The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport

Abstract

The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

Fig. 1

The flux of nizatidine (0.1 mM) across Caco-2 cell monolayers in the absorptive (AP–BL) and secretory (BL–AP) directions and the corresponding P _app values, in the presence or absence of the P-gp inhibitor verapamil (0.1 mM). Data presented as mean ± SD; each experimental group represents studies across three transwells (n = 3); **p < 0.01

Fig. 2

Inhibition of nizatidine (0.25 mM) transport in the secretory (BL–AP) direction by various verapamil concentrations (0.0001–5 mM). Data presented as a percentage of the uninhibited control. Each data point represents the mean ± SD of studies across three transwells (n = 3). The IC₅₀ value for verapamil on nizatidine transport was estimated by a nonlinear regression using GraphPad Prism 4.01

Fig. 3

The concentration dependence of nizatidine (0.05–10 mM) secretory (BL–AP) transport across Caco-2 cell monolayers in the absence of inhibitors (closed circle) and under complete P-gp inhibition (open circle). The data in the absence of inhibitors were well described by a model consisting of one saturable and one nonsaturable component, as described under the “Data Analysis” section. The conditions for complete P-gp inhibition (1 mM of verapamil) were identified from the lower plateau region of the concentration-dependent inhibitory effect of verapamil on nizatidine BL–AP transport. Each data point represents the mean ± SD of studies across three transwells (n = 3)

Fig. 4

The concentration dependency of nizatidine active transport mediated by P-gp in the secretory (BL–AP) direction across Caco-2 cell monolayers. The P-gp-mediated active transport rates were determined by subtracting the flux rate under complete P-gp inhibition from the flux rate under no inhibition (21). Apparent Michaelis–Menten parameters were estimated using GraphPad Prism 4.01. Data point are means ± SD of studies across three transwells (n = 3)

Fig. 5

The apparent permeability (P _app) of nizatidine (0.25 mM) in the absorptive (AP–BL; black bar) and the secretory (BL–AP; gray bar) directions, in the presence of various concentrations of different P-gp, MRP2, and BCRP inhibitors. Data presented as mean ± SD; each experimental group represents studies across three transwells (n = 3); Asterisk significantly different from control

Fig. 6

Analysis of P-gp, MRP2, and BCRP expression in the Caco-2 cells used in this paper by Western immunoblotting. P-gp was probed with the monoclonal antibody C219, MRP2 was probed with the monoclonal antibody M₂ III-6, and BCRP was probed with the monoclonal antibody BXP-21