Use of levofolic acid (5-mthf) for the preparation of a medicament for the treatment of psoriasis

Abstract

The application relates to a new use of levofolic acid (5-MTHF). Specifically disclosed is application of levofolic acid or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating and / or preventing psoriasis. Animal experiments show that 5-MTHF can significantly reduce IMQ-induced psoriasis-like skin inflammation of mice, reduce a PASI score, inhibit epidermal thickening, and down-regulate expression of inflammatory factors such as IL-17A and IL-1beta. The application provides a new medicine selection with high safety and oral administration for psoriasis treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a new pharmaceutical use of a known compound, specifically the use of 5-methyltetrahydrofolate (5-MTHF) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment and / or prevention of psoriasis. Background Technology

[0002] Psoriasis is an immune-mediated, chronic, relapsing, inflammatory skin disease with a global prevalence of approximately 2% to 3%, affecting over 7 million people in China. The disease is characterized by erythema, scaling, and thickened infiltration of the skin. Pathological features include excessive proliferation and abnormal differentiation of keratinocytes, inflammatory cell infiltration in the dermis, and vasodilation. Psoriasis not only affects patients' appearance and quality of life but is also closely associated with various systemic diseases such as metabolic syndrome, cardiovascular disease, and depression, imposing a significant economic burden on society.

[0003] Currently, the main treatments for psoriasis fall into three categories: topical medications, systemic therapies, and biologics. Mild psoriasis primarily relies on topical corticosteroids and vitamin D3 derivatives; however, long-term use of corticosteroids can lead to side effects such as skin atrophy, telangiectasia, and drug resistance. Traditional systemic therapies for moderate to severe psoriasis include methotrexate, cyclosporine A, and acitretin. While these drugs have some efficacy, long-term use can cause serious adverse reactions such as liver and kidney damage, bone marrow suppression, and teratogenicity. In recent years, targeted biologics (such as TNF-α inhibitors, IL-17 inhibitors, and IL-23 inhibitors) have significantly improved the treatment outcomes for moderate to severe psoriasis. However, biologics are expensive, require injection, and pose risks of immunogenicity and increased infection, leading to relapse in some patients after discontinuation. In the field of small molecule targeted drugs, the phosphodiesterase 4 (PDE4) inhibitor apremilast has problems with large gastrointestinal side effects and limited efficacy; the TYK2 inhibitor deuterocactitinib, although administered orally, is required by the FDA to add a black box warning (risk of serious cardiac events, cancer, thrombosis and death) to its JAK inhibitor category.

[0004] Folic acid is a key coenzyme factor in one-carbon metabolism, participating in fundamental biological processes such as nucleotide synthesis and DNA methylation. 5-Methyltetrahydrofolate (5-MTHF) is the main active form of folic acid in the body, which can be directly utilized by cells without the reduction step of dihydrofolate reductase (DHFR).

[0005] L-5-MTHF is a mature clinical drug that has been marketed in many countries and regions worldwide for many years, and is widely used as a pharmaceutical, dietary supplement, and functional food ingredient. Currently, 5-MTHF is mainly used clinically for the prevention and treatment of folic acid deficiency, as an adjunct to methotrexate chemotherapy to reduce its toxic side effects, and as an adjunct treatment for hyperhomocysteinemia. Due to its long clinical application and wide population coverage, 5-MTHF has accumulated a wealth of toxicological and safety data. Existing clinical studies and post-marketing surveillance data indicate that long-term use of 5-MTHF within the recommended dosage range has good safety and tolerability, with no reports of serious adverse events and no common safety issues associated with traditional psoriasis treatments such as immunosuppression, hepatotoxicity, nephrotoxicity, or myelosuppression. Furthermore, as an endogenous active form of folic acid, 5-MTHF can be utilized and eliminated through normal metabolic pathways in the body, without the risk of cumulative toxicity.

[0006] Developing levonorgestrel, a mature clinical drug, for the treatment of psoriasis falls under the "drug repurposing" research and development strategy. Compared to developing new chemical entities from scratch, "drug repurposing" has the following advantages:

[0007] (1) The preclinical safety data system is comprehensive, which can significantly shorten the toxicology study cycle and significantly reduce the uncertainty of drug interactions and long-term safety;

[0008] (2) Existing human pharmacokinetic and pharmacodynamic data can be used as a reference, and clinical dosing regimens can be designed directly based on them;

[0009] (3) Mature synthesis processes, quality standards, and formulation production technologies can be directly transferred, reducing the technical risks of industrialization and registration review. Therefore, the use of levofolic acid in the treatment of psoriasis has a realistic feasibility for rapid translation from the laboratory to the clinical setting.

[0010] In the field of dermatology, there are no reports of using 5-MTHF for the treatment of psoriasis.

[0011] Therefore, discovering drugs with high safety, oral administration, and novel mechanisms of action for the treatment of psoriasis remains an urgent clinical problem to be solved. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a new drug option for the treatment of psoriasis that is safe and can be administered orally.

[0013] This invention provides the use of 5-methyltetrahydrofolate or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of psoriasis.

[0014] In a preferred embodiment of the present invention, the 5-methyltetrahydrofolate is (6S)-5-methyltetrahydrofolate (i.e., L-5-MTHF), which is the natural active configuration of folic acid in the human body and has optimal bioavailability and safety.

[0015] In this invention, the routes of administration of the drug include, but are not limited to, oral administration, topical administration, and injection administration. In a preferred embodiment, the drug is an oral preparation selected from tablets, capsules, granules, powders, or oral liquid preparations. In another preferred embodiment, the drug is a topical preparation selected from creams, ointments, gels, lotions, patches, or sprays for the direct treatment of local skin lesions. In yet another preferred embodiment, the drug is an injectable preparation.

[0016] In this invention, the unit dose of 5-methyltetrahydrofolate is from about 0.1 mg to about 100 mg (calculated as free acid), preferably from about 1 mg to about 50 mg, more preferably from about 5 mg to about 30 mg. The specific dose can be appropriately adjusted according to the patient's age, weight, disease severity, and route of administration. Based on the mouse administration dose (25-50 mg / kg / day by gavage) and body surface area conversion, the equivalent daily oral dose for adults (60 kg) is approximately 5 mg to 30 mg. This dose range is far below the known upper limit of safe dose for 5-MTHF, exhibiting an excellent safety window.

[0017] In this invention, psoriasis encompasses all clinical subtypes, including but not limited to plaque psoriasis, guttate psoriasis, pustular psoriasis, and erythrodermic psoriasis.

[0018] The mechanism of action of 5-MTHF in treating psoriasis described in this invention may include one or more of the following aspects: (1) supplementing the deficiency of endogenous 5-MTHF in local skin tissue under the inflammatory state of psoriasis and restoring one-carbon metabolic homeostasis; (2) inhibiting the expression and activity of IL-1β, thereby reducing the inflammatory cascade response mediated by NLRP3 inflammasome; (3) inhibiting the expression of IL-17A, thereby blocking the core pro-inflammatory pathway mediated by Th17 cells; (4) downregulating the expression of pro-inflammatory mediators and chemokines (such as CXCL1 and CXCL12) derived from keratinocytes such as S100A8 and S100A9, and reducing the skin recruitment of inflammatory cells.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) This provides a novel therapeutic use for an endogenous substance (5-MTHF) with known high safety. As a naturally occurring active form of folic acid in the human body, 5-MTHF has been clinically validated for its safety. Its use in the treatment of psoriasis avoids the safety concerns associated with traditional immunosuppressants and biologics.

[0021] (2) Animal experiments showed that gavage administration of 5-MTHF could significantly reduce IMQ-induced psoriasis-like skin inflammation in mice: the total PASI score decreased significantly (P<0.01), the skin thickness of the ear was controlled from more than 450 μm in the model group to less than 300 μm, and epidermal hyperplasia and inflammatory cell infiltration were significantly improved.

[0022] (3) 5-MTHF can be administered orally, which significantly improves patient compliance compared to the injection method of biological agents.

[0023] (4) The mechanism of action of 5-MTHF is clear. It targets the two core pathogenic pathways of psoriasis, IL-1β and IL-17A, rather than a single cytokine inhibition, and has the unique advantage of multi-target regulation.

[0024] (5) In animal experiments, the weight of mice in the 5-MTHF intervention group was well maintained and no obvious toxic side effects were observed, which further verified its safety. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the grouping and modeling of mice in each example;

[0026] Figure 2 This is a comparison of the relative abundance of endogenous 5-MTHF in each group of mice in the examples;

[0027] Figure 3 This is a comparison of the macroscopic phenotypes of the skin on the backs of mice in each group, as shown in the examples.

[0028] Figure 4 This is a graph showing the change of PASI scores of mice in each group over time in the examples;

[0029] Figure 5 This is a comparison chart of the PASI scores (erythema) of each group of mice in the examples;

[0030] Figure 6 This is a comparison chart of the PASI scores (scalp) of each group of mice in the examples;

[0031] Figure 7 This is a graph showing the change in ear thickness in each group of mice in the examples;

[0032] Figure 8 This is a graph showing the changes in body weight of mice in each group in the examples;

[0033] Figure 9 The images shown are H&E stained sections of mouse skin tissue and epidermal thickness statistics for each group in the examples.

[0034] Figure 10 This is a comparison of the relative expression levels of inflammatory factors (Il17a, S100a8, S100a9, Il1b, Cxcl1, Cxcl12) mRNA in the skin lesions of mice in each group of the examples.

[0035] In the picture:

[0036] Significance was determined by ANOVA among multiple groups, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0037] The present invention will be further described in detail and completely below with reference to embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] Unless otherwise specified, the experimental methods in the following examples are generally performed under conventional conditions as described in the *Pharmacology Laboratory Manual* (3rd edition, Science Press, 2002). The method for constructing a psoriasis model using imiquimod (IMQ) is based on published literature (van der Fits L, et al. Imiquimod-induced psoriasis-likeskin inflammation in mice is mediated via the IL-23 / IL-17 axis. J Immunol. 2009;182(9):5836-5845) or as recommended by the manufacturer. Unless otherwise specified, all reagents involved in the examples of this invention are commercially available products and can be purchased through commercial channels.

[0039] The main materials involved in the embodiments of this invention are as follows:

[0040] 1. Laboratory animals

[0041] Specific pathogen-free (SPF) grade BALB / c male mice, 6-8 weeks old and weighing 20-23g, were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. and housed at the Laboratory Animal Center of Central South University. This study has passed the laboratory animal welfare ethics review of Central South University. The "3R" principle was strictly followed during the experiment to minimize animal suffering and discomfort.

[0042] 2. Experimental reagents

[0043] 5-Methyltetrahydrofolic acid (5-MTHF, (6S)-5-Methyltetrahydrofolic acid, purity ≥98%), imiquimod cream (5%, Sichuan Mingxin Pharmaceutical Co., Ltd.), TRIzol reagent (Invitrogen), reverse transcription kit (Vazyme), SYBR Green qPCR Master Mix (Bimake), primers synthesized by Shanghai Sangon Biotech Co., Ltd. LC-MS / MS grade solvent (Merck).

[0044] Example 1: Therapeutic effect of levofloxacin (5-MTHF) on IMQ-induced psoriasis-like skin inflammation

[0045] 1. Experimental Objective

[0046] Using an imiquimod (IMQ)-induced mouse psoriasis model, this study evaluated the therapeutic effect of 5-MTHF on psoriasis-like skin inflammation and explored its regulatory role in the local inflammatory microenvironment and core pathogenic factors of the skin.

[0047] 2. Experimental Methods

[0048] (1) Grouping and modeling of experimental animals ( Figure 1 )

[0049] Six-week-old male BALB / c mice were randomly divided into the following three groups, with eight mice in each group:

[0050] - Control group: Routinely fed, without induction, and given an equal volume of solvent by gavage daily.

[0051] - Model group (IMQ): Psoriasis-like dermatitis was induced by applying 5% imiquimod cream (62.5 mg / day) to the skin on the back of mice for 7 consecutive days.

[0052] - 5-MTHF intervention group (IMQ+5-MTHF): Starting 28 days before modeling, 5-MTHF (50 mg / kg, dissolved in deionized water) was administered by gavage at a fixed time every day until the end of modeling (day 35). From day 28 to 34 (i.e., days 29 to 35 after gavage administration), IMQ was applied to the skin on the back of the mice to establish the model, using the same method as the model group.

[0053] (2) Detection of endogenous 5-MTHF levels

[0054] Mice were sacrificed on day 35, and skin lesions on the backs of mice in each group were collected. The relative abundance of endogenous 5-MTHF in the tissues was detected by liquid chromatography-mass spectrometry (LC-MS / MS).

[0055] (3) Clinical Symptom Score (PASI score)

[0056] In a double-blind experiment, researchers scored the erythema, scaling, and thickness of the skin on the backs of mice daily on a scale of 0 to 4, and calculated the total score. The specific scoring criteria are shown in Table 1.

[0057] (4) Monitoring of physical indicators

[0058] Mice were weighed daily using a precision electronic balance, and changes in back skin thickness were precisely measured using a digital thickness gauge.

[0059] (5) Histopathological examination (H&E staining)

[0060] Mice were sacrificed on day 35, and skin tissue from the back was collected. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (4 μm thick), stained with hematoxylin and eosin (H&E), observed and photographed under an optical microscope, and the epidermal thickness was measured using ImageJ software.

[0061] (6) Detection of inflammatory factor mRNA expression (qRT-PCR)

[0062] Skin lesions from the backs of mice in each group were collected. Total RNA was extracted using the TRIzol method, and cDNA was synthesized via reverse transcription. Real-time quantitative PCR was then performed using the SYBR Green method. The genes detected were: Il17a, S100a8, S100a9, Il1b, Cxcl1, and Cxcl12. GAPDH was used as an internal reference gene, and the relative expression levels of each gene were calculated using the 2^-ΔΔCt method. Primer sequences are shown in Table 1.

[0063] Table 1 qRT-PCR primer sequences

[0064]

[0065] 3. Experimental Results

[0066] (1) Endogenous 5-MTHF is significantly downregulated with inflammation.

[0067] Figure 2 The image shows the experimental results of endogenous 5-MTHF, as shown below. Figure 2 As shown in the LC-MS / MS results, the normal control group mice contained high levels of endogenous 5-MTHF in their skin tissue, while the 5-MTHF content in the model group skin lesions significantly decreased after IMQ-induced psoriasis-like inflammation (P<0.001). This result indicates that there is a significant folate metabolism disorder in the local skin under psoriasis inflammatory conditions, and supplementing with exogenous 5-MTHF is of great significance in correcting the metabolic imbalance under inflammatory conditions.

[0068] (2) 5-MTHF significantly improved the macroscopic phenotype of the skin.

[0069] Figure 3 The image shows the results of the macroscopic phenotypic experiment, such as... Figure 3 As shown, macroscopic phenotypic observation revealed that the IMQ model group mice exhibited extreme redness and swelling on their backs, covered with thick white scales, presenting typical psoriasis-like skin lesions. The 5-MTHF intervention group mice showed significant reduction in skin lesions: the erythema area decreased, the scale thickness significantly decreased, and the overall severity of skin lesions was significantly lower than that of the model group.

[0070] (3) PASI score significantly decreased

[0071] Figure 4 The PASI score results are shown in the image. Figure 4 As shown, the PASI total score, scaling score, and erythema score of the IMQ model group increased rapidly with the number of days of modeling. The PASI total score of the 5-MTHF intervention group was significantly lower than that of the IMQ model group at all time points (P<0.01). Figures 5-6 For example, a graph showing the classification and scoring results. Figure 5-6 The scores of each sub-item (erythema, scaling) showed a consistent trend of improvement, demonstrating a significant anti-inflammatory protective effect.

[0072] (4) Changes in skin thickness and body weight

[0073] Figure 7 and Figure 8 The images show changes in mouse ear thickness and body weight, respectively. Figure 8 IMQ modeling caused a surge in dorsal skin thickness in the model group mice, increasing from approximately 100 μm to over 450 μm. The 5-MTHF intervention group maintained this thickness below 300 μm, a statistically significant difference (P < 0.01). Meanwhile, as... Figure 8 As shown, 5-MTHF intervention effectively alleviated the significant decrease in body weight in model mice caused by systemic inflammation, and the body weight of the intervention group mice was maintained at a level close to that of the normal control group.

[0074] (5) Improvement in histopathology

[0075] Figure 9 Image showing the results of H&E staining, as shown below. Figure 9 As shown, the IMQ model group mice exhibited significantly thickened epidermis with hyperkeratosis and parakeratosis, and numerous inflammatory cell infiltrations were observed in the dermis. In the 5-MTHF intervention group, the epidermal thickness was significantly reduced, keratinization abnormalities were significantly improved, and inflammatory cell infiltration in the dermis was significantly decreased.

[0076] (6) Downregulation of inflammatory factor expression

[0077] Figure 10The image shows the results of qPCR experiments for various inflammatory factors, such as... Figure 10 As shown, qRT-PCR results indicated that IMQ modeling significantly upregulated the mRNA levels of multiple inflammatory factors, including Il17a, S100a8, S100a9, Il1b, Cxcl1, and Cxcl12, in mouse skin lesions. After 5-MTHF intervention, the expression of all these inflammatory factors was significantly inhibited (P<0.05 or P<0.01). Among them, the downregulation of IL-17A (Il17a) and IL-1β (Il1b), which are core pathogenic pathways in psoriasis, was most significant. Simultaneously, the expression of their downstream effector molecules S100A8 and S100A9, as well as the chemokines CXCL1 and CXCL12, also decreased synchronously.

[0078] The above experimental results indicate that levofloxacin (5-MTHF) has a significant therapeutic effect on imiquimod-induced psoriatic skin inflammation. Its mechanism of action includes at least the following:

[0079] (1) Supplement the deficiency of endogenous 5-MTHF in the inflammatory state of psoriasis;

[0080] (2) Inhibits the IL-1β-mediated innate immune inflammatory pathway;

[0081] (3) Inhibit the IL-17A-mediated Th17 adaptive immune inflammatory pathway;

[0082] (4) Block the positive feedback loop of inflammation in S100A8 / S100A9-CXCL1 / CXCL12.

[0083] 5-MTHF has a high safety profile and can be administered orally, making it a valuable new drug for the treatment of psoriasis. Furthermore, animal studies using a long-term pretreatment and continuous concomitant therapy regimen at a dose of 25 mg / kg yielded conclusions consistent with the above experiments, validating the effectiveness of 5-MTHF at lower doses.

[0084] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made by those skilled in the art without departing from the scope of the technical solution of this application using the above-disclosed technical content should be considered as equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. Use of levofolic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of psoriasis.

2. The application according to claim 1, characterized in that, The levofolate mentioned is 5-methyltetrahydrofolate, specifically (6S)-5-methyltetrahydrofolate (levofolate, L-5-MTHF).

3. The application according to claim 1, characterized in that, The drug is an oral preparation, a topical preparation, or an injectable preparation.

4. The application according to claim 3, characterized in that, The oral preparations are selected from tablets, capsules, granules, powders, or oral liquid preparations.

5. The application according to claim 3, characterized in that, The topical preparation is selected from creams, ointments, gels, lotions, patches, or sprays.

6. The application according to any one of claims 1 to 5, characterized in that, The unit dose of the levofolic acid is 0.1 mg to 100 mg.

7. The application according to claim 1, characterized in that, The psoriasis referred to is psoriasis vulgaris, plaque psoriasis, guttate psoriasis, pustular psoriasis, or erythrodermic psoriasis.

8. The application according to claim 1, characterized in that, The drug exerts its therapeutic effect on psoriasis by supplementing the deficiency of endogenous levofolate and / or inhibiting the expression levels of IL-17A and / or IL-1β in skin tissue, and / or inhibiting the expression and activity of IL-1β, thereby alleviating the NLRP3 inflammasome-mediated inflammatory cascade; and / or blocking the core pro-inflammatory pathway mediated by Th17 cells; and / or downregulating the expression of pro-inflammatory mediators and chemokines derived from keratinocytes such as S100A8 and S100A9, thereby reducing the recruitment of inflammatory cells to the skin.

9. A medicine for treating psoriasis, characterized in that, The active ingredient in the drug is levofolic acid.

10. The medicament according to claim 9, characterized in that, The unit dose of the active ingredient of the drug is 0.1 mg to 100 mg.

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