Application of 6S-5-methyltetrahydrofolic acid or pharmaceutically acceptable salt thereof in preparation of medicine for treating or relieving dermatitis

Abstract

The invention provides application of 6S-5-methyltetrahydrofolic acid or pharmaceutically acceptable salt thereof in preparation of a medicine for treating or relieving dermatitis. The dermatitis comprises contact dermatitis caused by skin inflammation and allergy due to the fact that the skin makes contact with substances and traumatic dermatitis such as red, swollen and ecdysis occurring after epidermis is damaged.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to use of 6S-5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof in preparing a drug for treating or alleviating dermatitis. Background Art

[0002] 6S-5-Methyltetrahydrofolate calcium is the calcium salt of the main form of folic acid (L-5-methyltetrahydrofolate, L-5-MTHF) in the blood circulation. Ingestion of 6S-5-methyltetrahydrofolate calcium is easily and completely decomposed into Ca 2+ and L-5-MTHF ions, which are able to be absorbed into the human body through the intestinal mucosal cells. 6S-5-Methyltetrahydrofolate calcium is a folic acid substitute for human food and food supplements. There is evidence that supplementation with 6S-5-Methyltetrahydrofolate calcium can improve the body's folate status and prevent adverse symptoms associated with folate deficiency. In recent times, an increasing number of L-5-Methyltetrahydrofolate or 6S-5-Methyltetrahydrofolate calcium supplements have also been studied for the treatment of various diseases, such as Alzheimer's disease, depression, and other mental and neurological diseases.

[0003] Copper (Cu) is considered to be an essential trace element for maintaining the functions of organisms. However, the accumulation of Cu exceeding metabolic requirements or the imbalance of Cu homeostasis can lead to toxic effects. Copper has been widely used in building materials, the electronics industry, wood preservatives, water pipes, medical devices, and agricultural activities. Copper in the air is divided into natural sources such as dust, volcanoes, and forest fires, and anthropogenic sources such as copper smelters, steel production, and municipal incinerators. The copper content in the atmosphere ranges from 5-20 ng Cu / m3, and the average concentration in natural water is 4-10 μg Cu / L. Plumbers, welders, mechanics, and others who work with copper are at risk of copper poisoning. According to a large number of studies, excessive copper can cause various adverse health effects such as developmental toxicity, genotoxicity, immunotoxicity, hematotoxicity, nephrotoxicity, and neurotoxicity [1-4]. Oxidative damage and cell apoptosis have been shown to be important mechanisms of copper toxicology. Excessive copper can lead to lipid peroxidation, the formation of mitochondrial reactive oxygen species (ROS), and mitochondrial damage. Excessive copper can cause testicular damage in males, which has been confirmed by studies. The main function of the testicles is to produce sperm, androgens, and testosterone. Studies have reported that copper sulfate (CuSO 4 ) can impair male fertility, including sperm concentration, survival and motility[5].

[0004] Zebrafish is a well-established vertebrate model with a high degree of anatomical and genomic conservation with humans (70-80% similarity). Not only is zebrafish a cost-effective and amenable model for human disease physiology and pharmacological approaches, but it also shares important similarities with mammalian immunity, including the presence of neutrophils, macrophages, dendritic cells, and B and T cells. In addition, at the molecular level, zebrafish immunity also has conserved components, such as TLRs, complement proteins, proinflammatory cytokines, acute phase responder proteins, and novel immune receptors (NITRs). Copper sulfate is an irritant and corrosive, and environmental exposure of zebrafish to copper sulfate induces severe inflammatory responses. Effective drugs screened in this type of zebrafish model (including amodiaquine hydrochloride, afrozosine hydrochloride, and clonidine hydrochloride) also showed anti-inflammatory activity in a mouse model of skin inflammation. 6S-5-methyltetrahydrofolate calcium has a certain degree of irritation and corrosiveness, and zebrafish exposed to copper sulfate will induce severe inflammatory responses. Effective drugs screened in this type of zebrafish model (including amodiaquine hydrochloride, afrozosine hydrochloride, and clonidine hydrochloride) also showed anti-inflammatory activity in a mouse model of skin inflammation. 4 )-induced effects and related mechanisms in zebrafish have not been reported yet.

[0005] References [1] Goto N, Hara H, Kondo M, Yasuda N, Kamiya T, Okuda K, Adachi T. Hydrogen sulfide increases copper-dependent neurotoxicity via intracellularcopper accumulation. Metallomics. 2020 Jun 24;12(6):868-875. [2] Liu H, Guo H, Jian Z, Cui H, Fang J, Zuo Z, Deng J, Li Y, Wang X, Zhao L. Copper Induces Oxidative Stress and Apoptosis in the Mouse Liver.Oxid Med Cell Longev. 2020 Jan 11;2020:1359164. [3] Shah N, Khan A, Ali R, Marimuthu K, Uddin MN, Rizwan M, RahmanKU, Alam M, Adnan M, Muhammad, Jawad SM, Hussain S, Khisroon M. MonitoringBioaccumulation (in Gills and Muscle Tissues), Hematology, and GenotoxicAlteration in Ctenopharyngodon idella Exposed to Selected Heavy Metals. Biomed Res Int. 2020 Apr 13;2020:6185231. [4] Gaetke LM, Chow-Johnson HS, Chow CK. Copper: toxicologicalrelevance and mechanisms. Arch Toxicol. 2014 Nov;88(11):1929-38. [5] Babaei H, Abshenas J. Zinc therapy improves adverse effects oflong term administration of copper on epididymal sperm quality of rats. IranJ Reprod Med. 2013 Jul;11(7):577-82. Summary of the invention

[0006] The invention provides use of 6S-5-methyltetrahydrofolate and a pharmaceutically acceptable salt thereof in preparing a drug for treating or relieving dermatitis. The 6S-5-methyltetrahydrofolate can be used for treating or relieving dermatitis, thereby providing a new use of the 6S-5-methyltetrahydrofolate.

[0007] The dermatitis further includes contact dermatitis, which is inflammation and allergy of the skin caused by contact with substances, and traumatic dermatitis, such as redness, swelling and peeling caused by damage to the epidermis.

[0008] Furthermore, the substance that the skin contacts is metal or metal ions, and further, the substance that the skin contacts is copper or copper ions.

[0009] Based on the same inventive concept, the present application also provides a drug for treating or alleviating dermatitis, wherein the active ingredient of the drug includes 6S-5-methyltetrahydrofolate and / or a pharmaceutically acceptable salt thereof.

[0010] The present invention also provides the use of 6S-5-methyltetrahydrofolate and a pharmaceutically acceptable salt thereof in preparing a medicine for treating or alleviating the adverse symptoms caused by copper poisoning. 6S-5-methyltetrahydrofolate can be used for treating or alleviating the adverse symptoms caused by copper poisoning.

[0011] Further, the copper poisoning includes acute copper poisoning and chronic copper overdose.

[0012] Further, the adverse symptom caused by copper poisoning is testicular damage, and further, the adverse symptom is testicular damage caused by excessive copper leading to decreased male fertility.

[0013] The medicine of the present invention further comprises pharmaceutically acceptable excipients.

[0014] Furthermore, the excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, adhesives, wetting agents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0015] Optionally, the dosage form of the drug is any one of tablets, ointments, granules, capsules, dispersions, sprays and injections.

[0016] As a new source of folic acid, 6S-5-methyltetrahydrofolate is quite different from traditional synthetic folic acid in absorption and metabolism. Based on current research, it has shown efficacy that synthetic folic acid does not have in various fields. Based on this, the inventors of the present application used a zebrafish model to study the effects of 6S-5-methyltetrahydrofolate calcium on zebrafish after copper sulfate and tail cutting, and finally found that 6S-5-methyltetrahydrofolate calcium can significantly reduce the production of ROS induced by copper sulfate and inhibit the inflammatory response caused by the irritant substance copper sulfate in the environment. The present invention also found that 6S-5-methyltetrahydrofolate calcium also has an inhibitory effect on the inflammatory response caused by tissue damage. The above-mentioned effects and related mechanisms of 6S-5-methyltetrahydrofolate and its pharmaceutically acceptable salts have not been reported yet.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides a new use of 6S-5-methyltetrahydrofolate and a pharmaceutically acceptable salt thereof, and provides a new therapeutic drug option for patients with dermatitis. Currently, the use of dexamethasone acetate cream is a common method for treating itching and redness caused by dermatitis, but long-term and large-scale use of glucocorticoids or irregular use can lead to hormonal dermatitis. The technical solution of the present invention avoids the use of hormones, avoids the negative effects brought by the prior art, and achieves a technical effect similar to that of treating dermatitis.

[0018] Cu homeostasis is usually well maintained by effective regulatory mechanisms, and Cu toxicity caused by disturbed homeostasis is an important factor leading to many different symptoms and disease conditions. These disease states are usually related to the role of Cu as a redox-active transition metal, which may cause oxidative damage. Some rare diseases, such as Wilson's disease, are caused by the inability to metabolize copper normally, leading to chronic copper poisoning. 6S-5-methyltetrahydrofolate calcium can significantly reduce the production of reactive oxygen species induced by excessive Cu and reduce the damage caused by excessive copper. The present invention provides a new use and a drug for treating copper poisoning. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 MTHF-Ca versus CuSO 4 Effects of induced ROS production in zebrafish larvae. 3dpf larvae were exposed to CuSO 4 (20 μM) for 2 h, and then the embryo water was replaced with DEX (5 μg / mL) or MTHF-Ca (1, 2, and 4 mM) for 4 h, Ns indicates no significant difference, ** p < 0.01, *** P < 0.001, **** P < 0.0001.

[0020] Figure 2 MTHF-Ca alleviates the 4 Induced neutrophil recruitment in zebrafish model. 3dpf larvae were exposed to CuSO4 (20 μM) for 2 h, then washed and treated with DEX (5 μg / mL) or MTHF-Ca (1, 2, and 4 mM) for 4 h; (A) Representative fluorescent images and (B) statistical analysis of the number of recruited neutrophils in the trunk region (white dashed box) are shown; Data are expressed as the mean ± SD of three independent experiments; ****P < 0.0001, original magnification: ×40.

[0021] Figure 3 MTHF-Ca alleviates the recruitment of macrophages to the inflammatory response in the CuSO4-induced zebrafish model. 4(20 μM) for 2 h, followed by washing and treatment with DEX (5 μg / mL) or MTHF-Ca (1, 2, and 4 mM) for 4 h; (A) Representative fluorescence images and (B) statistical analysis showing the number of macrophages recruited in the trunk region (white dashed box); data are presented as the mean ± SD of three independent experiments; **p < 0.001, ****P < 0.0001; original magnification: ×40.

[0022] Figure 4 Effects of MTHF-Ca on the expression levels of genes related to the inflammatory pathway in zebrafish induced by CuSO4. 3 dpf larvae were exposed to CuSO 4 (20 μM) for 2 h, and then treated with MTHF-Ca (0, 4 mM) for 4 h, and total RNA was extracted from 50 zebrafish in each group. jnk , erk , NF-κB , p65 , TNF-α , il-1b and myd88 Statistical analysis of relative fold changes; data are presented as the mean ± SD of three independent experiments; ** p < 0.01, *** P < 0.001, **** P < 0.0001.

[0023] Figure 5 MTHF-Ca alleviates tail transection-induced neutrophil recruitment in zebrafish model. 3dpf larvae were subjected to tail transection and then treated with DEX (5μg / mL) or MTHF-Ca (1, 2, and 4 mM) for 4 h; (A) Representative fluorescent images and (B) statistical analysis showing the number of neutrophils recruited to the amputation site (white dashed box); Data are presented as mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001; Original magnification: ×40.

[0024] Figure 6MTHF-Ca attenuates macrophage recruitment to inflammatory response in tail transection-induced zebrafish model. 3dpf larvae were subjected to tail transection and then treated with DEX (5μg / mL) or MTHF-Ca (1, 2, and 4 mM) for 4 h; (A) Representative fluorescent images and (B) statistical analysis showing the number of macrophages recruited to the amputation site (white dashed box); Data are presented as mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, **** P < 0.0001; Original magnification: ×40. DETAILED DESCRIPTION

[0025] Example 1 Preparation of 6S-5-methyltetrahydrofolate calcium ointment Grind 100 mL of 10% nitroglycerin and lanolin in a mortar, add 700 g of vaseline in portions, grind into a paste, add 10 g of 6S-5-methyltetrahydrofolate calcium, 20 g of propylene glycol, and 3 g of paraben in portions, and then add an appropriate amount of purified water to obtain a uniform light yellow ointment.

[0026] Example 2 Preparation of 6S-5-Methyltetrahydrofolate Calcium Capsules 6000 mg of 6S-5-methyltetrahydrofolate calcium and 500 g of microcrystalline cellulose are uniformly mixed, prepared into granules, and poured into capsules to prepare a capsule preparation containing 30 mg of 6S-5-methyltetrahydrofolate calcium in each granule.

[0027] Experimental Example 1 6S-5-Methyltetrahydrofolate calcium to copper sulfate (CuSO 4 )-induced effects on zebrafish 1.1 Zebrafish maintenance and embryo collection Transgenic zebrafish [Tg (mpo: EGFP)] expressing green fluorescent protein in neutrophils and transgenic zebrafish [Tg (mpeg1: mcherry)] expressing red fluorescent protein in macrophages were obtained from the Chinese Zebrafish Resource Center. Adult zebrafish were maintained at 28.5°C and pH 7.4-7.6 in a 14:10 hour (h) light / dark photoperiod. Adult zebrafish were placed in breeding tanks with a 1:1 ratio of male to female overnight, embryos were generated by natural pairing, and cultured in embryo water (0.2 mg / mL Instant Oceans Salt in distilled water) at 28.5°C. The age of the larvae was expressed as hours post fertilization (hpf). All zebrafish studies were approved by the Institutional Animal Care and Use Committee of Nanjing Tech University.

[0028] 1.2 Determination of ROS content in zebrafish 3 dpf zebrafish larvae were randomly distributed into 24-well plates (20 larvae / well) and incubated with 20 μM CuSO 4 After 2 h of treatment, 20 μM CuSO 4 Washed off and treated with MTHF-Ca (0, 1, 2 and 4 mM) or dexamethasone (DEX, 5 μg / mL) for 4 h. After that, zebrafish larvae were treated with H2DCFDA solution (5 μM) at 28 °C in the dark for 40 min. ROS fluorescent probe (H2DCFDA) was purchased from Med Chem Express (Shanghai, China). Then the zebrafish larvae were washed, and the zebrafish larvae of each group were homogenized on ice, and then the supernatant was measured with a microplate reader (Synergy HT, BioTek, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The absorbance of the control group was considered to be 100% ROS production. The results were expressed as the effect of ROS production in the form of absorbance.

[0029] 1.3 MTHF-Ca in CuSO 4 Induced anti-inflammatory effects in zebrafish models Transgenic zebrafish [Tg(mpo: EGFP)] and [Tg(mpeg1: mcherry)] at 72 hpf were treated with 20 μM CuSO 4 The embryos were treated for 2 h and then randomly assigned to 24-well plates (20 embryos / well) containing MTHF-Ca (0, 1, 2, and 4 mM) or DEX (5 μg / mL) and cultured for 4 h in 1 mL of embryo water (distilled water containing 0.2 mg / mL instant sea salt). 4 )、CuSO 4 Induction group, CuSO 4 Induction +1mM MTHF-Ca group, CuSO 4 Induction +2mM MTHF-Ca group, CuSO 4 Induction +4mM MTHF-Ca group and a positive control group (CuSO 4 The migration and accumulation of neutrophils and macrophages in living larvae were observed and photographed under a fluorescence microscope (IX71, Olympus, Japan).

[0030] 1.4 Isolation of zebrafish total RNA and RT-PCR 3 dpf zebrafish larvae were treated with 20 μM CuSO 4After treatment for 2 h, the CuSO4 was washed off and treated with MTHF-Ca (0, 4 mM) or DEX (5 μg / mL) for 4 h. Total RNA was then extracted from 50 zebrafish larvae in each group. Single-stranded complementary DNA was synthesized according to the manufacturer's instructions (YiFeiXue, Nanjing, China). Real-time PCR was performed using the ABI7900-HT Fast Real-Time Fluorescence Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). The β-actin gene was used as an internal reference. The qRT-PCR thermal cycle program was as follows: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 30 s, and 72℃ for 30 s for 40 cycles. The relative quantification of the PCR results was determined by − ΔΔCT. The primer sequences are shown in Table 1.

[0031] Table 1 Primer sequences used in real-time PCR. 1.3 Results MTHF-Ca reduces the accumulation of ROS induced by CuSO4 in zebrafish larvae.

[0032] Results obtained in many experimental studies support the view that ROS-induced oxidative damage plays an important role in Cu toxicity. 4 A significant increase in ROS was observed in the zebrafish model induced by Figure 1 As shown, compared with the control group, exposure to CuSO 4 The accumulation of ROS increased by 41.77% after MTHF-Ca treatment and decreased in a dose-dependent manner (compared with CuSO 4 Compared with the induced group, the decreases were 14.73%, 17% and 27.2%, respectively.

[0033] MTHF-Ca reduces CuSO 4 Induced neutrophil and macrophage recruitment in zebrafish larvae.

[0034] CuSO 4 The zebrafish model of induced inflammation was also used to confirm the anti-inflammatory effect of MTHF-Ca. Figure 2 A and Figure 3 As shown in A, exposure to CuSO 4 After CuSO, a large number of neutrophils and macrophages were recruited to the neuromast. 4 After treatment, the levels of β-catenin and β-catenin increased by 4.05 and 1.75 times, respectively, compared with the corresponding control groups (Figures 2B and Figure 3B). However, compared with the CuSO4-induced group, DEX and MTHF-Ca significantly inhibited the migration of neutrophils and macrophages by 35.7% / 23.8%, 35.48% / 26.3%, 40.02% / 24.93%, and 47.25% / 33.93%, respectively ( Figure 2 B and Figure 3 B).

[0035] MTHF-Ca inhibits CuSO 4 Induced expression of inflammation-related cytokines in zebrafish larvae.

[0036] We investigated the anti-inflammatory effects of MTHF-Ca and whether it could inhibit CuSO 4 Induced expression of pro-inflammatory cytokines in zebrafish larvae. Figure 4 As shown, CuSO 4 After 2 hours of action, jnk The expression level of erk , NF-κB , p65 , TNF-α , il-1b The expression of CuSO increased by 1-3 times compared with the control group. 4 Induced zebrafish larvae myd88 The expression of CuSO increased by 0.53-fold. 4 mM MTHF-Ca inhibited the 4 Induced jnk , erk , NF-κB , myd88 , p65 , TNF-α and il-1b The expression of 40.73%, 22.05%, 45.32%, 34.17%, 30.55%, 27.7% and 51.16% (Figure 4).

[0037] Experimental Example 2 Effects of 6S-5-methyltetrahydrofolate calcium on tail-transected zebrafish 2.1 Zebrafish maintenance and embryo collection Transgenic zebrafish [Tg (mpo: EGFP)] expressing green fluorescent protein in neutrophils and transgenic zebrafish [Tg (mpeg1: mcherry)] expressing red fluorescent protein in macrophages were obtained from the Chinese Zebrafish Resource Center. Adult zebrafish were maintained at 28.5°C and pH 7.4-7.6 in a 14:10 hour (h) light / dark photoperiod. Adult zebrafish were placed in breeding tanks with a 1:1 ratio of male to female overnight, embryos were generated by natural pairing, and cultured in embryo water (0.2 mg / mL Instant Oceans Salt in distilled water) at 28.5°C. The age of the larvae was expressed as hours post fertilization (hpf). All zebrafish studies were approved by the Institutional Animal Care and Use Committee of Nanjing Tech University.

[0038] 2.2 Anti-inflammatory effect of MTHF-Ca in the zebrafish model induced by tail transection Transgenic zebrafish [Tg (mpo: EGFP)] and [Tg (mpeg1: mcherry)] at 72 hpf were subjected to tail transection at the same position under a microscope. Larvae were then carefully transferred to a 24-well plate (15 larvae / well). The experiment included a blank control group (no tail transection), a tail transection group, a tail transection + 1mM MTHF-Ca group, a tail transection + 2mM MTHF-Ca group, a tail transection + 4mM MTHF-Ca group, and a positive control group (tail transection + 5 µg / mL DEX), with 3 replicate wells in each group. At 4 h after drug administration, the migration and aggregation of neutrophils and macrophages in the tail transection site of each group were observed under a fluorescence microscope (IX71, Olympus, Japan).

[0039] 2.3 Results When tissue is damaged, an inflammatory response is triggered, and immune cells are recruited from the microcirculation to the damaged tissue, which is a key pathological process for inflammatory treatment. In this experiment, we used the zebrafish inflammation model induced by tail transection to dynamically observe the endogenous process of neutrophil and macrophage migration. Zebrafish larvae were exposed to embryonic water, 5μg / mL DEX, and different concentrations of MTHF-Ca (1, 2, and 4 mM) for 4 hours immediately after tail transection. The results showed that the number of neutrophils and macrophages recruited to the amputation site after tail transection was significantly increased by 5.28 times and 2.54 times, respectively, compared with the corresponding control group ( Figure 5 and Figure 6In contrast, DEX and MTHF-Ca treatments inhibited the recruitment of neutrophils and macrophages to the injury site in tail transection-induced zebrafish larvae, by 28.13% / 41.7%, 28.4% / 31.04%, 27.67% / 36.2%, and 33.19% / 45.75%, respectively, compared to the tail transection-induced group ( Figures 5B and 6B ).

Claims

1. Use of 6S-5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or alleviating dermatitis, characterized in that The dermatitis includes contact dermatitis, which is an inflammation and allergy of the skin caused by the skin contacting with a substance, and traumatic dermatitis, such as redness, swelling and peeling that occurs after the epidermis is damaged.

2. The use according to claim 1, characterized in that The substance that comes into contact with the skin is metal or metal ion.

3. The use according to claim 2, characterized in that The metal or metal ion is copper or copper ion.

4. A drug for treating or alleviating dermatitis, characterized in that: The active ingredients of the medicine include 6S-5-methyltetrahydrofolate and / or a pharmaceutically acceptable salt thereof.

5. A drug for treating or alleviating dermatitis according to claim 4, characterized in that: The dosage form of the medicine is ointment or spray.

6. Use of 6S-5-methyltetrahydrofolate and pharmaceutically acceptable salts thereof in the preparation of drugs for treating or alleviating adverse symptoms caused by copper poisoning.

7. The use according to claim 6, characterized in that The copper poisoning includes acute copper poisoning and chronic copper excess.

8. The use according to claim 6, characterized in that The adverse symptoms caused by copper poisoning are testicular damage.

9. The use according to claim 8, characterized in that The adverse symptom is decreased male fertility caused by testicular damage.

10. Use according to any one of claims 6 to 9 for preparing a drug, characterized in that The dosage form of the drug is any one of tablets, ointments, granules, capsules, dispersants, sprays and injections.

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