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]. 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.

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.

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]

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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.

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The Janus kinase (JAK) family of tyrosine kinases is associated with various cytokine receptors. JAK1 and JAK3 play particularly important roles in the immune response, and their inhibition is expected to provide targeted immune modulation. Several oral JAK inhibitors have recently been developed for treating autoimmune diseases, including rheumatoid arthritis (RA). Here, we investigated the pharmacological effects of peficitinib (formerly known as ASP015K), a novel, chemically synthesized JAK inhibitor. We found that peficitinib inhibited JAK1 and JAK3 with 50% inhibitory concentrations of 3.9 and 0.7 nM, respectively. Peficitinib also inhibited IL-2-dependent T cell proliferation in vitro and STAT5 phosphorylation in vitro and ex vivo. Furthermore, peficitinib dose-dependently suppressed bone destruction and paw swelling in an adjuvant-induced arthritis model in rats via prophylactic or therapeutic oral dosing regimens. Peficitinib also showed efficacy in the model by continuous intraperitoneal infusion. Area under the concentration versus time curve (AUC) at 50% inhibition of paw swelling via intraperitoneal infusion was similar to exposure levels of AUC at 50% inhibition via oral administration, implying that AUC might be important for determining the therapeutic efficacy of peficitinib. These data suggest that peficitinib has therapeutic potential for the oral treatment of RA.[1]

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In conclusion, peficitinib is a novel, orally available JAK inhibitor which demonstrates dose-dependent efficacy both in prophylactic and therapeutic dosing regimens in an AIA rat model in which the AUC, not Cmax or Ctrough, appears to underlie therapeutic efficacy. This finding suggests that this compound may have therapeutic potential for the oral treatment of RA, and indeed, peficitinib is currently in clinical development for this purpose. In addition, our findings suggest that the ex vivo IL-2-induced STAT5 phosphorylation assay may be effective as a pharmacodynamic marker to monitor the immune response to peficitinib.[1]

C18H23BRN4O2

407.304823160172

406.1

C, 53.08; H, 5.69; Br, 19.62; N, 13.76; O, 7.86

1353219-05-2

Peficitinib;944118-01-8

67998300

Solid powder

5

4

3

25

525

2

ZUVPMAPXNYGJQC-OGTXJUTESA-N

InChI=1S/C18H22N4O2.BrH/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);1H/t9?,10-,11+,14?,18?;

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

Peficitinib hydrobromide; ASP015K hydrobromide; ASP-015K hydrobromide; 1353219-05-2; Peficitinib hydrobromide [USAN]; U55XHZ5X6P; 1H-Pyrrolo(2,3-b)pyridine-5-carboxamide, 4-((anti-5-hydroxytricyclo(3.3.1.13,7)dec-2-yl)amino)-, hydrobromide (1:1); ASP 015K hydrobromide; Peficitinib hydrobromide

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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