In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494)

Abstract

Background: Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib.

Methods: Structure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines.

Results: Here, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting.

Conclusions: The data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile.

Keywords: Arthritis, rheumatoid; JAK inhibitor; Kinase; Selectivity.

Fig. 1

A. Upadacitinib modeled in the crystal structure of JAK1. A model of JAK1 complexed to upadacitinib is shown in blue. JAK2 (Protein Data Ban code: 2B7a) is overlaid in green (18). b. Chemical structure of (3S,4R)-3-ethyl-4-(3H-imidazo [1, 2-a] pyrrolo [2, 3-e] pyrazin-8-yl)-N-(2,2,2-trifluoroethyl) pyrrolidine-1-carboxamide (upadacitinib)

Fig. 2

Preclinical efficacy of upadacitinib in rat adjuvant induced arthritis model. a Exposure–response relationships for upadacitinib (solid symbols) and tofacitinib (open symbols) in a rat adjuvant-induced arthritis model. b Quantitative effect of upadacitinib on bone erosion as assessed by micro CT scanning. c and d Representative micro CT scans of the tarsal regions of vehicle (c) and adjuvant-induced arthritis rats treated with upadacitinib (d)

Fig. 3

Rat in vivo selectivity of tofactinib and upadacitinib. a. Exposure response curves of tofacitinib on inhibition of paw swelling in rat AIA (red circles), CD3⁻/CD16⁺/CD56⁺ NK cell inhibition (green triangles), and inhibition of Epo induced reticulocyte deployment (blue squares). Dotted gray vertical lines represent equivalent clinical exposures for 5 mg and 10 mg tofacitinib. b. Exposure response curves of upadacitinib on inhibition of paw swelling in rat AIA (red circles), CD3⁻/CD16⁺/CD56⁺ NK cell inhibition (green triangles), and inhibition of Epo induced reticulocyte deployment (blue squares). Dotted gray vertical lines represent equivalent clinical exposures for 6 mg and 12 mg upadacitinib

Fig. 4

a Effect on reticulocyte inhibition compared with inhibition of paw swelling in rat AIA for tofacitinib (red circles) and upadacitinib (blue circles) Green arrows denote 60 and 80% inhibition of paw swelling. b Effect on NK cell inhibition compared with inhibition of paw swelling in rat AIA for tofacitinib (red circles) and upadacitinib (blue circles). Green arrows denote 60 and 80% inhibition of paw swelling. Individual animal values are graphed with 95% confidence intervals represented with black dotted lines

Fig. 5

Ex vivo stimulation of whole blood from upadacitinib or placebo dosed healthy volunteers. Whole blood was drawn 1 h post upadacitinib or tofacitinib dose and stimulated with IL-6 (A) or IL-7 (B)