CN101129378A - Medicament spraying agent used for accelerating growth of hair - Google Patents

Abstract

The invention discloses a drug spray to accelerate the growth of hair, which comprises the following parts: minuodier, lecithine, vitamin E, natural essential oil and alcohol, wherein all or part of the components can be applied to prepare spray agent with alcohol liposome minuodier, which is covered by liposome to solve the solubility of minuodier and uniformity of drug in the water to strengthen the percutaneous absorption and drug effect after transmitting the drug into derma. The invention avoids side effect to reduce the toxicity for blood circulating system and stimulation for skin, which protects skin effectively.

Description

A medicinal spray for promoting hair growth

Technical Field

The invention belongs to the field of medicines, relates to a medicinal spray preparation, and particularly relates to a medicinal spray for promoting hair growth.

Background

Alopecia is a common clinical and frequently encountered disease, and although not life threatening, has a great influence on social interaction and mental health of patients. Most products currently on the market are ineffective, as demonstrated by the fact that the FDA only approved two therapies for treating alopecia ^[1] . At present, the hair tissue engineering and the hairy skin tissue engineering are not completely successful, and a plurality of difficulties still exist. Therefore, aiming at the intractable hair regeneration in skin beauty, the research and development of the medicine for treating the alopecia with high efficiency, specificity and small side effect has important significance for treating the alopecia.

Minoxidil (minoxidil) minoxidil ^[2] Belongs to a pyrimidine derivative (2, 4-diamino-3-hydroxy-6-piperidine pyrimidine), also known as Chang Ding or Ming le Xue Ding, and is the first external medicine approved by FDA for treating alopecia. Minoxidil induces hair growth and increases hair shaft diameter. 1996. The 2% minoxidil solution was formally approved by the FDA for the market as an over-the-counter drug for the treatment of alopecia caused by androgens in both men and women. The 5% topical minoxidil solution approved for marketing in 1998 remained as an over-the-counter drug, but was used only for male hair loss, the drug being sold under the trade name Rogine ^[3] . Schoolers at home and abroad ^[4~6] In-vivo and in-vitro experiments prove that the minoxidil has obvious hair growth promoting effect. The specific mechanism is not clear yet, and the minoxidil can directly promote cell proliferation and developmentPotassium ion channel, increase subcutaneous blood flow, increase papillary VEGF expression, and activate prostaglandin endoperoxidase (PGHS). Although the minoxidil external-use medicine of the common preparation has certain effect, the skin external-use medicine can act on hair follicles only through percutaneous absorption, but the transmittance of the skin external-use medicine is low (the subcutaneous absorption capacity is about 5 to 36 percent), the human body experiment result and the result of in vitro hair follicle culture have certain gap,meanwhile, adverse reactions of a cardiovascular system exist, the treatment time is long (9-12 weeks), and after the medicine is stopped for 6 months, new hairs gradually fall off, and the like. The key to improve the drug effect and reduce the adverse reaction is to improve the transdermal rate and the skin storage capacity. Therefore, the development of a high-efficiency, low-toxicity and hair follicle targeting local drug is urgently needed in clinic.

Liposome (liposome) hair disease treatment has historically presented two important problems: (1) the lesion is limited to hair, and the concentration of the drug reaching the local part of the skin is extremely limited through in vivo transportation, distribution and metabolism after systemic administration, so that the local part can reach the ideal treatment concentration and systemic toxic and side effects are caused; (2) some drugs have good curative effect when administered systemically, and once administered locally, the drug has little or no curative effect on local lesions due to poor transdermal absorption performance. Therefore, the research on an external medicinal preparation with good transdermal performance is the key point for solving the problems. The characteristics of liposome preparations themselves just meet this need. Liposome ^[7] Is a hollow pellet composed of phospholipid double-layer membrane. Compared with other formulations, the liposome can enable the medicament to have larger skin stratum corneum penetration amount, can be biodegraded, has no toxicity and does not generate skin irritation, and in recent years, the liposome is found to have the advantages of promoting transdermal absorption, keeping high concentration of the medicament on the skin and selectively targeting hair follicles, thereby being paid attention to and becoming a hotspot of research. The investment of international resources markets at present is most advocated, the most concerned is the nano material technology, and the liposome is the most mature, industrialized and commercialized entry point in the nano field at present. Because of its non-toxicity, high efficiency, wide application range and simple preparationSingle, no immunogenicity, easy realization of targeting and the like, and is widely concerned by clinicians and large pharmaceutical factories. The main routes of drug entry into the body through the skin are the following three: by penetrating cells, penetrating through intercellular spaces, and entering through openings in the cutaneous appendages (e.g., openings in hair follicles, sebaceous glands, sweat glands). The intercellular pathway is once considered to be the major pathway, while the follicular pathway is considered to function only as a bypass. Recent studies have seen different views, gradually emphasizing the importance of the third pathway, especially the important role played by the hair follicle in the transdermal process of drugs ^[8] . Research shows that the transdermal speed of the medicine passing through the opening of the hair follicle is obviously higher than that of the medicine passing through the cortex layer, in particular to the water-soluble medicine ^[9] . The liposome is used as a carrier of the drug, can promote the transdermal absorption of the drug and can also keep the high concentration of the drug in the skin. Liposome for promoting penetration of drug into skin cortexMay be associated with hydration, penetration mechanisms, fusion mechanisms, pilosebaceous pathways. The liposome has hair follicle affinity ^[10～12] The liposome-encapsulated anti-adriamycin monoclonal antibody is loaded into rat hair follicles, and alopecia caused by adriamycin is prevented, so that a new idea and a new method are provided for treating and preventing acquired alopecia caused by chemotherapy or radiotherapy. The characteristic of high selectivity of liposome loading suggests that the liposome has great potential in the aspect of practical and safe hair disease treatment.

The following are references given by the inventors:

[1]George Cotsarelis and Sarah E.Millar.Towards a molecular understanding of hair loss and its treatment[J].TRENDS in Molecular Medicine， 2001，7(7)：293～301。

[2]Lucky AW，Piacquadio DJ，Ditre CM，A randomized，placebo-controlled trial of 5％ and 2％ topical minoxidil solutions in the reatment of female pattern hair loss.J Am Acad Dermatol.2004 Apr；50(4)：541～553。

[3] schwann, butcher army wave, populus tremula, progress in the research of hair growth promoting drugs [ J ]. Journal of cosmeceutics in china, 2004.13 (5): 620 to 622.

[4] Experimental study of the effects of NGF and estrogen on ex vivo cultured human scalp hair follicles [ J ] cosmetic surgery, 2004, 20 (1): 48 to 50.

[5]Namazi MR，Handjani F.An immunologic mechanism for the therapeutic efficacy of minoxidil in alopecia areata.J Drugs Dermatol.2004 Mar-Apr；3(2)：129～130。

[6]Nippon.Hair growth effect of minoxidil[J].Yakurigaku Zasshi，2002， 119(3)：167～174。

[7] Chentao, wangjiuge, fujingduo, etc., the current research status and prospect of liposome pharmaceutical preparation [ J ]. World latest medicine, 2003, (2) 4:721 to 728.

[8]Lademann J，Otberg N，Richter H，et al.Follicular penetration. An important pathway for topicaily applied substances[J].Hautarzt， 2003，54 (4)：321～323。

[9]Ogiso T，Shiraki T，Okajima K，et al.Transfollicular drug delivery：penetration of drugs through human scalp skin and comparison of penetration between scalp and abdominal skins in vitro[J].J Drug Target，2002， 10(5)：369～378。

[10]Ciotti SN，Weiner N.Follicular liposomal delivery systems[J]..J Liposome Res，2002，12(1-2)：143～148。

[11] Butcher, wangyoucheng, populus, and the like, research progress of transdermal absorption of liposomes and targeting of hair follicles [ J ]. Journal of chinese plastic surgery, 2005.21 (3): 221 to 223.

[12]Hoffman RM.Gene and stem cell therapy of the hair follicle.Methods Mol Biol.2004：289：437～448。

Disclosure of Invention

The invention aims to provide a medicine spray for promoting hair growth, which aims to increase the solubility of a medicine and the permeability of skin cuticle by utilizing the complementary action of a liposome drug delivery technology, an antioxidant, a skin protective agent and a multi-element composite transdermal absorption permeation promoting agent, and has the effects of promoting transdermal absorption, targeting hair follicles, promoting hair growth and protecting skin.

The technical scheme adopted for realizing the invention is that,

the medicinal spray for promoting hair growth comprises the following effective components:

1) The dosage of the minoxidil used as the medicine is 0.5 to 20 percent (weight ratio);

2) The dosage of lecithin used as liposome coating material, transdermal absorption penetration enhancer and skin protectant is 50-80% (weight ratio);

3) Vitamin E as antioxidant, antioxidant and skin protection, in an amount of 0 to 0.4% (molar ratio)

4) Azone as transdermal absorption enhancer, with 0-0.2% (mol ratio);

5) The ethanol used as transdermal absorption penetration enhancer has the dosage of 10 to 40 percent (weight ratio);

6) The natural essential oil used as liposome packing material and transdermal absorption penetration enhancer is 0-10% (mol ratio);

the lecithin used as the liposome coating material, the transdermal absorption enhancer and the skin protective agent can be: one or more of egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, synthetic lecithin and sphingomyelin.

The natural essential oil used as the liposome coating material and the transdermal absorption enhancer can be: one or more of oleum Menthae Dementholatum, folium Eucalypti Globueli oil, oleum Rosae Rugosae, oleum Pini, oleum Caryophylli, thyme oil, lavender oil, oleum sesami, lemon oil, spearmint oil, bergamot oil, and oleum Santali albi.

The invention better solves the problem of water solubility of the medicine by liposome coating, enhances the transdermal absorption of the medicine and sends the medicine into the dermis layer to improve the concentration of the local medicine, obviously improves the medicine effect and enhances the action time of the medicine; meanwhile, the use of the traditional cosolvent with side effects is avoided, the medicine and the cosolvent entering the systemic blood circulation system can be reduced, and in addition, the phospholipid liposome not only reduces the stimulation to the skin, but also has good protection effect on the skin.

Drawings

FIG. 1 is a flow chart of the manufacturing process of the present invention;

FIG. 2 is a graph showing the particle size distribution of alcohol-containing liposomes detected by a laser scattering method;

FIG. 3 is a hollow lamellar vesicle (TEM 200, 000X) comprising alcoholic liposomes with single or multiple compartments;

FIG. 4 is a PE-Rhodamine alcoholic liposome transdermal condition (10X) under fluorescence microscope;

FIG. 5 shows the transdermal condition of calcein aqueous solution (10X) under fluorescence microscope;

fig. 6 is a transdermal condition of calcein-containing alcoholic liposomes (10 ×) under a fluorescence microscope;

FIG. 7 calcein blank CSLM (20 ×);

FIG. 8 is PE-Rhodamine blank CSLM (20 ×);

fig. 9 is a calcein aqueous solution transdermal condition CSLM (20 ×);

fig. 10 is a calcein-containing liposomal transdermal condition CSLM (20 ×);

FIG. 11 shows the transdermal profile CSLM (20X) of PE-Rhodamine alcoholic liposomes;

FIG. 12A, B, C are photographs of 1.4% liposomal minoxidil administered on days 1, 10, and 18

Fig. 12D is 1.4% liposomal minoxidil group skin administered for 18 days (HE x 200);

FIG. 13A, B, C are photographs of the 1.4% group of common minoxidil administered on days 1, 10, 18;

FIG. 13D is 1.4% normal minoxidil group skin (HE X200) administered for 18 days;

FIG. 14A, B, C are photographs of the placebo group taken on days 1, 10, 18;

FIG. 14D is blank control skin (HE X200) taken for 18 days;

FIG. 15 is the expression of VEGF in the sebaceous glands of hair follicles of the 1.4% alcoholic liposomal minoxidil group (SP X400) at day 18 of administration

FIG. 16 is the expression of VEGF in pilosebaceous follicles of the blank control group (SP X400) at day 18 of administration.

The present invention will be described in further detail with reference to the accompanying drawings and examples carried out by the inventors in accordance with the technical solutions of the present invention.

Detailed Description

1. The prescription, the preparation process and the process flow of the invention

1.1 inventive formulations

1) Best prescription

Name(s)	Molecular weight	Molar ratio (%)	Dosage (g)
				Egg phosphorusFat and oil	760		4.0
Minoxidil	209.25		1.0
				Azone compounds	281.48		0.002
Vitamin E	472		0.006
				Mint oil			0.06

2) The function of the auxiliary materials in the prescription

Ingredient name	Ingredient prescription effect
		Lecithin (lecithin) Minoxidil	Liposome coating material, transdermal absorption penetration promoter and skin protective agent Hair growth promoting medicine
Azone compounds	Transdermal absorption penetration enhancer
		Vitamin E	Antioxidant, skin protectant
Mint oil	Perfume, transdermal absorption enhancer, and antibacterial effect

1.2 preparation Process

The whole liposome production process is carried out under the protection of nitrogen.

1. Taking prescription amount of lecithin, minoxidil, azone and V _E Adding 20 ml-40 ml absolute ethyl alcohol (the dosage of the ethyl alcohol is controlled to be 10% -40% of the volume of the prepared liposome liquid medicine) into the medicines such as the peppermint oil and the like, and heating and stirring the mixture to be completely dissolved.

2. Slowly adding the ethanol medicine solution into 80-60 ml of purified water stirred at high speed (3000 r/min) by a shearing machine, and continuously stirring for 30min under the protection of nitrogen to obtain the multiphase liposome medicine solution.

3. Homogenizing the multiphase liposome medicine solution for 7-8 times by a high-pressure extruder under the pressure of 3-6MPa to obtain the large single-chamber single-phase liposome medicine solution with the particle size range of 150-250 nm. The final product should be a homogeneous milky solution without precipitation, delamination.

4. The product is bottled and stored at 2-15 deg.c in dark condition.

1.3 Process flow (see FIG. 1 for a preparation Process flow diagram of the present invention)

1) Firstly, preparing various prescription medicaments and auxiliary materials;

2) Adding ethanol, heating (40 + -2 deg.C), and stirring for 15min;

3) Collecting the ethanol medicinal solution;

4) Adding the ethanol medicinal solution into deionized water, and stirring with a shearing machine for 30min to obtain multiphase liposome medicinal liquid;

5) Homogenizing the multiphase liposome liquid medicine for 7-8 times by using an extruder to obtain the large single-chamber single-phase liposome liquid medicine.

2.1 examples

Example first part: dosage range of prescription of hair growth promoting medicine

The invention verifies the dosage range of the listed chemical synthesis hair growth promoting drugs in the prescription. Under the process and conditions of the invention, the components and the dosage of the liposome prescription are kept constant except that the dosage of lecithin is adjusted correspondingly according to the dosage of minoxidil, and any dosage of the chemically synthesized drug minoxidil within the content range of 0.5-20% can be ensured to be prepared into a stable final product due to the solubility problem of minoxidil. Although stable liposome solutions can be prepared with minoxidil content of less than 0.5%, the solution concentration does not reach the effective hair growth promoting concentration, and therefore, no medicinal significance is observed in the examples.

Example 1: the drug liposome product contains 1.5 percent of minoxidil

The prescription of the liposome comprises the following components:

name(s)	Molecular weight	Molar ratio (%)	Batch (g)
				Lecithin (lecithin)	760		4.0
Minoxidil	209.25		1.5

The prescription dose of lecithin and minoxidil is added into 40ml of ethanol, heated to 45 ℃ and stirred to be completely dissolved. And slowly adding the ethanol medicinal solution into 60ml of purified water stirred at high speed (3000 r/min) by a shearing machine, and continuously stirring for 30min under the protection of nitrogen to obtain the multiphase liposome medicinal solution. Homogenizing the multiphase liposome drug solution for 7 times by a high-pressure extruder under the pressure of 4 MPa-6 MPa and the temperature of not more than 60 ℃ in the homogenizing process to obtain the large single-chamber single-phase liposome drug solution with the particle size of 200nm +/-50 nm. The final product after dilution is a uniform milky solution without precipitation and delamination. The product is stored under the conditions of 2-15 ℃ and light protection after being filled.

Example 2: the drug liposome product contains 0.5% minoxidil

A pharmaceutical liposome prescription:

name (R)	Molecular weight	Molar ratio (%)	Batch (g)
				Lecithin (lecithin)	760	80	8.36
Minoxidil	209.25	20	0.5

Taking prescription dose of lecithin and minoxidil, adding 24ml of ethanol, heating and stirring until the lecithin and the minoxidil are completely dissolved. Slowly adding the ethanol medicinal solution into 76ml of purified water stirred at high speed (3000 r/min) by a shearing machine, and continuously stirring for 35min under the protection of nitrogen to obtain the multiphase liposome medicinal solution. Homogenizing the multiphase liposome medicinal solution for 6 times with a high pressure extruder under 4-6MPa at a temperature not higher than 60 deg.C to obtain large single-chamber single-phase liposome medicinal solution with particle diameter of 200nm + -35 nm. The final product is a uniform milky solution without precipitation and delamination. The product is stored under the conditions of 2-15 ℃ and light protection after being filled.

And (3) particle size detection:

adding 25 μ l of the prepared alcoholic liposome minoxidil into 25ml volumetric flask, diluting with physiological saline to constant volume, adding 250 μ l into small test tube, and loading on machine (NICOMP) ^TM 380PSS-NICOMP submicron particle sizer). The average was taken 3 times. The distribution of the particle size of the alcoholic liposomes was measured by laser scattering, and the mean particle size was 194.7nm (see FIG. 2). The particle size is relatively uniform, and the particle size of most of the liposome is distributed between 50nm and 500 nm.

The average particle size of 8 batches of samples was 195.25. + -.3 nm as determined by laser light scattering.

Laser scattering method for detecting particle size of 1% alcohol-containing liposome of different batches

Sample (I)	Average particle diameter (nm)
		1	199.3
2	192.6
		3	200.4
4	193.4
		5	195.7
6	192.8
		7	196.1
8	191.7
		Average	195.25±3

And (3) structure detection:

suspending the liposome with 10mmol/L ammonium acetate and 50mmol/L Tris solution (pH7.5), dropping the sample on copper mesh with support membrane, drying with filter paper, dropping ammonium acetate, drying with filter paper, naturally drying for 30min, and observing liposome structure with transmission electron microscope (HITACHI H-600).

1% of alcoholic liposome minoxidil (4% lecithin and 40% ethanol) is taken for observation under a transmission electron microscope, and the alcoholic liposome is a single-chamber or multi-chamber hollow thin-layer vesicle with uniform particle size (see figure 3). And (3) determining the embedding rate of the medicine:

1) Reversed-phase high performance liquid phase method:

chromatographic conditions are as follows: a chromatography column, alltech 18 (150 mm x 4.6mm,5 μm); mobile phase, methanol, water, glacial acetic acid (70: 30: 0.1) and 0.2g of decyl sodium sulfonate ion-pair reagent; the detection wavelength is 254nm; the sample volume is 20 mul, and the flow rate is 1ml/min; the column temperature was 37.0 ℃. The retention time of the minoxidil is about 6 min.

2) Linear test:

precisely weighing appropriate amount of minoxidil as reference substance, ultrasonically dissolving with methanol (chromatographic alcohol), making into solutions with concentrations of 0.001,0.01,0.05,0.25,0.75, and 1.25mg/ml, respectively sucking 20 μ l precisely, injecting into liquid chromatograph, and measuring. The 1g value of the peak area to the concentration is regressed to obtain a regression equation as follows: y =1.0039x + 7.6563 r2=0.9997, r =0.99985 (n = 8). Test results show that the sample injection amount of the minoxidil reference substance is in a good linear relation within the range of 0.001-1.25 mg/ml.

3) And (3) precision experiment:

taking the same reference substance solution for repeated sample injection for 5 times, recording the peak area value of minoxidil, and calculating RSD as follows: 0.51%, indicating that the method precision is good.

4) Stability test:

sampling the same sample test solution at 1h,2h,4h,6h and 8h respectively, recording the peak area value of minoxidil, and calculating RSD as: 0.75 percent, which indicates that the sample test solution has good stability within 8 hours.

5) The entrapment rate of the drug was determined by ultracentrifugation:

the alcoholic liposomal minoxidil was centrifuged at 20000rpm for 4h at 4 ℃ in a TL-100 ultracentrifuge. And detecting the suspended medicine amount and the precipitated medicine amount by a reversed-phase high-performance liquid phase method.

The minoxidil encapsulation efficiency was calculated by the following formula:

[(T-C)/T]×100％

wherein T is the sum of the amount of suspended drug and the amount of precipitated drug, and C is the amount of suspended drug.

Under the condition of the chromatogram, the phospholipid does not interfere the determination of the minoxidil, the peak shape of the minoxidil is good, and the analytical requirements are met.

The average encapsulation rate of the minoxidil in the alcohol-containing liposome of different batches is 90.4 +/-6.2 percent by detecting the encapsulation rate of the minoxidil by a high performance liquid chromatography through an ultracentrifugation method. The minoxidil encapsulation efficiency was calculated by the following formula:

[(T-C)/T]×100％。

HPLC method for detecting entrapment rate of minoxidil in alcohol-containing liposomes in different batches

Sample (I)	Encapsulation efficiency (%)
		1	84.5
2	83.6
		3	92.1
4	97.2
		5	98.1
6	96.2
		7	86.4
8	85.1
		Average	90.4±6.2

And (3) testing the stability:

and preliminarily judging the stability condition of the liposome by observing the characters and re-measuring the particle size 360 days after preparing the sample.

DLS detection of three batches of samples at different times shows that the particle size of the preparation is stable at room temperature for 1 year. The liquid appeared to be uniform milky without precipitation and delamination.

Method for detecting particle size stability nm of 1% alcoholic liposome by laser scattering method

)

Detection time after preparation (sky)	Batches of
				1	2	3
	1	-	194±4				195±6
30				-	198±2	199±7
	60	-	201±10				203±13
180				-	201±5	205±9
	360	205±8	-				-

Example second part: dosage range of each package material prescription of liposome

The invention verifies the dosage range of each packaging material of the listed liposome. Under the process and conditions of the invention, the use amount of 1% minoxidil is kept constant, and meanwhile, under the premise of ensuring that the total amount of lecithin, oleic acid, azone, vitamin E and peppermint oil is 85 mol%, any one of the use amounts of lecithin in the content range of 65 mol% -80 mol%, azone in the content range of 0-0.1 mol%, vitamin E in the content range of 0.05 mol% -0.2 mol% and peppermint oil in the content range of 0.05 mol% -0.3 mol% can be prepared into a stable final product.

Example 3: the drug liposome product contains 1% minoxidil

The prescription of the liposome comprises the following components:

name(s)	Molecular weight	Molar ratio (%)	Batch (g)
				Lecithin	760		4.0
Minoxidil	209.25		1.0
				Azone	281.48		0.002
Vitamin E	472		0.006
				Mint oil			0.06

Adding 24ml ethanol into prescription dosage of lecithin, minoxidil, azone, vitamin E and peppermint oil, heating and stirring until completely dissolved. Slowly adding the ethanol medicinal solution into 76ml of purified water stirred at high speed (3000 r/min) by a shearing machine, and continuously stirring for 35min under the protection of nitrogen, thus obtaining the multi-phase liposome medicinal solution. Homogenizing the multiphase liposome medicinal solution for 6 times with a high pressure extruder under 4-6MPa at a temperature not higher than 60 deg.C to obtain single-chamber single-phase liposome medicinal solution with particle size of 200nm + -35 nm. The final product is a uniform milky solution without precipitation and delamination. The product is stored under the conditions of 2-15 ℃ and light protection after being filled.

Example 4: the drug liposome product contains 0.5 percent of minoxidil:

a pharmaceutical liposome prescription:

name(s)	Molecular weight	Molar ratio (%)	Batch (g)
				Lecithin (lecithin)	760	80	8.36
Minoxidil	209.25	20	0.5
				Azone	281.48		0.002
Vitamin E	472		0.006
				Mint oil			0.06

Adding the prescription dose of lecithin, minoxidil, azone, vitamin E and peppermint oil into 40ml of ethanol, heating and stirring until the lecithin, the minoxidil, the azone, the vitamin E and the peppermint oil are completely dissolved. Slowly adding ethanol medicinal solution into 60ml of purified water stirred at high speed (3000 r/min) by a shearing machine, and continuously stirring for 35min under the protection of nitrogen gas to obtain the multi-phase liposome medicinal solution. Homogenizing the multiphase liposome medicinal solution for 6 times with a high pressure extruder under 4-6MPa at a temperature not higher than 60 deg.C to obtain single-chamber single-phase liposome medicinal solution with particle size of 200nm + -35 nm. The final product is a uniform milky solution without precipitation and delamination. The product is stored under the conditions of 2-15 ℃ and light protection after being filled.

Example third section: transdermal experimental model for detecting penetration promoting effect of alcohol-containing liposome minoxidil

Example 5:

recovery rate test:

taking blank C57BL/6 mouse in-vitro skin, uniformly crushing by a homogenizer, taking a proper amount, respectively adding quantitative minoxidil methanol (chromatographic alcohol) solutions according to different concentrations, ultrasonically mixing, standing for 2h, centrifuging for 10min at 7000g, and taking the supernatant. Extracting twice, mixing the extractive solutions, filtering with microporous membrane, and detecting the filtrate.

Sample	Added (μg)	Found (μg)	Recovery (％)	Averagerecovery (％)	RSD (％)
							25.24	25.13	99.56
Minoxidi	15.13 10.01 5.26 1.06 0.21 0.02 0.004	14.58 9.78 5.15 0.99 0.198 0.019 0.0038	96.36 97.70 97091 93.40 94029 0.95 0.95	96.15	2.10

Transdermal permeation assay of samples:

transdermal experiment operating procedure: c57BL/6 mice were sacrificed, the back villi were excised, the back skin was removed, the subcutaneous adipose tissue was scraped, and the pretreated skin was fixed on a percutaneous permeation device. Firstly, placing a magnetic stirrer in a receiving pool, then, flatly placing the prepared in-vitro rat skin at the upper end of the receiving pool with the cutin layer facing upwards, and then, fixing a release pool on the receiving pool by a clamp. Quantitatively adding 7ml of 1% alcohol-containing liposome minoxidil and 1% common minoxidil solution into a release tank, injecting normal saline into a receiving tank through a sampling tube by using an injector, exhausting air to make the other side of the skin closely contact with the receiving liquid, and stirring at constant temperature of 37 ℃ for 120 r/min. Sampling 0.5ml in the receiving pool for 2,4,8, 10, 12 and 24 hours respectively, filtering, and taking the subsequent filtrate for detection. Then 0.5ml of fresh receiving solution was added. After 24h, the skin for diffusion is taken out, after being evenly crushed by a homogenizer, a quantitative methanol (chromatographic alcohol) solution is added, ultrasonic mixing is carried out, standing is carried out for 2h, 7000g is centrifuged for 10min, and the supernatant is left. Extracting twice, mixing extractive solutions, filtering with microporous membrane, and detecting the filtrate. The experiment was repeated 5 times.

Injecting the solution into liquid chromatograph with 20 μ l each, and determining corresponding drug concentration C by external standard method _i The cumulative permeation quantity Q (mu g/cm) is calculated according to the cumulative permeation quantity formula ² )：

Wherein S is a permeation area (cm) ² )；C _n The sample concentration (μ g/ml) measured for the nth sampling point; c _i The sample concentration (μ g/ml) was determined for the ith sample point.

Cumulative skin penetration of minoxidil (n =5,

ug/cm.sup.2)

dosage forms	t/h
								2h	4h	6h	8h	12h	24h	Skin storage Volume (24 h)
	Alcohol-containing esters Plastid rice Nuodier (a Chinese character)	2.55±0.30	8.84±0.73	17.97±0.68	30.62±0.69	44.76±0.90	253.68±1.17								104.69±3.15
Common rice Nuodier (a medicine for treating diabetes)								0.89±0.17	1.65±0.14	3.50±0.33	5.85±0.32	10.35±0.57	26.70±1.13	34.82±1.19
	P	0.000211	1.15E-05	5.53E-07	1.07E-07	1.58E-07	1.25E-10								3.14E-07

The results indicate that the cumulative skin penetration of the drug-containing 2h,4h,6h,8h, 12h, 24h,1.0% alcohol-containing liposome minoxidil group and 1.0% common minoxidil group are compared (P < 0.01), and the statistical results show significant difference; compared with the skin storage capacity for 24 hours of a 1.0% alcohol-containing liposome minoxidil group and a 1.0% common preparation minoxidil group (P is less than 0.01), the statistical result is also significantly different.

From the above results, the following conclusions can be drawn: compared with the common preparation minoxidil group, the alcohol-containing liposome minoxidil group has obvious advantages in both accumulated skin permeation amount and skin storage amount. Therefore, the experimental result shows that the developed alcohol-containing liposome minoxidil has the characteristics of promoting transdermal absorption and skin accumulation.

Example fourth section: animal experiment model for detecting skin accumulation effect of alcohol-containing liposome minoxidil

Example 6:

animal experimental model a hair cycle animal model can be established using C57BL/6 mice or other murine lines.

Breeding experimental animals in a clean environment, and selecting back skin as a medicine applying area; the hair cycle of the mice was first synchronized by paraffin depilation. After 20 days, the hair follicles synchronously enter the resting period, and the back hairs of the mice are carefully shaved off by a blade, so that the backs of the mice are smooth, no injury or residual hairs exist, and the shaving area is about 2cm multiplied by 3cm. The groups were randomized into 2 groups and shaved and applied directly 2 times daily. After the application of the medicine for 1,2,4,8, 12 and 24 hours, taking a whole skin layer with the size of 0.5 x 0.8mm.sup.2 according to different areas, evenly crushing by a homogenizer, adding a quantitative methanol (tryptophol) solution, ultrasonically mixing, standing for 2 hours, centrifuging for 10 minutes at 7000g, and taking the supernatant. Extracting twice, mixing extractive solutions, filtering with microporous membrane, and detecting the filtrate.

The results suggest that the statistical results show significant differences in the skin storage capacity at various time points in the 1.0% alcoholic liposomal minoxidil group and the 1.0% prandial minoxidil group in the animal in vivo experimental model (P < 0.01). From the above results, it can be concluded that the alcohol-containing liposome minoxidil showed a significant advantage in skin storage capacity in the animal in vivo experimental model. Therefore, the experimental result also shows that the developed alcohol-containing liposome minoxidil has the characteristics of promoting transdermal absorption and skin accumulation.

Example fifth part: animal experiment model for detecting fluorescence accumulation of alcohol-containing liposome

Example 7: in vivo transdermal assay of fluorescently-labeled alcohol-containing liposomes

Preparing an alcoholic liposome according to the above method, wherein column separation is required after the preparation of the alcoholic liposome calcein, and the gel column removes unencapsulated free calcein to obtain a liposome calcein preparation completely encapsulated in the liposome; the same concentration of aqueous calcein solution was used as a control. And the alcohol-containing liposome system contains PE-Rhodamine with concentration of 0.56mg/ml.

The skin of the transdermal model can be the skin of the back of a white mouse or a mouse, and can also be the waste skin after human surgery.

Inbred C57BL/6 mice, females, 28 days old were bred to 44 days old, and 15 mice (body weight 18 + -0.5 g) with pink skin hair at resting stage were selected and randomly divided into 3 groups; carefully removing the hair on the back of the mouse by using a blade, shaving the hair, and directly coating a PE-Rhodamine-labeled alcohol-containing liposome, alcohol-containing liposome calcein and a calcein aqueous solution respectively. Taking 0.5 × 0.8mm according to different regions after 1,2,4,8, 12, and 24 hr respectively ² Placing the whole layer of large and small skins into a freezing microtome at the temperature of-25 ℃, precooling for 30min to harden the whole tissue block, observing the hair growth direction, trimming the tissue block to make the direction of the slice consistent with the longitudinal axis direction of the hair growth, then fixing the sample on a sample plate by using an embedding medium OCT (optical coherence tomography), continuously freezing for 30min, then starting to slice, repairing the redundant embedding medium and tissue block on the surface, adjusting the angle between the sample and a knife to make the direction of the slice consistent with the longitudinal axis direction of the hair growth, cutting out the sample with the thickness of 50 mu m, directly observing by using a fluorescence microscope and a laser confocal microscope, and taking a picture. Excitation wavelength is 488nm, emission wavelength is 520nm, emission intensity and anisotropy are used for correcting background fluorescence and scattering effect through blank specimen.

Under the same experimental conditions, the fat-soluble fluorescent marker PE-Rhodamine can enter the whole skin layer, and high aggregation is generated near hair follicles. The alcoholic liposome-containing calcein enters the skin deeper, more and more than the aqueous calcein solution, and more accumulates around the hair follicle. In FIG. 4,5,6, it is shown that after 24h under fluorescence microscope, the ester-soluble fluorescent marker PE-Rhodamine prepared from alcoholic liposomes was found to be concentrated in higher concentration in the whole skin layer, especially near the hair follicle. Compared with the unencapsulated naked calcein aqueous solution, the calcein serving as a water-soluble fluorescent marker prepared by the alcohol-containing liposome has obvious advantages in the amount and depth of penetrating into the whole skin layer through the skin, and the fluorescent agent is highly aggregated near hair follicles. No fluorescence was observed in the blank corrected skin specimens when observed under confocal laser microscopy (see FIGS. 7 and 8). After 24 hours, the fluorescence of the aqueous calcein solution was only present in the epidermis and there was a slight infiltration of the hair follicle opening (see fig. 9). Under the same experimental conditions, the calcein-containing liposome is not only positioned in epidermis and dermis layers, but also has high aggregation near hair follicles, is obviously superior to a naked calcein group in strength and depth (figure 10), and shows that water-soluble calcein can enter the deep layer of the skin through skin penetration by wrapping and forms high aggregation around the hair follicles. The liposome-prepared ester-soluble fluorescent marker PE-Rhodamine was found to aggregate in higher concentration in the whole skin layer, especially in the vicinity of hair follicle (FIG. 11), further indicating that the phospholipid component of the alcohol-containing liposome also can penetrate through the stratum corneum to reach the dermis layer and aggregate around the hair follicle.

The results show that: in an animal in-vivo experimental model, the alcohol-containing liposome system can carry fat-soluble and water-soluble medicines to enter the deep layer of the skin, hair follicle accumulation shows that the hair follicle sebaceous gland pathway can be an important delivery pathway of the alcohol-containing liposome, and targeted hair follicle delivery highlights the characteristics of the liposome.

Example section six: effect of Liposomal minoxidil Alcoholic on Hair growth in C57BL/6 mice

Example 8: 60 mice (weight 18 +/-0.5 g) with skin-color pink hair in a resting period are selected to be randomly divided into different groups when inbred line C57BL/6 mice, females and mice of 28 days old are bred to 44 days old; carefully removing the back hairs of the mouse by using a blade, and directly smearing minoxidil with different preparations after shaving the hairs; continuously observing the skin color change and the hair growth condition of the mouse and taking digital photos; respectively taking materials from each group of mice in the drug application area when the drugs are applied for 18 days and 35 days, preparing frozen sections after fixation, observing histological changes of hair follicles under a light microscope, and observing changes of ultrastructures under an electron microscope; the influence of each group of medicaments on the expression of VEGF is observed by adopting a method of combining digital photography and image analysis; the hair diameter was measured at the end of the experiment in each group of application areas.

1. Results of the skin irritation of the alcohol-containing Liposomal minoxidil on C57BL/6 mice

The evaluation results according to the skin irritation response score criteria and the skin irritation intensity evaluation criteria are as follows:

skin irritation response scoring criteria

Stimulus response conditions	Score value	Stimulus response conditions	Score value
				Erythema: no erythema Can be barely seen Is obviously visible Moderate to severe erythema Purplish red spot with eschar formation	0 1 2 3 4	Edema (edema) Without edema Can be barely seen Visible (high edge surrounding skin) The skin is raised by about 1cm and has clear outline Edema uplift more than 1cm	0 1 2 3 4

Skin irritation intensity scoring criteria

Mean value of	Evaluation of
		0～0.49 0.5～2.99 3.0～5.99 6.0～8.0	Has no irritation Light irritation Middle irritation Strong irritation property

The evaluation results show that no obvious irritation reaction such as erythema, edema and the like is seen on the skin of the drug-coated areas of the alcohol-containing liposome minoxidil group and the blank liposome group, and the integral value of the irritation reaction of each group is less than 0.49, which indicates that the liposome preparation has no obvious irritation to the intact skin of the mouse.

2. Effect and histological Change of Liposomal minoxidil-containing on C57BL/6 mouse skin

At 18 days of administration, the first 2 groups were in anagen phase and the skin was black, most of the hairs of mice in the group of 1.4% alcohol-containing liposome minoxidil had emerged (see fig. 12a, b and c), and histology found that the hair follicles were large in size, the hair papillae were completely surrounded by hair bulbs, which reached the deep layer of subcutaneous tissue, and significant melanogenesis was observed in the hair bulbs, and new hairs and inner root sheaths were found, and most of them were in the VI subphase of anagen phase (see fig. 12D).

While the skin of 1.4% minoxidil mice mostly darkened, but much hair did not grow out of the skin (see fig. 13a, b, c), histologically it was seen that hair follicles grew downward, with the terminal follicular epithelial cells (hair bulbs) partially surrounding the hair papilla, which was less porous and had less melanin production, much in anagen phase III subphase (see fig. 13D).

Most of the blank liposomes and blank control mice had pink skin (see FIG. 14A, B, C), and the number of hair follicles was small, the hair shaft ends were clubbed, the hair papillae was small, the lower ends of the hair follicles were degenerated, melanogenesis was rare, the hair papillae were moved up to the level of hair adhesion, and the hair follicles were mostly in the typical resting stage (see FIG. 14D)

Results of analysis of average optical Density value (OD) of skin color for each group: (

dn＝12)

Group of	1.4% alcoholic liposomal minoxidil	1.4% minoxidil	Blank liposomes	Blank control group
					OD	0.54±0.11	0.29±0.06	0.16±0.07	0.15±0.05

The results of the grey scale value analysis in the table above are also substantially consistent with the gross and histological hair follicle staging results above.

3. Effect of Liposomal minoxidil Alcoholic on the Hair cycle of C57BL/6 mice

Effect of Each group of drugs on the Hair cycle: (

dn＝12)

Group of	Applying the medicament to the skin opening Time to begin blackening (t 1)	The skin begins to turn black to open Initial graying time (t 2)
			1.4% alcohol-containing liposomal minoxDi Er	6.0±0.9	15.9±2.2
1.4% minoxidil	15.3±2.2	14.4±2.3
			Blank liposomes	23.8±3.0	12.3±2.4
Blank control group	23.3±2.4	11.9±2.5

The original groups of data are tested by Kolmogorov-Smirnov with P > 0.05, the total body of the samples of each group cannot be considered not to be subjected to normal distribution, each group of data is tested by Leven with P > 0.05, variance analysis is adopted, P < 0.01 is obtained, and the results are further compared pairwise, and are as follows:

comparison of the time (t 1) from the application of each group until the skin begins to turn black

	Group 1&4 groups of	Group 1&Group 3	Group 1&2 groups of	2 groups of&4 groups of	Group 3&4 groups of
						P value	P＜0.01	P＜0.01	P＜0.01	P＜0.01	P＞0.05

Comparison of the time from the beginning of blackening to the beginning of graying (t 2) of each group of skin

	Group 1&4 groups of	Group 1&Group 3	Group 1&2 groups of	2 groups of&4 groups of	3 groups of&4 groups of
						P value	P＜0.01	P＜0.01	P＞0.05	P＜0.05	P＞0.05

As can be seen from the results, the skin of mice in the 1.4% alcoholic liposomal minoxidil group and the 1.4% minoxidil group changed from pink to black (transition from resting phase to growth phase) by 17.3d (P < 0.01) and 8.0d (P < 0.01), respectively, compared with the blank control group, and the statistical results were significantly different; the total time of the skin in the black state is respectively prolonged by 4.0d (P is less than 0.01) and 2.5d (P is less than 0.05), and the statistical result shows that the total time is significantly different. Compared with the 1.4% minoxidil group, the 1.4% alcoholic liposome minoxidil group has the advantage that the time (t 1) for changing the skin color from pink to black is advanced by 9.3d, and the P is less than 0.01, so that the significant difference exists.

4. Effect of Liposomal minoxidil-containing on C57BL/6 mouse Hair diameter

Table 3-7 effect of each group of drugs on hair diameter: (

d)

Group of	1.4% alcoholic liposome minoxidil Er (Chinese character of 'Er')	1.4% minoxidil	Blank liposomes	Blank control group
					Hair diameter (μm)	60.31±5.64	54.26±6.09	52.72±6.80	48.98±5.25

The original groups of data are tested by Kolmogorov-Smirnov to obtain P & gt 0.05, the total body from which the samples of each group come cannot be considered to be in normal distribution, the original groups of data are tested by Leven to obtain P & gt 0.05, the square difference analysis is adopted to obtain P & lt 0.01, and the results are further compared pairwise, and are as follows:

tables 3-8 comparison of hair diameters for each group

	Group 1&4 groups of	Group 1&3 groups of	Group 1&2 groups of	2 groups of&4 groups of	3 groups of&4 groups of
						P value	P＜0.01	P＜0.01	P＜0.05	P＜0.05	P＞0.05

The results suggest that the hair diameter of the 1.4% alcohol-containing liposome minoxidil group (P < 0.01) and the 1.4% minoxidil group (P < 0.05) mice is larger than that of the blank control group, and the statistical results have significant differences. And the diameter of the 1.4% alcohol-containing liposome minoxidil mice is larger than that of the 1.4% minoxidil mice, and the statistical result shows significant difference (P is less than 0.05).

The minoxidil group can induce the hair follicles of the mice to change from the resting phase to the growth phase, accelerate the process, prolong the growth phase, thicken the diameters of the grown hairs, and induce the resting phase of the mice to change to the growth phase more quickly by the 1.4 percent alcohol-containing liposome minoxidil group, and the diameter is superior to that of the 1.4 percent minoxidil group, so the experimental results show the feasibility and the potential market prospect of the liposome preparation for treating alopecia.

5. Effect of Liposomal minoxidil Alcoholic on the expression of C57BL/6 mouse VEGF

At 18 days of administration, the minoxidil mice in each group had many hair follicles in the anagen phase, with positive expression sites mainly located in the cell membranes and cytoplasm in the pilosebaceous unit, while the blank liposome group and blank control group had many hair follicles in the typical telogen phase, with positive staining intensity and area of the pilosebaceous unit less than those in the first two groups (see fig. 15 and 16); after being taken for 35 days, the hair follicles in the resting stage are mostly in each group, and the hair follicle sebaceous unit is lighter in positive coloration and smaller in area.

The original groups of data are tested by Kolmogorov-Smirnov to obtain P more than 0.05, the total body of the samples of each group cannot be considered not to be in normal distribution, each group of data is tested by Leven to obtain P less than 0.05, after variable transformation is carried out, the Kolmogorov-Smirnov to obtain P more than 0.05 and the Leven to obtain P more than 0.05, variance analysis is adopted to obtain P less than 0.01, and pairwise comparison is further carried out, and the results are as follows:

comparison of the results of the average optical Density value (IOD) for the skin VEGF for each group (18 days)

	Group 1&4 groups of	Group 1&Group 3	Group 1&2 groups of	2 groups of&4 groups of
					P value	P＜0.01	P＜0.01	P＜0.05	P＜0.05

The results show that the IOD value of the positive expression of the VEGF in the skin of mice in a 1.4% alcohol-containing liposome minoxidil group (P < 0.01) and a 1.4% common preparation minoxidil group (P < 0.05) is greater than that in a blank control group at the time of taking the medicine for 18 days, and the statistical results are significantly different; the IOD value of the VEGF positive expression of the mouse skin in the 1.4% alcohol-containing liposome minoxidil group is greater than that in the 1.4% common preparation minoxidil group, and the statistical result has significant difference (P is less than 0.05); .

The following conclusions can be drawn according to the results: the liposome formulation was not significantly irritating to intact skin of mice. The minoxidil group can induce the hair follicle of the mouse to change from the resting period to the growth period, accelerate the process, prolong the growth period, thicken the diameter of the grown hair, and the effect of the 1.4% alcohol-containing liposome minoxidil group is better than that of the 1.4% minoxidil group. The alcoholic liposomal minoxidil may cause the change of the hair cycle of mice by up-regulating the expression of VEGF in the hair follicles of the mice and promoting the proliferation of cells around the hair follicles. Therefore, the experimental result shows the feasibility of using the alcoholic liposome minoxidil to treat alopecia.

In conclusion, the medicinal spray for promoting hair growth of the invention has the following four advantages:

1. effectively promoting hair growth:

the preparation contains minoxidil, and can induce the hair follicle of mouse to change from resting stage to growth stage, accelerate the process, prolong the growth stage, thicken the diameter of hair, and promote hair growth;

2. good transdermal performance, durable effect and relapse reduction:

the unique structure of the liposome and the natural and synthetic composite multi-element transdermal enhancer consisting of phospholipid, thin-loading oil, laurocapram and ethanol can achieve the effect which cannot be achieved by a single transdermal enhancer, so that the product has strong transdermal property, can deliver and store the medicament in the deep layer of the skin, can continuously exert the medicinal efficacy even after stopping administration, effectively reduces the relapse of alopecia, and can reduce the systemic toxicity caused by the medicament entering blood;

3. the ester-soluble fluorescent agent and the water-soluble fluorescent agent form high-concentration aggregation at the hair follicle part, and the characteristic that the developed alcohol-containing liposome has the characteristic of being hair follicle-philic is preliminarily shown.

4. Skin care effect:

the liposome has similar structure to biological membrane, and can be used as topical drug carrier to avoid skin irritation, and also has skin caring effect, and vitamin E antioxidant and skin nourishing effect to protect skin.

Claims (3)

1. A medicine spray for promoting hair growth is characterized in that the effective components and the percentage of the raw materials for preparing the spray are as follows: minoxidil: 0.5-20%, lecithin: 50 Percent to 80 percent, vitamin E:0 to 0.4%, azone: 0-0.2%, natural essential oil: 0 to 10 percent of ethanol, and the balance of ethanol, wherein the total amount of the raw materials is 100 percent.

2. The pharmaceutical spray for promoting hair growth of claim 1, wherein said lecithin is: one or more of egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, synthetic lecithin and sphingomyelin.

3. The pharmaceutical spray for promoting hair growth of claim 1, wherein said natural essential oil is: one or more of oleum Menthae Dementholatum, oleum Eucalypti, oleum Rosae Rugosae, oleum Pini, oleum Caryophylli, thyme oil, oleum Lavandula Angustifolia, oleum sesami, lemon oil, spearmint oil, bergamot oil, and oleum Santali albi.

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