Application of daidzin in preparation of medicine for preventing or treating atopic dermatitis

Abstract

The invention belongs to the technical field of medicines, discloses application of daidzein in preparation of atopic dermatitis medicines, and particularly discloses daidzein shown as a structural formula (I) and application of pharmaceutically acceptable salts of the daidzein in preparation of the atopic dermatitis prevention or treatment medicines. The invention also discloses combined application of daidzein and other medicines for atopic dermatitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of daidzein (CAS: 552-66-9) in the preparation of drugs for the prevention or treatment of atopic dermatitis. This invention includes the single or combined use of daidzein in the prevention or treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common, chronic, relapsing, inflammatory, and itchy skin disease characterized by erythema, papules, exudation, desquamation, and itching. It often begins in childhood and can persist into adulthood. S. (2021). Atopic Dermatitis. N Engl J Med, 384(12), 1136-1143). The prevalence of atopic dermatitis has been rising in recent years, with an adult prevalence of 2.1-4.9% and a child prevalence of 15%-20%, making it the skin disease with the highest disease burden (Laughter, MR, et al. (2021). The global burden of atopic dermatitis: lessons from the Global Burden of Disease Study 1990–2017. British Journal of Dermatology, 184(2), 304-309). Atopic dermatitis is a heterogeneous disease, which can be caused by a variety of factors such as genetics, skin barrier function, immune dysregulation, environmental factors, and microbial imbalance. Furthermore, there are significant individual differences in the clinical manifestations, severity, and treatment response of patients (Ziegler, SF (2021). Thymic Stromal Lymphopoietin, Skin barrier dysfunction, and the atopic dermatitis. Ann Allergy Asthma Immunol. doi:10.1016 / j.anai.2021.06.004).

[0003] Medication is an important means of treating atopic dermatitis. Topical medications are first-line therapy, including topical corticosteroids, calcineurin inhibitors, and phosphodiesterase 4 (PDE4) inhibitors. For severe cases, systemic therapy is used clinically, including antihistamines, immunosuppressants, JAK inhibitors, and monoclonal antibodies. S. (2021). Atopic Dermatitis. N Engl J Med, 384(12), 1136-1143; Atopic Dermatitis Collaborative Study Group (2020). Chinese Guidelines for Diagnosis and Treatment of Atopic Dermatitis (2020 Edition). Chin J Dermatol, 53(2), 81-88). Although there are multiple drugs available in the clinic, the survey results show that the satisfaction of atopic dermatitis patients with existing drugs is only 14%-42% (Oosterhaven, J. A. F., et al. (2021). Eczema control and treatment satisfaction in atopic dermatitis patients treated with dupilumab - a cross-sectional study from the BioDay registry. J Dermatolog Treat, 1-4.; Torrelo, A., et al. (2013). Health-related quality of life, patient satisfaction, and adherence to treatment in patients with moderate or severe atopic dermatitis on maintenance therapy: the CONDA-SAT study. Actas Dermosifiliogr, 104(5), 409-417). There is still a great need to develop new drugs for the treatment of atopic dermatitis.

[0004] Filaggrin (FLG) is an important protein for epidermis function, and its encoding gene is located on chromosome 1q21. FLG is first synthesized in the granular layer keratinocytes in the form of profilaggrin, which is highly phosphorylated and water-insoluble, with a mass of about 400 kDa, and its N-terminal is a calcium-binding domain and a nuclear localization element, followed by 10-12 FLG monomers in tandem, and finally a C-terminal related to degradation function. Profilaggrin is dephosphorylated and proteolyzed to form FLG monomers with a molecular weight of about 37 kD (Donovan, M., et al. (2020). Filaggrin and filaggrin2 processing are linked together through skin aspartic acid protease activation. PLoS One, 15(5), e0232679). Filaggrin plays an important role in the epidermal barrier function, including participating in the formation of the keratin envelope as an important molecule connecting keratin fibers, maintaining the physical barrier function of the epidermis; in addition, FLG is degraded by proteases in the stratum corneum to generate natural moisturizing factors (amino acids, pyrrolidone-5-carboxylic acid, and trans-urea acid, etc.), participating in skin hydration, maintaining the acidic environment of the stratum corneum, reducing the residence of microorganisms on the skin surface, and maintaining the normal function of the skin (Appendix Figure 1 )(Harding, C.R., et al. (2013). Filaggrin - revisited. International Journal of Cosmetic Science, 35(5), 412-423). Studies have shown that mutations in the filaggrin-encoding gene are highly associated with the onset of atopic dermatitis S. (2021). Atopic Dermatitis. N Engl J Med, 384(12), 1136-1143, over 500 FLG mutations have been identified (Gupta, J., & Margolis, D. J. (2020). Filaggrin gene mutations with special reference to atopic dermatitis. Curr Treat Options Allergy, 7(3), 403-413). FLG is a semi-dominant gene (Weidinger, S., et al. (2018). Atopic dermatitis. Nat Rev Dis Primers, 4(1), 1. doi:10.1038 / s41572-018-0001-z), approximately 10% of European descent and 3-6% of Asian descent people carry a mutated FLG gene on one chromosome, and the FLG expression level of such population is significantly reduced A. (2021). New Treatments for Atopic Dermatitis Targeting Skin Barrier Repair via the Regulation of FLG Expression. Journal of Clinical Medicine, 10(11), 2506). Meta-analysis shows that the odds ratio (OR) of AD incidence associated with FLG mutation is 3.1-4.8, and patients carrying FLG mutation gene have more severe and persistent disease (Drislane, C., & Irvine, A. D. (2020). The role of filaggrin in atopic dermatitis and allergic disease. Annals of Allergy, Asthma & Immunology, 124(1), 36-43).

[0005] In addition to the high correlation between FLG mutations and the incidence of atopic dermatitis, about 50%-90% of atopic dermatitis patients do not carry mutant FLG genes, however, the FLG expression in both lesion and non-lesion areas of all patients is lower than that of healthy people (Thyssen, J. P., et al. (2020). Filaggrin Expression and Processing Deficiencies Impair Corneocyte Surface Texture and Stiffness in Mice. Journal of Investigative Dermatology, 140(3), 615-623.e615), that is, atopic dermatitis is accompanied by low FLG expression. Since FLG is an important structural and functional protein for maintaining the epidermal barrier, the decrease of FLG expression in keratinocytes is not only an important cause of the occurrence of atopic dermatitis, but also an inevitable result of atopic dermatitis, so increasing the expression of FLG will help the skin barrier reconstruction of atopic dermatitis patients, and can be used for the prevention and treatment of wild type (FLG + / + ) and FLG single allele mutation genotype (FLG + / - ) AD patients.

[0006] Daidzin, Daidzin is an isoflavone compound, also known as daidzin, daidzin and soybean glycoside, which is mainly present in Pueraria lobata (Fabaceae) and soybean, and is one of the main active components of soybean isoflavones (Rosenberg Zand, et al., (2002). Flavonoids and steroid hormone-dependent cancers. Journal of Chromatography B, 777(1), 219-232). Daidzin is an inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH), and its inhibitory activity on ALDH-I (Ki: 20-40 nM) is higher than that on ALDH-II (Ki: 10-20 μM) (Keung & Vallee, (1993). Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase. Proc Natl Acad Sci USA, 90(4), 1247-1251). In vivo experimental results show that daidzin can delay ethanol absorption in rats, reduce alcohol poisoning, and reduce the alcohol concentration level in the blood (Xie et al., Daidzin, an Antioxidant Isoflavonoid, Decreases Blood Alcohol Levels and Shortens Sleep Time Induced by Ethanol Intoxication. Alcoholism: Clinical and Experimental Research, 18(6), 1443-1447).There are also studies showing that genistin can prevent 3,2'-dimethyl-4-amino biphenyl induced prostate cancer in rats (Kato et al., (2000). Suppressive Effects of Dietary Genistin and Daidzin on Rat Prostate Carcinogenesis. Japanese Journal of Cancer Research, 91(8), 786-791), and can also prevent early colon cancer induced by oxidative azomethane in rats (Thiagarajan, et al., (1998). Prevention of precancerous colonic lesions in rats by soy flakes, soy flour, genistein, and calcium 123. The American Journal of Clinical Nutrition, 68(6), 1394S-1399S). In addition, it is also found that genistin has anti-HIV activity (WO2015090201A1). No report has been found that genistin is used for treating atopic dermatitis.

[0007] The present application uses the silk aggregation protein promoter model to evaluate the natural product library, and finds that genistin can increase the expression of silk aggregation protein, and its activity is comparable to that of binimetinol, a newly marketed drug for treating atopic dermatitis. Genistin is the main active ingredient of soybean and has good safety, and we believe that genistin has high value for treating atopic dermatitis and has application prospects. The present application is an invention patent about new use of known compounds. SUMMARY

[0008] The technical problem solved by the present application is to provide the use of genistin and its pharmaceutically acceptable salt in the preparation of a drug for preventing or treating atopic dermatitis.

[0009] Specifically, to solve the technical problem of the present application, the following technical scheme is adopted:

[0010] The first aspect of the technical scheme of the present application provides the use of genistin and its pharmaceutically acceptable salt as shown in structural formula (I) in the preparation of a drug for preventing or treating atopic dermatitis,

[0011]

[0012] The pharmaceutically acceptable salt of the soybean glycoside includes pharmaceutically acceptable organic salt or inorganic salt, wherein the organic salt includes sulfonic acid salt, carboxylic acid salt, amino acid salt and fatty acid salt, and the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, hydrogen phosphate, dihydrogen phosphate and nitrate. Preferably, the salt is bisulfate, sulfate, hydrochloride and hydroiodide.

[0013] The sulfonic acid salt includes alkyl sulfonate containing 1-15 carbon atoms and benzene sulfonate, p-toluene sulfonate, o-toluene sulfonate and m-toluene sulfonate; the carboxylic acid salt includes tartarate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate and lactate; the amino acid salt includes glutamate and aspartate; and the fatty acid salt includes long chain fatty acid salt containing 2-18 carbon atoms.

[0014] The second aspect of the technical scheme of the present application provides a use of a pharmaceutical composition in the preparation of a medicine for preventing and treating atopic dermatitis, characterized in that the pharmaceutical composition contains soybean glycoside shown in structural formula (I) and pharmaceutically acceptable salt thereof and pharmaceutically acceptable carrier or excipient; the pharmaceutical composition can also contain other medicines for atopic dermatitis.

[0015]

[0016] The pharmaceutical composition can be prepared according to the methods known in the art. The compound of the present application or the pharmaceutical composition containing it can be administered in unit dose form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0017] The compound of the present application or the pharmaceutical composition containing it can be administered in unit dose form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0018] The administration dosage form can be liquid dosage form, solid dosage form or semi-solid dosage form. The liquid dosage form can be solution (including true solution and colloidal solution), emulsion (including o / w type, w / o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion and liniment, etc.; the solid dosage form can be tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, oral disintegrating tablet), capsule (including hard capsule, soft capsule, enteric-coated capsule), granule, powder, pellet, drop pill, suppository, film, patch, gas (powder) mist, spray, etc.; the semi-solid dosage form can be ointment, gel, paste, etc.

[0019] The compound of the present application can be prepared into common preparation, sustained release preparation, controlled release preparation, targeted preparation and various micro-particle drug delivery systems.

[0020] For preparing tablets of the compound of the present application, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0021] The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0022] For preparing capsules of the administration unit, the effective ingredient, the compound of the present application, can be mixed with diluents and glidants, and the mixture can be directly placed into hard capsules or soft capsules. Alternatively, the effective ingredient, the compound of the present application, can be first mixed with diluents, binders and disintegrants to prepare granules or pellets, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants and glidants used for preparing tablets of the compound of the present application can also be used for preparing capsules of the compound of the present application.

[0023] For preparing injections of the compound of the present application, water, ethanol, isopropyl alcohol, propylene glycol or their mixtures can be used as solvents, and appropriate amounts of solubilizers, co-solubilizers, pH adjustors and osmotic pressure adjustors commonly used in the art can be added. The solubilizers or co-solubilizers can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjustors can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjustors can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If lyophilized powder injections are prepared, mannitol, glucose, etc. can also be added as supporting agents.

[0024] In addition, if necessary, coloring agents, preservatives, flavors, taste correctors or other additives can also be added to the pharmaceutical preparation.

[0025] The inventors of the present application found that daidzin can alleviate the symptoms of atopic dermatitis by increasing the expression of filaggrin in epidermal cells. It can also be used in combination with other drugs for atopic dermatitis.

[0026] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present application can be administered by any known administration method.

[0027] The administration dose of the compound pharmaceutical composition of the present application can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route and dosage form, etc.

[0028] The compound or composition of the present application can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present application has a synergistic effect with other therapeutic drugs, the dose thereof should be adjusted according to the actual situation.

[0029] Beneficial technical effects

[0030] The inventors of the present application found that the natural product daidzin can increase the expression of filaggrin through the evaluation of natural product library, and its activity is comparable to that of the newly launched atopic dermatitis treatment drug binimetinobe. Daidzin is the main active ingredient of soybean, and has good safety. We believe that daidzin has high value for the treatment of atopic dermatitis and has application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 . Schematic diagram of filaggrin expression, maturation and metabolism

[0032] Figure 2 . Results of up-regulation of FLG gene transcription level of HaCaT cells by different concentrations of daidzin

[0033] Figure 3 . Results of the effect of daidzin on the viability of HaCaT cells

[0034] Figure 4 . Results of the effect of doxorubicin on the viability of HaCaT cells DETAILED DESCRIPTION

[0035] Example 1. Principle of the luciferase detection model of the upstream promoter of FLG gene

[0036] The promoter is located upstream of the transcription start site of a gene, and is a DNA sequence recognized and combined by transcription factors and RNA polymerase. The promoter can interact with transcription factors to control the initiation of transcription and regulate the degree of transcription.

[0037] The detection model adopts pGL4.11[luc2P] reporter gene plasmid for construction, which takes firefly luciferase gene as a reporter gene. The model uses human FLG gene upstream -2203 / +6 promoter to replace the promoter region of pGL4.11[luc2P], and expresses firefly luciferase when the promoter is activated, and the expression amount is proportional to the degree of activation of the promoter. At the same time, the detection model selects pGL4.74[hRluc TK] as an internal reference plasmid to correct the transfection efficiency, which uses HSV-TK promoter to regulate the transcription of sea pansy luciferase gene. By detecting the activity of firefly luciferase in cell lysate, and correcting with sea pansy luciferase activity, the degree of FLG gene transcription can be reflected. The detection model mainly detects the degree of up-regulation of FLG gene transcription by the compound.

[0038] The detection model co-transfects pGL4.11-(-2203 / +6) and pGL4.74 two plasmids into human immortalized keratinocytes HaCaT at a ratio of 20:1, digests the cells 6 hours after transfection and uniformly inoculates into 48-well plates, adds the compound 24 hours after inoculation, and detects the amount of firefly luciferase and sea pansy luciferase in the cell lysate using Luciferase Assay System luciferase detection kit (Promega Company) and Renilla Luciferase Assay System sea pansy luciferase detection kit (Promega Company) respectively 24 hours after treatment. By comparing with the solvent control group, the fold of up-regulation of FLG gene transcription by the compound is calculated.

[0039] Example 2. Principle of cell viability detection model

[0040] ATP plays an important role in various physiological processes of cells, and directly provides energy for the body, which is an important indicator reflecting cell viability and is positively correlated with the number of living cells. Therefore, by quantitatively detecting ATP in cell lysate, the number of living cells in the sample to be tested is reflected.

[0041] The model uses CellTiter Luminescent Cell Viability Assay luminescent cell viability assay kit (Promega Company) to detect ATP to quantitatively detect HaCaT cell viability, and evaluate the effect of the compound on HaCaT cell viability.

[0042] Example 3. Experimental method and results of detecting up-regulation of FLG gene transcription of HaCaT cells by different concentrations of soybean glycosides

[0043] HaCaT cells are inoculated at 1.8×10 6One cell was seeded into a 100 mm cell culture dish. After culturing for 24 hours, [the cells were then] used... HaCaT cells were co-transfected with 10 μg of pGL4.11-(-2203 / +6) plasmid and 0.5 μg of pGL4.74 internal control plasmid using transfection reagent (Polyplus). Six hours after transfection, cells in each dish were digested and divided into 3 × 10⁶ cells per well. 4 Cells were seeded into 48-well plates. 24 hours after seeding, daidzein was added at final concentrations of 1 μM, 3 μM, 10 μM, and 30 μM; benvitimod was used as a positive control; an equal volume of DMSO was added to the solvent control group. Three replicates were set up for each group. After 24 hours of treatment, the supernatant was discarded, and 50 μL of cell lysis buffer was added to each well. The cells were lysed on a horizontal shaker at 4°C for 15 min, then equilibrated to room temperature. 20 μL of the lysate was added to a 1.5 mL EP tube containing 30 μL of firefly luciferase substrate, vortexed, and the relative luciferase activity (RLUs) was measured using an FB12 single-tube chemiluminescence analyzer. Another 20 μL of the lysate from the same well was added to a 1.5 mL EP tube containing 30 μL of kidney luciferase substrate, vortexed, and the relative luciferase activity was measured using an FB12 single-tube chemiluminescence analyzer. The fold increase in FLG gene transcription in each experimental group was calculated using formula (1). The experimental data were analyzed using GraphPad Prism software.

[0044] (1) Increase factor = (Luciferase RLUs) 给药组 / Renilla RLUs 给药组 ) / (LuciferaseRLUs 溶剂对照组 / Renilla RLUs 溶剂对照组 )

[0045] The results showed that daidzein significantly increased the transcription level of the FLG gene in human keratinocyte HaCa T cells at final concentrations of 1-30 μM, and this effect was dose-dependent (see appendix). Figure 2 ).

[0046] Example 4. Detection of the effect of daidzein on cell viability

[0047] HaCaT cells were seeded into 96-well plates at a density of 8000 cells / well in 100 μL cell suspension and incubated at 37 °C, 5% CO2for 24 h. The next day, different concentrations of test compounds and positive control drug doxorubicin were added to the cells with equal amount of DMSO (0.1% v / v) as solvent control. After 48 h of continuous incubation, 100 μL of CellTiter-Glo reagent was added to each well, mixed well by shaking for 2 min, and incubated at room temperature for 10 min. The RLUs in each well were measured (Tang K, He S, Zhang X, et al. Tangeretin, an extract from Citrus peels, blocks cellular entry of arenaviruses that cause viral hemorrhagic fever. Antiviral Res. 2018, 160: 87-93). The cell survival rate of drug-treated wells was calculated with the RLUs value of DMSO solvent wells as 100%, and doxorubicin as positive control.

[0048] Cell survival rate (%) = fluorescence intensity 给药组 / fluorescence intensity 溶剂对照组 x 100%.

[0049] The experimental results showed that daidzin had no effect on the viability of HaCaT cells at the tested concentrations (dose-effect curve see Fig. 2), suggesting that daidzin has good safety. The dose-effect curve of the positive control compound doxorubicin is shown in Fig. 3. Figure 3 Figure 4 The experimental results showed that daidzin had no effect on the viability of HaCaT cells at the tested concentrations (dose-effect curve see Fig. 2), suggesting that daidzin has good safety. The dose-effect curve of the positive control compound doxorubicin is shown in Fig. 3.​

Claims

1. Use of soybean glycoside and its pharmaceutically acceptable salt as shown in structural formula (I) in the preparation of a medicament for preventing or treating atopic dermatitis.

2. Use according to claim 1, characterized in that, The pharmaceutically acceptable salt includes pharmaceutically acceptable organic salt or inorganic salt, wherein the organic salt includes sulfonic acid salt, carboxylic acid salt, amino acid salt and fatty acid salt, and the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, hydrogen phosphate, dihydrogen phosphate and nitrate.

3. Use according to claim 2, characterised in that, The sulfonic acid salt includes alkyl sulfonic acid salt containing 1-15 carbon atoms, benzene sulfonic acid salt, p-toluene sulfonic acid salt, o-toluene sulfonic acid salt, m-toluene sulfonic acid salt; the carboxylic acid salt includes tartaric acid salt, maleic acid salt, fumaric acid salt, citric acid salt, malic acid salt, cinnamic acid salt, benzoic acid salt, malonic acid salt, succinic acid salt, glutaric acid salt, adipic acid salt, pamoic acid salt and lactic acid salt; the amino acid salt includes glutamic acid salt and aspartic acid salt; and the fatty acid salt includes long chain fatty acid salt containing 2-18 carbon atoms.

4. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of atopic dermatitis, characterized in that, The pharmaceutical composition contains soybean glycoside and its pharmaceutically acceptable salt as shown in structural formula (I), and pharmaceutically acceptable carrier or excipient.

5. Use according to claim 4, characterised in that, The pharmaceutical composition further contains other atopic dermatitis treating drugs.

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