A microneedle for long-term administration of a polypeptide drug, and a preparation method and application thereof

Abstract

This invention discloses a microneedle for long-term administration of peptide drugs, its preparation method, and its application. The microneedle comprises a substrate and needle bodies arranged in an array on the substrate. Each needle body includes a base attached to the substrate and a needle tip located on the base. The needle tip contains a plurality of drug-loaded sustained-release microspheres with a particle size of less than 10 μm. In this microneedle, the drug-loaded sustained-release microspheres are located only in the needle tip portion. This microneedle exhibits high drug delivery efficiency, good puncture resistance, and good drug loading rate, effectively solving the problems of high frequency of administration, short half-life, and poor patient compliance associated with water-soluble peptide drugs.

Description

Technical Field

[0001] This invention relates to the field of microneedle technology. More specifically, it relates to a microneedle for long-term administration of peptide drugs, its preparation method, and its application. Background Technology

[0002] Water-soluble peptide drugs possess higher specificity, bioactivity, and lower biotoxicity, thus demonstrating greater potential in disease treatment. However, they also have certain limitations in administration, primarily manifested in poor stability, including short half-life, easy inactivation, and low oral bioavailability. Currently, because peptide drugs are inactivated in the gastrointestinal tract due to acidic pH and enzymatic degradation, they are mostly administered via parenteral routes, such as intravenous, intramuscular, or subcutaneous injection. However, even with parenteral administration, peptide drugs with complex tertiary and quaternary structures are still easily degraded and cleared by metalloproteinases and the kidneys. Therefore, the short half-life of peptide drugs necessitates frequent injections to maintain therapeutic concentrations. However, frequent injections significantly reduce patient compliance.

[0003] Micron-level particle delivery, hydrophobic polymer encapsulation, and chemical modification have been proven to improve the stability of peptides, thereby preventing their rapid degradation and potentially prolonging the controlled release of water-soluble peptide drugs. For example, AstraZeneca developed a drug called... This product is a long-acting microparticle suspension of exenatide for injection. Its advantage lies in reducing the previous twice-daily injection frequency to once a week, which to some extent reduces the frequency of administration and improves patient compliance. However, this product must be administered via subcutaneous injection, which is difficult for untrained patients to administer independently and easily, and may lead to secondary problems such as pain, needle phobia, and injection site infection.

[0004] Microneedling, as a novel drug delivery route, can significantly improve patient compliance by replacing subcutaneous injection with transdermal microneedling. Microneedles penetrate the skin barrier of the stratum corneum, reaching only the epidermis without damaging neurons in the dermis, thereby minimizing pain during administration and enhancing drug delivery efficiency.

[0005] Based on this, this invention proposes using microneedles to assist in the delivery of microparticles into the skin, thereby achieving sustained release of water-soluble peptide drugs. The microneedles penetrate the skin, forming channels in the stratum corneum. The microneedles then rapidly dissolve, allowing the microparticles to distribute subcutaneously and release the drug, thus achieving transdermal delivery. The drug-loaded microparticle-microneedle array effectively achieves sustained release of water-soluble peptide drugs, while reducing the frequency and difficulty of administration, improving administration efficiency and patient tolerance. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a microneedle for long-term administration of peptide drugs, its preparation method, and its application. This microneedle possesses high drug delivery efficiency, good puncture resistance, and good drug loading rate, effectively solving the problems of high frequency of administration, short half-life, and poor patient compliance associated with water-soluble peptide drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On one hand, the present invention provides a microneedle for long-term administration of peptide drugs, the microneedle comprising a substrate and needles arranged in an array on the substrate;

[0009] The needle body includes a needle body base that is coupled to the base, and a needle tip located on the needle body base;

[0010] The needle tip contains a plurality of drug-loaded sustained-release microspheres, and the particle size of the drug-loaded sustained-release microspheres is less than 10 μm.

[0011] In the microneedle, the drug-loaded sustained-release microspheres are located only at the tip of the needle.

[0012] It is understood that in the needle tip, the base of the needle body and the needle tip are connected to each other, but do not cover each other.

[0013] Furthermore, the drug-loaded sustained-release microspheres are insoluble polymer microspheres encapsulated with polypeptide drugs.

[0014] Furthermore, the mass ratio of the polypeptide drug to the polylactic acid-glycolic acid copolymer is (1-12):1. Under these conditions, the obtained drug-loaded microspheres have a more uniform particle size distribution, and the particle size can be controlled between 3-7 μm. Simultaneously, they exhibit good punctureability and high drug loading rate and drug delivery efficiency. For example, the mass ratio of the polypeptide drug to the polylactic acid-glycolic acid copolymer includes, but is not limited to, (2-5):1, (2-4):1, (2-3.5):1, (2-2.5):1, (2.5-5):1, (2.5-4):1, (2.5-3.5):1, (3.5-5):1, (3.5-4):1, etc., in which case the aforementioned effects are even better.

[0015] In this invention, the polypeptide drug is water-soluble. For example, the polypeptide drug is selected from one or more of exenatide, liraglutide, liximab, semaglutide, duraglutide, and abiglutide.

[0016] Furthermore, the insoluble polymer is selected as PLGA. Under these conditions, peptide drugs can be well encapsulated in the insoluble polymer, and the resulting drug-loaded sustained-release microspheres exhibit excellent drug delivery efficiency and sustained-release effect.

[0017] Furthermore, the particle size of the drug-loaded sustained-release microspheres is 3-10 μm, preferably 3-7 μm. Under these conditions, the drug-loaded microspheres can be concentrated at the tip of the layered microneedles, thereby ensuring that the microneedles have excellent drug delivery efficiency.

[0018] Furthermore, the mass percentage of drug-loaded sustained-release microspheres in the needle tip is 20-90%.

[0019] Furthermore, the needle tip also contains an excipient, and the drug-loaded insoluble polymer microspheres are uniformly dispersed in the excipient.

[0020] Preferably, in the needle tip, the mass ratio of drug-loaded sustained-release microspheres to excipients ranges from 1:20 to 6:1.

[0021] Furthermore, the excipient in the needle tip is selected from one or more of sucrose, polyvinylpyrrolidone, hyaluronic acid, dextran, and ethyl cellulose. Preferably, the excipient is sucrose. Under these conditions, the microneedle's excellent drug delivery efficiency can be ensured while maintaining good skin punctureability.

[0022] Furthermore, the needle tip also contains a surfactant, and the surfactant is uniformly dispersed in the excipient. The presence of the surfactant in the needle tip enables the drug-loaded sustained-release microspheres to be uniformly dispersed in the excipient.

[0023] Furthermore, the surfactant in the needle tip is selected from Tween. Exemplary Tween includes, but is not limited to, one or more selected from Tween 80, Tween 60, Tween 40 and Tween 20.

[0024] Furthermore, in the needle tip, the mass ratio of drug-loaded sustained-release microspheres to surfactant ranges from 5:4 to 30:1.

[0025] Furthermore, the materials of the substrate and the needle base are each independently selected from one or a mixture of several of ethyl cellulose, sucrose, and hyaluronic acid.

[0026] Furthermore, both the substrate and the base of the needle are made of a mixture of ethyl cellulose, sucrose, and hyaluronic acid. The preferred mass ratio of these three components is (0.5-3):1:(0.5-5), more preferably (1-3):1:(1-4), and even more preferably (1-2.5):1:(1-3). Under these conditions, the resulting microneedles exhibit good skin punctureability and maintain structural integrity even after puncture.

[0027] Furthermore, the height of the needle tip accounts for 20-50% of the total height of the needle body.

[0028] Furthermore, the preparation of the drug-loaded sustained-release microspheres includes the following steps:

[0029] The drug is dissolved in deionized water to obtain an internal aqueous phase solution containing the drug;

[0030] The insoluble polymer is dissolved in a good solvent to obtain a polymer oil phase solution that is incompatible with the aqueous phase solution.

[0031] The aqueous phase solution was added to the polymer oil phase solution, and ultrasonic treatment was performed in an ice bath to obtain an emulsion with monodisperse droplets.

[0032] The emulsion is added to an aqueous solution containing a surfactant and then sheared and mixed to obtain a water-in-oil-in-water emulsion.

[0033] The good solvent is stirred to evaporate, solidified and collected, and then washed and freeze-dried to obtain the drug-loaded sustained-release microspheres.

[0034] By using ultrasonic treatment in an ice bath to obtain an emulsion with monodisperse droplets (i.e., a primary emulsion), the size of the primary emulsion droplets can be effectively reduced, while the drug loading rate of the final drug-loaded sustained-release microspheres can be significantly improved. The particle size can also be effectively controlled within 3-10 μm, mainly within 3-8 μm.

[0035] By performing ultrasonic treatment in an ice bath, the problem of solvent evaporation caused by increased temperature during ultrasonic emulsification can be prevented.

[0036] Furthermore, in the preparation of the drug-loaded sustained-release microspheres, the drug concentration in the internal aqueous phase solution is 25-250 mg / ml, preferably 90-180 mg / ml, and more preferably 90-150 mg / ml. Under these conditions, a higher drug loading rate is achieved while ensuring the small particle size of the microspheres.

[0037] Furthermore, the good solvent is selected from one or more of dichloromethane, trichloromethane, ethyl acetate and dimethyl carbonate, preferably dichloromethane.

[0038] Furthermore, in the preparation of the drug-loaded sustained-release microspheres, the concentration of the insoluble polymer in the polymer oil phase solution is 30-150 mg / ml, preferably 65-100 mg / ml, and more preferably 90 mg / ml. Under these conditions, a higher drug loading rate is achieved while ensuring the small particle size of the microspheres.

[0039] Furthermore, the ultrasonic treatment has a power of 10-90W and a duration of 5-60s.

[0040] For example, the power of the ultrasonic treatment includes, but is not limited to, 30-90W, 30-60W, 30-50W, 30W, 60-90W, and 90W. Under these conditions, the size of the colostrum droplets can be better controlled, while improving the drug loading rate of the drug-loaded sustained-release microspheres.

[0041] For example, the ultrasonic treatment time includes, but is not limited to, 5-30s, 5-10s, 10-60s, 10-30s, 30-60s, 5s, 30s, 10s, and 60s. Under these conditions, the size of the colostrum droplets can be better controlled, while improving the drug loading rate of the drug-loaded sustained-release microspheres.

[0042] Furthermore, in the preparation of the drug-loaded sustained-release microspheres, the concentration of the surfactant relative to water in the aqueous solution containing the surfactant is 0.5-3 wt%, preferably 1 wt%.

[0043] Furthermore, in the preparation of the drug-loaded sustained-release microspheres, the surfactant is selected from polyvinyl alcohol.

[0044] Furthermore, the shearing and mixing is carried out in a high-speed homogenizer. Preferably, the rotation speed of the shearing and mixing is 6000-16000 rpm and the time is 30-120 s.

[0045] In another aspect, the present invention provides a method for preparing the microneedles as described above, the method comprising the following steps:

[0046] An aqueous solution containing the drug-loaded sustained-release microspheres is provided and mixed to obtain a needle tip solution;

[0047] The needle tip solution was dropped into a microneedle mold, vacuumed, and dried to obtain the needle tip.

[0048] The aqueous solution for forming the needle base and the aqueous solution for forming the base are respectively added to the mold and dried to form the needle base and the base.

[0049] Furthermore, the needle body base and the substrate can be integrally formed or formed separately.

[0050] Furthermore, in the needle tip solution, the relative water content of the drug-loaded sustained-release microspheres is 0.5-8 wt%, preferably 1-8 wt%. Under these conditions, the resulting microneedles have a high drug loading capacity. In some examples, the relative water content of the drug-loaded sustained-release microspheres in the needle tip solution includes, but is not limited to, 2-6 wt%, 3-6 wt%, 4-6 wt%, etc.

[0051] Furthermore, the aqueous solution containing the drug-loaded sustained-release microspheres also contains excipients and surfactants.

[0052] Furthermore, in the aqueous solution containing the drug-loaded sustained-release microspheres, the content of excipients relative to water is 1-10 wt%; for example, the content of excipients relative to water includes, but is not limited to, 2-10 wt%, 2-6 wt%, 3-7 wt%, etc.

[0053] Furthermore, in the needle tip solution, the content of surfactant relative to water is 0.01-1 wt%, preferably 0.1-0.5 wt%.

[0054] Furthermore, the preparation of the needle tip solution includes the following steps:

[0055] A surfactant and the drug-loaded sustained-release microspheres are added to an aqueous solution of the excipient, and after mixing, the needle tip solution in which the drug-loaded sustained-release microspheres are uniformly suspended is obtained.

[0056] In another aspect, the present invention provides a microneedle patch comprising microneedles as described above, and a liner bonded to a substrate in the microneedles.

[0057] The beneficial effects of this invention are as follows:

[0058] The microneedles provided in this invention contain sustained-release microspheres with a particle size of less than 10 μm, all concentrated at the needle tip. This results in microneedles with not only high and controllable drug loading rates, but also excellent drug delivery efficiency and sustained-release performance. These microneedles offer advantages such as simple preparation, sustained release of water-soluble peptide drugs, convenient transdermal drug delivery, extended drug half-life, reduced drug usage frequency, and improved patient compliance.

[0059] In the microneedle preparation method provided in this invention, ultrasound is preferably used as the colostrum preparation method. The prepared sustained-release microparticles (drug-loaded sustained-release microspheres) loaded with water-soluble polypeptide drugs have small particle size and high drug loading rate. The preparation method is simple, low-cost, and the drug loading rate is highly controllable.

[0060] In the microneedle preparation method provided in this invention, a mixture of small-diameter drug-loaded sustained-release microparticles, excipients, and surfactants is preferably used as the matrix material for the needle tip solution. This effectively concentrates the drug-loaded microparticles at the needle tip, achieving efficient transdermal delivery of the sustained-release microparticles. Simultaneously, by controlling the proportion of drug-loaded microparticles in the microneedle patch, a high degree of controllability in the drug loading of the microneedle patch is achieved. Attached Figure Description

[0061] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0062] Figure 1 The particle size distribution diagram of the drug-loaded microparticles prepared in Example 9 is shown.

[0063] Figure 2 The image shows a SEM image of the drug-loaded microparticles prepared in Example 9.

[0064] Figure 3 The image shows the morphology of the drug-loaded microparticles-dissolution layered microneedles in Example 18 under an optical microscope.

[0065] Figure 4 The image shows the morphology of the puncture results of the drug-loaded microparticle-dissolution layered microneedles in Example 18.

[0066] Figure 5 The image shows the fine structure morphology of the needle tip of the layered microneedles obtained in Example 30 under a laser confocal microscope.

[0067] Figure 6 The experimental results of the in vitro skin solubility test of the layered microneedles prepared in Example 18 are shown.

[0068] Figure 7 The in vitro drug release kinetics fitting curve of the layered microneedles prepared in Example 18 is shown.

[0069] Figure 8 The results of in vivo pharmacokinetic experiments in rats are shown for the layered microneedles prepared in Example 18 and drug-loaded microparticles with equal drug content administered subcutaneously.

[0070] Figure 9 The results of in vivo pharmacodynamic experiments using the layered microneedles prepared in Example 18, the drug-loaded microparticles of Example 11 with an equal amount of drug injected subcutaneously, and the blank microneedles applied to db / db mice are shown. Detailed Implementation

[0071] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0072] Example 1

[0073] Preparation and evaluation of water-soluble peptide drug-loaded microparticles using ultrasonic emulsification:

[0074] Prepare water-soluble polypeptide drug-loaded microparticles according to the following steps:

[0075] Preparation of water-soluble polypeptide drug-loaded microparticles: 9 mg exenatide was dissolved in 0.2 ml of deionized water as the inner aqueous phase;

[0076] Dissolve 90 mg PLGA (RESOMER RG 503H) in 1 ml dichloromethane as the oil phase;

[0077] The aqueous phase was added to the oil phase, and the mixture was ultrasonically treated for 30 seconds under ice bath conditions at a power of 60W to obtain the primary emulsion.

[0078] The obtained colostrum was added dropwise to 20 ml of an aqueous solution containing 1 wt% PVA and processed using a high-speed homogenizer at 10,000 rpm for 60 s.

[0079] Subsequently, the organic solvent was evaporated and the particles were solidified by stirring at room temperature for 3 hours.

[0080] Finally, the water-soluble polypeptide drug-loaded microparticles were obtained by washing three times with deionized water, centrifuging, collecting, and freeze-drying.

[0081] Colostrum particle size and particle size distribution test: Take 0.1 ml of colostrum, dilute 100x, and measure using a nanoparticle size potential analyzer (Mastersizer2000).

[0082] Drug-loaded microparticle size and particle size distribution test: Weigh an appropriate amount of drug-loaded microparticle powder from Example 1, wet and disperse it with 1wt% Tween 80 aqueous solution, and measure the particle size using a Bettersize 2600 laser particle size analyzer.

[0083] Drug loading and encapsulation efficiency of microspheres were measured: 5 mg of the drug-loaded microparticle powder from Example 1 was weighed, and 0.525 ml of acetonitrile was added. The mixture was vortexed at 3000 rpm / min for 30 min. Subsequently, 0.975 ml of 0.05 M potassium dihydrogen phosphate aqueous solution was added, and the mixture was vortexed at 3000 rpm / min for 30 min. The supernatant was filtered and the drug content was determined by high performance liquid chromatography. The drug loading and encapsulation efficiency of the microspheres were then calculated.

[0084] Microparticle drug loading rate = (actually measured mass of exenatide in microparticles / actual weighed mass of microparticles) × 100%;

[0085] The test results showed that the initial emulsion of the drug-loaded microparticles had a particle size of 298.11 nm and a PDI of 0.062; the particle size of the drug-loaded microparticles was 5.04 μm, the drug loading rate was 8.90%, and the encapsulation efficiency was 92.47%.

[0086] Comparative Example 1

[0087] Preparation of water-soluble peptide drug-loaded microparticles by vortex method:

[0088] Dissolve 9 mg of exenatide in 0.2 ml of deionized water as the internal aqueous phase;

[0089] Dissolve 90 mg PLGA (RESOMER RG 503H) in 1 ml dichloromethane as the oil phase;

[0090] The internal aqueous phase was added to the oil phase and treated with a vortex mixer at 1000 rpm for 30 seconds.

[0091] The obtained colostrum was added dropwise to 20 ml of an aqueous solution containing 1% PVA and processed using a high-speed homogenizer at 10,000 rpm for 60 s.

[0092] Subsequently, the organic solvent was evaporated and the particles were solidified by stirring at room temperature for 3 hours.

[0093] Finally, after washing three times with deionized water, centrifuging, and freeze-drying, water-soluble polypeptide drug-loaded microparticle powder was obtained.

[0094] The initial emulsion of the drug-loaded microparticles was tested and found to have a particle size of 3409.23 nm and a PDI of 0.970. The total particle size of the drug-loaded microparticles was 46.35 μm, and the drug loading rate was 1.34%.

[0095] Comparative Example 2

[0096] Preparation of water-soluble peptide drug-loaded microparticles by stirring method:

[0097] Dissolve 9 mg of exenatide in 0.2 ml of deionized water as the internal aqueous phase;

[0098] Dissolve 90 mg PLGA (RESOMER RG 503H) in 1 ml dichloromethane as the oil phase;

[0099] The aqueous phase was added to the oil phase and treated with a magnetic stirrer at 1000 rpm for 30 seconds.

[0100] The obtained colostrum was added dropwise to 20 ml of an aqueous solution containing 1% PVA and processed using a high-speed homogenizer at 10,000 rpm for 60 s.

[0101] Subsequently, the organic solvent was evaporated and the particles were solidified by stirring at room temperature for 3 hours.

[0102] Finally, after washing three times with deionized water, centrifuging, and freeze-drying, water-soluble polypeptide drug-loaded microparticle powder was obtained.

[0103] The initial emulsion of the drug-loaded microparticles was tested and found to have a particle size of 2909.23 nm and a PDI of 0.814. The total particle size of the drug-loaded microparticles was 103.98 μm, and the drug loading rate was 0.97%.

[0104] Comparative Example 3

[0105] Preparation of water-soluble peptide drug-loaded microparticles by homogenization:

[0106] Dissolve 10 mg of exenatide in 0.2 ml of deionized water as the internal aqueous phase;

[0107] 100 mg PLGA (RESOMER RG 503H) was dissolved in 1 ml of dichloromethane as the oil phase;

[0108] The internal aqueous phase was added to the oil phase and homogenized at 7000 rpm for 30 seconds.

[0109] The obtained colostrum was added dropwise to 20 ml of an aqueous solution containing 1% PVA and processed using a high-speed homogenizer at 10,000 rpm for 60 s.

[0110] Subsequently, the organic solvent was evaporated and the particles were solidified by stirring at room temperature for 3 hours.

[0111] Finally, after washing three times with deionized water, centrifuging, and freeze-drying, water-soluble polypeptide drug-loaded microparticle powder was obtained.

[0112] The initial emulsion of the drug-loaded microparticles was tested and found to have a particle size of 897.45 nm and a PDI of 0.523. The total particle size of the drug-loaded microparticles was 36.57 μm, and the drug loading rate was 4.26%.

[0113] Examples 2-8

[0114] Drug-loaded microparticles were obtained using different ultrasonic emulsification conditions. The comparative and example studies were conducted according to Example 1, except that the amount of exenatide added was 10 mg, the amount of PLGA added was 100 mg, and the ultrasonic power and time were different. Specific parameters of each component used, as well as the parameters of the resulting promulgated emulsion and drug-loaded microparticles, are shown in Table 1.

[0115] Table 1

[0116]

[0117]

[0118] The above results indicate that, compared with the preparation method in the comparative example, ultrasonic emulsification as a method for preparing colostrum can effectively reduce the size of colostrum droplets, significantly improve the drug loading rate of drug-loaded microparticles, and effectively control the particle size within 3-10 μm, mainly within 3-8 μm.

[0119] Example 9

[0120] Preparation of water-soluble polypeptide drug-loaded microparticles:

[0121] Dissolve 9 mg of exenatide in 0.2 ml of deionized water as the internal aqueous phase;

[0122] Dissolve 90 mg PLGA (RESOMER RG 503H) in 1 ml dichloromethane as the oil phase;

[0123] The aqueous phase was added to the oil phase and ultrasonically treated for 60 seconds at a power of 90W.

[0124] The obtained colostrum was added dropwise to 20 ml of an aqueous solution containing 1 wt% PVA and processed using a high-speed homogenizer at 13000 rpm for 60 s.

[0125] Subsequently, the organic solvent was evaporated and the particles were solidified by stirring at room temperature for 3 hours.

[0126] Finally, after washing three times with deionized water, centrifuging, and freeze-drying, water-soluble polypeptide drug-loaded microparticle powder was obtained.

[0127] The initial emulsion of the drug-loaded microparticles was tested and found to have a particle size of 119.34 nm and a PDI of 0.045. The final particle size of the drug-loaded microparticles was 5.46 μm, and the drug loading rate was 9.27%.

[0128] The particle size distribution of the drug-loaded microparticles is as follows: Figure 1 As shown, the SEM image of the drug-loaded microparticles is as follows. Figure 2 As shown.

[0129] Examples 10-17

[0130] Different ratios of peptide drugs to insoluble polymers were selected, and drug-loaded microparticles were prepared using ultrasonic emulsification, the same method as in Example 9. The differences in formulation and the parameters of the resulting colostrum and drug-loaded microparticles are shown in Table 2 below.

[0131] Table 2

[0132]

[0133] The above results indicate that using ultrasonic emulsification as the primary emulsion preparation method can effectively control the particle size of drug-loaded microparticles, keeping the particle size between 3 and 7 μm. Furthermore, by increasing the drug loading, ultrasonic emulsification can achieve a controllable drug loading rate of 9-24%, preferably within the range of 15-24%.

[0134] Example 18

[0135] A method for preparing soluble, layered microneedles loaded with polypeptide sustained-release microparticles includes the following steps:

[0136] 1) Preparation of microneedle tip matrix solution:

[0137] Take 5 mg of the drug-loaded microparticles from Example 11 above, add 10 mg of sucrose (excipient), 2 mg of Tween 20 (surfactant), and 1 ml of water, and vortex to mix.

[0138] 2) Preparation of the solution for the base of the microneedle body and the substrate:

[0139] Take 6.5g of ethyl cellulose, 6.5g of hyaluronic acid and 10g of sucrose, add 87g of water, stir well and then centrifuge to remove air bubbles;

[0140] 3) Preparation process of layered microneedles:

[0141] First, 30 μl of the needle tip solution was added to the surface of the PDMS mold, vacuumed for 30 min, and then dried at room temperature for 1 h to obtain the needle tip.

[0142] Subsequently, 90 μl of the microneedle body base and the base solution were added to the surface of the PDMS mold. After vacuuming for 30 min, the mold was dried at room temperature overnight to obtain layered microneedles.

[0143] 4) Various examination parameters for microneedles:

[0144] A. Integrity of microneedles: Observe the integrity of the microneedle patch using a stereomicroscope to check for any film curling, bending, breakage, or needle breakage.

[0145] Figure 3 The image shows the morphological observation results of the drug-loaded microparticle-dissolved layered microneedles in Example 18 under an optical microscope. The microneedle array contains 213 microneedles distributed at a depth of 0.75 cm. 2 On the circular patch, the microneedle height is 700μm.

[0146] B. Punctureability of Microneedles: Use skin from the back of a 1-month-old piglet, cut to a suitable area, and place the microneedles flat with the needle tips facing down. Then, apply a continuous pressure of 60N to the back of the microneedle patch for 30 seconds. After pressing, remove the microneedles from the skin, apply trypan blue staining solution to the microneedle application site, and after staining for 10 minutes, wipe the skin clean. Observing a neat array of blue needle holes indicates that the patch is punctureable.

[0147] Figure 4 The results of the layered microneedle puncture in Example 18 show that the microneedle patch puncture array in Example 18 is neat and clearly visible.

[0148] Following the above method, drug-loaded microparticles loaded with polypeptide sustained-release microparticles-soluble layered microneedles were prepared using the drug-loaded microparticles of Examples 10 and 12-17, respectively. These microneedles have the same excellent microneedle integrity and puncture resistance as the microneedles prepared using drug-loaded microspheres of Example 11.

[0149] Examples 19-24 and Comparative Examples 4-12

[0150] In Examples 19-24 and Comparative Examples 4-12, the needle tip liquid composition was the same as in Example 18. The effects of different excipients on the integrity and puncture resistance of the layered microneedle substrate were investigated. The specific formulations are shown in Table 3 below.

[0151] Table 3

[0152]

[0153]

[0154] The above results indicate that the mixture of ethyl cellulose, sucrose, and hyaluronic acid in the examples can prepare layered microneedles with excellent integrity and good puncture resistance. The layered microneedles prepared with PVA, HA, HPMC, and CS in the comparative examples showed edge curling and insufficient flatness; the layered microneedles prepared with sucrose, DEX, and PVP in the comparative examples were brittle and prone to breakage, therefore unsuitable as substrate excipients. The layered microneedles prepared with γ-PGA met the integrity requirements, but their puncture resistance was insufficient, making them unsuitable as substrate excipients.

[0155] Examples 25-29

[0156] The effect of different microparticle loading on the drug loading of layered microneedles was investigated: by changing the amount of microparticles added in the needle tip fluid, the effect of the amount of microparticles added in a single layered microneedle on the drug loading of the layered microneedles was investigated.

[0157] Determination of drug content in layered microneedles: Take one layered microneedle, add 0.35 ml of acetonitrile, vortex for 30 min; add 0.65 ml of ultrapure water, vortex for 30 min; after centrifugation at 10000 rpm for 10 min, take the supernatant and filter it into a liquid chromatography vial for HPLC determination.

[0158] The preparation method of the layered microneedles is the same as in Example 18. The drug-loaded microparticles are selected from Example 11. The specific formulation of the amount of drug-loaded microparticles added to the needle tip matrix solution is shown in Table 4 below:

[0159] Table 4

[0160] Group The amount of drug-loaded microparticles added relative to water in the needle tip solution / wt% Patch Drug Load μg / cm 2 ]] Example 25 2 109.05 Example 26 3 166.11 Example 27 4 236.19 Example 28 5 268.01 Example 29 6 326.14

[0161] The above results indicate that by changing the amount of drug-loaded microparticles added to the needle tip solution, the drug loading of the layered microneedle patch can be effectively controlled. With increasing amounts of drug-loaded microparticles in the needle tip solution, the drug loading per unit area of ​​the layered microneedle patch also increases, achieving a drug loading rate of 100-300 μg / cm² for a single microneedle. 2 The height is controllable.

[0162] In addition, the microneedles prepared in Examples 25-29 also have microneedle integrity and good puncture resistance.

[0163] Examples 30-33 and Comparative Examples 13-18

[0164] The effects of different tip excipient types and concentrations on the drug delivery efficiency of layered microneedles were investigated: the effects of sucrose, hyaluronic acid, PVP, and DEX as tip excipients on the drug delivery efficiency of layered microneedles were examined.

[0165] Determination of drug delivery efficiency of layered microneedles: One layered microneedle was pressed onto the skin of a one-month-old piglet after equilibration at room temperature for 30 minutes with a force of 60 N. The application time was controlled to be 20 minutes before the microneedle was removed. The removed microneedle residue was placed in a centrifuge tube, 0.35 ml of acetonitrile was added, and the mixture was vortexed for 30 minutes; then 0.65 ml of ultrapure water was added, and the mixture was vortexed for 30 minutes; after centrifugation at 10000 rpm for 10 minutes, the supernatant was collected, filtered into a liquid chromatography vial, and the drug content in the microneedle residue was determined by HPLC. The drug delivery efficiency of the layered microneedles was calculated as follows:

[0166] Drug delivery efficiency (%) = (mass of drug in residual patch / mass of drug in unused microneedle patch) × 100%.

[0167] The preparation method of the layered microneedles is the same as in Example 18. The drug-loaded microparticles are selected from Example 12. The amount and content of other components added in the needle tip matrix solution remain unchanged. The difference is that the needle tip excipient formulation is shown in Table 5 below.

[0168] Table 5

[0169] Group needle tip excipient type Concentration of needle tip excipient relative to water / wt% Drug delivery efficiency / % Example 18 sucrose 1 92.01 Example 30 sucrose 2 91.45 Example 31 sucrose 4 94.63 Example 32 sucrose 6 92.47 Example 33 sucrose 10 91.80 Comparative Example 13 Hyaluronic acid 4 49.63 Comparative Example 14 Hyaluronic acid 10 50.30 Comparative Example 15 PVP 4 54.15 Comparative Example 16 PVP 10 51.42 Comparative Example 17 DEX 4 65.39 Comparative Example 18 DEX 10 64.39

[0170] Figure 5 This is an image showing the fine structure of the needle tip of the layered microneedles of Example 30 as observed by a laser confocal microscope. It can be seen that the microparticles are well concentrated at the needle tip.

[0171] The drug delivery efficiency results show that, in the above examples, using sucrose as the tip excipient, the drug delivery efficiency remained above 90% even with changes in excipient concentration. In the comparative examples, hyaluronic acid and PVP as tip excipients resulted in drug delivery efficiencies of approximately 50%, and DEX as the tip excipient resulted in approximately 65%, both significantly lower than the drug delivery efficiency of the layered microneedles using sucrose as the tip excipient in the examples.

[0172] Examples 34-39 and Comparative Example 19

[0173] The effects of different surfactant types and concentrations on the drug delivery efficiency of layered microneedles were investigated: Examples 34-39 investigated the effects of Tween 20, Tween 40, Tween 60, and Tween 80 as surfactants in the needle tip solution, and Comparative Example 19, which did not contain any surfactant, on the drug delivery efficiency of layered microneedles.

[0174] Determination of drug delivery efficiency of layered microneedles: One layered microneedle was pressed onto the skin of a one-month-old piglet after equilibration at room temperature for 30 minutes with a force of 60 N. The application time was controlled to be 20 minutes before the microneedle was removed. The removed microneedle residue was placed in a centrifuge tube, 0.35 ml of acetonitrile was added, and the mixture was vortexed for 30 minutes; then 0.65 ml of ultrapure water was added, and the mixture was vortexed for 30 minutes; after centrifugation at 10000 rpm for 10 minutes, the supernatant was collected, filtered into a liquid chromatography vial, and the drug content in the microneedle residue was determined by HPLC. The drug delivery efficiency of the layered microneedles was calculated as follows:

[0175] Drug delivery efficiency (%) = (mass of drug in residual patch / mass of drug in unused microneedle patch) × 100%.

[0176] The preparation method of the layered microneedles is the same as in Example 18. The drug-loaded microparticles are selected from Example 13. In the needle tip matrix solution, the amount and content of other components remain unchanged. The difference lies in the formulation of the surfactant, as shown in Table 6 below.

[0177] Table 6

[0178] Group Surfactant type Surfactant concentration relative to water / wt% Drug delivery efficiency / % Example 34 Twain 20 0.1 91.80 Example 35 Twain 20 0.2 92.31 Example 36 Twain 20 0.4 92.44 Example 37 Twain 40 0.2 91.67 Example 38 Twain 60 0.2 91.22 Example 39 Twain 80 0.2 90.65 Comparative Example 19 No additions 0 64.15

[0179] The results above show that the addition of surfactants has a significant impact on the drug delivery efficiency of microneedles. Tween, as a surfactant, has a significant effect on the enrichment of drug-loaded particles in microneedles. Surfactants can significantly reduce the hydrophobic interactions of particles, thereby helping the drug-loaded particles to concentrate better on the needle tip, resulting in higher drug delivery efficiency of the microneedles. Different types and concentrations of Tween as surfactants have no significant effect on particle enrichment.

[0180] Example 40

[0181] Accelerated stability testing of drug-loaded microparticles-soluble layered microneedles:

[0182] The layered microneedles prepared in Example 28 were adhered to the surface of medical tape and packaged using blister packs and aluminum-plastic bags. The packaged microneedle patches were stored at 25°C for 3 months. Since the conventional storage temperature for peptide formulations is 4°C, 25°C was chosen as the accelerated stability condition. The exenatide content of the drug-loaded microparticle-soluble layered microneedles at 0 days and 3 months was determined using high-performance liquid chromatography (HPLC).

[0183] Liquid chromatography analysis showed that after 3 months, the drug content in the patch was 98.4% of that on day 0, indicating that loading exenatide into drug-loaded microparticles-soluble layered microneedles can maintain good drug stability.

[0184] Example 41

[0185] In vitro skin solubility test of drug-loaded microparticles-soluble layered microneedles:

[0186] The layered microneedles prepared in Example 28 were applied to the skin of 1-month-old piglets that had been activated at room temperature for 30 min. A vertical pressure of 60 N was applied for 30 s, and pressure-sensitive adhesive was then attached to a substrate. This process was maintained for 5, 10, and 20 min, respectively. At the specified time, the remaining microneedle patches were removed, and the height of the remaining microneedles was observed under a fluorescence microscope. The fluorescence intensity within the skin was also observed, thereby characterizing the in vitro skin solubility of the microneedles.

[0187] Figure 6 The results are from an in vitro skin solubility test of the layered microneedles prepared in Example 28. Observation of the residual patch morphology at 5 min, 10 min, and 20 min showed that the white needle tip portion of the residual patch gradually decreased with increasing application time, until almost no residual needle tips remained on the patch after 20 min. Further observation of the fluorescence of the drug in the skin showed that the fluorescence intensity gradually increased with increasing application time, indicating that the drug-loaded microparticles—the dissolving microneedles—can dissolve in the skin in a relatively short time, achieving effective drug retention.

[0188] Example 42

[0189] In vitro drug release assay of drug-loaded microparticles-soluble layered microneedles:

[0190] The layered microneedles prepared in Example 28 were placed in a dialysis bag and dispersed with 100 μl of receiving medium. The dialysis bag was then sealed with a string. The dialysis bag was suspended in a stoppered conical flask containing 4 ml of receiving solution (0.01 M PBS solution, pH 7.4). A magnetic stir bar was placed in the conical flask at 240 rpm, and the flask was placed in a constant temperature water bath at 37 ± 0.5 °C. At a specified time point, all the release medium was removed, and then the same volume of fresh release medium was added. The removed solution was centrifuged at 10,000 rpm for 10 min, and the concentration of exenatide was determined using high-performance liquid chromatography (HPLC). The cumulative drug release rate was then calculated.

[0191] Figure 7The in vitro drug release kinetics curves for the drug-loaded microparticle-layered dissolution microneedles prepared in Example 28 are shown. Analysis reveals that the layered microneedles of Example 18 release approximately 15% in one day, due to drug dissolution from the drug-loaded microparticle surface in contact with the medium. The cumulative weekly drug release rates for weeks 1, 2, 3, and 4 are 26.46±1.05%, 20.64±1.12%, 18.46±0.55%, and 7.19±0.85%, respectively. These results indicate that the drug-loaded microparticle-layered dissolution microneedles can achieve stable and slow drug release over 4 weeks, with a cumulative release rate exceeding 80%, demonstrating good and stable sustained-release effects for water-soluble peptide drugs.

[0192] Example 43

[0193] In vivo pharmacokinetic assays of drug-loaded microparticles-soluble layered microneedles:

[0194] SD rats approximately 8 weeks old (weighing approximately 250±20g) were selected as subjects for in vivo pharmacokinetic experiments using drug-loaded microparticles-soluble layered microneedles. First, after fixing the rats, their abdominal skin was depilated using a depilatory cream. Then, the microneedles were vertically pressed onto the rat skin with a force of 60N and held for 20 minutes before being removed. Blood samples were collected from the retro-orbital venous plexus of the rats at specified time points before and after drug administration using medical-grade blood. The samples were collected in EDTA anticoagulant tubes, centrifuged at 4℃ and 3000rpm for 10 minutes, and the supernatant plasma was collected and stored at -80℃. After collecting blood samples at all specified time points, the drug content in the plasma was determined.

[0195] Figure 8 The results of in vivo pharmacokinetic experiments in rats using the drug-loaded microparticle-dissolving layered microneedles of Example 28 and subcutaneous injection of drug-loaded microparticles with the same drug content are presented. It can be seen that the drug-loaded microparticle-dissolving layered microneedles maintained a stable blood drug concentration for 14 days, achieving a relative bioavailability of 89.71% compared to the reference formulation subcutaneously injected with the same drug content. This suggests that the drug-loaded microparticle-dissolving layered microneedles can serve as an alternative to conventional injection for transdermal drug delivery, with comparable therapeutic efficacy. Furthermore, the terminal elimination half-life (T1 / 2) and mean residence time (MRT) of the drug-loaded microparticle injection group were 309.6 ± 3.8 hours and 129.5 ± 8.8 hours, respectively, while those of the drug-loaded microparticle-dissolving layered microneedles group were 466.4 ± 12.2 hours and 178.8 ± 9.6 hours, respectively. This indicates that the drug-loaded microparticle-dissolving layered microneedles can maintain the blood drug concentration of the peptide drug more gently and sustainably than injection, prolonging the drug's presence and duration of action in plasma.

[0196] Example 44

[0197] In vivo pharmacodynamic testing of drug-loaded microparticles-soluble layered microneedles:

[0198] Diabetic (db / db) mice are a widely used model of type 2 diabetes, exhibiting symptoms of hyperglycemia. Therefore, db / db mice were selected for pharmacodynamic studies. In this study, animals were randomly assigned to three groups of six each. The first group received subcutaneous injections of drug-loaded microparticles. The second group received patches of drug-loaded microparticles-soluble layered microneedles, and the third group received patches of drug-free, blank microparticles-soluble layered microneedles. Mice were fasted overnight before the experiment, and baseline blood glucose levels were measured. For the second and third groups, abdominal hair was removed using depilatory cream, and then the corresponding microneedle patches were applied. Animals were provided with normal food during the day and fasted overnight to allow for fasting blood glucose measurement at predetermined time points. Fasting blood glucose levels were measured using a glucometer at predetermined time points (0, 2h, 8h, 1d, 2d, 3d, 5d, 7d, 10d, and 14d).

[0199] Figure 9 This study presents in vivo pharmacodynamic results in db / db mice using the drug-loaded microparticles-dissolving layered microneedles of Example 28, the drug-loaded microparticles of Example 11 with an equal amount of drug injected subcutaneously, and blank microneedles applied. In the control group, mice given blank microneedles maintained stable blood glucose levels within the range of 25-28 mmol / L with minimal fluctuations over the 14-day study, exhibiting a persistent hyperglycemic state. Blood glucose levels in both the drug-loaded microparticle-dissolving layered microneedle group and the drug-loaded microparticle group with an equal amount of drug injected subcutaneously were significantly reduced, falling below 20 mmol / L in the first 10 days of treatment, with no significant difference between the two groups. Further comparative analysis showed that the subcutaneous injection group exhibited a more severe hypoglycemic effect within the first 2 hours. This was because the free drug in the microparticles immediately came into contact with the tissue fluid after injection, thus rapidly entering the systemic circulation. Conversely, the drug-loaded microparticle-dissolving layered microneedles allowed for sustained release of microparticles into the epidermis via transdermal delivery, resulting in a more gradual decrease in blood glucose levels, thus minimizing the risk of severe hypoglycemia.

[0200] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A microneedle for long-term administration of polypeptide drugs, characterized in that, It includes a substrate and needles arranged in an array on the substrate; The needle body includes a needle body base that is coupled to the base, and a needle tip located on the needle body base; The needle tip contains a plurality of drug-loaded sustained-release microspheres, and the particle size of the drug-loaded sustained-release microspheres is less than 10 μm. In the microneedle, the drug-loaded sustained-release microspheres are located only at the tip of the needle.

2. The microneedle according to claim 1, characterized in that, The drug-loaded sustained-release microspheres are insoluble polymer microspheres encapsulated with polypeptide drugs; Preferably, the mass ratio of the polypeptide drug to the polylactic acid-glycolic acid copolymer is (1-12):1, more preferably (2-4):1; Preferably, the polypeptide drug is selected from one or more of exenatide, liraglutide, liximaglutide, semaglutide, duraglutide, and abiglutide; Preferably, the insoluble polymer is selected from PLGA.

3. The microneedle according to claim 1, characterized in that, The particle size of the drug-loaded sustained-release microspheres is 3-10 μm, preferably 3-7 μm.

4. The microneedle according to claim 1, characterized in that, The mass percentage of drug-loaded sustained-release microspheres in the needle tip is 20-90%. Preferably, the height of the needle tip accounts for 20-50% of the total height of the needle body.

5. The microneedle according to claim 1, characterized in that, The needle tip also contains an excipient, and the drug-loaded insoluble polymer microspheres are uniformly dispersed in the excipient; Preferably, in the needle tip, the mass ratio of drug-loaded sustained-release microspheres to excipients ranges from 1:20 to 6:1; Preferably, the excipient is selected from one or more of sucrose, polyvinylpyrrolidone, hyaluronic acid, dextran, and ethyl cellulose; Preferably, the needle tip further comprises a surfactant, and the surfactant is uniformly dispersed in the excipient; Preferably, in the needle tip, the mass ratio of drug-loaded sustained-release microspheres to surfactant ranges from 5:4 to 30:1; Preferably, the surfactant is selected from Tween.

6. The microneedle according to claim 1, characterized in that, The materials of the base and the needle base are each independently selected from one or a mixture of several of ethyl cellulose, sucrose, and hyaluronic acid; Preferably, the base and the needle base are both made of a mixture of ethyl cellulose, sucrose and hyaluronic acid, and the mass ratio of the three is preferably (0.5-3):1:(0.5-5), more preferably (1-3):1:(1-4).

7. The microneedle according to any one of claims 1-6, characterized in that, The preparation of the drug-loaded sustained-release microspheres includes the following steps: The drug is dissolved in deionized water to obtain an internal aqueous phase solution containing the drug; The insoluble polymer is dissolved in a good solvent to obtain a polymer oil phase solution that is incompatible with the aqueous phase solution. The aqueous phase solution was added to the polymer oil phase solution, and ultrasonic treatment was performed in an ice bath to obtain an emulsion with monodisperse droplets. The emulsion is added to an aqueous solution containing a surfactant and then sheared and mixed to obtain a water-in-oil-in-water emulsion. The good solvent is stirred to evaporate, solidified and collected, and then washed and freeze-dried to obtain the drug-loaded sustained-release microspheres; Preferably, the concentration of the drug in the aqueous phase solution is 25-250 mg / ml, more preferably 90-180 mg / ml, and most preferably 90-150 mg / ml; Preferably, the good solvent is selected from one or more of dichloromethane, trichloromethane, ethyl acetate and dimethyl carbonate, and more preferably dichloromethane; Preferably, the concentration of the insoluble polymer in the polymer oil phase solution is 30-150 mg / ml, more preferably 65-100 mg / ml, and even more preferably 90 mg / ml; Preferably, the ultrasonic treatment has a power of 10-90W and a duration of 5-60s; Preferably, in the aqueous solution containing the surfactant, the concentration of the surfactant relative to water is 0.5-3 wt%, preferably 1 wt%. Preferably, the surfactant is selected from polyvinyl alcohol; Preferably, the shearing and mixing is carried out in a high-speed homogenizer, and preferably, the rotation speed of the shearing and mixing is 6000-16000 rpm, and the time is 30-120 s.

8. The method for preparing microneedles according to any one of claims 1-7, characterized in that, Includes the following steps: An aqueous solution containing the drug-loaded sustained-release microspheres is provided and mixed to obtain a needle tip solution; The needle tip solution was dropped into a microneedle mold, vacuumed, and dried to obtain the needle tip. The aqueous solution for forming the needle base and the aqueous solution for forming the base are respectively added to the mold and dried to form the needle base and the base.

9. The preparation method according to claim 8, characterized in that, In the needle tip solution, the relative water content of the drug-loaded sustained-release microspheres is 0.5-8 wt%, preferably 1-8 wt%. Preferably, the aqueous solution containing the drug-loaded sustained-release microspheres further contains excipients and surfactants; Preferably, the surfactant content relative to water in the needle tip solution is 0.01-1 wt%, more preferably 0.1-0.5 wt%. Preferably, the preparation of the needle tip solution includes the following steps: A surfactant and the drug-loaded sustained-release microspheres are added to an aqueous solution of the excipient, and after mixing, the needle tip solution in which the drug-loaded sustained-release microspheres are uniformly suspended is obtained.

10. A microneedle patch, characterized in that, It comprises a microneedle as described in any one of claims 1-7, and a liner bonded to a substrate in the microneedle.

Images