Preparation method and application of andrographolide drug soluble microneedle

Abstract

The invention provides a preparation method and application of an andrographolide drug soluble microneedle, and the preparation method comprises the following steps: dissolving an andrographolide drug, a first matrix material PVP and a matrix material PVA in purified water, uniformly mixing and swelling to obtain a needle solution; dissolving a second matrix material PVP (Polyvinyl Pyrrolidone) and glycerol in purified water, uniformly mixing and swelling to obtain a matrix solution; and putting the needle head solution and the matrix solution into a mold to form the andrographolide drug soluble microneedle. An andrographolide drug is prepared into a needle solution, a matrix solution is prepared, the andrographolide drug soluble microneedle is formed, and experiments show that the andrographolide drug soluble microneedle has good biological safety and can effectively improve the penetrating ability to a cuticle barrier, namely, the drug transdermal absorption ability of the andrographolide drug can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to a preparation method and application of andrographolide-based drug soluble microneedles. Background Art

[0002] Andrographolide (ADR) is a natural diterpenoid lactone extract extracted from the traditional Chinese medicine Andrographis paniculata, which has a variety of pharmacological activities and broad clinical application prospects. However, ADR has poor solubility, the presence of P-gp efflux transport in the intestine, low membrane permeability, and poor gastrointestinal stability, resulting in low oral bioavailability. Therefore, clinically, the drug is mainly in the form of an injection of its similar components.

[0003] However, andrographolide-based component injections often cause adverse reactions such as rashes, dizziness, gastrointestinal reactions, and anaphylactic shock during clinical use, which are greatly restricted in clinical use.

[0004] Transdermal drug delivery can avoid first-pass metabolism, prevent drug degradation in the gastrointestinal tract, reduce toxicity and improve treatment efficiency, and maintain a stable drug level in the plasma. However, for andrographolide-based component injections, the stratum corneum barrier limits their transdermal absorption ability of drugs for transdermal drug delivery. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method and application of andrographolide-based drug soluble microneedles, aiming to solve the technical problem that for andrographolide-based component injections in the prior art, the stratum corneum barrier limits their transdermal absorption ability of drugs for transdermal drug delivery.

[0006] To achieve the above purpose, in the first aspect, the present invention provides a preparation method of andrographolide-based drug soluble microneedles, including the following steps:

[0007] Dissolve andrographolide-based drugs, the first matrix material PVP, and the matrix material PVA in purified water and mix them evenly to swell, so as to obtain a needle solution;

[0008] Dissolve the second matrix material PVP and glycerol in purified water and mix them evenly to swell, so as to obtain a matrix solution;

[0009] Place the needle solution and the matrix solution in a mold to form andrographolide-based drug soluble microneedles.

[0010] Further, the andrographolide-based drugs are andrographolide, dehydroandrographolide, sodium bisulfite andrographolide, andrographolide sulfonate, potassium sodium salt of dehydroandrographolide succinic acid half ester, and monopotassium salt of dehydroandrographolide succinic acid half ester.

[0011] Furthermore, the first matrix material PVP is PVP K30, PVP K60 or PVP K90.

[0012] Furthermore, the step of placing the needle solution and the matrix solution in a mold to form the soluble micro-needles of andrographolide drugs includes:

[0013] Pour the needle solution onto the mold, perform a first centrifugation treatment on the mold to enable the needle solution to fill the inside of the mold, remove the needle solution on the surface of the mold, and perform a first drying treatment to obtain a first-stage mold;

[0014] Pour the matrix solution onto the first-stage mold, perform a second centrifugation treatment on the first-stage mold to enable the matrix solution to enter the inside of the first-stage mold, remove the matrix solution on the surface of the first-stage mold, and perform a second drying treatment to obtain a second-stage mold;

[0015] Perform a demolding treatment on the second-stage mold to obtain the soluble micro-needles of andrographolide drugs.

[0016] Furthermore, the time of both the first centrifugation treatment and the second centrifugation treatment is 10 min, and the centrifugation speed of both the first centrifugation treatment and the second centrifugation treatment is greater than 5000 rpm.

[0017] Furthermore, the time of the first drying treatment is 0.5 h to 4 h, the time of the second drying treatment is 0.5 days to 2 days, and the temperature of both the first drying treatment and the second drying treatment is 25 °C.

[0018] Furthermore, the mixing ratio of the andrographolide drugs, the first matrix material PVP, the matrix material PVA and purified water is: 1:3:1:10, and the ratio of the second matrix material PVP, glycerol and purified water is: 3:0.03:5 to 3:0.3:5.

[0019] In a second aspect, the present invention provides an application of the soluble micro-needles of andrographolide drugs prepared by the method for preparing soluble micro-needles of andrographolide drugs as described in the first aspect above in improving the transdermal absorption rate.

[0020] In a third aspect, the present invention provides an application of the soluble micro-needles of andrographolide drugs prepared by the method for preparing soluble micro-needles of andrographolide drugs as described in the first aspect above in improving the bioavailability.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: By preparing the andrographolide drug into the needle solution and formulating the matrix solution to form the andrographolide soluble microneedles, experiments show that they have good biosafety and can effectively improve the penetration ability of the stratum corneum barrier, that is, can improve the transdermal absorption ability of andrographolide drugs. Description of the Drawings

[0022] Figure 1 It is a flowchart of the preparation method of andrographolide soluble microneedles in the embodiments of the present invention;

[0023] Figure 2 It is an optical microscope image of KDS-DMN in the embodiments of the present invention;

[0024] Figure 3 It is a scanning electron microscope image of KDS-DMN at different magnifications in the embodiments of the present invention, where Figure 3 A is the overall image, Figure 3 B is the partial image, Figure 3 C is the single needle image;

[0025] Figure 4 It is the membrane puncture rate of ADR-DMN, DAG-DMN, ASB-DMN, AS-DMN, KDS-DMN and PSD-DMN in each layer in the embodiments of the present invention (n = 3); Membrane puncture rate (n = 3);

[0026] Figure 5 It is the in vitro transdermal absorption results of different drug entities in the embodiments of the present invention (n = 3);

[0027] Figure 6 It is the in vitro transdermal absorption results of different ADR preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0028] Figure 7 It is the in vitro transdermal absorption results of different DAG preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0029] Figure 8 It is the in vitro transdermal absorption results of different ASB preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0030] Figure 9 It is the in vitro transdermal absorption results of different AS preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0031] Figure 10 It is the in vitro transdermal absorption results of different KDS preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0032] Figure 11 In vitro transdermal absorption results of different PSD preparations in the embodiments of the present invention (n = 3, * indicates p < 0.05);

[0033] Figure 12 Images of the application of soluble microneedles to pig skin at different times in the embodiments of the present invention;

[0034] Figure 13 Images of the application of soluble microneedles to the skin of live SD rats at different times in the embodiments of the present invention;

[0035] Figure 14 Images of the application of blank microneedles to healthy human skin at different times at different magnifications in the embodiments of the present invention, where Figure 14 A is the overall view, Figure 14 B is the partial view;

[0036] Figure 15 Accumulation of ASB-DMN in different skins in the embodiments of the present invention (n = 3);

[0037] Figure 16 Plasma concentration-time curves of ABS-MN, ABS-Gel and 2% Lau ABS-Gel in rats in the embodiments of the present invention (n = 7);

[0038] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0039] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0040] It should be noted that when an element is referred to as being "fixed to" another element, it can be directly on the other element or there can also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] Please refer to Figure 1 , in a first aspect, Embodiment 1 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, comprising the following steps:

[0043] S10: Dissolve andrographolide drugs, the first matrix material PVP, and the matrix material PVA in purified water and mix them evenly to swell, so as to obtain a needle solution;

[0044] The andrographolide drugs are andrographolide (ADR), and the first matrix material PVP (polyvinylpyrrolidone) is PVP K30. It should be noted that the andrographolide drugs, the first matrix material PVP, and the matrix material PVA (polyvinyl alcohol) need to swell sufficiently in purified water to form the needle solution.

[0045] The mixing ratio of the andrographolide drugs, the first matrix material PVP, the matrix material PVA, and purified water is: 1:3:1:10.

[0046] S20: Dissolve the second matrix material PVP and glycerol in purified water and mix them evenly to swell, so as to obtain a matrix solution;

[0047] The ratio of the second matrix material PVP, the glycerol, and purified water is: 3:0.03:5. It can be understood that the second matrix material PVP and the glycerol need to swell sufficiently in purified water to form the needle solution. In this embodiment, the second matrix material PVP is PVP K90.

[0048] S30: Place the needle solution and the matrix solution in a mold to form soluble microneedles of andrographolide drugs;

[0049] In this embodiment, the specification of the mold is a cylinder with a groove in the middle part. The height of the cylinder is 8 mm and the diameter is 24 mm. The groove is evenly distributed with 16*16 conical needle cavities with a diameter of 0.34 mm and a height of 0.8 mm. The distance between two conical needle cavities is 0.55 mm.

[0050] The step S30 includes:

[0051] S310: Pour the needle solution onto the mold, perform a first centrifugation treatment on the mold to enable the needle solution to fill the interior of the mold, remove the needle solution on the surface of the mold, and perform a first drying treatment to obtain a first-stage mold;

[0052] The time of the first centrifugation treatment is 10 min, and the centrifugation speed of the first centrifugation treatment is 5000 rpm. Since the specifications of the groove are small and there are smaller conical needle cavities therein, when pouring the needle solution, it is difficult for the needle solution to enter the conical needle cavity under normal conditions, thus making it difficult to form microneedles subsequently. Therefore, through the first centrifugation treatment, the needle solution can quickly fill the conical needle cavity. The time of the first drying treatment is 0.5 h to 4 h, and the temperature of the first drying treatment is 25 °C.

[0053] S320: Pour the matrix solution onto the first-stage mold, perform a second centrifugation treatment on the first-stage mold to enable the matrix solution to enter the interior of the first-stage mold, remove the matrix solution on the surface of the first-stage mold, and perform a second drying treatment to obtain a second-stage mold;

[0054] After pouring the matrix solution, the function of the second centrifugation treatment is the same as that of the first centrifugation treatment, and details will not be elaborated here. The centrifugation speed of the second centrifugation treatment is 5000 rpm, and the time of the second centrifugation treatment is 10 min. In this embodiment, the temperature of the second drying treatment is 25 °C, and the time of the second drying treatment is 0.5 days.

[0055] S330: Perform a demolding treatment on the second-stage mold to obtain andrographolide-class drug soluble microneedles.

[0056] It can be understood that the average height of the obtained andrographolide-class drug soluble microneedles is 800 μm, and their length can deliver the drug to the upper part of the dermis, while avoiding irritating the dermal nerves and not causing bleeding.

[0057] To explore the quality of the microneedles, taking the integrity of the microneedle needles, the integrity of the base, and the mechanical properties, which are closely related to penetrating the stratum corneum barrier, as evaluation indicators, a scoring standard for soluble microneedles is constructed, and the standard is shown in Table 1:

[0058] Table 1

[0059]

[0060] Embodiment 2 of the present invention provides a method for preparing andrographolide-class drug soluble microneedles, which is different from the method for preparing andrographolide-class drug soluble microneedles described in Embodiment 1 in that:

[0061] The first matrix material PVP (polyvinylpyrrolidone) is PVP K60.

[0062] Example 3 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0063] The first matrix material PVP (polyvinylpyrrolidone) is PVP K90.

[0064] Example 4 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0065] The ratio of the third matrix material, the glycerol and the purified water is: 3:0.3:5.

[0066] Comparative Example 1 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0067] The first matrix material PVP (polyvinylpyrrolidone) is PVP K120.

[0068] Comparative Example 2 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0069] The mixing ratio of the andrographolide drug, the first matrix material PVP, the matrix material PVA and the purified water is: 1:2:1:10.

[0070] Comparative Example 3 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0071] The ratio of the second matrix material PVP, the glycerol and the purified water is: 3:0.01:5.

[0072] Comparative Example 4 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0073] The andrographolide drug and the first matrix material PVP are both dissolved in purified water and mixed and swollen to obtain the needle solution. That is, in this comparative example, when preparing the needle solution, the matrix material PVA is not added.

[0074] Comparative Example 5 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0075] Dissolve the andrographolide drug and the matrix material PVA in purified water and mix well to swell, so as to obtain the needle solution. That is, in this comparative example, when preparing the needle solution, the first matrix material PVP is not added.

[0076] Comparative Example 6 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0077] Dissolve the second matrix material PVP in purified water and mix well to swell, so as to obtain the matrix solution. That is, in this comparative example, when preparing the matrix solution, glycerol is not added.

[0078] Prepare soluble microneedles of andrographolide drugs according to the methods for preparing soluble microneedles of andrographolide drugs described in Examples 1 to 4 and Comparative Examples 1 to 6 of the present invention, and conduct quality inspection on the prepared soluble microneedles of andrographolide drugs. The inspection results are shown in Table 2:

[0079] Table 2

[0080] Specific gravity needle-containing solution (W / V) Specific gravity matrix-containing solution (W / V) Needle integrity Substrate integrity Mechanical properties Example 1 30% PVP K30 + 10% PVA 60% PVP K90 + 0.6% glycerol 3 3 3 Example 2 30% PVP K60 + 10% PVA 60% PVP K90 + 0.6% glycerol 3 3 3 Example 3 30% PVP K90 + 10% PVA 60% PVP K90 + 0.6% glycerol 3 3 3 Example 4 30% PVP K30 + 10% PVA 60% PVP K90 + 6% glycerol 3 3 3 Comparative Example 1 30% PVP K120 + 10% PVA 60% PVP K90 + 0.6% glycerol 2 3 2 Comparative Example 2 20% PVP K30 + 10% PVA 60% PVP K90 + 0.6% glycerol 2 3 2 Comparative Example 3 30% PVP K30 + 10% PVA 60% PVP K90 + 0.1% glycerol 3 2 2 Comparative Example 4 10% PVA 60% PVP K90 + 0.6% glycerol 2 1 2 Comparative Example 5 30% PVP K30 60% PVP K90 + 0.6% glycerol 2 2 1 Comparative Example 6 30% PVP K30 + 10% PVA 60% PVP K90 2 1 1

[0081] It should be noted that in W / V in the table, W represents the weight of the materials used, and V represents the amount of purified water used. As can be seen from the above table, when using only the first matrix material PVP or the matrix material PVA to form the needle solution, without increasing the dosage, the integrity and mechanical properties of the formed needles are relatively poor. By combining the two, the dosage of excipients can be effectively reduced, the drug loading capacity in the microneedles can be increased, and at the same time, the integrity and mechanical properties of the needles can be improved; in the part of the matrix solution, the lack of glycerol will lead to a decline in the forming effect, a large number of bubbles in the formed substrate, and affect the subsequent mechanical properties; by controlling the overall component ratio, the production cost can be saved on the premise of ensuring the optimal needle integrity, substrate integrity and mechanical properties.

[0082] Example 5 of the present invention provides a method for preparing soluble microneedles of andrographolide drugs, which is different from the method for preparing soluble microneedles of andrographolide drugs described in Example 1 in that:

[0083] The time of the second drying treatment is 2 days.

[0084] Example 6 of the present invention provides a method for preparing soluble micro - needles of andrographolide drugs, which is different from the method for preparing soluble micro - needles of andrographolide drugs described in Example 1 in that:

[0085] The time of the second drying treatment is 1.5 days.

[0086] Comparative Example 7 of the present invention provides a method for preparing soluble micro - needles of andrographolide drugs, which is different from the method for preparing soluble micro - needles of andrographolide drugs described in Example 1 in that:

[0087] The time of the second drying treatment is 0.2 days.

[0088] Comparative Example 8 of the present invention provides a method for preparing soluble micro - needles of andrographolide drugs, which is different from the method for preparing soluble micro - needles of andrographolide drugs described in Example 1 in that:

[0089] The temperature of the second drying treatment is 40 °C.

[0090] Prepare soluble micro - needles of andrographolide drugs according to the method for preparing soluble micro - needles of andrographolide drugs described in Example 1, Example 5, Example 6 and Comparative Examples 7 - 8 of the present invention, and conduct quality inspection on the prepared soluble micro - needles of andrographolide drugs. The inspection results are shown in Table 3:

[0091] Table 3

[0092]

[0093] As can be seen from the above table, when the temperature of the second drying treatment is relatively high, it is easy to cause the needle tip to break, thereby greatly affecting the integrity of the needle tip, the integrity of the substrate and the mechanical properties. When the drying time does not reach the standard, it also has a certain impact on the overall quality.

[0094] Example 7 of the present invention provides a method for preparing soluble micro - needles of andrographolide drugs, which is different from the method for preparing soluble micro - needles of andrographolide drugs described in Example 1 in that:

[0095] The centrifugation speeds of the first centrifugation treatment and the second centrifugation treatment are 6000 rpm.

[0096] Comparative Example 9 of the present invention provides a method for preparing soluble micro - needles of andrographolide drugs, which is different from the method for preparing soluble micro - needles of andrographolide drugs described in Example 1 in that:

[0097] The centrifugation speeds of the first centrifugation treatment and the second centrifugation treatment are 3000 rpm.

[0098] The soluble micro - needles of andrographolide - type drugs were prepared by the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1, Example 7 and Comparative Example 9 of the present invention, and the quality of the prepared soluble micro - needles of andrographolide - type drugs was detected. The detection results are shown in Table 4 as follows:

[0099] Table 4

[0100]

[0101] As can be seen from the above table, when the rotation speed is greater than 5000 rpm, the integrity and mechanical properties of the soluble micro - needles are the best.

[0102] Example 8 of the present invention provides a method for preparing soluble micro - needles of andrographolide - type drugs, which is different from the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1 in that:

[0103] The andrographolide - type drug is dehydrated andrographolide (DAG).

[0104] Example 9 of the present invention provides a method for preparing soluble micro - needles of andrographolide - type drugs, which is different from the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1 in that:

[0105] The andrographolide - type drug is andrographolide sodium bisulfite (ASB).

[0106] Example 10 of the present invention provides a method for preparing soluble micro - needles of andrographolide - type drugs, which is different from the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1 in that:

[0107] The andrographolide - type drug is andrographolide sulfonate (AS).

[0108] Example 11 of the present invention provides a method for preparing soluble micro - needles of andrographolide - type drugs, which is different from the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1 in that:

[0109] The andrographolide - type drug is potassium sodium half - succinate of dehydrated andrographolide (PSD).

[0110] Example 12 of the present invention provides a method for preparing soluble micro - needles of andrographolide - type drugs, which is different from the method for preparing soluble micro - needles of andrographolide - type drugs described in Example 1 in that:

[0111] The andrographolide - type drug is monopotassium salt of half - succinate of dehydrated andrographolide (KDS).

[0112] The andrographolide - type drug soluble microneedles prepared by the preparation method of andrographolide - type drug soluble microneedles described in Embodiment 1, Embodiments 8 - 12 of the present invention are subjected to corresponding experiments to detect their effects. Please refer to Figure 2 and Figure 3 , taking KDS - DMN as an example, its overall morphology is complete, the microneedle body is pyramid - shaped, evenly distributed on the substrate, and the needle tip surface is smooth without defects.

[0113] Please refer to Figure 4 , the andrographolide - type drug soluble microneedles prepared in 6 embodiments all have good mechanical properties. They can not only penetrate a 4 - layer - thick sealing film, but also partially penetrate a 5 - layer one. Data shows that about 20% of the dissolved microneedles can reach a depth of 756 μm. Therefore, the dissolved microneedles can all penetrate the skin to transport the loaded drug to the deep layer of the skin without touching the capillaries under the dermis layer, thus causing no pain.

[0114] Please refer to Figure 5 , in vitro transdermal absorption of andrographolide - type drug raw materials: The in vitro transdermal release experiment uses the Franz transdermal diffusion method. The diameter of the connecting channel between the supply chamber and the receiving chamber of the diffusion cell is 0.75 cm. It is divided into 6 groups: ADR suspension group, DAG suspension group, ASB solution group, AS solution group, KDS solution group, and PSD solution group. The skin of SD rats is pre - hydrated with PBS for 30 min, transferred to filter paper to absorb moisture, and cut into appropriate sizes. The skin is directly fixed on the receiving cell of the transdermal cup equipped with a magnetic stirrer. The transdermal cup receiving cell is pre - filled with 15 ml of transdermal receiving solution. The diffusion cell is covered and clamped with a clip to make the receiving solution in full contact with the skin without air bubbles in the middle. Then, 200 μL of each group of solution or suspension is dropped on the stratum corneum side. A sealing film is covered on the supply chamber to prevent volatilization. The receiving medium is 15 mL of PBS solution containing 1% Tween 80. The temperature is controlled at 37 ± 0.2, and the receiving chamber is stirred at a speed of 280 rpm. 200 μL of the receiving solution is taken through the sampling port at 1, 2, 3, 4, 6, 8, 12, 24 h respectively, and an equal amount of receiving medium is immediately added to keep the volume stable. All groups are carried out in parallel 3 times. The amount - time curve of the cumulative permeation of each drug through the skin of SD rats is plotted. The permeation rate of all groups increases with time, but the amplitude is very small, and the cumulative permeation rate within 24 h is less than 15%. Among them, the KDS and ASB groups have the highest permeation rate at 24 h, both being 12.12%, and the PSD group is the lowest, with a 24 - h permeation rate of only 1.67%, hardly permeating through the skin. The 24 - h permeation rates of the DAG, AS, and ADR groups are 3.72%, 3.91%, and 5.77% respectively. The permeation rates of the 6 groups of drug raw materials are all very low, indicating that the percutaneous drug delivery permeation amounts of these six drugs are very poor in the conventional way.

[0115] Please refer to Figures 6 to 11In vitro transdermal absorption of andrographolide drugs in soluble microneedles: The in vitro transdermal absorption of andrographolide drugs in soluble microneedles in 6 examples was studied; meanwhile, the differences in drug transdermal absorption between commercially available solid microneedles and the soluble microneedles of the present invention were also compared.

[0116] a. In vitro transdermal absorption study of ADR preparations: It was divided into 4 groups: ADR solution (ADR), ADR microneedle tip solution (ADR-Oin), solid microneedle + ADR microneedle tip solution (ADR-SDMN), and ADR soluble microneedle (ADR-DMN). According to the Franz transdermal diffusion method, the amount-time curve of ADR accumulated through the skin of SD rats was obtained.

[0117] b. In vitro transdermal absorption study of DAG: It was divided into 4 groups: DAG solution (DAG), DAG microneedle tip solution (DAG-Oin), solid microneedle + DAG microneedle tip solution (DAG-SDMN), and DAG soluble microneedle (DAG-DMN). According to the Franz transdermal diffusion method, the amount-time curve of DAG accumulated through the skin of SD rats was obtained.

[0118] c. In vitro transdermal absorption study of ASB: It was divided into 4 groups: ASB solution (ASB), ASB microneedle tip solution (ASB-Oin), solid microneedle + ASB microneedle tip solution (ASB-SDMN), and ASB soluble microneedle (ASB-DMN). According to the Franz transdermal diffusion method, the amount-time curve of ASB accumulated through the skin of SD rats was obtained.

[0119] d. In vitro transdermal absorption study of AS: It was divided into 4 groups: AS solution (AS), AS microneedle tip solution (AS-Oin), solid microneedle + AS microneedle tip solution (AS-SDMN), and AS soluble microneedle (AS-DMN). According to the Franz transdermal diffusion method, the amount-time curve of AS accumulated through the skin of SD rats was obtained.

[0120] e. In vitro transdermal absorption study of KDS: It was divided into 4 groups: KDS solution (KDS), KDS microneedle tip solution (KDS-Oin), solid microneedle + KDS microneedle tip solution (KDS-SDMN), and KDS soluble microneedle (KDS-DMN). According to the Franz transdermal diffusion method, the amount-time curve of KDS accumulated through the skin of SD rats was obtained.

[0121] f. In vitro transdermal absorption study of PSD: It was divided into 4 groups: PSD solution (PSD), PSD microneedle tip solution (PSD-Oin), solid microneedle + PSD microneedle tip solution (PSD-SDMN), and PSD soluble microneedle (PSD-DMN). According to the Franz transdermal diffusion method, the amount-time curve of PSD cumulative permeation through the skin of SD rats was obtained. The results showed that among all andrographolide drugs, the transdermal absorption of the soluble microneedle group was the best.

[0122] Please refer to Figure 12 , the dissolution behavior after microneedle administration was evaluated in ex vivo porcine skin. The microneedles were removed at 0 min, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 1 h, and 2 h. As can be seen from the figure, the tip of the needle began to dissolve at 1 min, and about half of the height of the needle body dissolved after 10 min. After 1 h, almost the entire needle body of the microneedle was dissolved, and only a small part of the bottom of the needle body remained undissolved completely. After 2 h, it was observed that the microneedles were completely dissolved.

[0123] Please refer to Figure 13 , the dissolution behavior after microneedle administration was evaluated in the skin of live SD rats. The microneedles were removed at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. As can be seen from the figure, the tip of the needle began to dissolve at 0.5 h, and the middle part of the needle body was completely dissolved after 6 h. After 8 h, only a small part of the remaining edge of the needle body of the microneedle remained undissolved completely. After 12 h, it was observed that the microneedles were completely dissolved. At 24 h, only a thin layer of gelatinous base remained of the microneedles.

[0124] Please refer to Figure 14 , the dissolution behavior after microneedle administration was evaluated in the skin of healthy humans. The microneedles were removed at 0 min, 1 min, 2 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h. As can be seen from the figure, the tip of the needle began to dissolve at 1 min, and the tip part was completely dissolved after 1 h. Dissolution continued at 2 h along with a large number of filamentous parts. At the same time, the dissolution process also spread from the center where more force was applied in the middle to the edge. After 8 h, only a small part of the remaining edge of the needle body of the microneedle remained undissolved completely. After 12 h, it was observed that the microneedles were completely dissolved.

[0125] Please refer to Figure 15, 6-week-old live, 10-week-old live, and 10-week-old excised SD rat skins were selected. After 2 hours of microneedle drug administration, the drug accumulation in the skin was investigated. Taking andrographolide sodium bisulfite soluble microneedles (ASB-DMN) as an example, for the excised SD rat skin, the skin accumulation amount was always the largest, followed by 6-week-old SD rats, and finally 10-week-old SD rats. This phenomenon is considered to be related to the stratum corneum function and the water content in the skin. For rats of different ages, the stratum corneum of 6-week-old rats is thinner, and the microneedles are easier to penetrate, resulting in higher drug accumulation. The stratum corneum barrier function of excised rat skin is damaged, and the hindrance for drugs to enter the skin becomes relatively smaller. Moreover, the water content in excised rat skin is higher than that in live rat skin, and the microneedles are more likely to dissolve and release drugs when they enter the subcutaneous tissue.

[0126] Please refer to Figure 16 , taking andrographolide sodium bisulfite (ASB) as an example, the pharmacokinetic comparative study protocol for its different preparations was as follows: SD male rats weighing 190 - 210 g were selected and adaptively fed for one week. They were randomly divided into 3 groups and given soluble microneedles (ASB-DMN), ordinary transdermal gel (ASB-Gel), and gel containing 2% laurocapram (2% Lau-ASB-Gel) respectively. The administration dose of soluble microneedles was 18 mg / kg, and the administration dose of the gel was 80 mg / kg. At 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48 h after administration, approximately 0.3 mL of blood samples were collected from the orbital venous plexus and placed in EP tubes containing sodium heparin. After centrifugation at 4000 rpm for 10 min, an appropriate amount of plasma was transferred to a clean centrifuge tube and stored in a -20 °C refrigerator. The drug concentration in plasma was determined by HPLC-MS / MS method, and the pharmacokinetic parameters of different preparations were calculated and compared using DAS 3.3.0 software. The results showed that the Cmax and AUC of ASB-DMN were the largest, followed by 2% ASB-Lau-Gel, and the smallest was ASB-Gel. The absorption curves of ASB-DMN and 2% ASB-Lau-Gel were similar, both rising in the first 4 h to reach the maximum value and then decreasing, but the Cmax and AUC were much larger than those of 2% ASB-Lau-Gel. The Cmax and AUC of ASB-Gel were the smallest, and the absorption curve reached the maximum value at the first time point and then gradually decreased and remained at a relatively low level, with significant differences, indicating that adding 2% laurocapram could slightly improve the transdermal absorption of ABS (p < 0.05), but combining ASB with microneedle technology could significantly improve the bioavailability of ABS and promote transdermal absorption (p < 0.05). At the same time, the amount of drug detected in the rats in the ASB-DMN group at 24 h was much larger than that in the other two groups, indicating that after the microneedles opened the skin channels, the drugs were stored subcutaneously, ensuring the sustained release of the drugs.

[0127] Compare the skin irritation of andrographolide micro-needles and commercially available solid micro-needles. Among them, taking the soluble micro-needles of andrographolide sulfonate (AS-DMN) as an example, the solid micro-needles are disposable sterile skin rolling needles and dot needles. The specifications of the rolling needles are a diameter of 0.3 mm, a height of 750 μm, and 100 needles. The specifications of the dot needles are a diameter of 0.3 mm, a height of 750 μm, and 100 needles. The difference is that the 100 dot needles are evenly distributed on the cube cavity, and the 100 rolling needles are evenly distributed on the cylindrical cavity. After applying the soluble micro-needles and solid micro-needles (metal dot needles and metal rolling needles) to the abdominal skin of SD rats or the arm skin of healthy volunteers, the skin irritation was evaluated according to the scoring criteria shown in Table 5 at 0.5, 1, 2, 4, 6, 12, and 24 h:

[0128] Table 5

[0129]

[0130] The results are shown in Table 6, indicating that at 0.5 h after the soluble micro-needles were given to the rats, erythema and edema appeared on the abdominal skin of the rats; the edema recovered at 12 h, and the erythema recovered at 48 h. After the rats were applied with the two kinds of solid micro-needles, more severe redness, swelling and plaques appeared compared with the soluble micro-needles. The skin irritation index of the soluble micro-needles was 0-2, indicating that the irritation and skin damage caused by the soluble micro-needles were very slight:

[0131] Table 6

[0132]

[0133] After the soluble micro-needles were given to the arms of healthy adults, edema and a small amount of erythema appeared. The edema recovered at 4 h, and the erythema recovered at 24 h. After using the metal dot needles, micro-needle holes immediately appeared on the skin, and edema and a small amount of erythema appeared. The edema recovered at 4 h, and the erythema recovered at 24 h. After using the metal rolling needles, micro-needle holes were left on the skin at 1 min, local redness and swelling occurred after 15 min, and the redness and swelling basically disappeared within 12 h, and the arm recovered after 24 h. As can be seen from Table 7, the skin irritation index of the soluble micro-needles and the commercially available solid micro-needles was 0-2, indicating that the irritation and skin damage caused by the micro-needles were very slight:

[0134] Table 7

[0135]

[0136] The above results indicate that the soluble micro-needles in this application have no less safety in use than the commercially available solid micro-needles.

[0137] By preparing the andrographolide drug into the needle solution and formulating the matrix solution, the andrographolide drug soluble microneedles are formed. Experiments show that they have good biosafety and can effectively improve the penetration ability of the stratum corneum barrier, that is, the transdermal absorption ability of the andrographolide drug can be improved.

[0138] In a second aspect, the present invention provides an application of the andrographolide drug soluble microneedles prepared by the andrographolide drug soluble microneedle preparation method as described in the above embodiments in improving the transdermal absorption rate.

[0139] In a third aspect, the present invention provides an application of the andrographolide drug soluble microneedles prepared by the andrographolide drug soluble microneedle preparation method as described in the above embodiments in improving the bioavailability.

[0140] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0141] The above embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A preparation method of soluble micro-needles of andrographolide drugs, characterized in that, It includes the following steps: Dissolve the andrographolide drugs, the first matrix material PVP, and the matrix material PVA in purified water and mix well to swell, so as to obtain a needle solution; Dissolve the second matrix material PVP and glycerol in purified water and mix well to swell, so as to obtain a matrix solution; Place the needle solution and the matrix solution in a mold to form andrographolide drug soluble microneedles.

2. The method for preparing andrographolide soluble microneedles according to claim 1, characterized in that: The andrographolide drugs are andrographolide, dehydroandrographolide, andrographolide sulfite, andrographolide sulfonate, potassium sodium salt of dehydroandrographolide succinic acid half ester, and monopotassium salt of dehydroandrographolide succinic acid half ester.

3. The method for preparing andrographolide-soluble microneedles according to claim 1, characterized in that: The first matrix material PVP is PVP K30, PVP K60 or PVP K90.

4. The preparation method of the soluble micro needle of the andrographolide drug according to claim 1, characterized in that, The step of placing the needle solution and the matrix solution in a mold to form andrographolide drug soluble microneedles includes: Pour the needle solution onto the mold, perform a first centrifugation treatment on the mold to make the needle solution fill the inside of the mold, remove the needle solution on the surface of the mold, and perform a first drying treatment to obtain a first-stage mold; Pour the matrix solution onto the first-stage mold, perform a second centrifugation treatment on the first-stage mold to make the matrix solution enter the inside of the first-stage mold, remove the matrix solution on the surface of the first-stage mold, and perform a second drying treatment to obtain a second-stage mold; Perform a demolding treatment on the second-stage mold to obtain andrographolide drug soluble microneedles.

5. The preparation method of soluble micro needles of andrographolide drugs according to claim 4, characterized in that, The time of the first centrifugation treatment and the second centrifugation treatment is 10 min, and the centrifugation speed of the first centrifugation treatment and the second centrifugation treatment is greater than 5000 rpm.

6. The method for preparing andrographolide-soluble microneedles according to claim 4, characterized in that: The time of the first drying treatment is 0.5 h to 4 h, the time of the second drying treatment is 0.5 days to 2 days, and the temperature of the first drying treatment and the second drying treatment is 25 °C.

7. The method for preparing andrographolide-soluble microneedles according to claim 1, characterized in that: The mixing ratio of the andrographolide drugs, the first matrix material PVP, the matrix material PVA and purified water is: 1:3:1:10, and the ratio of the second matrix material PVP, glycerol and purified water is: 3:0.03:5 to 3:0.3:

5.

8. Application of andrographolide drug soluble microneedles prepared by the preparation method of andrographolide drug soluble microneedles according to any one of claims 1 to 7 in improving the transdermal absorption rate.

9. Application of andrographolide drug soluble microneedles prepared by the preparation method of andrographolide drug soluble microneedles according to any one of claims 1 to 7 in improving the bioavailability.

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