JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib

Abstract

Objective: Janus kinase inhibitors (JAKinibs) are efficacious in rheumatoid arthritis (RA) with variable reported rates of adverse events, potentially related to differential JAK family member selectivity. Filgotinib was compared with baricitinib, tofacitinib and upadacitinib to elucidate the pharmacological basis underlying its clinical efficacy and safety.

Methods: In vitro JAKinib inhibition of signal transducer and activator of transcription phosphorylation (pSTAT) was measured by flow cytometry in peripheral blood mononuclear cells and whole blood from healthy donors and patients with RA following cytokine stimulation of distinct JAK/STAT pathways. The average daily pSTAT and time above 50% inhibition were calculated at clinical plasma drug exposures in immune cells. The translation of these measures was evaluated in ex vivo-stimulated assays in phase 1 healthy volunteers.

Results: JAKinib potencies depended on cytokine stimulus, pSTAT readout and cell type. JAK1-dependent pathways (interferon (IFN)α/pSTAT5, interleukin (IL)-6/pSTAT1) were among the most potently inhibited by all JAKinibs in healthy and RA blood, with filgotinib exhibiting the greatest selectivity for JAK1 pathways. Filgotinib (200 mg once daily) had calculated average daily target inhibition for IFNα/pSTAT5 and IL-6/pSTAT1 that was equivalent to tofacitinib (5 mg two times per day), upadacitinib (15 mg once daily) and baricitinib (4 mg once daily), with the least average daily inhibition for the JAK2-dependent and JAK3-dependent pathways including IL-2, IL-15, IL-4 (JAK1/JAK3), IFNγ (JAK1/JAK2), granulocyte colony stimulating factor, IL-12, IL-23 (JAK2/tyrosine kinase 2) and granulocyte-macrophage colony-stimulating factor (JAK2/JAK2). Ex vivo pharmacodynamic data from phase 1 healthy volunteers clinically confirmed JAK1 selectivity of filgotinib.

Conclusion: Filgotinib inhibited JAK1-mediated signalling similarly to other JAKinibs, but with less inhibition of JAK2-dependent and JAK3-dependent pathways, providing a mechanistic rationale for its apparently differentiated efficacy:safety profile.

Keywords: antirheumatic agents; arthritis; cytokines; immune system diseases; rheumatoid.

Figure 1

Cytokine receptors are associated with distinct JAK pairing patterns. The JAK isoforms involved in each pathway vary according to the specific cytokine receptor and dictate downstream outcomes. Figure adapted from Winthrop. EPO, erythropoietin; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; JAK, Janus kinase; TYK2, tyrosine kinase 2.

Figure 2

(A) Experimental design and (B) data analysis procedures. Detailed methods are included in online supplemental materials. AUC-24h, area under the curve 0–24 hours; BARI, baricitinib; FIL, filgotinib; FSC-A, forward scatter area; HD, healthy donors; IC₅₀, half maximum inhibitory concentration; IL, interleukin; JAK, Janus kinase; JAKi, JAK inhibitor; MET, major metabolite of filgotinib (GS-829845); mono, monocytes; PBMCs, peripheral blood mononuclear cells; pSTAT, phosphorylated signal transducer and activator of transcription; RA, rheumatoid arthritis; SSC-A, side scatter area;TOFA, tofacitinib; UPA, upadacitinib.

Figure 3

Distinct JAKinib cellular selectivity for JAK heterodimeric cytokine signalling. Mean fold selectivity for each JAK–dimer pair normalised to inhibition of JAK1/TYK2 pathway (IFNα/pSTAT5 in monocytes). Cytokine/pSTAT pairs: JAK1/TYK2 (IFNα/pSTAT5 in monocytes); JAK1/2 (IL-6/pSTAT1 in monocytes); JAK1/3 (IL-15/pSTAT5 in NK cells); JAK1/2 (IFNγ/pSTAT1 in monocytes); JAK2/TYK2 (G-CSF/pSTAT3 in monocytes); and JAK2/2 (GM-CSF/pSTAT5 in monocytes). BARI, baricitinib; FIL, filgotinib; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; IFN, interferon; IL, interleukin; JAK, Janus kinase; JAKinib, JAK inhibitor; MET, major metabolite of filgotinib (GS-829845); NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription; TOFA, tofacitinib; TYK2, tyrosine kinase 2; UPA, upadacitinib.

Figure 4

Predicted cytokine inhibition profiles of JAKinibs at clinical doses. Pharmacodynamic pSTAT inhibition for JAKinibs at rheumatoid arthritis clinical doses over a 24-hour dose interval at steady state for selected cytokine stimulations based on in vitro blood measurements (n=7–10). (A) JAK1/JAK2 (IL-6/pSTAT1 in monocytes); (B) JAK1/TYK2 (IFNα/pSTAT5 in CD4+ T-cells); (C) JAK1/JAK2 (IFNγ/pSTAT1 in neutrophils); (D) JAK1/JAK3 (IL-4/pSTAT6 in CD4+ T-cells); (E) JAK2/TYK2 (G-CSF/pSTAT3 in monocytes); (F) JAK2/JAK2 (GM-CSF/pSTAT5 in monocytes). Dashed lines show 50% target inhibition. The average daily STAT inhibition and the number of hours per day JAKinib concentrations above 50% inhibition are indicated. FIL values include contribution from the major metabolite of FIL. *P<0.05, **P<0.01, ***P<0.001 higher vs FIL (200 mg); †P<0.05, ††P<0.01, †††P<0.001 lower vs FIL (200 mg). BARI, baricitinib; FIL, filgotinib; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; IC₅₀, half maximal inhibitory concentration; IFN, interferon; IL, interleukin; JAK, Janus kinase; JAKinib, JAK inhibitor; pSTAT, phosphorylated signal transducer and activator of transcription; TOFA, tofacitinib; TYK2, tyrosine kinase 2; UPA, upadacitinib.

Figure 5

Cytokine-induced pSTAT inhibition in the samples of patients with RA and in ex vivo stimulated blood from phase 1 studies of healthy volunteers. (A) Measured average inhibition of ex vivo stimulated pSTAT1 (IL-6/CD4+ T-cells) and pSTAT5 (GM-CSF/monocytes) over a 24-hour period in healthy volunteers receiving FIL (200 mg once daily) or placebo or from in vitro calculated values. The black bar indicates placebo-adjusted inhibition. FIL values include contribution of GS-829845. (B) Calculated pSTAT5 inhibition for JAKinibs at RA clinical doses over a 24-hour dose interval at steady state for IFNα/pSTAT5 in CD4+ T-cells in HDs (n=2) and patients with RA (n=3) based on in vitro measurements. (C) Heatmap of average daily percent STAT inhibition (area under the curve (AUC)–24 hours) by JAKinibs at clinical doses in patients with RA and matched HDs (based on in vitro whole blood measurements; n=2–3). BARI, baricitinib; FIL, filgotinib; GM-CSF, granulocyte macrophage colony stimulating factor; HDs, healthy donors; IFN, interferon; IL, interleukin; JAK, Janus kinase; JAKinib, JAK inhibitor; NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription; RA, rheumatoid arthritis; SD, standard deviation; TOFA, tofacitinib; TYK2, tyrosine kinase 2; UPA, upadacitinib.

Figure 6

Calculated cytokine inhibition of JAK-STAT signalling pathways at clinical doses with similar efficacy. (A) Calculated average target inhibition (AUC-24h±SD) over a 24-hour dose interval for selected JAKinibs for a given stimuli/cell type/pSTAT based on in vitro measurements in whole blood from healthy donors (n=7–10). *P<0.05 higher vs FIL 200 mg; †P<0.05 lower vs FIL 200 mg. (B–F) Calculated average target inhibition (AUC-24h±SD) over a 24-hour dose interval for selected JAKinibs at clinical doses with similar efficacy for a given stimulus/cell type/pSTAT based on in vitro measurements in whole blood from healthy donors (n=7–10). The clinical doses represented were FIL (200 mg), TOFA (5 mg), UPA (15 mg) and BARI (4 mg). (B) JAK1/TYK2, (C) JAK1/JAK2, (D) JAK1/JAK3, (E) JAK2/TYK2 and (F) JAK2/JAK2; FIL includes contribution of GS-829845. Dashed lines show 50% target inhibition. *P<0.05, **P<0.01, ***P<0.001 vs FIL (200 mg). AUC-24h, area under the curve 0–24 hours; BARI, baricitinib; FIL, filgotinib; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage colony stimulating factor; IFN, interferon; IL, interleukin; JAK, Janus kinase; JAKinib, JAK inhibitor; mono, monocyte; neut, neutrophil; NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription; TOFA, tofacitinib; TYK2, tyrosine kinase 2; UPA, upadacitinib.