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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

A Catenary Model to Study Transport and Conjugation of Baicalein, a Bioactive Flavonoid, in the Caco-2 Cell Monolayer: Demonstration of Substrate Inhibition

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (H.S., E.C.Y.C., K.S.P.); and School of Pharmacy (L.Z., Z.Z.) and Department of Pharmacology (G.L.), Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, People's Republic of China

The transport and metabolism of baicalein (Ba) was studied in vitro and in Caco-2 cells. Protein binding of Ba with Caco-2 lysate showed that Ba was bound to two classes of sites: a higher affinity, lower capacity site (K_A1 = 27.6 ± 4.7 µM^–1, n₁ = 10.6 ± 0.6 nmol/mg) and lower affinity, higher capacity site (K_A2 = 0.015 ± 0.0013 µM^–1, n₂ = 413 ± 21 nmol/mg). Incubation studies of Ba with Caco-2 lysate showed substrate inhibition of both glucuronidation and sulfation, with K_m values of 0.14 ± 0.034 and 0.015 ± 0.0053 µM, and K_I values of 6.75 ± 1.70 and 0.37 ± 0.16 µM, respectively. In the Caco-2 monolayer, Ba (8–47 µM) displayed good apparent permeabilities (P_app) across the membrane; P_app was found to be increased with elevated loading concentration in both the absorptive and secretory directions. However, the efflux ratio was less than unity, negating the involvement of apical efflux transporters. The concentration ratios of Ba sulfate (BS) and glucuronide (BG) decreased with increased loading Ba concentration, suggesting that BS and BG are apically excreted via transporters, likely breast cancer resistance protein and multidrug resistance-associated protein 2, respectively. Data fit to the catenary model, composed of basolateral, cellular, and apical compartments, showed a low cellular unbound fraction (0.0019 ± 0.00018), a high passive diffusion clearance (0.012 ± 0.00029 ml/min/mg), and substrate inhibition, with sulfation being more readily saturated and inhibited than glucuronidation, as evidenced by smaller K_m value (0.35 ± 0.078 versus 1.95 ± 0.57 µM) and K_I value (0.58 ± 0.20 versus 7.90 ± 1.10 µM); these patterns paralleled those observed in the lysate incubation studies. The results showed that the catenary model aptly predicts substrate inhibition kinetics and offers significant and mechanistic insight into the transport and atypical metabolism of drugs in the Caco-2 monolayer.

Address correspondence to. Dr. K. Sandy Pang, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, ON M5S 3M2, Canada. E-mail: ks.pang{at}utoronto.ca

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