CN114886843B

CN114886843B - Cetirizine hydrochloride liposome gel for treating androgenetic alopecia and preparation method and application thereof

Info

Publication number: CN114886843B
Application number: CN202210586110.5A
Authority: CN
Inventors: 袁进; 徐晓玲; 黄雪峰; 姜志辉; 萧伟斌; 季波; 杨晓敏; 胡利辉
Original assignee: Southern Theater Command General Hospital of PLA
Current assignee: Southern Theater Command General Hospital of PLA
Priority date: 2022-05-26
Filing date: 2022-05-26
Publication date: 2023-08-11
Anticipated expiration: 2042-05-26
Also published as: CN114886843A

Abstract

The application provides cetirizine hydrochloride liposome gel for treating androgenetic alopecia, and a preparation method and application thereof. The preparation method of cetirizine hydrochloride liposome gel comprises the following steps: s1: dissolving soybean lecithin and cholesterol in organic solvent to obtain medicinal membrane; s2: adding cetirizine hydrochloride solution into the medicinal membrane, and then hydrating, dispersing and granulating to obtain cetirizine hydrochloride liposome; s3: adding water into carbomer-940 to swell to prepare a gel matrix, adding cetirizine hydrochloride liposome, a humectant, a preservative and distilled water into the gel matrix, and uniformly stirring and then regulating the pH value to prepare the cetirizine hydrochloride liposome gel. The cetirizine hydrochloride liposome gel disclosed by the application can improve the storage capacity of cetirizine on scalp, so that the acting time of cetirizine is prolonged, and the purposes of optimizing the curative effect, improving the use compliance of AGA patients and the like are further achieved.

Description

Cetirizine hydrochloride liposome gel for treating androgenetic alopecia and preparation method and application thereof

Technical Field

The application relates to the technical field of pharmacy, in particular to cetirizine hydrochloride liposome gel for treating androgenetic alopecia and a preparation method and application thereof.

Background

In recent years, with the increase of life and work pressure, the phenomenon of alopecia is more and more common, and the mental pressure brought by the phenomenon has serious negative effects on life of people, so the demand for treating alopecia is increased. From clinical diagnosis, hair loss is largely classified into alopecia areata, androgenetic alopecia (AGA), drug-induced anagen alopecia, etc., with androgenetic alopecia being the most common. Currently guidelines recommend that men orally take finasteride and topical minoxidil and women orally take cyproterone, spironolactone and topical minoxidil. The treatment cycle of androgenetic alopecia is long, so that the medication compliance of patients is poor. The long-term use of oral drugs can act on the systemic system, thereby causing serious adverse reactions; the hair planting cost is high and is an invasive operation; spironolactone causes electrolyte disturbance and decreased sexual desire; externally applied minoxidil is an oily medicine which is not easy to clean, and has adverse reactions such as local hirsutism and the like after being used by female patients. Therefore, finding new drug action targets becomes a hotspot of current AGA treatment.

The first report in 2017 that the topical cetirizine ethanol solution has good curative effect on AGA, and the subsequent multiple clinical preliminary experiments also verify the curative effect of cetirizine on AGA. Cetirizine hydrochloride tablet is a second generation H1 receptor selective antagonist, and is used as an antiallergic agent for years in clinic, and the cetirizine hydrochloride has excellent safety. However, ethanol solutions have a strong irritation to the skin and are inconvenient to use; in particular, ethanol solutions are difficult to penetrate the stratum corneum of the scalp and have little storage effect within the scalp, thus potentially reducing the efficacy of cetirizine.

In view of this, the present application has been made.

Disclosure of Invention

The application aims to provide cetirizine hydrochloride liposome gel for treating androgenetic alopecia, and a preparation method and application thereof.

The application provides a preparation method of cetirizine hydrochloride liposome gel for treating androgenetic alopecia, which comprises the following steps:

s1: dissolving soybean lecithin and cholesterol in organic solvent to obtain medicinal membrane;

s2: adding cetirizine hydrochloride solution into the medicinal membrane, and then hydrating, dispersing and granulating to obtain cetirizine hydrochloride liposome;

s3: adding water into carbomer-940 to swell to prepare a gel matrix, adding cetirizine hydrochloride liposome, a humectant, a preservative and distilled water into the gel matrix, and uniformly stirring and then regulating the pH value to prepare the cetirizine hydrochloride liposome gel.

In the step S1, the mass ratio of the soybean lecithin to the cholesterol is (3-7): 1, preferably (5-7): 1, for example (250-350) mg:50mg; studies have shown that: the mass ratio of the soybean lecithin to the cholesterol is as high as 8:1, the Encapsulation Efficiency (EE) of the liposome is reduced (about EE 50%), and the mass ratio of the soybean lecithin to the cholesterol is (5-7): the encapsulation efficiency of the liposome is higher (EE 60% or more) in the 1-step, so the mass ratio of the soybean lecithin to the cholesterol is preferably (5-7): 1.

further, in step S1, the organic solvent is chloroform; the preparation method of the medicinal film comprises the following steps: removing chloroform by rotary evaporation under reduced pressure in 45-55deg.C water bath, and making into medicinal membrane.

In the step S2, the preparation method of the cetirizine hydrochloride solution includes: dissolving cetirizine hydrochloride in a PBS solution to prepare a cetirizine hydrochloride solution; wherein the mass volume ratio of cetirizine hydrochloride to PBS solution is (20-30) mg: (5-15) mL. Studies have shown that: the dosage of cetirizine hydrochloride has a certain influence on the entrapment rate of the liposome, the entrapment rate of the liposome has an increasing trend along with the dosage of cetirizine hydrochloride, the entrapment rate of cetirizine hydrochloride is lower (about EE 40%) when the dosage of cetirizine hydrochloride is 5mg, and the entrapment rate of cetirizine hydrochloride is highest (above EE 60%) when the dosage of cetirizine hydrochloride is 30mg, so the dosage of cetirizine hydrochloride is preferably 20-30mg; in addition, the use amount of the PBS solution has a certain influence on the encapsulation efficiency of the liposome, and the result shows that the encapsulation efficiency of the liposome is highest when the use amount of the PBS solution is 10mL, and the encapsulation efficiency is lowest when the use amount is 20mL (about EE 40%), so the use amount of the PBS solution is preferably 5-15mL.

Further, in the step S2, hydration is carried out under the stirring condition, wherein the stirring speed is 200-300rpm, the hydration temperature is 30-70 ℃, and the hydration time is 0.5-1.5h; dispersing under ultrasonic condition, wherein the ultrasonic power is 2000-2200W, and the dispersing time is 4-6min; the finishing comprises the following steps: the pellets were sized by extrusion filtration through a 0.45 μm filter. Studies have shown that: the hydration temperature has no obvious influence on the entrapment rate of the liposome, the entrapment rate of the liposome does not generate obvious change due to the increase of the hydration temperature, and the hydration temperature can be set to be 30-70 ℃; the hydration time has a certain influence on the entrapment rate of the liposome, and the result shows that the entrapment rate of the liposome is in a decreasing trend along with the increase of the hydration time, and the hydration time is 0.5-1.5h, so that the higher entrapment rate is achieved, and the preferable hydration time is 0.5-1.5h.

Preferably, the preparation method of the cetirizine hydrochloride liposome comprises the following steps: dissolving soybean lecithin 250mg and cholesterol 50mg in 10mL chloroform, and removing chlorine by rotary evaporation under reduced pressure in water bath to obtain a medicinal membrane; 5mL of PBS solution dissolved with 25mg of cetirizine hydrochloride is added into the medicine membrane, the medicine membrane is magnetically stirred and hydrated for 30min in a water bath at 50 ℃, then cooled to room temperature, and after ultrasonic dispersion, the medicine membrane is extruded and filtered by a filter membrane with the thickness of 0.45 mu m to carry out granule finishing, thus obtaining the cetirizine hydrochloride liposome.

In the step S3, the preparation method of the gel matrix includes: adding distilled water into carbomer-940, stirring for 2-4 hr, and fully swelling at 3-5deg.C for 20-28 hr to obtain gel matrix; wherein the mass content of carbomer-940 in the gel matrix is 3-5%.

Further, in step S3, the humectant is glycerin, and the mass ratio of the gel matrix to the glycerin is 5g: (1.8-2.2) g, preferably 5g:2g; the preservative is ethylparaben, and the mass ratio of the gel matrix to the ethylparaben is 5g: (15-20) mg, preferably 5g: (15-17) mg, further preferably 5g:16mg.

In addition, in the step S3, the mass-volume ratio of the gel matrix to the cetirizine hydrochloride liposome is (2.5-5) g:10mL, preferably (4.5-5) g:10mL, more preferably 5g:10mL; the mass content of carbomer-940 in the cetirizine hydrochloride liposome gel is 0.5-1%, preferably 1%. Studies have shown that: when the mass fraction of carbomer-940 is about 1%, the liposome gel has better dispersibility, proper viscosity, good formability and easy spreading; the problem of poor formability exists when the mass fraction of the carbomer-940 is about 0.25%, and the problem of caking, thicker and difficult spreading exists when the mass fraction of the carbomer-940 is about 1.5%. In particular, in the preparation process of the gel matrix, a blank gel matrix with Gao Kabo m content (such as 4%) is prepared firstly, and then a proper amount of liposome gel is prepared, so that the liposome gel with 1% carbomer content is obtained, gel matrix loss in the preparation process is avoided, and the preparation efficiency is improved.

Further, in step S3, adjusting the pH value includes: triethanolamine was added dropwise to adjust the pH to 6.5. Studies have shown that: the viscosity of the gel is increased along with the increase of the pH value, when the pH value is 7.5, the viscosity of the gel is too high to be spread, the gel is difficult to spread, the phenomenon of agglomeration occurs, cetirizine hydrochloride liposome is not easy to disperse, and the overall quality of the gel is poor; when the pH value of the gel is 4.5, the viscosity of the gel is lower, the texture is thinner, and the gel is not easy to adhere to the skin; when the pH value of the gel is 6.5, the viscosity of the gel is moderate, the whole appearance of the gel is uniform and fine and is easy to spread, and the pH value is close to the pH value of the skin surface, so that the irritation of the external gel preparation to the skin can be reduced.

Preferably, the preparation method of the cetirizine hydrochloride liposome comprises the following steps: adding 10mL of cetirizine hydrochloride liposome, 2g of glycerol and 16mg of ethylparaben into 5g of 4% gel matrix, adding distilled water until the total mass is 20g, stirring uniformly, adding a proper amount of triethanolamine, regulating the pH to 6.5, and stirring uniformly to obtain cetirizine hydrochloride liposome gel.

The cetirizine hydrochloride liposome prepared by the preparation method is a pale yellow semitransparent semisolid preparation, and is fine, smooth and uniform and free of agglomerated particles; the pH value is stabilized at 6.5, and the pH has small irritation to human skin; the appearance is uniform after centrifugation, and layering or agglomeration phenomenon is avoided; the liposome has stable properties, and no solidification or layering phenomenon exists after the liposome is placed at different temperatures. In addition, the encapsulation efficiency of cetirizine hydrochloride liposomes is >50%; the drug loading rate of cetirizine hydrochloride liposome is more than 5%.

The application also provides cetirizine hydrochloride liposome gel for treating androgenetic alopecia, which is prepared according to the preparation method.

The application also provides application of the cetirizine hydrochloride liposome gel in preparing medicines for treating androgenetic alopecia.

The cetirizine hydrochloride liposome gel is used as an external preparation for treating androgenetic alopecia (AGA), so that the local drug concentration can be improved, and the occurrence rate of adverse reactions can be reduced; meanwhile, the gel is convenient to use and easy to clean, can effectively increase medication compliance of patients, and has remarkable treatment effect.

The implementation of the application has at least the following advantages:

1. aiming at the defects of ethanol solution, the application takes liposome which is easy to penetrate through skin cutin layer and safe and nontoxic as a carrier, and prepares a novel cetirizine hydrochloride liposome gel which is convenient to use, safe and nontoxic, skin-targeted and provides a preliminary preparation research basis for clinical application by virtue of the advantages of good biocompatibility, no stimulation, easy cleaning and the like of the gel;

2. the application establishes a stable and reliable liposome gel preparation method and a quality evaluation system, improves liposome encapsulation efficiency and transdermal absorptivity of liposome gel, and can improve the storage capacity of cetirizine on scalp, thereby prolonging the action time of cetirizine, achieving the purposes of optimizing curative effect and improving the use compliance of AGA patients.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is an electron microscope of cetirizine hydrochloride liposomes prepared in example 1 of the present application;

FIG. 2 is a graph showing the particle size and potential distribution of cetirizine dihydrochloride liposomes prepared in example 1 of the present application; wherein: a: particle size diagram; b: a potential profile;

FIG. 3 shows the results of stability test of cetirizine dihydrochloride liposome gel prepared in example 1 of the present application;

FIG. 4 is a graph showing the skin color change of the mice of test example 3 according to the present application; wherein: a: cetirizine hydrochloride liposome gel group, b: minoxidil group, c: blank control group.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1

The preparation method of cetirizine hydrochloride liposome gel for treating androgenetic alopecia of this embodiment comprises the following steps:

1) Preparation of cetirizine hydrochloride liposomes

Precisely weighing 250mg of soybean lecithin and 50mg of cholesterol, adding 10mL of chloroform to dissolve in a eggplant-shaped bottle, then removing chloroform by reduced pressure rotary evaporation for about 10min under the water bath condition of 50 ℃ to obtain a medicinal membrane, adding 5mL of PBS dissolved with 25mg of cetirizine hydrochloride raw material medicine into the medicinal membrane, magnetically stirring and hydrating for 0.5h at 250rpm in the water bath of 50 ℃, then cooling to room temperature, dispersing nanoparticle uniform liposome at 2100W for 5min, extruding and filtering by using a filter membrane of 0.45 mu m to obtain coarse cetirizine hydrochloride liposome, and preserving at 4 ℃ for standby.

2) Preparation of cetirizine hydrochloride Liposome gel

Precisely weighing 2.0g of carbomer-940 powder into a conical flask, adding distilled water to 50g, stirring on a magnetic stirrer for 3h, and then placing in a refrigerator at 4 ℃ to fully swell for 24h to obtain a blank gel matrix with the mass fraction of 4%.

Precisely weighing 5g of the prepared 4% blank gel matrix, adding 10mL of cetirizine hydrochloride liposome solution into a beaker, adding 2g of glycerin as a humectant, adding 16mg of ethylparaben as a preservative, adding distilled water to 20g, uniformly stirring, finally adding a proper amount of triethanolamine to adjust the pH value to 6.5, and uniformly stirring to obtain the cetirizine hydrochloride liposome gel, wherein the liposome gel has good dispersibility, proper viscosity, good molding shape and easy coating.

The encapsulation efficiency and drug loading rate of cetirizine hydrochloride liposomes were determined as follows:

HPLC content determination:

chromatographic conditions: chromatographic column: c18 (4.6X250 mm,5 μm); mobile phase: acetonitrile: water = 40:60 A) is provided; flow rate: 1mL/min; column temperature: 25 ℃; the ultraviolet detection wavelength is as follows: 232.11nm; sample injection amount: 20 μl. Linear regression is carried out on the mass concentration (c) of the cetirizine hydrochloride reference substance by using the peak area (A), so that the regression equation of the cetirizine hydrochloride standard curve is A=34.21×c+25.85, and the correlation coefficient r is obtained ² ＝0.9996。

Measurement of liposome entrapment (EE) and Drug Loading (DL):

precisely sucking 100 mu L of cetirizine hydrochloride liposome solution, adding 900 mu L of methanol solution, swirling for 5min, centrifuging for 30min at 12000r/min after ultrasonic treatment for 5min, precisely sucking 20 mu L of supernatant after centrifugation, sampling in HPLC, and measuring the total amount W2 of the encapsulated drug in the prepared liposome in HPLC according to chromatographic conditions under 1.1 item; precisely sucking 1000 mu L of cetirizine hydrochloride liposome solution, centrifuging at 12000r/min for 30min, taking 100 mu L of supernatant after centrifuging, adding methanol to 1000 mu L, swirling for 5min, taking 20 mu L, sampling by HPLC, and measuring the free drug amount W1 of the encapsulated drug in the prepared liposome by sampling in HPLC according to the chromatographic condition of 1.1.

Encapsulation efficiency (%) and drug loading (%) were calculated according to the following formulas:

encapsulation efficiency (%) = (W2-W1)/w2×100%

Drug loading (%) = (W2-W1)/w3×100%

In the above formula: w3 is the sum of the total amount of materials and the total amount of medicines.

The encapsulation efficiency and the drug loading rate of the cetirizine hydrochloride liposome are shown in tables 1 and 2.

Example 2

1) Preparation of cetirizine hydrochloride liposomes

Precisely weighing 250mg of soybean lecithin and 50mg of cholesterol, adding 10mL of chloroform to dissolve in a eggplant-shaped bottle, then removing chloroform by reduced pressure rotary evaporation for about 10min under the water bath condition of 45 ℃ to obtain a medicinal membrane, adding 5mL of PBS solution in which 20mg of cetirizine hydrochloride crude drug is dissolved into the medicinal membrane, hydrating for 0.5h under the magnetic stirring of 200rpm in the water bath of 70 ℃, cooling to room temperature, dispersing nanoparticle uniform liposome by 2200W ultrasound for 5min, extruding and filtering by a filter membrane of 0.45 mu m to obtain coarse cetirizine hydrochloride liposome, and preserving at 4 ℃ for standby.

2) Preparation of cetirizine hydrochloride Liposome gel

Precisely weighing 2.0g of carbomer-940 powder into a conical flask, adding distilled water to 50g, stirring on a magnetic stirrer for 2h, and then placing in a refrigerator at 4 ℃ to fully swell for 28h to obtain a blank gel matrix with the mass fraction of 4%.

Precisely weighing 5g of the prepared 4% blank gel matrix, adding 10mL of cetirizine hydrochloride liposome solution into a beaker, adding 2g of glycerin as a humectant, adding 16mg of ethylparaben as a preservative, adding distilled water to 20g, uniformly stirring, finally adding a proper amount of triethanolamine to adjust the pH value to 6.5, and uniformly stirring to obtain the cetirizine hydrochloride liposome gel.

The encapsulation efficiency of cetirizine hydrochloride liposomes was determined as in example 1 and the results are shown in table 1.

Example 3

1) Preparation of cetirizine hydrochloride liposomes

Precisely weighing soybean lecithin 350mg and cholesterol 50mg, adding 10mL of chloroform to dissolve in a eggplant-shaped bottle, then removing chloroform by reduced pressure rotary evaporation for about 10min under the water bath condition of 45 ℃ to obtain a medicinal membrane, adding 15mL of PBS solution in which 25mg of cetirizine hydrochloride raw material medicine is dissolved into the medicinal membrane, hydrating for 0.5h under magnetic stirring at 200rpm in the water bath of 70 ℃, cooling to room temperature, dispersing nanoparticle uniform liposome by 2200W ultrasound for 5min, extruding and filtering by using a filter membrane of 0.45 mu m to obtain coarse cetirizine hydrochloride liposome, and preserving at 4 ℃ for standby.

2) Preparation of cetirizine hydrochloride Liposome gel

Example 4

1) Preparation of cetirizine hydrochloride liposomes

Precisely weighing 300mg of soybean lecithin and 50mg of cholesterol, adding 10mL of chloroform to dissolve in a eggplant-shaped bottle, then removing chloroform by reduced pressure rotary evaporation for about 10min under the water bath condition of 55 ℃ to obtain a medicinal membrane, adding 5mL of PBS solution dissolved with 25mg of cetirizine hydrochloride raw material medicine into the medicinal membrane, magnetically stirring and hydrating for 1.5h at 300rpm in the water bath of 30 ℃, cooling to room temperature, dispersing nanoparticle uniform liposome at 2000W for 5min, extruding and filtering by using a filter membrane of 0.45 mu m to obtain coarse cetirizine hydrochloride liposome, and preserving at 4 ℃ for standby.

2) Preparation of cetirizine hydrochloride Liposome gel

Precisely weighing 2.0g of carbomer-940 powder into a conical flask, adding distilled water to 50g, stirring for 4h on a magnetic stirrer, and then placing in a refrigerator at 4 ℃ to fully swell for 20h to obtain a blank gel matrix with the mass fraction of 4%.

Example 5

1) Preparation of cetirizine hydrochloride liposomes

Precisely weighing soybean lecithin 350mg and cholesterol 50mg, adding 10mL of chloroform to dissolve in a eggplant-shaped bottle, then removing chloroform by reduced pressure rotary evaporation for about 10min under the water bath condition of 50 ℃ to obtain a medicinal membrane, adding 5mL of PBS dissolved with 30mg of cetirizine hydrochloride raw material medicine into the medicinal membrane, magnetically stirring and hydrating for 1h at 250rpm in the water bath of 50 ℃, cooling to room temperature, dispersing nanoparticle uniform liposome by 2100W ultrasound for 5min, extruding and filtering by using a filter membrane of 0.45 mu m to obtain coarse cetirizine hydrochloride liposome, and preserving at 4 ℃ for standby.

2) Preparation of cetirizine hydrochloride Liposome gel

Comparative example 1

This comparative example is substantially the same as example 4 except that the preparation method of cetirizine hydrochloride liposome is different.

The preparation method of cetirizine hydrochloride liposome of the comparative example is as follows:

precisely weighing soybean lecithin 150mg and cholesterol 50mg, adding 10mL of absolute ethyl alcohol to dissolve in a beaker, dissolving 10mg of cetirizine hydrochloride crude drug in 10mL of PBS solution, preheating at 50 ℃, sucking the ethanol solution by a syringe, rapidly injecting the ethanol solution under the PBS liquid level, magnetically stirring at constant temperature of 50 ℃ for a certain time, volatilizing the absolute ethyl alcohol, cooling to room temperature, ultrasonically dispersing nanoparticle uniform liposome for 5min, extruding and filtering by a filter membrane of 0.45 mu m, and finishing particles to obtain cetirizine hydrochloride liposome crude product, and preserving at 4 ℃ for standby.

TABLE 1 results of encapsulation efficiency determination of cetirizine hydrochloride liposomes

Cetirizine hydrochloride liposome	Encapsulation efficiency (%)
		Example 1	74.14
Example 2	74.06
		Example 3	69.35
Example 4	65.39
		Example 5	63.03
Comparative example 1	41.54

Table 1 the results show that:

compared with the conventional preparation method of the comparative example 1, the cetirizine hydrochloride liposome prepared by the preparation method of each embodiment of the application has higher encapsulation efficiency.

Test example 1

1. Preparation reproducibility investigation of cetirizine hydrochloride liposome

Three batches of cetirizine hydrochloride liposome samples were prepared in parallel by the method of example 1, and the reproducibility of the preparation was examined by using the encapsulation efficiency and the drug loading as examination indexes, and the results are shown in table 2.

Table 2 reproducibility of cetirizine dihydrochloride liposome preparation method

Sequence number	EE(％)	DL(％)
			1	74.91	5.13
2	72.79	5.14
			3	74.62	5.10

Table 2 the results show that:

the average encapsulation rate of three batches of cetirizine hydrochloride liposome solutions prepared by the preparation method is 74.11 percent, and the RSD value is 1.55 percent; the average drug loading was 5.12% and RSD value was 0.41%. Therefore, the preparation method has simple and reasonable process and stable and reliable result.

2. Investigation of physical and chemical Properties of cetirizine hydrochloride Liposome

1. Morphology of Liposome

A small amount of cetirizine hydrochloride liposome prepared in example 1 was taken, diluted with PBS, added dropwise onto a copper sheet, subjected to negative dyeing with 1% sodium phosphotungstate solution and air-dried, and the microscopic morphology of the cetirizine hydrochloride liposome was observed by a Transmission Electron Microscope (TEM), and the result is shown in fig. 1.

From the electron microscope of fig. 1, the liposomes observed under TEM are uniformly distributed, have smooth surfaces and round-like appearance, and can be observed as a distinct phospholipid bilayer structure, the interior is shaded, and the liposomes can be seen to successfully encapsulate cetirizine hydrochloride.

2. Particle size and potential of liposomes

The three batches of cetirizine hydrochloride liposome solutions prepared in example 1 were respectively absorbed in appropriate amounts, diluted by appropriate factors, and the particle size distribution, zeta potential and polydispersity were measured by a dynamic light scattering instrument, and the results are shown in fig. 2.

The results in FIG. 2 show that the liposome particle size was (174.6.+ -. 1.2) nm, the polydispersity index (PDI) was 0.263.+ -. 0.013, and the zeta potential (-43.2.+ -. 1.6) mV.

3. Stability factor investigation of cetirizine hydrochloride liposome gel

1. Appearance property investigation

Three batches of cetirizine hydrochloride liposome gel prepared in example 1 were taken in a small transparent beaker and the appearance was observed, and the results are shown in fig. 3.

The results in figure 3 show that cetirizine hydrochloride liposome gel is a pale yellow semi-solid preparation, and is fine, uniform and free of agglomerated particles.

2. pH of gel

Three batches of cetirizine hydrochloride liposome gel prepared in example 1 were taken, 1g each, and 20mL of distilled water was added and stirred uniformly, so that the gel was fully dispersed in distilled water, and the pH value was measured in a pH meter. The result shows that the pH value of the cetirizine hydrochloride liposome gel is about 6.5, the cetirizine hydrochloride liposome gel is relatively stable, and the pH in the range has small irritation to human skin.

3. Centrifugal experiment

Three batches of cetirizine hydrochloride liposome gel prepared in example 1 were taken, each 1g was placed in a 2mL centrifuge tube, centrifuged at 3500r/min for 30min, and taken out for observation. The results in FIG. 3 show that cetirizine hydrochloride liposome gel has uniform appearance after centrifugation and no delamination or agglomeration phenomenon.

4. Temperature experiment

The three batches of cetirizine hydrochloride liposome gels prepared in example 1 were taken out and observed after being placed at-20 deg.c and 25 deg.c for 48 hr and at 60 deg.c for 6 hr. The results of figure 3 show that the three batches of cetirizine hydrochloride liposome gels prepared are all free from coagulation or layering phenomenon after being placed at the temperature of-20 ℃ and the temperature of 25 ℃ and the temperature of 60 ℃ for a corresponding time, and the properties of the prepared liposome gel are relatively stable.

Test example 2 irritation investigation

1. Preparation of the drugs used in the experiments

Cetirizine hydrochloride liposome gel is prepared by the method of example 1, and is placed at normal temperature for standby after sealing.

2. New Zealand rabbit skin preparation

6 healthy New Zealand rabbits for experiments in an animal experiment center are taken, local rabbit hair on the back of the rabbits are cut short by surgical scissors respectively, then, the two sides of the spine on the back of the rabbits are shaved by an electric animal shaving instrument (the shaving area is 3cm multiplied by 3 cm), and the skin exposure of the rabbits in the back shaving area can be clearly observed. The complete skin group needs to observe whether red swelling or damage occurs in the shaved area after 24 hours, and if so, the skin irritation test of the complete skin group cannot be performed. After shaving, the damaged skin group uses a sterile syringe needle to mark the shaved area in a cross shape until blood oozes out.

3. Skin irritation test with single administration

The test is mainly based on: technical guidelines for chemical drug irritation, allergy and hemolysis studies.

New Zealand white rabbits were randomly divided into a whole skin group and a broken skin group, each group having 3 animals. Vehicle controls were set up and autologous controls on the left and right sides of the syngeneic body were used. Directly coating one side of cetirizine hydrochloride liposome gel; the other side is coated with excipient (blank gel) for comparison, then covered with medical gauze, fixed by adhesive tape, each rabbit is fed in a single cage, the application time is not less than 4 hours, after 4 hours, the gauze and the adhesive tape are removed, the administration part is cleaned by warm water and wiped dry, and then the condition of whether red spots or oedema exist at the coated part is observed and recorded by naked eyes respectively at 1, 24, 48 and 72 hours.

4. Skin irritation test with multiple administrations

On the basis of the completion of the skin irritation test by single administration, vehicle controls were set according to the same group, and autologous controls on the left and right sides of the same body were used. Directly coating one side of cetirizine hydrochloride liposome gel; the other side is coated with excipient (blank gel) for comparison, then covered with medical gauze, fixed with adhesive tape, and each rabbit is fed in a single cage, the application time is not less than 4 hours, after 4 hours, the gauze and the adhesive tape are removed, the administration part is cleaned with warm water and wiped dry, then whether erythema and edema appear on the administration part is observed, and cetirizine hydrochloride liposome gel and excipient are continuously administered for 7 days.

5. Evaluation of results

The skin irritation test was performed in a single dose, the average score of the integral of skin response of each group of test substance and vehicle or vehicle at each observation time point was calculated, and the irritation intensity was evaluated according to the skin irritation response scoring criteria. The skin irritation test is carried out for multiple times, the integral mean value of each group at each observation time point is calculated firstly, then the integral mean value of each animal irritation every day in the observation period is calculated, and the irritation intensity evaluation is carried out according to the skin irritation intensity evaluation standard.

6. Scoring criteria

Skin irritation response scoring criteria: no erythema, mild erythema, moderate erythema, severe erythema, mauve erythema to mild eschar formation, 0,1,2,3,4 points respectively; no edema, mild edema, moderate edema, severe edema and severe edema were counted as 0,1,2,3,4 points, respectively.

Stimulation score = (integral of erythema + integral of edema)/animal count.

The skin irritation intensity evaluation criteria are shown in Table 3.

TABLE 3 skin irritation intensity evaluation criteria

7. Statistical method

Statistical analysis was performed on experimental data using GraphPad prism 8 software. For related dataThe comparison between groups is expressed by adopting a one-factor analysis of variance, and the comparison between groups is expressed by adopting a Tukey test. The test level α=0.05, p < 0.05, i.e. the difference is statistically significant.

8. Test results

The skin irritation test results are shown in Table 4.

TABLE 4 skin irritation results for cetirizine hydrochloride liposome gels

Table 4 experimental results show that:

after single administration and multiple administration, no obvious abnormal phenomenon is seen at the tested part of the whole skin group of the New Zealand rabbit; the damaged skin group showed slight erythema after scratch modeling, two showed slight edema and one showed slight erythema at the scratched sites observed 1h after a single administration, but the duration was short, the edema was dissipated at 24 hours after administration, and the damaged skin group showed no erythema and edema after 7 days of continuous administration.

Test example 3 drug efficacy investigation of cetirizine dihydrochloride Liposome gel

1. Establishing a model of hair loss

24 SPF-class C57BL/6 healthy mice with the age of 4 weeks are purchased and are fed into an SPF-class environment (the temperature is 25+/-1 ℃ and the humidity is 50+/-5%) of an animal experiment center of a general hospital in a south war zone, and fed for 2-3 weeks, and the mice normally eat and feed water in the feeding process, so that the experiment mice adapt to the experiment environment, and whether the eating of the mice is abnormal or not is closely observed in the period. When 24 experimental mice reached 7 weeks of age, subsequent experiments were performed.

The day before the experiment starts, 24 male C57BL/6 healthy mice with no abnormal back hair growth are subjected to back dehairing treatment, the hair in the central area of the back of the mice is removed by an animal electric dehairing instrument, and then the residual back villi is further treated by dehairing paste according to the operation instructions, wherein the dehairing area is about 2 multiplied by 2cm ² . In order to avoid long-time adhesion and irritation of the depilatory cream to the skin of the back of the mouse, the depilatory cream should be used for cleaning the depilatory part in time, and then the back of the mouse is wiped by a sterile cotton ball. The method is used for preventing the electric dehairing instrument from scratching the back skin of the experimental mouse, and the damage of the back skin can influence the growth of back hair so as to influence the observation of experimental results.

2. Experimental grouping

24C 57BL/6 male healthy mice with dehaired backs and intact skin at dehaired positions were selected and randomly divided into 3 groups of 8 mice each, a blank control group, a minoxidil group and a cetirizine hydrochloride liposome gel group (prepared by the method of example 1), and then 8 mice in each cage were labeled according to the toe-cut method.

3. Preparation of cetirizine hydrochloride liposome gel

Three batches of cetirizine dihydrochloride liposomes were prepared using the procedure of example 1.

4. Therapeutic administration

The experimental treatment dosing was started 24 hours after the mice were dehaired. Coating cetirizine hydrochloride liposome gel prepared in the example 1 on the back dehairing part of a mouse, then packaging the administration part with a preservative film, and cleaning and wiping the administration part with physiological saline after packaging for 30min each time, wherein the daily dose is 1 g/time; the minoxidil group used 5% minoxidil tincture for external use, 1 mL/time daily; the blank was applied with saline over the back and dehaired 1 mL/time daily. All three groups were dosed continuously for 18 days, during the experiment mice were fed at a temperature (25.+ -. 1) ℃ and a relative humidity (50.+ -. 5)%, and fed according to normal life habits.

5. Observation index

Skin color change and hair growth condition of the back dehairing part of the mice are observed in the experimental process, and photographing recording is carried out every day. In the administration process, whether the skin of the administration part of the mice has symptoms of contact dermatitis such as red swelling, blisters or festers and the like is closely observed, and meanwhile, whether the life habit of the mice is changed or not is also noted.

6. Statistical method

7. Experimental results

The effect of cetirizine dihydrochloride liposome gel on mouse skin color graying and hair growth time is shown in table 5 and fig. 4.

TABLE 5 time to skin color change in mice

As can be seen from the results in Table 5, the cetirizine hydrochloride liposome gel group showed statistical differences (P < 0.05) in the skin color greying and hair growth time of the mice compared with minoxidil group and blank control group, indicating that cetirizine hydrochloride has a promoting effect on hair growth of the mice. The mice do not have the symptoms of contact dermatitis such as red swelling, blisters or ulcers in the administration process.

From the photographic observations of fig. 4, it can be seen that, after 5-6 days of continuous administration, the cetirizine dihydrochloride liposome gel group mice begin to appear as larger areas of gray skin, the minoxidil group also appears as lighter gray plaques, and the blank group mice have no gray skin. After 9 days of continuous administration, the skin of the mice of the cetirizine hydrochloride liposome gel group is basically completely grey and more villi grow out; the skin of the dorsum of minoxidil group mice was also substantially fully greyed; the backs of the mice in the blank group appeared grey but uneven, and four of the mice had a large area of pale red skin.

Therefore, the cetirizine hydrochloride liposome gel prepared by the application is used as an external preparation, so that the local application concentration can be improved, the occurrence rate of adverse reactions can be reduced, the application compliance of patients can be effectively increased, and the treatment effect on AGA is remarkable.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. A method for preparing cetirizine hydrochloride liposome gel for treating androgenetic alopecia, which is characterized by comprising the following steps:

s3: adding water into carbomer-940 to swell to prepare a gel matrix, adding cetirizine hydrochloride liposome, a humectant, a preservative and distilled water into the gel matrix, and uniformly stirring and then regulating the pH value to prepare cetirizine hydrochloride liposome gel;

in the step S1, the mass ratio of the soybean lecithin to the cholesterol is (3-7): 1, an organic solvent is chloroform;

in the step S2, hydration is carried out under the stirring condition, wherein the stirring speed is 200-300rpm, the hydration temperature is 30-70 ℃, and the hydration time is 0.5-1.5h; dispersing under ultrasonic condition, wherein the ultrasonic power is 2000-2200W, and the dispersing time is 4-6min;

in the step S3, the humectant is glycerin, and the mass ratio of the gel matrix to the glycerin is 5g: (1.8-2.2) g; the preservative is ethylparaben, and the mass ratio of the gel matrix to the ethylparaben is 5g: (15-20) mg.

2. The preparation method according to claim 1, wherein in step S1, the mass ratio of soybean lecithin to cholesterol is (5-7): 1.

3. the method according to claim 1, wherein in step S1, the method for preparing a drug film comprises: removing chloroform by rotary evaporation under reduced pressure in 45-55deg.C water bath, and making into medicinal membrane.

4. The preparation method according to claim 1, wherein in step S2, the preparation method of cetirizine hydrochloride solution comprises: dissolving cetirizine hydrochloride in a PBS solution to prepare a cetirizine hydrochloride solution; wherein the mass volume ratio of cetirizine hydrochloride to PBS solution is (20-30) mg: (5-15) mL.

5. The method according to claim 1, wherein in step S2, the finishing comprises: the pellets were sized by extrusion filtration through a 0.45 μm filter.

6. The method according to claim 1, wherein in step S3, the method for preparing a gel matrix comprises: adding distilled water into carbomer-940, stirring for 2-4 hr, and swelling at 3-5deg.C for 20-28 hr to obtain gel matrix.

7. The preparation method according to claim 6, wherein the mass content of carbomer-940 in the gel matrix is 3-5%.

8. The preparation method according to claim 1, wherein in step S3, the mass-to-volume ratio of the gel matrix to cetirizine hydrochloride liposome is (2.5-5) g:10mL.

9. The preparation method according to claim 1, wherein in step S3, the mass content of carbomer-940 in the cetirizine dihydrochloride liposome gel is 0.5-1%.

10. The preparation method according to claim 9, wherein in step S3, the mass content of carbomer-940 in the cetirizine dihydrochloride liposome gel is 1%.

11. The method according to claim 1, wherein in step S3, the adjusting of the pH value comprises: triethanolamine was added dropwise to adjust the pH to 6.5.

12. Cetirizine hydrochloride liposome gel for the treatment of androgenic alopecia, characterized in that it is prepared according to the preparation method of any of claims 1-11.

13. Use of a cetirizine dihydrochloride liposome gel according to claim 12 for the preparation of a medicament for the treatment of androgenic alopecia.