Pharmaceutical composition for promoting wound healing and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a pharmaceutical composition for promoting wound healing and a preparation method thereof. Meanwhile, the invention provides a pharmaceutical composition containing the polypeptide and capable of effectively promoting wound healing and application of the polypeptide to preparation of a medicine for promoting wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a pharmaceutical composition for promoting wound healing and a preparation method thereof. Background Art

[0002] The skin is directly exposed to many internal and external environments that can affect its structure and function, so it is extremely vulnerable to damage. Rapid repair of the skin after injury is an important prerequisite for reestablishing the body's internal environmental homeostasis and normal physiological function. Wound healing mainly includes four stages: hemostasis, inflammatory response, cell proliferation and tissue remodeling. The physiological regulation of skin wound healing is a complex process that relies on the interaction between multiple cell types and mediators in a highly complex physiological healing process. Therefore, it may be affected by many factors, leading to the transformation into chronic wounds or non-healing skin ulcers. The aging population, the rising incidence of diabetes and obesity have all led to an increase in the prevalence of chronic wounds and a sharp increase in medical expenses. Chronic wounds are prone to secondary infections, water and electrolyte disorders, septic shock, multiple organ failure, and even death. Therefore, the timely and effective reconstruction of the structural and functional integrity after skin injury is crucial to human health and survival.

[0003] Traditional wound repair drugs can be divided into two categories: small molecule compounds extracted from plants and growth factor proteins represented by epidermal growth factor (EGF). Although these drugs can promote wound healing, they also have disadvantages. For example, the former class of compounds are unstable and have relatively low activity, while the latter class of growth factors are expensive, require strict storage conditions, and are prone to cause hypertrophic scars. In addition, stem cells are also considered to be an attractive alternative for wound repair. However, under the harsh inflammatory environment, the activity of stem cells in the wound bed is poor, which often reduces the therapeutic potential of the cells. Therefore, the development of new and promising interventions for the treatment of chronic skin wounds is undoubtedly urgent. Summary of the invention

[0004] In view of the prior art, the purpose of the present invention is to provide a polypeptide CA-608, a composition thereof and its use in promoting wound healing. The polypeptide of the present 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 preparing a drug for promoting wound healing.

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

[0008] The polypeptide of the present invention or a pharmaceutical composition comprising the polypeptide may be topically administered to an individual.

[0009] The pharmaceutical composition of the present invention comprises the polypeptide, which is formulated into a preparation for local administration.

[0010] In some embodiments, the topical formulations include liquids, ointments, gels, slurries, creams, lotions, powders, and latexes.

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

[0012] In some embodiments, examples of gel forming 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, alginic acid, natto gum, aloe vera, bentonite, carbomer, carboxymethylcellulose, ethylcellulose, gelatin, elastin, hydroxypolyamide, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, poloxamer, polyvinyl alcohol, sodium alginate, tragacanth gum, xanthan gum, gelatin, carboxyvinyl polymer, starch, water-swellable hydrocolloid, carrageenan, hyaluronate, agarose, alginate, acrylate, and ammonium acryloyldimethyltaurate / VP copolymer.

[0013] In some embodiments, the gel forming 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 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 present invention relates to a method for preparing an aqueous phase-enhanced sodium-ion battery comprising: providing a sodium-ion battery of at least one embodiment of the present invention; wherein the sodium-ion battery is in the range of about 160 Pa·s to about 200 Pa·s, about 0.05 Pa·s to about 160 Pa·s, about 0.05 Pa·s to about 140 Pa·s, about 0.05 Pa·s to about 120 Pa·s, about 0.05 Pa·s to about 100 Pa·s, about 0.05 Pa·s to about 80 Pa·s, about 0.05 Pa·s to about 60 Pa·s, about 0.05 Pa·s to about 40 Pa·s, about 0.05 Pa·s to about 20 Pa·s, about 0.05 Pa·s to about 10 Pa·s, about 0.05 Pa·s to about 5.0 Pa·s, about 0.05 Pa·s to about 3.0 Pa·s or about 0.05 Pa·s to about 1.0 Pa·s.

[0016] In some embodiments, the amount of the 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 gel forming 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 administered topically via a spray device, a dressing, or a patch.

[0019] Another aspect of the present invention also provides use of a pharmaceutical composition comprising the polypeptide for preparing a drug for promoting wound healing.

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

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

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

[0023] The polypeptide of the present invention has obvious wound healing activity, can be used to accelerate wound healing of the skin surface, and reduce scar formation. Therefore, the polypeptide of the present invention has excellent wound healing activity, can be used for medicines with skin repair function, and has certain clinical application value. In addition, the polypeptide CA-608 provided by the present invention is easy to synthesize, low in cost, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1A-1C The results shown are that peptide CA-608 promotes the growth of HaCaT ( Figure 1A )、HFF( Figure 1B )、HUVEC( Figure 1C ) cell proliferation.

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

[0026] Figure 3 The results shown are that the peptide CA-608 accelerates the healing of full-thickness wounds in mice.

[0027] Figure 4 The results shown are that the hydrogel loaded with peptide CA-608 accelerates the healing of full-thickness wounds in mice. DETAILED DESCRIPTION

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

[0029] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ± 1%.

[0030] The invention described herein may include one or more ranges of values ​​(e.g., size, concentration, etc.). The range of values ​​will be understood to include all values ​​within the range, including the values ​​defining the range, and values ​​adjacent to the range, which result in the same or substantially the same result as the values ​​adjacent to the values ​​defining the boundaries of the range directly. For example, it will be appreciated by those skilled in the art that a 10% change in the upper or lower limit of the range may be completely appropriate and encompassed by the present invention. More specifically, the change in the upper or lower limit of the range will be 5%, or as is generally recognized in the art, whichever is greater.

[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 does not heal within the normal time period that heals in an otherwise healthy individual. Chronic wounds are those that fail to heal due to the individual's health; for example, where the individual has poor circulation or a disease such as diabetes, or where the individual is taking medications that inhibit the normal healing process. In some cases, chronic wounds can remain for weeks, months, or even years without healing. 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 is formed suddenly and heals quickly due to an accident, trauma or external violence. These wounds usually occur in a short period of time, resulting in skin tearing, cutting or rupture, and most of them heal significantly 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, "promote" or "increase" or "promoting" or "increasing" are used interchangeably herein. 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 cell, tissue, or individual can also be compared before and after treatment. In some embodiments, the increase in the treated cell, tissue, or individual is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or more compared to an untreated cell, tissue, or individual.

[0035] As used herein, "treatment," "treating," or "amelioration" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefits and / or prophylactic benefits.

[0036] As used herein, the term "promoting wound healing" refers to enhancing wound healing as compared to wound healing that would be observed in an untreated wound.

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

[0038] "Subjects" refer to all animals including humans that have been injured or may be injured. Animals include not only humans but also cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats and other mammals that need to treat similar but not limited symptoms.

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

[0040] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier. Such carriers include, but are not limited to: normal saline, buffered normal 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 the present invention are composed of one or more excipients commonly used in common standard formulations, such as surfactants, solubilizers, stabilizers, emulsifiers, thickeners, and preservatives. Such excipients are well known to those skilled in the art.

[0041] The pharmaceutical composition of the present invention can be administered by one or more pharmaceutically acceptable carriers in a manner suitable for the selected administration, including systemic or local administration, enteral or parenteral administration such as injection, infusion or implantation, oral, transdermal or topical administration. In some embodiments of the present invention, the composition is prepared as a pharmaceutically acceptable carrier, and the preparation includes liquid, ointment, gel, slurry, cream, emulsion, powder and latex and any applicable form. In some embodiments, the preparation is locally administered by a spray device, dressing or patch.

[0042] The pharmaceutical composition of the present invention also comprises a gel-forming agent to form an external gel product having a viscosity ranging from about 0.05 Pa·s to about 200 Pa·s.

[0043] The pharmaceutical composition of the present invention is 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 buffer components, non-limiting examples of which are tromethamine, 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 effect against important pathogens (e.g., Staphylococcus aureus or Pseudomonas aeruginosa), to prevent overgrowth of any strains that develop resistance during long-term treatment, or to expand the antimicrobial spectrum to include non-bacterial pathogens, including but not limited to fungi.

[0045] The composition of the present invention can be applied daily or intermittently, and the frequency of application can be once a day or 2 to 3 times. If each of the two active ingredients is a single preparation, the frequency of application can be the same or different from each other. In addition, the composition of the present invention can be used alone or in combination with other wound healing drugs. Taking all of the above factors into account, it is important to apply it at the lowest dose without side effects to achieve the best therapeutic effect, which can be easily 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 for the remaining life cycle of an individual in need. In other embodiments, the patient is treated with a 14-day treatment regimen using the pharmaceutical composition of the present invention.

[0046] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0047] Example 1 Preparation of polypeptide

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

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

[0050] Wound re-epithelialization and granulation tissue formation are crucial for wound healing, which requires the participation of keratinocytes, fibroblasts, endothelial cells and other cells. Therefore, the proliferation and cell migration activities 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 3 Cells / well), after the cells adhered to the wall, 10 μL of gradient diluted peptide CA-608 was added, and the final concentrations were 0, 2.5, 5, 10, and 20 μg / mL, respectively, and cultured in a constant temperature incubator at 37°C and 5% CO2 for 16 hours. 10 μL of CCK-8 solution (Yisheng Biological, item number: 40203ES60) was added to each well, and incubated for 1 hour at 5% CO2, 37°C, away from light. The absorbance at 450nm was measured with an enzyme marker, and a zeroing well was set at the same time. Cell proliferation activity was calculated according to the following formula: Cell activity (%) = [experimental well-zeroing well] / [control well-zeroing well] × 100%, where the experimental well is the absorbance of the well with cells, CCK-8 solution and peptide CA-608 added, the zeroing well is the absorbance of the well with culture medium and CCK-8 solution added; the control well is the absorbance of the well with cells and CCK-8 solution added.

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

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

[0054] The cell scratch test was used to detect the effect of polypeptide CA-608 on HUVEC cell migration. HUVEC cells were inoculated into 6-well plates. When the cells were cultured to about 90% confluency, the serum-free medium was replaced for cell starvation. After 24 hours, a 200μL pipette tip was used to scratch to simulate a wound. After scratching, the plate was washed 3 times with PBS to remove the scratched cells. Serum-free medium containing a final concentration of 20μg / mL polypeptide CA-608 was added to the experimental group for culture. Serum-free medium was added to the control group for culture, and photos were taken at 0, 12, 24, and 48 hours to record the migration of cells.

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

[0056] Example 4. Detection of the activity of polypeptide CA-608 in promoting skin wound healing in vivo

[0057] Balb / c mice were randomly divided into a control group and a treatment group, with 5 mice in each group. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital, the hair was removed, and the skin was disinfected with 75% medical alcohol. A full-thickness skin trauma wound with a diameter of about 6 mm was made on the back of the mouse using surgical scissors. After the mice woke up, they were placed in a mouse cage for breeding. The control group and the treatment group were treated with sterile PBS and 250μg / mL peptide CA-608 every 12 hours, respectively. The area and wound healing rate were calculated using Image J software.

[0058] like Figure 3 As shown in the figure, compared with the control group, topical application of peptide CA-608 can significantly accelerate the wound healing process of mice. At 4d, 8d, and 12d after trauma, the wound healing rates of mice treated with topical peptide CA-608 reached 36%, 64%, and 94%, respectively, while the control group only had 17%, 33%, and 65.5%. It can be clearly seen that the wounds of mice treated with peptide CA-608 were almost completely healed. After trauma, no abnormal behavior or death of mice was found, indicating that peptide CA-608 has good safety.

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

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

[0061] serial number Gel former CA-608 Ultrapure water pH A 0.5% (w / w) xanthan gum 0.002%(w / w) The remainder Approx. 7.0 B 0.2% (w / w) xanthan gum 0.005%(w / w) The remainder Approx. 7.0 C 0.1% (w / w) methylcellulose 0.0002%(w / w) The remainder Approx. 6.5 D 2% (w / w) Aloe 0.05%(w / w) The remainder Approx. 6.0

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

[0063] Example 6. Detection of the activity of hydrogel loaded with polypeptide CA-608 in promoting skin wound healing in vivo

[0064] Balb / c mice were randomly divided into a control group and a treatment group, with 5 mice in each group. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital, the hair was removed, and the skin was disinfected with 75% medical alcohol. A full-thickness skin trauma wound with a diameter of about 6 mm was made on the back of the mouse using surgical scissors. After the mice woke up, they were placed in a mouse cage for breeding. The control group and the treatment group were treated with sterile PBS, peptide CA-608, carbomer, and hydrogel B loaded with peptide CA-608 every 12 hours. The area and wound healing rate were calculated using Image J software.

[0065] like Figure 4 As shown, compared with the control group, topical application of hydrogel B loaded with peptide CA-608 can significantly accelerate the wound healing process in mice. At 8 days after trauma, the wound healing rates of mice treated with topical application of 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-6081 according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, which is formulated into a preparation for topical administration.

4. The pharmaceutical composition according to claim 3, wherein the topical preparation comprises a liquid, an ointment, a gel, a slurry, a cream, an emulsion, a powder and a latex.

5. The pharmaceutical composition according to claim 4, wherein the preparation for topical administration is a gel.

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

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

8. Use of the polypeptide according to claim 1 in preparing a medicament for promoting wound healing.

9. Use of the pharmaceutical composition according to any one of claims 2 to 7 for preparing a medicament for promoting wound healing.

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