Topical preparations of fulvic acid used to relieve atopic dermatitis

Abstract

This invention patent relates to the protection of novel topical formulations containing fulvic acid, specifically its efficacy in alleviating Th2-type inflammation such as atopic dermatitis, and its mechanism of action. Studies have found that it can inhibit the levels of Th2-type inflammatory factors CCL17 and CCL22 at both the cellular and in vivo levels, with this inhibition exerted through regulation of the p38MAPK / JNK signaling pathway. In in vivo experiments, topical application of fulvic acid significantly improved itching and skin manifestations in a mouse model of 4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis. The mechanism of action was verified by blocking the aforementioned pathway in a TNF-α / IFN-γ-induced epidermal cell inflammation model induced by fulvic acid. This patented research provides new insights and treatment options for the prevention and treatment of Th2-type inflammation such as atopic dermatitis, and offers scientific support for the development of new therapeutic methods for Th2-type inflammation that can replace glucocorticoids and targeted drugs, as well as for the development of effective, safe, and economical new drugs. It has significant scientific value and great clinical application prospects.

Description

Technical Field

[0001] This invention patent relates to protecting a novel topical formulation of fulvic acid, as well as its novel efficacy in relieving atopic dermatitis and its mechanism of action. Studies have found that it can reduce the levels of Th2 inflammatory factors CCL17 and CCL22 at both cellular and in vivo levels, and this effect is achieved by inhibiting the p38MAPK / JNK signaling pathway. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, complex, and refractory inflammatory skin disease caused by an abnormal Th2 immune response. It is characterized by pruritic, eczematous skin lesions, and its pathogenesis involves Th2-type inflammation. Helper T cells differentiate into Th2 cells, and the immune inflammatory response generated by Th2 cells and their secreted cytokine population is called Th2-type inflammation. AD affects approximately 11-20% of children and 5-10% of adults, significantly impacting the quality of life and mental health of patients and their families. AD involves disruption of the epidermal barrier, allowing external antigens to enter and inducing keratinocytes to produce cytokines such as thymic activation-regulated chemokine (TARC / CCL17), macrophage-derived chemokine (MDC / CCL22), and thymic stromal lymphopoietin (TSLP). These inflammatory factors recruit immune cells to the skin, leading to the inflammatory manifestations of AD.

[0003] Previous studies have shown that CCL17 and CCL22 are closely related to the severity of Alzheimer's disease (AD). CCL17 and CCL22 are members of the chemokine family that can specifically bind to CCR4 on Th2-phenotype CD4+ T cells and play an important role in Th2 migration. Furthermore, research has indicated that downregulating the production of CCL17 and CCL22 by keratinocytes is an effective target for treating Th2-type inflammatory skin diseases such as AD.

[0004] Current clinical treatment options for Alzheimer's disease (AD) include glucocorticoids, calcineurin inhibitors, and cyclosporine, but long-term use of these all produce skin and systemic side effects with significant risks, especially in children. In recent years, targeted therapies have been applied to AD, but drugs such as dupilumab have numerous adverse clinical reactions, particularly in pediatric patients, and their safety and high cost severely limit their application. Therefore, there is a strong market demand for the development of new, safe, effective, and economical mechanistic treatments.

[0005] The Ming Dynasty's "Compendium of Materia Medica" records that black gold stone (whose main active ingredient is fulvic acid) is used for knife wounds, accidental ingestion of gold and silver, and excessive menstruation. Its hemostatic, blood-activating, antibacterial, antiviral, and anti-ulcer effects have been confirmed. However, there are currently no reports on the treatment of Alzheimer's disease with fulvic acid.

[0006] Main References

[0007] 1. Talamonti M, Galluzzo M, Silvaggio D, et al. Quality of Life andPsychological Impact in Patients with Atopic Dermatitis. J Clin Med. 2021.21;10(6):1298.

[0008] 2. Langan SM, Irvine AD, Weidinger S. Atopic dermatitis. Lancet.2020.1;396 (10247):345-360.

[0009] 3. Horikawa T, Nakayama T, et al. IFN-gamma-inducible expression ofthymus and activation-regulated chemokine / CCL17 and macrophage-derivedchemokine / CCL22 in epidermal keratinocytes and their roles in atopicdermatitis. Int Immunol. 2002;14(7):767-73.

[0010] 4. Zhao Y, Paderu P, Delmas G, et al. Carbohydrate-derived fulvicacid is a highly promising topical agent to enhance healing of woundsinfected with drug-resistant pathogens. J Trauma Acute Care Surg. 2015;79(4Suppl 2):S121-9.

[0011] 5. Chien SJ, Chen TC, Kuo HC, et al. Fulvic acid attenuateshomocysteine-induced cyclooxygenase-2 expression in human monocytes. BMCComplement Altern Med. 2015.13;15:61.

[0012] 6. Yamada P, Isoda H, Han JK, et al. Inhibitory effect of fulvic acidextracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3and KU812 cells. Biosci Biotechnol Biochem. 2007;71(5):1294-305.

[0013] 7. Jayasooriya RGPT, Dilshara MG, Kang CH, et al. Fulvic acidpromotes extracellular anti-cancer mediators from RAW 264.7 cells, causing tocancer cell death in vitro. Int Immunopharmacol. 2016;36:241-248.

[0014] 8. An HJ, Kim JY, Kim WH, et al.Therapeutic effects of bee venom andits major component, melittin, on atopic dermatitis in vivo and in vitro. BrJ Pharmacol. 2018 ;175(23):4310-4324.

[0015] 9. Mehta NN, Teague HL, Swindell WR, et al. IFN-γ and TNF-α synergism may provide a link between psoriasis and inflammatory atherogenesis. Sci Rep. 2017 23;7(1):13831.

[0016] 10. Saeki H, Tamaki K. Thymus and activation regulated chemokine(TARC) / CCL17 and skin diseases. J Dermatol Sci. 2006;43(2):75-84.

[0017] 11. Miodovnik M, Koren R, Ziv E, Ravid A. The inflammatory response of keratinocytes and its modulation by vitamin D: the role of MAPK signalingpathways. J Cell Physiol. 2012;227(5):2175-83.

[0018] 12. Kong L, Liu J, Wang J, et al. Icariin inhibits TNF-α / IFN-γinduced inflammatory response via inhibition of the substance P and p38-MAPKsignaling pathway in human keratinocytes. Int Immunopharmacol. 2015;29(2):401-407. Summary of the Invention

[0019] The purpose of this invention patent is to protect the novel topical formulation containing fulvic acid, as well as its novel efficacy in relieving atopic dermatitis and its mechanism of action. The invention is as follows:

[0020] Topical application of FA solution significantly alleviated itching and skin lesions caused by DNCB-induced atopic dermatitis in mice. This effect was achieved by inhibiting the p38 MAPK / JNK signaling pathway at the lesion site, thereby reducing the levels of CCL17 and CCL22. FA solution also reduced the levels of CCL17 and CCL22 in a TNF-α / IFN-γ-induced epidermal cell inflammation model. Similar results were obtained by blocking these pathways with channel inhibitors, thus validating its mechanism of action.

[0021] The technical solution adopted in this invention

[0022] This invention uses DNCB to induce Balb / c mice to establish two models: a mouse atopic dermatitis model and a TNF-α / IFN-γ-induced epidermal Th2-type inflammatory cell model. It clarifies the antipruritic, anti-inflammatory, and immunomodulatory effects of FA, as well as its upstream signaling pathway regulation mechanism, and identifies its target as CCL17 and CCL22.

[0023] Acquisition of FA and formulation of topical preparations

[0024] Referring to the "Analysis and Standards of Humic Acid Products", gray-black fulvic acid solid powder was obtained from lignite in Xundian, Yunnan Province using the ion exchange method. After purification, it was dissolved in double-distilled water for later use.

[0025] This topical solution is prepared from the following main oil and aqueous phase components by weight percentage: FA 0.5–5.5%; glycerin 5–7.5%; butylene glycol 1–5%; 1,2-hexanediol 1–3%; capryloyl hydroxamic acid 1–7.5%; caprylic / capric triglyceride 1–1.5%; hydrogenated lecithin 1–1.5%; triethanolamine 1–1.5%; and carbomer 0.1–1.5%. The balance is deionized water.

[0026] After weighing and mixing the water and oil phases as described above, heat to 85°C, stirring slowly during heating until all solids in the mixture are completely melted. The contents of the container should be clear and transparent, with clear separation of the oil and water phases. Homogenize the emulsion using a homogenizer at 2500 rpm for 4 minutes. Then stop heating and slowly cool with cold water, followed by defoaming in a water bath. Clean the storage tank thoroughly, spray with 75% alcohol, drain, and irradiate under a UV lamp for 20 minutes. Store the solution for later use.

[0027] The scope of this patent protection is as described above, but not limited to, including all topical dosage forms such as lotions, creams, ointments, film-forming agents, sprays, mouthwashes, etc. The patent-protected formulations are as described above, but not limited to this scope.

[0028] 2.1 Effects of different concentrations of FA on the viability of human keratinocytes (HaCaT)

[0029] HaCaT cells were treated with FA at concentrations of 10–1000 µg / ml for 24 h using the Cell Counting Kit 8 (CCK-8) method. No cytotoxicity was observed even at concentrations as high as 900 µg / ml. Figure 1 Therefore, the experimental concentrations of FA were determined to be 200 and 500 µg / ml.

[0030] 2.2 FA reduces the mRNA expression of pro-inflammatory factors and chemokines in TNF-α / IFN-γ stimulated HaCaT cells.

[0031] TNF-α / IFN-γ is widely used in altase inhibitory syndrome (AD) cell model. TNF-α / IFN-γ acts on HaCaT cells, stimulating the production of chemokines and cytokines, including IL-6, IL-8, CCL17, and CCL22. HaCaT cells were pretreated with FA for 30 min, followed by stimulation with TNF-α / IFN-γ for 20 h. RT-qPCR was then used to detect whether FA affected the mRNA expression of these cytokines and chemokines. The experiment showed that FA treatment significantly reduced the expression levels of CCL17 and CCL22 mRNA. Figure 2 A). Furthermore, the inhibitory effect of FA was dose-dependent, with higher concentrations showing more significant inhibition. FA also downregulated the mRNA expression levels of IL-6, IL-8, and CCL2. Immunocytochemistry (ICC) analysis indicated that FA inhibited the protein expression levels of CCL17 and CCL22 in TNF-α / IFN-γ-induced HaCaT cells. Figure 2 B).

[0032] 2.3 FA inhibits the MAPK signaling pathway in TNF-α / IFN-γ stimulated HaCaT cells

[0033] MAPK phosphorylation is an important pathway in the development of inflammatory responses, promoting inflammation by generating inflammatory mediators. Furthermore, the MAPK signaling pathway, including JNK, is closely related to TNF-α / IFN-γ-stimulated signal transduction, and could serve as a therapeutic target for Alzheimer's disease (AD). Western blot (WB) analysis was used to analyze the phosphorylation of p38MAPK, JNK, and ERK proteins. The phosphorylation levels of JNK and p38 were significantly increased in the TNF-α / IFN-γ-stimulated model group. Dexamethasone (Dex), a commonly used glucocorticoid for treating AD, was used as a positive control at 10 µM. Compared with the model group, Dex significantly inhibited the phosphorylation of p38MAPK and JNK. FA treatment at a concentration of 200 µg / ml mildly inhibited p38MAPK (…). Figure 3 A) JNK ( Figure 3The phosphorylation level of B) was significantly inhibited at 500 µg / ml, showing a dose-dependent effect, which could significantly inhibit the phosphorylation of p38MAPK and JNK. Figure 3 ).

[0034] 2.4 p38 MAPK and JNK inhibitors confirm the mechanism by which FA inhibits the expression of CCL17 and CCL22.

[0035] SB202190 and SP600125 are inhibitors of p38 MAPK and JNK, respectively. After incubating HaCaT cells with SB202190 and SP600125 for 30 min, the above signaling pathways were blocked, and HaCaT cells were stimulated with TNF-α / IFN-γ at 10 ng / ml. The results showed that SB202190 significantly inhibited the expression levels of CCL17 and CCL22 mRNA. Figure 4 A); SP600125 also has a mild inhibitory effect on the expression of CCL17 and CCL22 mRNA. Figure 4 B). The expression levels of CCL17 and CCL22 mRNA in the FA treatment group did not change significantly. This result suggests that FA mainly inhibits the expression levels of CCL17 and CCL22 by suppressing the p38 MAPK signaling pathway.

[0036] 2.5 Topical application of FA can significantly alleviate AD-like symptoms and skin manifestations induced by DNCB in BALB / c mice.

[0037] DNCB stimulation of mouse ear skin induced AD-like skin lesions. The experimental protocol is as follows: Figure 5 As shown in Figure A. In vivo experiments were conducted at FA concentrations of 1 mg / ml and 5 mg / ml, respectively. In the DNCB-induced group, 1% DNCB was dissolved in acetone / olive oil (3:1) and applied to both ears of mice. Dexamethasone was used as a positive control at a concentration of 0.2 mg / kg. Changes in ear skin lesions and ear thickness were recorded on day 14. Figure 5 As shown in Figure B, repeated DNCB stimulation can induce severe AD-like lesions, with significant local erythema, edema, and lichenification. On day 4 of the experiment, the skin thickened, exhibiting severe redness, swelling, inflammation, and desquamation. After one week of treatment, significant improvement in redness and desquamation of the auricular lesions was observed after topical application of FA. Figure 5 B), ear thickness and swelling were significantly relieved. Figure 5 C). During subsequent treatment, the differences were more significant compared to the control and model groups. The treatment effect of the high-dose group was significantly more significant than that of the low-dose group, showing a dose-dependent effect. H&E staining histopathological changes indicated that FA treatment significantly improved the inflammatory manifestations of the ear skin. Figure 5 B). Epidermal hyperplasia ( Figure 5 Both D) and dermal inflammatory infiltration were significantly reduced ( Figure 5E). High-dose FA treatment showed no significant difference compared to the dexamethasone positive control.

[0038] 2.6 Topical application of FA significantly reduced serum CCL17 and CCL22 levels in DNCB-stimulated BALB / c mice.

[0039] Serum CCL17 and CCL22 levels in DNCB-induced BALB / c mice treated with FA were detected using ELISA. FA treatment significantly reduced serum CCL17 (… Figure 6 A) and CCL22 level ( Figure 6 B) The differences were more significant compared to the control group and the model group. The treatment effect of the high-dose group was significantly more significant than that of the low-dose group and was dose-dependent. The efficacy of the high-concentration group was comparable to that of the dexamethasone group. These results indicate that FA intervention can effectively reduce serum CCL17 and CCL22 levels and exert a targeted anti-inflammatory effect.

[0040] The beneficial effects of this invention are the protection of fulvic acid and its novel topical formulations, as well as its novel efficacy in inhibiting atopic dermatitis and its mechanism of action. This patented study clarifies that topical FA effectively alleviates DNCB-stimulated AD dermatitis changes in mice by regulating the p38 MAPK and JNK signaling pathways and inhibiting the expression of TNF-α / IFN-γ-induced CCL17 and CCL22. This patented study provides new ideas and treatment options for the prevention and treatment of Th2-type inflammation such as AD, and provides scientific support for the development of new treatments for Th2-type inflammation that can replace glucocorticoids and targeted drugs, as well as for the development of effective, safe, and economical new drugs. It has significant scientific value and great clinical application prospects. Attached Figure Description

[0041] Figure 1 HaCaT cell viability was assessed after treatment with different concentrations of fatty acid (FA) (10, 100, 200, 400, 500, 600, 700, 800, 900, and 1000 µg / ml). Under 5% CO2 and 37°C conditions, there was no significant difference in HaCaT cell viability after 24 hours of treatment with different concentrations of FA. The experiment was repeated at least three times under the same conditions.

[0042] Figure 2Effects of FA on TNF-α / IFN-γ-stimulated HaCaT cell mRNA expression levels. HaCaT cells were pretreated with FA (200 and 500 µg / ml) for 30 min, followed by treatment with TNF-α / IFN-γ (10 ng / ml) for 20 h (qRT-PCR) or 30 min (ICC). (A) FA treatment significantly reduced the expression levels of CCL17 and CCL22 mRNA, while also reducing the mRNA expression levels of IL-6, IL-8, and CCL2. (B) FA significantly inhibited the protein expression levels of CCL17 and CCL22. Image scale: 100 µM. Data are presented as mean ± standard deviation (n=3), and experiments were repeated at least three times. * P <0.05,** P <0.01, *** P <0.001, **** P < 0.0001, ns indicates that the difference is not significant.

[0043] Figure 3 Fatty acid (FA) inhibited TNF-α / IFN-γ-induced phosphorylation levels of (A)p38 MAPK and (B)JNK in HaCaT cells. Cells were pretreated with different concentrations of FA (200 and 500 µg / ml) for 30 min, followed by treatment with TNF-α / IFN-γ (10 ng / ml) for 30 min. Western blot analysis showed a significant decrease in p38 MAPK and JNK phosphorylation levels after FA intervention. Data are presented as mean ± standard deviation (n=3), and experiments were repeated at least three times. P <0.05,** P <0.01, ns indicates that the difference is not significant.

[0044] Figure 4 Inhibitors of the p38 MAPK and JNK signaling pathways confirmed that FA suppressed TNF-α / IFN-γ-induced CCL17 and CCL22 expression. Cells were pretreated with SB202190 (5 µM) and SP600125 (10 µM) for 30 min, and the results were observed. (A) SB202190 significantly inhibited CCL17 and CCL22 mRNA expression levels; (B) SP600125 also had a mild inhibitory effect on CCL17 and CCL22 mRNA expression. Data are expressed as mean ± standard deviation (n=3), and experiments were repeated at least three times. * P <0.05, *** P <0.001, ns indicates that the difference is not significant.

[0045] Figure 5Topical application of fatty acids (FA) alleviated DNCB-induced AD skin lesions and histological changes in BALB / c mice. (A) Schematic diagram of animal experiments; (B) FA treatment significantly alleviated DNCB-induced AD skin lesions and histological changes in the mouse ear; (C) Ear thickness in mice decreased significantly; (D) Epidermal thickness at the lesion site decreased significantly; (E) Dermal thickness at the lesion site decreased significantly due to the disappearance of inflammatory edema. Data are expressed as mean ± standard deviation (n=4), and experiments were repeated at least three times. P <0.01, *** P <0.001, **** P <0.0001, ns indicates that the difference is not significant.

[0046] Figure 6 FA intervention reduced serum CCL17 and CCL22 levels in BALB / c-stimulated mice. Serum CCL17 and CCL22 levels were measured by ELISA on day 15. FA treatment significantly reduced serum CCL17 (A) and CCL22 levels (B). Data are presented as mean ± standard deviation (n=4), and experiments were repeated at least three times. P <0.05,** P <0.01, **** P <0.0001, ns indicates that the difference is not significant.

Claims

1. A topical humic acid preparation for relieving atopic dermatitis, characterized in that, It is composed of the following components by weight percentage: FA 0.5-5.5%; glycerol 5-7.5%; butylene glycol 1-5%; 1,2-hexanediol 1-3%; capryloyl hydroxamic acid 1-7.5%; caprylic / -capric triglyceride 1-1.5%; hydrogenated lecithin 1-1.5%; triethanolamine 1-1.5%; carbomer 0.1-1.5%; and the balance being deionized water. The topical preparation is a topical solution preparation; The preparation method of the topical solution is as follows: weigh and mix the above components, heat to 85°C, and slowly stir until the solids in the mixture are completely melted; homogenize and emulsify using a homogenizer at a speed of 2500 rpm for 4 minutes; after homogenization, stop heating, slowly cool with cold water, defoam in a water bath, and store in a jar to obtain the topical solution. The concentration of fulvic acid is 200 μg / mL or 500 μg / mL.

Images