Comparison of baricitinib, upadacitinib, and tofacitinib mediated regulation of cytokine signaling in human leukocyte subpopulations

Abstract

Background: The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level.

Methods: Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC50) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC50 and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type.

Results: Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC50 values and increased time above IC50 translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees.

Conclusions: Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF.

Keywords: Baricitinib; Cytokine; Janus kinase; Phosphorylated signal transducer and activator of transcription; Potency; Receptor kinase signaling; Rheumatoid arthritis; Selectivity; Tofacitinib; Upadacitinib.

Conflict of interest statement

IBM has received grants and personal fees from AbbVie, Eli Lilly and Company, Janssen, Novartis, Pfizer, and Roche. NLB, REH, WLM, SN, RAO, GR, TPR, XZ, and SHZ are employees and shareholders of Eli Lilly and Company. JL was an employee and shareholder of Eli Lilly and Company when contributions to this article were made. He is now an employee of PDD4Patients LLC. TW is an employee and shareholder of Primity Bio. PCT has received grants and personal fees from Eli Lilly and Company, Galapagos, and UCB. He has received grants from Abide Therapeutics and Celgene and personal fees from AbbVie, Biogen, GSK, Janssen, Novartis, Pfizer, and Sandoz.

Figures

Fig. 1

JAKi exposure curves for select cytokines. The number of hours per day JAKi concentrations are above IC50 values is shown for IL-21/pSTAT3 in NK cells (a), IL-3/pSTAT5 in monocytes (b), GM-CSF/pSTAT5 in monocytes (c), IL-6/pSTAT3 in CD4+ T cells (d), and IFN-γ/pSTAT1 in monocytes (e). Panels a–e include the average daily percent STAT inhibition. Protein binding was accounted for in the calculation of hours per day above IC50 and average daily percent STAT inhibition, and the IC50 values were corrected for the compound-bound proportion. *p < 0.01, **p < 0.001, ***p < 0.0001 compared to baricitinib 2 mg; †p < 0.01, ††p < 0.001, †††p < 0.0001 compared to baricitinib 4 mg. Bari, baricitinib; BID, twice daily; GM-CSF, granulocyte-macrophage colony-stimulating factor; h, hours; IC50, half maximum inhibitory concentration; IFN, interferon; IL, interleukin; JAK, Janus kinase; JAKi, JAK inhibitor; NK, natural killer; pSTAT, phosphorylated STAT; QD, once daily; STAT, signal transducer and activator of transcription; Tofa, tofacitinib; Upa, upadacitinib