JAK3 (IC50 = 0.7 nM); JAK1 (IC50 = 3.9 nM); JAK2 (IC50 = 5 nM); Tyk2 (IC50 = 4.8 nM)

In a concentration-dependent manner, peficitinib hydrobromide (0-100 nM; 3 days) suppresses T cell proliferation driven by IL-2[1]. With a mean IC50 of 124 nM in rat whole blood and a mean IC50 of 127 nM in human cells, peficitinib hydrobromide (10-1000 nM) suppresses IL-2-induced STAT5 phosphorylation in a concentration-dependent manner[1].

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In vitro activity of peficitinib[1]
The structure of peficitinib is shown in Fig. 1. Peficitinib inhibited JAK activity in a concentration-dependent manner with IC50 values of 3.9 nM (JAK1), 5.0 nM (JAK2), 0.7 nM (JAK3), and 4.8 nM (TYK2) (Table 1). Under the same assay conditions, tofacitinib exhibited comparable IC50 values of 3.7 nM (JAK1), 3.1 nM (JAK2), 0.8 nM (JAK3), and 16 nM (TYK2). Both compounds exhibited the most potent inhibitory activity on JAK3. Selectivity for JAK3 over JAK2 was approximately 7-fold greater for peficitinib and 4-fold for tofacitinib.

In an adjuvant-induced arthritic rat model, peficitinib hydrobromide (1-30 mg/kg; po; once daily for 24 days) exhibits dose-dependent effectiveness in both preventative and therapeutic dosage regimens[1].
In the study of oral treatment, peficitinib exhibited a prophylactic effect on paw swelling with an ED50 of 2.7 mg/kg and bone destruction in paws of rats with AIA[1].

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Notably, peficitinib dose-dependently improved paw swelling in rats with AIA and suppressed ex vivo IL-2-induced STAT5 phosphorylation. More interestingly, the plasma concentration inducing 50% inhibition of paw swelling in the infusion pump experiments was 29.0 ng/mL (88.9 nM), which appears to be consistent with the IC50 of STAT5 phosphorylation in the in vitro whole blood assay (124 nM, Fig. 3B). These findings further suggest that the extent of JAK1/3-mediated STAT5 phosphorylation might correlate with pathogenesis of AIA. Taken together, these data suggest that the ex vivo IL-2-induced STAT5 phosphorylation assay might be useful as a pharmacodynamic marker, with the inhibitory effect of STAT5 phosphorylation in whole blood assay being predictive of the effect of peficitinib in the AIA model[1].
Additionally, IL-2-induced STAT5 phosphorylation was observed in human whole blood, and peficitinib concentration-dependently inhibited STAT5 phosphorylation with a similar IC50 value to rat whole blood. This result suggests that interspecific differences in the mode of STAT5 phosphorylation by peficitinib do not exist between rats and humans[1].

Kinase assay[1]
Human JAK1, 2, 3 and TYK2 kinase-domains were purchased commercially, and assays were 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 was dissolved in dimethyl sulfoxide. The reaction was initiated by adding the kinase domain, followed by incubation at room temperature for 1 h. Kinase activity was measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2 or Biotin-IRS-Substrate using HRP-conjugated anti-phosphotyrosine antibody using a phosphotyrosine-specific ELISA. TYK2 kinase assay of peficitinib was performed with modification of the ATP concentration to 10 μM.

Cell Proliferation Assay[1]
Cell Types: Splenocytes from male Lewis rats
Tested Concentrations: 0 -100 nM
Incubation Duration: 3 days
Experimental Results: Inhibited IL-2-induced T cell proliferation in a concentration-dependent manner with an IC50 of 10 nM.

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Proliferation of rat T cells[1]
Splenocytes from male Lewis rats were 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 were cultured with Concanavalin A for 24 h at 37 °C to induce IL-2 receptor expression. Splenocytes were then incubated with IL-2 and peficitinib or tofacitinib at designated concentrations in 96-well tissue culture plates. After 3-day incubation, alamarBlue® was added to each of the test wells, followed by 4–6 h incubation. Fluorescence intensity was measured at an excitation wavelength of 545 nm and an emission wavelength of 590 nm. All experiments were performed in triplicate, and experiments were performed either four times or once for assays using peficitinib or tofacitinib, respectively. For each individual, wells cultured with cells and medium alone were prepared for the blanks, and IL-2 stimulated cells without JAK inhibitors were prepared for the controls. To calculate the % inhibition of JAK inhibitors, blanks and controls were designated as 100% and 0% inhibition, respectively.

Animal/Disease Models: Seven-weeks-old female Lewis rats, adjuvant-induced arthritis (AIA) model[1]
Doses: 1, 3, 10, and 30 mg/kg
Route of Administration: Oral administration, one time/day for 24 days
Experimental Results: Dramatically inhibited the increase in paw volume at doses of 1 mg/kg or greater with an ED50 value of 2.7 mg/kg (95% confidence interval: 1.5–4.2 mg/kg). Dramatically decreased the bone destruction score at 10 mg/kg or greater and almost fully ameliorated both paw swelling and bone destruction scores at 30 mg/kg.

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For the oral administration regimen, four of the adjuvant-injected groups received peficitinib (1, 3, 10, and 30 mg/kg) dissolved in 0.5% methylcellulose (MC) once daily. Rats in the normal and control groups received 0.5% MC alone. Two different dosing regimens, prophylactic and therapeutic, were employed using the same set of rats described above. For the prophylactic dosing regimen, rats received peficitinib or 0.5% MC once daily for 24 days starting the day after adjuvant injection. Body weight was measured on days 4, 10, 15, 21, and 25 post-adjuvant injection, and paw volume was measured on days 10, 15, 21, and 25 post-adjuvant injection. For the therapeutic dosing regimen, adjuvant-injected rats were grouped evenly based on body weight and increase in left hind paw volume at day 15. Rats received peficitinib or 0.5% MC from day 15–24, and body weight and paw volume were measured on days 15, 18, 21, and 25 post-adjuvant injection.[1]
In the prophylactic and therapeutic dosing regimens, rats were sacrificed on day 25 after measuring body weight and paw volume, and the left hind paw of each animal was collected. In the intraperitoneal infusion regimen, peficitinib was dissolved in polyethyleneglycol and an equal volume of 500 mM acetic acid at concentrations of 1, 2, and 4 mg/mL, and was administered at day 9 post-adjuvant injection via intraperitoneal infusion using an osmotic infusion pump which was implanted under sterile conditions with isoflurane anesthesia. Dosage levels were calculated as approximately 0.12, 0.24, and 0.48 mg/day per body or 0.75, 1.5, and 3 mg/kg/day per unit body weight. Rats in the normal and control groups underwent pump implantation surgery and vehicle was administered. Body weight and paw volume were measured on days 0, 9, 15, 18, 21 and 24 post-adjuvant injection.[1]

Pharmacokinetic data from a 4-week repeated oral dose study of peficitinib at 3 mg/kg using female SD rats showed a maximum plasma concentration (Cmax) of 367 ng/mL, AUC of 834 ng h/mL, and trough concentration (Ctrough) of 2.9 ng/mL (unpublished observations). Taken together with pharmacokinetic data from the present study, at ED50, the Cmax was estimated as 330 ng/mL, AUC as 751 ng h/mL, and Ctrough as 2.6 ng/mL.[1]
In our examination of the effects of continuous infusion, the plasma levels of peficitinib administered as an intraperitoneal infusion were determined, and the EC50 of paw swelling was estimated as 29.0 ng/mL. AUC was therefore calculated as 696 ng h/mL (29.0 ng/mL × 24 h) – a similar exposure level to that of orally administered peficitinib, even though the transition of plasma level differed between continuous infusion and oral administration. These findings suggest that the efficacy of peficitinib on paw swelling in the AIA model was dependent on AUC rather than Cmax or Ctrough, providing a potentially important insight for the design of a clinical dosing regimen.[1]

[1]. A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J Pharmacol Sci. 2017 Jan;133(1):25-33.

Peficitinib has been used in trials studying the treatment and basic science of Psoriasis, Pharmacodynamics, Drug Interactions, Colitis, Ulcerative, and RHEUMATOID ARTHRITIS, among others.

C18H22N4O2

326.39

326.174

C, 66.24; H, 6.79; N, 17.17; O, 9.80

944118-01-8

Peficitinib hydrobromide;1353219-05-2;Peficitinib hydrochloride;1353219-06-3

57928403

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.777

3.26

4

4

3

24

525

2

C1[C@@H]2CC3(C[C@@H](C2NC4=C5C=CNC5=NC=C4C(=O)N)CC1C3)O

DREIJXJRTLTGJC-JQCLMNFQSA-N

InChI=1S/C18H22N4O2/c19-16(23)13-8-21-17-12(1-2-20-17)15(13)22-14-10-3-9-4-11(14)7-18(24,5-9)6-10/h1-2,8-11,14,24H,3-7H2,(H2,19,23)(H2,20,21,22)/t9?,10-,11+,14?,18?

4-[[(1R,3S)-5-hydroxy-2-adamantyl]amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.66 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 (7.66 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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