Human basic fibroblast growth factor gel with wound surface bacteriostasis and its preparation method

Abstract

This invention discloses a recombinant human (or bovine) basic fibroblast growth factor gel with wound-healing and antibacterial functions, and its preparation method. Recombinant human (or bovine) basic fibroblast growth factor gel is a genetically engineered topical drug used clinically to promote healing of burn wounds, chronic wounds, and fresh wounds, with significant efficacy. However, its drawbacks include limited antibacterial properties and inability to be used simultaneously with chemical disinfectants such as iodine and ethanol, which, while promoting wound healing, are not conducive to controlling wound infection. Research has found that protamine sulfate or polylysine, with high safety and good antibacterial properties, can exert good antibacterial and anti-infection functions without negatively affecting the bioactivity and stability of recombinant human (or bovine) basic fibroblast growth factor. This represents an advanced method for preparing recombinant human (or bovine) basic fibroblast growth factor gel with wound-healing and antibacterial functions, enabling this type of drug to better exert its clinical effects and expand its application scope.

Description

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a recombinant human basic fibroblast growth factor gel with wound antibacterial activity and its preparation method. Technical Background

[0002] Basic fibroblast growth factor (bFGF) is a trace protein found in the human body, belonging to the fibroblast growth factor family. It is a key multicellular tissue repair factor that significantly promotes cell growth related to wound repair, including promoting capillary regeneration, accelerating granulation tissue formation, and accelerating nerve fiber regeneration. More than 90% of the cells in subcutaneous tissue (deep skin) are fibroblasts, and fibroblast proliferation is a core component of wound healing. Therefore, bFGF is an excellent deep skin repair factor with broad biological activity against various cell types derived from the mesoderm and ectoderm. Clinically, recombinant human basic fibroblast growth factor gel is a topical preparation with significant repair effects on ulcers, wounds, and tissue damage. It is used for burn wounds (including superficial second-degree, deep second-degree, and granulation wounds), chronic non-healing wounds (including oral ulcers, cervical erosion, residual wounds, vascular ulcers, diabetic foot ulcers, and pressure sores), and surgical trauma (including skin grafts, gynecological surgeries, orthopedic surgeries, and anorectal surgeries), with significant therapeutic effects. It can also improve tissue structure and strength, reduce the formation of pathological scars, and make the healing effect closer to that of normal skin. Due to its significant therapeutic effects and minimal side effects, it is widely used.

[0003] Recombinant human basic fibroblast growth factor gel has significant clinical advantages, but some problems and shortcomings have gradually emerged in its long-term use, such as: (I) Limited antibacterial ability and infection control effect on wounds. As human basic fibroblast growth factor is a large molecule protein drug, it will denature and affect its biological activity when used in combination with commonly used chemical disinfectants such as iodine, hydrogen peroxide, and ethanol. Infection control of patient wounds is a key focus of clinical treatment, and the microbial load and degree of infection of the wound have an important impact on the healing process. The inability to help control wound infection while promoting wound healing is a key factor restricting the further expansion of the clinical application of this type of healing-promoting drug. (II) To prevent contamination and deterioration during its storage period and multiple uses, small molecule chemical preservatives are often added to its preparations, which is not conducive to the health of patients. (III) Due to the molecular structure characteristics of human basic fibroblast growth factor, its stability in aqueous preparations is not ideal, with a shelf life of only 18 months, which reduces the convenience of use and limits its application in a wider range. (iv) There is still room for improvement in the therapeutic effect of human basic fibroblast growth factor preparations.

[0004] Regarding questions (i) and (ii): The moist environment and devitalized tissue of burns, chronic non-healing wounds, and fresh wounds provide a suitable environment for the infection and colonization of various microorganisms (Tredget, E, E, et al. Epidemiology of Infections with Pseudomonas aeruginosa in Burn Patients: The Role of Hydrotherapy[J]. Clinical Infectious Diseases, 1992.). Therefore, wound infection is a common complication, which can lead to sepsis, septic shock, multiple organ dysfunction syndrome (MODS), and even death in severe cases (Kwang Chear Lee, Kavita Joory, Naiem S. Moiemen. History of burns: The past, present and the future[J]. Burns & Trauma, 2014, 2(4):169-180.). Multiple animal studies on infected burn wounds have shown that a high microbial load leads to a significant inflammatory response in the wound and significantly increases the wound healing time in model animals (e.g., Shen Juan, Jin Xiaobao, Ding Jing, et al. Construction of a mouse model of burn wound infection [J]. Chinese Journal of Experimental Animals, 2013(03):65-69.). Multiple clinical studies have shown that when using human basic fibroblast growth factor (rGF) to treat burns and chronic wounds, combining it with antibiotics to control wound infection can accelerate healing and achieve better therapeutic effects. For example, Li Gongying et al., in exploring the clinical efficacy of topical recombinant human basic fibroblast growth factor combined with mupirocin in treating residual burn wounds infected with methicillin-resistant Staphylococcus aureus (MRSA), found that the combined use of topical rGF and mupirocin effectively killed MRSA in infected wounds, significantly accelerated wound healing, and further improved the therapeutic effect. (Li Gongying. Clinical study on topical recombinant human basic fibroblast growth factor combined with mupirocin for the treatment of residual wounds and MRSA infection in burn patients [J]. Strait Pharmaceutical Journal, 2019, 031(001):110-111.). Pan Debiao, Ye Guanxiong, et al., when exploring the effect of combined application of recombinant human basic fibroblast growth factor and sulfadiazine on the healing process of chronic refractory wounds, found that the combined application of recombinant human basic fibroblast growth factor and sulfadiazine significantly shortened the wound healing time and improved the wound healing rate compared with the conventional treatment group and the single drug group.(Pan Debiao, Ye Guanxiong, Wu Chengjun, et al. The repair effect of recombinant human basic fibroblast growth factor combined with silver sulfadiazine cream on the healing process of chronic refractory wounds [J]. Journal of Wenzhou Medical University, 2014, 44(4).). Sun Chunyan, Wang Liping, et al. found in their observation of the combined treatment of burns with recombinant human basic fibroblast growth factor and silver sulfadiazine that the two drugs, when applied to burn wounds, fully exerted their effects of promoting tissue repair, antibacterial, anti-inflammatory and astringent properties, which could accelerate wound healing, improve the healing rate and reduce patient pain. (Sun Chunyan, Wang Liping. Observation on the combined treatment of burns with recombinant human basic fibroblast growth factor and silver sulfadiazine [J]. Shanxi Medical Journal, 2012, 03(v.41):71-71.). These studies all used antibiotics to exert a synergistic anti-infection effect and avoided the simultaneous use of bFGF and chemical disinfectants. However, antibiotics are well-known for causing drug resistance, affecting the immune system, and damaging the liver and kidneys, and their excessive use has long been discouraged in clinical practice. Therefore, simultaneously addressing the needs for wound antibacterial and anti-infection treatment while using human basic fibroblast growth factor remains a challenge.

[0005] Protamine is a basic protein mainly found in the mature sperm of fish (such as salmon, trout, and herring). It typically consists of 30-50 amino acids, with a molecular weight ranging from 3000 to 10000. It is rich in arginine, soluble in water and olefinic acids, but poorly soluble in organic solvents such as ethanol and acetone. It exhibits good stability and does not coagulate upon heating. Numerous studies indicate that, compared to chemically synthesized preservatives, protamine sulfate is a highly effective, thermally stable, and extremely safe natural preservative. It also demonstrates better antibacterial properties at neutral and alkaline pH levels. Protamine has been increasingly widely used in the pharmaceutical, food and other industries. Related literature reports include: Preliminary study on the intervention effect of protamine on Staphylococcus aureus biofilm [D]. Huazhong Agricultural University, 2014. Li Wenru; Antibacterial mechanism of protamine and its application in food preservation [J]. Bulletin of Microbiology, 2007, 34(4):0795-0798; Wu Leiyan, Peng Chaoying. Protamine, a new food preservative [J]. Modern Food Science and Technology, 2006, 22(2):263-266; Luan Jinshui, Wang Nanzhou, Zhong Liren, et al. Study on the food preservation properties of salmon protamine [J]. Natural Product Research and Development, 2000, 12(3); Lou Fanli. Zhong Liren, Lü Minzhu, Zhang Hewen, et al. Antibacterial mechanism of protamine [J]. Journal of Fisheries of China, 2001, 25(2), etc. Protamine sulfate is also a pharmaceutical excipient listed in the pharmacopoeia. In biphasic insulin products, under suitable conditions, it can co-crystallize with insulin molecules through electrostatic interactions, resulting in a sustained-release effect. The concentration used in this regard is 0.2~0.4 mg / ml. It is also a procoagulant, used to neutralize the anticoagulant effect of heparin or to treat spontaneous bleeding. Its single intravenous dose can reach 50~100 mg / dose, with a high safety profile. Years of extensive clinical application have also proven its safety. Protamine is metabolized in the human body into essential amino acids, and has no toxic side effects at commonly used doses. It is a pure natural molecule with good antibacterial properties and high safety, capable of replacing chemical preservatives during bFGF storage and repeated use, and also possesses the potential to exert synergistic antibacterial and anti-infective effects during treatment.

[0006] Regarding questions (iii) and (iv), human basic fibroblast growth factor (bFGF) only possesses biological activity in its monomeric state. Its peptide chain contains four cysteine ​​residues located at positions 34, 78, 96, and 101, respectively. The cysteine ​​residues at positions 78 and 96 do not form disulfide bonds and exist in free form. When bFGF monomers approach each other, they bind through the free cysteine ​​residues, forming polymers and losing their activity. This is the main reason for the poor stability of bFGF in aqueous media, and this has been experimentally confirmed. Researchers have obtained a site-directed mutagenesis mutant with Ser at positions 78 and 96, which has the same spatial structure and mitogenic activity as natural bFGF, but is less likely to form polymers (Fox GM, Schiffer SG, Rohde MF, et al. Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine ​​replaced by serine[J]. Journal of Biological Chemistry, 1988, 263(34):18452-8.). Furthermore, the common characteristic of proteins in aqueous media is their easy degradation, which is also an important factor affecting their stability. Although carbomer in gelling agents exists in a cross-linked network, it cannot completely prevent the migration and aggregation of bFGF molecules in the medium, nor can it provide sufficient protection against the self-degradation of protein molecules.

[0007] Recombinant human basic fibroblast growth factor (RBGF) is a single-chain protein with a molecular weight of approximately 17 kDa. Its active functional site is mainly concentrated in the C-terminal 146 amino acid region, comprising a heparin-binding domain and an FGF receptor-binding domain. It is fundamentally basic and rich in basic amino acid residues, such as the heparin-binding domain, which includes an N-terminal region (Lys27-Arg31) and two C-terminal regions (Arg161-Lys119) and (Lys128-Lys138) rich in basic amino acids. These positively charged basic amino acid residues bind to the negatively charged heparin-binding domain to exert their effect (Heath WF, Cantrell AS, Mayne NG, et al. Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity.[J]. Biochemistry, 1991, 30(22):5608.). Although bFGF is also a basic protein, its structure contains regions rich in non-basic amino acids, such as the N-terminal region rich in Ser, Gly, and Pro, from positions 1 to 17. Therefore, it can still form a weak, reversibly separable complex with protamine, which is rich in basic amino acids throughout its entire sequence, through charge interaction under suitable conditions. Because the heparin-binding region of bFGF is rich in positively charged basic amino acid residues, protamine will not bind to the functional sites of bFGF, thus not affecting its activity. Based on these structural characteristics, protamine has the potential to be a good protective agent for human basic fibroblast growth factor. Patent CN102357242B uses human serum albumin as a protein protective agent for recombinant bovine basic fibroblast growth factor, but human serum albumin does not have antibacterial function and does not possess a molecular conformation that allows for reversible binding to bFGF. The reversible weak binding between protamine and bFGF may result in better inclusion when used as a protectant, thereby better restricting the migration of bFGF monomers in aqueous media, preventing them from forming polymers and becoming inactive, and also providing a dual antibacterial effect.

[0008] However, like other FGFs, bFGF's biological activity is mediated through a dual-receptor system. Its receptors consist of two parts: tyrosine kinase receptors (FGFRs) and heparin sulfate proteoglycans (HSPGs). bFGF must bind to heparin to form a complex before acting on the FGFRs on the cell membrane to exert its full biological activity (Zheng, Liu, & Wang. Dual-receptor system of fibroblast growth factor. Progress in Biochemistry and Biophysics, 1998, 25: 122-125.). Without the assistance of heparin or heparin sulfate, its binding affinity to FGFRs is significantly reduced. Furthermore, heparin-bound bFGF is less prone to degradation, thus prolonging its half-life and duration of action. Protamine also binds to heparin, competitively inhibiting its activity and reducing bFGF's biological activity. Therefore, targeted design is needed to reduce the competitive binding of protamine to heparin, the active ligand of bFGF. For example, developing heparin-like molecules with relatively stronger binding affinity to bFGF and pre-binding them to bFGF to form a complex could better resist the influence of protamine. The main structural feature of heparin is the repeated chain of disaccharide units linked by (1→4) glycosidic bonds between glucosamine and iduronic acid; while the binding of bFGF to heparin involves a basic amino acid-rich region with an N-terminus (Lys27-Arg31) and two C-termini (Arg16l-Lys119) and (Lys128-Lys138), and the space of its binding surface is relatively small. Therefore, it is speculated that truncated heparin-like molecules or small-molecule heparin should also be able to bind to bFGF as ligands, promoting its full activity. S. Faham, RE Hileman, et al. obtained uniformly long heparin fragments after digestion and purification. Their research showed that these fragments not only bound to bFGF to form a complex but also exhibited bFGF-promoting activity comparable to that of intact heparin (S. Faham, RE Hileman, JR Fromm, RJ Linhardt, DC Rees. Heparin Structure and Interactions with Basic Fibroblast Growth Factor[J]. Science, 1996, 271(5252).). Through crystal structure analysis, they determined that the eight sugar residues along the helical axis of the heparin molecule roughly correspond to the binding width with bFGF, approximately 34 Å. Therefore, theoretically, more than one bFGF molecule can bind to a single heparin molecule. Based on this, we hypothesize that shorter heparin fragments, upon binding to bFGF, increase the difficulty of protamine binding, thus avoiding competitive inhibition. Furthermore, such complexes can also promote the binding of bFGF to (FGFR)s, exhibiting bFGF-promoting activity comparable to that of intact heparin.

[0009] bFGF is a basic protein with an isoelectric point of approximately 9.6-9.8, and is sensitive to trypsin, chymotrypsin, and serine proteases. Experiments show that bFGF can be stored for several years at -70℃, only one week at 4℃, and is inactivated after 1 minute at 60℃, demonstrating the significant impact of temperature on bFGF stability. The temperature at human wound sites is generally around 37℃, at which temperature the activity of various proteases sensitive to bFGF is often highest, which is detrimental to the stability of the bFGF molecule. Wound sites are also environments where immune cells such as leukocytes, neutrophils, and mast cells accumulate, secreting even larger amounts of proteases, which further hinders the stability of bFGF during treatment. The concentration of human basic fibroblast growth factor in each gram of gel is typically 3-15 μg; the low-dose nature of the drug further amplifies the negative impact of protease attack on the treatment efficacy. While bFGF is a low-dose drug, a certain concentration must be maintained during use to achieve optimal therapeutic effects. In a study conducted by Wang Xiaobing et al. on the effects of recombinant human basic fibroblast growth factor (bFGF) on the proliferation of isolated human epidermal cells, it was found that bFGF at concentrations of 1–100 ng / ml had a significant proliferative effect on cultured human epidermal cells, with the best effect achieved at 100 ng / ml. The proliferative effect decreased with further increases in bFGF concentration (Wang Xiaobing, Wang Xiaojian, Zhang Baolin. Effects of different concentrations of recombinant human basic fibroblast growth factor on the proliferation of isolated human epidermal cells [J]. Chinese Journal of Drugs and Clinical Use, 2008, 8(012):969-970.). In summary, the stability of bFGF in the wound environment and the maintenance of its dosage are key to further improving its therapeutic efficacy.

[0010] Studies have shown that free bFGF has a short-lasting effect on Schwann cell proliferation, while polyethylene glycol bFGF sustained-release microspheres can promote Schwann cell proliferation over a longer period (Perrone L, Peluso G, Melone MA. RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cell morphological changes[J]. Journal of Cellular Physiology, 2010, 217(1):60-71.). Other studies have shown that bFGF-polylactic acid-glycolic acid copolymer microspheres prepared using a double emulsion method can stably release bFGF in synovial fluid for more than 10 days (Hu Junyu, Duan Hong, Zou Yuanwen, et al. Degradation and drug release properties of sustained-release basic fibroblast growth factor microspheres in rabbit knee synovial fluid[J]. Chinese Journal of Tissue Engineering Research and Clinical Rehabilitation, 2008, 12(23):4401). -4405.), but microsphere formulations only provide sustained release and cannot protect the released free human basic fibroblast growth factor (bFGF). Furthermore, sustained-release microsphere formulations are not suitable for use on wound sites. However, when protamine, another basic protein, is present in a much higher concentration than bFGF in the formulation, it can form a protective background in the wound microenvironment, shielding bFGF from various proteases and thus prolonging the duration of action of free bFGF. The binding of heparin fragments to bFGF also creates a blocking effect, making bFGF less susceptible to degradation and helping to maintain its drug concentration and increase its half-life (B, Boilly, and, et al. FGF signals for cell proliferation and migration through different pathways[J]. Cytokine & GrowthFactor Reviews, 2000.). These factors all contribute to improving the therapeutic effect of human basic fibroblast growth factor gel. In addition, wound healing generally involves three processes: 1. Local inflammatory response stage; 2. Cell proliferation and differentiation stage; 3. Tissue remodeling and reconstruction stage. The first two stages are crucial in influencing the healing cycle. In stage 1, blood clotting at the affected site is crucial, while in stage 2, fibroblast proliferation and angiogenesis are the main focus. The procoagulant effect of protamine sulfate on the wound surface, combined with the fibroblast-promoting effect of bFGF, may further enhance the therapeutic efficacy of human basic fibroblast cytokine gel. Summary of the Invention

[0011] The purpose of this invention is to provide a recombinant human basic fibroblast growth factor gel. Through extensive literature and technical data research, and targeted design and research, the inventors have developed a recombinant human basic fibroblast growth factor gel that solves the problems existing in the prior art, such as short shelf life, inability to simultaneously exert healing-promoting and antibacterial effects on wounds, and the presence of certain chemical preservatives. Furthermore, it achieves superior therapeutic effects.

[0012] First, to achieve the goal of accelerating wound healing while simultaneously inhibiting bacterial growth in the recombinant human basic fibroblast growth factor (BGF) gel, a large number of chemical substances with disinfectant properties, including ethanol, povidone-iodine, phenol, benzalkonium chloride, benzalkonium bromide, p-chloro-m-xylenol, and chlorobutanol, were screened for compatibility. The results showed that most small-molecule antibacterial agents either affected the biological activity of BGF or the properties of the gel, and therefore could not achieve synergistic effects with BGF. Chloro-m-xylenol had a relatively small impact on the activity of BGF, but its antibacterial duration was short, and long-term use had certain toxic side effects on humans and was prone to the development of drug-resistant bacteria; therefore, it was not considered. Antibiotic use easily leads to drug resistance, and clinical practice advocates for reduced use; therefore, it was not considered in this study. Further expanding the research scope revealed a surprising finding: protamine and polylysine, both basic proteins similar to human basic fibroblast growth factor (bFGF), had relatively little impact on bFGF bioactivity. Furthermore, at certain doses, they produced sufficient antibacterial and anti-infection effects on the wound surface without negatively affecting the gel's properties. It was difficult to find a less desirable alternative.

[0013] Subsequently, through experiments, it was confirmed that protamine in the formulation exhibits excellent antibacterial properties within a dosage range of 0.1–50 mg / g. It demonstrates good bactericidal efficacy against Staphylococcus aureus (26003), Escherichia coli (44102), Pseudomonas aeruginosa (10104), and Candida albicans (98001), and also shows some inhibitory effect against Aspergillus niger (99003). Its performance meets the Class A standard in the pharmacopoeia. Furthermore, due to the relatively large molecular weight of protamine, generally between 3000 and 10000, it does not diffuse easily, which is beneficial for its long-term retention on the wound surface, thus exerting a sustained antibacterial effect. The anti-infection ability of protamine-containing human basic fibroblast cytokine gel on wounds was investigated using a protamine-free human basic fibroblast cytokine gel as a reference. Both formulations were applied to wounds in a randomly grouped Sprague Dawley (SD) rat burn infection model. After a certain period of time, skin tissue samples were taken for microbial load testing. Experimental results showed that protamine sulfate also exhibited good inhibitory and bactericidal effects against microorganisms such as Staphylococcus aureus and Pseudomonas aeruginosa in rat wounds. Therefore, the introduction of protamine sulfate can provide sufficient preservative protection during product storage, removing small-molecule chemical preservatives such as ethylparaben from the human basic fibroblast growth factor gel product. During the treatment phase, it can remain on the wound for an extended period, exerting a long-lasting antibacterial effect on various microorganisms, reducing the microbial load, and forming a synergistic effect with human basic fibroblast growth factor, thus promoting better wound healing. This was confirmed in wound treatment experiments using a burn infection animal model established in SD rats.

[0014] Human basic fibroblast growth factor (bFGF) is only biologically active in its monomeric state, where cysteine ​​residues at positions 78 and 96 do not form disulfide bonds and exist in free form. In aqueous media, it easily loses its biological activity through intermolecular polymerization of free cysteine ​​residues, and it is also prone to degradation and inactivation in water-rich environments. Although carbomer in the gel exists in a cross-linked network, it cannot completely prevent the migration and aggregation of bFGF molecules in the medium, nor can it protect against the self-degradation of protein molecules. Although bFGF is also a basic protein, its sequence contains regions rich in non-basic amino acids, such as the exposed region at positions 1-17 at the N-terminus rich in Ser, Gly, and Pro. Therefore, it can still form a reversibly separable complex with protamine, which is rich in basic amino acids throughout its sequence, under suitable conditions. In vitro bioactivity assays using the Balb / c 3T3 cell line confirmed a relatively weak binding strength between the two molecules. This weak binding is precisely what facilitates the release of the active drug molecule bFGF during use. Experimental results also showed that the presence of protamine only caused a slight rightward shift in the bFGF release curve, without any substantial impact. Further increasing the amount of protamine in the study, allowing it to encapsulate the bFGF molecule, significantly enhanced the stability of human basic fibroblast growth factor in aqueous media. This is because the encapsulation effectively restricts the migration of bFGF monomers in aqueous media, preventing them from forming polymers and becoming inactive. Simultaneously, its spatial shielding and locking effect also makes it less prone to self-degradation of bFGF molecules. Therefore, subsequent accelerated stability and long-term stability tests showed that the recombinant human basic fibroblast growth factor gel provided by this invention has a shelf life of up to 30 months, significantly better than the 18 months of current similar products.

[0015] However, further in-depth research revealed that the presence of protamine sulfate has a certain downregulating effect on the biological activity of human basic fibroblast growth factor (bFGF), negatively impacting therapeutic efficacy. After thorough experimental research and analysis, the underlying cause of this problem was identified. The active site of bFGF is mainly concentrated in the C-terminal 146 amino acid region, including binding sites, heparin binding sites, and homodimerization sites. In the human body, bFGF first binds to heparin to form a complex, and then further binds to the extracellular region of (FGFR)s (i.e., the receptors for bFGF) on the cell membrane, initiating the activation of intracellular tyrosine kinases. After a series of intracellular signal transductions, bFGF exerts its pro-proliferative activity. Even when bFGF does not bind to heparin to form a complex, it can still bind to (FGFR)s to exert its biological activity, but the binding affinity is reduced, leading to decreased activity. Simultaneously, bFGF without heparin binding is more prone to degradation, thus shortening its half-life and affecting therapeutic efficacy. The heparin-binding region of the bFGF molecule is characterized by its high content of basic amino acids. Therefore, protamine competitively binds to heparin, antagonizing the binding of bFGF to heparin molecules. Further research and development were conducted to address this challenge. Through literature review and in vitro experiments, it was found that pre-incubating bFGF with digested and purified heparin fragments or low-molecular-weight heparin to form a complex significantly reduced the competitive binding of protamine to heparin-like molecules. Furthermore, as the sugar chain length and molecular weight of the heparin-like molecules decrease, the affinity of protamine for competitive binding further declines. In vitro affinity studies based on BIACORE showed that the affinity of protamine for complexes composed of low-molecular-weight heparin (such as enoxaparin) and bFGF is significantly lower than that for complexes composed of unfractionated heparin (UFH) and bFGF, and far lower than its affinity for heparin monomers. Protamine's affinity for complexes composed of heparin fragments with molecular weights below 3500 Da and bFGF is slightly lower than that for complexes composed of low molecular weight heparin (LMWH) and bFGF. Therefore, once bFGF is co-incubated with LMWH or heparin fragments (with molecular weights below 3500 Da) to form a complex, it is difficult for protamine to competitively displace bFGF even in the presence of protamine in the system.

[0016] Subsequently, bFGF was incubated with Enoxaparin and heparin fragments with a molecular weight less than 3500 Da, respectively. Activity tests using Balb / c 3T3 cell lines showed high activity retention, with no significant difference from the activity results of bFGF monomer under conventional detection methods. This indicates that after bFGF forms a complex with the LMWH / heparin fragment, it can exert almost complete biological activity under their synergistic effect. Therefore, the gel preparation process was designed to first incubate bFGF and the LMWH / heparin fragment at 5–45°C to form a primary complex, and then blend it with protamine to induce reversible weak binding and inclusion, forming a secondary complex. After administration to the affected area, the secondary complex diffuses into the body fluids. Due to the dilution effect of the body fluids and the difference in binding force, the weakly bound protamine dissociates from the primary complex, releasing the inclusion effect. The primary complex of bFGF and the LMWH / heparin fragment is released into the lesion to exert its pro-healing biological activity, while the dissociated protamine exerts its antibacterial and background protective effects at the affected site. Although the dissociated protamine may also compete for binding with heparin-like substances in the body, the bFGF molecule has already formed a more robust complex with the LMWH / heparin fragment, and therefore is unaffected by this in vivo, allowing it to exert its full biological activity. The LMWH / heparin fragment, in this design, only acts as a coenzyme-like complex in one step of the signaling pathway; therefore, its dosage is extremely low, only 1×10⁻⁶. -6 The drug is applied at a low level, and the application site is the local epidermis, therefore it will not bring any unexpected pharmacological effects or safety risks. To improve the therapeutic effect of the newly developed human basic fibroblast growth factor gel formulation, further research and optimization of other formulation components were conducted to obtain an ideal release rate of bFGF. Experimental results showed that the presence of glycine, sodium chloride, and glycerol in the system, with concentrations of 0.5%–2.5%, 0.1%–1.5%, and 2%–10% respectively, was more conducive to the dissociation of protamine and bFGF, allowing the active molecules in the drug to exert their activity more quickly at the affected area and achieving a good drug release rate.

[0017] The binding of the LMWG / heparin fragment to bFGF also makes bFGF less susceptible to degradation in the wound microenvironment, helping to maintain its drug concentration and increase its half-life. Simultaneously, protamine, a basic protein present in much higher concentrations than bFGF in the formulation, can form a protective background in the wound microenvironment, shielding bFGF from attack by various proteases and thus prolonging the duration of action of free bFGF. Using commercially available human basic fibroblast cytokine gel as a reference, a study was conducted on the wound dose maintenance of the human basic fibroblast cytokine gel provided by this invention in an animal model of burn wounds. The test product and reference product were applied to the wounds of randomly grouped animals with deep second-degree burns. After a certain period, tissue samples were taken from the affected area, and the changes in bFGF content were detected and compared using an established ELISA method. The experimental results showed that the active ingredient in the human basic fibroblast cytokine gel provided by this invention had a significantly longer retention time in the wound microenvironment.

[0018] In summary, the human basic fibroblast growth factor gel provided by this invention not only promotes wound healing but also achieves a synergistic function of wound antibacterial activity, with a lower microbial load further contributing to faster wound healing. Simultaneously, the active ingredient bFGF provides multi-dimensional protection at the affected area, delaying the degradation by numerous proteases at approximately 37°C, and significantly improving its stability, concentration maintenance, and duration of action. Furthermore, the potential hemostatic effect of protamine sulfate also benefits the wound healing process. Therefore, these synergistic functions surprisingly produce a significant therapeutic enhancement effect. In a burn animal model established using SD rats, the human basic fibroblast growth factor gel provided by this invention significantly shortened the scab formation time and wound healing time compared to the current control group using human basic fibroblast growth factor gel. It demonstrates a clear advantage in wound healing rate and healing time. Overall, protamine sulfate is also a superior choice compared to poly-L-lysine.

[0019] Based on the above research and development work, a new formulation of human basic fibroblast growth factor preparation and its preparation process with many significant advantages have been obtained. The technical solution of this invention is as follows:

[0020] The gel formulation contains: human basic fibroblast growth factor, protamine, glycine, sodium chloride, glycerol, pH adjuster, active cofactor, and gel matrix, with water as the solvent.

[0021] Furthermore, the human basic fibroblast growth factor contained in the formulation is a recombinant human basic fibroblast growth factor obtained by fermentation, expression, and purification using genetically engineered bacteria such as Escherichia coli or yeast. Its dosage in the formulation is between 1000 IU / g and 20000 IU / g.

[0022] Furthermore, the protamine in the formulation includes protamine sulfate, protamine hydrochloride, and other salt forms. The dosage used is 0.1~50 mg / g.

[0023] Furthermore, the amount of glycine used in the formulation is 5~25 mg / g.

[0024] Furthermore, the amount of sodium chloride used in the formulation is 1~15 mg / g.

[0025] Furthermore, the amount of glycerol used in the formula is 20~100mg / g.

[0026] Furthermore, the pH adjuster in the formulation is selected from one or more of hydrochloric acid, sulfuric acid, sodium hydroxide, and triethanolamine, to adjust the pH to the range of 6.0 to 9.0.

[0027] Furthermore, the active cofactor in the formulation is low molecular weight heparin or a heparin molecular fragment. The molecular weight of the heparin molecular fragment is below 3500 Da, and the dosage is 0.1~20 μg / g. The low molecular weight heparin is selected from one or more of Enoxaparin, Dalteparin, and Nadroparin, and the dosage is 0.1~20 μg / g.

[0028] Furthermore, the gel matrix in the formulation is selected from one or more of carbomer, methylcellulose, hydroxypropyl methylcellulose, cellulose derivatives, cross-linked acrylic polymers, poloxamer, alginate, sodium hyaluronate, gellan gum, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol. The dosage is 2~50 mg / g.

[0029] Furthermore, the aqueous solvent in the formulation is water for injection or purified water that meets the quality standards stipulated in the Chinese Pharmacopoeia.

[0030] The recombinant human basic fibroblast growth factor gel with antibacterial function described in this invention can be prepared by the following methods and steps:

[0031] ①. Dissolve recombinant human basic fibroblast growth factor in water for injection or purified water at a dosage of 1000 IU / g-20000 IU / g in the final product. Then add 0.0001‰~0.02‰ of active cofactor, incubate at 5~45℃ for more than 0.5h, and filter sterilely through 0.45μm or 0.22μm filter membranes to obtain solution A.

[0032] ②. Dissolve 0.01-5.0% of protamine sulfate by weight in water for injection or purified water, and then filter it through a double-layer filter membrane of 0.45μm and 0.22μm to obtain solution B.

[0033] ③. Dissolve 0.5%~2.5% glycine, 0.1%~1.5% sodium chloride, 2%~10% glycerol, and an appropriate amount of triethanolamine that can control the pH of the final product between 6.0 and 9.0 in water for injection or purified water, and sterilize at 121~124℃ to obtain solution C.

[0034] ④. Dissolve 0.2% to 5.0% of the total weight of the gel matrix in water for injection, stir until fully dissolved to prepare a gel solution, and sterilize at 121 to 124°C to obtain solution D.

[0035] ⑤. Mix the sterilized solutions A and B separately with solutions C and D, and then mix them in an emulsifying mixing tank. After homogenization, the recombinant human basic fibroblast growth factor gel of this invention is obtained.

[0036] Beneficial effects

[0037] The beneficial effects of this invention are as follows: The recombinant human basic fibroblast growth factor gel provided, through the design of a secondary complex based on affinity differences, significantly improves the stability of bFGF in aqueous media, extending its shelf life to 30 months, which is significantly better than the 18 months of current commercially available products. Furthermore, while ensuring the bioactivity of bFGF, it simultaneously achieves the synergistic effects of accelerating wound healing and inhibiting wound microorganisms and preventing infection. While accelerating wound healing, it can reduce the microbial load and assist the human immune system in fighting microbial infections at the wound site. At the same time, the active ingredient bFGF receives multi-dimensional protection at the affected area, delaying the degradation by a large number of proteases at temperatures around 37°C, and its stability and duration of action are also significantly improved. In addition, the hemostatic effect that protamine may bring is also beneficial to the wound healing process. These synergies also bring surprisingly significant therapeutic enhancement effects. Furthermore, the newly developed formulation abandons small-molecule chemical preservatives, which is more beneficial to the health of patients. In summary, the technical solution provided by this invention solves the problems existing in current products, eliminates several shortcomings, and improves the overall performance of the product. This will further expand the clinical application scope and convenience of recombinant human basic fibroblast growth factor, improve its therapeutic benefits and clinical application value, and provide patients with a better option. Attached Figure Description

[0038] Figure 1 The trend of bioactivity changes in the recombinant human basic fibroblast growth factor in the long-term stability test of the examples and comparative examples in Test Example 5. Detailed Implementation

[0039] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can intuitively understand the advantages and effects of the present invention based on the disclosed embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0040] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Unless otherwise specified, the experimental methods used in the embodiments are considered conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Example

[0041] This invention provides a method for preparing a recombinant human basic fibroblast growth factor gel:

[0042] Based on the potency test results of recombinant human basic fibroblast growth factor stock solution, and according to the total feed amount of 1.3×10 8 IU was added to 2340g of water for injection, and then 0.26g of Enoxaparin was weighed and added, and stirred until completely dissolved. The mixture was incubated at 18~22℃ for 3h, and then sterilized by filtration through a double-layer PVDF membrane of 0.45μm and 0.22μm, and labeled as solution A.

[0043] Weigh 52g of protamine sulfate, add 3380g of water for injection, stir until completely dissolved, and then filter sterilely through a 0.45μm, 0.22μm double-layer PVDF filter membrane. Label this solution as solution B.

[0044] Weigh 910g of glycerol, 312g of sodium chloride, and an appropriate amount of triethanolamine, add them to 5850g of water for injection, stir to dissolve, and prepare an aqueous solution of glycerol, sodium chloride, and triethanolamine. Sterilize at 121~124℃ for 30~35min and label it as solution C.

[0045] Weigh 312g of glycine and add it to 11700g of water for injection, stirring until completely dissolved. Then weigh 260g of carbomer and add it to the solution, stirring until fully dissolved to prepare a carbomer gel solution. Sterilize at 121~124℃ for 30~35min and label it as solution D.

[0046] After sterilizing solutions A, B, C, and D separately, they are added sequentially to an emulsifying mixing tank. Approximately 150g of sterile water for injection is added to adjust the total volume to 26000g. The final concentrations of the excipients are: 1.2% glycine, 0.2% protamine sulfate, 1.0% carbomer, 0.01‰ enoxaparin, 1.2% sodium chloride, and 3.5% glycerol. The final concentration of recombinant human basic fibroblast growth factor is 5000 IU per gram. An appropriate amount of triethanolamine is added to adjust the pH to 7.40-7.50. The emulsifying tank stirring device is turned on at a speed of 60 rpm for at least 30 minutes to obtain the semi-finished product. The qualified semi-finished product is then packaged using a sealing and filling machine to obtain the recombinant human basic fibroblast growth factor gel provided by this invention.

[0047] Comparative Example

[0048] The preparation method of the gel in this comparative example is the same as that in the example, except that the formulation is different. The gel in this comparative example, by weight percentage, comprises: 1.0% carbomer, 0.2% polysorbate 80, 0.1% ethylparaben, 5.0% glycerol, and recombinant human basic fibroblast growth factor at a final concentration of 5000 IU per 1g, with an appropriate amount of triethanolamine to adjust the pH to 7.40-7.50. The solvent is water for injection, as in both examples.

[0049] Test Example 1

[0050] Key pharmaceutical quality tests of recombinant human basic fibroblast growth factor gel in examples and comparative examples:

[0051] (1) Appearance: Visual inspection revealed that both were colorless, transparent, viscous gels.

[0052] (2) pH value: The pH value of the gel in the example was 7.47, and the pH value of the gel in the comparative example was 7.44.

[0053] (3) Viscosity: The viscosity of the gel in the example was 12.8 Pa·S and the viscosity of the gel in the comparative example was 13.5 Pa·S.

[0054] (4) Sterility: The sterility test was performed according to the sterility test method of the 2015 edition of the Chinese Pharmacopoeia. No sterile growth was observed in the gel test tubes of the examples and comparative examples, which met the requirements.

[0055] Test Example 2

[0056] Bioactivity testing of recombinant human basic fibroblast growth factor gel in examples and comparative examples:

[0057] Preparation of standard solution: Take one vial of recombinant human basic fibroblast growth factor standard, reconstitute it according to the instructions, and dilute it with maintenance medium to a concentration of 40 IU per ml. Perform a 4-fold serial dilution in a 96-well cell culture plate, with 8 dilutions and 2 wells for each dilution. All procedures should be performed under aseptic conditions.

[0058] Preparation of test solution: Accurately weigh approximately 0.5 g of the test sample into a sterile glass test tube, add 5 ml of sterile physiological saline, mix well, centrifuge at 1000 rpm for 10 min, collect the supernatant, and dilute with maintenance medium to a concentration of 40 IU per ml. Perform a 4-fold serial dilution in a 96-well cell culture plate, creating 8 dilutions, with 2 wells for each dilution. All procedures should be performed under aseptic conditions.

[0059] Viability assay: Balb / c 3T3 cells were cultured in complete medium at 37°C and 5% CO2, with a cell concentration controlled at 1.0 × 10⁻⁶ cells / mL. 5 ~5.0×10 5 Cells / ml, passaged for 24-36 hours for biological activity assay. Discard the culture medium in the culture flask, digest and collect the cells, and prepare 5.0 × 10⁶ cells / ml using complete culture medium. 4 ~1.3×10 5 Cell suspensions of 100 μL per cell culture medium were seeded into 96-well cell culture plates. The plates were incubated at 37°C and 5% CO2 for 24 h. Then, maintenance medium was added, and the plates were incubated at 37°C and 5% CO2 for 28–32 h. The maintenance medium was discarded, and standard solutions and test samples were added to each well (100 μL). The plates were incubated at 37°C and 5% CO2 for 64–72 h. At the end of the incubation period, 20 μL of MTT solution was added to each well, and the plates were incubated at 37°C and 5% CO2 for 5–6 h. 100 μL of lysis buffer was added to each well, and the plates were mixed thoroughly. The absorbance was measured at 570 nm using a microplate reader with 630 nm as the reference wavelength, and the results were recorded.

[0060] Results Calculation: Experimental data were processed using a computer program or linear regression method, and the results were calculated using the following formula:

[0061] Biological activity of the test sample (IU / ml) = Pr × Ds × Es / (Dr × Er)

[0062] In the formula, Pr represents the biological activity of the standard, in IU / ml; Ds represents the pre-dilution factor of the test sample; Dr represents the pre-dilution factor of the standard; Es represents the half-effective dose dilution factor of the test sample equivalent to the standard; and Er represents the half-effective dose dilution factor of the standard.

[0063] The bioactivity of 10 batches of recombinant human basic fibroblast growth factor gel prepared according to the procedures of the examples and comparative examples was tested and analyzed using this bioactivity assay method. The test data are shown in Table 1. The results show that the bioactivity of each batch was within 80%-150% of the labeled amount, meeting the quality standard requirements. No batches exceeded the standard limits, and the quality of each batch was relatively stable.

[0064] Table 1. Summary of bioactivity test results of human basic fibroblast growth factor

[0065]

[0066] The results of the two sets of experiments were subjected to normality tests and t-tests, and the results are shown in Table 2.

[0067] Table 2 Statistical Analysis of Bioactivity Comparison

[0068]

[0069] Independent Samples Test

[0070]

[0071] The results showed that the sig value was 0.209 > 0.05, indicating that the variances were homogeneous. The two-tailed probability p-value was 0.539 > 0.05, indicating that the recombinant human basic fibroblast growth factor gel formulation of the examples had no significant difference in biological activity compared with the comparative example.

[0072] Test Example 3

[0073] Antibacterial efficacy test of recombinant human basic fibroblast growth factor gel in the examples:

[0074] The "Antimicrobial Efficacy Test Method" in General Chapter 1121 of Part IV of the 2015 edition of the Chinese Pharmacopoeia must be performed in a liquid state; the antimicrobial efficacy of protamine sulfate cannot be examined in a gel state. Carbomer is a loosely structured, inert resin that has no significant inhibitory or growth-promoting effect on microorganisms. Therefore, the antimicrobial efficacy test of protamine sulfate was conducted without the addition of carbomer. A carbomer-free test sample was prepared according to the formulation in the examples with a protamine sulfate concentration of 2.0‰. Two sets of carbomer-free test samples were then prepared simultaneously with protamine sulfate concentrations of 1.0‰ and 0.5‰.

[0075] The test strains included Staphylococcus aureus (26003), Escherichia coli (44102), Pseudomonas aeruginosa (10104), Candida albicans (98001), and Aspergillus niger (99003), at a concentration of 10 μL per 1 ml of test sample. 5 ~10 6CFU inoculation was performed directly onto each test bacterium, with one test bacterium inoculated into each container. The inoculation was thorough, ensuring even distribution of the test bacteria in the sample. The sample was stored at 20–25°C, protected from light. At 6 hours, 24 hours, and 7 days post-inoculation, 1 ml of the sample was taken from each inoculated container to determine the bacterial count. Tryptic-soybean agar was used for bacterial microorganism determination, and Sabouraud dextrose agar was used for fungal microorganism determination. Bacterial counts were determined according to Section 1105 of the 2015 edition of the Chinese Pharmacopoeia, Part IV, "Microbial Limit Tests for Non-Sterile Products: Microbial Counting Method". The results of the antimicrobial efficacy tests are shown in Tables 3 and 4.

[0076] Table 3. Results of bacterial count test of antibacterial efficacy.

[0077]

[0078] Table 4 Summary of Antibacterial Efficacy Test Results

[0079]

[0080] Note NR: The test bacteria did not resume growth.

[0081] The experimental results showed that the test sample containing protamine sulfate had excellent bactericidal effects against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, and also had a certain inhibitory effect on Aspergillus niger. The antibacterial efficacy increased with the increase of the protamine sulfate concentration. The antibacterial efficacy was significantly better at concentrations of 1.0 mg / ml and 2.0 mg / ml than at 0.5 mg / ml. After inoculating the specified test bacteria into the solution, at concentrations of 1.0 mg / ml and 2.0 mg / ml, no growth of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans was detected at 24 hours, and the bacterial counts of these four bacteria decreased by more than 6.0 log units at 6 hours. While it could not completely kill Aspergillus niger, its performance basically met the Class A standard in the pharmacopoeia. The Aspergillus niger bacterial count decreased by more than 2 log units on day 7. Aspergillus niger is also a rare pathogen that causes wounds.

[0082] The test results showed that protamine sulfate has a broad-spectrum antibacterial effect, and the dosage of 0.2% has a good killing effect on microorganisms.

[0083] Test Example 4

[0084] Tests on the antibacterial activity of recombinant human basic fibroblast growth factor gel against infected wounds (Examples and Comparative Examples):

[0085] Establishment of a deep second-degree burn model: Sixty SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at 40 mg / kg. The backs were shaved and routinely disinfected. A self-made 3 cm diameter burn board was applied to both sides of the spine on the back of the rats simultaneously for 9 seconds to obtain a model rat meeting the criteria of deep second-degree burns: deep dermal damage, remaining skin appendages, slightly thicker surface tissue due to deterioration, smaller or thinner blisters, and slightly lower skin temperature (histological observation after HE staining). The rats were housed individually in cages at an ambient temperature maintained at 20–25℃. All cages, bedding, feed, and water used for housing were autoclaved. Rats with relatively good mental state and appetite were selected after the burns and randomly divided into 6 groups of 8 rats each, half male and half female.

[0086] Establishment of a burn wound infection model: Based on the established deep second-degree burn model, common conditionally pathogenic bacteria of the skin, Staphylococcus aureus and Pseudomonas aeruginosa, were selected to further establish a burn wound infection model.

[0087] Staphylococcus aureus (26003) and Pseudomonas aeruginosa (10104) were recovered and inoculated onto agar medium. The cultures were incubated at 35°C for 18–22 h. Colonies were then picked up with an inoculation loop and inoculated onto blood culture medium, and incubated for another 18–22 h. A 1×10⁻⁶ culture medium was prepared using the dilution method. 9 Fresh bacterial suspension at CFU / ml. In rats of groups 1, 2, and 3 after injury, one side of the wound was inoculated with Staphylococcus aureus suspension, while the other side was treated with an equal volume of physiological saline as a control. The wounds were covered with sterile gauze. In rats of groups 4, 5, and 6 after injury, one side of the wound was inoculated with Staphylococcus aureus suspension, while the other side was treated with an equal volume of physiological saline as a control. The wounds were covered with sterile gauze.

[0088] Groups 1 and 4 wounds were treated with human basic fibroblast cytokine gel as described in the example; groups 2 and 5 wounds were treated with human basic fibroblast cytokine gel as described in the comparative example; groups 3 and 6 wounds were covered with ordinary gauze, and the wounds were disinfected and the gauze was changed every 3 days. In each group of 8 rats, a small amount of tissue was taken from the wound on days 3, 6, and 9 post-injury. The tissue was weighed, minced, and mixed with PBS buffer at a ratio of 1:10. The mixture was thoroughly homogenized using an electric stirrer. The homogenate was diluted 1:10, and 0.1 mL of the diluted solution was inoculated onto nutrient agar plates and incubated at 35–37°C for 18–24 hours. The number of bacteria under the scab was then counted. Bacterial count (CFU / g) = colony count (CFU) × dilution factor / tissue weight (g). The experimental results are shown in Table 5.

[0089] Table 5. Changes in wound microbial load at different time points during dressing change treatment in SD rats (x±s, n=8)

[0090]

[0091] Experimental results show that the recombinant human basic fibroblast growth factor gel in the examples has a significant inhibitory effect on Staphylococcus aureus and Pseudomonas aeruginosa in infected wounds, demonstrating a clear advantage.

[0092] Test Example 5

[0093] Stability test of recombinant human basic fibroblast growth factor gel in examples and comparative examples:

[0094] Long-term stability tests were conducted on three batches of recombinant human basic fibroblast growth factor gels from the examples and three batches from the comparative examples, using bioactivity as an indicator.

[0095] Three consecutive batches of recombinant human basic fibroblast growth factor gel prepared according to the method described in the example (batch numbers: S8, S9, S10) and three consecutive batches of recombinant human basic fibroblast growth factor gel prepared according to the comparative method (batch numbers: D8, D9, D10) were simultaneously placed in a refrigerated environment at 2–8°C. Samples were taken at the end of the 0th, 3rd, 6th, 9th, 12th, 18th, 24th, and 30th months of storage for bioactivity testing and comparison. The experimental results are shown in Table 6, and the trend of bioactivity changes is shown in the appendix. Figure 1 .

[0096] The results showed that under long-term stability test conditions stored at 2–8°C, the biological activity of the comparative recombinant human basic fibroblast growth factor was all below the lower limit of the quality standard (80% of the labeled amount) by the 30th month, and two batches were close to the 80% lower limit by the 24th month. However, the biological activity of the example recombinant human basic fibroblast growth factor still fully met the quality standard requirements by the 30th month. The rate of decline in biological activity was significantly slower, and the stability was better, essentially achieving a shelf life of 30 months.

[0097] Table 6 Summary of Bioactivity Test Results in Long-Term Trials

[0098]

[0099] Test Example 6

[0100] Comparative experiment on the healing-promoting effects of recombinant human basic fibroblast growth factor gel in the examples and comparative examples:

[0101] Establishment of a deep second-degree burn model: Sixty SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at 40 mg / kg. The backs were shaved and routinely disinfected. A self-made 3 cm diameter burn board was applied to both sides of the spine on the rats' backs for 9 seconds to induce burns, obtaining rats meeting the criteria for deep second-degree burns: deep dermal damage, remaining skin appendages, slightly thickened superficial tissue due to deterioration, small or thin blisters, and slightly lower skin temperature (histological observation after HE staining). The rats were housed individually at an ambient temperature maintained at 20–25℃. All cages, bedding, feed, and water used were autoclaved. Rats with relatively good mental state and appetite were selected after burns and randomly divided into three groups of 10 rats each (half male and half female).

[0102] After modeling, the wounds in group 1 were treated with human basic fibroblast cytokine gel as described in the example; the wounds in group 2 were treated with human basic fibroblast cytokine gel as described in the comparative example; and the wounds in group 3 were covered with ordinary gauze. The wounds were disinfected and the gauze was changed every 3 days. Wound healing was considered to be achieved when the epithelialization rate was >95%. The scab formation time and final healing time of the wounds in each group of rats were recorded. Data are expressed as mean ± standard deviation (X±S). SPSS 18.0 software was used for t-tests, and one-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant. The experimental results are shown in Table 7.

[0103] Table 7. Scabbing time and healing time for each group of experiments (n=10)

[0104]

[0105] The comparison of experimental results shows that the wound healing time and scab formation time were the shortest in the example group of rats; the control group was second; and the wound scab formation and healing time were the longest in the untreated group. The differences among the three groups were statistically significant (P < 0.05). Under the combined effects of these multiple mechanisms, the human basic fibroblast growth factor gel in the example achieved faster wound healing and demonstrated superior therapeutic efficacy.

Claims

1. A recombinant human basic fibroblast growth factor gel with wound antibacterial activity, characterized in that, Composed of the following components: recombinant human basic fibroblast growth factor, protamine sulfate, glycine, sodium chloride, glycerol, pH adjuster, active cofactor, and gel matrix, using purified water or water for injection as solvent; wherein the active cofactor is a heparin fragment or low molecular weight heparin, the heparin fragment having a molecular weight below 3500 Da, and the low molecular weight heparin selected from one or more of enoxaparin, dalteparin, and nadroparin; wherein the amount of recombinant human basic fibroblast growth factor is 1000~20000. IU / g; the amount of protamine sulfate is 0.01%~5.0% w / w; the amount of glycine is 0.5%~2.5% w / w; the amount of sodium chloride is 0.1%~1.5% w / w; the amount of glycerol is 2%~10% w / w; the amount of active cofactor is 0.0001‰~0.02‰ w / w; the amount of gel matrix is ​​0.2%~5.0% w / w; the pH value of the gelling agent is 6.0~9.

0.

2. The gelling agent according to claim 1, characterized in that, The pH adjuster is selected from one or more of hydrochloric acid, sulfuric acid, sodium hydroxide, and triethanolamine.

3. The gelling agent according to claim 1, characterized in that, The gel matrix is ​​selected from one or more of the following: carbomer, methylcellulose, hydroxypropyl methylcellulose, cross-linked acrylic polymer, poloxamer, alginate, sodium hyaluronate, gellan gum, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol.

4. A method for preparing the recombinant human basic fibroblast growth factor gel according to claim 1, characterized in that, Includes the following steps: 1) Dissolve recombinant human basic fibroblast growth factor in water at a dosage of 1000~20000 IU / g in the final product, then add 0.0001‰~0.02‰ of active cofactor, incubate at 5~45℃ for more than 0.5h, and then filter sterile through 0.45μm and 0.22μm filter membranes to obtain solution A; 2) Dissolve 0.01% to 5.0% of protamine sulfate by weight in an aqueous solvent, and then filter it through 0.45μm and 0.22μm filter membranes for sterilization. This is solution B. 3) Dissolve 0.5%~2.5% glycine, 0.1%~1.5% sodium chloride, 2%~10% glycerol, and a pH adjuster that can control the pH of the final product at 6.0~9.0 in an aqueous solvent, and sterilize at 121~124℃ to obtain solution C. 4) Dissolve 0.2-5.0% of the total weight of the gel matrix in an aqueous solvent, stir until fully dissolved, and sterilize at 121-124℃ to obtain solution D. 5) Mix solutions A, B, C, and D separately, then add them sequentially to an emulsifying mixing tank, add water solvent to the total volume, homogenize, and obtain the human basic fibroblast growth factor gel.

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