Sun Huadong, Zhang Li, Chow Edwin Chiu Yuen, Lin Ge, Zuo Zhong, Pang K Sandy
Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, ON M5S 3M2, Canada.
J Pharmacol Exp Ther. 2008 Jul;326(1):117-26. doi: 10.1124/jpet.108.137463. Epub 2008 Apr 2.
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 microM(-1), n(1) = 10.6 +/- 0.6 nmol/mg) and lower affinity, higher capacity site (K(A2) = 0.015 +/- 0.0013 microM(-1), n(2) = 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 microM, and K(I) values of 6.75 +/- 1.70 and 0.37 +/- 0.16 microM, respectively. In the Caco-2 monolayer, Ba (8-47 microM) 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 microM) and K(I) value (0.58 +/- 0.20 versus 7.90 +/- 1.10 microM); 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.
在体外和Caco-2细胞中研究了黄芩苷（Ba）的转运和代谢。Ba与Caco-2裂解物的蛋白结合表明，Ba与两类位点结合：一类是高亲和力、低容量位点（K(A1) = 27.6 +/- 4.7 microM(-1)，n(1) = 10.6 +/- 0.6 nmol/mg），另一类是低亲和力、高容量位点（K(A2) = 0.015 +/- 0.0013 microM(-1)，n(2) = 413 +/- 21 nmol/mg）。Ba与Caco-2裂解物的孵育研究表明，葡萄糖醛酸化和硫酸化均存在底物抑制，K(m)值分别为0.14 +/- 0.034和0.015 +/- 0.0053 microM，K(I)值分别为6.75 +/- 1.70和0.37 +/- 0.16 microM。在Caco-2单层细胞中，Ba（8 - 47 microM）在跨膜时表现出良好的表观渗透率（P(app)）；在吸收和分泌方向上，P(app)均随加载浓度升高而增加。然而，外排率小于1，排除了顶端外排转运体的参与。硫酸钡（BS）和葡萄糖醛酸苷（BG）的浓度比随加载Ba浓度的增加而降低，表明BS和BG可能分别通过乳腺癌耐药蛋白和多药耐药相关蛋白2从顶端排出。数据拟合由基底外侧、细胞和顶端区室组成的链状模型，结果显示细胞内未结合分数较低（0.0019 +/- 0.00018），被动扩散清除率较高（0.012 +/- 0.00029 ml/min/mg），且存在底物抑制，硫酸化比葡萄糖醛酸化更容易饱和和受抑制，这可通过较小的K(m)值（0.35 +/- 0.078对1.95 +/- 0.57 microM）和K(I)值（0.58 +/- 0.20对7.90 +/- 1.10 microM）得到证明；这些模式与裂解物孵育研究中观察到的一致。结果表明，链状模型能够恰当地预测底物抑制动力学，并为Caco-2单层细胞中药物的转运和非典型代谢提供重要的机制性见解。
