CN113499434B

CN113499434B - Shampoo for treating few hairs and alopecia

Info

Publication number: CN113499434B
Application number: CN202110974075.XA
Authority: CN
Inventors: 余兵生; 杨洋
Original assignee: Haibishi Hainan Industrial Group Co ltd
Current assignee: Haibishi Hainan Industrial Group Co ltd
Priority date: 2021-08-24
Filing date: 2021-08-24
Publication date: 2023-10-31
Anticipated expiration: 2041-08-24
Also published as: CN113499434A

Abstract

The invention provides a shampoo for treating few hairs and alopecia, which contains a monoclonal antibody specific to 5 alpha reductase and hesperidin, and experiments prove that the shampoo can well promote the proliferation of mouse hairs and has a good application value.

Description

Shampoo for treating few hairs and alopecia

Technical Field

The invention relates to the field of medicines or cosmetics, in particular to shampoo for treating few hairs and alopecia.

Background

Alopecia is increasingly high in clinical proportion and various in forms. The etiology of alopecia mainly includes androgenetic alopecia and alopecia areata. Hair follicles in androgenic alopecia patients are inherently androgen sensitive and can inhibit their metabolism due to aggregation of 5α -dihydrotestosterone to the hair follicle. Alopecia areata is also considered to be associated with immune disorders, in addition to neurological disorders, and is an organ-specific disease mediated by inflammatory cells. The treatment of alopecia comprises systemic and local treatment, the finasteride oral administration and the minoxidil tincture external application have definite curative effects, and the high-concentration corticosteroid hormone external application has definite curative effects.

The clinically common hair loss is androgenetic alopecia. Androgenetic alopecia (AGA) has a family history and is an androgen-dependent hereditary alopecia. About 90% of all hair loss. In recent years, it is widely considered that the disease is an androgen-dependent polygenic hereditary disease, the incidence of white men is highest, and the incidence increases with age. Men usually develop after puberty, and women usually develop after menopause. The androgen level of the hair follicle of the patient suffering from alopecia is similar to that of normal people, the hair follicle of the patient is sensitive to androgens, and the metabolism of the hair follicle can be inhibited due to the aggregation of 5 alpha-dihydrotestosterone in the hair follicle. According to the theory of viscera in traditional Chinese medicine, the etiology of alopecia takes deficiency of liver and kidney as the principal, and damp-heat, blood heat and blood stasis as the principal. Seborrheic alopecia is mainly caused by deficiency, excess or deficiency-excess inclusion, and long-term psychological imbalance, germ infection, eating disorder and mental stress can induce or aggravate illness. Because of invasion of damp-heat into skin, malnutrition, loose striae and striae, stagnation of collaterals and unfavorable biochemical actions of essence and blood cause unstable hair root, and alopecia is caused. Dampness-heat in spleen and stomach manifests as sweet and fat, wet hair, oily scales, itchiness hair and hair loss; blood deficiency and wind dryness are manifested as dry hair, thin and loose hair, scales piling up and scalp itching.

In recent years, with the development of molecular biology, the pathogenesis of AGA, in particular the relationship between androgens and AGA, has become more apparent. This will result in the transition of the end hair follicle of the AGA patient to the microfollicular, thereby causing AGA. There are two isozymes for 5α reductase, namely type I5α reductase and type II 5α reductase. Type I5α reductase is mainly distributed in the liver, kidneys and sebaceous glands, and type ii 5α reductase is mainly present in gonadal tissue and hair follicles of the scalp. The function of 5α reductase is to convert testosterone to dihydrotestosterone, which has a greater affinity for ARP than testosterone. The above mechanism becomes an important basis for the current development of drugs for the treatment of AGA.

Currently 5 alpha reductase inhibitors are mainly of the following type. Finasteride: is steroid compound and is II type 5 alpha reductase inhibitor. Is used for treating the prostate diseases initially, and the commodity name is prostate protection. Recently, the FDA in the united states passed lmg/tablet finasteride, a commodity available under the name of milbeol, for the treatment of AGA. Finasteride is well absorbed in the gastrointestinal tract and has a biological half-life of 5-6 hours. Through metabolism of liver, urine and feces are excreted. The primary effect of finasteride is to reduce circulating dihydrotestosterone concentration by inhibiting production of dihydrotestosterone by the prostate. The extent of effect of lmg finasteride and 5mg finasteride on dihydrotestosterone in the scalp and testosterone in serum was similar. l553 AGA patients (18-4 l years old), oral finasteride or placebo, lmg/d, treatment period 1 year, with 1215 patients continuing the double blind trial 2 years. Comprehensive evaluations of efficacy were made by hair count, bi-directional evaluation by patients and researchers, and by panelists comparing the pre-and post-photographs. The results indicate that lmg finasteride promotes hair growth, while the control group continued to lose hair. WO9704002 discloses that analogues of finasteride have similar inhibitory activity to 5α reductase as Dutasteride and have promise for the treatment of AGA.

However, at present, the types of inhibitors for 5 alpha reductase are not enough, and the types of drugs to be selected are not enough, so that the drugs are worthy of further research.

Disclosure of Invention

The present invention overcomes the deficiencies of the prior art and provides improved medicaments and methods for treating bursts.

The invention provides a medicine box for treating alopecia, which is in a liquid dosage form, and the effective components of the medicine box are glucosyl hesperidin and the monoclonal antibody.

Further, the kit also contains one or more wetting agents selected from ethanol, glycerol or propylene glycol.

Hesperidin (also called hesperidin or hesperidin) is mainly present in pericarp, fruit juice and seed of citrus plants of the family Rutaceae, especially at maximum in pericarp, at a content of about 3%. Hesperidin is a dihydroflavonoid compound, is nontoxic and cannot be synthesized by human body. Clinically, the traditional Chinese medicine composition is often used as an auxiliary medicine and a hemostatic for treating hypertension. The hesperidin has the same efficacy as vitamin P, can be used together with vitamin C to enhance the effect of the vitamin C, has stronger antiviral and antibacterial effects, and can inhibit the reproduction of influenza viruses in large dosage; can inhibit tyrosinase which causes skin blackening, and can be used for treating dermatoses such as black spot and freckle. Has effects in maintaining normal permeability of blood vessel, and preventing and treating capillary hemorrhage, gingival hemorrhage, etc. Can strengthen capillary resistance, enhance elasticity and toughness of capillary, make capillary permeate normally, and can improve blood flow of scalp capillary by combining with vitamin C, which is beneficial to hair growth. Consistent with the action mechanism of minoxidil.

The monoclonal antibody adopted by the invention has the effect of inhibiting 5 a-reductase, inhibits the conversion of excessive androgens into dihydrotestosterone, reduces excessive skin grease induced by dihydrotestosterone factors, can radically solve androgenetic alopecia, and improves the 5 a-reductase inhibition effect as an external preparation of the 5 a-reductase inhibitor; clinically, the water-soluble hesperidin can strengthen the resistance of capillaries, strengthen the elasticity and toughness of the capillaries, ensure that the capillaries are penetrated normally, and can improve alopecia by being matched with monoclonal antibodies.

In one aspect, the invention provides an inhibitor of 5α reductase that is a monoclonal antibody specific for 5α reductase.

Further, the light and heavy chain variable regions of the monoclonal antibody are shown in SEQ ID NO:2 and 3.

Furthermore, the monoclonal antibody of the invention performs bioinformatics analysis according to amino acid sequences of human and mouse type II 5 alpha reductase, comprehensively analyzes secondary structure, antigenicity, hydrophilicity and hydrophobicity, accessibility and flexibility of PCT protein, performs analysis scoring on each section, and selects a region with high score as a B cell epitope region. Specifically, technical parameters such as beta rotation angle prediction by Chou & Fasman, antigen surface accessibility prediction by Emini method, protein flexibility prediction by Karplus & Schulz method, kolaska & Tongaonkar protein antigenicity analysis, parker method protein hydrophobicity analysis, bepipred linear antigen epitope prediction and the like are utilized to obtain an epitope peptide fragment of II type 5 alpha reductase, and the epitope peptide fragment is prepared by immunized mice.

Furthermore, the invention provides a hair restorer for treating alopecia, which comprises a monoclonal antibody specific to 5 alpha reductase and hesperidin, and further comprises light and heavy chain variable regions of the monoclonal antibody are respectively shown in SEQ ID NO:2 and 3;

the hair growth water can be gel preparation, and the specific preparation method comprises the following steps:

(1) Dissolving DOPE in chloroform to obtain 50 mg/mL solution ^-1 Adding triethylamine into the solution, and adding a proper amount of 200 mg.mL of the solution ^-1 PEG-(Pnp) ₂ Chloroform solution, argon shield, stir and incubate overnight at 25 ℃. Evaporating under reduced pressure to remove chloroform, and adjusting pH to 2.0 (HCl solution) to 0.15mol.L ^-1 Ultrasonic obtaining Pnp-PEG in NaCl solution ₃₀₀₀ DOPE micelle, sephadex LH 20 column for separation and purification, and chloroform extraction of Pnp-PEG ₃₀₀₀ DOPE to obtain the target product Pnp-PEG3000-DOPE;

(2) Weighing Pnp-PEG ₃₀₀₀ DOPE is dissolved in ethanol, and the monoclonal antibody prepared by the invention is dissolved in sodium dihydrogen phosphate buffer solution with pH of 5.3, pnp-PEG is added ₃₀₀₀ Injecting DOPE ethanol solution into sodium dihydrogen phosphate buffer salt, regulating pH to 8.0 with 1moL/L sodium hydroxide solution, and incubating at room temperature for 2.5h to allow the monoclonal antibody to be fully connected with Pnp-PEG3000-DOPE to obtain primary conjugate of the monoclonal antibody;

(3) Weighing phospholipid, cholesterol, pnp-PEG3000-DOPE, dissolving in chloroform, placing into an eggplant-shaped flask, and rotary evaporating at 40deg.C for 60 r.min ^-1 Removing the organic solvent at a rotating speed, introducing nitrogen by a nitrogen purging instrument, adding sodium dihydrogen phosphate buffer solution with pH of 7.5 and primary conjugate of monoclonal antibody, and 130 r.min ^-1 Removing membrane, incubating to obtain antibody modified liposome, performing ultrasonic treatment for 3min with ultrasonic breaker for 2s at intervals of 3s and power of 300W, and finishing particles with 400nm using a liposome extruder;

(4) Weighing carbomer 940, ethylparaben, adding antibody modified liposome solution, and swelling at 4deg.C in refrigerator to obtain a solution of 1mol.L ^-1 And regulating the pH value to 7.0-7.5 by sodium hydroxide, and uniformly grinding to obtain the antibody modified liposome transdermal gel preparation.

Furthermore, the invention provides a pharmaceutical composition for treating alopecia, which comprises a monoclonal antibody specific to 5 alpha reductase and hesperidin, and further comprises light and heavy chain variable regions of the monoclonal antibody are respectively shown in SEQ ID NO:2 and 3.

Furthermore, the preparation form of the medicament for treating alopecia is a liquid preparation form.

Further, the pharmaceutical excipients of the liquid dosage form include a wetting agent selected from one or more of ethanol, glycerol or propylene glycol.

Furthermore, the preparation form of the medicament for treating alopecia is an ointment preparation form.

Further, the administration route of the alopecia treating medicine is external skin administration.

As non-limiting examples, the 5α reductase inhibitor may be formulated as a composition comprising a moisturizing lotion, a cream, a sunscreen, gel, an ointment, foam, spray, etc. (which may include cosmetic formulations) that is configured to be applied at or near the hairline of an individual (male or female) to treat or prevent hair loss. The composition can be applied to skin, and can be applied to skin or absorbed into skin, massage in skin, comb or brush hair, etc.

The efficacy of a treatment to treat or prevent androgenic alopecia can be determined by monitoring the hair density on a given area of the subject's body, e.g., a given area of the scalp. If the rate of hair loss is reduced, e.g., by 10% or more, after treatment, the treatment is effective for preventing hair loss. If the hair density increases after treatment, for example by 5% or more, for example by 10% or more, and despite ongoing treatment, the treatment is also considered to be effective for the treatment and/or prevention of androgenic alopecia. As noted above, it is expected that all forms of hair loss would benefit from the techniques described herein, thereby facilitating the treatment or prevention of Yu Xiong hormonal hair loss.

The term "alopecia" of the present invention may refer to alopecia areata, androgenetic alopecia, male pattern alopecia, female alopecia, cicatricial alopecia, centralistic cicatricial alopecia, frontal fibrotic alopecia, chemotherapy-induced alopecia.

The pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier or excipient used in the preparation of generally safe pharmaceutical compositions, which is non-toxic and neither biologically nor undesirable, and includes carriers or excipients acceptable for veterinary use and human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable carrier/excipient" includes one or more than one such excipient.

Advantageous effects

The invention provides a pharmaceutical composition for treating alopecia, which contains a monoclonal antibody specific to 5 alpha reductase and hesperidin, and experiments prove that the pharmaceutical composition can well promote mouse hair proliferation and has good application value.

Drawings

FIG. 1 shows the results of specific detection of monoclonal antibody

FIG. 2A graph of hair length treatment results

FIG. 3 is a graph showing the number of hair follicles treated with mab

FIG. 4 is a graph showing the results of the combination therapy of hair follicle count

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 type II 5 alpha reductase hyperimmune screening

Bioinformatics analysis is carried out according to amino acid sequences of human and mouse type II 5 alpha reductase, secondary structure, antigenicity, hydrophilicity and hydrophobicity, accessibility and flexibility of PCT protein are comprehensively analyzed, analysis and scoring are carried out on each section, and a region with high score is selected as a B cell epitope region. Specifically, technical parameters such as beta rotation angle prediction by Chou & Fasman, antigen surface accessibility prediction by Emini method, protein flexibility prediction by Karplus & Schulz method, protein antigenicity analysis by Kolaska & Tongaonkar method, protein hydrophobicity analysis by Parker method, bepipred linear antigen epitope prediction and the like are utilized to obtain an epitope peptide fragment of II type 5 alpha reductase; the epitope peptide fragments synthesized chemically are as follows: N-cvsganafliniew-C (SEQ ID NO: 1), which introduces cysteine C at its N-terminus during chemical synthesis to increase its cross-linking ability with BSA, followed by cross-linking with BSA. Cross-linking (> 60%) resulted in a type II 5α reductase epitope peptide fragment in which vsganflgeiew was 100% matched to human and mouse type II 5α reductase.

EXAMPLE 2 preparation of type II 5 alpha reductase monoclonal antibodies

First immunization the immunogen synthesized in example 1 was diluted to lmg/ml with 0.01M phosphate buffer pH7.4 and mixed with Freund's complete adjuvant in an emulsion with 50. Mu.g of immunogen per mouse. The mice were inoculated subcutaneously on the abdomen and subcutaneously on the back. The same amount of immunogen is emulsified and mixed with Freund's incomplete adjuvant uniformly every 2-3 weeks for enhancing immunity, the total immunization is carried out for 4 times, the eyesockets of immunized mice are collected for 1 week after the last immunization, serum is centrifugally separated, an indirect ELISA method is adopted, an enzyme label plate is coated with the immunogen, and the immune titer is respectively detected. Mice with higher immunization cost effectiveness are screened, and 100 mug immunogen is used for intraperitoneal injection on the 10 th day of final immunization, so as to perform impact immunization, and conventional cell fusion is performed three days after impact immunization.

Cell fusion: subculturing NS0 cells with RPMI-1640 2d before fusion, and culturing feeder cells with HAT 1 d; on day 3 after the super-immunization of the mice used for fusion, the eyeball bloodletting method was taken, and blood was collected and serum was separated. Mice were then sacrificed by cervical removal, and after ethanol soaking, spleens were picked up on an ultra clean bench and spleen cells were prepared. Spleen cells were fused with NS0 cells in 50% PEG-4000. The fused cells were suspended in HAT medium pre-warmed at 37℃and added to 96-well cell culture plates containing feeder cells at 100. Mu.L per well. The cell culture plates were incubated in a constant temperature cell incubator containing 5% CO2 at 37 ℃. Incubate for 14d, and change fluid on days 4,7, and l0, respectively.

Screening of positive hybridoma cell strains: incubation day 14 began with RPMI-1640 and screening. The fusion cell supernatants were diluted with phosphate buffer (typically at 1:5), and positive well screening was performed by indirect ELISA and blocking ELISA. And the wells with high titer, high sensitivity and vigorous cell growth are selected for the expansion culture. Through 3 limiting dilution cloning, the hybridoma cell strain 3F6 which stably secretes the anti-type II 5 alpha reductase mAb is finally screened out and then frozen.

The anti-type II 5 alpha reductase mAb is prepared by adopting an in-vivo induced abdominal water method. Amplifying and culturing the screened positive hybridoma cell strain 3F 6; healthy BALB/c mice are selected, sterilized paraffin liquid is injected from the abdominal cavity according to the amount of 0.5 mL/mouse, 107 cloned anti-phytase positive hybridoma cells are injected from the abdominal cavity after 10d, and ascites is extracted after the abdomen of the mice becomes larger. Purifying and concentrating ascites by octanoic acid/saturated ammonium sulfate salting-out method, detecting mAb content to 1mg/mL by using DU-600 nucleic acid-protein analyzer, and freezing at-20deg.C for use.

Example 3 identification of antibodies

(1) Affinity identification

Antibody affinity refers to the degree of tightness with which an antibody binds to an epitope, and the stronger the affinity, the stronger the binding, is one of the most important indicators for evaluating the properties of an antibody. The affinity constants of the antibodies were determined by kinetic methods using the fortebioactetk 2 instrument from PALL corporation. As a result, the affinity constant of the antibody was 7.2X10-10, which gave a good affinity.

(2) Monoclonal antibody specific detection

The specificity detection of the monoclonal antibody adopts an ELISA indirect assay method, uses an immunogenic peptide as a positive control, and uses 2 mug/ml BSA, type II 5 alpha reductase and type I5 alpha reductase to identify the specificity of the monoclonal antibody. The results are shown in FIG. 1.

As can be seen from the results of FIG. 1, the monoclonal antibody prepared by the invention has better binding specificity and can specifically bind to the type II 5 alpha reductase.

(3) Monoclonal antibody sequence identification

And the light and heavy chain sequences of the antibody are identified and obtained by adopting a conventional monoclonal antibody light and heavy chain sequence identification method in the field, wherein the sequences of the light and heavy chain sequences of the antibody are respectively shown as SEQ ID NO:2 and 3.

Light chain variable region (SEQ ID NO: 2)

DIVITQRPALSAASPGEKVTITCNNLVELCRVAIMWYQQKSGISPKPWIYYAVHDGAGVPARFSGSGSGTSYSLTITSMEAEDAATYYCLWHGETVRLFGAGTKLELK

Heavy chain variable region SEQ ID NO:3

EVQLEESATELAGPGASVKLSCKASGYIFSWGWRCWIKQRPGQGLEWIGWGWRCWVWKVMQICDHGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGECREFDHWGLGTTLAVSS。

Example 4 Effect of monoclonal antibody on 5 alpha reductase Activity

3 clean female SD rats were removed from the neck and sacrificed after overnight fast, and the livers were taken out at 0deg.C and weighed and minced, and 1 was prepared with pre-chilled buffer (0.32 mol/L sucrose, 1mmol/L dithiothreitol, 20mmol/L sodium phosphate, pH 6.5) in a glass homogenizer: 4 homogenizing, sequentially centrifuging at 0deg.C for 10000g, 15min and 100000g for 1 hr to obtain 5. Reductase suspension, packaging, and storing at-80deg.C. NADPH acts as a hydrogen donor for catalyzing testosterone to generate DHT by using 5. NADPH has maximum light absorption at 340nm and is continuously consumed in the reaction process, so that the enzyme activity can be indirectly reacted according to the light absorption value of NADPH. A blank control group (testosterone+phosphate buffer solution+NADPH+5α reductase), a positive control group (testosterone+phosphate buffer solution+NADPH+5α reductase+finasteride) and a drug group (testosterone+phosphate buffer solution+NADPH+5α reductase+monoclonal antibody) were set, and the inhibition effect of the monoclonal antibody on 5α reductase was measured respectively, wherein the addition amount of 5α reductase enzyme solution was 100. Mu.l, the final testosterone concentration was 25. Mu.mol/L, and the final NADPH concentration was 100. Mu.mol/L. The results are shown in Table 1.

TABLE 1 IC50 of 5 a-reductase Activity-inhibiting monoclonal antibodies

Pharmaceutical set	IC50(μg/mL)
		Positive control group	0.051
Monoclonal antibody	0.017

From the results shown in Table 1, the monoclonal antibodies prepared by the invention have better 5 a-reductase activity inhibition effect.

EXAMPLE 5 preparation of transdermal preparation of monoclonal antibody

Dissolving DOPE 32. Mu. Mol in chloroform to obtain 50 mg/mL solution ^-1 80 mu L of triethylamine is added into the solution, and a proper amount of 200 mg/mL of triethylamine is added ^-1 PEG-(Pnp) ₂ Chloroform solution, argon shield, stir and incubate overnight at 25 ℃. Evaporating under reduced pressure to remove chloroform, and adjusting pH to 2.0 (HCl solution) to 0.15mol.L ^-1 Ultrasonic obtaining Pnp-PEG in NaCl solution ₃₀₀₀ DOPE micelle, sephadex LH 20 column for separation and purification, and chloroform extraction of Pnp-PEG ₃₀₀₀ DOPE to obtain a target product Pnp-PEG3000-DOPE, and repeating the experiment to obtain the target product for subsequent experiments.

100mg of Pnp-PEG was weighed out ₃₀₀₀ DOPE is dissolved in ethanol, 50mg of the monoclonal antibody prepared by the invention is dissolved in sodium dihydrogen phosphate buffer with pH of 5.3, pnp-PEG is added ₃₀₀₀ And (3) injecting the DOPE ethanol solution into sodium dihydrogen phosphate buffer salt, regulating the pH value to 8.0 by using 1moL/L sodium hydroxide solution, and performing incubation reaction for 2.5h at room temperature to fully connect the monoclonal antibody with Pnp-PEG3000-DOPE so as to obtain the monoclonal antibody primary conjugate.

400mg of phospholipid, 130mg of cholesterol, 9mg of Pnp-PEG3000-DOPE and 1.5mg of monoclonal antibody primary conjugate are weighed.

Phospholipid, cholesterol and Pnp-PEG ₃₀₀₀ Dissolving DOPE in chloroform, placing into 500mL eggplant-shaped flask, forming film at 40deg.C, and rotary evaporating for 60 r.min ^-1 Removing the organic solvent at a rotating speed, introducing nitrogen for 20min by using a nitrogen purging instrument, adding 10mL of sodium dihydrogen phosphate buffer solution with pH of 7.5 and the primary conjugate of the monoclonal antibody, and 130 r.min ^-1 Removing membrane, incubating for 40min to obtain antibody modified liposome, performing ultrasonic treatment for 3min with ultrasonic breaker for 2s at intervals of 3s and power of 300W, and granulating with liposome extruder at 400 nm. The predicted encapsulation efficiency in response to the liposome was 70.24%, and the measured value was (74.58.+ -. 3.40)%. The invention shows that the antibody modified liposome with better encapsulation efficiency is prepared.

Carbomer 940.2 g, ethylparaben 0.002g and 10mL of 10mg/mL antibody modified liposome solution were weighed, and after swelling in a refrigerator at 4deg.C, 1 mol.L ^-1 And regulating the pH value to 7.0-7.5 by sodium hydroxide, and uniformly grinding to obtain the antibody modified liposome transdermal hair tonic.

EXAMPLE 6 treatment of mouse alopecia

Establishment of a mouse alopecia model: about 20g of female C57BL/6J mice are evenly smeared on the backs of the mice with a proper amount of 6% sodium sulfide solution, the area is about 2cm multiplied by 3cm, and the mice are immediately washed with water and wiped dry after 2min of action. After 10% chloral hydrate was intraperitoneally injected into anesthetized mice, the backs were shaved, and the shaving range was: bilateral to the anterior axillary line, head to the under ear, tail to the tail root. After shaving, the edges of the shaving part are protected by a preservative film and a thin foam plate. The constant temperature water bath kettle maintains the water temperature to 95 ℃, the back of the mouse is immersed in the water bath for 2S, the 40% TBSA I degree scald is caused, the water at the back of the mouse is wiped after the water is discharged, the wound surface is protected by smearing 1% iodophor on the scald part, and no hair is left in the smearing area, so that the model is used as a model of the hair loss caused by the dermis injury.

Mice were randomly divided into 3 groups of 10 animals each, each of which was an antibody-treated group (0.1 g of antibody-modified liposome transdermal hair-growing agent), a negative control group (purified water), and a positive control group (0.1 mL of 1% finasteride solution). The depilatory areas were each smeared with the above solutions 2 times daily. The mice were observed daily for skin color changes and recorded on regular photographs from the time the hair loss model was established until the hair was completely covered by the back skin.

Hair length was measured on day 16 starting from the growth of hair from the back of the mice. Each group of mice took 3 plucked parts, 10 plucked hairs per part, the plucked hairs were measured with a vernier caliper, the hair length was recorded, and statistical analysis was performed. The results are shown in FIG. 2. Compared with the negative control group, the positive control group and the antibody treatment group have shortened hair growth time. On day 16 of the application, the hair of the depilatory region of the mice of the antibody treatment group is completely covered with skin, and the experimental group is significantly different from the negative control group, wherein P is less than 0.05.

Optical observation and counting of mouse skin section hair follicles: mice were sacrificed by cervical dislocation on day 16 after dehairing, the hair was drawn parallel to the spine at the dehairing site, the newly grown hair was cut off, then the skin was trimmed to a 1cm wide strip, flatly attached to cardboard, 10% formalin was fixed for 30h, tap water was used to wash out formalin, gradient alcohol dehydration, paraffin embedding, blocking, slicing, making the slice direction consistent with the longitudinal axis direction of hair growth, the slice thickness was 6 μm, hematoxylin-eosin staining, 3 low power fields (. Times.100) were taken under an optical microscope, hair follicles were counted, and statistical analysis was performed. The results are shown in FIG. 3, and compared with 41.35+ -1.02 hair follicles in the negative control group, the number of hair follicles in the antibody-treated group reaches 55.24 + -2.95, and the number is obviously increased, so that the effect is better than that of the positive control group. The hair growth experiment of mice proves that the antibody can promote the hair growth speed of dehaired mice, the hair follicle number of the treated part is obviously different from that of a negative control, and the hair growth effect is good.

Example 7 monoclonal antibody combination therapy experiment

The mice were randomly divided into 4 groups of 10 animals each, each of which was an antibody-treated group (0.1 g of antibody-modified liposome transdermal hair-growing agent), a negative control group (purified water), and an antibody-combined treatment group (0.25% glucosyl hesperidin solution+0.1 g of antibody-modified liposome transdermal hair-growing agent), and 0.25% hesperidin-treated group. Each group was smeared with the above solution 2 times a day, 50. Mu.L each time. Mice were sacrificed by cervical dislocation on day 16 after dehairing, the hair was drawn parallel to the spine at the dehairing site, the newly grown hair was cut off, then the skin was trimmed to a 1cm wide strip, flatly attached to cardboard, 10% formalin was fixed for 36h, tap water was used to wash out formalin, gradient alcohol dehydration, paraffin embedding, blocking, slicing, making the slice direction consistent with the longitudinal axis direction of hair growth, the slice thickness was 6 μm, hematoxylin-eosin staining, 3 low power fields (. Times.100) were taken under an optical microscope, hair follicles were counted, and statistical analysis was performed. The results are shown in fig. 4, and the number of hair follicles in the mice in the combination treatment group reaches 62.13±2.21, which shows that the combination treatment has a very good synergistic treatment effect, compared with the single antibody treatment group and the hesperidin group.

The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention 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 invention.

Sequence listing

<110> Yichunyu biological products Co., ltd

<120> shampoo for treating hair loss and alopecia

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Cys Val Ser Gly Ala Asn Phe Leu Gly Glu Ile Ile Glu Trp

1 5 10

<210> 2

<211> 108

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Asp Ile Val Ile Thr Gln Arg Pro Ala Leu Ser Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Asn Asn Leu Val Glu Leu Cys Arg Val

20 25 30

Ala Ile Met Trp Tyr Gln Gln Lys Ser Gly Ile Ser Pro Lys Pro Trp

35 40 45

Ile Tyr Tyr Ala Val His Asp Gly Ala Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Thr Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Trp His Gly Glu Thr Val

85 90 95

Arg Leu Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 3

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Glu Val Gln Leu Glu Glu Ser Ala Thr Glu Leu Ala Gly Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Trp Gly

20 25 30

Trp Arg Cys Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Gly Trp Arg Cys Trp Val Trp Lys Val Met Gln Ile Cys Asp

50 55 60

His Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Glu Cys Arg Glu Phe Asp His Trp Gly Leu Gly Thr Thr Leu

100 105 110

Ala Val Ser Ser

115

Claims

1. A kit for treating hair loss, the kit comprising a monoclonal antibody specific for 5α reductase and hesperidin, the monoclonal antibody having light and heavy chain variable regions as set forth in SEQ ID NO:2 and 3, wherein the hesperidin is glucosyl hesperidin, and the dosage of the antibody and the glucosyl hesperidin is 1:1.

Use of a monoclonal antibody to 5α reductase in the manufacture of a kit for treating hair loss, the kit consisting of a monoclonal antibody specific for 5α reductase and hesperidin, the monoclonal antibody having light and heavy chain variable regions as set forth in SEQ ID NO:2 and 3, wherein the hesperidin is glucosyl hesperidin, and the dosage of the antibody and the glucosyl hesperidin is 1:1.

3. The use according to claim 2, wherein the dosage form of the monoclonal antibody and hesperidin in the kit is a liquid dosage form.

4. The use according to claim 3, wherein the pharmaceutical excipients in the liquid dosage form comprise wetting agents.

5. The use according to claim 4, wherein the wetting agent is selected from one or more of ethanol, glycerol or propylene glycol.