GLP-1RA gel preparation for treating diabetic foot ulcer and preparation method thereof

Abstract

The invention relates to the technical field of medicine, in particular to a GLP-1RA gel preparation for treating diabetic foot ulcer and a preparation method thereof.The preparation is a composition of GLP-1RA and poloxamer temperature-sensitive gel, and poloxamer 407, poloxamer 188 and purified water are sequentially added into a Schottky bottle according to the part ratio of the components of the poloxamer temperature-sensitive gel; placing the Schottky bottle in an ice bath, and fully stirring to obtain a temperature-sensitive gel matrix; liraglutide or loxenatide and the temperature-sensitive gel matrix are stirred and mixed according to the mass ratio, and the GLP-1RA gel preparation is prepared. The GLP-1RA gel preparation is good in biocompatibility, has good elasticity and plasticity, can be tightly attached to a wound, effectively accelerates ulcer repair and shortens ulcer healing time.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a GLP-1RA gel preparation for treating diabetic foot ulcers and a preparation method thereof. Background Art

[0002] Diabetic foot ulcer (DFU) is one of the serious complications of diabetes patients. 19%-34% of diabetic patients may eventually develop diabetic foot ulcer. DFU is closely related to the morbidity, mortality and amputation rate of diabetic patients. The pathogenesis of diabetic foot is complex, so its clinical treatment is also very difficult. Commonly used clinical treatment methods mainly include anti-infection, local decompression and debridement, vascular reconstruction when necessary, and in recent years, hyperbaric oxygen, dressings, negative pressure drainage, cell growth factors, stem cell transplantation, etc.

[0003] Glucagon-like peptide-1 receptor agonists (GLP-1RA) are a class of drugs that produce hypoglycemic effects by stimulating glucagon-like peptide-1 (GLP-1) receptors and exerting incretin effects. In the prior art, GLP-1RA is mainly administered by subcutaneous injection, such as the administration of soluble microneedles. In the final animal experiment, it was found that the soluble microneedles of GLP-1RA were easily broken free by mice, so additional bandages were required for pressure dressing in animal experiments. In reality, the wounds of patients with diabetic foot ulcers are irregular, and microneedle treatment at the wounds of patients has poor fit. However, pressure dressing of diabetic wounds is not conducive to wound healing and recovery. The gel preparation can prolong the retention time of the drug at the administration site to achieve sustained release of the drug. It does not contain organic solvents and has less systemic toxicity. It also has good elasticity and plasticity, and can fit tightly with the wound to isolate bacteria. Summary of the invention

[0004] To this end, the present invention provides a GLP-1RA gel preparation for treating diabetic foot ulcers and a preparation method thereof, so as to overcome the problems of poor compliance and poor treatment effect caused by subcutaneous injection / soluble microneedle administration in the prior art.

[0005] To achieve the above objectives, on the one hand, the present invention provides a GLP-1RA gel preparation for treating diabetic foot ulcers, comprising:

[0006] The preparation is a composition of GLP-1RA and poloxamer thermosensitive gel.

[0007] Furthermore, the poloxamer thermosensitive gel consists of poloxamer 407, poloxamer 188 and purified water.

[0008] Furthermore, the poloxamer thermosensitive gel contains 5-10 parts of poloxamer 407, 1-3 parts of poloxamer 188, and 22-35 parts of purified water.

[0009] Furthermore, the GLP-1RA is liraglutide.

[0010] Furthermore, the mass ratio of the liraglutide to the poloxamer thermosensitive gel is 1:20678.

[0011] Furthermore, the GLP-1RA is loxenatide.

[0012] Furthermore, the mass ratio of the loxenatide to the poloxamer thermosensitive gel is 1:37525.

[0013] On the other hand, the present invention also provides a method for preparing a GLP-1RA gel preparation for treating diabetic foot ulcers, comprising the following steps:

[0014] Step S1, adding the poloxamer 407, poloxamer 188 and purified water into a Schott bottle in sequence according to the ratio of the components of the poloxamer thermosensitive gel;

[0015] Step S2, placing the Schott bottle in an ice bath and stirring thoroughly to obtain a thermosensitive gel matrix;

[0016] Step S3, stirring and mixing the liraglutide or loxenatide with the thermosensitive gel matrix according to a mass ratio to prepare a GLP-1RA gel preparation.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a new drug, which for the first time applies the GLP-1RA gel preparation to the treatment of diabetic foot ulcers. Animal experimental results show that GLP-1RA accelerates the repair of ulcers and shortens the ulcer healing time by promoting angiogenesis and promoting the transformation of macrophages from M1 to M2.

[0018] The GLP-1RA gel preparation has good biocompatibility, is easy to apply, has good elasticity and plasticity, can fit tightly to the wound, and prevent bacterial invasion. In addition, the biological dressing has porosity, which is conducive to gas exchange, and can effectively absorb wound exudate, keep the wound in a moist environment, accelerate the proliferation and migration of vascular endothelial cells and fibroblasts, stimulate them to secrete more growth factors, promote angiogenesis and the formation of granulation tissue, thereby accelerating the healing of ulcers and improving their healing quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1An implementation roadmap for investigating the therapeutic effectiveness of the GLP-1RA liraglutide gel formulation in the C57BL / 6 mouse wound model in the present invention;

[0020] Figure 2 This is a graph showing the results of the GLP-1RA liraglutide gel dosage form in the present invention on the healing of wounds in a C57BL / 6 mouse wound model;

[0021] Figure 3 This is a comparison chart of the wound treatment effects of the GLP-1RA liraglutide solution and liraglutide gel formulation (dressing change every day / dressing change every 2 days) on the wound surface model of C57BL / 6 mice according to the embodiment of the present invention;

[0022] Figure 4 This is a result diagram of the changes in dermal thickness and epithelial maturity of a C57BL / 6 mouse wound model treated with the GLP-1RA liraglutide gel dosage form of the present invention for 10 days;

[0023] Figure 5 This is a graph showing the CD31 positive cell rate level results of the GLP-1RA liraglutide gel dosage form in the present invention in the treatment of the wound model of C57BL / 6 mice on the 10th day;

[0024] Figure 6 This is a diagram showing that the GLP-1RA liraglutide gel dosage form of the present invention can promote the transformation of the phenotype of macrophages from M1 to M2 in the treatment of wounds in a C57BL / 6 mouse wound model;

[0025] Figure 7 (a) is a graph showing the results of the scratch method for detecting the migration ability of GLP-1RA loxenatide on human keratinocyte cell line HaCaT cells in an embodiment of the present invention;

[0026] Figure 7 (b) is a graph showing the results of detecting the proliferation ability of GLP-1RA loxenatide on human keratinocyte cell line HaCaT cells using the CCK-8 method in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Those skilled in the art will appreciate that the method of the present invention for determining a single parameter mentioned above may be to select the value with the highest proportion as the preset standard parameter based on data distribution, use weighted summation to use the obtained value as the preset standard parameter, substitute each historical data into a specific formula and use the value obtained by using the formula as the preset standard parameter or other selection methods, as long as the method of the present invention can clearly define different specific situations in the single determination process through the obtained values.

[0029] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0030] See also Figures 1 to 7 As shown, they are respectively an implementation roadmap for exploring the therapeutic effectiveness of the GLP-1RA liraglutide gel formulation in the C57BL / 6 mouse wound model according to the embodiment of the present invention; a result diagram of the healing of the wound in the C57BL / 6 mouse wound model by the GLP-1RA liraglutide gel formulation according to the embodiment of the present invention; a comparison diagram of the therapeutic effects of the GLP-1RA liraglutide solution and the liraglutide gel formulation (dressing change every day / dressing change every 2 days) on the wound in the C57BL / 6 mouse wound model according to the embodiment of the present invention; and a comparison diagram of the dermal thickness and epithelial maturity changes of the wound in the C57BL / 6 mouse wound model after 10 days of treatment with the GLP-1RA liraglutide gel formulation according to the embodiment of the present invention. ; The result graph of the CD31 positive cell rate level on the 10th day of the wound of the C57BL / 6 mouse wound model treated with the GLP-1RA liraglutide gel dosage form of the embodiment of the present invention; The result graph of the GLP-1RA liraglutide gel dosage form of the embodiment of the present invention in treating the C57BL / 6 mouse wound model can promote the phenotype of macrophages to change from M1 to M2; The result graph of the scratch method detection of the migration ability of the human keratinocyte HaCaT cells of the GLP-1RA loxenatide in the embodiment of the present invention; The result graph of the CCK-8 method detection of the proliferation ability of the human keratinocyte HaCaT cells of the GLP-1RA loxenatide in the embodiment of the present invention.

[0031] The GLP-1RA gel preparation for treating diabetic foot ulcers according to the embodiment of the present invention and the preparation method thereof include:

[0032] Example 1

[0033] 1) Prescription

[0034]

[0035] 2) Preparation process

[0036] (a) adding the prescribed amount of poloxamer 407, poloxamer 188 and purified water into a 100 ml Schott bottle in sequence, and stirring in an ice bath until completely dissolved to obtain a thermosensitive gel matrix;

[0037] (b) The prescribed amount of thermosensitive gel matrix and liraglutide were added to a 50 ml Schott bottle in sequence and stirred at room temperature for 5 min until completely dissolved to obtain liraglutide thermosensitive gel.

[0038] Example 2

[0039] 1) Prescription

[0040]

[0041] 2) Preparation process

[0042] (a) adding the prescribed amount of poloxamer 407, poloxamer 188 and purified water into a 100 ml Schott bottle in sequence, and stirring in an ice bath until completely dissolved to obtain a thermosensitive gel matrix;

[0043] (b) The prescribed amount of thermosensitive gel matrix and polyethylene glycol loxenatide injection were sequentially added into a 50 ml Schott bottle and stirred at room temperature for 5 min until completely dissolved to obtain loxenatide thermosensitive gel.

[0044] Verification Example 1:

[0045] The therapeutic efficacy of GLP-1RA liraglutide was investigated in a C57BL / 6 mouse wound model.

[0046] 1. Establishment of wound model in C57BL / 6 mice

[0047] Twelve 7-week-old SPF male C57BL / 6J mice, weighing 16-18g, were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd., with an animal certificate number of NO.110011221102791978. After arriving at the SPF animal room, the mice were first raised in the quarantine room for 3-5 days, and then transferred to the feeding room for normal feeding (feeding conditions: temperature: 20-25℃; daily temperature difference <3℃; relative humidity 40%-70%; 12-hour light and dark alternation, free access to food and water). After the mice were randomly divided into groups, they were anesthetized by intraperitoneal injection of tribromoethanol at a dose of 20ml / kg. After anesthesia, all mice were depilated on the back, and two wounds 1 cm apart were prepared on the back of each mouse with a sterile 6mm skin biopsy punch.

[0048] Experimental Group:

[0049] (1) Daily topical application of GLP-1RA liraglutide solution (2 μg / cm 2 );

[0050] (2) Gel group I: daily topical application of GLP-1RA liraglutide gel (2 μg / cm 2 );

[0051] (3) Gel II group: Topical application of GLP-1RA liraglutide gel (2 μg / cm 2 );

[0052] Control group:

[0053] Apply a gel dressing topically with an equal amount of sterile saline daily.

[0054] 2. Experimental results

[0055] (1) GLP-1RA promotes wound healing

[0056] During the healing process, the GLP-1RA gel formulation significantly accelerated wound healing (P<0.05). Figure 2 shown.

[0057] Comparison of the efficacy of GLP-1RA solution and gel

[0058] During the healing process, both GLP-1RA solution and gel type can significantly promote wound healing compared with the control group (* represents P<0.05, ** represents P<0.01). Compared with the GLP-1RA solution group, Gel I group (dressing changed every day) and Gel II group (dressing changed every two days) can significantly promote wound healing (# represents P<0.05). There was no significant difference between Gel I group and II group in promoting wound healing. Therefore, the use of GLP-1RA gel can achieve dressing changes every two days without affecting the efficacy, such as Figure 3 shown.

[0059] (3) GLP-1RA increases dermal production and angiogenesis

[0060] HE staining showed that compared with the control group, after 10 days of treatment with GLP-1RA gel, the wound size was significantly reduced, and the dermal thickness and epithelial maturity were significantly increased. Figure 4 shown.

[0061] In addition, immunofluorescence was used to detect the positive cell rate of CD31, a characteristic marker of capillaries, to determine whether the GLP-1RA gel formulation can promote wound healing by promoting angiogenesis. The results showed that in the control group and the experimental group, over time, the CD31 positive cell rate level on day 10 was higher than that on day 0, and the CD31 positive rate in the experimental group was significantly higher than that in the control group, indicating that the GLP-1RA gel formulation may play a role in promoting angiogenesis. Figure 5 shown.

[0062] (3) GLP-1RA regulates macrophage polarization

[0063] The mechanism was explored in a mouse model to see whether the use of GLP-1RA gel formulation could promote the transformation of macrophage phenotype from M1 to M2. Western blot was used to detect the M1 / M2 macrophage levels, and it was found that on the 5th day, the expression of M1 marker iNOS was significantly reduced in the GLP-1RA group compared with the control group, while the expression of M2 marker YM-1+2 was significantly increased, indicating that GLP-1RA intervention can indeed significantly promote the transformation of macrophages from M1 to M2. Figure 6 shown.

[0064] The animal experiment of the present invention illustrates for the first time that the use of GLP-1RA gel formulations in wounds can promote macrophage transformation. This evidence will provide a basis for the use of GLP-1RA gel formulations in patients with diabetic wounds and help increase the treatment options for diabetic wounds.

[0065] Verification of Example 2 GLP-1RA Losenatide's ability to promote wound healing in vitro

[0066] The scratch method and CCK-8 method were used to detect the migration and proliferation ability of human keratinocytes (HaCaT cells) with or without loxenatide under normal glucose conditions to evaluate the wound healing ability of loxenatide. Figure 7 (a) shown.

[0067] The results of the scratch test showed that compared with the control group, at each designated time point, the addition of loxenatide significantly reduced the blank area of ​​the scratch, indicating that loxenatide can significantly promote the migration of HaCaT cells. The CCK-8 experiment found that the OD value of the loxenatide group was significantly higher than that of the control group, indicating that loxenatide can promote the proliferation of HaCaT cells. Therefore, it is believed that GLP-1RA loxenatide has the ability to promote wound healing in vitro. Figure 7 (b) as shown.

[0068] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A GLP-1RA gel preparation for treating diabetic foot ulcers, characterized in that: The preparation is a composition of GLP-1RA and poloxamer thermosensitive gel.

2. The GLP-1RA gel preparation for treating diabetic foot ulcers according to claim 1, characterized in that: The poloxamer thermosensitive gel consists of poloxamer 407, poloxamer 188 and purified water.

3. The GLP-1RA gel preparation for treating diabetic foot ulcers according to claim 2, characterized in that: The poloxamer thermosensitive gel contains 5-10 parts of poloxamer 407, 1-3 parts of poloxamer 188 and 22-35 parts of purified water.

4. The GLP-1RA gel preparation for treating diabetic foot ulcer according to claim 1, characterized in that: The GLP-1RA is liraglutide.

5. The GLP-1RA gel preparation for treating diabetic foot ulcers according to claim 4, characterized in that: The mass ratio of the liraglutide to the poloxamer thermosensitive gel is 1:20678.

6. The GLP-1RA gel preparation for treating diabetic foot ulcers according to claim 1, characterized in that: The GLP-1RA is loxenatide.

7. The GLP-1RA gel preparation for treating diabetic foot ulcers according to claim 6, characterized in that: The mass ratio of the loxenatide to the poloxamer thermosensitive gel is 1:37525.

8. A method for preparing the GLP-1RA gel preparation for treating diabetic foot ulcers according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step S1, adding the poloxamer 407, poloxamer 188 and purified water into a Schott bottle in sequence according to the ratio of the components of the poloxamer thermosensitive gel; Step S2, placing the Schott bottle in an ice bath and stirring thoroughly to obtain a thermosensitive gel matrix; Step S3, stirring and mixing the liraglutide or loxenatide with the thermosensitive gel matrix according to a mass ratio to prepare a GLP-1RA gel preparation.

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