Application of ACC inhibitor in preparation of hair growth product

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an effect of ACC in hair growth and application of an ACC inhibitor in preparation of hair growth products. The method is characterized in that ACC is acetyl coenzyme A carboxylase and is a key enzyme in a liver tissue fatty acid synthesis process. The ACC inhibitor comprises but not limited to an ACC small molecule compound inhibitor, an ACC monoclonal antibody, ACC small interfering RNA, an ACC mimic peptide inhibitor, ACC mimic antibody protein, ACC antisense oligonucleotide or an ACC vaccine, and is applied to preparation of hair growth products. A systematic or external ACC inhibitor product can be further developed and is used for promoting hair growth and treating alopecia diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to the role of acetyl-CoA carboxylase (ACC) in promoting hair growth, and the application of ACC inhibitors in the preparation of hair growth products. Background Art

[0002] Alopecia areata (AA) is a non-scarring alopecia. Epidemiological surveys in China show that the prevalence of male androgenetic alopecia is 21.3%, and that of female is 6.0%. Alopecia areata usually presents as sudden hair loss patches. In severe cases, it can affect the entire scalp, and at this time it is called alopecia totalis (AT). When it affects all the hairs on the whole body including axillary hair and pubic hair, it is called alopecia universalis (AU), which can easily have a serious impact on the appearance and psychology of patients. Abnormal or unstable autoimmune function and neuropsychiatric factors are considered important related factors. The earlier alopecia areata is intervened for treatment, the higher the cure rate. Minoxidil can promote skin blood vessel dilation, improve local blood circulation, and promote hair growth, and it is a common topical drug for treating alopecia areata. Severe alopecia areata often uses glucocorticoids, mainly including prednisolone, compound betamethasone, etc., which can be taken orally, topically or intradermally injected. For patients who are not suitable for glucocorticoid drugs, immunosuppressants can be used for treatment. Common drugs include cyclosporine and methotrexate. Glucocorticoids and immunosuppressants have many side effects.

[0003] Androgenetic alopecia (AGA), also known as androgenic alopecia, seborrheic alopecia (SA), male pattern hair loss or hereditary hair loss, is an androgen-dependent hereditary hair loss. Male hair loss often presents a horseshoe-shaped appearance. The skin at the hair loss area is shiny, with pores shrinking or leaving a little fine vellus hair remaining. The speed, scope and severity of hair loss are affected by genetics and individuals. In women, it is mostly diffuse hair loss occurring on the top of the head. The etiology and pathogenesis of androgenetic alopecia are not yet clear. Generally, it is considered that androgens and their receptors play a key role in the occurrence of this disease. Under normal physiological conditions, androgens play a certain stimulating and promoting role in the growth and development of hair in the body, but can induce hair loss in certain specific parts; testosterone is the main androgen in the body, which is converted into dihydrotestosterone by 5α-reductase. The latter can cause the transformation of terminal hair into vellus hair, ultimately leading to hair loss. Androgenetic alopecia is a refractory type of hair loss disease, and the animal model of this disease is usually used as a representative model of hair loss diseases. Minoxidil is a non-specific drug for treating hair loss and is the first-line topical drug approved by the FDA for treating hair loss. However, it may cause hirsutism on the face and limbs during use, and the therapeutic effect gradually disappears after discontinuation. Finasteride is a type II 5α-reductase selective inhibitor. The FDA has approved oral finasteride for treating androgenetic alopecia, which can continuously improve hair growth. However, finasteride has adverse reactions such as sexual dysfunction, transient reduction of sperm and abnormal gynecomastia in men. Teratogenic effects have been found in animal experiments, so it is not suitable for children and women of childbearing age. Cimetidine needs to be taken continuously for 5 months or longer, and the side effects are gynecomastia, impotence, reduced libido, etc. Oral contraceptives: mainly include norgestimate, levonorgestrel (levonorgestrel), norethindrone, ethynodiol diacetate, norgestimate (norgestimate), ethynodiol diacetate and ethynodiol diacetate, etc., which are often used to treat female AGA. Hair will improve after 6 to 12 months of treatment.

[0004] Hair loss caused by anti-tumor drugs is the most common anagen effluvium. While eliminating rapidly dividing cancer cells, anti-tumor drugs also attack rapidly dividing cells around hair follicles, causing hair loss. Hair loss treatment is difficult, especially androgenetic alopecia, which is prone to recurrence. Therefore, more safe and effective drugs and products for treating hair loss need to be found.

[0005] Acetyl-CoA carboxylase (ACC) is a key enzyme in the process of fatty acid synthesis in liver tissue. Acetyl-CoA carboxylase is a biotin enzyme, which is divided into two types: homomeric and heteromeric. It can catalyze fatty acid synthesis and is the rate-limiting enzyme in the process of fatty acid synthesis, widely existing in the biological world. Acetyl-CoA carboxylase in animals belongs to the homomeric type and is divided into two subtypes, ACC1 and ACC2. The genebank No: 35761 of ACC.

[0006] ACC inhibitors include small molecule inhibitors of ACC, ACC monoclonal antibodies, ACC mimetic peptides (polypeptide inhibitors), mimetic antibody proteins, ACC small interfering RNAs (ACC siRNAs), antisense oligonucleotide inhibitors, etc. PF-05175157 from Pfizer is a broad-spectrum and effective ACC inhibitor, which has inhibitory effects on human ACC1, human ACC2, rat ACC1 and rat ACC2. Firsocostat (GS-0976) from selleck is a reversible ACC inhibitor, which has strong inhibitory effects on human ACC1 and human ACC2; ND-646 is an allosteric inhibitor of the ACC enzyme, which can prevent the dimerization of ACC subunits, thereby inhibiting fatty acid synthesis and having inhibitory effects on human ACC1 and human ACC2. CP-610431 from MedChemExpress is a reversible ATP non-competitive isozyme and a non-selective acetyl-CoA carboxylase (ACC) inhibitor, which can inhibit ACC1 and ACC2. CP-640186 hydrochloride from MedChemExpress is an orally active and cell-permeable acetyl-CoA carboxylase (ACC) inhibitor. MK-4074 from MedChemExpress is a liver-specific inhibitor of acetyl-CoA carboxylase. hACC2-IN-1 is an effective inhibitor of acetyl-CoA carboxylase 2 (ACC2). ACC1 / 2-IN-2 (compound PF-3) is an effective ACC1 / 2 inhibitor. Summary of the Invention

[0007] The problems to be solved by the present invention are: to clarify the role of the ACC gene in the pathogenesis of hair loss and the application of ACC inhibitors in the preparation of products for treating hair loss. By establishing animal models of alopecia areata or androgenetic alopecia, experimental studies of the present invention have found that ACC plays a very crucial role in the pathogenesis of hair loss and have found the application of ACC inhibitors in the preparation of products for promoting hair growth.

[0008] Technical Solution of the Present Invention

[0009] C57BL / 6 mice are animal models widely used at home and abroad for studying the hair cycle. The hair cycles of each hair follicle in humans are asynchronous, while these mice can exhibit unique hair cycle synchrony, so they are often used as hair research models. Melanocytes in the trunk skin of these mice only exist in hair follicles and synthesize melanin only during the growth phase. During the growth phase of hair, since melanocytes in the hair bulb continuously produce melanin and transfer it to the hair follicle keratinocytes, the skin appearance shows black. During the regression phase, melanin production decreases and the skin becomes grayish-black. During the resting phase, due to the shrinkage and disappearance of the hair bulb, the hair follicle stops producing melanin and the skin turns pink. After the hair in the resting phase is plucked, a highly synchronous new hair cycle can be induced locally, and the histological changes are consistent with the natural cycle of these mice. Therefore, the change of the hair cycle can be inferred from the change of skin color.

[0010] In this invention, an experimental androgenetic alopecia model was established by subcutaneous injection of testosterone propionate injection solution, and it was found that knocking out the ACC gene could significantly promote hair growth in mice with androgenetic alopecia model and reduce the damage to subcutaneous hair follicles and sebaceous glands. This invention also established a mouse non-specific alopecia model by wax depilation and confirmed that various ACC inhibitors had obvious promoting effects on hair growth. In addition, through the experiment on the rat androgenetic alopecia model, it was found that the hair growth rate of the rats in the ACC inhibitor group was significantly faster than that of the model control group. The above experiments confirmed that ACC inhibitors play a role in the treatment of alopecia diseases.

[0011] In the preparations that can significantly block ACC in this invention, representative ACC small molecule inhibitors, ACC monoclonal antibodies, ACC mimetic peptides (polypeptide inhibitors), mimetic antibody proteins, ACC small interfering RNAs (ACC siRNAs) and antisense oligonucleotide inhibitors that can be purchased on the current market were respectively selected, and the animal alopecia models were treated by subcutaneous injection or topical skin application and compared with the blank control group. The results showed that the curative effects of the ACC inhibitor groups were significantly better than those of the model control group, and no obvious adverse reactions occurred in each group. The experiments confirmed that systemic or topical ACC inhibitors had obvious promoting effects on hair growth.

[0012] In summary, we found that knocking out the ACC gene could significantly promote hair growth, and ACC inhibitors could reverse alopecia caused by various reasons and had obvious promoting effects on hair growth. Based on the above research, this invention relates to the application of ACC inhibitors (blockers) in the preparation of products for treating alopecia diseases, wherein the ACC is Acetyl CoA carboxylase, a biotin enzyme encoded by the ACC gene; the alopecia diseases include androgenetic alopecia, alopecia areata or alopecia caused by various treatments of diseases, etc.

[0013] The ACC inhibitor described in the present invention can be any product or method that can inhibit the expression or secretion of the ACC gene by conventional molecular biology or medicinal chemistry means, such as, but not limited to, knocking out or silencing the ACC gene by existing molecular biology techniques; in some embodiments, it can also be or use an ACC inhibitor. Preferably, the above ACC inhibitor (blocker) is an ACC small molecule compound, an ACC monoclonal antibody, an ACC small RNA inhibitor, an ACC mimetic peptide, an ACC mimetic antibody protein, an ACC antisense oligonucleotide, or an ACC vaccine.

[0014] In some examples, the ACC small molecule inhibitors described in the present invention include, but are not limited to, the product PF-05175157 from Selleck, chemical formula: C 24 H 27 N3O 2， Molecular weight: 405.49, structural formula: (CAS: 1301214-47-0); or the synthetic product Firsocostat (GS-0976, NDI-010976, ND-630) from Selleck, chemical formula: C 28 H 31 N3O8S, molecular weight: 569.63, structural formula: (CAS: 1434635-54-7); or the synthetic product ND-646 from Selleck, chemical formula: C 28 H 32 N4O7S, molecular weight: 568.64, structural formula: (CAS: 1434639-57-2); or CP-640186 from MedChemExpress, chemical formula: C 30 H 36 ClN3O3, molecular weight: 522.08, structural formula: (CAS: 591778-70-0). MK-4074 from MedChemExpress, chemical formula: C 33 H 31 N3O6, molecular weight: 565.62, structural formula: (CAS: 1039758-22-9). hACC2-IN-1, chemical formula: C 23 H 32 N2O4S, molecular weight: 432.58, structural formula: (CAS: 192323-14-1). ACC1 / 2-IN-2, chemical formula: C 24 H 25 N3O3, molecular weight: 403.47, structural formula: (CAS: 1031411-94-5).

[0015] In some examples, the ACC monoclonal antibody inhibitors described in the present invention include, but are not limited to, the acyl-CoA carboxylase recombinant monoclonal antibody of Wenzhou Kemiao Biotechnology, AJ1010a of the Antibody Library of Dingxiangtong, and RT1015 of Huaan Biotechnology.

[0016] In some examples, the Acc1 and -2 antisense oligonucleotide inhibitors ASO of the present invention are as follows: ISIS-338292, 5′-CGTGGGATGCCTTCTGCTCT-3′ (position 5116-5136bp NM_022193); ISIS-189594, 5′-GAGTCCTCTGCTGACTGGC-3′ (241-261bp AB004329); and ISIS-362037, 5′-CTCCATCTGGGTTTTCGCTG-3′ (1530-1550bp NM_022193 and 1950-1970bp AB004329).

[0017] As is well known to those skilled in the art, based on the above mechanism of action of ACC, ACC inhibitors also have a therapeutic effect on other alopecia diseases. ACC inhibitors can be used alone or in combination with other drugs or treatment methods, including traditional drugs, physical therapy methods, and skin care products, etc.

[0018] The present invention also provides a pharmaceutical composition, which is composed of the monomer described in the present invention or its pharmaceutically acceptable salt as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier.

[0019] The monomer or composition described in the present invention can be prepared into any dosage form permitted in pharmacy, for example, a preparation suitable for any administration method such as topical application to the skin and hair, oral administration, parenteral administration, intraperitoneal administration, intravenous administration, intraarterial administration, transdermal administration, sublingual administration, intramuscular administration, rectal administration, buccal administration, intranasal administration, inhalation administration, vaginal administration, intraocular administration, topical administration, subcutaneous administration, intralipid administration, intra-articular administration, intraperitoneal administration, or intrathecal administration.

[0020] In a preferred embodiment, the dosage form described in the present invention is a solution, lotion, tincture, spirit, aerosol, oil, emulsion, ointment, film-forming agent, gel, tablet, granule, oral liquid, capsule, dropping pill, enema, film, or injection.

[0021] The "product" described in the present invention can be any preparation suitable for administration to a patient according to the methods commonly used in the pharmaceutical field, such as drugs, cosmetics, toiletries, and health products. The product described in the present invention can be administered to mammals, such as rats, mice, livestock, humans, etc.; in some specific examples, it is humans.

[0022] The beneficial effects of the present invention compared with the prior art are as follows: The present invention provides a new treatment method for hair loss diseases. Through the disclosure of the present invention, system or topical ACC inhibitor (blocker) products can be further prepared, and then monomeric new drugs or compound preparations containing various ACC inhibitors can be developed for the treatment of various types of hair loss diseases, including androgenetic alopecia or alopecia areata. Existing clinical trials have proved that drugs containing such ACC inhibitors have significant curative effects and good tolerance, and can provide a series of new products with good curative effects and safety for the market. Description of the Drawings

[0023] Figure 1 On the 45th day of modeling, the hair growth of mice in each group was observed. The hair growth of the ACC- / - mouse group was faster than that of the model group, and there was no significant difference compared with the negative control.

[0024] Figure 2 The immunohistochemical results showed that the expression of VEGF protein in the skin hair follicles of the model group decreased. Compared with the model group, the expression of VEGF protein in the ACC- / - group increased significantly, and there was no significant difference compared with the negative control. Specific Embodiments:

[0025] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0026] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include the plural forms.

[0027] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. Except for the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention, and all belong to the protection scope of the present invention.

[0028] Example 1 Effect of ACC gene knockout on androgenetic alopecia (AGA)

[0029] 1. Experimental methods

[0030] 1.1 Animal grouping and model establishment

[0031] Experimental animals: SPF-grade C57BL / 6 mice, C57BL / 6-ACC- / - mice (using CRISPR gene editing technology to knockout the ACC gene and establish an ACC gene knockout mouse model).

[0032] The mice with different genotypes were divided into three groups: the negative control group of C57BL / 6 mice, the model group of C57BL / 6 mice, and the model group of C57BL / 6-ACC- / - mice, with 8 mice in each group, 4 males and 4 females. The back of each mouse was depilated as the observation area. Except for the negative control group, the other groups of mice were subcutaneously injected with testosterone propionate injection [8 ml / (kg·d)] at the back of the neck once a day for 60 consecutive days to establish an AGA model. After 30 days of continuous subcutaneous injection of testosterone propionate, the mice gradually showed hair loss, indicating that the androgenetic alopecia model was successfully established. The hair growth was observed.

[0033] 1.2 Observation indicators and test methods

[0034] In the scalp follicles of the alopecia area of AGA patients, the expression of vascular endothelial growth factor (VEGF) is lower than that of normal people. In this experiment, the expression of VEGF protein in the skin of the observation area on the back of the mice was detected by immunohistochemistry. Every 15 days, 10 hairs were plucked from the observation area on the back of each mouse, and the hair length was measured with a vernier caliper. After the experiment, the skin of the experimental observation area was taken for routine tissue dehydration, paraffin embedding, staining, and light microscopy to observe the pathological changes of the skin follicles and sebaceous glands of the mice, and immunohistochemistry was used to detect the expression of vascular endothelial growth factor (VEGF) protein in the skin of each group. Semi-quantitative analysis was performed on the lesions of each group. The grading criteria are as follows: The cells of the skin dermis tissue and the subcutaneous hair follicles and sebaceous gland structures are normal, recorded as "-": There is no hyperplasia in the skin dermis, the lesions of the hair follicles and sebaceous glands are limited, and there is no inflammation in the subcutaneous tissue, recorded as "±": There is no obvious hyperplasia in the skin dermis tissue, the hair follicles are significantly cystic, the sebaceous glands have no obvious hyperplasia, and there is no inflammation in the subcutaneous tissue, recorded as "+": The segmental hyperplasia of the skin dermis tissue is not obvious, a small number of hair follicles have cystic changes, the sebaceous glands have mild hyperplasia and hypertrophy, and there is no obvious inflammation in the subcutaneous tissue, recorded as "++": The cells of the skin dermis tissue have segmental hyperplasia of different degrees, some hair follicles are cystic, showing uneven hair follicle sizes and no cells in the peripheral part. The sebaceous glands have hyperplasia, and there are fewer cell nuclei in the hyperplastic glandular bodies. There is mild inflammatory hyperplasia in the subcutaneous tissue of individual mice, recorded as "+++".

[0035] 2. Experimental results

[0036] 2.1 Effect on mouse hair growth

[0037] The hair lengths of the C57BL / 6-ACC- / - mouse model group were longer than those of the C57BL / 6 mouse model group on the 15th, 30th, 45th, and 60th days after modeling, and the differences were statistically significant (P<0.01). See Table 1. The hair growth conditions of mice in each group on the 45th day after modeling are shown in Figure 1 .

[0038] Table 1 Hair growth lengths of mice in each group

[0039]

[0040] * P<0.01 compared with the C57BL / 6 mouse model group

[0041] 2.2 Effect on the morphology of hair follicles in the superficial dermis of the skin tissue in the observation area of mice

[0042] In the C57BL / 6 mouse model group, some skin dermal tissue cells showed segmental thickening to varying degrees, and subcutaneous lymphocytes increased; some subcutaneous hair follicles in mice showed obvious cystic changes, with different sizes of hair follicles, mild fibrosis around them, disappearance of cells around the hair follicles or obvious reduction in the number of cell layers, increased number of sebaceous glands, hypertrophy of some glands, obvious reduction in the number of nuclei in the hypertrophied glands, and reduction in the number of normal hair follicles. The immunohistochemical results in various tissues of the skin showed a decrease in the expression of VEGF protein. The lesions of skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands in the C57BL / 6-ACC- / - mouse model group were alleviated to varying degrees compared with those in the C57BL / 6 mouse model group, and the number of damaged hair follicles in the skin was significantly reduced. See Table 2. The immunohistochemical results showed that the expression of VEGF protein in the skin hair follicles of the model group decreased. Compared with the model group, the expression of VEGF protein in the ACC- / - mouse model group increased, and there was no significant difference compared with the negative control. See Figure 2 .

[0043] Table 2 Effects of each group on mouse skin hair follicles and sebaceous glands (number)

[0044] Group — ± + ++ +++ P value Negative control group 8 0 0 0 0 <0.01 C57BL / 6 mouse model group 0 0 1 2 5 —— C57BL / 6-ACC- / - mouse model group 0 4 3 1 0 <0.01

[0045] * P<0.01 compared with the mouse model group

[0046] 3. Experimental conclusions

[0047] Knockout of the ACC gene can significantly promote hair growth in mice with androgenetic alopecia models and reduce the damage to subcutaneous hair follicles and sebaceous glands.

[0048] Example 2 Effect of ACC inhibitor on mouse hair growth

[0049] 1. Experimental method

[0050] 1.1 Materials

[0051] 1.1.1 ACC Small Molecule Inhibitor 1: PF-05175157 from Selleck, chemical formula: C 24 H 27 N3O 2， Molecular weight: 405.49 (CAS: 1301214-47-0); ACC Small Molecule Inhibitor 2: Firsocostat (GS-0976, NDI-010976, ND-630) from Selleck, chemical formula: C 28 H 31 N3O8S, molecular weight: 569.63, CAS: 1434635-54-7; ACC Small Molecule Inhibitor 3: ND-646 from Selleck, chemical formula: C 28 H 32 N4O7S, molecular weight: 568.64, CAS: 1434639-57-2; ACC Small Molecule Inhibitor 4: CP-610431 from MedChemExpress, chemical formula: C 30 H 37 N3O2, molecular weight: 471.63, (CAS: 591778-83-5); ACC Small Molecule Inhibitor 5: CP-640186 from MedChemExpress, chemical formula: C 30 H 36 ClN3O3, molecular weight: 522.08 (CAS: 591778-70-0); ACC Small Molecule Inhibitor 6: MK-4074 from MedChemExpress, chemical formula: C 33 H 31 N3O6, molecular weight: 565.62 (CAS: 1039758-22-9); ACC Small Molecule Inhibitor 7: hACC2-IN-1, chemical formula: C 23 H 32 N2O4S, molecular weight: 432.58 (CAS: 192323-14-1). ACC Small Molecule Inhibitor 8: ACC1 / 2-IN-2, chemical formula: C 24 H 25 N3O3, molecular weight: 403.47 (CAS: 1031411-94-5). Preparation method of sample topical solution: 60% ethanol is respectively mixed with appropriate amounts of the above ACC inhibitors to prepare solutions with different concentrations.

[0052] 1.1.2 ACC monoclonal antibodies 1: Recombinant monoclonal antibody of acyl-CoA carboxylase from Wenzhou Kemiao Biotechnology; ACC monoclonal antibody 2: AJ1010a from the Antibody Library of Dingxiangtong; ACC monoclonal antibody 3: RT1015 from Huaan Biotechnology Co., Ltd.

[0053] 1.1.3 Antisense oligonucleotide inhibitors of Acc-1 and -2, ASO, are as follows: ISIS-338292, 5′-CGTGGGATGCCTTCTGCTCT-3′ (position 5116 - 5136bp NM_022193); ISIS-189594, 5′-GAGTCCTCTGCTGACTGGC-3′ (241 - 261bp AB004329); and ISIS-362037, 5′-CTCCATCTGGGTTTTCGCTG-3′ (1530 - 1550bp NM_022193 and 1950 - 1970bp AB004329).

[0054] 1.1.4 siRNA-ACC: ACC small interfering RNA, synthesized by Shanghai Sangon Biotech Co., Ltd.

[0055] 1.1.5 Positive control group: 2% minoxidil solution, produced by the Institute of Dermatology, Chinese Academy of Medical Sciences.

[0056] 1.2 Animal grouping and model establishment

[0057] SPF-grade C57BL / 6 mice were selected, numbered according to body weight, and divided into groups by the random permutation table method: Compound Group 1 (applying 2% PF-05175157 solution on the skin), Compound Group 2 (applying 2% Firsocostat solution on the skin), Compound Group 3 (applying 2% ND-646 solution on the skin), Compound Group 4 (applying 2% CP-610431 solution on the skin), Compound Group 5 (applying 2% CP-640186 solution on the skin), Compound Group 6 (applying 2% MK-4074 solution on the skin), Compound Group 7 (applying 2% hACC2-IN-1 solution on the skin), Compound Group 8 (applying 2% hACC2-IN-2 solution on the skin), Monoclonal Antibody Group 1 (subcutaneously injecting Kemin Bio ACC monoclonal antibody, 15 mg / kg per week), Monoclonal Antibody Group 2 (subcutaneously injecting AJ1010a, 15 mg / kg per week), Monoclonal Antibody Group 3 (subcutaneously injecting RT1015, 15 mg / kg per week), Acc-1 and -2 Antisense Oligonucleotide Inhibitor ASO Group (applying 2% antisense oligonucleotide solution on the skin, with the same sequence as above), Positive Control Group (applying 2% minoxidil solution on the skin), Negative Control Group (applying 60% ethanol on the skin). There were 10 mice in each group, with 5 males and 5 females. After the mice were anesthetized with ether, a mixture of rosin / paraffin (1:1) was melted and applied to the back. After it solidified and hardened, it was removed. The back of the mouse was considered clean when it was smooth, without injury and hair roots, and the depilated area was about 3 cm × 4 cm. From the second day after depilation, the corresponding drugs were applied to the depilated area, twice a day, 0.5 mL per mouse each time.

[0058] 1.3 Observation Indicators and Test Methods

[0059] 1.3.1 Macroscopic Observation

[0060] From the second day after drug application, observe the daily color change of the skin at the hair-pulling site on the back of the mice to determine the growth status of hair follicles and visually observe the hair growth in the depilated area with the naked eye. Score the new hair growth status in the depilated area of each mouse once a day. Reserve 5 mice in each group for macroscopic observation for 40 days, and record the hair growth on the back.

[0061] 1.3.2 Histological Observation Experiment

[0062] On the 17th day, 5 mice in each group were sacrificed by cervical dislocation. Samples were taken from the same part of the back parallel to the spine, fixed in 10% formaldehyde, dehydrated, embedded in paraffin, sectioned, stained with HE, sealed with neutral gum, and the histological changes of hair follicles were observed under a light microscope and the hair follicles were morphologically staged. According to the international hair cycle scoring method, the following scores were given to hair follicles in each stage: stage VI of growth was 100 points, early regression was 200 points, middle regression was 300 points, and late regression was 400 points. 50 hair follicles were randomly selected from each mouse to determine the cycle of hair follicles in each group, and the average hair cycle score and the percentages of hair follicles in stage VI of growth, early regression, middle regression, and late regression were calculated.

[0063] 1.4 Statistical methods

[0064] The experimental data were statistically processed using SPSS 16.0 system software. The statistical variables of the experimental data were expressed as (x±s), and χ2 test and t test were used. The α value was taken bilaterally. P>0.05 indicated no significant difference, P<0.05 indicated a significant difference, and P<0.01 indicated a highly significant difference.

[0065] 2. Experimental results

[0066] 2.1 Macroscopic observation of the changes in the hair removal sites of mice

[0067] In the negative control group, the back skin of mice changed from pink to black on the 7th day after hair removal, and then changed from black to gray-black around the 19th day after hair removal. In the inhibitor group and the positive control group, the back skin of mice changed from pink to black around the 6th day after hair removal, and then changed from black to gray-black around the 17th day after hair removal. As shown in Table 3, there was no significant difference in the time of skin color turning black and gray-black and the duration of black color in the hair removal areas of mice in each inhibitor group compared with the positive control group (P>0.05 for all), and there were significant differences compared with the negative control group (P<0.01 for all). It is suggested that ACC inhibitors have an obvious effect of prolonging the growth phase of hair follicles.

[0068] Table 3 Changes in the skin color of the hair removal areas on the backs of mice in each group (days)

[0069]

[0070]

[0071] Note: *P<0.05 compared with the negative control group for all.

[0072] 2.2 Hair growth in the hair removal areas on the backs of mice

[0073] Around the 11th day after hair plucking, new hair appeared in the hair-plucked area on the back of the mice in the negative control group, and around the 37th day, the hair length in the hair-plucked area was the same as that in the non-experimental area. Around the 7th day after hair plucking, new hair appeared in the hair-plucked area on the back of the mice in the inhibitor group and the positive control group, and around the 19th day, the hair length in the hair-plucked area was the same as that in the non-experimental area. As shown in Table 4, there was no significant difference in the new hair growth rate between the inhibitor group and the positive control group (P>0.05 for both), and there were significant differences between the inhibitor group and the negative control group (P<0.01 for both). It is suggested that the inhibitor has an obvious effect on promoting hair growth.

[0074] Table 4 Hair growth time (days) in the hair-plucked area on the back of mice in each group

[0075]

[0076] Note: *There were significant differences compared with the negative control group (P<0.01 for both)

[0077] 3. Experimental conclusion

[0078] The ACC inhibitor has an obvious effect of prolonging the hair follicle growth period in mice, thus promoting hair growth.

[0079] Example 3 Effects of various ACC inhibitors on androgenetic alopecia (AGA) rat models

[0080] 1. Experimental method

[0081] 1.1 Materials

[0082] 1.1.1 ACC small molecule inhibitor 1: PF-05175157 from Selleck, chemical formula: C 24 H 27 N3O 2， Molecular weight: 405.49 (CAS: 1301214-47-0); ACC small molecule inhibitor 2: Firsocostat (GS-0976, NDI-010976, ND-630) from Selleck, chemical formula: C 28 H 31 N3O8S, molecular weight: 569.63, CAS: 1434635-54-7; ACC small molecule inhibitor 3: ND-646 from Selleck, chemical formula: C 28 H 32 N4O7S, molecular weight: 568.64, CAS: 1434639-57-2; ACC small molecule inhibitor 4: CP-610431 from MedChemExpress, chemical formula: C 30 H 37N3O2, molecular weight: 471.63, (CAS: 591778-83-5); ACC small molecule inhibitor 5: CP-640186 from MedChemExpress, chemical formula: C 30 H 36 ClN3O3, molecular weight: 522.08 (CAS: 591778-70-0); ACC small molecule inhibitor 6: MK-4074 from MedChemExpress, chemical formula: C 33 H 31 N3O6, molecular weight: 565.62 (CAS: 1039758-22-9); ACC small molecule inhibitor 7: hACC2-IN-1, chemical formula: C 23 H 32 N2O4S, molecular weight: 432.58 (CAS: 192323-14-1). ACC small molecule inhibitor 8: ACC1 / 2-IN-2, chemical formula: C 24 H 25 N3O3, molecular weight: 403.47 (CAS: 1031411-94-5). Preparation method of the sample topical solution: Mix 60% ethanol with appropriate amounts of the above ACC inhibitors to prepare solutions with different concentrations.

[0083] 1.1.2 ACC monoclonal antibody 1: Recombinant monoclonal antibody of acyl-CoA carboxylase from Kemiiao Biotech Co., Ltd. in Wenzhou; ACC monoclonal antibody 2: AJ1010a from the Antibody Library of Dingxiangtong; ACC monoclonal antibody 3: RT1015 from Huaan Biotech.

[0084] 1.1.3 Acc1 and -2 antisense oligonucleotide inhibitors ASO are as follows: ISIS-338292, 5′-CGTGGGATGCCTTCTGCTCT-3′ (position 5116 - 5136bp NM_022193); ISIS-189594, 5′-GAGTCCTCTGCTGACTGGC-3′ (241 - 261bp AB004329); and ISIS-362037, 5′-CTCCATCTGGGTTTTCGCTG-3′ (1530 - 1550bp NM_022193 and 1950 - 1970bp AB004329).

[0085] 1.1.4 Positive control group: 5% minoxidil tincture (trade name: Mandy, produced by Zhejiang Wansheng Pharmaceutical Co., Ltd.).

[0086] 1.2 Animal grouping and model establishment

[0087] SPF-grade Wistar rats were selected and divided into groups using a random permutation table method: Compound Group 1 (skin applied with 5% PF-05175157 solution), Compound Group 2 (skin applied with 5% Firsocostat solution), Compound Group 3 (skin applied with 5% ND-646 solution), Compound Group 4 (skin applied with 2% CP-610431 solution), Compound Group 5 (skin applied with 5% CP-640186 solution), Compound Group 6 (skin applied with 5% MK-4074 solution), Compound Group 7 (skin applied with 5% hACC2-IN-1 solution), Compound Group 8 (skin applied with 5% hACC2-IN-2 solution), Monoclonal Antibody 1 Group (subcutaneously injected with Kemiao Bio ACC monoclonal antibody, 10 mg / kg per week), Monoclonal Antibody 2 Group (subcutaneously injected with AJ1010a, 10 mg / kg per week), Monoclonal Antibody 3 Group (subcutaneously injected with RT1015, 10 mg / kg per week), Acc1 and -2 Antisense Oligonucleotide Inhibitor ASO Group (skin applied with 5% antisense oligonucleotide solution, same sequence as above), Positive Control Group (skin applied with 5% minoxidil solution), Negative Control Group (skin applied with 75% ethanol), Model Control Group (skin applied with 75% ethanol), with 10 rats in each group, 5 males and 5 females.

[0088] Before the experiment, a 4 cm x 5 cm area on the back of each rat was depilated as the observation area. Except for the Negative Control Group, rats were subcutaneously injected with testosterone propionate injection [5 ml / (kg·d)] at the back of the neck once a day for 60 consecutive days to establish an AGA model. After 4 weeks of continuous subcutaneous injection of testosterone propionate, rats gradually showed hair loss, and the remaining hair became thin and brittle, indicating the successful establishment of the androgenetic alopecia model. During the modeling, the corresponding drug groups of rats were given drugs by skin application or subcutaneous injection in the observation area of the back. The application dose was 1 mL / (rat·time), once a day; for subcutaneous injection, once a day. The Negative Control Group and the Model Control Group were applied with excipient (75% ethanol solution), 1 mL / (rat·time), once a day, for 60 consecutive days.

[0089] 1.3 Observation Indicators and Test Methods

[0090] Hair samples were taken from the back observation area of each rat every 15 days during the administration period, and the hair length was measured using a vernier caliper. After 60 days of administration, the skin samples from the experimental observation area were taken for routine tissue dehydration, paraffin embedding, HE staining, and light microscopy examination to observe the histopathological changes of hair follicles and sebaceous glands in the rat skin. Semi-quantitative analysis was performed on the lesions in each group. The grading criteria were as follows: The skin dermis tissue cells and subcutaneous hair follicles and sebaceous gland structures were normal, recorded as "-"; There was no hyperplasia in the skin dermis, the lesions of hair follicles and sebaceous glands were limited, and there was no inflammation under the skin, recorded as "±"; There was no obvious hyperplasia in the skin dermis tissue, obvious cystic changes in hair follicles, no obvious hyperplasia in sebaceous glands, and no inflammation under the skin, recorded as "+"; There was segmental hyperplasia in the skin dermis tissue, which was not obvious, cystic changes in a small number of hair follicles, mild hyperplasia and hypertrophy of sebaceous glands, and no obvious inflammation under the skin, recorded as "++"; There was segmental hyperplasia of different degrees in the skin dermis tissue cells, cystic changes in some hair follicles, showing uneven hair follicle sizes and no cells in the peripheral part, hyperplasia of sebaceous glands, fewer cell nuclei in the hyperplastic glandular bodies, and mild inflammatory hyperplasia under the skin in individual rats, recorded as "+++".

[0091] 2. Experimental results

[0092] 2.1 Effects on rat hair growth

[0093] The hair lengths of rats in the ACC inhibitor group were longer than those in the model control group on the 15th, 30th, 45th, and 60th days of administration, and the differences were statistically significant (P < 0.01). See Table 5

[0094] Table 5 Effects of each group on rat hair growth length

[0095]

[0096] * The differences were statistically significant compared with the model control group (P < 0.01 for all)

[0097] 2.2 Effects on the morphology of hair follicles in the superficial dermis of the skin tissue in the rat observation area

[0098] In the model group, some skin dermis tissue cells in rats had segmental thickening of different degrees, there was mild lymphatic keratinization under the skin in rats, mild fibrosis around, the cells around the hair follicles disappeared or the cell layers were significantly reduced, and there seemed to be calcified substances stained blue in the cavity. The number of sebaceous glands increased, some glands were hypertrophied, the cell nuclei in the hypertrophied glands were significantly reduced, and the number of normal hair follicles decreased. The lesions of skin dermis tissue cells, subcutaneous hair follicles, and sebaceous glands in the ACC inhibitor group and minoxidil tincture group were alleviated to varying degrees compared with the model group. The number of damaged hair follicles in the skin of rats in the ACC inhibitor group was significantly reduced compared with the model control group (P < 0.01 for all). Compared with the model control group, the lesions of skin dermis tissue cells, subcutaneous hair follicles, and sebaceous glands in the ACC inhibitor group and minoxidil tincture group were significantly alleviated (P < 0.01 for all). See Table 6

[0099] Table 6 Effects of each group on hair follicles and sebaceous glands of rat skin (rats)

[0100] Group Number — ± + ++ +++ P value * Model control group 10 0 0 2 4 4 — Negative control group 10 10 0 0 0 0 <0.01 Positive control group 10 3 4 2 1 0 <0.01 Compound group 1 10 1 5 3 1 0 <0.01 Compound group 2 10 3 5 1 1 0 <0.01 Compound group 3 10 2 5 2 1 0 <0.01 Compound group 4 10 2 6 1 2 0 <0.01 Compound group 5 10 3 3 3 1 0 <0.01 Compound group 6 10 1 4 3 2 0 <0.01 Compound group 7 10 3 3 2 2 0 <0.01 Compound group 8 10 2 4 3 1 0 <0.01 Monoclonal antibody group 1 10 0 4 4 2 0 <0.01 Monoclonal antibody group 2 10 2 5 2 1 0 <0.01 Monoclonal antibody group 3 10 2 4 2 2 0 <0.01 Antisense oligonucleotide group 10 2 3 3 2 0 <0.01 siRNA-ACC group 10 2 4 3 1 0 <0.01 Combined treatment group 1 10 5 3 1 1 0 <0.01 Combined treatment group 2 10 5 4 1 0 0 <0.01

[0101] Note: * indicates significant difference compared with the model control group (P < 0.01 for all)

[0102] 3. Experimental conclusions

[0103] ACC inhibitors can significantly promote hair growth in rats with androgenetic alopecia model and reduce the damage to subcutaneous hair follicles and sebaceous glands.

Claims

1. Use of an ACC inhibitor in the preparation of a hair growth product, characterized in that The ACC is acetyl-CoA carboxylase, including ACC1 and ACC2.

2. The application according to claim 1, characterized in that The hair loss diseases include, but are not limited to, androgenetic alopecia, seborrheic alopecia, alopecia areata, or drug-induced alopecia.

3. The application according to claim 1 and 2, characterized in that, The ACC inhibitors include, but are not limited to, small molecule compound inhibitors of ACC, ACC monoclonal antibodies, ACC small interfering RNAs, ACC mimetic peptide inhibitors, ACC mimetic antibody protein inhibitors, ACC antisense oligonucleotides, or ACC vaccines.

4. The application according to any one of claims 1 to 3, characterized in that, The ACC inhibitor is a small molecule compound inhibitor of ACC, an ACC monoclonal antibody, an ACC small interfering RNA, an ACC mimetic peptide inhibitor, or an ACC antisense oligonucleotide.

5. The application according to claims 1 to 3, characterized in that The small molecule compound inhibitors of ACC include, but are not limited to, compounds with structural formulas as shown in Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, and Formula VII; 6. The application according to claims 1 to 3, characterized in that Use of an ACC inhibitor and a pharmaceutically acceptable carrier in the preparation of a hair growth product.

7. The application according to claims 1 to 3, characterized in that, The ACC inhibitor can be used alone or in combination with other treatment methods, products, or drugs in the preparation of a hair growth product.

8. The application according to any one of claims 1 to 3, characterized in that, The ACC inhibitor and the composition described in Claims 6-7 can be prepared into any pharmaceutically acceptable dosage form for use in a hair growth product.

9. The application according to claims 1 to 3, characterized in that The ACC inhibitor and the composition described in Claims 6-7 can be prepared for any pharmaceutically acceptable administration method for use in a hair growth product.

Images