range of measurement uncertainty and experimental error, numerical values ​​approximating the endpoints or recorded values ​​may be used without altering the technical essence. The upper and lower limits of any range, the endpoints of different ranges, and the single-point data, representative values, or thresholds listed herein can be combined to generate new sub-ranges, which are considered clearly disclosed and directly applicable. Different measurement methods such as mass fraction, parts by weight, volume fraction, molar ratio, and concentration can be substituted for each other after equivalent conversion; unless otherwise specified, the significant figures and rounding rules of numerical values ​​shall be interpreted according to applicable testing standards or industry-standard specifications.

[0033] This invention provides a human growth hormone-Fc fusion protein injection formulation, comprising: 10-30 mg / mL human growth hormone-Fc fusion protein, 1-5 mg / mL polysorbate 80, 0.1-0.3 mg / mL disodium edetate, and sodium chloride to adjust the osmotic pressure to 280-320 mOsm / kg. The buffer system is a 5-20 mM citrate buffer or phosphate buffer, and the buffer system maintains the pH of the injection solution at 6.5-7.5.

[0034] Human growth hormone-Fc fusion protein (Fc-GH) is a biological drug obtained through recombinant technology. It combines human growth hormone and the Fc fragment of immunoglobulin, extending its half-life and enhancing its circulation time in the body by introducing the Fc fragment. The Fc fragment allows the fusion protein to bind to Fc receptors, reducing the rate at which it is cleared by the immune system, thereby reducing the frequency of administration and ensuring a longer duration of efficacy in treating growth hormone deficiency. It promotes bone and muscle growth and regulates metabolic processes in the body.

[0035] Polysorbate 80 is a nonionic surfactant that helps stabilize proteins and prevent their aggregation by reducing the surface tension of solutions. It works by coating the protein surface, reducing nonspecific interactions between proteins, and preventing protein aggregation or precipitation. Particularly during the storage and use of protein solutions, polysorbate 80 effectively improves protein solubility and stability, ensuring that drugs maintain their activity and efficacy during long-term storage.

[0036] Disodium edetate is a potent metal chelating agent; it can react with metal ions (such as Fe) in aqueous solutions. 2+ Cu 2+ (e.g., sodium edetate) forms a stable complex, preventing metal ion-catalyzed oxidation reactions and thus reducing the risk of protein degradation. Metal ions can accelerate protein oxidation, leading to structural changes and loss of function. By adding disodium edetate, the interference of these metal ions can be effectively reduced, extending the protein's shelf life and ensuring its stability and activity.

[0037] Sodium chloride, as an osmotic pressure regulator, can adjust the ion concentration in a solution to match the physiological osmotic pressure of the human body. Cells and tissues in the human body require an isotonic environment to prevent cell rupture or shrinkage caused by osmotic pressure differences. By adding an appropriate amount of sodium chloride, it is ensured that the injected solution will not cause osmotic imbalance in cells or blood vessels after injection, thereby reducing local irritation and discomfort.

[0038] The primary function of buffer systems is to maintain pH stability in solutions, preventing adverse effects of pH fluctuations on proteins. Changes in pH can affect protein structure and function, leading to loss of biological activity or degradation. Citrate buffers and phosphate buffers are commonly used to maintain pH stability, ensuring that the pH of injection solutions remains within the ideal range, thereby guaranteeing protein solubility, stability, and biological activity. Buffers absorb external acids and bases, reducing the magnitude of pH changes and providing an ideal environment for proteins, thus extending their shelf life.

[0039] In the examples, reduced glutathione was selected from aladdin, G105426 glutathione (reduced) 70-18-8, with a purity of 98%. Sodium thiogluconate, CAS No.: 10593-29-0, 1-Thio-β-D-glucose sodium salt.

[0040] Polysorbate 80(II): Nanjing Well Pharmaceutical Group Co., Ltd. Sodium chloride: Tianjin Haiguang Technology Development Co., Ltd. Disodium edetate: Hunan Ercon Pharmaceutical Co., Ltd. Poloxamer 188: Nanjing Well Pharmaceutical Group Co., Ltd. Arginine: Shanghai Xiehe Amino Acid Co., Ltd. Mannitol: Hunan Jiudian Hongyang Pharmaceutical Co., Ltd.

[0041] The human growth hormone-Fc fusion protein (Fc-GH) was prepared using the method described in Example 5 of the applicant's invention patent CN110256575A.

[0042] Example 1

[0043] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM citrate buffer, and the pH value of the system is controlled at 7.0.

[0044] Preparation method: Prepare 10 mM citrate buffer, adjust the pH to 7.0, dissolve human growth hormone-Fc fusion protein in the buffer and control its concentration to 20 mg / mL, add polysorbate 80 and control its concentration to 3 mg / mL, then add disodium edetate and control its concentration to 0.2 mg / mL, and add sodium chloride to adjust the osmotic pressure to 300 mOsm / kg.

[0045] Example 2

[0046] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM phosphate buffer, and the pH value of the system is controlled at 7.0.

[0047] Example 3

[0048] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, 0.10 mg / mL reduced glutathione, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM citrate buffer, and the pH value of the system is controlled at 7.0.

[0049] Preparation method: Prepare 10 mM citrate buffer, adjust the pH to 7.0, dissolve human growth hormone-Fc fusion protein in the buffer and control its concentration to 20 mg / mL, add polysorbate 80 and control its concentration to 3 mg / mL, then add disodium edetate and control its concentration to 0.2 mg / mL, add reduced glutathione and control its concentration to 0.10 mg / mL, and add sodium chloride to adjust the osmotic pressure to 300 mOsm / kg.

[0050] Example 4

[0051] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, 0.10 mg / mL sodium thiogluconate, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM citrate buffer, and the pH value of the system is controlled at 7.0.

[0052] Preparation method: Prepare 10 mM citrate buffer, adjust the pH to 7.0, dissolve human growth hormone-Fc fusion protein in the buffer and control its concentration to 20 mg / mL, add polysorbate 80 and control its concentration to 3 mg / mL, then add disodium edetate and control its concentration to 0.2 mg / mL, add sodium thiogluconate and control its concentration to 0.10 mg / mL, and add sodium chloride to adjust the osmotic pressure to 300 mOsm / kg.

[0053] Example 5

[0054] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, 0.05 mg / mL reduced glutathione, 0.05 mg / mL sodium thiogluconate, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM citrate buffer, and the pH of the system is controlled at 7.0.

[0055] Preparation method: Prepare 10 mM citrate buffer, adjust the pH to 7.0, dissolve human growth hormone-Fc fusion protein in the buffer and control its concentration to 20 mg / mL, add polysorbate 80 and control its concentration to 3 mg / mL, then add disodium edetate and control its concentration to 0.2 mg / mL, add reduced glutathione and sodium thiogluconate and control their concentrations to 0.05 mg / mL, and add sodium chloride to adjust the osmotic pressure to 300 mOsm / kg.

[0056] Comparative Example 1

[0057] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL poloxamer 188, 0.2 mg / mL arginine, sodium chloride to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM phosphate buffer, and the pH value of the system is controlled at 7.0.

[0058] Comparative Example 2

[0059] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL poloxamer 188, mannitol to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM phosphate buffer, and the pH value of the system is controlled at 7.0.

[0060] Comparative Example 3

[0061] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, 0.2 mg / mL disodium edetate, mannitol to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM phosphate buffer, and the pH value of the system is controlled at 7.0.

[0062] Comparative Example 4

[0063] The specific formulation of the human growth hormone-Fc fusion protein injection is as follows: 20 mg / mL human growth hormone-Fc fusion protein, 3 mg / mL polysorbate 80, mannitol to adjust the osmotic pressure to 300 mOsm / kg, the buffer system is 10 mM phosphate buffer, and the pH value of the system is controlled at 7.0.

[0064] Test Example 1

[0065] The samples of the examples and comparative examples were placed under accelerated conditions (25℃±2℃, 60%±5%RH) for different times, and the purity of high performance liquid chromatography, charge heterogeneity and electrophoretic purity were tested. ;

[0066] Examples 3 and 4, based on Example 1, added small-molecule antioxidants such as reduced glutathione or sodium thiogluconate, further improving the stability and activity of the human growth hormone-Fc fusion protein injection. These antioxidants reduce protein oxidative damage by scavenging free radicals and peroxides, particularly preventing disulfide bond breakage and maintaining protein structural stability. Furthermore, they prevent metal-catalyzed degradation reactions by binding to metal ions, thereby improving protein purity and activity. The addition of antioxidants also enhances the protein's biostability, ensuring its efficacy under different storage conditions and further improving the long-term stability and clinical efficacy of the formulation. Example 5 is superior to Examples 3 and 4, mainly because reduced glutathione, through its thiol group, utilizes strong reducing properties to scavenge free radicals and peroxides, preventing protein oxidative damage, particularly avoiding disulfide bond oxidative breakage and maintaining protein structural stability. Meanwhile, the thiol group of sodium thiogluconate binds to metal ions, preventing metal-induced oxidation reactions and further improving protein purity and activity. The synergistic effect of both not only enhances protein stability but also improves the long-term efficacy of the formulation.

[0067] The charge heterogeneity results in Table 3 show that, under 25℃ conditions, the isoelectric points are all within the acceptable range of 5.5-6.5.

[0068] Test Example 2

[0069] Rats were divided into four groups: a model control group (MOD), a teriparatide control group (POS), a bone peptide injection control group (COI), a high-dose group (HIG), a medium-dose group (MID), and a low-dose group (LOW), with 16 rats of each group (half male and half female). All rats were anesthetized with Supra-50, shaved, and disinfected. A transverse tibial fracture model was constructed via open surgery. The surgical procedure included incising the skin to expose the muscle, separating the muscle to expose the tibia, horizontally sawing the tibia with a hacksaw, opening the knee joint cavity and drilling holes at specific points, fixing the tibia with Kirschner wires, and finally suturing the muscle and skin and disinfecting. Postoperative anti-inflammatory treatment with ampicillin sodium was administered for three consecutive days after model establishment. On the third day, during the intervention treatment phase, the model group received only saline injections, the teriparatide and bone peptide injection groups received daily injections of teriparatide and bone peptide injections, and the Fc-GH dose groups received the corresponding dose of Fc-GH every seven days for a total of four injections. CT scans were performed every seven days during the experiment. On day 28 after administration (28D), rats were anesthetized with Sutacetin 50 and sacrificed, and tibia samples were collected for preservation.

[0070] Model control group (MOD): 0.2 mL of normal saline was injected weekly; normal saline: 0.9% sodium chloride solution.

[0071] Teriparatide control group (POS): Teriparatide daily injection dose was 20µL; Teriparatide injection (Xinfutai Pro), manufacturer: Sinopharm (Suzhou) Pharmaceutical Co., Ltd., specification: 20µg: 80µL, 2.4mL / vial.

[0072] Bone peptide injection control group (COI): The daily injection dose of bone peptide was 3.2 mg / kg; Compound bone peptide injection (Guqiang), manufacturer: Nanjing Xinbai Pharmaceutical Co., Ltd., specification: 2 mL: 30 mg.

[0073] High-dose group (HIG): Human growth hormone Fc fusion protein injection preparation of Example 1, administered at a dose of 3.6 mg / kg per rat.

[0074] Medium-dose group (MID): Human growth hormone Fc fusion protein injection preparation of Example 1, the dosage was 1.8 mg / kg per rat.

[0075] Low-dose group (LOW): Human growth hormone Fc fusion protein injection preparation of Example 1, administered at a dose of 0.9 mg / kg per rat.

[0076] I. Results of in vivo CT scan 3D modeling analysis of tibial CT scans of rats at 28 days post-surgery clearly showed that the HIG group exhibited significant healing, while the MOD group showed obvious fractures. Although the POS and COI groups had healed, incomplete healing areas were observed. The MID and LOW groups showed healing levels similar to those of the POS and COI groups. (See details...) Figure 1 .

[0077] II. Results of Safranin-Fix-Green Staining of Tibial Pathological Sections Pathological examination revealed obvious fracture sites in both the HIG and MOD groups on day 7 (7D), with cartilage protecting the injury site. On day 14 (14D), the MOD group still showed cartilage tissue, while in the HIG group, some cartilage at the fracture site calcified into new bone. On day 21 (21D), the MOD group showed only partial cartilage calcification, while the HIG group showed completely calcified new bone tissue at the injury site, with no fracture site, and the recovery speed was significantly faster than the model group. See details. Figure 2 .

[0078] Test Example 3

[0079] Rats were divided into four groups: blank control group (CTL), model control group (MOD), positive control group (POS), high-dose group (HIG), medium-dose group (MID), and low-dose group (LOW), with 20 rats of each group (half male and half female). Except for the blank control group, all other groups were anesthetized with salbutamol (Salvastatin 50), their dorsal hair was removed, and they were treated with 100°C hot water for 10 seconds to control the burn area to 4cm × 4cm, thus establishing a rat skin burn model. During the intervention treatment phase, the blank control group and the model group received only saline injections, the positive control group received recombinant human epidermal growth factor (rhEGF) gel, and the Fc-GH dose groups received injections of the corresponding dose of Fc-GH every 7 days, for a total of 2 injections over a period of 21 days. During the experiment, the modified Vancouver Scar Score (MVSS) was used to periodically score the wound in four aspects: pigmentation, vascular distribution, flexibility, and thickness. After the experiment, rats were anesthetized with Sutacetin 50 and sacrificed. Skin samples were taken from the injured area on the back and the samples were stained with hematoxylin and eosin (HE). Key factors in the signaling pathways related to epidermal damage repair were detected.

[0080] Blank control group (CTL): 0.2 ml of normal saline was injected weekly; normal saline: 0.9% sodium chloride solution.

[0081] Model control group (MOD): 0.2 ml of physiological saline was injected weekly; physiological saline: 0.9% sodium chloride solution.

[0082] Positive control group (POS): The positive control group consisted of EGF gel, a topical preparation; human epidermal growth factor (EGF), manufacturer: Guilin Huanowei Gene Pharmaceutical Co., Ltd., specification: 100,000 IU (200µg) / 20g / vial, dosage: 0.1g / cm³. 2 .

[0083] High-dose group (HIG): Human growth hormone Fc fusion protein injection preparation of Example 1, administered at a dose of 3.6 mg / kg per rat.

[0084] Medium-dose group (MID): Human growth hormone Fc fusion protein injection preparation of Example 1, the dosage was 1.8 mg / kg per rat.

[0085] Low-dose group (LOW): Human growth hormone Fc fusion protein injection preparation of Example 1, administered at a dose of 0.9 mg / kg per rat.

[0086] I. Evaluation of the appearance of burn wound tissue By simultaneously scoring the wound in four aspects—pigmentation, vascular distribution, flexibility, and thickness—using a modified Vancouver Scar Rating Scale (MVSS), the results showed that the Fc-GH high-dose treatment group exhibited a significant difference in scoring compared to the model group on day 14 (14D) (P<0.05); and a highly significant difference on day 21 (21D) (P<0.01). The positive control group also showed a difference compared to the model group on day 21 (P<0.05). See details... Figure 3 and Figure 4 . Figure 3 Apparent results showed no significant difference in damage among the groups after modeling (D1); D21 showed that the high-dose group was significantly better than the other groups; the differences were fully reflected in the apparent quantitative scoring. Figure 4 The 14D scores showed a significant difference between the high-dose group and the model group (*, P<0.05); the 21D scores showed an extremely significant difference between the high-dose group and the model group (**, P<0.01), and a significant difference between the positive control group and the model group (*, P<0.05).

[0087] II. Pathological HE staining results The results of HE staining of rat skin in each group are shown below. Figure 5 , Figure 6 .like Figure 5 As shown, tissue samples taken on day 7 revealed that the skin structure of rats in the CTL group was intact, with collagen fibers arranged neatly and tightly, and a clear epidermal-dermal boundary; rats in the MOD, POS, HIG, MID, and LOW groups showed diffuse inflammatory cell infiltration, significantly damaged epidermal structure, and severe skin lesions; compared with Figure 5 compared to Figure 6 In the 21D HIG, POS, MID, and LOW groups, the epidermal structure of mice had begun to recover, but some epidermal thickening remained. The HIG and POS groups showed the best recovery in skin structure, with more orderly collagen fiber arrangement and significantly improved diffuse inflammatory infiltration, more closely resembling the CTL group. Fc-GH can accelerate the recovery of rat skin tissue and reduce damage and inflammation.

[0088] III. Results of ELISA Detection of Relevant Factors IGF-1 and IGFBP-3, as the first-line effectors of GH in vivo, directly reflect the effects of growth hormone in the body through their serum concentrations. In the 21-day experimental samples, the levels of IGF-1 and IGFBP-3 in the Fc-GH group were significantly different from those in the model group, positive control group, and blank control group (P<0.01). See details... Figure 7 .

[0089] TGF-β is a key regulator of scar formation, promoting fibroblast proliferation and collagen deposition (especially type I and type III collagen), thus affecting the quality of tissue repair after burns. Its immunomodulatory effects suppress excessive inflammation in the early stages and promote tissue remodeling in the later stages, but overexpression may lead to pathological scars (such as hypertrophic scars or keloids). During epithelial repair, TGF-β1 regulates keratinocyte migration and affects epidermal regeneration. This study assesses the risk of tissue fibrosis after burns or the effectiveness of interventions in improving scarring by detecting this factor. In the analysis of samples from 7D and 21D, the high-dose treatment group showed a significant difference from the model group (P<0.05) after 7D, but no difference from the positive control group. After 21D, all treatment groups showed highly significant differences from the model group (P<0.01). See details... Figure 8 .

[0090] This analysis shows that Fc-GH promotes TGF-β1 expression and accelerates healing in the early stage of injury treatment, and downregulates the expression of this factor in the later stage of the experiment because the injury has basically healed.

[0091] VEGF expression promotes angiogenesis, providing oxygen and nutrients to burn wounds and playing a crucial role in tissue regeneration. Simultaneously, VEGF expression can increase vascular permeability, leading to early edema after burns, but it also creates conditions for inflammatory cell infiltration and repair. The expression of this factor reflects the regenerative capacity of blood vessels after burns, and this study aims to evaluate the potential value of Fc-GH in promoting angiogenesis or treating edema.

[0092] Analysis of samples from 7D and 21D showed that, after 7D results analysis, the high-dose administration group differed significantly from the model group (P<0.05), but not from the positive control group; after 21D results analysis, all administration groups showed highly significant differences from the model group (P<0.01). See details. Figure 9 .

[0093] TNF-α is rapidly released by macrophages and other cells after a burn, serving as an initial signal for the pro-inflammatory response and recruiting other inflammatory cells (such as neutrophils). At low concentrations, it promotes the clearance of necrotic tissue, but persistently high expression can exacerbate tissue damage (such as apoptosis and oxidative stress). In severe burns, TNF-α may enter circulation, triggering systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS).

[0094] TNF-α detection is used to assess the intensity of the inflammatory response after burns or the regulatory effect of anti-inflammatory treatment on excessive inflammation. Analysis of 7-day and 21-day samples showed significant differences between the high-dose treatment group and the model group (P<0.05), but no difference between the high-dose treatment group and the positive control group; see details... Figure 10 .

[0095] This analysis shows that Fc-GH can significantly inhibit the inflammatory response during the injury treatment process, and promote the repair of damage by reducing the expression of TNF-α at a low level.

Claims

1. A human growth hormone Fc fusion protein injection formulation, characterized in that, The injectable formulation comprises: human growth hormone-Fc fusion protein, polysorbate 80, disodium edetate, small molecule antioxidant, and a buffer system; The concentration of the human growth hormone-Fc fusion protein is 10-30 mg / mL; The concentration of polysorbate 80 is 1-5 mg / mL; The concentration of the disodium edetate is 0.1-0.3 mg / mL; The concentration of the small molecule antioxidant is 0.05-0.15 mg / mL; The small molecule antioxidant is a compound of reduced glutathione and sodium thiogluconate, wherein the mass ratio of reduced glutathione to sodium thiogluconate is 1-4:1-4.

2. The human growth hormone Fc fusion protein injection formulation as described in claim 1, characterized in that, The buffer system is a 5-20 mM citrate buffer or phosphate buffer, and the buffer system maintains the pH of the injection solution at 6.5-7.

5.

3. The human growth hormone Fc fusion protein injection formulation as described in claim 1 or 2, characterized in that, Sodium chloride was used to adjust the osmotic pressure to 280-320 mOsm / kg.

4. The method for preparing the human growth hormone Fc fusion protein injection formulation according to any one of claims 1-3, characterized in that, include: Human growth hormone-Fc fusion protein, polysorbate 80, disodium edetate, and small molecule antioxidants were added to the buffer system.

5. The use of the human growth hormone Fc fusion protein injection formulation as described in any one of claims 1-3 in the preparation of drugs for fractures or burns.

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