Use of an atx inhibitor for the preparation of a medicament for the treatment of urticaria and pharmaceutical compositions thereof

Abstract

The application provides three new ATX inhibitors as applications for preparing drugs for treating urticaria. According to the animal experiment results, whether oral administration or external administration has a significant therapeutic effect on the urticaria mice, and no obvious toxic side effects are observed; and the application provides a new direction for treating urticaria.

Description

Technical Field

[0001] This invention relates to the application of a novel ATX inhibitor in the preparation of drugs for treating urticaria and its pharmaceutical composition, belonging to the field of medicine. Background Technology

[0002] Chronic urticaria is a skin disease characterized primarily by wheals and / or angioedema accompanied by itching, with a course lasting longer than 6 weeks. It is a common disease worldwide, with an overall prevalence of approximately 4.4%. Significant regional differences exist in prevalence, with a prevalence of 1.4% in Asian populations, higher than 0.5% in Europe and 0.1% in North America, and the prevalence continues to rise annually. It significantly impacts patients' quality of life, including learning, work, sleep, and mental health, resulting in a substantial economic and social burden. To date, the etiology and pathogenesis of chronic spontaneous urticaria are not fully understood. According to the EAACI / GA2LEN / EDF / WAO international guidelines, standard-dose second-generation antihistamines are first-line treatment, quadruple-dose second-line treatment is second-line treatment, and omalizumab, cyclosporine, and montelukast are primarily third-line treatments. Some patients still do not receive effective treatment, impacting their quality of life; therefore, there is an urgent need to develop drugs with novel targets and mechanisms to meet clinical needs.

[0003] Autotaxin (ATX), also known as exonucleotide pyrophosphatase phosphodiesterase 2 (Enpp2), is a secretory lysophospholipase D (lysoPLD) that primarily hydrolyzes extracellular lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), thereby activating G protein-coupled receptors and inducing various cellular responses, such as cell proliferation, migration, and cytokine production. This signaling pathway is associated with metabolic and inflammatory disorders, and inhibiting this pathway has a positive effect on the treatment of related diseases. In healthy individuals, ATX is normally expressed in various tissues and body fluids. ATX levels are significantly elevated in several human cancers (renal cell carcinoma, bladder cancer, and lung cancer, etc.) (doi:10.1007 / s12032-016-0836-7; doi:10.1158 / 0008-5472.CAN-17-3797). Several small-molecule ATX inhibitors have been developed in recent years, revealing their significant effects in improving pulmonary fibrosis and inhibiting gastrointestinal malignancies (DOI:10.1016 / j.iotech.2023.100384; DOI:10.1186 / s13046-023-02780-4). Furthermore, ATX also plays a role in the pathogenesis of autoimmune diseases, arthritis, chronic hepatitis, obesity, and impaired glucose homeostasis.

[0004] Discovering more effective treatments has become an urgent clinical problem. Summary of the Invention

[0005] To address the problems existing in the prior art, one of the objectives of this invention is to provide an ATX inhibitor, the structural formula of which is shown in Formula I:

[0006]

[0007] The compound with structural formula I has the chemical formula C18H16N4O2S2 and the chemical name 2-(3-{[4-methyl-5-(thiophen-2-yl)-1,2,4-triazol-3-yl]thio}propyl)isoindole-1,3-dione, 2-(3-{[4-methyl-5-(thiophen-2-yl)-1,2,4-triazol-3-yl]sulfanyl}propyl)isoindole-1,3-dione; referred to as AF-399 / 41668442 in subsequent texts.

[0008] The specific use of the above-mentioned ATX inhibitor is: the application of 2-(3-{[4-methyl-5-(thiopurin-2-yl)-1,2,4-triazol-3-yl]thio}propyl)isoindole-1,3-dione in the preparation of drugs for treating urticaria.

[0009] One object of the present invention is to provide a use for an ATX inhibitor, the structural formula of which is shown in Formula II:

[0010]

[0011] The chemical formula of the compound with structure II is C. 17 H 10 ClN3S, chemically named 2Z-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-3-pyridin-3-ylprop-2-enenitrile, (2Z)-2-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-3-pyridin-3-ylprop-2-enenitrile; referred to as AI-219 / 34612004 in subsequent texts.

[0012] The specific use of the aforementioned ATX inhibitor is in the preparation of drugs for treating urticaria using 2-[4-(4-chlorophenyl)-1,3-thiazolyl-2-yl]-3-pyridin-3-ylprop-2-enonitrile.

[0013] A third objective of this invention is to provide an ATX inhibitor, the structural formula of which is shown in Formula III:

[0014]

[0015] Accordingly, the chemical formula of Formula III is C 16 H 15 ClN2OS, chemically named 3-[3-(4-chlorophenyl)sulfanylpropyl]-1H-benzimidazol-2-one, hereinafter abbreviated as AI-204 / 31685055.

[0016] The specific use of the aforementioned ATX inhibitor is in the preparation of drugs for treating urticaria, specifically 3-[3-(4-chlorophenyl)sulfonylpropyl]-1H-benzimidazole-2-one.

[0017] Preferably, the above-mentioned urticaria is chronic urticaria.

[0018] Preferably, the above-mentioned urticaria is chronic spontaneous urticaria.

[0019] Preferably, the above-mentioned drug is an oral drug; in a more preferred embodiment, the oral drug dose in the urticaria mouse model is 20 mg / kg to 40 mg / kg.

[0020] Compared with existing technologies, ATX inhibitors represent a novel target. Animal experiments show that the three ATX inhibitors provided in this invention have significant therapeutic effects on urticaria mice. All three ATX inhibitors (AI-204 / 31685055, AF-399 / 41668442, AI-219 / 34612004) significantly reduced vascular permeability after PCA modeling (3.53 vs 2.21, P < 0.001; 3.53 vs 2.32, P < 0.01; 3.53 vs 2.00, P < 0.001) and inhibited mast cell degranulation induced by modeling (66.04% vs 39.85%, P < 0.01; 66.04% vs 39.85%, P < 0.01). The inhibitory effects of relative mRNA expression of IL13 (38.09%, P<0.01; 66.04% vs 32.22%, P<0.001), and CCL2 (3.5fold, P<0.001; 6.9fold, P<0.001; 4.9fold, P<0.001), and CCL2 (3.5fold, P<0.001; 6.9fold, P<0.001; 6.0fold, P<0.00) were observed. 01) The relative inhibitory effect on CCL3 mRNA expression (2.5fold, P<0.05; 2.7fold, P<0.01, 3.6fold, P<0.01), and the relative inhibitory effect on PGD2 mRNA expression (2.4fold, P<0.01; 3.9fold, P<0.001, 6.0fold, P<0.0001); AI-219 / 34612004 also inhibited plasma histamine release (374.9 pg / ml vs 131.6 pg / ml, P<0.05). This invention provides a new direction for the treatment of urticaria. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the drug administration process for each group of mice in the examples;

[0022] Figure 2 This is a comparison of the size and color intensity of the blue spots on the ears of mice in each experimental group in the example.

[0023] Figure 3 This is a comparison of the standardized blue dye results after formamide extraction on the ears of mice in each experimental group in the examples;

[0024] Figure 4 This is a comparison chart of histamine expression levels in mice from different experimental groups in the examples;

[0025] Figure 5 This is a comparison chart of the percentage of degranulated mast cells in mice in each experimental group in the examples.

[0026] Figure 6 This is a comparison chart of the percentage of degranulated mast cells in mice in each experimental group in the examples.

[0027] Figure 7 This is a diagram showing the comparison of IL-13 expression levels in mice from different experimental groups in the examples.

[0028] Figure 8 This is a graph showing the comparison of CCL2 expression levels in mice from different experimental groups in the examples;

[0029] Figure 9 This is a graph showing the comparison of CCL3 expression levels in mice from different experimental groups in the examples.

[0030] Figure 10 This is a diagram showing the comparison of PGD2 expression levels in mice from different experimental groups in the examples.

[0031] In the picture:

[0032] Significance was determined by the Wilcoxon rank-sum test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PCA: Passive cutaneous anaphylaxis; 5055_PCA: AI-204 / 31685055; 8422_PCA: AF-399 / 41668442; 2004_PCA: AI-219 / 34612004. Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, the experimental methods in the following examples are generally performed under conventional conditions as described in *Pharmacology Laboratory Manual, Third Edition*, Science Press, 2002, or as recommended by the manufacturer. Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0035] The main materials involved in the embodiments of this invention are as follows:

[0036] 1. Laboratory animals

[0037] Specific pathogen-free (SPF) grade BAL B / 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.

[0038] 2. Experimental reagents

[0039]

[0040]

[0041] Example

[0042] I. PCA Animal Model

[0043] Passive cutaneous anaphylaxis, also known as PCA reaction, is a method to test the high sensitivity of antibodies or antigens by detecting a local allergic reaction caused by antibodies that bind to tissues of the same or different animal species.

[0044] IgE is an important mediator of skin allergic reactions. It has a strong affinity for tissue cells. When IgE antibodies enter the body, the Fc terminus of IgE binds to specific receptors on the surface of mast cells in the skin, forming an IgE complex that sensitizes mast cells. When attacked by an antigen, the antigen binds to the Fab terminus of IgE on the mast cell surface, leading to a change in the conformation of the IgE molecule. This causes local mast cells to release allergic mediators, triggering an allergic reaction and increasing local vascular permeability. Skin allergic reactions often manifest as symptoms such as urticaria, itching, and edema. Using IgE as an sensitizer can easily induce skin allergic reactions.

[0045] The PCA procedure in this embodiment is as follows: IgE is subcutaneously injected into mice. After 3-5 hours, binding antibodies bind to mast cells in the tissue, while other antibodies diffuse away. 16-24 hours later, an antigen solution containing Evans blue dye is injected into the inner canthal vein. The antibodies react with the antibodies attached to the mast cells at the IgE injection site, releasing histamine or other active factors and causing a local allergic reaction, specifically a local allergic reaction at the IgE injection site, i.e., the ear. In this allergic area, due to increased vascular permeability, the blue dye leaks into the tissue, causing a blue spot on the skin. The extent or intensity of the blue spot is then measured as an indicator of the reaction intensity.

[0046] II. Animal experiments on the efficacy of treatment for urticaria

[0047] 1. Experimental groups and administration methods

[0048] Mice, 10 mice in each experimental group, were subjected to PCA modeling after administration as listed in the table below:

[0049]

[0050]

[0051] Dosage such as Figure 1 As shown.

[0052] 2. Therapeutic efficacy trials

[0053] After the above steps were performed, each experimental group underwent PCA modeling on day 4 to observe the severity of the local allergic reaction of the drug in the mouse ear and to evaluate the effect of each experimental group.

[0054] 2.1 Effects of blue spots on the ear

[0055] Figure 2 Comparison photos of blue spots in the ears of each experimental group, such as... Figure 2 As shown, compared with the PCA modeling group, the blue spots on the ears of the ATX inhibitor treatment groups were significantly reduced.

[0056] 2.2 Effects of ear vascular leakage

[0057] Evans dye was extracted from the ears of mice in each experimental group using formamide, and the OD value was recorded. The OD value indicates the amount of dye exudation, i.e., the degree of blood vessel leakage in the ear. The results were standardized according to the weight of the mouse ears, and the comparison results are as follows: Figure 3 As shown, the results indicated that the degree of vascular leakage in the ear was significantly reduced in each ATX inhibitor treatment group compared to the PCA group (P < 0.01).

[0058] 2.3 Effects on histamine expression levels

[0059] Histamine levels in mouse ear tissue were detected using an ELISA kit, and the results are as follows: Figure 4 As shown, the results indicated that histamine levels in the ATX inhibitor AI-219 / 34612004 treatment group were lower than those in the PCA group (P < 0.05).

[0060] 2.4 Inhibition of mast cell degranulation

[0061] The experimental methods were referenced from the literature Hou YB, Ji K, Sun YT, Zhang LN, Chen JJ. CDK4 / 6 inhibitor palbociclib suppresses IgE-mediated mast cell activation. J Transl Med. 2019;17(1):276. Published 2019 Aug 20. doi:10.1186 / s12967-019-2026-9.

[0062] After toluidine blue staining of ear tissue from mice in each experimental group, the degree of inhibition of mast cell degranulation was assessed by photographing under a 400× optical microscope. The results are as follows: Figure 5 and Figure 6As shown, the results indicated that the percentage of mast cell degranulation was lower in the ATX inhibitor treatment group than in the PCA group (P < 0.01).

[0063] 2.5 Effects on Cytokine Expression Levels

[0064] 2.5.1 Tissue RNA Extraction

[0065] (1) Transfer mouse ear tissue to tissue homogenization tubes, add 1 mL Magzol RNA extractant and two enzyme-free steel balls to each tube, homogenize on a tissue homogenizer for 30 s, cool for 30 s, repeat 3 times, and let stand at room temperature for 10 min.

[0066] (2) Add 200uL of chloroform to the tube, shake for 15s, let stand at room temperature for 2min, and centrifuge at 12000rpm for 10min at 4℃.

[0067] (3) Label the new enzyme-free EP tube and transfer the supernatant after centrifugation into the new EP tube, about 450-500 uL.

[0068] (4) Add 500uK of isopropanol to each tube, invert and mix 30 times, let stand for 10 min, and centrifuge at 12000 rpm for 10 min at 4℃.

[0069] (5) Discard the supernatant after centrifugation. When you see white clumps at the bottom of the EP tube, add 1 mL of 75% ethanol (prepared with DEPC water) to each tube, gently invert 10 times, and centrifuge at 12000 rpm for 10 min at 4°C.

[0070] (6) Discard the supernatant after centrifugation, add 1 mL of 75% ethanol again to wash and centrifuge to discard the supernatant.

[0071] (7) Dry the RNA at room temperature until it becomes transparent and gelled. Add an appropriate amount of RNase-free water to dissolve the RNA. After thoroughly mixing, measure the concentration using a Nanodrop 8000 instrument and store at -80℃.

[0072] 2.5.2 Reverse Transcription

[0073] Prepare a 20 μL reverse transcription reaction system in an RNase-free water centrifuge tube.

[0074] (1) Residual genomic DNA removal: Add 500 ng RNA, 3 uL 5×g DNA digester Mix, add RNase-free water to quantify to 15 uL, mix quickly with a shaker, centrifuge for 15 s, and incubate at 42 ℃ for 2 min in a PCR instrument.

[0075] (2) Add 4×HifairⅢSuperMix plus 5uL directly to the reaction tube of the previous step, mix quickly with a shaker and centrifuge for 15s.

[0076] (8) Reverse transcription using a PCR instrument: 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min. Store the product at -80℃.

[0077] RT-qPCR reaction system

[0078] Table 7 RT-qPCR reaction system

[0079]

[0080] PCR reaction procedure

[0081]

[0082] Melting curve analysis (65–95℃), with plate readings every 2℃;

[0083] 72℃ for 7 minutes;

[0084] The program has ended.

[0085] (3) Primer list

[0086] Primer sequences

[0087]

[0088] 2.5.3 Inflammation Expression Level

[0089] RNA was extracted from ear tissues of mice in each experimental group after homogenization, and then reverse transcribed into cDNA. Mouse primers IL13, CCL2, CCL3, and PGD2 were designed to identify the expression levels of inflammatory factors in the mouse ear. Results are as follows: Figure 7-10 As shown in the RT-qPCR results, compared with the PCA group, the ATX inhibitor treatment groups significantly reduced the expression levels of IL13, CCL2, CCL3, and PGD2 after modeling (p < 0.05).

[0090] The above experimental results indicate that ATX inhibitors (AI-204 / 3168505, AF-399 / 41668442, AI-219 / 34612004) significantly reduced vascular permeability after PCA modeling (3.53 vs 2.21, P < 0.001; 3.53 vs 2.32, P < 0.01; 3.53 vs 2.00, P < 0.001) and inhibited mast cell degranulation induced by modeling (66.04% vs 39.85%, P < 0.01; 66.04% vs 38.09%, P < 0.01; 66.04% vs 38.09%, P < 0.01). The inhibitory effects of relative mRNA expression of IL13 (32.22%, P < 0.001), CCL2 (3.5 fold, P < 0.001, 6.9 fold, P < 0.0001, 4.9 fold, P < 0.0001), and CCL3 (3.5 fold, P < 0.001, 6.9 fold, P < 0.0001, 6.0 fold, P < 0.0001) on the expression of CCL2 and CCL3 were observed. The inhibitory effect on RNA expression (2.5fold, P<0.05; 2.7fold, P<0.01, 3.6fold, P<0.01), and the inhibitory effect on the relative mRNA expression of PGD2 (2.4fold, P<0.01; 3.9fold, P<0.001, 6.0fold, P<0.0001); in addition, AI-219 / 34612004 also inhibited the release of plasma histamine (374.9 pg / ml vs 131.6 pg / ml, P<0.05).

[0091] 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. The use of a compound of formula I in the preparation of a medicament for treating chronic urticaria, wherein the structural formula of formula I is shown below: I The chemical formula of the compound with structural formula I is C 18 H 16 N4O2S2, chemical name: 2-(3-{[4-methyl-5-(thiopurin-2-yl)-1,2,4-triazol-3-yl]thio}propyl) isoindole-1,3-dione.

2. The use according to claim 1, characterized in that, The chronic urticaria mentioned refers to chronic spontaneous urticaria.

3. The use according to claim 1, characterized in that, The drug is an oral medication.

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