WO2024146091A1

WO2024146091A1 - Glp-1 receptor agonist microneedle and preparation method therefor

Info

Publication number: WO2024146091A1
Application number: PCT/CN2023/103061
Authority: WO
Inventors: 高云华; 刘寒; 周泽荃
Original assignee: 中科微针（北京）科技有限公司
Priority date: 2023-01-03
Filing date: 2023-06-28
Publication date: 2024-07-11

Abstract

The present application relates to a GLP-1 receptor agonistic drug microneedle composition, which comprises a GLP-1 receptor agonist drug, a protective agent selected from a zinc-containing pharmaceutical compound, and a microneedle excipient, wherein the mass ratio of the protective agent to the GLP-1 receptor agonist drug is 0.1:1-2:1. The composition provided in the present application can be used in the microneedle to improve the stability of the GLP-1 receptor agonist drug during high-temperature storage. Further disclosed in the present application are a microneedle prepared from the composition, and a preparation method therefor and the use thereof.

Description

[Corrected 26.09.2023 according to Rule 91] A GLP-1 receptor agonist drug microneedle composition, microneedles prepared therefrom, and preparation method and application thereof

Technical Field

[Corrected 26.09.2023 in accordance with Article 91]
The present invention relates to the field of microneedle technology, and more specifically, to a GLP-1 receptor agonist drug microneedle composition, a microneedle prepared therefrom, and a preparation method and application thereof.

Background technique

Diabetes is a chronic disease that often leads to serious secondary diseases. It has the characteristics of high prevalence, easy inheritance, many complications and incurable. Glucagon-like peptide 1 (GLP-1) is a new target for blood sugar regulation and is increasingly attracting people's attention. GLP-1 receptor agonists have fewer adverse reactions during use, and their effect of stimulating insulin secretion is glucose concentration-dependent, which is not easy to induce hypoglycemia. They are safer than insulin, and can reduce food intake and delay gastric emptying, which is beneficial for weight control and can protect pancreatic β cells. They are widely developed and applied in clinical practice. At present, the dosage form of GLP-1 receptor agonists used in clinical practice is mainly injection, which has a long use cycle. This dosage form has problems such as inconvenient operation, infection at the injection site, poor patient compliance, and poor drug accessibility caused by cold chain transportation and storage conditions of biological drugs. Therefore, it is necessary to develop new dosage forms to achieve convenient administration of GLP-1 receptor agonists and long-term stable storage at room temperature.

Compared with other transdermal delivery methods, microneedles are a new transdermal delivery technology. After applying it to the skin, it can break through the stratum corneum-skin barrier and penetrate the epidermis only in a minimally invasive manner without damaging the neurons in the dermis, thereby minimizing the pain associated with transdermal administration, showing improved skin permeability and enhancing transdermal administration. This can overcome the discomfort, needle phobia and side effects that traditional injections bring to patients, while being painless and non-invasive, making administration simple and convenient, and improving patient compliance. With the improvement of microneedle preparation technology, microneedle technology has achieved remarkable results in the field of medicine. The development of microneedle technology is conducive to the realization of cold chain transportation and storage of temperature-sensitive drugs, which can greatly reduce transportation costs.

[Corrected 26.09.2023 in accordance with Article 91]
Currently, most GLP-1 receptor agonist drug preparations on the market are injections. Novo Nordisk has an oral semaglutide product, which has a relatively low bioavailability of only 0.5-1%, so the active ingredients required are much higher than those of injection preparations, and the price is relatively high. Due to stability issues, most existing GLP-1 receptor agonist drug injection preparations need to be stored at low temperatures. The existing multi-dose packaging form is difficult to ensure low-temperature storage conditions during transportation, which affects the quality of the drug and further affects the efficacy.

At present, there are literatures that disclose the formulation of dissolving microneedles loaded with exenatide. However, although the long-term storage stability of microneedles was evaluated in these studies, there were differences in stability, so it is necessary to screen and evaluate the matrix materials loaded with biological drugs. Therefore, from the perspective of the stability of biological drugs and the particularity of microneedle dosage forms, it is necessary to conduct customized microneedle dosage form stability studies on GLP-1 receptor agonist drugs.

Summary of the invention

[Corrected 26.09.2023 in accordance with Article 91]
Based on the above facts, the purpose of the present invention is to provide a GLP-1 receptor agonist drug microneedle composition, a microneedle prepared therefrom, and a preparation method and application thereof, so as to solve the problems of high transportation cost, inconvenient administration, and strict storage conditions of GLP-1 receptor agonist drugs.

In order to achieve the above object, the present invention adopts the following technical solutions:

[Corrected 26.09.2023 in accordance with Article 91]
In one aspect, the present invention provides a GLP-1 receptor agonist drug microneedle composition, the composition comprising:

GLP-1 receptor agonist drugs;

A protective agent selected from zinc-containing pharmaceutical compounds; and

Microneedle excipients;

Wherein, the mass ratio of the protective agent to the GLP-1 receptor agonist drug is 0.1:1 to 2:1.

In the technical solution of the present invention, the protective agent in the above-mentioned amount can well improve the stability of GLP-1 receptor agonist drugs (including room temperature and high temperature stability).

Furthermore, the protective agent is selected from one or more of zinc sulfate, zinc chloride, zinc citrate, zinc oxide or zinc gluconate.

Furthermore, the mass ratio of the protective agent to the GLP-1 receptor agonist drug is 0.4:1 to 0.8:1. In this case, the stabilizing effect of the GLP-1 receptor agonist drug is better.

Furthermore, the GLP-1 receptor agonist drug is selected from one or more of exenatide, liraglutide, lixisenatide, semaglutide, dulaglutide or albiglutide.

Furthermore, the microneedle excipient mainly comprises one or more of dextran or polyvinyl alcohol, which can significantly improve the storage stability of GLP-1 receptor agonist drugs in microneedle preparations compared with other excipients.

Furthermore, the mass ratio of the GLP-1 receptor agonist drug to the microneedle excipient is 1:200 to 1:2.

[Corrected 26.09.2023 in accordance with Article 91]
In another aspect, the present invention provides a GLP-1 receptor agonist drug microneedle prepared from a raw material comprising the composition as described above.

Furthermore, the microneedle comprises a substrate and a needle body located on the substrate; wherein at least the needle body portion is prepared from a raw material comprising the composition.

Further, the microneedle is an integrated microneedle or a layered microneedle;

When the microneedle is a layered microneedle, the microneedle excipient of the base portion is polyvinyl alcohol, and the microneedle excipient of the needle body portion is dextran.

[Corrected 26.09.2023 in accordance with Article 91]
In another aspect, the present invention provides a method for preparing the GLP-1 receptor agonist drug microneedle as described above, comprising the following steps:

preparing an aqueous solution comprising a GLP-1 receptor agonist drug, a protective agent and a microneedle excipient;

[Corrected 26.09.2023 in accordance with Article 91]
The aqueous solution is placed in a microneedle mold and dried to obtain the GLP-1 receptor agonist drug microneedle.

Furthermore, in the aqueous solution, the solid content of the microneedle excipient is 5% to 40%.

[Corrected 26.09.2023 in accordance with Article 91]
In another aspect, the present invention provides a microneedle patch comprising the GLP-1 receptor agonist drug microneedles as described above.

Furthermore, the microneedle patch also includes a backing.

The beneficial effects of the present invention are as follows:

The microneedles provided by the present invention can basically realize the room temperature transportation and storage of GLP-1 receptor agonist drug microneedles, greatly reducing the use cost of such drug preparations. And can be widely used in different transdermal drug delivery preparations, including but not limited to the preparation of integrated microneedles and layered microneedles.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific implementation modes of the present invention will be further described in detail below in conjunction with the accompanying drawings.

FIG. 1 shows a microscope image of the integrated microneedle loaded with exenatide in Example 1.

FIG2 and FIG3 respectively show the process of preparing the layered dissolving microneedles and the side view microscope image of the microneedle tip loaded with FITC-EXT (FITC-labeled exenatide) after successful preparation.

FIG. 4 shows the cumulative drug release diagrams of Examples 28 to 31, in which different drug release rates are caused by different Dex-40 contents at the needle tip.

FIG. 5 shows the cumulative drug release diagram of Examples 30, 32 to 34, in which different drug release rates are caused by different PVA contents in the substrate.

Detailed ways

In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

Example 1

Exenatide integrated microneedle

The integrated microneedles of Exenatide were prepared as follows:

(1) Preparation of microneedle matrix solution: Prepare a microneedle matrix solution with a solid content of 40% dextran-40 (Dex-40) and a solid content of 0.2% exenatide. The ratio of dextran-40 to exenatide is 200:1. Use a pipette to add 0.8 mL of ultrapure water to a centrifuge tube, then use a pipette to transfer 0.4 mL of exenatide solution (10 mg/mL stock solution concentration) to the centrifuge tube and mix well. Weigh 0.8 g of Dex-40 and add it to the centrifuge tube, stirring until completely dissolved. Centrifuge at 4°C to remove bubbles to obtain a microneedle preparation solution.

(2) Preparation of microneedles: 50 μL of the microneedle preparation solution obtained above was pipetted with a liquid adding gun and dripped onto the PDMS microneedle mold. The mold was evacuated under negative pressure for 10 min, and the microneedles were dried at room temperature and then demolded. The stereomicroscope image of the obtained integrated microneedles loaded with exenatide is shown in Figure 1. The theoretical drug content of the entire microneedle is 100 μg.

(3) Drug content detection in the 4th week of the accelerated experiment: The microneedle patch was packaged in blisters and aluminum-plastic bags and stored in a 40°C/75% RH environment for 4 weeks. The exenatide content in the integrated microneedle at day 0 and week 4 was determined using high performance liquid chromatography.

The results showed that the remaining percentage of the exenatide microneedles after being placed at 40°C/75% RH for 4 weeks was 97.93% (the ratio of the actual exenatide content measured at 4 weeks to 0 days).

Comparative Examples 1 to 8, Examples 2 to 3

Different microneedle excipients were selected to prepare integrated microneedles loaded with exenatide.

Table 1 lists the integrated dissolving microneedles prepared by the microneedle excipients used by the inventors in Comparative Examples 1 to 8 and Examples 2 to 3. The microneedle excipients in the comparative examples include sodium hyaluronate 20,000 molecular weight (HA-2), sodium hyaluronate 80,000 molecular weight (HA-8), sodium carboxymethyl cellulose (CMC), sodium alginate (SA), polyvinyl pyrrolidone (PVP K90), polyvinyl pyrrolidone (PVP K30), hydroxypropyl methylcellulose (HPMC) and ethyl cellulose (EC). The microneedle excipients in the examples include polyvinyl alcohol (PVA) and dextran-40 (Dex-40). The preparation methods of the integrated dissolving microneedles in the comparative examples and the examples are all implemented according to Example 1. The solid content of the microneedle excipients and the solid content of exenatide contained are prepared according to the ratio in Table 1, and the content of exenatide in the microneedles is detected according to the same method as in Example 1, and the remaining percentage is calculated. The results are shown in Table 1.

Table 1 Microneedle prescriptions and experimental results of Comparative Examples 1 to 8 and Examples 2 to 3

The above results show that the integrated microneedles prepared using different matrix materials in Comparative Examples 1 to 8 cannot maintain the activity of exenatide well, while polyvinyl alcohol and dextran can maintain the activity of exenatide well.

Comparative Examples 9 to 21, Example 4

Integrated microneedles prepared by adding different protective agents.

Prepare the integrated microneedles containing protective agents according to the following steps:

(1) Preparation of integrated microneedle matrix solution: First, directly prepare 35 mL of HA-2 solution containing exenatide, with a solid content of 20% for HA-2 and 0.2% for exenatide; weigh 28 g of ultrapure water in a centrifuge tube, add the weighed 0.07 g of exenatide to the centrifuge tube to dissolve, then add the weighed 7 g of HA-2, stir until completely dissolved, centrifuge at 4°C to remove bubbles, and set aside. The prepared microneedle matrix solution was dispensed into 15 centrifuge tubes, with 2 g in each centrifuge tube, and then according to Table 2 below, the weighed protective agent was added to the centrifuge tubes, stirred evenly, and centrifuged at 4°C to remove bubbles to obtain the microneedle preparation solution.

(2) Preparation of microneedles: 50 μL of the microneedle preparation solution obtained above was pipetted with a liquid adding gun and added dropwise to the PDMS microneedle mold. The mold was vacuumed for 10 min, and the microneedles were dried at room temperature and then demolded. The theoretical drug content of the entire microneedle was 100 μg.

(3) Drug content detection at the 8th week of the accelerated experiment: The microneedle patch was packaged in blisters and aluminum-plastic bags and stored at 50°C for 8 weeks. The exenatide content in the integrated microneedles at day 0 and week 8 was determined using high performance liquid chromatography.

Table 2 Prescriptions and experimental results of microneedles containing protective agents in comparative examples 9 to 21 and example 4

As can be seen from the results in Table 2 above, in the storage condition of 50°C for 8 weeks in the accelerated experiment, Comparative Example 9 is shown as a control without adding any protective agent. The integrated microneedles prepared by adding different proportions of different protective agents in Comparative Examples 10 to 21 did not improve the activity of exenatide during storage, and even reduced the activity of exenatide. The addition of trehalose and sucrose in Comparative Examples 10 to 15 had almost no effect on the stability of exenatide during the storage of microneedles, but when the content was added in large amounts, the stability of exenatide would be reduced; in Comparative Examples 16 to 21, as the content of PVP C17 and mannitol added gradually increased, the stability of exenatide during the storage of microneedles would gradually decrease, and only a very small amount of addition would have little effect on the stability of exenatide. In Example 4, the addition of zinc sulfate can significantly improve the stability of exenatide during high-temperature storage of microneedles. This is because it reacts with exenatide to increase the stability of exenatide during the storage of microneedles, but as the content of zinc sulfate increases, the recovery rate will decrease accordingly. The test results of the zinc sulfate to exenatide content ratio of 5:1 are not presented here because the exenatide cannot be completely released due to the complexation reaction.

Comparative Examples 22 to 25, Example 5

Dextran integrated microneedles prepared by adding different divalent metal salts.

The integrated microneedles containing divalent metal salts were prepared according to the following steps:

(4) Preparation of integrated microneedle matrix solution: The solid content of dextran is 30%, the solid content of exenatide is 0.3%, and the solid content of divalent metal salt is 0.3%; first, 36 mg of exenatide is weighed into a centrifuge tube, and 3 mL of ultrapure water is added thereto to dissolve it, to obtain a 12 mg/mL exenatide stock solution; then, 6 mg of calcium chloride, magnesium chloride, copper chloride, and zinc chloride are weighed into a centrifuge tube, and 0.9 mL of ultrapure water is added thereto, and then 0.5 mL of exenatide stock solution is added thereto, and then 0.6 g of dextran is added, and the mixture is stirred until completely dissolved, and centrifuged at 4°C to remove bubbles, to obtain a microneedle preparation solution. The control was made by not adding divalent metal salt.

(5) Preparation of microneedles: 50 μL of the microneedle preparation solution obtained above was pipetted with a liquid adding gun and added dropwise to the PDMS microneedle mold. The mold was vacuumed for 10 min, and the microneedles were dried at room temperature and then demolded. The theoretical drug content of the entire microneedle was 150 μg.

(6) Drug content detection in the first and second weeks of the accelerated experiment: The microneedle patch was packaged in blisters and aluminum-plastic bags and stored in a 50°C environment for 2 weeks. The exenatide content in the integrated microneedles on day 0 and week 1 and week 2 was determined by high performance liquid chromatography, and the remaining percentage (ratio of the exenatide content actually measured in week 1 or 2 to that on day 0) was calculated.

Table 3 Comparative Examples 22-25 and Example 5 Microneedle Prescriptions and Experimental Results Containing Divalent Metal Salts

From the results in Table 3 above, it can be seen that the integrated dextran microneedles are stored under the accelerated test conditions of 50°C for 1 week and 2 weeks. Comparative Example 22 is shown as a control without the addition of any divalent metal salts. The integrated microneedles prepared by adding calcium chloride in Comparative Example 23 slightly reduce the activity of exenatide during storage. The magnesium chloride added in Comparative Example 24 has little effect on the activity of EXT. The addition of copper chloride in Comparative Example 25 will rapidly reduce the stability of exenatide. In Example 5, the addition of zinc chloride can improve the stability of exenatide when the microneedles are stored at high temperatures.

Embodiments 6 to 19

Integrated microneedles prepared by adding zinc sulfate in different proportions.

The integrated microneedles containing different proportions of zinc sulfate were prepared according to the following steps:

(1) Preparation of integrated microneedle matrix solution:

Directly prepare 20 mL of PVA solution with a solid content of 20%: weigh 16 g of ultrapure water into a centrifuge tube, add 4 g of PVA into the centrifuge tube, place it in an 80°C oven to heat and swell, stir every half an hour until completely dissolved, centrifuge to remove bubbles, and set aside.

Directly prepare 30 mL of Dex-40 solution with a solid content of 30%: weigh 21 g of ultrapure water into a centrifuge tube, add 9 g of Dex-40 into the centrifuge tube, stir until completely dissolved, centrifuge to remove bubbles, and set aside.

Directly prepare 2 mL of exenatide solution with a concentration of 0.06 g/mL: weigh 0.12 g of exenatide into a centrifuge tube, add 1.88 mL of ultrapure water with a pipette and shake to dissolve, centrifuge at 4°C to remove bubbles, and set aside.

The prepared PVA matrix solution was dispensed into 5 centrifuge tubes, with 2.9 g in each centrifuge tube. 100 μL of the prepared exenatide solution was added using a pipette. Then, according to Table 3 below, the corresponding zinc sulfate was added to each centrifuge tube. The mixture was stirred until completely mixed. The mixture was centrifuged at 4°C to remove bubbles to obtain PVA microneedle preparation solutions containing different proportions of zinc sulfate.

The prepared Dex-40 matrix solution was dispensed into 9 centrifuge tubes, with 2.85 g in each centrifuge tube. 150 μL of the prepared exenatide solution was then added using a pipette. According to Table 3 below, the corresponding zinc sulfate was added to each centrifuge tube. The mixture was stirred until completely mixed. The mixture was centrifuged at 4°C to remove bubbles to obtain Dex-40 microneedle preparation solutions containing different proportions of zinc sulfate.

(2) Preparation of microneedles: 50 μL of the microneedle preparation solution obtained above was pipetted with a liquid adding gun and dripped onto the PDMS microneedle mold. The mold was vacuumed for 10 min, and the microneedles were dried at room temperature and then demolded. The theoretical drug content of the whole microneedle prepared by PVA material was 100 μg, and the theoretical drug content of the whole microneedle prepared by Dex-40 material was 150 μg.

(3) Drug content detection on the 5th and 10th days of the accelerated experiment: The microneedle patch was packaged in blisters and aluminum-plastic bags and stored in an environment of 60°C for 10 days. The exenatide content in the integrated microneedles on days 0, 5, and 10 was determined by high performance liquid chromatography.

Table 4: Prescriptions and experimental results of integrated microneedles containing different proportions of zinc sulfate in Examples 6 to 19

It can be seen from the above table that the microneedles prepared by these two matrix materials can keep exenatide stable during high temperature storage, and the stability of Dex-40 material to exenatide is stronger than that of PVA. In the PVA excipient, when the ratio of zinc sulfate to exenatide is in the range of 0.5:1 to 2:1, the protective agent zinc sulfate has better stability for exenatide. In the Dex-40 excipient, when the ratio of zinc sulfate to exenatide is in the range of 0.2:1 to 1:1, the protective agent zinc sulfate has better stability for exenatide; more preferably, when the ratio of zinc sulfate to exenatide is in the range of 0.4:1 to 0.8:1, the protective agent zinc sulfate has better stabilizing effect on exenatide. Therefore, when making layered needles, Dex-40 is selected as the needle tip material for loading exenatide.

Examples 20 to 27

Layered dissolving microneedles of exenatide.

The following steps were followed to prepare the exenatide layered dissolving microneedles:

(1) Preparation of layered microneedle matrix solution:

Preparation of needle tip solution 1: Accurately weigh 0.006 g of exenatide in a centrifuge tube, add 1.4 mL of ultrapure water with a pipette to dissolve it, then add 0.6 g of Dex-40, stir until completely dissolved, centrifuge to remove bubbles, and obtain needle tip solution 1 with a Dex-40 solid content of 30% and an exenatide solid content of 0.3%.

Preparation of needle tip liquid 2: Accurately weigh 0.0018 g of zinc sulfate in a centrifuge tube, add 1 g of the prepared needle tip liquid 1 to the centrifuge tube, stir until completely dissolved, centrifuge to remove bubbles, and obtain a needle tip liquid 2 with a Dex-40 solid content of 30%, an exenatide solid content of 0.3%, and a zinc sulfate solid content of 0.18% (the ratio of zinc sulfate to exenatide is 0.6:1).

Preparation of base liquid 1: Weigh 10.5g ultrapure water into a centrifuge tube, add 4.5g PVA into the centrifuge tube, place it in an oven at 80℃ to heat and swell, stir every half an hour until it is completely dissolved, centrifuge to remove bubbles, and obtain base liquid 1 with a PVA solid content of 30%.

Preparation of base liquid 2: Accurately weigh 0.009 g of zinc sulfate in a centrifuge tube, add 5 g of the prepared base liquid 1 into the centrifuge tube, stir until completely uniform, centrifuge to remove bubbles, and obtain base liquid 2 with a PVA solid content of 30% and a zinc sulfate solid content of 0.18%.

Preparation of base solution 3: Weigh 4.2 g ultrapure water into a centrifuge tube, add 1.8 g Dex-40 into the centrifuge tube, stir until completely dissolved, centrifuge to remove bubbles, and obtain base solution 3 with a Dex-40 solid content of 30%.

Preparation of base solution 4: Accurately weigh 0.0054 g of zinc sulfate in a centrifuge tube, add 3 g of the prepared base solution 3 into the centrifuge tube, stir until completely uniform, centrifuge to remove bubbles, and obtain base solution 4 with a Dex-40 solid content of 30% and a zinc sulfate solid content of 0.18%.

(2) Preparation of microneedles: Prepare microneedles according to the combination method in Table 4 below. Use a liquid adding gun to take 5 μL of the microneedle tip liquid 1 and the tip liquid 2 obtained above and drop them onto the PDMS microneedle mold unit. After the mold is vacuumed under negative pressure for 5 minutes, the microneedle tip is naturally dried at room temperature for 30 minutes; then 50 μL of the microneedle base liquid 1-4 obtained above is dropped respectively, and the mold is vacuumed under negative pressure for 10 minutes. The microneedles are naturally dried at room temperature and then demolded. The theoretical drug content of the obtained layered microneedles is 15 μg.

(3) Drug content detection on the 5th and 10th days of the accelerated experiment: The microneedle patch was packaged in blisters and aluminum-plastic bags and stored in an environment of 60°C for 10 days. The exenatide content in the layered microneedles on days 0, 5, and 10 was determined by high performance liquid chromatography.

Table 5 Layered dissolving microneedle prescriptions and experimental results of Examples 20 to 27

From Table 5 we can see that:

Result 1: Prescriptions 5 and 6 have the same needle tip solution (Dex-40+Exenatide+Zinc Sulfate), the base solution of Prescription 5 does not contain zinc (PVA), and the base solution of Prescription 6 contains zinc (PVA+Zinc Sulfate). There is no significant difference in the remaining percentage of Exenatide obtained by the two groups, P>0.5. Therefore, after the needle tip solution contains zinc ions, the presence or absence of zinc ions in the base solution without Exenatide has little effect on the stability of Exenatide in the needle tip.

Result 2: For the same needle tip solution of prescriptions 7 and 8 (Dex-40+Exenatide+Zinc Sulfate), the base solution of prescription 7 does not contain zinc (Dex-40), and the base solution of prescription 8 contains zinc (Dex-40+Zinc Sulfate). There is no significant difference in the remaining percentage of Exenatide between the two groups, P>0.5. Therefore, the presence or absence of zinc ions in the base solution without Exenatide has little effect on the stability of Exenatide in the needle tip.

For results 1 and 2, it can also be concluded that Dex-40 as a base material can better stabilize the activity of exenatide in the needle tip than PVA as a base material.

Result 3: Prescriptions 1 and 2 have the same needle tip fluid (Dex-40 + exenatide), the base fluid of prescription 1 does not contain zinc (PVA), and the base fluid of prescription 2 contains zinc (PVA + zinc sulfate). The remaining percentage of exenatide obtained by the two groups is significantly different, P < 0.001. This shows that in the absence of zinc ions in the needle tip fluid, the presence of zinc ions in the base fluid will improve the stability of exenatide in the needle tip to a certain extent.

Result 4: The same prescriptions 3 and 4 had the same needle tip fluid (Dex-40 + exenatide), the base fluid of prescription 3 did not contain zinc (Dex-40), and the base fluid of prescription 4 contained zinc (Dex-40 + zinc sulfate). The remaining percentage of exenatide obtained by the two groups was significantly different, P < 0.01. This still shows that in the absence of zinc ions in the needle tip fluid, the presence of zinc ions in the base fluid will improve the stability of exenatide in the needle tip to a certain extent.

For results 3 and 4, it was also concluded that Dex-40 as a base material could stabilize the activity of exenatide in the needle tip better than PVA as a base material.

A method for preparing FITC-EXT loaded layered dissolving microneedles

(1) The needle tip solution is an aqueous solution containing 10% Dex-40 and 2 mg/ml FITC-EXT;

(2) The base liquid is a 30% PVA aqueous solution;

(3) Preparation of microneedles As shown in Figure 2, 15uL of the above needle tip liquid was aspirated and placed on a microneedle mold (needle height 500μm, needle spacing 500μm) (as shown in step (a) in Figure 2), the mold was vacuumed at negative pressure for 5 minutes, and then the excess solution on the mold surface was removed with an acrylic plate (as shown in step (b) in Figure 2), and naturally dried at room temperature for 30 minutes, and the residual components on the surface were removed with tape (as shown in step (c) in Figure 2) to obtain a needle tip layer; then 40μL of the above-obtained microneedle base liquid was added dropwise (as shown in step (d) in Figure 2), the mold was vacuumed at negative pressure for 10 minutes (as shown in step (e) in Figure 2), and then it was placed in a drying cabinet to dry overnight and then demolded (as shown in step (f) in Figure 2), to obtain a layered dissolved microneedle loaded with FITC-EXT (Figure 3).

Embodiments 28 to 34

Effect of Dex-40 content at the needle tip or PVA content at the base on drug utilization in layered dissolving microneedles.

(1) Preparation of layered dissolution microneedle matrix solution:

Preparation of 10 mg/ml exenatide stock solution: accurately weigh 35 mg of exenatide into a centrifuge tube, add 3.5 mL of ultrapure water with a pipette to dissolve, and shake to dissolve to obtain 10 mg/ml exenatide stock solution.

Preparation of needle tip fluid: weigh 0.1, 0.2, 0.6 and 0.8 g of Dex-40 respectively into a centrifuge tube, add 0.4, 0.3, 0.4 and 0.2 ml of water respectively with a pipette, then add 0.5, 0.5, 1.0 and 1.0 ml of exenatide stock solution, stir until completely dissolved, to obtain the needle tip fluids of Examples 29 to 32. The needle tip fluids of Examples 33 to 35 are the same as those of Example 31.

Preparation of base liquid: weigh 7.5, 7, 6.5 and 18 g of water into centrifuge tubes respectively, then add 2, 2.5, 3 and 12 g of PVA powder respectively. Place in an oven at 80°C to heat and swell. Stir every half hour until completely dissolved. Centrifuge at 5000 rpm for 10 min to remove bubbles to obtain base liquids with PVA solid contents of 25%, 30%, 35% and 40%.

(2) Preparation of microneedles: 15 μL of the microneedle tip liquid obtained above was transferred with a pipette and added dropwise to the PDMS microneedle mold unit. The mold was vacuumed under negative pressure for 5 min, and then the excess solution on the mold surface was removed with an acrylic plate. The mold was naturally dried at room temperature for 30 min, and the residual components on the surface were removed with tape (to improve drug utilization); then 40 μL of the microneedle base liquid obtained above was added dropwise, the mold was vacuumed under negative pressure for 10 min, and then placed in a drying cabinet to dry overnight before demolding.

(3) Determination of drug content in the body and base of microneedles: Use a scalpel to scrape off the body of the microneedle and collect it in a centrifuge tube, and collect the base in a new centrifuge tube at the same time. Add 1 mL of PBS solution with pH = 7.4 to each centrifuge tube, vortex for 1 hour, centrifuge at 10,000 rpm for 10 minutes, and take the supernatant for HPLC analysis; calculate the actual drug content using the standard curve of the day.

(4) Drug utilization rate = drug content in needle tip/(drug content in needle tip + drug content in base) × 100%

Table 6 Formulations and experimental results of layered dissolving microneedles in Examples 28 to 34

From Table 6 we can get:

Result 1: In Examples 28 to 31, when the solid content of the base PVA was the same, the effect of the solid content of Dex-40 in the needle tip solution on the drug utilization rate was investigated. The results showed that with the increase of the Dex-40 content, the drug utilization rate tended to decrease slightly.

Result 2: In Examples 30, 32 to 34, when the solid content of Dex-40 was the same, the effect of the solid content of PVA in the base fluid on the drug utilization was investigated. The results showed that with the increase of the PVA content, the drug utilization was significantly improved, which indicates that the base viscosity has a greater effect on the diffusion of the drug in the needle tip to a certain extent.

In vitro cumulative drug release test

The in vitro cumulative drug release test used SD male rats, and the subtraction method was used to calculate the drug passage rate at different time points = (whole microneedle drug content - residual patch drug content) / whole microneedle drug content. The microneedle patches obtained in Examples 28 to 34 were subjected to in vitro cumulative release tests. One day before the experiment, the abdominal hair of the rats was removed and removed with a depilatory cream. During the experiment, the microneedles of each prescription were pressed on the abdominal skin of the rats for 30 seconds, and the residual patches were removed at 0.5, 1, 2, 5, 10, 60, 240, 480 and 720 minutes, respectively, and collected in a centrifuge tube. 1 mL of PBS solution with pH = 7.4 was added, vortexed for 1 hour, centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken for HPLC analysis of the residual microneedle patch drug content. The resulting in vitro cumulative release curves are shown in Figures 3 and 4. As can be seen from Figure 4, when the Dex-40 content is 10%, the exenatide release rate can reach 63.36±8.84% when the application is 0.5min; and when the Dex-40 content is 40%, the exenatide release rate is only 38.25±3.58% when the application is 0.5min. This is because as the Dex-40 content at the needle tip increases, the height of the needle tip increases when the drug content is the same, and the drug is more dispersed, resulting in a large difference in early release. After 2 minutes of application, the exenatide release rate of these prescriptions basically tends to be flat, and the release difference is small. After the Dex-40 containing a large amount of drug at the needle tip is quickly dissolved, the remaining is the PVA material containing a small amount of drug (due to diffusion) that begins to dissolve. The material dissolves slowly in the skin, so the amount of drug released in the later stage tends to be flat. As can be seen from Figure 5, when the PVA content is 25%, the exenatide release rate is 30.37±10.12% after 0.5min of application; and when the PVA content is 40%, the exenatide release rate can reach 50.94±9.31% after 0.5min of application. Similarly, after 1h of application, the exenatide release rate of these prescriptions tends to increase slowly. This shows that if the PVA content of the substrate is low, it will cause more drug diffusion from the needle tip to the substrate, which will lead to slow release of the drug. When the PVA content increases to more than 35%, the difference in the cumulative release curve of exenatide is not obvious, so the microneedle substrate content should be ensured to be above 35%.

Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

[Corrected 26.09.2023 in accordance with Article 91]
A GLP-1 receptor agonist drug microneedle composition, characterized in that the composition comprises:

GLP-1 receptor agonist drugs;

A protective agent selected from zinc-containing pharmaceutical compounds; and

Microneedle excipients;

Wherein, the mass ratio of the protective agent to the GLP-1 receptor agonist drug is 0.1:1 to 2:1.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle composition according to claim 1, characterized in that the protective agent is selected from one or more of zinc sulfate, zinc chloride, zinc citrate, zinc oxide or zinc gluconate; and/or

The mass ratio of the protective agent to the GLP-1 receptor agonist drug is 0.4:1 to 0.8:1.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle composition according to claim 1, characterized in that the GLP-1 receptor agonist drug is selected from one or more of exenatide, liraglutide, lixisenatide, semaglutide, dulaglutide or albiglutide.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle composition according to claim 1, characterized in that the microneedle excipient mainly comprises one or more of dextran or polyvinyl alcohol.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle composition according to claim 1, characterized in that the mass ratio of the GLP-1 receptor agonist drug to the microneedle excipient is 1:200 to 1:2.
[Corrected 26.09.2023 in accordance with Article 91]
A GLP-1 receptor agonist drug microneedle, characterized in that it is prepared from a raw material comprising the composition according to any one of claims 1 to 5.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle according to claim 6, characterized in that the microneedle comprises a substrate and a needle body located on the substrate; wherein at least the needle body portion is prepared from a raw material comprising the composition.
[Corrected 26.09.2023 in accordance with Article 91]
The GLP-1 receptor agonist drug microneedle according to claim 7, characterized in that the microneedle is an integrated microneedle or a layered microneedle;

When the microneedle is a layered microneedle, the microneedle excipient of the base portion is polyvinyl alcohol, and the microneedle excipient of the needle body portion is dextran.
[Corrected 26.09.2023 in accordance with Article 91]
The method for preparing the GLP-1 receptor agonist drug microneedle according to any one of claims 6 to 8, characterized in that it comprises the following steps:

preparing an aqueous solution comprising a GLP-1 receptor agonist drug, a protective agent and a microneedle excipient;

The aqueous solution is placed in a microneedle mold and dried to obtain the GLP-1 receptor agonist drug microneedle.
[Corrected 26.09.2023 in accordance with Article 91]
A microneedle patch, characterized in that it comprises the GLP-1 receptor agonist drug microneedles as described in any one of claims 6 to 8.