Preparation method of minoxidil liniment

Abstract

The present application relates to the technical field of minoxidil, in particular to a preparation method of minoxidil liniment. The preparation method comprises the following steps: preparing a raw material solution containing minoxidil by using glacial acetic acid solution, adding water ophrydis volatile oil, polyethylene glycol 400 and propylene glycol, and adding soybean lecithin to prepare the external use nanometer minoxidil liniment. The minoxidil liniment forms micro-nanoparticles and can be stored for a long time. In the prior art, ethanol is used as an auxiliary material, and the minoxidil liniment provided by the present application has no skin irritation in single administration and multiple administrations, and the skin irritation is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of minoxidil, in particular to a preparation method of minoxidil liniment. BACKGROUND

[0002] Minoxidil, finasteride, platelet-rich plasma, low-level laser therapy, stem cell therapy and hair follicle transplantation are the main methods for treating AGA at present. Among them, minoxidil can strengthen the blood supply of hair follicles, thereby increasing the oxygen and nutrient transport of cells, shortening the resting period and prolonging the growth period, and is considered to be the most effective drug treatment, and has been established as the first-line treatment for AGA.

[0003] Due to the low water solubility of minoxidil, the currently marketed external preparations are minoxidil liniment and formulations prepared with ethanol and propylene glycol as solvents, but they mainly have the following deficiencies. ① Poor compliance: the ethanol solvent is easy to volatilize and crystallize on the skin surface, which is not conducive to the absorption of the drug by the skin; after volatilization, it will leave a thick propylene glycol / water residue, which may cause the user's aversion; it stays in the skin for a short time, and it is difficult to play a sustained therapeutic effect. ② Sensitization: due to the evaporation of ethanol and the formation of minoxidil crystals, it can cause adverse reactions of the skin, including itching, skin rash, dandruff and allergic contact dermatitis, and some patients have the phenomenon of rapid increase of hair loss. ③ Inconvenient administration: the adhesion of the solution is poor, and it is difficult to fix the dose of administration. Due to the poor skin penetration ability of minoxidil, the effectiveness of minoxidil as a hair growth drug is limited, and some studies have reported that minoxidil is more easily absorbed through the local route, so a more novel and suitable preparation is needed for the local administration of minoxidil. SUMMARY

[0004] Therefore, the present application provides a preparation method of minoxidil liniment to at least partially solve one of the above-mentioned existing defects.

[0005] In a first aspect, the present application provides a preparation method of minoxidil liniment, comprising:

[0006] Dissolving minoxidil with an aqueous solution of glacial acetic acid, and obtaining a raw material solution after filtration;

[0007] Adding water celery volatile oil, polyethylene glycol 400 and propylene glycol to the raw material solution, and stirring with a homogenizer for 30 min to obtain a uniform mixed solution;

[0008] Adding soybean lecithin to the mixed solution, and homogenizing for 30 min to obtain the external nano minoxidil liniment.

[0009] In the first aspect, the content of minoxidil in the raw material solution is not less than 0.25 g / L.

[0010] In the first aspect, the proportion of the volatile oil of water-leek in the raw material liquid is 1g:10-15mL, the proportion of the polyethylene glycol 400 in the raw material liquid is 1g:3-8mL, and the proportion of the propylene glycol in the raw material liquid is 1g:1-5mL.

[0011] In the first aspect, the concentration of the glacial acetic acid aqueous solution is 50-80%.

[0012] In the first aspect, the concentration of the glacial acetic acid aqueous solution is 70%, the content of minoxidil in the raw material liquid is 0.36g / L, the proportion of the volatile oil of water-leek in the raw material liquid is 1g:12.5mL, the proportion of the polyethylene glycol 400 in the raw material liquid is 1g:5mL, the proportion of the propylene glycol in the raw material liquid is 1g:2mL, and the weight ratio of the soybean lecithin to minoxidil is 2:1.

[0013] In the first aspect, the concentration of the glacial acetic acid aqueous solution is 70%, the content of minoxidil in the raw material liquid is 0.52g / L, the proportion of the volatile oil of water-leek in the raw material liquid is 1g:10mL, the proportion of the polyethylene glycol 400 in the raw material liquid is 1g:3mL, the proportion of the propylene glycol in the raw material liquid is 1g:1mL, and the weight ratio of the soybean lecithin to minoxidil is 2:1.

[0014] In the first aspect, the concentration of the glacial acetic acid aqueous solution is 80%, the content of minoxidil in the raw material liquid is 0.36g / L, the proportion of the volatile oil of water-leek in the raw material liquid is 1g:12.5mL, the proportion of the polyethylene glycol 400 in the raw material liquid is 1g:5mL, the proportion of the propylene glycol in the raw material liquid is 1g:2mL, and the weight ratio of the soybean lecithin to minoxidil is 1.8:1.

[0015] In the first aspect, the preparation method of the volatile oil of water-leek comprises the following steps:

[0016] The water-leek is crushed into fine powder, and then mixed with a mixed solvent of anhydrous ethanol and ethyl acetate according to a liquid-to-material ratio of 25:1 (mL / g). The extraction is performed by ultrasonic method at an extraction temperature of 60℃ for 50 minutes. The extraction liquid is filtered, and then dried under reduced pressure. After adding anhydrous sodium sulfate and anhydrous magnesium sulfate, the mixture is filtered to obtain the volatile oil of water-leek.

[0017] In the second aspect, the minoxidil preparation prepared by the preparation method of the first aspect has a particle size of 72.45-78.37nm, a PDI of 0.24-0.26, a Zeta potential of-51.42--50.68mV, and a loading capacity of minoxidil of 92.36-94.62%.

[0018] In a third aspect, the present application provides the use of the minoxidil preparation prepared by the preparation method of the first aspect in the preparation of a hair loss medicine.

[0019] Compared with the prior art, the present application has at least one of the following beneficial effects:

[0020] Compared with the prior art, the present application uses ethanol as an auxiliary material, uses water celery volatile oil as an auxiliary material of the preparation, and uses glacial acetic acid as a dissolution medium, thereby improving the solubility of minoxidil and facilitating the formation of micro-nano preparations. Through the stability study of the preparation, it is found that the particle size, PDI, Zeta potential and content of the nano preparation formed by the preparation do not change significantly, which indicates that the minoxidil preparation provided by the present application forms micro-nano particles and can be stored for a long time.

[0021] In addition, it is proved by cell experiments that the minoxidil preparation provided by the present application can increase the membrane fluidity of HaCaT cells, which indicates that it has the effect of reducing the skin epidermal barrier to facilitate the transdermal absorption of drugs. The minoxidil preparation directly acts on the skin of male SD rats and has a better transdermal effect.

[0022] In addition, compared with the prior art which uses ethanol as an auxiliary material, the minoxidil preparation provided by the present application has no skin irritation in single and multiple dosing, thereby reducing skin irritation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure shows the particle size results of the minoxidil preparations provided by examples 1-3 and comparative examples 2-4 respectively after being stored at-4±1℃, 25±1℃ and 60±1℃ for 3 months.

[0024] Figure 2 The figure shows the PDI results of the minoxidil preparations provided by examples 1-3 and comparative examples 2-4 respectively after being stored at-4±1℃, 25±1℃ and 60±1℃ for 3 months.

[0025] Figure 3 The figure shows the Zeta potential results of the minoxidil preparations provided by examples 1-3 and comparative examples 2-4 respectively after being stored at-4±1℃, 25±1℃ and 60±1℃ for 3 months.

[0026] Figure 4 The figure shows the minoxidil content results of the minoxidil preparations provided by examples 1-3 and comparative examples 1-4 respectively after being stored at-4±1℃, 25±1℃ and 60±1℃ for 3 months.

[0027] Figure 5 The figure shows the results of the detection of the membrane fluidity of HaCaT cells by the minoxidil preparations prepared by examples 1-3 and comparative examples 1-4 respectively.

[0028] Figure 6 The 24h unit area cumulative permeation amount of minoxidil in the formulations provided by Examples 1 to 3 and Comparative Examples 1 to 4 is shown.

[0029] Figure 7 The steady state transdermal rate of minoxidil in the formulations provided by Examples 1 to 3 and Comparative Examples 1 to 4 is shown.

[0030] Figure 8 The lag time of minoxidil in the formulations provided by Examples 1 to 3 and Comparative Examples 1 to 4 is shown. DETAILED DESCRIPTION

[0031] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not intended to limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from the prior art.

[0032] It should be noted that the terms "first", "second", and the like in the description and claims of the present application and the above-described drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or a chronological sequence, nor do they necessarily limit the subsequent technical features. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0033] In order to better understand the present application without limiting the scope of the present application, all numbers representing amounts, percentages, and other numerical values used in the present application are understood to be modified by the word "about" in all instances. Therefore, unless otherwise specifically indicated, the numerical parameters listed in the specification and the attached claims are approximations. They can vary depending on the desired properties sought to be obtained by the ideal properties. Each numerical parameter should be considered as being at least as accurate as the rounding methods used by the numerical data reported and obtained by conventional rounding methods.

[0034] I. Raw materials

[0035] 1. Minoxidil, CAS: 38304-91-5, purity: 99% HPLC, Hangzhou Baishensheng Biological Technology Co., Ltd.

[0036] 2. Preparation of volatile oil of water crowfoot

[0037] Water crowfoot (commercially available) was ground into fine powder using a grinder. 10.0 g of water crowfoot powder was accurately weighed into an extraction beaker. A mixed solvent of anhydrous ethanol and ethyl acetate (volume ratio of ethanol to ethyl acetate was 6:4) was added according to a liquid-to-material ratio of 25:1 (mL / g). The extraction was performed using ultrasonic method at an extraction temperature of 60°C for 50 min. The extraction liquid was filtered, and the filtrate was dried under reduced pressure. After adding 1.9 g of anhydrous sodium sulfate and 0.52 g of anhydrous magnesium sulfate, the mixture was filtered to obtain 0.73 g of volatile oil of water crowfoot.

[0038] 3. Detection of components of volatile oil of water crowfoot

[0039] A gas chromatography-mass spectrometry (GC / MS) instrument (American HPGC6890 / MS5973) was used. The chromatographic conditions were as follows: HP-5MS (30 m x 0.25 mm x 0.25 μm) elastic quartz capillary column, carrier gas: helium. The vaporization chamber temperature was 240°C, the interface temperature was 230°C, the column temperature was 50°C, which was increased to 90°C at a rate of 5°C / min, then to 160°C at a rate of 10°C / min, followed by 230°C at a rate of 14°C / min, and maintained for 2 min. The mass spectrometry conditions were as follows: ionization source: EI, electron energy: 70 eV; ion source temperature: 230°C; scanning mass range: 30-500 amu; multiplier voltage: 1.40 kV.

[0040] The volatile oil of water crowfoot was determined according to the above experimental conditions. The quantification of compounds was calculated according to the relative percentage of peak area by peak area normalization. The chemical components in the volatile oil of water crowfoot were identified by comparing the mass spectrometry information obtained by GC / MS with the standard spectrum by searching the NIST98.L database. The results showed that the volatile oil of water crowfoot prepared by the above method contained 12.78% 1-methyl-3-(1-methylethyl)-benzene, 15.36% 1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene, 7.93% 3-carene, 6.59% β-phellandrene, 7.5% β-pinene, 8.13% β-myrcene, 6.52% 1-methyl-4-(1-methylethylidene)-cyclohexene, 4.96% 2-methyl-6-methylene-3,7-octadien-2-ol, 7.35% 2,6-dimethyl-1,5,7-octatrien-3-ol, 6.78% 1,9-dimethyl-7-(1-methylethyl) octahydro naphthalene, and 8.09% 7-methyl-4-methylene-1-(1-methylethyl)-octahydro naphthalene.

[0041] Preparation and performance study of minoxidil preparation

[0042] Comparative Example 1:

[0043] Table 1 Minoxidil preparation formula

[0044]

[0045] Preparation of minoxidil preparation:

[0046] (1) Weighing: according to the batch production instruction, respectively weigh the prescription amount of raw material minoxidil 15.00 kg (after drying and pure, weigh the feeding), auxiliary material ethanol 90.0 L (95% pure, convert to weight according to density, weigh the feeding), auxiliary material propylene glycol 150.0 L (after conversion to weight according to density, weigh the feeding), and 10% of the batch volume of purified water 30.00 kg.

[0047] (2) Preparation: take the prescription amount of ethanol, propylene glycol and 10% of the batch volume of purified water, add them into the preparation tank 1 in turn, start the stirring paddle, and stir for 5 minutes; take the prescription amount of raw material minoxidil, add it into the above-mentioned mixed solvent preparation tank, stir for 5 minutes to disperse the raw material in the solvent, start the emulsification homogenization pump, and homogenize for 60 minutes, then draw the liquid into the preparation tank 2 after homogenization, start the stirring until the raw material is completely dissolved, and take sample for content detection; calculate the water addition amount according to the intermediate content, and add purified water to constant volume to the theoretical capacity.

[0048] (3) Filtration: start the emulsification homogenization pump to make the liquid circulate backflow for 15 minutes; after the liquid circulation backflow is completed, start the emulsification homogenization pump to make the liquid pass through the stainless steel layered filter (filter material: PP microporous filter membrane; pore size: 0.8 μm) to the transfer tank. Start the stirring of the transfer tank and at the same time start the emulsification homogenization pump to make the liquid circulate backflow; take sample for quality inspection of intermediate product: the inspection indexes are appearance, relative density, ethanol amount, propylene glycol amount, content and microbial limit.

[0049] (4) Filling: assemble the metering pump and filling head of the liquid filling machine, after the empty machine is tested normally, put the medicine bottles into the conveying turntable and convey them to the bottle dial, put the bottle caps into the oscillation hopper, adjust the filling capacity, when the filling capacity is qualified, carry out the filling production, the capacity control is 61 ml ± 1 ml / bottle; during the filling process, the capacity and sealing property should be checked every 30 minutes, the sealing property checking method is: the cap should be screwed tightly, and it should not leak liquid when inverted.

[0050] Comparative Example 2:

[0051] Soybean lecithin, minoxidil, cholesterol and Tween 80 were precisely weighed in a beaker, and then a proper amount of anhydrous ethanol was added to dissolve them completely under heating and ultrasonic treatment. The mass ratio of soybean lecithin to minoxidil was 3:1, the mass ratio of soybean lecithin to cholesterol was 4:1, and the mass ratio of minoxidil to Tween 80 was 4:18.

[0052] The dissolved solution was slowly and evenly injected into the preheated 5 mL deionized water under constant stirring with a syringe, and magnetic stirring was continued for 30 min after the injection was completed. The prepared suspension was rotary evaporated at 45°C to remove ethanol, and then was ultrasonically treated (20%, 130 W) in an ice bath for 20 min with 3 s "on" and 3 s "off". The obtained vesicle suspension was filtered through 0.80 μm, 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain a minoxidil vesicle sample, which was stored in a refrigerator at 4°C.

[0053] Comparative Example 3

[0054] Soybean lecithin, minoxidil, cholesterol and Tween 80 were precisely weighed in a dry round-bottom flask, and then a small amount of chloroform was added to dissolve them. The solvent was evaporated under reduced pressure at 45°C with a rotary evaporator until a uniform double lipid layer was formed on the wall of the flask, and then the mixture was dried under vacuum overnight to completely evaporate the organic solvent. The mass ratio of soybean lecithin to minoxidil was 3:1, the mass ratio of soybean lecithin to cholesterol was 4:1, and the mass ratio of minoxidil to Tween 80 was 4:18.

[0055] The next day, the deposited film was hydrated with 5 g of deionized water for 1 h at a temperature higher than the phase transition temperature of phospholipid (about 44°C) and a rotation speed of 75 rpm, and the obtained lipid vesicles were fully expanded at room temperature (22-25°C) for 2 h, and then were ultrasonically treated (20%, 130 W) in an ice bath for 20 min with 3 s "on" and 3 s "off". Finally, the obtained vesicle system was further squeezed through 0.80 μm, 0.45 μm and 0.22 μm microporous filter membranes under controlled pressure (controlled manually) to obtain uniform lipid vesicles, which were stored in a refrigerator at 4°C.

[0056] Example 1

[0057] The preparation method of the minoxidil liniment provided in the example comprises the following steps:

[0058] (1) Minoxidil was dissolved in a 70% aqueous acetic acid solution, and then the raw material solution was obtained after filtration;

[0059] (2) The volatile oil of Nolana coerulea, polyethylene glycol 400 and propylene glycol were added to the raw material solution, and then a uniform mixed solution was obtained by stirring with a homogenizer for 30 min;

[0060] (3) adding soybean lecithin into the mixed solution, homogenizing for 30 min to obtain the nano minoxidil liniment for external use.

[0061] In the embodiment, the content of minoxidil in the raw material solution is 0.36 g / L, the ratio of the volatile oil of water ageratum to the raw material solution is 1 g:12.5 mL, the ratio of polyethylene glycol 400 to the raw material solution is 1 g:5 mL, the ratio of propylene glycol to the raw material solution is 1 g:2 mL, and the weight ratio of soybean lecithin to minoxidil is 2:1.

[0062] Embodiment 2

[0063] The preparation method of the minoxidil liniment provided in the embodiment comprises:

[0064] (1) dissolving minoxidil with 70% acetic acid aqueous solution, and filtering to obtain a raw material solution;

[0065] (2) adding the volatile oil of water ageratum, polyethylene glycol 400 and propylene glycol into the raw material solution, and stirring for 30 min with a homogenizer to obtain a uniform mixed solution;

[0066] (3) adding soybean lecithin into the mixed solution, homogenizing for 30 min to obtain the nano minoxidil liniment for external use.

[0067] In the embodiment, the content of minoxidil in the raw material solution is 0.52 g / L, the ratio of the volatile oil of water ageratum to the raw material solution is 1 g:10 mL, the ratio of polyethylene glycol 400 to the raw material solution is 1 g:3, the ratio of propylene glycol to the raw material solution is 1 g:1 mL, and the weight ratio of soybean lecithin to minoxidil is 2:1.

[0068] Embodiment 3

[0069] The preparation method of the minoxidil liniment provided in the embodiment comprises:

[0070] (1) dissolving minoxidil with 80% acetic acid aqueous solution, and filtering to obtain a raw material solution;

[0071] (2) adding the volatile oil of water ageratum, polyethylene glycol 400 and propylene glycol into the raw material solution, and stirring for 30 min with a homogenizer to obtain a uniform mixed solution;

[0072] (3) adding soybean lecithin into the mixed solution, homogenizing for 30 min to obtain the nano minoxidil liniment for external use.

[0073] The content of minoxidil in the raw material solution is 0.36 g / L. The proportion of water ageratum volatile oil in the raw material solution is 1 g:12.5 mL, the proportion of polyethylene glycol 400 in the raw material solution is 1 g:5 mL, the proportion of propylene glycol in the raw material solution is 1 g:2 mL, and the weight ratio of soybean lecithin to minoxidil in this embodiment is 1.8:1.

[0074] Comparative Example 4:

[0075] The preparation method of the minoxidil liniment provided in this embodiment comprises:

[0076] (1) Dissolve minoxidil with 20% acetic acid aqueous solution, and filter to obtain a raw material solution;

[0077] (2) Add water ageratum volatile oil, polyethylene glycol 400 and propylene glycol to the raw material solution, and stir with a homogenizer for 30 min to obtain a uniform mixed solution;

[0078] (3) Add soybean lecithin to the mixed solution, and homogenize for 30 min to obtain the external use nano minoxidil liniment.

[0079] The content of minoxidil in the raw material solution is 0.36 g / L. The proportion of water ageratum volatile oil in the raw material solution is 1 g:12.5 mL, the proportion of polyethylene glycol 400 in the raw material solution is 1 g:5 mL, the proportion of propylene glycol in the raw material solution is 1 g:2 mL, and the weight ratio of soybean lecithin to minoxidil in this embodiment is 1.8:1.

[0080] 1. Determination of particle size, PDI and Zeta potential

[0081] The particle size, PDI and Zeta potential were determined using a Malvern laser particle size analyzer at a scattering angle of 90°, an equilibrium time of 60 s and a temperature of 25±0.2℃. Before determination, all samples were diluted 10 times with deionized water to avoid multiple scattering effects. Each sample was measured in triplicate, and the results were expressed as mean±standard deviation (SD).

[0082] The minoxidil niosomes prepared in the above examples and comparative examples were precisely weighed, filtered through a filter membrane, added with methanol and ultrasonicated for 5 min, diluted with an appropriate multiple of sample injection by HPLC chromatography (Prabagar Balakrishnan et al. Formulation and in vitro assessment of minoxidil niosomes for enhanced skin delivery [J]. International Journal of Pharmaceutics, 2009, 377(1): 1-8), filtered through a 0.22 μm microporous filter, and analyzed to calculate the total amount of minoxidil W' in the niosomes. The drug loading of the niosomes DL = W' / W, wherein W is the added amount of minoxidil niosomes.

[0083] The results are shown in Table 2. The minoxidil niosomes prepared in Comparative Example 1 had no obvious nanoparticles (‘-’ in the table), the particle size, polydispersity index (PDI) and Zeta potential of the niosomes prepared in Comparative Examples 2-3 were higher than those of Examples 1-3, and the drug loading was lower than that of Examples 1-3. The drug loading of Comparative Example 4 was lower than that of Examples 1-3. Therefore, the minoxidil niosomes prepared in Examples 1-3 had better microscopic properties.

[0084] Table 2

[0085] Embodiment Particle size (nm) PDI Zeta potential (mV) DL (%) Example 1 72.45 0.24 -50.68 92.36 Example 2 78.37 0.25 -51.42 93.54 Example 3 74.86 0.26 -50.78 94.62 Comparative Example 1 － － － 34.68 Comparative Example 2 90.36 0.32 -36.35 76.83 Comparative Example 3 110.96 0.29 -31.14 81.64 Comparative Example 4 75.47 0.23 -49.73 46.72

[0086] 2. Stability study of minoxidil niosomes

[0087] The minoxidil niosomes provided by Examples 1-3 and Comparative Examples 1-4 were centrifuged at 10,000 rpm for 30 min, and whether there was precipitation and stratification was observed. The tested formulations were completely sealed and stored at different temperatures (-4±1℃, 25±1℃ and 60±1℃) for 3 months. The physical and chemical stability of the niosomes was evaluated by taking the sample at 0 days as a control. The physical stability was evaluated by appearance, particle size, PDI and Zeta potential; the chemical stability was determined by measuring the content of minoxidil in the niosomes by HPLC (as a percentage of the content at 0 days).

[0088] The results showed that the color of the minoxidil niosomes provided by Comparative Examples 2-3 gradually changed to light yellow during storage at 25±1℃ and 60±1℃ for 3 months, while the minoxidil niosomes provided by Examples 1-3, Comparative Example 1 and Comparative Example 4 did not have this phenomenon.

[0089] Figure 1 The particle size results of the minoxidil niosomes provided by Examples 1-3 and Comparative Examples 2-4 after storage at -4±1℃, 25±1℃ and 60±1℃ for 3 months, respectively, are shown.Figure 2 PDI results of minoxidil solutions provided by Examples 1-3 and Comparative Examples 2-4 respectively after being stored at -4±1℃, 25±1℃ and 60±1℃ for 3 months are shown. Figure 3 Zeta potential results of minoxidil solutions provided by Examples 1-3 and Comparative Examples 2-4 respectively after being stored at -4±1℃, 25±1℃ and 60±1℃ for 3 months are shown. Figure 4 Minoxidil content results of minoxidil solutions provided by Examples 1-3 and Comparative Examples 1-4 respectively after being stored at -4±1℃, 25±1℃ and 60±1℃ for 3 months are shown. It can be seen that, after being stored at -4±1℃, 25±1℃ and 60±1℃ for 3 months, the particle size, PDI, Zeta potential and content of minoxidil solutions provided by Examples 1-3 and Comparative Examples 2-4 respectively are less affected under the conditions of -4±1℃ and 25±1℃, while there is a certain impact under the condition of 60±1℃. Relative to minoxidil solutions provided by Comparative Examples 1-4 respectively, the particle size, PDI, Zeta potential and content of minoxidil solutions provided by Examples 1-3 respectively are not significantly affected, and the minoxidil solutions provided by Examples 1-3 can be better stored for a long time.

[0090] III. Cell Experiments

[0091] 1. Cells

[0092] Human immortalized epidermal cells (HaCaT cells) were purchased from the Basic Medicine Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences, and were cultured in a 37℃, 5% CO2 incubator using MEM / EBSS medium (containing 10% fetal bovine serum, 100 U / mL of penicillin-streptomycin mixture).

[0093] 2. CCK-8 Test

[0094] Test samples: minoxidil solutions prepared by Examples 1-3 and Comparative Examples 1-4 respectively. Control sample: azone. Logarithmic phase HaCaT cells were collected, and 7.0×10 3The cells were seeded in 96-well plates at a density of 3 wells per 100 μL of complete medium. Blank (PBS), control (1% DMSO in medium) and drug groups were set up. After incubation at 37°C in 5% CO2for 24 h, serially diluted test samples (0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 8.0 mg / mL, 15.0 mg / mL) and control samples (0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.08 mg / mL, 0.15 mg / mL and 0.3 mg / mL) were added. After 24 h of drug treatment, the medium was removed and 110 μL of medium containing 10 μL of CCK-8 reagent (Dojindo) was added and incubated at 37°C for 2 h. The absorbance (A) of each well was then measured at 450 nm using a microplate reader. The cell survival rate was calculated as follows: survival rate (%) = (A drug group - A blank group) / (A control group - A blank group) x 100%. The half maximal inhibitory concentration (IC50) of the drug was calculated based on the cell survival rates of serially diluted drugs.

[0095] The half maximal inhibitory concentration of aminon calculated based on the cell survival rate was 0.042 mg / mL. The half maximal inhibitory concentrations of the minoxidil formulations provided in Examples 1 to 3 were 2.033 mg / mL, 2.145 mg / mL and 2.116 mg / mL, respectively. The half maximal inhibitory concentrations of the minoxidil formulations provided in Comparative Examples 1 to 4 were 1.826 mg / mL, 2.159 mg / mL, 2.325 mg / mL and 2.075 mg / mL, respectively. These results indicated that the minoxidil formulations had low toxicity to HaCaT cells.

[0096] 3. Measurement of cell membrane fluidity

[0097] The effects of minoxidil formulations and the positive penetration enhancer aminon on the membrane fluidity of HaCaT cells were measured using fluorescence recovery after photobleaching (FRAP). Logarithmic phase HaCaT cells were collected and seeded at a density of 1 x 10 5The cell density of HaCaT cells was inoculated in a laser confocal culture dish, and 1.5 mL of complete culture medium was added to each well. Blank control group (normal culture medium), control group (culture medium containing 1% DMSO), and minoxidil agent (2.0 mg / mL) and azone drug group (0.042 mg / mL) provided by Examples 1-3 and Comparative Examples 1-4 were set, and after 24 h of incubation at 37°C in a 5% CO2 environment, the culture medium was removed and the culture medium containing the above corresponding drugs was added, and the CO2 incubator was incubated for 24 h. Then the culture medium of each group was removed and washed with PBS for 3 times, 500 μL of 2 μg / mL NBD-C6-HPC fluorescent probe solution was added to each well and incubated in a CO2 incubator for 0.5 h, then washed with PBS for 3 times, and then placed under a laser confocal microscope for testing. The instrument parameters of the laser confocal instrument were set as follows: excitation wavelength 488 nm, receiving wavelength 530 nm; laser bleaching wavelength 488 nm, power 100%, bleaching time 900 ms; scanning frequency 1.65 s, and total scanning time 120 s. The fluorescence bleaching animation at the bleaching position was recorded, and the cell membrane fluidity was characterized by the fluorescence recovery rate (R). The greater the fluorescence recovery rate, the greater the cell membrane fluidity. R = (F2-F1) / (F1-F0) x 100%, F2 is the fluorescence recovery value after fluorescence bleaching, F1 is the fluorescence value before fluorescence bleaching, and F0 is the instantaneous fluorescence value after bleaching.

[0098] The results are shown in Table 1. Figure 5 As shown in Table 1, there was no significant difference in the fluorescence recovery rate between the control group and the blank group, indicating that the culture medium and DMSO had little effect on the membrane fluidity of HaCaT cells. The 2.0 mg / mL minoxidil agent provided by Examples 1-3 could significantly improve the fluorescence recovery rate, and the improvement effect was comparable to that of azone; the 2.0 mg / mL minoxidil agent provided by Comparative Examples 1-4 had a lower improvement effect on the fluorescence recovery rate. Therefore, the minoxidil agent provided by the present application can increase the membrane fluidity of HaCaT cells, suggesting that it has the effect of reducing the skin epidermal barrier to facilitate the transdermal absorption of drugs.

[0099] III. In vitro transdermal experiment

[0100] 1. Preparation of isolated rat skin

[0101] Male SD rats (Shibefu (Beijing) Experimental Animal Technology Co., Ltd.) were sacrificed, the abdominal fur of the rats was carefully shaved with a shaver, and the abdominal skin was peeled off. The subcutaneous fat tissue and adhesions were carefully removed on a glass dish using cotton, and then washed with PBS.

[0102] 2. Transdermal test

[0103] A TK-24B drug transdermal diffusion tester was used, the upper chamber was a diffusion chamber, and the lower chamber was a receiving chamber. The volume of the receiving chamber was 8 mL, and the effective diffusion area was 3.14 cm2 The in-vitro mouse skin is fixed between two chambers, the horny layer faces the diffusion cell, and is fixed with a clamp. 2 mL of the minoxidil preparation provided by Examples 1-3 and Comparative Examples 1-4 is respectively added to the upper chamber, and a 3% polyoxyethylene (20) oil ether solution in phosphate buffered saline (PBS) is used as the receiving liquid (pH = 7.2-7.4) in the lower chamber. Air bubbles are excluded, and the liquid surface is completely in contact with the in-vitro mouse skin. The magnetic stirring speed is set to 200 r / min, the water bath temperature is controlled at (32±0.1)℃, and 1 mL of the receiving liquid is removed at 2, 4, 6, 8, 10, 12, 22 and 24 h, while an equal volume of fresh receiving liquid is added. After the sample is filtered through a 0.22 μm microporous filter, 10 μL of the filtrate is injected into the HPLC chromatography for detection of the minoxidil content. The drug transdermal rate, cumulative transdermal amount and lag time are respectively determined and calculated, and each group of experiments is performed in triplicate.

[0104] The cumulative transdermal amount of the drug is calculated according to the following formula.

[0105]

[0106] wherein Q (μg / cm 2 ) is the cumulative transdermal amount per unit area, V is the volume of the diffusion cell (8 mL), Vi is the volume of each sampling, Cn and Ci are the drug concentrations in the receiving liquid at the nth and ith sampling, respectively, and A is the diffusion area (3.14 cm 2 ). The cumulative transdermal amount is plotted against time, and the slope of the linear part (6-12 h) is the steady-state transdermal rate (Jss, μg / cm 2 ·h), and the intersection of the reverse extension line of the linear part with the X axis is the lag time (Tlag, h).

[0107] Figure 6-8 The 24 h cumulative transdermal amount per unit area, the steady-state transdermal rate and the lag time of minoxidil in the preparation provided by Examples 1-3 and Comparative Examples 1-4 are respectively shown. As can be seen from the figures, the 24 h cumulative transdermal amount per unit area and the steady-state transdermal rate of minoxidil in the preparation provided by Examples 1-3 are higher than those in Comparative Examples 1-4, and the lag time is lower than that in Comparative Examples 1-4. It can be known that the minoxidil preparation provided by the present application directly acts on the skin of male SD rats, and has a better transdermal effect.

[0108] Four, skin administration and irritation evaluation method

[0109] The skin irritation of the minoxidil preparation prepared in the above examples and comparative examples is further investigated.

[0110] 1. Single administration experiment

[0111] The guinea pigs (healthy guinea pigs, half male and half female, weighing 300-350 g, purchased from Changyang Xishan Breeding Farm in Beijing) were washed with warm water and dried before administration. 0.5 g of the minoxidil preparation was applied to both sides, and physiological saline was applied to the physiological saline group. The preparation was evenly applied to the test area, and then covered with sterile gauze patches and fixed with medical bandages. After 4 h of administration, the preparation on the administration site was removed, and the administration site was washed and wiped with warm water. At 1 h, 24 h, 48 h and 72 h after the removal of the test preparation, the skin of the administration site was observed for erythema, edema and the like. The results were scored according to the skin irritation reaction scoring standard in Table 3 for the skin at each time point, the average score was calculated according to the following formula, and the skin irritation intensity was evaluated according to Table 4. The average score of the skin irritation reaction = (total score of erythema reaction + total score of edema reaction) / number of animals in the group

[0112] 2. Multiple administration experiment

[0113] The administration method was the same as above. Another group of guinea pigs was taken, and 0.5 g of the preparation was administered once a day at the same time and the same site for 7 consecutive days. At 1 h, 24 h, 48 h and 72 h after the removal of the preparation at the last administration, the skin of the administration site was observed for erythema, edema and the like, and evaluated according to Tables 3 and 4.

[0114] Table 3 Skin irritation reaction scoring standard

[0115]

[0116] Table 4 Skin irritation intensity evaluation standard

[0117] Intensity Score No irritation 0～0.49 Mild irritation 0.50～2.99 Moderate irritation 3.0～5.99 Strong irritation 6.0～8.0

[0118] Table 5 Skin irritation intensity results

[0119]

[0120] As shown in Table 5, the minoxidil preparation provided by the present application has no skin irritation after single administration and multiple administrations. The minoxidil preparation provided by Comparative Example 1 has a mild irritation reaction 1 h after multiple administrations, and the reaction is eliminated only after 72 h. The minoxidil preparation provided by Comparative Example 3 has a mild irritation reaction 1 h after multiple administrations, and the irritation reaction is eliminated only after 48 h.

[0121] In summary, the present application uses ethanol as an excipient, uses water celery volatile oil as an excipient of the preparation, and uses glacial acetic acid as a dissolving medium, thereby improving the solubility of minoxidil and facilitating the formation of micro-nano preparations. Through the stability study of the preparation, it is found that the particle size, PDI, Zeta potential and content of the nano preparation formed do not change significantly, indicating that the minoxidil preparation provided by the present application forms micro-nano particles and can be stored for a long time.

[0122] In addition, it is proved by cell experiments that the minoxidil preparation provided by the present application can increase the membrane fluidity of HaCaT cells, suggesting that it has the effect of reducing the skin epidermal barrier to facilitate the transdermal absorption of drugs. The minoxidil preparation directly acts on the skin of male SD rats, and has a better transdermal effect.

[0123] In addition, compared with the prior art using ethanol as an excipient, the minoxidil preparation provided by the present application has no skin irritation in single and multiple dosing, thereby reducing the skin irritation.

[0124] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for preparing a minoxidil liniment, characterized by, The application relates to a minoxidil external-use nanometer liniment, which comprises the following steps: Minoxidil is dissolved in an aqueous solution of glacial acetic acid with a concentration of 50-80%, and a raw material solution is obtained after filtration; the content of minoxidil in the raw material solution is not less than 0.25 g / L; Water celery volatile oil, polyethylene glycol 400 and propylene glycol are added into the raw material solution, and a homogeneous mixed solution is obtained after stirring for 30 min by using a homogenizer; Soybean lecithin is added into the mixed solution, and external-use nanometer minoxidil liniment is obtained after homogenization for 30 min; The preparation method of the water celery volatile oil comprises the following steps: water celery is crushed into fine powder, a mixed solvent of anhydrous ethanol and ethyl acetate is added according to a liquid-material ratio of 25 mL:1 g, ultrasonic extraction is carried out at an extraction temperature of 60 DEG C, the extraction time is 50 min, the extraction liquid is filtered, the filtrate is dried under reduced pressure, anhydrous sodium sulfate and anhydrous magnesium sulfate are added and filtered, and the water celery volatile oil is obtained. The water celery volatile oil contains 12.78% 1-methyl-3- (1-methylethyl) -benzene, 15.36% 1-methyl-4- (1-methylethyl) -1, 4-cyclohexadiene, 7.93% 3-carene, 6.59% beta-ophuiene, 7.5% beta-pinene, 8.13% beta-myrcene, 6.52% 1-methyl-4- (1-methylethyl) -cyclohexene, 4.96% 2-methyl-6-methylene-3, 7-octadiene-2-ol, 7.35% 2, 6-dimethyl-1, 5, 7-octatriene-3-ol, 6.78% 1, 9-dimethyl-7- (1-methylethyl) -octahydro-naphthalene and 8.09% 7-methyl-4-methylene-1- (1-methylethyl) -octahydro-naphthalene.

2. The production method according to claim 1, characterized by, The proportion of the water celery volatile oil in the raw material solution is 1g:10-15mL, the proportion of the polyethylene glycol 400 in the raw material solution is 1g:3-8mL, the proportion of the propylene glycol in the raw material solution is 1g:1-5mL, and the weight ratio of the soybean lecithin to minoxidil is 1-2:

1.

3. The preparation method according to claim 2, characterized in that The concentration of the aqueous solution of glacial acetic acid is 70%, the content of minoxidil in the raw material solution is 0.36g / L, the proportion of the water celery volatile oil in the raw material solution is 1g:12.5mL, the proportion of the polyethylene glycol 400 in the raw material solution is 1g:5mL, the proportion of the propylene glycol in the raw material solution is 1g:2mL, and the weight ratio of the soybean lecithin to minoxidil is 2:

1.

4. The preparation method according to claim 2, characterized in that The concentration of the aqueous solution of glacial acetic acid is 70%, the content of minoxidil in the raw material solution is 0.52g / L, the proportion of the water celery volatile oil in the raw material solution is 1g:10mL, the proportion of the polyethylene glycol 400 in the raw material solution is 1g:3mL, the proportion of the propylene glycol in the raw material solution is 1g:1mL, and the weight ratio of the soybean lecithin to minoxidil is 2:

1.

5. The preparation method according to claim 2, characterized in that The concentration of the glacial acetic acid aqueous solution is 80%, the content of minoxidil in the raw material solution is 0.36 g / L, the proportion of the volatile oil of water cress in the raw material solution is 1 g:12.5 mL, the proportion of the polyethylene glycol 400 in the raw material solution is 1 g:5 mL, the proportion of the propylene glycol in the raw material solution is 1 g:2 mL, and the weight ratio of the soybean lecithin to minoxidil is 1.8:

1.

6. The minoxidil liniment prepared by the method of any one of claims 1 to 5, characterized in that, The minoxidil liniment has a particle size of 72.45-78.37 nm, a PDI of 0.24-0.26, a Zeta potential of-51.42--50.68 mV, and a loading of minoxidil of 92.36-94.62%.

7. Use of the minoxidil liniment prepared by the preparation method of any one of claims 1-6 in the preparation of a hair loss medicine.

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