A poorly soluble drug / stevioside solid dispersion, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical formulation technology, and relates to a poorly soluble drug / stevioside solid dispersion, its preparation method, and its application. The poorly soluble drug / stevioside solid dispersion comprises a poorly soluble drug and a solubilizing excipient, steviol glycoside, with a mass ratio of the poorly soluble drug to steviol glycoside of 1:(5-20). The poorly soluble drug is paclitaxel, silymarin, cannabidiol, betamethasone acetate, capsaicin, and minoxidil. This invention discloses the solubilizing effect of steviol glycoside on six poorly soluble drugs and its application in the field of microneedles. Stevioside serves as both a solubilizing excipient and a matrix material for microneedles, achieving a dual purpose.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, and particularly relates to a poorly soluble drug / stevioside solid dispersion, its preparation method, and its application. Background Technology

[0002] Throughout the history of drug development, most drugs have been poorly soluble, such as paclitaxel (BCS class IV), silymarin (BCS class II), cannabidiol (BCS class II), betamethasone acetate (BCS class II), capsaicin (BCS class II), and minoxidil (BCS class II). Low solubility is a key factor limiting their formulation development and bioavailability. To overcome this obstacle, many methods have been developed to improve the solubility of poorly soluble drugs, such as solid dispersion technology, reducing drug particle size, cyclodextrin inclusion, self-emulsifying drug delivery systems, and liposome formulations. Further research is needed to further improve the solubility and bioavailability of poorly soluble drugs by altering the pH of the formulation and using co-solvents.

[0003] Among poorly soluble drugs, minoxidil (MXD) was initially used as a peripheral vasodilator to treat refractory hypertension. However, after two weeks of oral treatment, patients experienced the side effect of hirsutism. This clinical observation led to the subsequent development of minoxidil into a topical formulation to promote hair growth. Currently, topical minoxidil is the only over-the-counter drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of androgenetic alopecia (AGA), including a 5% foaming formulation and two 5% and 2% liniments. Pharmacokinetic studies have shown that only about 1.4% of topical minoxidil is absorbed through the skin. MXD is classified as a Class II drug with low solubility and high permeability in the biopharmaceutics classification system (BCS). Therefore, poor water solubility is a limiting factor for the bioavailability of MXD, and improving the solubility of MXD is expected to improve its bioavailability.

[0004] Clinically, there is a lack of effective treatments for androgenetic alopecia, and minoxidil tincture, while a commonly used drug, has limited efficacy and numerous drawbacks in clinical application. The tincture formulation contains a large amount of propylene glycol as a co-solvent and absorption enhancer, along with ethanol. While this partially addresses the issue of minoxidil's poor water solubility and skin penetration, these organic solvents can irritate the skin, easily causing side effects such as dermatitis, itching, and scaling. Therefore, improving the solubility of MXD is crucial. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a poorly soluble drug / stevioside solid dispersion, its preparation method, and its application. The specific technical solution is as follows:

[0006] The first objective of this invention is to provide a poorly soluble drug / stevioside solid dispersion comprising a poorly soluble drug and a solubilizing excipient steviol glycoside, wherein the mass ratio of the poorly soluble drug to steviol glycoside is 1:(5-20); wherein the poorly soluble drug is paclitaxel, silymarin, cannabidiol, betamethasone acetate, capsaicin, and minoxidil.

[0007] The solubilizing excipient stevioside used in this invention has a solubilizing effect on six poorly soluble drugs: paclitaxel, silymarin, cannabidiol, betamethasone acetate, capsaicin, and minoxidil.

[0008] Furthermore, the poorly soluble drug is minoxidil, and the preferred mass ratio of minoxidil to stevioside is 1:15.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned poorly soluble drug / stevioside solid dispersion, the preparation steps of which are as follows:

[0010] The poorly soluble drug and stevioside were placed together in anhydrous ethanol organic solvent and dissolved completely by sonication. Then, the mixture was dried under reduced pressure using a rotary evaporator to obtain a solid dispersion of the poorly soluble drug / stevioside.

[0011] The poorly soluble drug / stevioside solid dispersion after vacuum drying in a rotary evaporator can be removed with a scraper, then sieved, collected in an EP tube, sealed with sealing film, and stored at 4°C for later use.

[0012] Furthermore, the mass-to-volume ratio of the poorly soluble drug to the solvent is (2-3) mg:1 ml.

[0013] Furthermore, the frequency of the ultrasound is 60-80Hz, and the ultrasound duration is 20-60min.

[0014] Furthermore, the water bath temperature of the rotary evaporator is 40-60℃, the vacuum degree is 0.08-0.1Mpa, and the time is 50-70min.

[0015] A third objective of this invention is to provide the application of the above-mentioned poorly soluble drug / stevioside solid dispersion in the preparation of soluble microneedles.

[0016] Furthermore, the method for preparing the soluble microneedles includes the following steps:

[0017] (1) Preparation of needle tip solution: Accurately weigh a certain amount of poorly soluble drug / stevioside solid dispersion and dissolve it in anhydrous ethanol or water. Stir and mix well, centrifuge at 4000-5000 rpm for 3-10 min to remove air bubbles, and calculate the needle tip solution with a certain concentration of poorly soluble drug / stevioside solid dispersion based on the total mass of the solid dispersion; the concentration of poorly soluble drug / stevioside solid dispersion is 1-3 g / mL.

[0018] (2) Preparation of backing solution: Weigh a certain amount of polyvinylpyrrolidone K-90 (PVP K90) and dissolve it in anhydrous ethanol or water. After stirring and mixing, swell in a water bath at 55-65℃ for 2-4 hours. Centrifuge at 4000-5000 rpm for 3-10 minutes to remove air bubbles and obtain a 10%-30% (w / v) PVP K90 backing solution.

[0019] (3) Microneedle preparation: Microneedles are prepared by a two-step centrifugation method. The needle tip solution is filled into the microneedle mold and centrifuged once at 4000-5000 rpm for 3-10 min. Excess drug solution outside the needle tip is scraped off with a scraper. Then, the backing solution is added and centrifuged a second time at 4000-5000 rpm for 3-10 min. The microneedles are dried at 40-60℃ for 2-4 h and then demolded to obtain soluble microneedles.

[0020] The microneedle negative mold is replicated from the microneedle metal positive mold and is used to prepare microneedle patches; the microneedle metal positive mold is made by laser etching technology, such as... Figure 1 As shown in Figure a, the microneedle array consists of 15×15 metal microneedles. The needles are pyramidal in shape, with a height of 600 μm, a spacing of 800 μm, and a base diameter of 280 μm. Both the male and female metal molds for the microneedles can be reused, which greatly reduces the manufacturing cost of the microneedles.

[0021] The microneedle negative mold fabrication process is as follows: Polydimethylsiloxane (PDMS) and a curing agent (such as polydimethyl-methylhydrosiloxane) are weighed in a ratio of 1:1 to 20:1, stirred for 5 minutes, and then placed in a vacuum drying oven. A vacuum of 0.06 MPa is applied at room temperature for 30 minutes to remove air bubbles generated during stirring, yielding a mixed material. This mixed material is then poured onto a microneedle metal positive mold, placed in an oven, and dried at 60°C for 1 hour. After demolding, the PDMS negative mold is obtained. Figure 1 As shown in b, the mold is milky white overall, with a smooth and intact surface and no residual air bubbles inside.

[0022] Furthermore, the poorly soluble drug is minoxidil, and the solvent is water.

[0023] Soluble microneedles use water as a solvent, avoiding the skin irritation side effects caused by the use of organic solvents.

[0024] Furthermore, the concentration of the needle tip solution minoxidil / stevioside solid dispersion is preferably 1.5 g / (1 ml water).

[0025] Furthermore, the backing solution is preferably 30% (w / v) PVP K90.

[0026] The minoxidil / stevioside solid dispersion soluble microneedles significantly improve the release and penetration behavior of minoxidil, increase the amount of drug retained in the skin, and have a significant effect on normal resting hair follicles and atrophied resting hair follicles. It can effectively reverse the hair follicle cycle and improve hair loss symptoms.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention discloses the solubilizing effect of stevioside on six poorly soluble drugs: paclitaxel, silymarin, cannabidiol, betamethasone acetate, capsaicin, and minoxidil. The invention also employs a thin-film evaporation method to prepare solid dispersions. Using apparent solubility as an indicator, a significant linear correlation was found between the apparent solubility of five poorly soluble drugs (paclitaxel, silymarin, cannabidiol, betamethasone acetate, and capsaicin) after solubilization and the stevioside ratio. The solubility of minoxidil increased with increasing stevioside ratio, reaching a maximum of approximately 47.41 mg / mL at a drug-to-excipient ratio of 1:15, which is 18.42 times that of the MXD raw material.

[0029] This invention is the first to apply stevioside to the field of microneedling. Poorly soluble drug / stevioside solid dispersions can be used to prepare soluble microneedles. Stevioside serves as both a solubilizing excipient and a matrix material for the microneedles, achieving a dual purpose. Soluble microneedles can pierce the stratum corneum of the skin, delivering drugs painlessly, non-invasively, or minimally invasively, reducing drug delivery costs, effectively promoting drug penetration, significantly improving drug release and penetration behavior, and increasing bioavailability to enhance therapeutic efficacy. It particularly significantly improves the release and penetration behavior of minoxidil, increasing drug retention in the skin. It has a significant effect on both normal and atrophied telogen follicles, effectively reversing the hair follicle cycle and improving hair loss symptoms, providing a novel strategy for the treatment of androgenetic alopecia. Furthermore, soluble microneedles can use water as a solvent, avoiding the skin irritation side effects caused by the use of organic solvents. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the microneedle mold of the present invention;

[0031] a. Metal male mold; b. PDMS female mold;

[0032] Figure 2 Line graphs showing the apparent solubility of each group of poorly soluble drugs in this invention (n=3);

[0033] The image on the right is a magnified view of the dotted line area in the left image.

[0034] Figure 3 This is a powder diagram of the MXD / STE-SDP of the present invention;

[0035] Figure 4 The apparent solubility curves of MXD / STE-SDP for each proportion of the formulation of this invention (n=3);

[0036] Figure 5 This is the DSC spectrum of the present invention;

[0037] a.MXD / STE-SDP; b.MXD / STE-PM; c.STE; d.MXD;

[0038] Figure 6 This is the PXRD pattern of the present invention;

[0039] a.MXD / STE-SDP; b.MXD / STE-PM; c.STE; d.MXD;

[0040] Figure 7 For the present invention STE 1 H-NMR spectra and their assignments;

[0041] Figure 8 Different concentrations of steviol glycosides in D2O according to the present invention 1 H-NMR spectrum;

[0042] Figure 9 This is a graph showing the chemical shift of the proton of stevioside H-20 as a function of mass concentration.

[0043] Figure 10 This is a graph showing the chemical shift of the proton of stevioside H-17 as a function of mass concentration.

[0044] Figure 11 The log[C] of steviol glycoside H-20 of this invention t (δ obs -δ mon )] for log[C t (δ mic -δ obs Create a diagram;

[0045] Figure 12 The 2D of 2mg / ml STE of this invention 1 HH NOESY spectrum;

[0046] Figure 13 The 2D of 30mg / ml STE of this invention 1 HH NOESY spectrum;

[0047] Figure 14 This is a structural diagram of the MXD of the present invention;

[0048] Figure 15 This invention relates to the 2D process of MXD / STE-SDP in D2O. 1 H- 1 H NOESY spectrum;

[0049] Figure 16 This is a flowchart of the MXD / STE-SDP microneedle fabrication process of the present invention;

[0050] Figure 17 The present invention relates to two types of MXD / STE microneedles;

[0051] a. MXD / STE-PM anhydrous ethanol microneedles; b is a magnified view of a portion of the needle in figure a.

[0052] c. MXD / STE-SDP water-soluble microneedles; d is a magnified view of a portion of the needle in image c.

[0053] Figure 18 This is a schematic diagram illustrating the selection of the second centrifugation conditions in this invention;

[0054] a.4500rpm, 3min; b.4500rpm, 5min;

[0055] c.4500rpm, 10min; d.3000rpm, 3min;

[0056] Figure 19 To investigate the drying method of this invention;

[0057] a: Drying method (1); b. Drying method (2)

[0058] Figure 20 The drug loading capacity of the microneedles with different concentrations of needle tip solution according to the present invention (n=3);

[0059] Figure 21 Characterization of the microneedle morphology of the present invention;

[0060] a. Front view of the microneedle; b. Microscopic image of the needle body (100×);

[0061] Figure 22 This is a schematic diagram of the mechanical strength test of the microneedles of the present invention;

[0062] Figure 23 The pressure-deformation test curve of the microneedle of this invention (n=3);

[0063] Figure 24 This is a diagram showing the skin insertion of the microneedles according to the present invention;

[0064] a. Trypan blue stained skin sample; b. H&E stained section;

[0065] Figure 25 These are SEM images of the microneedles inserted into the skin at different time points according to the present invention;

[0066] abc represent SEM images before microneedle insertion, 3 minutes after insertion, and 5 minutes after insertion, respectively.

[0067] Figure 26 Images from the skin irritation test of this invention;

[0068] abcde represent skin surface images at 0, 0.5, 1, 2, and 6 hours after microneedle removal, respectively; Figure 27 This is a schematic diagram of the diffusion cell of the transdermal transdermal device of the present invention;

[0069] Figure 28 This is the transdermal release curve of the present invention (n=3);

[0070] Figure 29 This shows the hair growth of the blank control mice and the model mice during the modeling period of this invention;

[0071] Figure 30 The condition of hair follicles 14 days after modeling according to this invention;

[0072] a. Normal mice; b. Model mice;

[0073] Figure 31 This shows the hair growth on the backs of mice in each group during the administration period of this invention;

[0074] Figure 32 The area of ​​newly grown hair in each group of mice in this invention (n=6);

[0075] Figure 33 The hair growth rate of the mice in this invention (n=6);

[0076] Figure 34 H&E stained sections (100×) of newly grown hair areas in mice of each group in this invention;

[0077] a. Model group; b. Liniment group; c. STE blank microneedle group; d. MXD / STE-SDP microneedle group;

[0078] Figure 35 The number of hair follicles in the newly grown hair sites of mice in each group of the present invention was statistically analyzed (n=4);

[0079] Figure 36 H&E stained sections (100×) of hairless areas of mice in each group of the present invention;

[0080] a. Model group; b. Liniment group; c. STE blank microneedle group; d. MXD / STE-SDP microneedle group;

[0081] Figure 37 The number of hair follicles in the non-hair-grown areas of mice in each group of mice in this invention is statistically analyzed (n=4). Detailed Implementation

[0082] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0083] Example:

[0084] 1. Solubilizing effect of steviol glycosides on poorly soluble drugs

[0085] Preparation of poorly soluble drug / stevioside solid dispersion: Weigh approximately 50 mg of poorly soluble drug and weigh different amounts of steviol glycoside (STE) according to the drug-excipient ratios of 1:5, 1:10, 1:15, and 1:20 shown in Table 1. Place the drug and excipients together in a 50 mL eggplant-shaped flask, add 20 mL of anhydrous ethanol, and sonicate for about 30 min until completely dissolved. Dry the mixture using a rotary evaporator under reduced pressure (vacuum degree 0.08-0.1 MPa) in a 50 °C water bath for 1 h. After removing the mixture with a scraper, pass it through an 80-mesh sieve three times and collect it in an EP tube. Seal the tube with sealing film and store it at 4 °C for later use.

[0086] Test method: Using the apparent solubility of the poorly soluble drug as the evaluation index, add 0.3 mL of distilled water to an EP tube, then add an appropriate amount of the poorly soluble drug / stevioside solid dispersion (equivalent to 1 mg of the poorly soluble drug). Stir manually until homogeneous, and then shake for 24 hours at 37±0.5℃ and 600 rpm in a constant temperature mixer. Centrifuge the solution at 14000 rpm for 10 min. Pipette the supernatant solution and analyze according to chromatographic conditions. Plot the apparent solubility on the ordinate and the drug-excipient ratio on the abscissa. The results are shown below. Figure 2 As shown, the apparent solubility of the five poorly soluble drugs all increased with the increase of the amount of steviol glycoside in the solid dispersion. We can intuitively observe a significant linear correlation between the apparent solubility of the drug after solubilization and the STE ratio.

[0087] Table 1. Proportions of Solid Dispersion Formulations

[0088]

[0089] 2. Preparation of MXD / STE-SDP

[0090] 2.1 Screening of the ratio of pharmaceutical excipients

[0091] Preparation of MXD / STE-SDP: Accurately weigh 50 mg of minoxidil (MXD). Weigh different amounts of STE at drug-excipient ratios (mass ratio) of 1:5, 1:10, 1:15, and 1:20. Place all ingredients in a 50 mL round-bottom flask, add 20 mL of anhydrous ethanol, and sonicate for approximately 30 min until completely dissolved. Dry under reduced pressure in a 50 °C water bath for 1 h. Remove the product with a scraper and pass it through an 80-mesh sieve three times. Collect the product in an EP tube, seal with sealing film, and store at 4 °C until use. The resulting solid dispersion is shown below. Figure 3 As shown.

[0092] Screening method: Using the apparent solubility of minoxidil (MXD) as the evaluation index, 0.3 mL of distilled water was added to an EP tube, followed by MXD / STE-SDP. The mixture was manually stirred until homogeneous and then shaken at 37±0.5℃ and 600 rpm for 24 h in a constant temperature mixer. The solution was then centrifuged at 14000 rpm for 10 min. The supernatant was pipetted and analyzed according to chromatographic conditions. The apparent solubility was plotted on the ordinate, and the drug-excipient ratio on the abscissa. The results are shown below. Figure 4 As shown, the solubility of MXD increases with the increase of STE ratio, reaching a maximum of about 47.41 mg / mL when the drug-to-excipient ratio is 1:15, which is 18.42 times that of MXD raw material. Therefore, subsequent experiments used Formulation 3 (drug-to-excipient ratio 1:15) with the highest apparent solubility for microneedle development.

[0093] 2.2 Characterization of MXD / STE-SDP

[0094] 2.2.1 DSC diagram:

[0095] Using a NETZSCH DSC 3500 differential scanning calorimeter, appropriate amounts of physical mixtures of STE and MXD (MXD / STE-PM), MXD / STE-SDP, MXD raw material, and STE were weighed and placed in aluminum crucibles, which were then sealed with lids. An empty crucible was used as a reference for the experiment.

[0096] Operating conditions: heating rate 10℃ / min, scanning range 40-300℃. Results are as follows: Figure 5 As shown, both MXD API and MXD / STE-PM have obvious endothermic peaks around 190℃, which are characteristic endothermic peaks of MXD, indicating that MXD exists in crystalline form in the physical mixture. However, the characteristic endothermic peak of MXD disappears in MXD / STE-SDP, verifying that MXD exists in an amorphous state in the solid dispersion.

[0097] 2.2.2 PXRD plot

[0098] Phase analysis of MXD / STE-SDP was performed using powder X-ray diffraction, and PXRD analysis was performed on MXD / STE-PM, MXD / STE-SDP, MXD active pharmaceutical ingredient, and STE, respectively.

[0099] Operating conditions: CuKa graphite monochromator diffraction; tube current 40mA; high voltage intensity 40kV; grazing angle θ 3-40°; scanning speed 4° / min; emission, scattering, and receiving slits 1°, 1°, and 0.2°, respectively. Results are as follows: Figure 6 As shown, the crystal diffraction peaks of MXD active pharmaceutical ingredient are very clear, with two main characteristic peaks appearing at diffraction angles 2θ of 16.569° and 19.726°, along with other scattered medium-intensity fingerprint diffraction peaks, confirming its crystal structure. The diffraction pattern of STE shows broad and short diffuse peaks. MXD / STE-PM exhibits the main characteristic peaks of the MXD crystal structure, but the diffraction peak intensity is significantly weakened, and other fingerprint peaks are not obvious. The diffraction pattern of MXD / STE-SDP is diffuse, further confirming that MXD exists in an amorphous state in the solid dispersion.

[0100] 3. Study on the solubilization mechanism of steviol glycoside solid dispersion

[0101] To further explore the solubilization mechanism of STE, one-dimensional and two-dimensional NMR techniques were used to characterize MXD / STE-SDP.

[0102] 3.1 Stevioside 1 H-NMR signal attribution

[0103] Figure 7 For STE 1 The 1H NMR spectrum and the assignment of each peak were obtained. Approximately 30 mg of STE was weighed, dissolved in D2O, sonicated for 10 min, filtered, and transferred to an NMR tube for analysis. The graph shows that steviol glycosides have three β-configured glycosyl groups. The terminal protons are δ 5.36 (1H, d, J = 5.0 Hz, H-1'), 4.79 (1H, s, H-1”), and 4.61 (1H, s, H-1”'). δ 5.01 (1H, s, H-17) and 4.85 (1H, s, H-17) represent the characteristic hydrogen signal at position 17 of the double bond. The range of δ 3.15-3.83 represents glycosyl protons, and the range of δ 0.8-2.2 represents terpene ring proton signals, of which δ 1.15 (3H, s, H-18) and 0.80 (3H, s, H-20) represent two methyl proton signals.

[0104] 3.2 Chemical shift changes of different concentrations of steviol glycosides

[0105] Take an appropriate amount of STE powder, accurately weigh 1g and place it in a microcentrifuge tube. Accurately add 1mL of D2O and sonicate to dissolve, preparing a high-concentration stock solution. Dilute sequentially to prepare concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, and 50 mg / mL. -1 The sample solution is then obtained and transferred to an NMR tube for measurement. For example... Figure 8 As shown, the peak signal of steviol glycoside protons becomes increasingly broad with increasing concentration, reaching 20 mg / mL. -1 Multiplets of the proton portion of the terpene ring can be clearly observed to gradually overlap; the chemical shift values ​​of different types of protons of steviol glycosides all changed, and the hydrophilic protons showed two changes: the chemical shift values ​​of terminal protons shifted to higher fields, while some protons shifted to lower fields, indicating that the aggregation of the hydrophilic portion was relatively complex; the chemical shift values ​​of terpene ring protons (such as protons on double bonds and methyl groups) all shifted to lower fields, and the changes were relatively simple, suggesting that steviol glycosides formed micelles with terpene ring protons as the core.

[0106] 3.3 Critical micelle concentration of steviol glycosides

[0107] Changes in the microenvironment of a compound lead to changes in the chemical shift of protons. Therefore, the critical molecular weight (CMC) of steviol glycosides can be estimated by monitoring these changes. The micellar artifact model considers the CMC to be a critical point between monomers and aggregates. If the exchange rate between steviol glycoside monomers and aggregates is faster than the NMR timescale, the detected chemical shift value is a weighted average of the two. That is, the detected chemical shift value δ... obs When aggregation occurs:

[0108] δ obs =δ mon C mon / C t +δ mic C mic / C t

[0109] Where, δ mon δ mic Chemical shifts of monomers and aggregates, C mon C mic and C t These represent monomer concentration, aggregate concentration, and total concentration, respectively.

[0110] According to the law of mass action, when the solution concentration does not reach the critical point CMC between monomers and aggregates, no self-aggregation occurs, and the surfactant in the solution exists in the form of monomers, i.e., C < CMC. Therefore:

[0111] C t =C mon

[0112] δ obs =δ mon

[0113] When the actual concentration of the solution exceeds the critical point (CMC), aggregation begins, and the molecules in the solution simultaneously exist as monomers and aggregates, i.e., C... t >CMC, then C mon =CMC, and remain constant, then

[0114] C t =C mon +C mic

[0115] C mic =C t -C mon =C t -CMC

[0116] δ obs =δ mic -CMC / C(δ mic -δ mon )

[0117] In the study of the critical micelle concentration of steviol glycosides, δ obs Plotting 1 / C, we get two straight lines; the intersection of these two lines is CMC. For example... Figure 9 , Figure 10 These are stevioside H-20 and H-17, respectively. 1 The ¹H-NMR chemical shift values ​​change with steviol glycoside concentration. (Based on steviol glycoside H-20) 1 Fitting the 1 / C ratio to the 1H-NMR chemical shift values, the concentration at the intersection of the two straight lines was found to be 14.66 mg·mL. -1 , by stevioside H-17 1 Fitting the 1 / C values ​​to the 1H-NMR chemical shift values ​​yielded a concentration of 14.16 mg·mL at the intersection of the two straight lines. -1 Therefore, the critical micelle concentration of steviol glycoside aqueous solution is approximately 14 mg·mL⁻¹. -1 .

[0118] 3.4 Aggregate number of steviol glycosides

[0119] The aggregation number can also be determined based on the change in proton chemical shift with mass concentration. Assuming the law of mass action applies and micelle formation follows a single-step equilibrium model, the aggregation number can be expressed as:

[0120]

[0121] Where n is the aggregation number, A and An represent the steviol glycoside monomer and aggregate, respectively, and K is the aggregation equilibrium constant.

[0122] K = [An][A] - n

[0123] C t =C mon +C mic

[0124] By combining the formula with the derivation, we can obtain...

[0125] [A] = C t (δ mic -δ obs ) / (δ mic -δ mon )

[0126] [An] = C t (δ obs -δ mon ) / (δ mic -δ mon )

[0127] log[C t (δ obs -δ mon )]=nlog[C t (δ mic -δ obs ]+lognK+(1-n)×log(δ mic -δ mon )

[0128] log[C t (δ obs -δ mon )] for log[C t (δ mic -δ obs Linear fitting yields two straight lines, and the slope of the fitted line is the N of the aggregate. agg .like Figure 11 As shown, two straight lines with different slopes can be fitted based on the changes in the chemical shift value of steviol glycosides. The intersection of the two lines is the CMC. Therefore, the two slopes represent the N values ​​when the steviol glycoside concentration is below the CMC and above the CMC, respectively. agg Below the CMC, steviol glycoside has an N1 of approximately 1, existing as a monomer; above the CMC, steviol glycoside has an N2 of approximately 6, existing as a hexamer.

[0129] 3,5-Steviol 1 H- 1 H ROESY diagram

[0130] Based on the internuclear NOE effect, 1 H- 1The intensity and position of relevant cross peaks on the H ROESY spectrum can reflect the strength and arrangement of intermolecular interactions in aggregates, thus providing information about the spatial structure of the aggregates. For example... Figure 12 , 13 Stevioside 2 g·mL -1 30g·mL -1 of 1 H- 1 H ROESY spectrum, Figure 12 There are almost no signal-related peaks in it, while Figure 13 It can be seen that δ5.36 (1H, s, H-1') is correlated with glycoproton δ3.30, glycoproton is correlated between δ3.15 and 3.83, terpene ring proton is correlated between δ0.8 and 2.2, and δ5.01 (1H, s, H-17) and 4.85 (1H, s, H-17) are correlated with terpene ring proton between δ1.8 and 2.2.

[0131] 3.6 MXD / STE-SDP 1 H- 1 H ROESY diagram

[0132] Weigh approximately 45 mg of MXD / STE-SDP, dissolve it in D2O, sonicate for 10 min, filter, and transfer it to an NMR tube for analysis. Figure 14 The diagram shows the molecular structure of MXD. The chemical shifts of H-2′, H-3′, and H-4′ in MXD are at δ3.32, δ1.43, and δ1.50, respectively. Figure 15 2D in water by MXD / STE 1 H- 1 In the H ROESY spectrum, we observed that the protons in the first half of MXD all shifted to higher fields and exhibited strong interactions with the protons of the STE terpene rings. Simultaneously, the NOE effect between the terpene ring protons of the STE molecules weakened or disappeared. This suggests that the originally aggregated STE molecules increased their intermolecular distance due to encapsulating MXD, leading to a weakening or disappearance of the intermolecular interactions between STE molecules. This resulted in NOE correlation peaks between multiple alkane hydrogens in the MXD molecule and the hydrophobic terpene hydrogens of the STE molecules, while the NOE effect between glycoprotein protons was enhanced. It is speculated that MXD was encapsulated within a hydrophobic cavity formed by multiple STE molecules connected end-to-end, thereby achieving the purpose of solubilizing MXD.

[0133] 4. Preparation of MXD / STE-SDP microneedles

[0134] 4.1 Solvent Selection

[0135] Using microneedle appearance as an evaluation index, we screened MXD / STE-PM anhydrous ethanol microneedles and MXD / STE-SDP water-soluble microneedles.

[0136] like Figure 16 The MXD / STE-SDP water-soluble microneedles mentioned above:

[0137] (1) The apparent solubility of the MXD / STE-SDP formulation with a drug-to-excipient ratio of 1:15 was a needle tip solution (the concentration of the solid dispersion was approximately 2.4 g / mL);

[0138] (2) The backing solution is a 10% (w / v) aqueous solution of PVP K90;

[0139] (3) Microneedles were prepared using a two-step centrifugation method. 150 μL of needle tip solution was filled into a PDMS mold, and the mixture was centrifuged once at 4500 rpm for 5 min. The residue was scraped off with a scraper. Figure 16 Remove excess drug solution outside the needle tip; then add 700 μL of backing solution, centrifuge at 4500 rpm for 5 min for a second time; dry at 50℃ for 3 h, and demold.

[0140] MXD / STE-PM anhydrous ethanol microneedles:

[0141] (1) Add anhydrous ethanol to the stevioside / minoxidil solid dispersion with a drug-excipient ratio of 1:15, and sonicate for 2 hours to dissolve it, so as to prepare a 5% MXD (w / v) needle tip solution.

[0142] (2) Backing solution: 10% (w / v) PVP K90 anhydrous ethanol solution;

[0143] (3) Microneedles were prepared by two-step centrifugation. 150 μL of needle tip solution was filled into the PDMS mold and centrifuged at 4500 rpm for 5 min for the first centrifugation. Excess drug solution outside the needle tip was scraped off with a scraper. Then 700 μL of backing solution was added and centrifuged at 4500 rpm for 5 min for the second centrifugation. The microneedles were dried at 50℃ for 3 h and then demolded.

[0144] The results are as follows Figure 17 The results showed that the microneedles prepared with anhydrous ethanol had visible needle tip defects. Figure 17 (a, 17b) are thin and fragile, and cannot form a complete needle structure. In contrast, STE microneedle patches prepared with water as a solvent have a good appearance and intact needles. Figure 17 (c, 17d). Although anhydrous ethanol is an organic solvent with relatively low harm to the human body and is often used as a solubilizer and penetration enhancer in topical skin preparations, it can still cause skin irritation and damage with prolonged use. This study uses STE as a new microneedle material, and the preparation process does not require the use of organic solvents, making it safer and more reliable.

[0145] 4.2 Selection of the first centrifugation time

[0146] Using needle integrity as an indicator, the effects of centrifugation times of 3, 5, and 10 min on needle tip integrity were investigated. Higher centrifugation speeds resulted in greater centrifugal force, which was more conducive to needle tip filling. Therefore, a maximum centrifugation speed of 4500 rpm was fixed. The results are shown in Table 2. Insufficient centrifugation time led to incomplete needle bodies after demolding. To ensure complete filling of the mold by the high-viscosity needle tip solution, a centrifugation time of 10 min was selected.

[0147] Table 2. Effect of centrifugation time on needle tip integrity

[0148]

[0149] 4.3 Selection of Second Centrifugation Time and Speed

[0150] Secondary centrifugation after filling with the backing material can affect drug diffusion into the backing. 1 mg of Rhodamine B was accurately weighed and added to 1 mL of needle tip solution to prepare a needle tip solution containing 0.1% (w / v) of the model drug. After one centrifugation, excess drug solution was carefully scraped off, and any excess dried drug powder around the edges was taped to prevent interference with observation. The backing was a 10% PVP K90 solution. The effects of secondary centrifugation time and speed on drug diffusion were investigated according to the conditions in Table 3. The results are as follows: Figure 18 This indicates that as the secondary centrifugation time increases, the color of the backing solution becomes increasingly dark, meaning that the diffusion of Rhodamine B into the backing solution gradually worsens. At 4500 rpm for 3 minutes, the drug concentrates at the needle tip and diffuses almost nothing into the backing solution; after 5 minutes, slight diffusion occurs; and after 10 minutes, severe diffusion occurs. Decreasing the centrifugation speed has no significant effect on diffusion. Therefore, the backing centrifugation conditions were adjusted to 4500 rpm for 3 minutes.

[0151] Table 3 Secondary centrifugation conditions

[0152]

[0153] 4.4 Screening of drying methods after centrifugation

[0154] Microneedles were prepared using the adjusted centrifugation method. The appearance of the microneedles was used as an indicator, and they were dried in a 50°C oven. The drying methods were as follows: (1) after one centrifugation, the needle tip was dried for 30 min, followed by a second centrifugation and then another 3 h of drying; (2) after a second centrifugation, the entire microneedles were dried for 3 h. After demolding, the microneedles were as follows: Figure 19 As shown, the microneedles prepared by method (1) are filled with small air bubbles and the needle body is missing, while method (2) can obtain microneedle patches with good appearance. Therefore, the drying method is fixed as centrifugation followed by drying in an oven at 50°C for 3 hours.

[0155] 4.5 Screening of backing solution concentration

[0156] The concentration of the backing solution affects drug distribution within the microneedle. In this study, 0.1% Rhodamine B was added to the needle tip solution, and the effects of 10%, 20%, and 30% (w / v) PVP K90 backing solutions on drug diffusion were investigated. The results showed that with 10% PVP K90 as the backing, the entire backing was stained pink by the Rhodamine B in the needle tip. At a concentration of 20% PVP K90, the color lightened, and diffusion improved slightly. With a 30% PVP K90 backing solution, the color was concentrated at the needle tip, indicating that Rhodamine B was mainly concentrated there, and diffusion into the backing was significantly reduced. Further increasing the PVP K90 concentration would result in excessive viscosity, hindering backing solution filling and increasing the likelihood of introducing large amounts of air bubbles that are difficult to expel. Therefore, a 30% PVP K90 solution was chosen as the optimal backing solution concentration.

[0157] 4.6 Screening of MXD / STE-SDP microneedle tip solution concentration

[0158] Using the apparent solubility of MXD as an indicator, MXD / STE-SDP was dissolved in 1 ml of water to prepare tip solutions with concentrations of 1 g / mL, 1.5 g / mL, 2 g / mL, 2.5 g / mL, and 3 g / mL. The backing solution was 30% PVP K90. Microneedles were prepared according to the above method. The needle tips were scraped off with surgical scissors, placed in an EP tube, and 1 mL of methanol was added. The solution was vortexed to dissolve and filtered through a 0.45 μm organic filter membrane. The results are as follows. Figure 20 As shown, the drug loading of the prepared microneedles was highest when the concentration of the needle tip solution was 1.5 g / mL, which was about 23.34 μg.

[0159] 4.7 Optimal Prescription and Processing

[0160] The needle tip solution concentration was 1.5 g / mL, and the backing solution was 30% PVP K90. The microneedle patch was prepared using a two-step centrifugation method: 150 μL of needle tip solution was filled into a PDMS mold, and centrifuged at 4500 rpm for 10 min for the first centrifugation; excess solution was scraped off with a scraper. 700 μL of backing solution was added, and the patch was centrifuged at 4500 rpm for 3 min for the second centrifugation. The patch was then dried in a 50℃ oven for 3 h, and demolded to obtain the microneedle patch.

[0161] 5. Characterization and basic performance evaluation of MXD / STE-SDP microneedles

[0162] 5.1 Morphological Characterization

[0163] The morphology of MXD / STE-SDP microneedles was characterized using stereomicroscopy. The overall appearance of the microneedles was good, and the microneedle characterization was as follows: Figure 21 The microneedles have a good overall appearance. In (b), it can be observed that the microneedles have a good shape, a smooth and flat surface, and a sharp tip.

[0164] 5.2 Mechanical Strength Test

[0165] See the schematic diagram of the microneedle mechanical strength test. Figure 22 Cut 5×5 arrays of small patches from the microneedle patch to be tested at random locations. Place them on the stage above the pressure testing machine base with the needle tips facing upwards. Set the downward movement speed of the pressure testing machine probe to 30 mm / min. When the probe contacts the needle tip, the downward movement speed is reduced to 5 mm / min. Stop the test when the sensor detects a pressure of 30 N. Output the pressure and deformation data, and calculate the average pressure on each needle. The results are as follows: Figure 23 As shown in the figure, the pressure of the microneedle did not show a significant inflection point during the test, proving that no breakage occurred. At the end of the test, the maximum force reached 1.66 N / needle, which meets the requirements for skin penetration. The above test results prove that the MXD / STE-SDP microneedle has good mechanical strength.

[0166] 5.3 Examination of puncture capability

[0167] Preparation of trypan blue staining solution: Weigh 4g of trypan blue, dissolve it in 100mL of water, filter to obtain a 4% (w / v) trypan blue solution, take 1mL, dilute with PBS to a 0.4% (w / v) trypan blue staining solution, and store at 4℃ for later use.

[0168] Preparation of 4% paraformaldehyde fixative: Accurately weigh an appropriate amount of paraformaldehyde and add it to PBS phosphate buffer. Stir continuously at 60°C with a heating stirrer until completely dissolved. Add an appropriate amount of 1mol / L NaOH to adjust the pH to 7.4 to prepare a 4% (w / v) paraformaldehyde fixative. Store at 4°C for later use.

[0169] Trypan blue staining experiment: Isolated porcine skin was removed from freezing, cut to a suitable size, thawed at room temperature, and then immersed in PBS phosphate buffer for 20 minutes. After removal, the surface moisture was blotted with absorbent paper, and the skin was placed on an 8-layer sealing film. The sealing film simulated subcutaneous tissue and prevented skin curling. A self-made MXD / STE-SDP microneedle was placed flat on the skin with the needle tip facing down, and the tip was pressed vertically with the thumb for 5 minutes to penetrate the skin. The microneedle patch was removed, and a few drops of trypan blue staining solution were immediately applied to the skin, spreading it evenly. After 10 minutes, excess dye was washed off with PBS, photographed, and the penetration rate was calculated. The results are shown below. Figure 24 a.

[0170] HE staining experiment: A piece of ex vivo skin with microneedle patches was taken, and immediately after removing the microneedles, it was placed in an EP tube containing 4% paraformaldehyde fixative for 3 days. The fixed skin tissue underwent a series of operations including dehydration, embedding, sectioning, dewaxing, and staining to prepare hematoxylin-eosin (H&E) stained longitudinal sections of the skin. The sections were then viewed and photographed under an upright microscope. (See attached image). Figure 24 b.

[0171] Blue dots, representing micropores left by the microneedles, are clearly visible in the trypan blue images of ex vivo skin, indicating the excellent skin penetration capability of the MXD / STE-SDP microneedles. The percentage of micropores out of the total number of microneedles (225) shows a penetration rate as high as 94%. H&E staining of the sections confirms that the microneedles successfully penetrated the stratum corneum and reached below the epidermis. In conclusion, the MXD / STE-SDP microneedles developed in this study possess excellent mechanical properties and can be used for transdermal drug delivery.

[0172] 5.4 Microneedle Dissolution Experiment

[0173] After acclimatizing C57 mice for 2 days, the hair on their backs was shaved short with a razor, and then a suitable amount of depilatory cream was applied. After about 10 minutes, the hair was wiped clean with a damp paper towel. The dissolution experiment was performed the next day. Microneedles were inserted into the skin on the back of the mice with thumb pressure and held in place with clamps to maintain pressure. The microneedle patches were removed at 3 and 5 minutes after microneedle application and immediately placed in a drying oven to dry for 30 minutes. The images were then photographed under a scanning electron microscope. The results are as follows: Figure 25 As shown, the needle tip dissolved 3 minutes after insertion into the skin, and almost the entire needle dissolved after 5 minutes, with only a small amount remaining at the base where it connects to the backing. The experimental results indicate that MXD / STE-SDP microneedles can rapidly dissolve under the influence of the interdermal fluid within the skin, and STE is a high-performance soluble microneedle material.

[0174] 5.5 Skin irritation test

[0175] The mouse skin pretreatment was the same as in section "5.4" above, and a skin irritation test was performed the following day. Mice were placed in an air anesthesia machine and removed after immobilization. A microneedle patch was then inserted into the mouse's back skin using thumb pressure, and fixed with clips for 5 minutes. After removing the microneedle patch, photographs of the treatment site were taken at 0, 0.5, 1, 2, and 6 hours post-removal to observe for skin irritation such as erythema and edema. Results were as follows: Figure 26 As shown. In the initial stage after the insertion, there may be mild erythema. This may be due to the mild irritation caused by the microneedles piercing the stratum corneum and entering the skin, or the pressure from the pressure, as shown by the indentation at the edge of the microneedle patch in the upper left corner. However, no edema occurred, and the erythema gradually disappeared after 30 minutes. After 1 hour, the micropores were completely closed and disappeared, with almost no irritation. It can be seen that the MXD / STE-SDP microneedles are highly safe and non-irritating to the skin.

[0176] 5.6 In vitro permeation experiment

[0177] A schematic diagram of the diffusion cell of a transdermal transdermal device is shown below. Figure 27As shown, after thawing isolated pig skin, skin samples were cut into groups based on the area of ​​the contact surface between the supply and receiving chambers. These groups consisted of three samples each of MXD / STE-SDP microneedles and a 2% MXD topical solution. The samples were equilibrated with PBS solution for 20 minutes. The microneedle patch was placed vertically and flat on the skin, and the skin was penetrated by pressing with the thumb for 5 minutes. 2 μL of the topical solution (containing approximately 40 μg of MXD) was precisely pipetted and added to the skin in the supply chamber of the topical solution group. 8 mL of transdermal release medium at a water bath temperature was pre-added to the receiving chamber, and a stir bar was placed inside. The treated skin was quickly fixed between the supply and receiving chambers. The diffusion cell was placed in the transdermal transdermal apparatus, with the water bath temperature set to 32 ± 0.2℃ and the rotation speed set to 250 rpm. The sampling port and supply chamber were sealed with plastic wrap to prevent liquid evaporation. If air bubbles were present between the skin and the receiving chamber, the diffusion cell could be gently inverted towards the sampling port to drain them. At 1, 2, 4, 6, 8, 10, 12, 16, and 24 hours, 300 μL of the receiving solution was collected, and an equal volume of fresh receiving solution at constant temperature was added simultaneously. The collected receiving solution was filtered through a 0.45 μm filter, stored in a liquid chromatography vial at 4 °C, and analyzed by HPLC. The results are shown below. Figure 28 The microneedle group exhibited rapid drug release within 2 hours because the microneedles can penetrate the stratum corneum, delivering the drug directly to the epidermis or superficial dermis. After dissolution and release, the release rate slowed slightly from 2 to 6 hours, but became almost uniform after 6 hours. The cumulative penetration rate reached 85.21% at 24 hours, demonstrating high transdermal delivery efficiency. The topical application group showed slow drug release in the first 2 hours, followed by a faster release rate. This process is consistent with the need for topical applications to first disrupt the lipid structure of the stratum corneum with organic solvents to achieve effective drug delivery. However, the release rate gradually slowed from 10 to 24 hours, with a cumulative penetration rate of approximately 68.15% at 24 hours, significantly lower than that of the MXD / STE-SDP microneedles.

[0178] Pharmacodynamic studies of 6 MXD / STE-SDP microneedles

[0179] 6.1 Androgenetic alopecia mouse model

[0180] The purchased testosterone propionate injection solution was in a 1mL:25mg specification, while the daily dosage for mice was 5mg / (kg·d). Therefore, the original drug concentration needed to be diluted. Considering the poor absorption of subcutaneously injected drugs, and taking into account the overall dosage volume, the testosterone propionate injection solution was diluted with injection-grade soybean oil to a final concentration of 2mg / mL. Specifically, 1mL of the original injection solution was taken into a 15mL EP tube, 11.5mL of injection-grade soybean oil was added, and the mixture was vortexed for 3-5 minutes to mix thoroughly. Mice were dehaired, and 24 hours later, mice with pink skin and no broken skin were selected for modeling. Testosterone propionate was subcutaneously injected daily at a dose of 5mg / (kg·d) per mouse.

[0181] 6.2 Modeling Grouping and Scheme

[0182] After 14 days of continuous subcutaneous injection of testosterone propionate, mice without new hair growth on their backs were randomly divided into two groups: a model group and a normal group. Mice with intact skin after hair removal were selected for the model group. The model group received slow subcutaneous injections of 2.5 mg / mL testosterone propionate daily, with a dosage of approximately 0.04 mL, for 14 consecutive days. In normal mice, hair follicles gradually transition from the resting phase to the anagen phase after hair removal; hair was allowed to grow naturally without further treatment. Figure 29 This study shows the hair growth in mice 14 days after modeling. Normal mice showed bluish skin on their backs at day 7, indicating the hair follicles were gradually entering the anagen phase and pigmentation was deepening. By day 14, large areas of new hair had appeared. However, the modeling group mice showed no new hair growth; their back skin was pinkish-white, indicating the telogen phase of hair growth, and they were experiencing seborrhea-like dandruff shedding, consistent with the pathological characteristics of androgenetic alopecia.

[0183] 6.3 Biological Sample Collection and Evaluation

[0184] Starting from the first day of testosterone propionate injection, photographs were taken of the backs of the model mice and blank control mice on days 0, 7, and 14. After 14 days of modeling, three model mice and three normal mice were taken, and the skin from the backs of the two groups of mice was cut into 1x1cm pieces, fixed with 4% (w / v) paraformaldehyde for 2-3 days, and stained with H&E. Figure 30 Skin sections from normal mice and model mice were stained. The results show that subcutaneous injection of testosterone propionate prolonged the telogen phase, delayed the anagen phase, and resulted in follicle atrophy and sparse hair, consistent with the pathological characteristics of androgenetic alopecia, thus proving the successful establishment of the androgenetic alopecia model.

[0185] 6.4 Grouping of Pharmacodynamic Studies

[0186] Twenty-four hours after hair removal, mice with intact, undamaged back skin were selected for model establishment. Mice were subcutaneously injected with testosterone propionate daily. After 14 days, mice with successfully established pinkish-white back skin were selected for pharmacodynamic studies. Mice were randomly divided into four groups: model group, MXD topical application group, STE blank microneedle group, and MXD / STE-SDP microneedle group, with six mice in each group. All mice continued to receive subcutaneous injections of testosterone propionate every morning to maintain the pathological state of androgenetic alopecia. For the STE blank microneedle group and the MXD / STE-SDP microneedle group, each mouse received one corresponding microneedle patch daily. The mice were anesthetized with an air anesthesia machine for approximately 3 minutes, and the microneedle patch was inserted into the back skin with thumb pressure and maintained for 5 minutes. The patch remained on the skin until it naturally detached. Mice in the MXD topical application group were sprayed with approximately 167 μL of 2% commercially available MXD topical solution once daily, applied and massaged into the back to promote absorption.

[0187] 6.5 Pharmacodynamic Evaluation Methods

[0188] Starting from day 0, the hair growth on the backs of mice in each group was photographed and recorded weekly. The area of ​​newly grown hair per week for each mouse was calculated using ImageJ software, and a curve showing the area of ​​newly grown hair and the hair growth rate for each group was plotted. After 35 days of experimentation, mice were euthanized by spinal cord dislocation. Skin from the back areas with newly grown hair and the pinkish-white areas from the areas without newly grown hair were harvested, fixed, and stained with H&E. Hair follicle growth was observed under a microscope. For each group of skin, four fields of view were randomly selected under a 100x microscope to count hair follicles, and one-way ANOVA and plotting were performed using Origin software.

[0189] Figure 31 The study investigated hair growth in model mice after different drug administration groups. In the control group, only a few hairs appeared at 35 days, and the growth was minimal, demonstrating the sustained inhibitory effect of testosterone propionate on hair follicle growth. Mice in the STE blank microneedle group showed a few black spots at 21 days, and bluish-gray new hair appeared at 35 days, presumably due to the stimulating effect of the microneedles on hair follicles, but this showed no significant improvement compared to the model group. Mice in the topical treatment group showed small bluish-black patches on their skin at 14 days, but new hair growth was slow, covering part of their backs after 35 days. In contrast, the MXD / STE-SDP microneedle group showed extensive new hair growth at 14 days, with very dark skin pigmentation in this area, and the new hair growth area increased rapidly, covering a large area of ​​the backs by 35 days. Figure 32 , 33 The MXD / STE-SDP microneedle group mice showed large areas of new hair growth around day 14, and maintained a considerable growth rate, reaching a hair growth area of ​​6.44 ± 1.14 cm² at day 35. 2 The growth rate of the MXD topical treatment group reached 67.48±8.07%, while the MXD topical treatment group only showed a significant hair growth trend around day 21, and the growth was slow, with lower new hair area and hair growth rate, reaching only 2.24±0.84 cm at the end of the 35-day trial. 2 (25.74±6.57)%. Figure 34 H&E-stained sections of newly grown hair in mice 40 days after drug administration; in the model mice, hair follicles were sparsely distributed in the superficial dermis, with incomplete atrophy and central cavities lacking melanin. The same situation occurred in the STE microneedle group, where hair follicles were clearly still in the resting phase. In the MXD topical group, hair follicles in the newly grown hair area were sparsely distributed in the superficial and dermal layers, with a darker color, but the follicles were smaller; while... Figure 34 The d-axis showed that the hair follicles in the MXD / STE-SDP microneedle group were large and intact, and very dark in color, with a large amount of melanin deposition in the center, distributed extensively in the dermis and superficial dermis, with a number of up to 42.25±9.22. Figure 35 Statistical analysis of hair follicle counts in areas of newly growing hair in mice; Figure 36 H&E-stained sections of hairless areas from mice in each group were prepared. Figure 37 The number of hair follicles in the non-hair-growing areas of mice in each group was statistically analyzed. In the model group and the STE microneedle group, hair follicles were sparsely distributed on the superficial skin, with very light pigmentation and indistinct shapes, indicating they were still in the resting phase. The MXD topical treatment group showed a significant increase in the number of hair follicles, but they were still only distributed in the superficial dermis, were small, and exhibited poor growth. The MXD / STE-SDP microneedle group had a greater number of hair follicles, distributed in the dermis, with deeper follicles showing darker pigmentation and intact structure, indicating that MXD / STE-SDP microneedles can promote the transition of hair follicles from the resting phase to the anagen phase. MXD / STE-SDP microneedles exhibit excellent drug release behavior and skin retention in vitro, and demonstrate superior anti-AGA and hair growth-promoting effects in vivo.

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a poorly soluble drug / stevioside solid dispersion in the preparation of soluble microneedles, characterized in that, The method for preparing the soluble microneedles includes the following steps: (1) Preparation of needle tip solution: Dissolve the poorly soluble drug / stevioside solid dispersion in a solvent, stir and mix well, centrifuge at 4000-5000 rpm for 3-10 min to remove air bubbles, and prepare needle tip solution; The poorly soluble drug is minoxidil, the solvent is water, and the concentration of the poorly soluble drug / stevioside solid dispersion in the needle tip solution is 1.5 g / mL; The mass ratio of the poorly soluble drug minoxidil to stevioside is 1:15; The method for preparing the poorly soluble drug / stevioside solid dispersion is as follows: the poorly soluble drug and stevioside are placed together in anhydrous ethanol organic solvent, completely dissolved by ultrasonication, and then dried under reduced pressure using a rotary evaporator to obtain the poorly soluble drug / stevioside solid dispersion; (2) Preparation of backing solution: Dissolve polyvinylpyrrolidone K-90 in water, stir and mix well, swell in a water bath at 55-65℃ for 2-4 hours, centrifuge at 4000-5000rpm for 3-10 minutes to remove air bubbles, and obtain a backing solution of 30% w / v PVPK90. (3) Microneedle preparation: Microneedles were prepared by a two-step centrifugation method. The needle tip solution was filled into the microneedle mold and centrifuged once at 4000-5000 rpm for 5-10 min. Excess drug solution outside the needle tip was scraped off with a scraper. Then, the backing solution was added and centrifuged a second time at 4000-5000 rpm for 3-5 min. The microneedles were dried at 40-60℃ for 2-4 h and then demolded to obtain soluble microneedles.

Images