Background
Peficitinib (ASP015K) is an oral Janus kinase (JAK) inhibitor. It inhibits enzyme activities of JAK1, JAK2, JAK3 and Tyk2 with IC50 values of 3.9, 5.0, 0.71 and 4.8 nmol/L, respectively. It showed moderate selectivity for JAK3 inhibition [1].
JAK family of non-receptor protein tyrosine kinases includes JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). JAK1, JAK2, JAK3 and Tyk2 are critically important in haematopoietic cells and immune cells [1].
Erythropoietin regulates haematopoiesis (the production of blood cells) [2]. Treatment with ASP015K inhibited the human T-cell proliferation induced by IL-2. And this effect was greater than the effect to inhibit EPO-induced human erythroleukemia cell proliferation. In human whole blood, ASP015K concentration-dependently inhibited STAT5 phosphorylation (pSTAT5) [3].
In healthy volunteers, STAT5-P was dose-dependently inhibited by ASP015K. When the doses of ASP015K were 60, 120, 200, or 300 mg, the mean peak percentage inhibitions of STAT5-P were 84%, 85%, 92%, and 93%, respectively. Plasma ASP015K inhibited STAT5-P with an EC50 value of 48 ng/mL, a shape factor (γ) of ~1.2, and an estimated Emax close to 100%. On days 1, 7, and 14, at ~2 hours after treatment with ASP015K at multiple doses, the peak of median percentage of STAT5-P inhibition appeared [4].
参考文献:
[1].? Takeuchi T, Tanaka Y, Iwasaki M, et al. Efficacy and safety of the oral Janus kinase inhibitor peficitinib (ASP015K) monotherapy in patients with moderate to severe rheumatoid arthritis in Japan: a 12-week, randomised, double-blind, placebo-controlled phase IIb study. Annals of the rheumatic diseases, 2015: annrheumdis-2015-208279.
[2].? Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades. Nature, 2001, 412(6847): 641-647.
[3].? Yamazaki S, Morio H, Inami M, et al. THU0101 ASP015K: A Novel Jak Inhibitor Demonstrated Potent Efficacy in Adjuvant-Induced Arthritis Model in Rats. Annals of the Rheumatic Diseases, 2013, 72(Suppl 3): A197-A197.
[4].? Zhu T, Valluri U, Lewand M, et al. THU0256 Pharmacodynamics of ASP015K, A Novel Janus Kinase Inhibitor, in Healthy Volunteers. Annals of the Rheumatic Diseases, 2013, 72(Suppl 3): A252-A252.
Protocol
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Kinase experiment:
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Human JAK1, JAK2, JAK3, TYK2-domains assays performed using streptavidin-coated 96-well plates. Reaction mixture contained 15 mM Tris-HCl (pH 7.5), 0.01% Tween 20, 2 mM dithiothreitol, 10 mM MgCl2, 250 nM Biotin-Lyn-Substrate-2 (for JAK1, 2 and 3) or Biotin-IRS1-Substrate (for TYK2), and ATP (at final concentrations of 200 μM [JAK1], 10 μM [JAK2], 8 μM [JAK3], and 4 μM [TYK2]). Peficitinib or tofacitinib is dissolved in DMSO. The reaction is initiated by adding the kinase domain, followed by incubation at room temperature for 1 h. Kinase activity is measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2 or Biotin-IRS-Substrate using HRP-conjugated anti-phosphotyrosine antibody (HRP-PY-20) using a phosphotyrosine-specific ELISA. TYK2 kinase assay of Peficitinib is performed with the ATP concentration of 10 μM[1].
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Cell experiment:
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Splenocytes from male Lewis rats are suspended in RPMI1640 supplemented with 10% fetal bovine serum and 50 μM 2-mercaptoethanol at a density of 1.5 × 106 cells/mL. Rat splenocytes are cultured with Concanavalin A for 24 h at 37°C to induce IL-2 receptor expression. Splenocytes are then incubated with IL-2 and Peficitinib or tofacitinib at designated concentrations in 96-well tissue culture plates. After 3-day incubation, alamarBlue? is added to each of the test wells, followed by 4-6 h incubation. Fluorescence intensity is measured at an excitation wavelength of 545 nm and an emission wavelength of 590 nm. All experiments are performed in triplicate, and experiments are performed either four times or once for assays using Peficitinib or tofacitinib, respectively. For each individual, wells cultured with cells and medium alone are prepared for the blanks, and IL-2 stimulated cells without JAK inhibitors are prepared for the controls. To calculate the % inhibition of JAK inhibitors, blanks and controls are designated as 100% and 0% inhibition, respectively[1].
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Animal experiment:
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Rats[1]Seven-weeks-old female Lewis rats are used for the adjuvant-induced arthritis (AIA) model. Body weight and left hind paw volume of each rat are measured (MK-101PR volume meter), and the values are used to assign animals to one of six groups (n = 10). Arthritis is induced on day 0 in five of these groups by injecting a suspension of Mycobacterium tuberculosis H37 RA strain (0.5 mg/rat) in liquid paraffin into the right hind foot pad. The remaining group is not injected with adjuvant (normal group, n = 10). For the oral administration regimen, four of the adjuvant-injected groups receive Peficitinib (1, 3, 10, and 30 mg/kg) dissolved in 0.5% methylcellulose (MC) once daily. Rats in the normal and control groups receive 0.5% MC alone[1].
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参考文献:
[1]. Ito M, et al. A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J Pharmacol Sci. 2017 Jan;133(1):25-33.
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