Ca(2+) release-activated Ca(2+) (CRAC) channels; store-operated Ca2+ entry (SOCE)

Without changing the expression of CD11b and GFAP, YM-58483 can lower the levels of P-ERK and P-CREB. Moreover, intestinal IL-1β, TNF-α, and PGE2 production are inhibited by YM-58483 [1]. In a one-way mixed solution reaction (mLR), YM-58483 and cyclosporine A both inhibit T cell growth with IC50 values of 330 and 12.7 nM, respectively [2]. With IC50 values of 460 and 310 nM, YM-58483 suppresses the synthesis of IgE-stimulated leukotriene (LT) and histamine in RBL-2H3 cells (a gradient alkaline dilution cell line). With IC50 values of 125 and 148 nM, respectively, YM-58483 also suppresses the production of IL-5 and IL-13 in human peripheral blood cells triggered by phytohemagglutinin-P (PHA), a mechanism that is around five times less potent than prednisolone [3]. In human whole blood cells stimulated with phytohemagglutinin, YM-58483 reduces the production of IL-5 and IL-4 in murine Th2 T cell clones (D10.G4) stimulated with leucine. The IC50 value is similar to the published CRAC channel inhibition value (about 100 nM) [4].

Intrathecal injection of YM-58483 at doses of 300 μM (1.5 nmol) and 1000 μM (10 nmol) produced strong central analgesic effects on SNL [1]. , YM-58483 (1-30 mg/kg, po) and cyclosporine A (1-30 mg/kg, po) suppress donor anti-cytotoxic T (CTL) activity and IFN-γ production, and Reduce the amount of donor T cells in the spleen, specifically donor CD8+ T cells. YM-58483 (1-10 mg/kg, po) and cyclosporine A (2, 10 mg/kg, po) reduce the delayed-type hypersensitivity (DTH) response elicited by sheep red blood cells (SRBC) [2]. M-58483 (30 mg/kg, elbow) significantly decreased ovalbumin (OVA)-induced delayed-type contractions in OVA-sensitized guinea pigs, whereas YM-58483 (3-30 mg/kg, face) and Prednisolone (100 mg/kg, facial) considerably and totally inhibited the airway hyperresponsiveness (AHR) generated by OVA [3]. YM-58483 buffers eosinophils caused by breathing into the airways and lowers IL-4 and cysteine foam leukotriene levels in activated airways induced by active sensitization of brown Norwegian Scotch centers. Pathway YM-58483 prevents powerful induction of late death and eosinophilic respiration in actively sensitized guinea pigs [4].

PHA-induced IL-5 and IL-13 production from whole human peripheral blood[3]
PHA was used as a stimulator to mimic the initial responses the antigens evoke in T-lymphocytes. Human peripheral blood was obtained from healthy volunteers. Heparinized human peripheral whole blood was diluted in RPMI1640 medium with penicillin-streptomycin at a rate of 1:3.5. Diluted blood (450 μl) was pretreated with various concentrations of YM-58483(BTP2) (10–1000 nM) or prednisolone (1–100 nM), then stimulated with PHA (10 μg/ml) in 24-well plates (total volume: 500 μl) for 48 h at 37 °C. The plates were centrifuged (250g, for 10 min at 4 °C), and then the supernatants were harvested and frozen at −20 °C until use. The levels of IL-5 and IL-13 in the supernatant were measured ELISA using an anti-IL-5 monoclonal antibody and a human IL-13 ELISA Kit DuoSet, respectively.

DNP-induced histamine release and LTs production in RBL-2H3 cells[3]
RBL-2H3 cells, a rat basophilic leukemia cell line with a phenotype similar to that of mucosal mast cells, were purchased from the American Type Culture Collection, and maintained as monolayer cultures in RPMI 1640 medium with 10% FBS, 25 mM HEPES, 2 g/l NaHCO3, and penicillin (100 U/ml)/streptomycin (100 μg/ml) in a 5% CO2-humidified atmosphere. Mediator release from RBL-2H3 cells was induced using DNP and a method described previously, with minor modifications. RBL-2H3 cells were cultured with monoclonal anti-DNP IgE (100 ng/ml) in a 1000-ml spinner culture bottle at a density of 3×105 cells/ml for 24 h. After washing, the cells that had been primed with IgE (8×10~5 cells) in an assay buffer (5 mM HEPES, 140 mM NaCl, 5 mM KCl, 0.6 mM MgCl2, 1 mM CaCl2, 5 mM d(+)-glucose, pH 7.4), were stimulated with the antigen, DNP-BSA (0.1–100 ng/ml), in 96-well plates (total volume: 200 μl) at 37 °C for 20 min. After stimulation, the cells were centrifuged (250g, 10 min at 4 °C), and then the supernatants were removed and frozen at −0 °C until use. The levels of histamine and LTC4/D4/E4 (LTs) in the supernatant were determined using histamine ELISA kits and LTC4/D4/E4 EIA kits, respectively. To measure the effect of the test compounds, IgE-primed cells were pretreated with various concentrations of YM-58483(BTP2) (30–3000 nM), or prednisolone (10 μM), and then stimulated with DNP-BSA (30 ng/ml).

Induction of graft-versus-host disease (GVHD)[3]
For the in vivo experiments, YM-58483(BTP2) was suspended in 0.5% methylcellulose solution for oral administration at a volume of 10 ml/kg.
Induction of GVHD was performed according to a previously described method [27]. Single-cell suspensions were prepared in HBSS from the spleen of female C57BL/6 mice (donor strain, H-2b) and female BDF1 mice (recipient strain, H-2b/d). The spleen cell suspensions were filtered through 70 μm-pore sterile nylon mesh, and then, to lyse the erythrocytes, resuspended in 3 ml of Tris ammonium chloride (0.26 M NH4Cl, 0.017 M Tris, pH 7.6), agitated for 2 min at room temperature, and then washed twice with HBSS. For the induction of GVHD, female BDF1 recipient mice were given a injection of spleen cells (5 × 10~7 cells) from sex- and age-matched C57BL/6 in the tail vein on day 0. As a negative control, female BDF1 mice were injected with sex- and age-matched syngeneic (BDF1) mice spleen cells (5 × 10~7 cells). As a normal control, female BDF1 mice were injected with 200 μl HBSS alone. YM-58483(BTP2) and cyclosporine A were administered orally once daily from day 0 to day 9 (10 consecutive days). Spleen cells from GVHD, negative control, and normal mice were used to examine anti-host CTL activity and the ability to produce IFN-γ on day 10. Cell subsets were also identified on day 10.

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YM-58483, has analgesic effects on neuropathic pain, but its mechanism is unclear. This experiment performed on spinal nerve ligation (SNL)-induced neuropathic pain model in rats tries to explore the mechanism, whereby YM-58483(BTP2) attenuates neuropathic pain. The left L5 was ligated to produce the SNL neuropathic pain model in male Sprague-Dawley rats. The withdrawal threshold of rats was measured by the up-down method and Hargreaves' method before and after intrathecal administration of YM-58483 and vehicle. The SOCCs in the spinal dorsal horn were located by immunofluorescence. The expression of phosphorylated ERK and phosphorylated CREB, CD11b, and GFAP proteins in spinal level was tested by Western blot, while the release of proinflammatory cytokines (IL-1β, TNF-α, PGE2) was measured by enzyme-linked immunosorbent assay (ELISA). Intrathecal YM-58483 at the concentration of 300 μM (1.5 nmol) and 1000 μM (10 nmol) produced a significant central analgesic effect on the SNL rats, compared with control + vehicle (n = 7, P < 0.001). However, both could not prevent the development of neuropathic pain, compared with normal + saline (P < 0.001). Immunofluorescent staining revealed that Orai1 and STIM1 (the two key components of SOCCs) were located in the spinal dorsal horn neurons. Western blot showed that YM-58483 could decrease the levels of P-ERK and P-CREB (n = 10, #P < 0.05), without affecting the expression of CD11b and GFAP (n = 10, #P > 0.05). YM-58483 also inhibited the release of spinal cord IL-1β, TNF-α, and PGE2, compared with control + vehicle (n = 5, #P < 0.001). The analgesic mechanism of YM-58483 may be via inhibiting central ERK/CREB signaling in the neurons and decreasing central IL-1β, TNF-α, and PGE2 release to reduce neuronal excitability in the spinal dorsal horn of the SNL rats.[1]

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YM-58483/BTP2 is a blocker of store-operated Ca2+ entry (SOCE), which regulates the activation of non-excitable cells such as lymphocytes. YM-58483(BTP2) has been reported to inhibit cytokine production and proliferation in T cells, and to be useful as a probable medicinal candidate for treatment of bronchial asthma. The present study investigated the pharmacological profile and therapeutic potential of YM-58483 in relation to cell-mediated immune responses. In the mouse graft-versus-host disease (GVHD) model, YM-58483 (1-30 mg/kg, p.o.) and cyclosporine A (1-30 mg/kg, p.o.) inhibited donor anti-host cytotoxic T lymphocyte (CTL) activity and IFN-gamma production, and also reduced the number of donor T cells, especially donor CD8+ T cells, in the spleen. YM-58483 and cyclosporine A inhibited T cell proliferation in a one-way mixed lymphocyte reaction (MLR) with IC50 values of 330 and 12.7 nM, respectively. Additionally, YM-58483 (1-10 mg/kg, p.o.) and cyclosporine A (2, 10 mg/kg, p.o.) inhibited the sheep red blood cell (SRBC)-induced delayed type hypersensitivity (DTH) response. These results suggest that the inhibition of SOCE leads to the prevention of antigen-induced T cell responses, which participate in autoimmune diseases such as autoimmune hepatitis and rheumatoid arthritis.[2]

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T cells play a regulatory role in the pathogenesis of various immune and allergic diseases, including human asthma. Recently, it was reported that a pyrazole derivative, YM-58483(BTP2), potently inhibits Ca(2+) release-activated Ca(2+) (CRAC) channels and interleukin (IL)-2 production in T cells. We investigated the effects of YM-58483(BTP2) on T helper type 2 (Th2) cytokine production in vitro and antigen-induced airway asthmatic responses in vivo. YM-58483 inhibited IL-4 and IL-5 production in a conalbumine-stimulated murine Th2 T cell clone (D10.G4.1), and IL-5 production in phytohemagglutinin-stimulated human whole blood cells with IC(50) values comparable to those reported for its CRAC channel inhibition (around 100 nM). YM-58483 inhibited antigen-induced eosinophil infiltration into airways, and decreased IL-4 and cysteinyl-leukotrienes content in inflammatory airways induced in actively sensitized Brown Norway rats. Furthermore, orally administered YM-58483 prevented antigen-induced late phase asthmatic bronchoconstriction and eosinophil infiltration in actively sensitized guinea pigs. These data suggest that the inhibition of Ca(2+) influx through CRAC channel leads to the prevention of antigen-induced airway inflammation, probably via the inhibition of Th2 cytokine production and inflammatory mediators release. YM-58483 may therefore be useful for treating airway inflammation in bronchial asthma.[4]

[1]. The Central Analgesic Mechanism of YM-58483 in Attenuating Neuropathic Pain in Rats. Cell Mol Neurobiol. 2016 Oct;36(7):1035-43.

[2]. Characterization of YM-58483/BTP2, a novel store-operated Ca2+ entry blocker, on T cell-mediated immune responses in vivo. Int Immunopharmacol. 2008 Dec 20;8(13-14):1787-9.

[3]. The suppressive effects of YM-58483/BTP-2, a store-operated Ca2+ entry blocker, on inflammatory mediator release in vitro and airway responses in vivo. Pulm Pharmacol Ther. 2008;21(2):360-9.

[4]. YM-58483, a selective CRAC channel inhibitor, prevents antigen-induced airway eosinophilia and late phase asthmatic responses via Th2 cytokine inhibition in animal models. Eur J Pharmacol. 2007 Apr 10;560(2-3):225-33.

YM-58483/BTP-2, 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, blocks the store-operated Ca2+ entry (SOCE) that mediates the activation of non-excitable cells. This study investigated the pharmacological profile and therapeutic potential of YM-58483 as anti-asthma drug. YM-58483 inhibited DNP antigen-induced histamine release from and leukotrienes (LTs) production in IgE-primed RBL-2H3 cells, a rat basophilic leukemia cell line, with IC50 values of 460 and 310 nM, respectively. Prednisolone did not inhibit either of these responses. YM-58483 also inhibited phytohemagglutinin-P (PHA)-stimulated IL-5 and IL-13 production in human peripheral blood cells with IC50 values of 125 and 148 nM, respectively, which is approximately 5 times less potent than prednisolone. YM-58483 (30 mg/kg, p.o.) significantly suppressed ovalbumin (OVA)-induced bronchoconstriction in OVA-sensitized guinea pigs, whereas prednisolone did not. YM-58483 (3-30 mg/kg, p.o.) and prednisolone (100mg/kg, p.o.) both significantly and completely suppressed airway hyperresponsiveness (AHR) caused by OVA exposure. Since YM-58483 inhibits two major characteristic symptoms of bronchial asthma, namely bronchoconstriction and AHR via the suppression of inflammatory mediator and cytokine production, SOCE inhibition is a potential approach for treatment.[3]

C15H9F6N5OS

421.3214

421.043

C, 42.76; H, 2.15; F, 27.06; N, 16.62; O, 3.80; S, 7.61

223499-30-7

223499-30-7;

2455

White to off-white solid powder

1.6±0.1 g/cm3

1.608

3.77

1

11

3

28

568

0

XPRZIORDEVHURQ-UHFFFAOYSA-N

InChI=1S/C15H9F6N5OS/c1-7-12(28-25-23-7)13(27)22-8-2-4-9(5-3-8)26-11(15(19,20)21)6-10(24-26)14(16,17)18/h2-6H,1H3,(H,22,27)

N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide

YM-58483; YM 58483; YM58483; BTP 2; 223499-30-7; YM-58483; btp2; CRAC Channel Inhibitor, BTP2; N-[4-[3,5-bis(trifluoromethyl)pyrazol-1-yl]phenyl]-4-methylthiadiazole-5-carboxamide; N-(4-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide; N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide; CHEMBL262766; BTP-2; BTP2.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~296.69 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)