O1-2.2 Sparse instrumental variables: an integrative approach to biomarker validation

Abstract

Dapsone activates CYP2C9-mediated metabolism in various expression systems and is itself metabolized by CYP2C9 to its hydroxylamine metabolite. Studies were conducted with expressed CYP2C9 to characterize the kinetic effects of dapsone (0–100 μM) on (<i>S</i>)-flurbiprofen (2–300 μM), (<i>S</i>)-naproxen (10–1800 μM), and piroxicam (5–900 μM) metabolism in 6 × 6 matrix design experiments. The influence of (<i>S</i>)-flurbiprofen on dapsone hydroxylamine formation was also studied. Dapsone increased the Michaelis-Menten-derived <i>V</i>_max of flurbiprofen 4′-hydroxylation from 12.6 to 20.6 pmol/min/pmol P450, and lowered its <i>K</i>_m from 28.9 to 10.0 μM, suggesting that dapsone activates CYP2C9-mediated flurbiprofen metabolism without displacing flurbiprofen from the active site, supporting a two-site model describing activation. Similar results were observed with piroxicam 5′-hydroxylation, as<i>V</i>_max was increased from 0.08 to 0.20 pmol/min/pmol P450 and <i>K</i>_m was decreased from 183 to 50 μM in the presence of dapsone. In addition, the kinetic profile for naproxen was converted from biphasic to hyperbolic in the presence of dapsone, while exhibiting similar decreases in<i>K</i>_m and increases in<i>V</i>_max. Kinetic parameters were also estimated using the two-site binding equation, with α values &lt;1 and β values &gt;1, indicative of activation. Additionally, dapsone hydroxylamine formation was measured from incubations containing flurbiprofen, exhibiting a kinetic profile that was minimally affected by the presence of flurbiprofen. Overall, these results suggest that dapsone activates the metabolism of multiple substrates of CYP2C9 by binding within the active site and causing positive cooperativity, thus lending further support to a two-site binding model of P450-mediated metabolism.