Microneedle patch for treating diabetic lower limb ischemic lesions and application thereof

Abstract

A microneedle patch for treating diabetic lower limb ischemia includes an outer shell layer made of a mixture of glucagon-like peptide-1 (GLP-1)(32-36)a from incretin and a first polymer, and a core layer made of PEG-GLP-1(32-36)a, a photoinitiator, and a second polymer. This microneedle patch features a "burst release followed by sustained release" characteristic, allowing the drug to rapidly enter the body to reach an effective therapeutic concentration and maintaining the drug concentration within the effective therapeutic window for a long period, thus maximizing the therapeutic effect.

Description

Technical Field

[0001] This invention relates to a drug-loaded device, and more particularly to a microneedle patch that loads a short peptide of glucagon-like peptide-1 (e.g., GLP-1(32-36)amide) into a microneedle to achieve differential release of the short peptide, which is beneficial for its application in the treatment of diabetic lower limb ischemia. Background Technology

[0002] With changing lifestyles and an aging population, the number of people with diabetes is increasing year by year. According to data from the Global Diabetes Association in 2022, more than 500 million people worldwide have diabetes, and nearly half of them are not receiving effective treatment. Once blood sugar is not effectively controlled, diabetes will continue to worsen and cause a series of complications, including damage to blood vessels and nerves, and in later stages, it can even lead to blindness, amputation, or death.

[0003] Lower limb ischemia in diabetic patients is the most common chronic vascular complication. Its cause is the activation of oxidative stress signaling pathways in a high-glucose environment, leading to the production of large amounts of reactive oxygen species (ROS) that damage vascular endothelial cells, thus causing lower limb ischemia. Currently, no drugs have been found that can effectively improve lower limb ischemia in diabetic patients besides surgical treatment. However, due to anatomical limitations, the symptoms of lower limb ischemia in some patients do not improve effectively after surgical treatment, and amputation may still be necessary. Therefore, there is an urgent need for a highly effective and low-toxicity drug formulation to effectively treat lower limb ischemia in diabetic patients.

[0004] Glucagon-like peptide-1 (GLP-1(32-36)amide, or GLP-1(32-36)a, as a breakdown product of glucagon-like peptide-1 (GLP-1), has gradually come into the public eye with the discovery of GLP-1's glucose-dependent regulatory properties. Early studies suggested that only GLP-1 derived from intestinal L cells could exert its glycemic regulatory effects and improve the prognosis of diabetes-related complications in vivo. However, with further research, the biological effects of various GLP-1 breakdown products have been gradually discovered. In recent years, GLP-1(32-36)a has been found to inhibit apoptosis of pancreatic β-cells in vitro, thereby increasing glucose distribution in diabetic mice. It also shows effects in increasing basal energy expenditure and inhibiting weight gain in diabetic mice. However, there are no reports on the role of GLP-1(32-36)a in treating diabetic lower limb ischemia. Our previous research found that GLP-1(32-36)a can promote endothelial cell repair and angiogenesis, effectively improving lower limb ischemia in diabetic mice. However, as a short peptide (molecular weight 570 Da) composed of 5 amino acids, GLP-1(32-36)a has an extremely short half-life in vivo, often resulting in degradation before it can exert its therapeutic effect. Therefore, relying on current subcutaneous injection methods, multiple injections are usually required to ensure the peptide's biological effects are achieved. Furthermore, after lower limb ischemia, it is difficult for the drug to reach effective concentrations at the most distal parts of the limb. To reduce the suffering caused by multiple injections, developing a long-acting, locally administered sustained-release formulation is a pressing issue that needs to be addressed.

[0005] Microneedles are arrays of multiple tiny needles, typically less than 300 μm in diameter and ranging in length from 200 μm to 2000 μm. Compared to traditional transdermal drug delivery systems, microneedles offer the advantage of rapidly delivering large biomolecules that are poorly absorbed by the gastrointestinal tract, such as proteins, peptides, antibodies, vaccines, RNA, and DNA. Microneedles can be classified into five categories based on their manufacturing process and material properties: solid microneedles, coated microneedles, soluble microneedles, hydrogel microneedles, and hollow microneedles. Among these, hydrogel microneedles are currently a hot research topic in microneedle drug delivery systems. Hydrogel microneedles are microneedles with a hydrogel matrix. Upon insertion into the skin, this array of microneedles rapidly absorbs interstitial fluid, causing the hydrogel to swell and create continuous, unobstructed channels within the gel. Drugs then penetrate and diffuse into the skin tissue through the tissue fluid. A key advantage is the ability to control the release rate of large molecule drugs by adjusting the cross-linking density of the hydrogel fibers. Previous experiments revealed that GLP-1(32-36)a has an extremely small molecular weight, making it difficult to achieve controlled release of the short peptide using hydrogel microneedles alone. Therefore, there is an urgent need to prepare a macromolecular prodrug that can be metabolized in vivo to assist hydrogel microneedles in achieving long-acting sustained release. Summary of the Invention

[0006] One object of the present invention is to provide a microneedle patch carrying a short peptide of glucagon-like peptide-1, which enables differential release of the short peptide and maintains it within the effective therapeutic window for a long period of time.

[0007] Another objective of this invention is to provide a microneedle patch containing differentially released glucagon-like peptide-1 short peptide for use in the preparation of medical devices for treating diabetic lower limb ischemia.

[0008] Another object of the present invention is to provide a medical device comprising a microneedle patch for differentially releasing glucagon-like peptide-1 short peptide.

[0009] The microneedle patch of glucagon-like peptide-1 (GLP-1) of the present invention comprises core-shell microneedles, in which GLP-1(32-36)a is loaded, giving GLP-1(32-36)a a "burst release followed by sustained release" characteristic. That is, after the microneedles are inserted into the skin, the GLP-1(32-36)a loaded in the outer shell layer is rapidly released to reach an effective therapeutic concentration, while the GLP-1(32-36)a loaded in the inner core layer is released slowly, allowing the drug to be maintained within the effective therapeutic window for a long time.

[0010] The outer shell of the GLP-1(32-36)a microneedles is made by mixing GLP-1(32-36)a with a polymer. Suitable polymers include, but are not limited to, polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronic acid, and polyvinylpyrrolidone, which are used alone or in combination in this invention, with polyvinylpyrrolidone being preferred.

[0011] The core layer of the GLP-1(32-36)a microneedles is made of PEG-modified GLP-1(32-36)a (PEG-GLP-1(32-36)a), a photoinitiator, and a polymer. Polymers used to prepare the core include, but are not limited to, polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronic acid, polyvinylpyrrolidone, and methacrylic silk fibroin, methacrylic chitosan, methacrylic gelatin, and methacrylic hyaluronic acid. These compounds are used alone or in combination in this invention, with methacrylic gelatin being preferred.

[0012] PEG has a molecular weight of 1w to 100w.

[0013] Photoinitiators include one or more of the following: 2-hydroxy-2-methyl-1-phenylpropanone, 1-hydroxycyclohexylphenyl ketone, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-dimethylamino-2-benzyl-1-[4-(4-morpholino)phenyl]-1-butanone, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and methyl benzoylcarbamate.

[0014] PEG-GLP-1(32-36)a crosslinking agents such as, but not limited to, polyethylene glycol-disulfide-succinimide carbonate, polyethylene glycol-succinimide carbonate, polyethylene glycol-disulfide-polyethylene glycol-succinimide carbonate, etc., are prepared and used alone or in combination in this invention. Among them, polyethylene glycol-disulfide-succinimide carbonate and polyethylene glycol-succinimide carbonate are preferred.

[0015] Each microneedle is disposed on a substrate layer to form a microneedle patch. The substrate layer is also composed of polymers, such as, but not limited to, polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronic acid, and polyvinylpyrrolidone, which are used alone or in combination in this invention, with polyvinylpyrrolidone being preferred.

[0016] Multiple GLP-1(32-36)a microneedles are arranged in a regular pattern. Generally, the distance between each microneedle is equal. The shape of the microneedles includes, but is not limited to, conical and prismatic shapes, with a preference for conical shapes. The needle length ranges from 100 μm to 1000 μm. The diameter of the needle base surface ranges from 30 μm to 500 μm.

[0017] One embodiment of a GLP-1(32-36)a microneedle patch has a needle length ranging from 100μm to 1000μm.

[0018] Another embodiment of the GLP-1(32-36)a microneedle patch has a needle body base surface diameter ranging from 30μm to 500μm.

[0019] Another implementation of the GLP-1(32-36)a microneedle patch involves setting 1 to 3 microneedles within a length range of 1 mm, with 1 to 2 microneedles being preferred.

[0020] Another implementation of the GLP-1(32-36)a microneedle patch, with an area of ​​1 cm² 2 Within the range, set 10 to 100 microneedles, with a preference for 50 to 100 microneedles.

[0021] The microneedle patch preparation process of this invention is a "dual-mold injection method". To facilitate understanding of the preparation method by those skilled in the art, the following preparation process is provided by way of example, and not limitation:

[0022] (1) Preparation of PEG-GLP-1(32-36)a

[0023] First, an aqueous solution of GLP-1(32-36)a (pH=7.8) and a crosslinking agent solution (pH=7.8) were prepared. Then, the crosslinking agent solution was added dropwise to the GLP-1(32-36)a aqueous solution to initiate the crosslinking reaction at 4℃. The desired PEG-GLP-1(32-36)a solution was separated using a dextran gel column chromatography method. Finally, lyophilized PEG-GLP-1(32-36)a powder was obtained.

[0024] (2) Preparation of long-lasting microneedle patches loaded with GLP-1(32-36)a

[0025] Prepare a polymer solution containing PEG-GLP-1(32-36)a and a photoinitiator (i.e., working solution 1), a polymer solution for the substrate layer (i.e., working solution 2), and a polymer solution containing GLP-1(32-36)a (i.e., working solution 3);

[0026] Then, working solution 1 is added to the prefabricated first microneedle patch mold. By centrifugation or vacuum method, working solution 1 is completely introduced into the hole of the first microneedle mold. Excess liquid that does not enter the needle hole is scraped off and dried at room temperature overnight.

[0027] Next, working solution 2 is added to the first microneedle patch mold, and centrifugation or vacuum is continued to ensure that working solution 2 completely enters the hole of the first microneedle mold. After drying at room temperature overnight, the microneedles are peeled off and cured with ultraviolet light to obtain the core structure of the PEG-GLP-1(32-36)a-loaded microneedle patch. Then, working solution 3 is added to the pre-made second microneedle patch mold, and centrifugation or vacuum is used to ensure that working solution 3 completely enters the hole of the microneedle mold. The core structure of the PEG-GLP-1(32-36)a-loaded microneedle patch is then pressed into the second microneedle patch mold and dried at room temperature to obtain the GLP-1(32-36)a-loaded microneedle patch, which has the characteristic of differential release of GLP-1(32-36)a.

[0028] The PEG-GLP-1(32-36)a contained in working solution 1 was 1-30 wt%.

[0029] The concentration of the polymer contained in working solution 1 is 5–30 wt%.

[0030] The polymer contained in working solution 2 has a concentration of 5-30 wt% and is used to make the base layer of the patch.

[0031] The concentration of GLP-1(32-36)a in working solution 3 is 1-5 wt%.

[0032] The concentration of the polymer contained in working solution 3 is 5–30 wt%.

[0033] The GLP-1(32-36)a microneedles of the present invention have the characteristics of "burst release followed by sustained release", which not only allows the drug to enter the body quickly to reach an effective therapeutic concentration, but also maintains the drug concentration in the body within the effective therapeutic window for a long time, thereby maximizing the therapeutic effect of the drug. Attached Figure Description

[0034] Figure 1 A schematic diagram of the route for preparing PEG-GLP-1(32-36)a;

[0035] Figure 2 A schematic diagram of the preparation of GLP-1(32-36)a microneedle patch;

[0036] Figure 3 The image shows the appearance of the GLP-1(32-36)a microneedle patch; where A is a stereomicroscopic image of the GLP-1(32-36)a microneedle and B is a confocal image of the GLP-1(32-36)a microneedle. The scale bar in the image is 200 μm.

[0037] Figure 4 The displacement versus load-bearing capacity curves of the GLP-1(32-36)a microneedles;

[0038] Figure 5 The image shows a skin biopsy image with GLP-1(32-36)a microneedles, with the scale bar at 100 μm.

[0039] Figure 6 The images show the in vitro release curves of the GLP-1(32-36)a microneedle patch, where A is the release curve of GLP-1(32-36)a in the outer shell layer of the GLP-1(32-36)a microneedle patch; and B is the release curve of GLP-1(32-36)a in the entire GLP-1(32-36)a microneedle patch.

[0040] Figure 7 Blood flow diagrams of GLP-1(32-36)a formulation for treating lower limb ischemia in diabetic patients. In this diagram, A is the Doppler blood flow diagram of each group of animals treated with GLP-1(32-36)a formulation; B is the blood flow statistics diagram of each group of animals treated with GLP-1(32-36)a formulation.

[0041] Figure 8The pharmacokinetic curves of the GLP-1(32-36)a formulation are shown below. In this diagram, A is the pharmacokinetic curve of the subcutaneous injection of GLP-1(32-36)a; and B is the pharmacokinetic curve of the microneedles loaded with GLP-1(32-36)a. Detailed Implementation

[0042] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0043] Example 1: GLP-1(32-36)a polyvinyl alcohol microneedle patch

[0044] First, aqueous solutions containing 2% (w / v) GLP-1(32-36)a (pH = 7.8) and 2% (w / v) PEG were prepared separately. 20K -Disulfide bond-succinimide carbonate solution (NHS-SS-PEG) 20K ); then, PEG 20K A disulfide-succinimide carbonate solution was added dropwise to the above GLP-1(32-36)a aqueous solution under magnetic stirring to carry out a crosslinking reaction at a temperature of 4°C (synthetic route see [link]). Figure 1 The desired GLP-1(32-36)a-PEG was further separated by dextran gel column chromatography. 400 Solution; finally, GLP-1(32-36)a-PEG was obtained by lyophilization. 20K Freeze-dried powder.

[0045] Formulated with 3% (w / v) GLP-1(32-36)α-PEG 20K 20% (w / v) polyvinyl alcohol 1788 (PVA) aqueous solution (core solution), 0.1% (w / v) GLP-1 (32-36)a and 30% (w / v) polyvinylpyrrolidone K90 (PVP) ethanol solution (shell solution) and 15% (w / v) PVP ethanol solution (base layer solution);

[0046] Subsequently, the core solution was added to a microneedle mold with 100 conical pores, each 600 μm deep, 250 μm in maximum diameter, and 500 μm between tips. Centrifugation or vacuum was used to ensure the core solution completely entered the pores of the microneedle mold. Excess liquid not entering the pores was scraped off, and the mold was dried overnight at room temperature. Then, the base layer solution was added to the microneedle patch mold, and centrifugation or vacuum was continued to ensure the base layer solution completely entered the pores of the microneedle mold. The mold was dried overnight at room temperature, and the microneedles were peeled off to obtain GLP-1(32-36)a-PEG loaded with PEG. 20K The core structure of the microneedle patch (carrying GLP-1(32-36)a-PEG) 20K Microneedle patch); then, the shell solution is added to a microneedle mold with 100 conical holes, each 800 μm deep, 300 μm in maximum diameter, and 500 μm between needle tips. The shell solution is then completely inserted into the microneedle mold holes using centrifugation or vacuum methods. GLP-1(32-36)a-PEG is then loaded... 20K The microneedle patch is pressed into the above mold and dried at room temperature to obtain the final polyvinyl alcohol microneedle patch loaded with GLP-1(32-36)a. For the specific preparation route, see [link to preparation method]. Figure 2 .

[0047] Example 2: GLP-1(32-36)a gelatin microneedle patch

[0048] First, aqueous solutions containing 2% (w / v) GLP-1(32-36)a (pH = 7.8) and 2% (w / v) PEG were prepared separately. 20K - Succinimide carbonate solution; then, PEG 20K A succinimide carbonate solution was added dropwise to the above-mentioned GLP-1(32-36)a aqueous solution under magnetic stirring to initiate a crosslinking reaction at 4°C. The desired GLP-1(32-36)a-PEG was further separated by dextran gel column chromatography. 20K Solution; finally, GLP-1(32-36)a-PEG was obtained by lyophilization. 20K Freeze-dried powder.

[0049] Formulated with 3% (w / v) GLP-1(32-36)α-PEG 20K0.5% (w / v) 2-hydroxy-2-methyl-1-phenylpropanone, 5% (w / v) chitosan, 15% (w / v) methacrylic gelatin in a 4% (v / v) aqueous acetic acid solution (core solution), containing 0.1% (w / v) GLP-1 (32-36)a and 30% (w / v) polyvinylpyrrolidone K90. The core solution was prepared by mixing a K90 (PVP) ethanol solution (shell solution) with a 15% (w / v) PVP ethanol solution (base layer solution). Then, the core solution was added to a microneedle mold with 100 conical pores, each 600 μm deep, with a maximum pore diameter of 250 μm and a tip distance of 500 μm. The core solution was then completely incorporated into the microneedle mold pores using centrifugation or vacuum. Excess liquid not incorporated into the pores was scraped off, and the mixture was dried overnight at room temperature. Next, the base layer solution was added to the microneedle patch mold, and centrifugation or vacuum was continued to ensure complete incorporation. The mixture was dried overnight at room temperature, the microneedles were peeled off, and UV curing was performed to obtain GLP-1(32-36)a-PEG. 20K The core structure of the microneedle patch (carrying GLP-1(32-36)a-PEG) 20K Microneedle patch); then, the shell solution is added to a microneedle mold with 100 conical holes of 800 μm depth, 300 μm maximum hole diameter, and 500 μm tip distance. The shell solution is then completely inserted into the microneedle mold holes using centrifugation or vacuum methods. GLP-1(32-36)a-PEG is then loaded... 20K The core structure of the microneedle patch is pressed into the above mold and dried at room temperature to obtain the final gelatin microneedle patch loaded with GLP-1(32-36)a.

[0050] Example 3: GLP-1(32-36)a hyaluronic acid microneedle patch

[0051] First, aqueous solutions containing 2% (w / v) GLP-1(32-36)a (pH = 7.8) and 2% (w / v) PEG were prepared separately. 20K - Succinimide carbonate solution; then, PEG 20K - Succinimide carbonate solution was added dropwise to the above GLP-1(32-36)a-PEG being magnetically stirred. 20K The cross-linking reaction was carried out in aqueous solution at a reaction temperature of 4°C. The desired GLP-1(32-36)a-PEG was separated using a dextran gel column chromatography method. 20K Solution; finally, GLP-1(32-36)a-PEG was obtained by lyophilization. 20K Freeze-dried powder.

[0052] Formulated with 3% (w / v) GLP-1(32-36)α-PEG20K 0.5% (w / v) 2-hydroxy-2-methyl-1-phenylpropanone, 5% (w / v) chitosan, 5% (w / v) sodium methacrylate in 4% (v / v) acetic acid aqueous solution (core solution), 0.1% (w / v) GLP-1 (32-36)a and 30% (w / v) polyvinylpyrrolidone K90 (PVP) in ethanol solution (outer shell solution) and 15% (w / v) PVP in ethanol solution (base layer solution).

[0053] Subsequently, the core solution was added to a microneedle mold with 100 conical pores, each 600 μm deep, 250 μm in maximum diameter, and 500 μm between tips. Centrifugation or vacuum was used to ensure the core solution completely entered the pores of the microneedle mold. Excess liquid not entering the pores was scraped off, and the mold was dried overnight at room temperature. Then, the base layer solution was added to the microneedle patch mold, and centrifugation or vacuum was continued to ensure the base layer solution completely entered the pores of the microneedle mold. The mold was dried overnight at room temperature, the microneedles were peeled off, and the mixture was cured with UV light to obtain GLP-1(32-36)a-PEG. 20K The core structure of the microneedle patch (carrying GLP-1(32-36)a-PEG) 20K Microneedle patch); then, the shell solution is added to a microneedle mold with 100 conical holes, each 800 μm deep, 300 μm in maximum diameter, and 500 μm between needle tips. The shell solution is then completely inserted into the microneedle mold holes using centrifugation or vacuum methods. GLP-1(32-36)a-PEG is then loaded... 20K The core structure of the microneedle patch is pressed into the above mold and dried at room temperature to obtain the final hyaluronic acid microneedle patch loaded with GLP-1(32-36)a.

[0054] 2.1 Observation of the appearance and morphology of GLP-1(32-36)a polyvinyl alcohol microneedle patch

[0055] Sample: Core loaded with FITC-GLP-1(32-36)a-PEG prepared according to Example 1 20K The outer shell contains a microneedle patch containing 6G of Rhodamine.

[0056] Experimental methods:

[0057] 1. Fluorescent stereomicroscope

[0058] The morphology of the microneedles was observed using a stereomicroscope (S6D, Leica, Germany).

[0059] 2. Confocal microscope

[0060] The kernel containing FITC-GLP-1(32-36)a-PEG was examined using a confocal microscope (SEM, JSM-6330F, Tokyo, Japan). 20K We observed and analyzed the internal structure and drug distribution of the microneedles by placing a microneedle patch containing 6g of rhodamine on its outer shell.

[0061] Experimental results:

[0062] Figure 3 The morphological characteristics of the microneedles are shown, revealing their sharp tips. (Stereoscopic view) Figure 3 A) It can be clearly observed that the prepared microneedles are core-shell microneedles, with the drug mainly distributed at the tip. The core height of the microneedle is 607 μm, the base diameter is approximately 282 μm, the outer shell height is approximately 810 μm, and the base diameter is approximately 325 μm. There is a certain gap between the outer shell layer and the core layer, and the core layer can be uniformly and completely coated. Figure 3 B).

[0063] 2.2 Mechanical property determination of microneedle patches

[0064] Samples: GLP-1(32-36)a long-lasting polyvinyl alcohol microneedle patches and GLP-1(32-36)a-PEG-loaded microneedle patches prepared according to Example 1. 20K Microneedle patch.

[0065] Experimental methods:

[0066] Double-sided tape is used to attach the backing layer of the microneedles and fix them to the metal stage of the pressure-tension detector. The tips of the microneedles face the probe of the instrument. The probe presses down on the microneedles, and the displacement and force curves are recorded and plotted. The elastic modulus of the two types of microneedles is obtained by calculating the slope of the curve.

[0067] Experimental results:

[0068] Through the mechanical curve ( Figure 4 Calculations show that the elastic modulus of GLP-1(32-36)a-polyvinyl alcohol microneedles is 82 MPa, slightly less than that of GLP-1(32-36)a-PEG. 20K Microneedles (elastic modulus of 90 MPa).

[0069] Example 4: Skin Insertion Performance of Microneedle Patches

[0070] Sample: GLP-1(32-36)a polyvinyl alcohol microneedle patch prepared according to Example 1.

[0071] Experimental methods:

[0072] Balb / C mice (male, 20±1g) were used as an animal model. Hair on the outer thighs of the Balb / C mice was shaved using a razor and depilatory cream, and the exposed skin surface was cleaned with ethanol. GLP-1(32-36)a polyvinyl alcohol microneedle patches were vertically inserted into the dorsal skin of the rats, held for 5 minutes, and then peeled off. Mice were euthanized by cervical dislocation, the skin was peeled off, and the microneedle insertion sites were cut, embedded, and frozen in liquid nitrogen. Sections were prepared to a thickness of 5 μm and placed on silane-coated slides. Skin sections were observed under an inverted microscope (IX-71, Olympus, Tokyo, Japan).

[0073] Experimental results:

[0074] The depth of microneedle insertion into the skin is crucial to drug delivery and treatment efficacy. Figure 5 The results showed that the microneedles penetrated to a depth of approximately 210 μm into the skin, demonstrating that the microneedles can be effectively inserted into the skin, thus laying a theoretical foundation for the in vitro transdermal release of microneedles.

[0075] Experimental Example 5: In Vitro Release Experiment of Microneedle Patches

[0076] Sample: GLP-1(32-36)a polyvinyl alcohol microneedle patch prepared according to Example 1

[0077] Experimental methods:

[0078] 1. Determination of drug content in microneedle bodies

[0079] The needles and base layer of a 0.03 g GLP-1(32-36)a microneedle patch were separated using a scalpel. The needles were collected and dissolved overnight in 3 ml of 4% (v / v) acetic acid aqueous solution, then filtered through a 0.22 μm microporous membrane. The content of the short peptide drug in the filtrate was determined by liquid chromatography-mass spectrometry (LC-MS).

[0080] Chromatographic conditions: An Agilent C18 column (100 mm × 2.1 mm, 1.7 μm) was used. The mobile phase was 0.1% (w / v) formic acid solution and acetonitrile. The flow rate was 0.3 ml / min and the injection volume was 5 μl.

[0081] 2. In vitro release experiment of microneedle bodies

[0082] The separated needles were placed in a vial containing 1 ml of physiological saline, ensuring the microneedles were completely submerged in the 32°C saline solution, and magnetically stirred at 300 rpm. At predetermined time points, 0.1 ml of sample solution was removed, filtered through a 0.22 μm filter, and an equal volume of isothermal fresh physiological saline was added. The GLP-1(32-36)a and GLP-1(32-36)a-PEG in the filtrate were determined using the liquid chromatography-mass spectrometry (LC-MS) method described above. 20K The amount.

[0083] Experimental results:

[0084] The GLP-1(32-36)a in the outer shell was cumulatively released at 75±7.8% within 15 min and 89±4.2% within 60 min, indicating that the drug in the outer shell would rapidly dissolve in the skin after insertion. Figure 6 A). 95±10.8% of the drugs in the microneedles were released after 400 hours, demonstrating that the prepared microneedles have a long-lasting sustained-release effect. Figure 6 B).

[0085] Experimental Example 6: Pharmacodynamic Study of GLP-1 (32-36) Polyvinyl Alcohol Microneedle Patch

[0086] Sample: GLP-1(32-36)a polyvinyl alcohol microneedle patch prepared according to Experimental Example 1.

[0087] Experimental methods:

[0088] Balb / C mice (male, 20±1g) were selected as model animals, and diabetic mice were induced by injecting streptozotocin (70mg / kg). Subsequently, the aorta in the right leg of the mice was cut and ligated with surgical sutures to establish a diabetic mouse model of lower limb ischemia. Eighteen of these mice (male, 21±1g) were selected and divided into three groups: model group, subcutaneous injection group (10μg GLP-1(32-36)a was injected subcutaneously into each mouse daily for 30 days), and GLP-1(32-36)a-loaded polyvinyl alcohol microneedle group (350μg GLP-1(32-36)a was injected into each mouse via microneedle once within 30 days), with 6 mice in each group. The back hair of mice was shaved with a razor. The blood perfusion of the right hind limb was measured before modeling using a hind limb blood flow Doppler. The model group received no treatment. The blood perfusion of the healthy and affected hind limbs of the mice was measured at predetermined time points. The improvement of hind limb blood flow perfusion in different groups of mice was compared.

[0089] Experimental results:

[0090] pass Figure 7 A observed that blood flow in the right leg of the mouse was significantly reduced after the surgery, proving that the diabetic lower limb ischemia model was successfully established. Figure 7A and Figure 7 B indicates that after 30 days of treatment, there was a significant difference in blood perfusion between the treatment group and the model group, demonstrating that GLP-1(32-36)a has a significant therapeutic effect on diabetic lower limb ischemia symptoms. The treatment effect of the microneedle group was significantly better than that of the subcutaneous injection group, proving the long-term effect of microneedles and their advantage of maximizing the therapeutic effect of drugs. In addition, the GLP-1(32-36)a-loaded polyvinyl alcohol microneedle patches prepared in this study used all approved pharmaceutical excipients, and the preparation process is simple and conducive to production transformation.

[0091] Pharmacokinetic Study of GLP-1 (32-36) Polyvinyl Alcohol Microneedle Patch in Experiment Example 7

[0092] Sample: GLP-1(32-36)a polyvinyl alcohol microneedle patch prepared according to Experimental Example 1.

[0093] Balb / C mice (male, 20±1g) were selected as the model animals, and diabetic mice were induced by injecting streptozotocin (70mg / kg). Subsequently, the aorta in the right leg of these mice was severed and ligated with surgical sutures to establish a diabetic mouse model of lower limb ischemia. Twelve male diabetic mice (21±1g) were selected and divided into two groups: a subcutaneous injection group and a GLP-1(32-36)a polyvinyl alcohol microneedle group, with six mice in each group. The hair on the right leg of the mice was shaved with a razor, and the medication was administered at this location.

[0094] Blood samples were collected at predetermined time points, centrifuged at 1500g for 10 min, and 100 μL of the supernatant plasma was collected. Then, 100 μL of acetonitrile was added to completely precipitate the plasma proteins before analysis. The LC-MS method and chromatographic conditions described in Example 4 were used to determine the blood drug concentrations of each group of drugs in mice. The results are as follows: Figure 8 A and Figure 8 B.

[0095] Compared with GLP-1(32-36)a subcutaneous injection, the microneedle patch of this embodiment not only achieved a considerable peak concentration in plasma after administration, but also exhibited slower and more stable drug metabolism, resulting in a longer duration of drug retention in plasma.

Claims

1. The application of a microneedle patch loaded with glucagon-like peptide-1 short peptide in the preparation of a medical device for treating diabetic lower limb ischemia, characterized in that... The aforementioned microneedle-based core-shell type includes: The outer shell layer is made of GLP-1(32-36)a mixed with polyvinylpyrrolidone; The core layer is made of PEG-GLP-1(32-36)a, a photoinitiator, and polyvinyl alcohol.

2. The application according to claim 1, characterized in that... The molecular weight of the PEG is 1w to 100w.

3. The application according to claim 1, characterized in that... The length of the microneedles ranges from 100μm to 1000μm.

4. The application according to claim 1, characterized in that... The diameter of the base surface of the microneedle ranges from 30μm to 500μm.

5. The application according to claim 1, characterized in that... Area is 1cm 2 Within a certain range, 10 to 100 of the aforementioned microneedles are set.

6. The application according to claim 1, characterized in that... Within a length range of 1 mm, 1 to 3 microneedles are provided.

7. The application according to claim 1, characterized in that... Microneedle patches are prepared as follows: The first working solution is added to the prefabricated first microneedle patch mold, and the first working solution is allowed to enter the hole of the first microneedle mold by centrifugation or vacuum method, and then dried at room temperature overnight; The second working solution is added to the first microneedle patch mold. The solution is centrifuged or vacuumed to allow it to enter the hole of the first microneedle mold. After drying at room temperature overnight, the microneedles are peeled off and cured with ultraviolet light to obtain the core structure of the PEG-GLP-1(32-36)a microneedle patch. Then, the third working solution is added to the pre-made second microneedle patch mold, and centrifugation or vacuum method is used to allow the third working solution to enter the microneedle mold hole. Then, the core structure of the PEG-GLP-1(32-36)a loaded microneedle patch is pressed into the second microneedle patch mold and dried at room temperature to obtain the GLP-1(32-36)a loaded microneedle patch. The first working solution contains 1~30 wt% PEG-GLP-1(32-36)a. The concentration of polyvinylpyrrolidone in the first working solution is 5-30 wt%. The concentration of GLP-1(32-36)a in the third working solution is 1~5 wt%; The concentration of polyvinyl alcohol in the third working solution is 5-30 wt%.

8. A medical device, characterized in that... The microneedle patch comprising the glucagon-like peptide-1 short peptide as described in any one of claims 1 to 7.

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