A pharmaceutical composition for promoting wound healing and a preparation method thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a medicine composition for promoting wound healing and a preparation method thereof. The present application also provides a medicine composition for effectively promoting wound healing containing the polypeptide and the use of the polypeptide in the preparation of the medicine for promoting wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for promoting wound healing and its preparation method. Background Technology

[0002] Skin is directly exposed to numerous internal and external environments that can affect its structure and function, making it highly susceptible to injury. Rapid repair after skin injury is a crucial prerequisite for restoring homeostasis and normal physiological function. Wound healing primarily involves four stages: hemostasis, inflammatory response, cell proliferation, and tissue remodeling. The physiological regulation of skin wound healing is a complex process, relying on the interactions between various cell types and mediators within a highly complex physiological healing process. Therefore, it can be influenced by many factors, leading to chronic trauma or non-healing skin ulcers. Population aging, the rising incidence of diabetes and obesity have all contributed to the increasing prevalence of chronic trauma and a sharp increase in medical costs. Chronic trauma easily leads to secondary infections, water and electrolyte imbalances, septic shock, multiple organ failure, and even death. Therefore, timely and effective reconstruction of the structural and functional integrity of the skin after injury is vital for human health and survival.

[0003] Traditional wound healing drugs can be divided into two main categories: small molecule compounds extracted from plants and growth factor proteins, represented by epidermal growth factor (EGF). While these drugs can promote wound healing, they also have drawbacks. For example, the former type of compounds is unstable and has relatively low activity, while the latter type of growth factors is expensive, requires strict storage conditions, and is prone to causing hypertrophic scarring. In addition, stem cells are considered an attractive alternative for wound repair. However, in the harsh inflammatory environment, stem cells in the wound bed exhibit poor activity, which often reduces their therapeutic potential. Therefore, developing novel and promising preventative measures for treating chronic skin trauma is undoubtedly urgent. Summary of the Invention

[0004] In view of the existing technology, the purpose of this invention is to provide a polypeptide CA-608, its composition and its application in promoting wound healing. The polypeptide of this invention has a good therapeutic effect on wound healing, good safety and significant efficacy.

[0005] The present invention first provides a polypeptide CA-608, characterized in that the amino acid sequence of the polypeptide is AGNIPEFVHNFIRCKFC, as shown in SEQ ID NO:1.

[0006] Another aspect of the present invention also provides the use of the polypeptide CA-608 in the preparation of a wound-healing medicament.

[0007] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the polypeptide CA-608 and a pharmaceutically acceptable carrier.

[0008] The polypeptides of the present invention, or pharmaceutical compositions comprising said polypeptides, can be applied topically to an individual.

[0009] The present invention comprises a pharmaceutical composition containing the aforementioned polypeptide, which is formulated into a formulation for topical application.

[0010] In some embodiments, the topically applied formulation includes liquids, ointments, gels, slurries, creams, emulsions, powders, and latexes.

[0011] In some embodiments, the present invention provides a composition comprising about 0.1% (w / w) to about 2% (w / w) of a gelling agent and about 0.0001% (w / w) to about 0.05% (w / w) of a polypeptide CA-608, wherein the amino acid sequence of said polypeptide is as shown in SEQ ID NO:1.

[0012] In some embodiments, examples of gelling agents include (but are not limited to) polyethylene glycol (PEG) diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, chitosan, hyaluronic acid, collagen, fibrin, gum arabic, alginate, natto gum, aloe vera, bentonite, carbomer, carboxymethyl cellulose, ethyl cellulose, gelatin, elastin, hydroxypolyamide, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, magnesium aluminum silicate, methyl cellulose, poloxamer, polyvinyl alcohol, sodium alginate, tragacanth gum, xanthan gum, gelatin, carboxyvinyl polymers, starch, water-swellable hydrocolloids, carrageenan, hyaluronic acid, agarose, alginate, acrylates, and ammonium acryloyl dimethyl taurate / VP copolymers.

[0013] In some embodiments, the gelling agent is carbomer, xanthan gum, methylcellulose, or aloe vera.

[0014] In some embodiments, the composition has a pH in the range of about 5.5 to about 7.5. In some embodiments, the pH is in the range of about 6.0 to about 7.5, about 6.5 to about 7.5, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.5 to about 7.0, about 5.5 to about 6.5, or about 6.0 to about 6.5.

[0015] In some embodiments, the composition has a viscosity in the range of about 0.05 Pa·s to about 200 Pa·s. In some embodiments, the viscosity is in the range of about 0.1 Pa·s to about 200 Pa·s, about 0.5 Pa·s to about 200 Pa·s, about 1.0 Pa·s to about 200 Pa·s, about 5 Pa·s to about 200 Pa·s, about 10.0 Pa·s to about 200 Pa·s, about 20.0 Pa·s to about 200 Pa·s, about 40.0 Pa·s to about 200 Pa·s, about 60.0 Pa·s to about 200 Pa·s, about 80.0 Pa·s to about 200 Pa·s, about 100.0 Pa·s to about 200 Pa·s, about 120 Pa·s to about 200 Pa·s, about 140 Pa·s to about 200 Pa·s, about... The range is from 160 Pa·s to about 200 Pa·s, from about 0.05 Pa·s to about 160 Pa·s, from 0.05 Pa·s to about 140 Pa·s, from 0.05 Pa·s to about 120 Pa·s, from 0.05 Pa·s to about 100 Pa·s, from 0.05 Pa·s to about 80 Pa·s, from 0.05 Pa·s to about 60 Pa·s, from 0.05 Pa·s to about 40 Pa·s, from 0.05 Pa·s to about 20 Pa·s, from 0.05 Pa·s to about 10 Pa·s, from 0.05 Pa·s to about 5.0 Pa·s, from 0.05 Pa·s to about 3.0 Pa·s, or from 0.05 Pa·s to about 1.0 Pa·s.

[0016] In some embodiments, the amount of immunomodulatory protein is in the range of about 0.0001% (w / w) to about 0.03% (w / w), about 0.0001% (w / w) to about 0.01% (w / w), about 0.0001% (w / w) to about 0.03% (w / w), about 0.0001% (w / w) to about 0.01% (w / w), about 0.0001% (w / w) to about 0.005% (w / w), about 0.0001% (w / w) to about 0.003% (w / w), about 0.0001% (w / w) to about 0.001% (w / w), or about 0.0001% (w / w) to about 0.0005% (w / w).

[0017] In some embodiments, the amount of gelling agent is in the range of about 0.5% (w / w) to about 2.0% (w / w), about 0.5 (w / w) to about 1.5% (w / w), about 0.5 (w / w) to about 1.2% (w / w), about 0.5 (w / w) to about 1.0% (w / w), about 0.1 (w / w) to about 1.5% (w / w), about 0.1 (w / w) to about 1.0% (w / w), about 0.1 (w / w) to about 0.5% (w / w), about 1.0% (w / w) to about 2.0% (w / w), or about 1.5% (w / w) to about 2% (w / w).

[0018] In some embodiments, the pharmaceutical composition is applied topically via a spray device, dressing, or patch.

[0019] Another aspect of the invention also provides the use of a pharmaceutical composition comprising the polypeptide in the preparation of a wound-healing medicament.

[0020] In some embodiments, the wound is a chronic wound or an acute wound.

[0021] In some embodiments, the chronic wound includes diabetic ulcers, pressure sores, and tropical ulcers.

[0022] In some embodiments, the acute wound includes surgical incisions, traumatic wounds, and burn wounds.

[0023] The polypeptide of this invention exhibits significant wound-healing activity, which can be used to accelerate the healing of skin wounds and reduce scarring. Therefore, the polypeptide of this invention possesses excellent wound-healing activity and can be used in pharmaceuticals with skin-repairing functions, thus having certain clinical application value. Furthermore, the polypeptide CA-608 provided by this invention is easy to synthesize, low in cost, and easy to promote and apply. Attached Figure Description

[0024] Figure 1A-1C The results shown indicate that peptide CA-608 promotes HaCaT ( Figure 1A ), HFF Figure 1B ), HUVEC Figure 1C Cell proliferation.

[0025] Figure 2 The results show that the peptide CA-608 promotes HUVEC cell migration and has scratch repair activity.

[0026] Figure 3 The results show that the peptide CA-608 accelerates the healing of full-layer skin injuries in mice.

[0027] Figure 4 The results show that the CA-608-loaded polypeptide hydrogel accelerates wound healing in mice with full-layer skin injury. Detailed Implementation

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0029] Except as provided in the operational examples or otherwise indicated, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0030] The invention described herein may include one or more ranges of values ​​(e.g., size, concentration, etc.). A range of values ​​will be understood to include all values ​​within that range, including the value defining the range and values ​​adjacent to the range that result in the same or substantially the same result as the value directly adjacent to the value defining the boundary of the range. For example, those skilled in the art will understand that a 10% variation in the upper or lower limit of the range may be perfectly suitable and covered by this invention. More specifically, the variation in the upper or lower limit of the range will be 5%, or, as is generally recognized in the art, the greater of the two.

[0031] The term "wound healing" refers to the partial or complete restoration of tissue integrity.

[0032] The term "chronic wound" refers to a wound that fails to heal within the normal timeframe that would otherwise heal in a healthy individual. Chronic wounds are those that fail to heal due to an individual's health condition; for example, poor circulation or a disease such as diabetes, or medications that inhibit the normal healing process. In some cases, chronic wounds can remain unhealed for weeks, months, or even years. Examples of chronic wounds include (but are not limited to) diabetic ulcers, pressure sores, and tropical ulcers.

[0033] The term "chronic wound" refers to a wound that forms suddenly and heals relatively quickly, caused by an accident, trauma, or external violence. These wounds typically occur within a short period, resulting in skin tears, cuts, or ruptures, and most heal noticeably within 7–10 days. Examples of acute wounds include (but are not limited to) surgical incisions, traumatic wounds, and burn wounds.

[0034] As used herein, the terms “promote,” “increase,” “promoting,” and “increasing” are used interchangeably. These terms refer to an increase in a measured parameter in a treated cell, tissue, or individual compared to an untreated cell, tissue, or individual. The same cells, tissue, or individual may also be compared before and after treatment. In some embodiments, the increase in the treated cell, tissue, or individual compared to an untreated cell, tissue, or individual is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, or more times.

[0035] As used herein, the terms “treatment” or “improvement” are used interchangeably. These terms refer to the means of achieving a beneficial or desired outcome (including, but not limited to, therapeutic and / or preventative benefits).

[0036] As used in this article, the term "promoting wound healing" refers to improving wound healing compared to the wound healing that would be observed in an untreated wound.

[0037] The term "effective amount" herein refers to a sufficient quantity of the polypeptide that, according to the invention, provides the desired therapeutic effect or induces a specific type of response. The required effective amount varies between individuals and is determined based on disease state, physical condition, age, sex, individual species, and weight, etc. However, an appropriate effective amount can be synthesized by standard methods or in any manner commonly used or known to those skilled in the art. For example, the pharmaceutical composition can be administered at daily doses ranging from 0.0001 mg / kg to 1000 mg / kg, specifically from 0.001 mg / kg to 100 mg / kg.

[0038] "Subjects" refers to all animals, including humans, that are injured or may be injured. Animals include not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar, but not limited, symptoms.

[0039] In this application, unless otherwise specifically stated, the use of the singular includes the plural. In this application, unless otherwise stated, the use of “or” means “and / or”. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not restrictive. Additionally, unless otherwise specifically stated, terms such as “element” or “component” cover elements and components that include one unit as well as elements and components that include more than one subunit. Additionally, the use of the term “part” can include a portion of a part or an entire portion. Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the specified integer or group of integers, but not to exclude any other integer or group of integers.

[0040] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier. This carrier includes, but is not limited to, physiological saline, buffered physiological saline, glucose, water, glycerol, ethanol, propylene glycol, polyoxyethylene castor oil, nanoparticles, liposomes, polymers, and combinations thereof. In addition to standard carriers, the pharmaceutical compositions of this invention comprise one or more excipients commonly used in common standard formulations, such as surfactants, solubilizers, stabilizers, emulsifiers, thickeners, and preservatives. These excipients are well known to those skilled in the art.

[0041] The pharmaceutical compositions of the present invention can be administered via one or more pharmaceutically acceptable carriers in a manner suitable for a chosen route of administration, including systemic or local administration, via enteral or parenteral administration such as injection, infusion or implantation, oral administration, transdermal or local administration. In some embodiments of the invention, the compositions are prepared as pharmaceutically acceptable carriers, and the formulations comprise liquids, ointments, gels, slurries, creams, emulsions, powders, latexes, and any applicable form. In some embodiments, the formulation is applied locally via a spray device, dressing, or patch.

[0042] The pharmaceutical compositions of the present invention also include a gelling agent to form a topical gel product with a viscosity in the range of about 0.05 Pa·s to about 200 Pa·s.

[0043] The pharmaceutical compositions of the present invention are formulated to a pH of 5.5 to 7.5. In one embodiment, the pH of the aqueous medium can be adjusted by low concentrations of suitable biocompatible buffering agents, non-limiting examples of which are glycerol, sodium carbonate and sodium bicarbonate, and sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0044] The pharmaceutical compositions of the present invention may also contain one or more additional antimicrobial agents to enhance their bactericidal activity against important pathogens (e.g., Staphylococcus aureus or Pseudomonas aeruginosa), prevent the overgrowth of any strains that develop resistance during long-term treatment, or broaden the antimicrobial spectrum to include non-bacterial pathogens, including (but not limited to) fungi.

[0045] The compositions of the present invention can be administered daily or intermittently, with a frequency of once daily or two to three times daily. If each of the two active ingredients is a single formulation, their frequency of administration can be the same or different. Furthermore, the compositions of the present invention can be used alone or in combination with other wound healing agents. Considering all the foregoing factors, it is important to administer the lowest possible dose to achieve optimal therapeutic effect without side effects, which can be readily determined by those skilled in the art. In some embodiments, the dosing regimen is repeated, for example, once, twice, three times, or more; for example, repeated over the remaining lifespan of the individual in need. In other embodiments, the patient is treated with the pharmaceutical compositions of the present invention using a 14-day treatment regimen.

[0046] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0047] Example 1: Preparation of polypeptides

[0048] The polypeptide shown in SEQ ID NO: 1 (sequence: AGNIPEFVHNFIRCKFC) was synthesized by Dankang Biotechnology Co., Ltd. Mass spectrometry and high-performance liquid chromatography analysis confirmed that the obtained product was the target polypeptide compound. The polypeptide compound was a white powder with a purity ≥98%. The lyophilized powder was dissolved in physiological saline to prepare a polypeptide solution with a concentration of 250 μg / ml, which was then sterilized by filtration membrane before use.

[0049] Example 2. Peptide CA-608 promotes the proliferation of HaCaT, HFF, and HUVEC cells.

[0050] Reepithelialization and granulation tissue formation are crucial for wound healing, requiring the joint participation of cells such as keratinocytes, fibroblasts, and endothelial cells. Therefore, the proliferation and migration of keratinocytes, fibroblasts, and endothelial cells are particularly important for wound repair.

[0051] Human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT), and human skin fibroblasts (HFF) were seeded into 96-well plates (5 × 10⁻⁶ cells per well). 3 Cells were cultured in wells at 10 μL each (cells / well). After cell adhesion, serially diluted CA-608 peptide was added to each well, with final concentrations of 0, 2.5, 5, 10, and 20 μg / mL. The wells were incubated at 37°C with 5% CO2 for 16 h. 10 μL of CCK-8 solution (Yisheng Biotechnology, catalog number: 40203ES60) was added to each well, and the wells were incubated at 37°C with 5% CO2 for 1 h in the dark. The absorbance at 450 nm was measured using a microplate reader, with a zeroing well also included. Cell viability was calculated using the following formula: Cell viability (%) = [Experimental wells - Zeroing wells] / [Control wells - Zeroing wells] × 100%, where the experimental wells contain the absorbance of cells, CCK-8 solution, and CA-608; the zeroing wells contain the absorbance of culture medium and CCK-8 solution; and the control wells contain the absorbance of cells and CCK-8 solution.

[0052] The results are as follows Figure 1A-1C As shown, peptide CA-608 can promote HaCaT ( Figure 1A ), HFF Figure 1B ), HUVEC Figure 1C Cell proliferation exhibits a dose-dependent effect.

[0053] Example 3. Peptide CA-608 promotes HUVEC cell migration

[0054] The effect of peptide CA-608 on HUVEC cell migration was detected using a cell scratch assay. HUVEC cells were seeded into 6-well plates and cultured to approximately 90% confluence. Cells were then starved using serum-free medium. After 24 h, scratches were created using a 200 μL pipette tip to simulate wounds. After scratching, the plates were washed three times with PBS to remove the scratched cells. The experimental group was cultured in serum-free medium containing a final concentration of 20 μg / mL of peptide CA-608. The control group was cultured in serum-free medium. Cell migration was recorded by photographing at 0, 12, 24, and 48 h.

[0055] The results are as follows Figure 2 As shown, peptide CA-608 significantly enhanced the migration of HUVECs. Compared with the control group, the scratch repair rates of HUVECs treated with peptide CA-608 were 58.85% and 89.55% at 24 h and 48 h, respectively, while the repair rates of the control group were 43.90% and 55.10%.

[0056] Example 4. In vivo assay of the skin wound healing-promoting activity of peptide CA-608

[0057] Balb / c mice were randomly divided into a control group and a treatment group, with five mice in each group. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital, their hair was removed, and the skin was disinfected with 75% medical alcohol. A full-thickness skin wound of approximately 6 mm in diameter was then made on the back of each mouse using surgical scissors. After the mice regained consciousness, they were placed in cages for rearing. The control group and the treatment group were treated with sterile PBS and 250 μg / mL of peptide CA-608, respectively, every 12 hours. The wound area and healing rate were calculated using ImageJ software.

[0058] like Figure 3 As shown, compared with the control group, topical application of peptide CA-608 significantly accelerated the wound healing process in mice. At 4, 8, and 12 days post-traumatic injury, the wound healing rates of mice treated with topical peptide CA-608 reached 36%, 64%, and 94%, respectively, while the control group only achieved 17%, 33%, and 65.5%, clearly demonstrating that the wounds of mice treated with peptide CA-608 healed almost completely. No abnormal behavior or death was observed in the mice after trauma, indicating that peptide CA-608 has good safety.

[0059] Example 5. Preparation of hydrogel loaded with peptide CA-608

[0060] Examples of hydrogel formulations are listed in the table below.

[0061] serial number gelling agent CA-608 Ultrapure water pH A 0.5% (w / w) xanthan gum 0.002% (w / w) The remaining part Approximately 7.0 B 0.2% (w / w) xanthan gum 0.005% (w / w) The remaining part Approximately 7.0 C 0.1% (w / w) methylcellulose 0.0002% (w / w) The remaining part Approximately 6.5 D 2% (w / w) Aloe Vera 0.05% (w / w) The remaining part Approximately 6.0

[0062] The polypeptide CA-608 having SEQ ID NO:1 was added to ultrapure water and then mixed thoroughly. Subsequently, a gel-forming agent was added to the resulting mixture with continuous stirring until a gel was formed. The resulting gel was incubated in a refrigerator at 4°C for at least 16 hours. The resulting gel was then stored in a suitable container.

[0063] Example 6. In vivo assay of the skin wound healing-promoting activity of hydrogel loaded with peptide CA-608

[0064] Balb / c mice were randomly divided into a control group and a treatment group, with five mice in each group. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital, their hair was removed, and the skin was disinfected with 75% medical alcohol. A full-thickness skin wound of approximately 6 mm in diameter was made on the back of each mouse using surgical scissors. After the mice regained consciousness, they were housed in cages. The control and treatment groups were treated every 12 hours with sterile PBS, peptide CA-608, carbomer, and hydrogel B loaded with peptide CA-608, respectively. The wound area and healing rate were calculated using ImageJ software.

[0065] like Figure 4 As shown, compared with the control group, topical application of hydrogel B loaded with peptide CA-608 significantly accelerated the wound healing process in mice. Eight days post-traumatic injury, the wound healing rates in mice treated with topical PBS, peptide CA-608, carbomer, and hydrogel B loaded with peptide CA-608 reached 31.35%, 60.30%, 36.75%, and 83.55%, respectively.

Claims

1. A polypeptide CA-608, which consists of the amino acid sequence shown in SEQ ID NO:

1.

2. A pharmaceutical composition comprising an effective amount of the polypeptide CA-608 as described in claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, wherein it is formulated into a formulation for topical application.

4. The pharmaceutical composition according to claim 3, wherein the topically applied formulation comprises a liquid, ointment, gel, powder, or latex.

5. The pharmaceutical composition according to claim 4, wherein the topically applied formulation is a gel.

6. The pharmaceutical composition of claim 5, wherein the gel comprises 0.1% (w / w) to 2% (w / w) of a gelling agent and 0.0001% (w / w) to 0.05% (w / w) of a polypeptide CA-608.

7. The pharmaceutical composition according to claim 5, wherein the gelling agent used in the gel is carbomer, xanthan gum, methylcellulose, or aloe vera.

8. Use of the polypeptide according to claim 1 in the preparation of a wound-healing medicament.

9. Use of the pharmaceutical composition according to any one of claims 2-7 in the preparation of a wound-healing medicament.

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