Primary Human Cell BioMAP® Profiling of Methotrexate, Tocilizumab, Adalimumab, and Tofacitinib Reveals Different Mechanisms of Action With Distinct Phenotypic Signatures

Version:: V12013
File Name/Number:: 2013 ACR/ARHP Conference
Year:: 2013

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Background/Purpose
Proinflammatory cytokines cause joint inflammation and destruction in rheumatoid arthritis (RA) as exemplified by the therapeutic success of inhibiting tumor necrosis factor alpha (TNFa) by adalimumab (ADA) and interleukin (IL)-6 receptor (IL-6R) by tocilizumab (TCZ). Such biologics and small molecule inhibitors, including methotrexate (MTX) and the recently approved tofacitinib (TOF), a Janus kinase inhibitor, have diverse and potential overlapping biologic effects. The purpose of this study was to utilize the human primary cell–based BioMAP® platform to identify similar and discriminating biological activities of TCZ, ADA, MTX, and TOF.

Methods
BioMAP systems model complex signaling networks in primary human cell types and have been used extensively to validate compounds and targets, identify mechanisms of action and potential toxicities, and determine phenotypic signatures (Berg EL et al. J Pharmacol Toxicol Methods. 2006;53:67-74). TCZ, ADA, MTX, and TOF were profiled across a panel of 14 cell-based BioMAP systems containing early-passage primary human cells cultured alone or as cocultures with different stimulus combinations. Compounds were profiled at concentrations that would cover their reported clinical plasma exposure for the respective approved dosing regimen. To examine the effects of compounds on IL-6 trans-signaling, parallel experiments were conducted in the presence of exogenous soluble IL-6R (sIL-6Ra). Compound-mediated perturbations of protein-based and clinically relevant biomarker readouts and other cellular events (eg, proliferation, cell cytotoxicity) were used to generate a biological activity plot (ie, BioMAP profile), which serves as a multisystem signature of the activity for each compound.

Results
TCZ significantly inhibited expression of P-selectin (4H system), IL-8 (SAg system), TNFa (BT and HDFSAg systems), IP-10 and IL-17A (HDFSAg system), and CD69 (LPS and /Mphg systems),consistent with anti-inflammatory and immunomodulatory effects. In general, there was little overlap in the BioMAP profiles for TCZ, ADA, MTX, and TOF. TOF disrupted IL-17F, but not IL-17A, production inthe HDFSAg system and showed dose-dependent selectivity; the phenotypic signatures of TOF at
clinically relevant doses (<1 μM) were distinct from those at higher concentrations. sIL-6Ra induced additional activities in the BioMAP, including inflammation-related, novel immunomodulatory, and tissue/matrix remodeling effects. Importantly, TCZ, but not ADA or TOF, completely blocked all sIL-6Ra–related activities at concentrations corresponding to clinical plasma exposure levels.

Conclusion
TCZ, ADA, MTX, and TOF have markedly unique BioMAP signatures, indicating that these compounds exert different mechanisms of action. IL-6 signaling in BioMAP requires the presence of sIL-6Ra for functional trans-signaling, which was fully reversed by TCZ, but not by ADA or TOF, at concentrations consistent with reported clinical plasma exposures. The relevance of these findings requires confirmation in clinical studies.