Abstract
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 (KA1 = 27.6 ± 4.7 μM–1, n1 = 10.6 ± 0.6 nmol/mg) and lower affinity, higher capacity site (KA2 = 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 Km values of 0.14 ± 0.034 and 0.015 ± 0.0053 μM, and KI 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 (Papp) across the membrane; Papp 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 Km value (0.35 ± 0.078 versus 1.95 ± 0.57 μM) and KI 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.
Footnotes
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This work is supported by Canadian Institutes of Health Research New Emerging Team Grant NET457250 (to K.S.P.) and CUHK 2041299 and CUHK 478607 (to Z.Z.) from the Research Grants Council of the Hong Kong SAR, China.
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H.S. and L.Z. made equal contributions to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.137463.
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ABBREVIATIONS: MRP, multidrug resistance-associated protein; Ba, baicalein; BG, baicalein-7-O-β-glucoronide; UGT, UDP-glucuronosyltransferase; BS, baicalein sulfate; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; A, apical; B, basolateral; Papp, apparent permeability; EfR, efflux ratio; n1, n2, the number of binding sites in the first and second class of binding sites, respectively; CL′int,sec and CL′d2{BG or BS}, net efflux clearance of the Ba conjugate (BG or BS) at the apical and basolateral membranes, respectively; CLd1 and CLd2, CLd4 and CLd3, influx and efflux, passive diffusion clearance at the basolateral and apical membrane, respectively; CLinflux and CLinflux, transporter-mediated influx and efflux intrinsic clearances at the basolateral membrane; CLabs and CLint,sec, transporter-mediated intrinsic clearances for absorption and efflux at the apical membrane, respectively; Vap, Vcell, and Vbaso, volumes of the apical, cellular, and basolateral compartment, respectively; fap, fcell, and fbaso, unbound fractions of drug in apical, cellular, and basolateral compartment, respectively; , and , maximum velocity of glucuronidation and sulfation, respectively; and , Michaelis-Menten constant for glucuronidation and sulfation, respectively; and , inhibition constant for glucuronidation and sulfation, respectively; ABL, aqueous boundary layer.
- Received February 3, 2008.
- Accepted April 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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