Preparation and application of minoxidil eutectic crystal and liposome co-loaded with minoxidil eutectic crystal and nucleic acid

Abstract

The invention discloses preparation and application of minoxidil eutectic and liposome co-loaded with minoxidil eutectic and nucleic acid. According to the invention, minoxidil and a proper acidic ligand substance form a eutectic substance under a certain condition, so that the original solubility and percutaneous permeability of minoxidil medicine are greatly improved. The invention finds that minoxidil eutectic and androgen receptor (AR) targeted small interfering RNA (siRNA) have a synergistic effect in anti-alopecia treatment, anti-alopecia active substances with two different anti-alopecia action mechanisms are combined, and the liposome is prepared by a microfluidic technology, so that the anti-alopecia effect is greatly improved, and the low-dose and high-efficiency anti-alopecia effect is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-hair loss drugs, and particularly relates to preparation and application of a minoxidil cocrystal and a liposome co-loaded with a nucleic acid. Background Art

[0002] According to the World Health Organization, about 1.6 billion people out of the world's 7.7 billion people face the problem of hair loss, which means that on average, about 1 in 4 people suffer from hair loss, and most of them are people with androgenic alopecia. Currently, the two most recognized types of drugs for anti-hair loss treatment are topical minoxidil and oral finasteride. Although spironolactone, cyproterone acetate and ethinyl estradiol also have anti-hair loss effects, they are only suitable for women. Minoxidil and finasteride may cause side effects such as sexual dysfunction, headache, anxiety, contact dermatitis, hirsutism, breast tenderness and swelling in the treatment of hair loss. The drugs take a long time of 3 to 6 months to work, and long-term use is required to maintain the anti-hair loss effect. Discontinuation of the drug will lead to recurrent hair loss. Hair transplantation is an invasive and expensive surgical treatment method with the risk of postoperative pain and infection. Low-energy laser has a certain effect in moderate and mild androgenic alopecia, but the effect varies from person to person, and there are adverse effects such as local pain, redness, swelling, erosion or dandruff. Although platelet-rich plasma treatment is a new anti-hair loss treatment method, it has harsh storage conditions, high treatment costs, and side effects such as local pain, itching, and edema. It is currently only used in small-scale research and has not yet been popularized. Therefore, drug treatment is still the first choice for anti-hair loss treatment. Among them, minoxidil has poor water and oil solubility, and is often solubilized by a large amount of polyols to facilitate its application.

[0003] Therefore, the current trend is to reduce the frequency and dosage of use, improve the efficacy and reduce the side effects of anti-hair loss materials. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of high frequency of use, large dosage or many side effects in the prior art, and to provide a minoxidil cocrystal and a preparation method thereof, and an anti-hair loss composition and a preparation method thereof. The minoxidil cocrystal prepared by the present invention greatly improves the solubility and transdermal permeability of ordinary minoxidil in water, and expands the selection types of anti-hair loss drugs.

[0005] In order to achieve the above object, the first aspect of the present invention provides a minoxidil co-crystal, wherein the structure of the minoxidil co-crystal is as follows:

[0006]

[0007] Wherein, X is an organic acid.

[0008] The invention greatly improves the water solubility and transdermal permeability of ordinary minoxidil through the cocrystal of organic acid and ordinary minoxidil.

[0009] The second aspect of the present invention provides a method for preparing a minoxidil cocrystal, wherein the preparation method comprises: dissolving an organic acid in a first solvent to obtain an acidic ligand, dissolving minoxidil in a second solvent to obtain a minoxidil suspension, then adding the acidic ligand to the minoxidil suspension for reaction, and removing the first solvent and the second solvent after the reaction to obtain the minoxidil cocrystal.

[0010] The method for preparing eutectic provided by the invention is simple, highly operable, easy to scale up for production, and uses no toxic or harmful solvents.

[0011] The third aspect of the present invention provides an anti-hair loss composition, wherein the anti-hair loss composition comprises: the minoxidil co-crystal described in the first aspect of the present invention or the minoxidil co-crystal prepared according to the preparation method described in the second aspect of the present invention.

[0012] The anti-hair loss composition provided by the invention has good permeability, does not contain toxic solvents, and does not contain traditional ether additives, and has better safety.

[0013] A fourth aspect of the present invention provides a method for preparing an anti-hair loss composition, wherein the preparation method comprises:

[0014] After dissolving the cationic salt and polyethyleneimine in a portion of ethanol, dissolving the phospholipid, cholesterol and preservative in another portion of ethanol, and then mixing them to obtain an alcohol phase;

[0015] Dividing the DEPC water into three parts, dissolving taurine in one part of the DEPC water, dissolving androgen receptor-targeted si-RNA in the second part of the DEPC water, dissolving minoxidil cocrystal in the third part of the DEPC water, and then mixing them to obtain an aqueous phase;

[0016] The alcohol phase and the water phase are mixed by a microfluidic method to obtain an anti-hair loss composition.

[0017] The anti-hair loss composition prepared by the invention does not use toxic solvents, and the prepared lipid nanoparticles have uniform particle sizes and are monodisperse, have high drug encapsulation efficiency, and have good stability, and can realize continuous scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0019] Figure 1 is the H NMR spectrum (deuterated DMSO) of the minoxidil cocrystal and citric acid of Preparation Example 1;

[0020] Figure 2is the NMR carbon spectrum (deuterated DMSO) of the minoxidil cocrystal and citric acid of Preparation Example 1;

[0021] Figure 3 is the Fourier transform infrared spectra of the minoxidil cocrystal and minoxidil and citric acid of Preparation Example 1;

[0022] Figure 4 It is the XRD crystal diffraction of minoxidil cocrystal and minoxidil and citric acid in Preparation Example 1;

[0023] Figure 5 The minoxidil co-crystal and the transdermal retention test results of minoxidil in Preparation Example 1;

[0024] Figure 6 The minoxidil co-crystal and the transdermal penetration concentration test results of minoxidil in Preparation Example 1;

[0025] Figure 7 This is the appearance of minoxidil and the minoxidil eutectic prepared in Preparation Example 1-5 after dissolution;

[0026] Figure 8 It is the excitation / absorption fluorescence scan of FAM-labeled nucleic acid;

[0027] Fig. 9 is the linear regression line between the fluorescence intensity and concentration of a series of concentrations of nucleic acid labeled with FAM;

[0028] Fig.10 is the relative expression level graph of AR gene;

[0029] Fig.11 is a graph of the relative expression level of the TGF-β2 gene;

[0030] Fig.12 is the relative expression level graph of β-catenin gene;

[0031] Fig.13 is the relative expression level graph of iL-1α gene;

[0032] Fig.14 is a graph of the relative expression level of the DDK-1 gene;

[0033] Fig.15 It is a gel electrophoresis test diagram (from left to right: control, comparative example 3, comparative example 5, embodiment 7); DETAILED DESCRIPTION

[0034] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] The first aspect of the present invention provides a minoxidil co-crystal, wherein the structure of the minoxidil co-crystal is as follows:

[0036]

[0037] Wherein, X is an organic acid.

[0038] The invention greatly improves the water solubility and transdermal permeability of ordinary minoxidil through the cocrystal of organic acid and ordinary minoxidil.

[0039] The prepared minoxidil cocrystals should be stored dry in the presence of phosphorus pentoxide.

[0040] In some embodiments, preferably, the organic acid is citric acid, lactic acid, lactobionic acid, gluconic acid or tartaric acid, which is different from strongly acidic inorganic acids that directly form salts, while long-chain organic acids are incompatible with minoxidil and cannot form eutectics.

[0041] The second aspect of the present invention provides a method for preparing a minoxidil cocrystal, wherein the preparation method comprises: dissolving an organic acid in a first solvent to obtain an acidic ligand, dissolving minoxidil in a second solvent to obtain a minoxidil suspension, then adding the acidic ligand to the minoxidil suspension for reaction, and removing the first solvent and the second solvent after the reaction to obtain the minoxidil cocrystal.

[0042] The method for preparing the eutectic provided by the present invention is simple, highly operable, easy to scale up for production, and uses no toxic or harmful solvents. Nitrogen filling protection can be performed as needed during the reaction.

[0043] In some embodiments, preferably, the molar ratio of the organic acid to minoxidil is 1-1.5: 1. The molar ratio of the organic acid to minoxidil can be 1: 1, 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1. Within this range, too much acidic ligand will not cause skin irritation due to excessive acidity, and too little acidic ligand will not cause the inability to completely convert minoxidil into a eutectic form.

[0044] In some embodiments, preferably, the reaction time is 2-8h and the reaction temperature is 20-60°C. The reaction temperature affects the time required for the reaction. The reaction temperature can be any value between any two of 20°C, 30°C, 40°C, 50°C, and 60°C, and the reaction time can be any value between any two of 2h, 2.5h, 3h, 3.5h, 4h, 5h, 6h, 7h, and 8h.

[0045] In some embodiments, preferably, the organic acid is citric acid, lactic acid, lactobionic acid, gluconic acid and / or tartaric acid.

[0046] In some embodiments, preferably, the mass ratio of the organic acid to the first solvent is 1:8-15. The first solvent is used to dissolve the organic acid and make the obtained acidic ligand concentration appropriate. The mass ratio of the organic acid to the first solvent can be any value between any two numbers of 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0047] In some embodiments, preferably, the first solvent is water, or a mixture of water and ethanol in a volume ratio of 1:1-2.

[0048] In some embodiments, preferably, the mass ratio of minoxidil to the second solvent is 1:5-20. The second solvent is used to dissolve minoxidil, and the mass ratio of minoxidil to the second solvent can be any value between any two numbers of 1:5, 1:7, 1:9, 1:10, 1:12, 1:14, 1:15, 1:17, 1:18, 1:20.

[0049] In some embodiments, preferably, the second solvent is water.

[0050] In some embodiments, preferably, the acidic ligand is added to the minoxidil suspension at a rate of 2-5 mL / min. The addition rate of the acid-washed ligand is related to whether it can fully contact with the minoxidil suspension, thereby affecting the purity and yield of the obtained eutectic. The addition rate of the acidic ligand can be any value between any two of 2 mL / min, 3 mL / min, 4 mL / min, and 5 mL / min.

[0051] In some embodiments, preferably, the method for removing the first solvent and the second solvent is vacuum distillation at 40-60° C. The solvent used in the present invention is safe and non-toxic and can be easily removed. The vacuum distillation temperature can be any value between any two of 40° C., 50° C., and 60° C.

[0052] In some embodiments, preferably, the preparation method further comprises: removing the first solvent and the second solvent and then vacuum drying for 4-12 hours to obtain the minoxidil cocrystal.

[0053] The third aspect of the present invention provides an anti-hair loss composition, wherein the anti-hair loss composition comprises: the minoxidil co-crystal described in the first aspect of the present invention or the minoxidil co-crystal prepared according to the preparation method described in the second aspect of the present invention.

[0054] The anti-hair loss composition provided by the invention has good permeability, does not contain toxic solvents, and does not contain traditional ether additives, and has better safety.

[0055] In some embodiments, preferably, the anti-hair loss composition further comprises: androgen receptor-targeted si-RNA, cationic salts, phospholipids, cholesterol, polyethyleneimine, preservatives, alcohol solvents, taurine, and DEPC water. Using alcohol as a solvent can help improve permeability, polyethyleneimine is used to encapsulate nucleic acids to form small particles, phospholipids, cholesterol, and cations are used to form liposomes and encapsulate the outer layer of nucleic acid small particles, and taurine is used to maintain an acidic environment to prevent si-RNA from being destroyed by enzymes and improve stability.

[0056] In some embodiments, preferably, based on 100 g of the anti-hair loss composition, it includes: 0.05-5 g of minoxidil cocrystal, 0.3-3.4 mg of androgen receptor-targeted si-RNA, 5-1000 mg of phospholipids, 1-100 mg of cholesterol, 0.1-2.5 mg of cationic salts, 0.4-8.4 mg of polyethyleneimine, 0-0.5 g of preservatives, 10-100 mg of taurine, and a total volume of 100 mL of alcohol solvent and DEPC water, wherein the alcohol solvent is 20-80 mL.

[0057] Based on 100g of the anti-hair loss composition, the minoxidil cocrystal can be any value between any two of 0.05g, 0.1g, 0.3g, 0.5g, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, and 5g; the androgen receptor-targeted si-RNA can be 0.3mg, 0.34mg, 0.4mg, 0.6mg, 0.8mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 3.5g, 4g, 4.5g, and 5g. Any value between any two numbers in 4mg; phospholipids can be any value between any two numbers in 5mg, 10mg, 50mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg; cholesterol can be 1mg, 5mg, 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg , 100mg; cationic salt can be any value between any two numbers in 0.1mg, 0.13mg, 0.15mg, 0.18mg, 2mg, 2.2mg, 2.5mg; polyethyleneimine can be any value between any two numbers in 0.4mg, 0.42mg, 0.5mg, 1mg, 1.5mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 8.4mg; antiseptic The dosage can be any value between any two of 0g, 0.1g, 0.2g, 0.3g, 0.4g, and 0.5g; the dosage of taurine can be any value between any two of 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, and 100mg; the dosage of the alcohol solvent can be any value between any two of 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, and 80mL.

[0058] In some embodiments, preferably, the mass ratio of the phospholipid to cholesterol is 1-10: 1. Phospholipids and cholesterol are used to form liposomes with cations to encapsulate nucleic acids, and the mass ratio of the two can be any value between any two numbers of 1: 1, 1: 2, 1: 5, 1: 6, 1: 7, 1: 9, 1: 10.

[0059] In some embodiments, preferably, the molar ratio of the cationic salt, the unit derived from ethyleneimine monomer of polyethyleneimine and the nucleotide unit of androgen receptor targeted si-RNA is 0.5-5:5-40:1. Wherein, polyethyleneimine is polymerized from ethyleneimine monomer, polyethyleneimine comprises a plurality of repeated units derived from ethyleneimine monomer, and the androgen receptor targeted si-RNA used in the present invention comprises a plurality of nucleotide units, wherein one nucleotide unit comprises a sense strand and an antisense strand. The molar ratio of the cationic salt, the unit derived from the ethyleneimine monomer of polyethyleneimine, and the nucleotide unit of the androgen receptor targeting si-RNA can be any value between any two numbers of 0.5:5:1, 1:5:1, 2:5:1, 3:5:1, 4:5:1, 5:5:1, 0.5:8:1, 0.5:10:1, 0.5:20:1, 0.5:30:1, 0.5:40:1, 2:8:1, 2:10:1, 0.5:20:1, 2:30:1, 2:40:1, 5:8:1, 5:10:1, 5:20:1, 5:30:1, 5:40:1.

[0060] In some embodiments, preferably, the sense strand of the androgen receptor targeting si-RNA is: 5'-CUGCUACUCUUCAGCAUUATT-3', and the antisense strand is: 5'-UAAUGCUGAAGAGUAGCAGTT-3'.

[0061] In some embodiments, preferably, the phospholipids include one or more of phospholipid PC90, phospholipid PC75, phospholipid PC50, phospholipid H90, phospholipid H75, phospholipid P100, and phospholipid P75.

[0062] In some embodiments, preferably, the average molecular weight of the polyethyleneimine is 10000-30000 kDa. The average molecular weight of polyethyleneimine affects the encapsulation efficiency, and the molecular weight can be any value between any two numbers of 10000 kDa, 15000 kDa, 20000 kDa, 25000 kDa, and 30000 kDa.

[0063] In some embodiments, preferably, the preservative includes one or more of pentanediol, hexylene glycol, glycerol and butylene glycol.

[0064] In some embodiments, preferably, the alcohol solvent is ethanol.

[0065] In some embodiments, preferably, the concentration of DEPC water is 0.5-0.2 v / v%. DEPC water is enzyme-free sterile water, and its volume concentration can be 0.05 v / v%, 0.1 v / v%, 0.15 v / v%, 0.2 v / v%, and can be purchased from the market and refrigerated at 2-8°C for later use, or can be prepared as follows:

[0066] Prepare DEPC water in proportion and stir at 50-80℃ for 2-8h. Then sterilize by high pressure, cool and store at 2-8℃ for later use.

[0067] In some embodiments, preferably, the cationic salt is a quaternary ammonium salt.

[0068] In some embodiments, preferably, dodecyldimethylbenzylammonium chloride, tetradecyldimethylbenzylammonium chloride, hexadecyldimethylbenzylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, behenyltrimethylammonium chloride, didodecyldimethylammonium chloride, ditetradecyldimethylammonium chloride, dihexadecyldimethylammonium chloride, dioctadecyldimethylammonium chloride and their corresponding ammonium bromide salts are one or more. The above ammonium chloride salts are also applicable after being replaced with the corresponding ammonium bromide salts.

[0069] A fourth aspect of the present invention provides a method for preparing an anti-hair loss composition, wherein the preparation method comprises:

[0070] Dissolving the cationic salt and polyethyleneimine in a portion of the alcohol solvent, dissolving the phospholipid, cholesterol and preservative in another portion of the alcohol solvent, and then mixing them to obtain an alcohol phase;

[0071] Dividing the DEPC water into three parts, dissolving taurine in one part of the DEPC water, dissolving androgen receptor-targeted si-RNA in the second part of the DEPC water, dissolving minoxidil cocrystal in the third part of the DEPC water, and then mixing them to obtain an aqueous phase;

[0072] The alcohol phase and the water phase are mixed by a microfluidic method to obtain an anti-hair loss composition.

[0073] The anti-hair loss composition prepared by the invention is essentially a liposome and has two levels of transdermal permeability.

[0074] In some embodiments, preferably, the flow rate ratio of the alcohol phase to the aqueous phase is 1: 1-4. The flow rate ratio of the alcohol phase to the aqueous phase affects the particle size of the prepared liposomes. Generally, the smaller the particle size, the better the permeability. The flow rate ratio of the alcohol phase to the aqueous phase can be any value between any two numbers of 1: 1, 1: 2, 1: 3, and 1: 4.

[0075] In some embodiments, preferably, the total flow rate of the alcohol phase and the aqueous phase is 8-24 mL / min. The total flow rate of the alcohol phase and the aqueous phase can be any value between any two numbers of 8 mL / min, 12 mL / min, 16 mL / min, 20 mL / min, and 24 mL / min.

[0076] In some embodiments, preferably, the anti-hair loss composition further comprises an emulsifier, a preservative, a thickener, etc. The use of the emulsifier, preservative, and thickener meets the relevant requirements of daily chemical products. The thickener can be carbomer, AVC, etc., and the preservative can be a polyol, such as pentanediol, hexanediol, glycerol, butylene glycol, etc. The anti-hair loss composition can also be used in combination with other nutrients, such as further adding amino acids, etc.

[0077] In some embodiments, preferably, the method of using the anti-hair loss composition comprises invasive and / or non-invasive transdermal delivery.

[0078] In some embodiments, preferably, the anti-hair loss composition further comprises one or more of polyvinyl alcohol, xanthan gum, sodium hyaluronate, polyvinyl pyrrolidone, amino acid, and thickener. There is no special requirement for the selection of polyvinyl alcohol, and commercially available products are all acceptable. The anti-hair loss composition of the present invention is combined with biocompatible materials such as polyvinyl alcohol, xanthan gum, sodium hyaluronate, and polyvinyl pyrrolidone to obtain a microneedle for preventing hair loss, or an anti-hair loss essence used in conjunction with a mechanical microneedle, or an anti-hair loss essence product, an anti-hair loss ointment, an anti-hair loss oil control gel drug, etc. used alone. The amount of polyvinyl alcohol, xanthan gum, sodium hyaluronate, and polyvinyl pyrrolidone is used as needed, and meets the relevant industry standards.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] 1. The present invention forms a eutectic substance with minoxidil and a suitable ligand substance under certain conditions, which greatly improves the solubility and percutaneous permeability of ordinary minoxidil in water, avoids the adverse reactions caused by the large amount of alcohol ether cosolvents used in traditional preparations, and expands the selection types of anti-hair loss drugs;

[0081] 2. The preparation of the anti-hair loss composition provided by the present invention does not involve the use of toxic solvents, and the prepared lipid nanoparticles have uniform particle size and are monodisperse, with high drug encapsulation efficiency and good stability, and can achieve continuous scale-up production;

[0082] 3. The anti-hair loss composition prepared by the present invention can be administered through minimally invasive microneedle invasive or non-invasive transdermal administration, which greatly improves drug compliance and reduces metabolic problems and potential side effects caused by the common injection method of current nucleic acid drugs / cosmetics;

[0083] 4. The anti-hair loss composition prepared by the present invention develops potassium ion channels through minoxidil, inhibits calcium ion influx, and promotes the expression of vascular endothelial growth factor. The androgen receptor-targeted siRNA produces a silencing effect on the expression of the androgen receptor, inhibits the binding of dihydrotestosterone to the androgen receptor, and further cascades to produce an anti-hair loss effect. The combination of two different anti-hair loss mechanisms has good compatibility and a synergistic effect, which improves the anti-hair loss effect, achieves a low-dose and high-efficiency anti-hair loss effect, and avoids repeated medication.

[0084] According to a particularly preferred embodiment of the present invention, based on 100g of the anti-hair loss composition, it includes: 0.2-5g of minoxidil cocrystal, 0.34-3.4mg of androgen receptor-targeted si-RNA, 5-100mg of phospholipids, 20-100mg of cholesterol, 0.5-2.5mg of cationic salts, 0.42-4.3mg of polyethyleneimine, 0-0.5g of preservatives, 10-50mg of taurine, an alcohol solvent with a total volume of 100mL and DEPC water, wherein the alcohol solvent is 20-80mL.

[0085] The present invention will be described in detail below through examples.

[0086] The sources of the main raw materials used in the following examples and comparative examples are shown in Table 1, and those not mentioned are common commercially available products.

[0087] Table 1 List of raw materials

[0088]

[0089] The molecular weight of the nucleic acid (si-RNA) of the present invention is 13300, and the molecular weight of each nucleotide unit is 332.5; polyethyleneimine includes about 295 units derived from ethyleneimine monomers, and the molecular weight of each unit is 34.

[0090] In the following examples and comparative examples, PDI was measured by laser particle size analyzer; minoxidil cocrystal was characterized by H-NMR, C-NMR, Fourier transform infrared and XRD; transdermal permeability was measured by Franz diffusion cell method; encapsulation efficiency was measured by ultrafiltration centrifugation method; particle size and Zeta potential were measured by laser particle size analyzer; and stability was measured by appearance observation method.

[0091] 1. Preparation of Minoxidil Cocrystal

[0092] Preparation Example 1

[0093] At room temperature, 20.2 g of citric acid (with a molar ratio of 1.05:1 to minoxidil) was dissolved in 202 g of water to obtain an acidic ligand, and then 20.9 g of minoxidil was dispersed in 104.5 g of water and stirred to prepare a minoxidil suspension, and then the acidic ligand was added dropwise to the minoxidil suspension at a rate of 2.5 mL / min, and the mixture was stirred until transparent and then heated. After reacting at 45 ° C for 8 h, the water was removed by reduced pressure distillation at 60 ° C, and vacuum dried for 12 h to obtain a minoxidil eutectic product, which was finally dried and stored in phosphorus pentoxide.

[0094] The minoxidil cocrystal prepared in Preparation Example 1 was characterized by H-NMR, C-NMR, FT-IR and XRD. Figure 1-4 shown.

[0095] Figure 1 The NMR hydrogen spectra (deuterated DMSO) of the minoxidil cocrystal and citric acid of Preparation Example 1 are shown in the figure. The upper part is the NMR hydrogen spectrum of citric acid, and the lower part is the NMR hydrogen spectrum of the minoxidil cocrystal of Preparation Example 1. By comparison, it can be seen that in the NMR hydrogen spectrum, the displacement of the hydrogen spectrum of the minoxidil cocrystal relative to that of citric acid disappears, indicating that the hydrogen of the carboxylic acid in the citric acid disappears or is shielded due to the formation of the cocrystal with minoxidil.

[0096] Figure 2 The NMR carbon spectra (deuterated DMSO) of the minoxidil cocrystal and citric acid in Preparation Example 1, the upper part is the NMR hydrogen spectrum of citric acid, and the lower part is the NMR hydrogen spectrum of the minoxidil cocrystal in Preparation Example 1. By comparison, it can be seen that compared with citric acid, the carbon spectrum of the minoxidil cocrystal has obvious splitting changes at 174.99, 171.75, 72.91, and 43.14, and the chemical shift also has a certain shift, indicating that the changes may be caused by the formation of a cocrystal between citric acid and minoxidil.

[0097] Figure 3 The Fourier transform infrared spectra of minoxidil cocrystal and minoxidil and citric acid in Preparation Example 1 show that compared with citric acid, minoxidil cocrystal has a higher peak at 3495 cm -1 and 3292cm -1 The OH stretching vibration of the minoxidil eutectic is shielded, compared with minoxidil, which has a wavelength of 3200-3500 cm -1 There are multiple absorption peaks, which are due to the shielding of the symmetric and asymmetric stretching vibrations of the two primary amines. -1 and 2847cm -1 There was no significant change in the CH2 stretching vibration at the position, indicating that the carboxyl and amino groups of citric acid and minoxidil interacted with each other.

[0098] Figure 4The XRD patterns of the minoxidil cocrystal of Preparation Example 1 and minoxidil and citric acid show that the XRD crystal diffraction absorption peaks of minoxidil and citric acid monomers are significantly different from those of the minoxidil cocrystal in position and absorption intensity, and the minoxidil cocrystal has obvious diffraction absorption peaks, indicating that minoxidil and citric acid form a cocrystal.

[0099] The minoxidil co-crystal prepared in Preparation Example 1 was further tested for its transdermal penetration enhancing effect. The method is as follows:

[0100] The Franz diffusion cell method was used to analyze the transdermal permeation and retention of different samples. Panama pig skin was used as the permeation object. The minoxidil content was detected by HPLC. The transdermal permeation concentration and transdermal retention of minoxidil or minoxidil eutectic solution (dissolved in PBS buffer, the concentration of minoxidil or minoxidil eutectic was 0.2wt%) at the same concentration were tested at 4h, 8h, and 16h. The results are shown in Figure 5 and Figure 6 ,in Figure 5 is the retention of the two, Figure 6 is the osmotic concentration of the two.

[0101] analyze Figure 5 and 6 It can be seen that as time goes by, the transdermal permeation concentration and transdermal retention of minoxidil cocrystal (minoxidil citrate cocrystal) relative to minoxidil are increasing. The transdermal permeation concentration and transdermal retention of minoxidil cocrystal (minoxidil citrate cocrystal) relative to minoxidil are increased by 3.95 times and 6.70 times at the 16h time point, respectively. Therefore, minoxidil cocrystal has a good transdermal penetration enhancement effect, which can greatly improve its own transdermal permeation and retention concentration, thereby providing the possibility of achieving a hair loss prevention effect of reducing the amount and increasing the efficiency.

[0102] The above experiments prove that the co-crystal of organic acid and minoxidil can be prepared by the method provided by the present invention, and the obtained co-crystal has good permeability.

[0103] Preparation Example 2

[0104] At room temperature, 40.6 g of lactobionic acid (with a molar ratio of 1.1:1 to minoxidil) was dissolved in 200 g of water and stirred and heated to 60° C. to obtain an acidic ligand, and 20.9 g of minoxidil was dispersed in 50 g of water and stirred to prepare a minoxidil suspension, and then the acidic ligand was dropwise added to the minoxidil suspension at a rate of 4.5 mL / min, stirred and heated, the temperature was 60° C., under nitrogen protection, reacted for 4 hours, and then the water was removed by reduced pressure distillation at 60° C., vacuum dried for 8 hours to obtain a minoxidil eutectic product, and finally dried and stored in phosphorus pentoxide.

[0105] Preparation Example 3-5

[0106] The method of Preparation Example 1 is referred to, except that citric acid is replaced by lactic acid, tartaric acid and gluconic acid in sequence.

[0107] The minoxidil co-crystals prepared in the above Preparation Examples 1-5 and minoxidil alone were dissolved in water respectively, and their solubility was tested. The results are shown in Table 2.

[0108] Table 2 Solubility test results

[0109] name Solubility (room temperature, water) Appearance Minoxidil <2mg / mL Precipitation, not completely dissolved Preparation Example 1 Cocrystal >80 mg / mL Slightly yellow transparent Preparation Example 2 Cocrystal >80 mg / mL Yellow transparent Preparation Example 3 Cocrystal >50 mg / mL Slightly yellow transparent Preparation Example 4 Cocrystal >50 mg / mL Colorless and transparent Cocrystal of Preparation Example 5 >80 mg / mL Yellow transparent

[0110] As can be seen from Table 2, compared with pure minoxidil, the solubility of the cocrystals prepared in Preparation Examples 1-5 is significantly improved, the solubility is increased by 25 times or even more than 40 times. The appearance of minoxidil and its cocrystal after dissolution is shown in Table 2. Figure 7 As shown in the figure, from left to right, the 6 bottles dissolved are minoxidil, the cocrystal of Preparation Example 5, the cocrystal of Preparation Example 2, the cocrystal of Preparation Example 3, the cocrystal of Preparation Example 1, and the cocrystal of Preparation Example 4. The improved solubility of the cocrystal prepared by the present invention is conducive to the use of high-dose minoxidil. At the same time, the minoxidil cocrystal can improve the transdermal penetration performance of minoxidil without affecting the chemical bonds of minoxidil, which is conducive to exerting its anti-hair loss effect.

[0111] 2. Microfluidic Parameter Selection

[0112] In order to obtain an anti-hair loss composition with better penetration effect, an experiment was conducted according to the anti-hair loss composition of the following composition, and the obtained product is referred to as liposome:

[0113] Phospholipid (PC90) 500 mg, cholesterol 10 mg, dodecyltrimethylammonium chloride 1.3 mg, polyethyleneimine 1.72 mg, minoxidil cocrystal prepared in Preparation Example 1 0.1 g, nucleic acid (siRNA) 0.34 mg, taurine 10 mg, ethanol and DEPC water total volume is 100 mL, the specific volumes of the two are shown in Table 2.

[0114] Dissolving dodecyltrimethylammonium chloride and polyethyleneimine in one portion of ethanol, dissolving phospholipid and cholesterol 1,2-hexanediol in another portion of ethanol, and mixing the two portions of ethanol mixture to obtain an alcohol phase;

[0115] The DEPC water was divided into three parts, taurine was dissolved in one part of the DEPC water, and the androgen receptor-targeted si-RNA was dissolved in the second part of the DEPC water, and the minoxidil cocrystal was dissolved in the third part of the DEPC water, and then they were mixed to obtain an aqueous phase.

[0116] The microfluidic device parameters were set as follows: the initial volume of 0.5 mL was discarded, the total flow rate of the alcohol phase and the water phase was 12 mL / min, the samples were collected and the particle size and dispersion coefficient (particle size distribution index, PDI) of the samples were tested using a laser particle size analyzer. The results are shown in Table 3.

[0117] Table 3 Particle size and particle size distribution index of samples at different alcohol / water flow rates

[0118] Volume ratio of ethanol and DEPC water Alcohol / water flow rate ratio Particle size, nm PDI, % 20:80 1:4 88.6 12.1 33:67 1:2 124.5 10.4 50:50 1:1 156.4 16.3 67:33 2:1 208.3 18.6 80:20 4:1 281.0 19.2

[0119] From the results in Table 3, it can be seen that when the alcohol / water phase flow rate is relatively low (the volume of the water phase is larger), the particle size of the prepared liposomes decreases, the particle size is monodisperse, and the PDI is <20%. Generally, the smaller the particle size, the easier it is to penetrate the skin, but more water phase will dilute the sample concentration. Therefore, it is more appropriate to select an alcohol / water phase flow rate ratio of 1:1-4.

[0120] Furthermore, based on the alcohol phase and aqueous phase prepared as above, the microfluidic parameters were set: initial discard volume 0.5 mL, alcohol phase to aqueous phase flow rate ratios 1:1, 1:1, 1:1, 1:4, 1:4, 1:4, and total flow rates corresponding to 8 mL / min, 16 mL / min, 24 mL / min, 8, 16 mL / min, and 24 mL / min, respectively. The particle size and polydispersity coefficient of the obtained lipid nanoparticles were tested by a laser particle size analyzer, and the results are shown in Table 4.

[0121] Table 4 Particle size results under different total flow rate and alcohol / water phase flow rate ratio parameters

[0122]

[0123]

[0124] It can be seen from Table 4 that when the flow rate ratio of the alcohol phase to the aqueous phase is moderate, the total flow rate increases and the particle size does not change significantly. When the flow rate ratio of the alcohol phase to the aqueous phase is small, the total flow rate increases and the liposome particle size becomes larger. The larger the aqueous phase / alcohol phase ratio leads to a greater change in particle size. However, when the microfluidic setting has different flow rate ratios or different total flow rates, the prepared particle sizes are all monodisperse.

[0125] 3. Preparation of anti-hair loss composition

[0126] The preparation methods of the following Examples 1-11 are as follows:

[0127] Dissolving dodecyltrimethylammonium chloride and polyethyleneimine in one portion of ethanol, dissolving phospholipid and cholesterol 1,2-hexanediol in another portion of ethanol, and mixing the two portions of ethanol mixture to obtain an alcohol phase;

[0128] The DEPC water was divided into three parts, taurine was dissolved in one part of the DEPC water, and the androgen receptor-targeted si-RNA was dissolved in the second part of the DEPC water, and the minoxidil cocrystal was dissolved in the third part of the DEPC water, and then they were mixed to obtain an aqueous phase.

[0129] The microfluidic device parameters were set as follows: the initial volume of 0.5 mL was discarded, the total flow rate of the alcohol phase and the aqueous phase was 16 mL / min, and the flow rate ratios of the alcohol phase and the aqueous phase of Examples 1-11 were: 1:4, 1:4, 2:3, 2:3, 1:4, 2:3, 3:7, 1:1, 3:7, 3:7, 1:4. The compositions of each example are shown in Table 5.

[0130] The preparation methods of Comparative Examples 1-6 are as follows:

[0131] The method of Example 1-11 was followed, except that the flow rate ratio of the alcohol phase to the water phase was 1:4.

[0132] The preparation method of Comparative Example 7 was carried out by a conventional solvent array method, as follows:

[0133] Organic phase: 1 mL of dihexadecyldimethylammonium chloride (540 mg / L) diluted with ethanol and 1 mL of polyethyleneimine (860 mg / L) diluted with ethanol were mixed with phospholipids, cholesterol, 1,2-hexanediol and ethanol (18 mL), stirred evenly at room temperature, and the volatile solvent was removed by reduced pressure distillation at 40°C to obtain an organic phase;

[0134] Aqueous phase: dilute and dissolve the minoxidil eutectic prepared in Preparation Example 1 with 20 mL of DEPC water, dissolve by stirring at room temperature to obtain a minoxidil eutectic solution, dilute the nucleic acid with 1 mL of DEPC water to obtain a nucleic acid solution, dilute taurine with the remaining DEPC water to obtain a taurine solution, dissolve by stirring at room temperature, and place in a refrigerator at 2-8°C for later use;

[0135] The minoxidil eutectic solution and taurine solution in the aqueous phase were added to the organic phase after the solvent was removed by rotary evaporation, hydrated and stirred in a 60°C water bath for 1 hour, then ultrasonically treated for 1 hour, cooled to room temperature and filtered three times with a 0.45 μm filter membrane, and finally the nucleic acid solution was added and mixed to obtain a comparison sample, which was then refrigerated at 2-8°C.

[0136] The compositions of Comparative Examples 1-7 are shown in Table 5.

[0137] Table 5 Composition of each embodiment and comparative example

[0138]

[0139]

[0140] Table 5 (Continued)

[0141]

[0142]

[0143] 1. The samples prepared in Examples 1-11 and Comparative Examples 1-7 were tested for their particle size, Zeta potential, nucleic acid encapsulation efficiency and appearance stability after one month, wherein the particle size and Zeta potential were tested by a laser particle size analyzer. When observing the appearance stability, the presence of flocculation, precipitation and / or stratification was considered unstable, otherwise it was stable. The nucleic acid encapsulation efficiency test method was as follows:

[0144] The encapsulation efficiency was tested by ultrafiltration centrifugation. The principle is: by using FAM-labeled siRNA (FAM-siRNA), the relative fluorescence intensity represents the relative content of siRNA, and the encapsulation efficiency is determined by comparing the fluorescence intensity of the original solution before ultrafiltration centrifugation with the fluorescence intensity after ultrafiltration centrifugation. The specific operation is as follows: first, a series of standard concentration samples are prepared with FAM-siRNA, and the fluorescence intensity of the series of standard concentration samples is measured to obtain a series of fluorescence intensities. The series of intensities should be curve-fitted with the concentrations of the series of standard concentration samples to obtain a linear regression line for subsequent calculations. Figure 8 and Fig. 9 They are the excitation / absorption fluorescence scanning diagram of FAM-labeled nucleic acid and the linear regression line of fluorescence intensity and concentration.

[0145] Accurately pipette 3 mL of the sample to be tested (the si-RNA in which has been labeled with FAM in advance), add it into a Millipore 20kd ultrafiltration tube, centrifuge it at 5000 r / min for 30 min at 4°C, collect 0.5 mL of the filtrate after filtration, dilute it to 5 mL with DEPC water, use the Flexstation3 cell calcium flow detection system to detect the fluorescence intensity at a wavelength of 492 nm, and calculate the concentration through the linear regression curve. The concentration at this time is the free fluorescence concentration C.

[0146] Take the same 3 mL of the sample to be tested (the si-RNA in which has been labeled with FAM in advance), add 0.5 mL of 10% Triton X-100 solution, vortex for 3 minutes, add DEPC water to make up to 5 mL, detect the fluorescence intensity, and calculate the concentration through the linear regression curve. The concentration at this time is the total fluorescence concentration Ctotal. The nucleic acid encapsulation efficiency is calculated as follows:

[0147] Nucleic acid encapsulation efficiency = (C 总 -C 游 ) / C 总 ×100%.

[0148] The results of the above tests are shown in Table 6.

[0149] Table 6 Particle size, Zeta potential, nucleic acid encapsulation efficiency test and stability results

[0150] project Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Particle size nm 126.8 136.7 178.2 195.4 117.0 212.4 PDI% 15.4 9.7 (Twin Peaks) 22.5 (Twin Peaks) 10.6 (Twin Peaks) 8.6 15.9 (Twin Peaks) Zeta potential mV 13.3 23.2 17.4 20.7 15.5 19.2 Nucleic acid encapsulation rate% 99.72 99.94 99.54 99.60 98.72 99.94 Appearance stability Stablize Stablize Stablize Stablize Stablize Stablize project Example 7 Example 8 Example 9 Example 10 Embodiment 11 Comparative Example 1 Particle size nm 181.5 230.2 213.8 179.6 121.3 156.3 PDI% 11.3 16.9 10.4 8.8 21.5 (Twin Peaks) 26.3 Zeta potential mV 15.1 18.3 16.5 16.3 15.4 18.1 Nucleic acid encapsulation rate% 99.45 99.06 99.88 99.67 99.70 99.53 Appearance stability Stablize Stablize Stablize Stablize Stablize Stablize project Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Particle size nm 69.2 390.4 231.8 156.2 268.4 340.8 PDI% 16.5 27.6 (Twin Peaks) 18.0 10.4 28.3 (three peaks) 33.5 (Twin Peaks) Zeta potential mV 18.3 3.2 16.8 17.5 17.9 18.3 Nucleic acid encapsulation rate% / 68.58 86.42 99.84 89.50 82.7 Appearance stability Stablize Unstable Stablize Stablize Stablize Unstable

[0151] As can be seen from Table 6, the particle size of the samples of Examples 1-11 is in the range of 100-300nm, and has a weak positive charge characteristic, the absolute value of Zeta potential is 10-25mV, and generally has a good encapsulation rate for nucleic acids, the encapsulation rate is>98%, the stability is good within one month, and there is no flocculation or precipitation. Among them, phospholipids with relatively high phosphatidylcholine content, such as PC90, P100, and H90, are monodispersed nanoparticles prepared by microfluidics, while the nanoparticles prepared by phospholipids with low phosphatidylcholine content are bimodal distribution, that is, a small and a large particle size distribution, and the small particle size is <10nm, which may be due to the small particle size produced by the impurity content in the phospholipids. Liposomes prepared by systems without cationic salts or traditional methods are unstable and will produce flocculation precipitation. The encapsulation rate of liposomes prepared by systems without phospholipids, systems without cationic salts, systems without polyethyleneimine, or traditional methods for ribonucleic acid is low relative to the encapsulation rate prepared by microfluidics.

[0152] 2. Detection of the relative expression levels of genes (AR, TGF-β2, DKK-1, β-catein, iL-1α) in HFDPC cells by RT-PCR

[0153] Configure the RT-qPCR reaction system as shown in Table 7.

[0154] Table 7 RT-qPCR reaction system

[0155]

[0156]

[0157] The experimental operation method is as follows:

[0158] (1) Collect cell pellets and use the TRIZOL method to extract total RNA from tissues;

[0159] (2) According to the RNA concentration, take 1 μg RNA template and 1 μL oligo(dT) 18 Primers were supplemented with RNase-free H2O to 12 μL, reacted in a constant temperature metal bath at 65°C for 5 min, and then immediately placed on ice for precooling;

[0160] (3) Take 4 μL of 5× reaction buffer, 1 μL of Ribolock RNase inhibitor, 2 μL of 10 mM dNTP mix, and 1 μL of RevertAid M-Mulv (200 U / μL), respectively, mix them, add them to the pre-cooling system in step (2), react at a constant temperature metal bath at 42°C for 60 min, then react at 70°C for 5 min, and freeze the product cDNA at -80°C for later use;

[0161] (4) Take 0.4 μL of the cDNA (50 ng / μL) synthesized in step (3) above, add 2×KAPA qPCR MasterMix Universal, 10 μL; 10 μM Forward primer, 0.4 μL; 10 μM Reverse primer, 0.4 μL; RNase-free H2O, 8.8 μL and mix;

[0162] (5) Amplification detection was performed using a fluorescent quantitative PCR instrument (because the product was less than 300 bp, a two-step method was used for amplification). The reaction conditions were: ① pre-denaturation: 95°C / 3 min; ② denaturation: 95°C / 3 s, annealing / extension: 60°C / 30 s, ② a total of 40 cycles; according to the Ct value, 2 -△△Ct Relative mRNA levels.

[0163] The AR (androgen receptor) gene is the target gene for siRNA silencing. After inhibiting AR expression, dihydrotestosterone cannot bind to AR, thereby inhibiting the subsequent hair loss problem. It is the upstream source of the target gene for controlling hair loss. After DHT modeling, the androgen receptor gene is strongly expressed. The AR gene has different changes after treatment in different embodiments or comparative examples. Among them, Example 7 and Comparative Example 1, Comparative Example 5, and Comparative Example 6 are all significantly reduced, while Comparative Examples 2-4 have no significant effect on the expression of androgen receptor AR (see Fig.10 ), indicating that systems without nucleic acid, ionic liquid or polyethyleneimine are ineffective, and minoxidil cocrystal has no inhibitory effect on androgen receptor.

[0164] The TGF-β2 gene is a growth inhibitor for most types of cells and an inducer of the catagen phase in the hair cycle. It is possible that it induces the abnormal hair cycle of AGA in the early catagen phase, inhibits the proliferation of epithelial cells and stimulates the synthesis of specific apoptotic proteases. The test results show that the TGF-β2 gene is significantly increased after modeling, while the addition of the system of Example 7 and Comparative Examples 1-6 significantly reduces its expression level. Comparative Example 2 containing only minoxidil citrate cocrystal also has a significant effect, and the effects of Example 7 and Comparative Example 5 containing both minoxidil citrate cocrystal and nucleic acid are the best (see Fig.11), indicating that minoxidil cocrystal and nucleic acid have a synergistic effect on TGF-β2 gene expression.

[0165] The β-catenin gene is in the skin hair follicle tissue, and the Wnt signal regulates the growth cycle of hair matrix cells. Once the Wnt signal transduction is blocked, the hair follicles will enter the degeneration phase prematurely, leading to hair loss. Experiments have shown that Wnt signal activation can promote the hair follicles to restart the growth phase from the resting phase and promote new hair production. The test results show that the expression of the β-catenin gene in the model group is blocked, while Example 7 and Comparative Example 5 have significantly increased its expression, and Comparative Examples 1 and 6 also have significant improvements (see Fig.12 ), indicating that the effect of minoxidil citrate without minoxidil is reduced, while minoxidil citrate alone has no effect, indicating that there is also a synergistic effect on β-catenin gene expression.

[0166] The expression of inflammatory chemokine iL-1α gene was significantly increased by more than 10 times after modeling, while Example 7, Comparative Example 1, Comparative Example 5 and Comparative Example 6 all had a significant effect on reducing the expression of inflammatory factors, while Comparative Examples 2-4 were ineffective (see Fig.13 ), indicating that the effect of nucleic acid requires the delivery of ionic liquid and polyethyleneimine, while minoxidil citrate cocrystal has no significant inhibitory effect, indicating that the anti-hair loss mechanism of minoxidil cocrystal is not anti-inflammatory. Therefore, nucleic acid and minoxidil cocrystal for AR in this study have different anti-hair loss mechanisms.

[0167] The DDK1 gene is a typical inhibitor of the Wnt / β-catenin signaling pathway. It mainly competitively binds to the LRP5 / 6 receptor with the Wnt protein, antagonizes the activity of the Wnt / β-catenin signaling pathway, regulates cell proliferation, differentiation and carcinogenesis, affects cell apoptosis, tumor cell invasion and metastasis, and in the anti-hair loss effect, DKK-1 can inhibit the growth of outer root sheath cells and trigger apoptotic cell death. The test results show that the DDK-1 gene expression in the model group is strong, while Example 7 and Comparative Examples 1-6 have a significant effect of reducing its gene expression level (see Fig.14 ). This indicates that both minoxidil cocrystal and nucleic acid have effects on DDK-1.

[0168] In summary, nucleic acid and minoxidil cocrystals have similar inhibitory effects on the expression of DDK-1 and TGF-β2 genes, while nucleic acid has a significant inhibitory effect on AR, β-catenin and iL-1α genes, and promotes the expression of β-catenin, while minoxidil in minoxidil cocrystal has no effect. Therefore, by combining nucleic acid and minoxidil cocrystal, there is a significant synergistic effect on the expression of genes related to anti-hair loss.

[0169] 3. Gel electrophoresis

[0170] The stability of the newly prepared pure siRNA samples and the samples of Comparative Example 3, Comparative Example 5 and Example 7 (all with a concentration of 25 nmol / L) at 4° C. for one month was tested by 2.5 wt % polyacrylamide gel electrophoresis.

[0171] Test results see Fig.15 (The first row in the figure is for positioning, and the test results are shown in the second row. From left to right in the second row are the test results of blank siRNA samples (pure siRNA stored in cryopreservation, with the best stability), Comparative Example 3, Comparative Example 5 and Example 7). The results show that a certain amount of nucleic acid residues can be detected in Example 7, while no nucleic acid is present in Comparative Example 3 and Comparative Example 5. Therefore, cationic ionic liquids or taurine have an effect on the stability of nucleic acids, and the test feedback shows that the stability of the example samples is higher.

[0172] 4. Anti-hair loss human efficacy

[0173] The raw materials of the above Examples 1-11 and Comparative Examples 2 and 3 were prepared into gel products (based on the total amount of the extremely easy gel product, the composition from Examples 1-11 or Comparative Examples 2 and 3 was 10 wt%, carbomer U21 0.4 wt%, thickener AVC 0.3 wt%, xanthan gum 0.1 wt%, hexylene glycol 0.5 wt%, p-hydroxyacetophenone 0.5 wt%, arginine 0.2 wt%, ethanol 5 wt%, and deionized water as the balance), and minoxidil (2 wt%) and the eutectic from Preparation Example 1 (2 wt%) were used for comparison.

[0174] Test steps: 200 volunteers aged 20-55 years with androgenic alopecia, alopecia areata or hair loss were screened, including 132 males and 68 females. The subjects were randomly divided into 20 groups, with 10 people in each group. The samples were randomly divided into groups for blind testing and applied to the scalp. The frequency of use was gradually reduced: 1 time / day (first 2 weeks), 4 times / week (3-4 weeks), 2 times / week (after 4 weeks). It can be used in the morning and evening, and the hair needs to be cleaned before use. Pump 3 to 5 times of sample solution (about 2-5mL) each time and apply it to the scalp where the hair is falling out, and massage until absorbed. Use it continuously for 2 months. No other cosmetics, anti-hair loss drugs or health products that may affect the results were used during the test. The hair follicle hair analyzer was used before and after use to detect the changes in hair follicle density at the hair loss site (area 5cm×5cm), and the average value was taken. The results are shown in Table 8. At the same time, the improvement of the subjects' hair loss symptoms before and after use was evaluated. The evaluation criteria are as follows:

[0175] 1) Excellent - hair loss stops and healthy hair regrows in the hairless areas;

[0176] 2) Good: Hair loss is significantly improved, and some new hair grows in the hairless areas;

[0177] 3) Effective: Hair loss is improved to a certain extent, and a small amount of new hair grows in some hairless areas;

[0178] 4) Ineffective - no significant relief of hair loss and no change in hairless areas.

[0179] In addition, the results of volunteers’ self-assessment of self-effectiveness are shown in Table 9.

[0180] Table 8 Changes in average hair follicle density (number / cm 2 )

[0181]

[0182]

[0183] The results in Table 8 show that under the condition of using once a day and gradually reducing the frequency to twice a week, the anti-hair loss supramolecular nucleic acid composition of the present invention has a good anti-hair loss effect. The changes in the number of hair follicles in Examples 2, 4, 5 and 10 are significantly improved compared with the control example of using minoxidil and the cocrystal of minoxidil citric acid (2 wt %) alone, and the cocrystal of minoxidil and citric acid (2 wt %) has a better effect of promoting hair follicle generation than minoxidil of the same concentration. Comparative Example 1 does not contain minoxidil cocrystal, but has a good anti-hair loss effect by virtue of the anti-hair loss nucleic acid; Comparative Example 2 does not contain nucleic acid, and the minoxidil cocrystal content in the sample is low, so there is no obvious anti-hair loss effect; Comparative Example 3 does not contain ionic liquid, and the delivery effect of nucleic acid is poor, resulting in a poor anti-hair loss effect; Comparative Example 4 does not contain polyethyleneimine, and the poor delivery effect of nucleic acid also leads to a poor anti-hair loss effect; Comparative Example 5 does not contain taurine, and it has been proved that taurine has an effect on the long-term stability of nucleic acid in the sample in terms of stability. Therefore, the long-term stability of nucleic acid in the raw material is reduced, resulting in a reduced anti-hair loss effect; Comparative Example 6 does not contain phospholipids, resulting in the inability to form a smaller liposome structure, poor permeability, and poor anti-hair loss effect; Comparative Example 7 is a minoxidil cocrystal and nucleic acid co-loaded liposome prepared by a traditional thin film evaporation method, which has a low encapsulation rate, a large particle size, and a worse anti-hair loss effect than the liposome prepared by the present invention. Compared with Comparative Example 1 in which minoxidil cocrystal 2% is used alone or nucleic acid is used alone, minoxidil cocrystal and nucleic acid co-loaded liposomes have better anti-hair loss effect. Therefore, minoxidil cocrystal and AR target nucleic acid have a synergistic effect in anti-hair loss treatment. The supramolecular liposomes prepared by the two through microfluidic technology have better penetration-enhancing effect and stability, which greatly improves the anti-hair loss effect of nucleic acid and minoxidil cocrystal, achieving a low-dose and high-efficiency anti-hair loss effect.

[0184] Table 9 Self-assessment of hair loss improvement

[0185]

[0186]

[0187] The results of the volunteers' self-assessment of hair loss in Table 9 show that the proportion of people whose hair loss is significantly improved in each embodiment is higher than that in the case of minoxidil alone or comparative example 3, especially in the case of high-concentration nucleic acid, embodiments 2, 4 and 5 all report significant effects, indicating that the composition obtained in the present invention has a good anti-hair loss effect.

[0188] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A minoxidil eutectic, characterized in that: The structural formula of the minoxidil co-crystal is as follows: Wherein, X is an organic acid.

2. The minoxidil cocrystal according to claim 1, wherein The organic acid is citric acid, lactic acid, lactobionic acid, gluconic acid or tartaric acid.

3. A method for preparing minoxidil cocrystal, characterized in that: The preparation method comprises: dissolving an organic acid in a first solvent to obtain an acidic ligand, dissolving minoxidil in a second solvent to obtain a minoxidil suspension, then adding the acidic ligand into the minoxidil suspension to react, and after the reaction is completed, removing the first solvent and the second solvent to obtain a minoxidil eutectic.

4. The preparation method according to claim 3, wherein The molar ratio of the organic acid to minoxidil is 1-1.5:1; And / or, the reaction time is 2-8h, and the reaction temperature is 20-60°C; And / or, the organic acid is citric acid, lactic acid, lactobionic acid, gluconic acid and / or tartaric acid.

5. The preparation method according to claim 3 or 4, wherein The mass ratio of the organic acid to the first solvent is 1:8-15; And / or, the first solvent is water, or a mixture of water and ethanol in a volume ratio of 1:1-2; And / or, the mass ratio of the minoxidil to the second solvent is 1:5-20; And / or, the second solvent is water; and / or, the acidic ligand is added to the minoxidil suspension at a rate of 2-5 mL / min; and / or, the method for removing the first solvent and the second solvent is distillation under reduced pressure at 40-60° C.; And / or, the preparation method further comprises: removing the first solvent and the second solvent and then vacuum drying for 4-12 hours to obtain the minoxidil cocrystal.

6. An anti-hair loss composition, characterized in that: The anti-hair loss composition comprises: the minoxidil co-crystal according to claim 1 or 2 or the minoxidil co-crystal prepared by the preparation method according to any one of claims 3 to 5.

7. The anti-hair loss composition according to claim 6, wherein The anti-hair loss composition further comprises: androgen receptor-targeted si-RNA, cationic salt, phospholipid, cholesterol, polyethyleneimine, preservative, alcohol solvent, taurine and DEPC water; Preferably, based on 100 g of the anti-hair loss composition, it comprises: 0.05-5 g of minoxidil cocrystal, 0.3-3.4 mg of androgen receptor-targeted si-RNA, 5-1000 mg of phospholipids, 1-100 mg of cholesterol, 0.1-2.5 mg of cationic salts, 0.4-8.4 mg of polyethyleneimine, 0-0.5 g of preservatives, 10-100 mg of taurine, and a total volume of 100 mL of alcohol solvent and DEPC water, wherein the alcohol solvent is 20-80 mL; Preferably, the mass ratio of phospholipid to cholesterol is 1-10:1; Preferably, the molar ratio of the cationic salt, the unit derived from the ethyleneimine monomer of polyethyleneimine, and the nucleotide unit of the androgen receptor targeting si-RNA is 0.5-5:5-40:

1.

8. The anti-hair loss composition according to claim 7, wherein the sense strand of the androgen receptor-targeted si-RNA is: 5'-CUGCUACUCUUCAGCAUUATT-3', and the antisense strand is: 5'-UAAUGCUGAAGAGUAGCAGTT-3'; and / or, the phospholipid comprises one or more of phospholipid PC90, phospholipid PC75, phospholipid PC50, phospholipid H90, phospholipid H75, phospholipid P100, and phospholipid P75; And / or, the average molecular weight of the polyethyleneimine is 10000-30000 kDa; And / or, the preservative includes one or more of pentanediol, hexylene glycol, glycerol and butylene glycol; And / or, the alcohol solvent is ethanol; And / or, the concentration of DEPC water is 0.05-0.2 v / v%; And / or, the cationic salt is a quaternary ammonium salt; Preferably, the quaternary ammonium salt comprises: One or more of dodecyldimethylbenzylammonium chloride, tetradecyldimethylbenzylammonium chloride, hexadecyldimethylbenzylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, behenyltrimethylammonium chloride, didodecyldimethylammonium chloride, ditetradecyldimethylammonium chloride, dihexadecyldimethylammonium chloride, dioctadecyldimethylammonium chloride and their corresponding ammonium bromide salts.

9. A method for preparing the anti-hair loss composition according to claim 7, characterized in that: The preparation method comprises: Dissolving the cationic salt and polyethyleneimine in a portion of the alcohol solvent, dissolving the phospholipid, cholesterol and preservative in another portion of the alcohol solvent, and then mixing them to obtain an alcohol phase; Dividing the DEPC water into three parts, dissolving taurine in one part of the DEPC water, dissolving androgen receptor-targeted si-RNA in the second part of the DEPC water, dissolving minoxidil cocrystal in the third part of the DEPC water, and then mixing them to obtain an aqueous phase; The alcohol phase and the water phase are mixed by a microfluidic method to obtain an anti-hair loss composition; Preferably, the flow rate ratio of the alcohol phase to the water phase is 1:1-4; Preferably, the total flow rate of the alcohol phase and the aqueous phase is 8-24 mL / min.

10. Use of the anti-hair loss composition according to claim 7, characterized in that: The application mode of the anti-hair loss composition includes invasive and / or non-invasive transdermal delivery; Preferably, the anti-hair loss composition further comprises one or more of polyvinyl alcohol, xanthan gum, sodium hyaluronate, polyvinyl pyrrolidone, amino acid, and thickener.

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