Enzyme potency and selectivity assays
Active recombinant human catalytic domains of JAK1 (aa 845–1142) and JAK3 (aa 811–1103) were prepared in house and expressed in SF9 cells as a glutathione s transferase (GST) fusion and purified by glutathione affinity chromatography. Active human TYK2 (aa880–1185) was purified in house and contains an N-terminal histidine-tag and C-terminal FLAG tag. It was purified by immobilized metal ion affinity chromatography. Recombinant kinase domain of JAK2 was purchased from Millipore (Burlington, MA). Peptides Biotin-TYR2 (Biotin-(Ahx)-AEEEYFFLFA-amide) and Biotin-TYR1 (Biotin-(Ahx)-GAEEEIYAAFFA-COOH were purchased from New England Peptide (Gardner, MA). Reactions were carried out at 100 μM ATP in the presence of inhibitor and 2 μM peptide. For competition assays, the JAK1 IC50 of upadacitinib was determined in the presence of varying amounts of ATP (0.01-1 mM) equal to and greater than the ATP Km for the kinase. ATP competitiveness was evaluated using the Cheng-Prusoff equation. Inhibitors that are ATP competitive will display changes in the IC50 consistent with the theoretical values derived from the Cheng-Prusoff equation at varying ATP concentrations.
Ba/F3 cellular potency and selectivity assays
The TEL-JAK2, TEL-JAK3, TEL-TYK2, and BCR-JAK1 Ba/F3 engineered cell lines were purchased from Advanced Cellular Dynamics (San Diego, CA). Cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin-glutamine and 0.5 μg/ml puromycin (ThermoFisher Scientific, Waltham, MA).
For measurement of phosphorylation of signal transducer and activator of transcription 5 (pSTAT5), cells were washed and resuspended in Hank’s balanced salt solution at a density of 2 X 107 cells/mL. Five microliters of cell suspension were added to a 384-well, low-volume, white-walled polystyrene plate (Perkin Elmer, Hopkinton, MA) that contained 5 μL of compound (in a 11 point [1:3] titration series). Cells were incubated with compound (final DMSO concentration 0.5%) for 30 min at 37 °C before proceeding with pSTAT5 detection. pSTAT5 was measured with the SureFire pSTAT5 Assay kit (Perkin Elmer, Hopkinton, MA) per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision (Perkin Elmer, Hopkinton, MA).
Cytokine potency assays
IL-6 and Oncostatin M (OSM) induced STAT3 phosphorylation was assessed in the human erythroleukemia TF-1 cell line. Erythropoietin-induced STAT5 phosphorylation was assessed in the human UT-7 cell line. IL-2 and IL-15 induced STAT5 phosphorylation was assessed in activated human T-cells. Detection of phosphorylated STATs was accomplished with the SureFire pSTAT5 or pSTAT3 Assay kit (Perkin Elmer, Hopkinton, MA) per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision (Perkin Elmer, Hopkinton, MA). IFNγ induced STAT1 phosphorylation was assessed in the CD14+ monocyte population in human PBMC by flow cytometry. CD14 BV421 was purchased from BD Biosciences (San Jose, CA). STAT1 PE (pY705) was purchased from ThermoFisher Scientific (Waltham, MA). IL-4 and IL-13 induced STAT6 phosphorylation and IL-31 induced STAT3 phosphorylation were assessed in adult human epithelial keratinocytes (HEKa, ThermoFisher Scientific, Waltham, MA) by flow cytometry. STAT6 PE (pY641) and STAT3 PE (Y705) were purchased from BD Biosciences (San Jose, CA).
Rat adjuvant-induced arthritis (AIA) model
Arthritis was induced in female Lewis rats (weight, 125 – 150 g) (Charles River, Portage, MI) by a single intradermal injection of 0.1 mL of microbacterium tuberculosis emulsion into the right hind footpad (Day 0). Rats were dosed as indicated orally by gavage twice a day (BID) for 10 days (Day7 – Day17) post immunization with either vehicle or study drug. To evaluate the severity of arthritis, paw swelling was evaluated with a water displacement plethysmograph (Ugo Basile North America Inc. PA) every other day up to Day 17. On Day 17, all rats were exsanguinated by cardiac puncture under isolfuorane anesthesia. Left rear paws were scanned using a μCT (SCANCO Medical, Southeastern, PA, Model #μCT40). Bone volume and density were determined in a 360 μm vertical section encompassing the tarsal section of the paw.
Reticulocyte deployment assays
Naïve male Lewis rats were injected intravenously with either PBS or 1000 IU of epoetin α (Procrit®, Janssen Products, LP, Horsham, PA) for two consecutive days. Reticulocytes were measured on day 4 by flow cytometry using thiozole orange as a dye as previously described . Dose responses of either upadacitinib or tofacitinib were dosed 30 min prior to the first Epo injection and then once every 12 h subsequently for 3 days.
NK cell analysis
Sprague Dawley rats were dosed orally with either upadacitinib or tofacitinib at doses indicated for 14 days. Blood was collected and stained using BD MultiTest IMK kit (BD Biosciences, San Jose, CA) per manufacturer’s instructions. NK cell numbers were determined by using FlowJo analysis software (FlowJo, LLC, Ashland, OR) and by examining the CD3−/CD16+/CD56+ population. The number of cells/μL was calculated by using the following equation: (# events in cell population/# of events in absolute bead count region) × (# beads per test/test volume), with the value beads per test indicated on the BD Trucount tube label.
A direct maximum enhancement model was the most predictive for defining the efficacious concentration range and human efficacious dose. Efficacious area under the concentration-versus-time curve (AUC) was based on paw swelling on the last day of the study plotted against the cumulative plasma concentration of upadacitinib or tofacitinib over 12 h (AUC0–12).
Clinical ex vivo stimulation assays
For each subject, blood was collected by venipuncture into 2 mL sodium heparin tubes at 0, 1, 6, and 12 h post upadacitinib or tofacitinib dose. Recombinant human IL-6 (400 ng/ml), or IL-7 (400 ng/ml), (R&D Systems, Minneapolis, MN) was added to blood and incubated for 10 min at 37°C. Surface antibodies were added (CD14-APC, CD3-fluorescein isothiocyanate [FITC]; BD Biosciences, San Jose, CA) and incubated on ice for an additional 20 min. Samples were lysed (Lyse/Fix, BD Biosciences, San Jose, CA) and incubated for 10 min at 37°C. Samples were washed and stored at − 70°C. For intracellular staining, samples were thawed, washed, and resuspended in BD Perm buffer III (BD Biosciences, San Jose, CA) on ice for 30 min. Samples were washed and stained with pSTAT5-PE or pSTAT3-PE (BD Biosciences, San Jose, CA) for 60 min at room temperature and then analyzed immediately on a FACSCalibur. Geometric means were determined using FlowJo analysis software. Percent inhibition of relevant STAT phosphorylation was calculated as follows: (1-(Induction of pSTAT at 1 h – baseline pSTAT at 0 h) / (Induction of pSTAT at 0 h – baseline pSTAT at 0 h)*100.
In the AIA experiments, significant differences were determined by comparing each treatment group to vehicle with Dunnett’s multiple comparison test. P < 0.05 was considered indicative of statistically significant differences among groups.