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

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 (K A1 ϭ 27.6 Ϯ 4.7 M Ϫ1 , n 1 ϭ 10.6 Ϯ 0.6 nmol/mg) and lower affinity, higher capacity site (K A2 ϭ 0.015 Ϯ 0.0013 M Ϫ1, n 2 ϭ 413 Ϯ 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 Ϯ 0.034 and 0.015 Ϯ 0.0053 M, and K I values of 6.75 Ϯ 1.70 and 0.37 Ϯ 0.16 M, respectively. In the Caco-2 monolayer, Ba (8 -47 M) 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 Ϯ 0.00018), a high passive diffusion clearance (0.012 Ϯ 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 Ϯ 0.078 versus 1.95 Ϯ 0.57 M) and K I value (0.58 Ϯ 0.20 versus 7.90 Ϯ 1.10 M); 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.Flavonoids are polyphenolic compounds that are widely distributed in both edible plant and derived foods (Yang et al., 2001). Flavonoids possess anticarcinogenic activity and other beneficial effects, including the prevention of oxidation of low-density lipoproteins and development of coronary heart disease. These activities have aroused increasing interest among scientists (Yang et al., 2001). However, several studies have demonstrated that the oral bioavailabilities of flavonoids are low and associated with extensive first-pass metabolism, including glucuronidation, sulfation, and methylation (Kroon et al., 2004;Manach and Donovan, 2004 ABBREVIATIONS: MRP, multidrug resistance-associated protein; Ba, baicalein; BG, baicalein-7-O-␤-glucoronide; UGT, UDP-glucuronosyltransferase; BS, baicalein sulfate; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; A, apical; B, basolateral; P app , apparent permeability; EfR, efflux ratio; n 1 , n 2 , the number of binding sites in the first and second class of binding sites, respectively; CLЈ int,sec and CLЈ d2 {BG or BS}, net efflux clearance of...

In vitro and in situ evaluation of herb–drug interactions during intestinal metabolism and absorption of Baicalein

“…The in vitro metabolism study by C-2C lysate was conducted as described in our previous study (Sun et al, 2008). A final concentration of 5 M of B was pre-incubated with C-2C lysate with a final protein concentration of 1.34 mg/ml at 37 • C for 10 min.…”

“…Similar to other flavonoid glucuronides, BG undergoes hydrolysis by intestinal glucuronidase or intestinal microflora to release its aglycone B, which will then be absorbed (Zhang et al, 2007c). The absorbed B undergoes extensive first-pass intestinal and hepatic Phase II metabolism, including glucuronidation (>90%), catalyzed by the enzyme UDP-glucuronosultransferase (UGT) and less significant sulfation, catalyzed by sulfatransferase (SULT) (Zhang et al, 2007b;Sun et al, 2008), resulting in its conjugated metabolites, BG and Baicalein 7-O-sulfate (BS) (Fig. 1).…”

Pharmacokinetic Interplay of Phase II Metabolism and Transport: A Theoretical Study

“…8,[24][25][26] For simplicity, it was assumed that transport of aglycone (D) across the cell membrane was mainly driven by passive diffusion, which is true for most UGT/SULT substrates. 11,27 Diffusional clearances CL d1 and CL d2 denote passive transport CLs on the apical membrane, whereas CL d3 and CL d4 denote passive transport CLs on the basolateral membrane. Unless specified, it was assumed that the CLs were equal for both influx and efflux at both apical and basolateral membranes (CL d1 = CL d2 = CL d3 = CL d4 = CL d ).…”

In Vitro Approaches to Evaluate Placental Drug Transport by Using Differentiating JEG‐3 Human Choriocarcinoma Cells

“…The TEER was measured across the Millicell Ò 24-well inserts in a 24-well culture plate using a Millicell Ò -ERS device (Millipore Co.) [22,23]. The measurements were taken after two washes with phosphate-buffered saline at room temperature and corrected for the resistance of the collagen-coated membrane using measurements taken in the absence of any cells.…”