Active efflux kinetics of etoposide from rabbit small intestine and colon.

The aim of the present study was to investigate the directional transport kinetics of etoposide in rabbit intestinal tissues using side-by-side diffusion chambers. Etoposide is a routinely used mixed-mechanism 'efflux' inhibitor; however, its absorptive and secretory transport kinetics in rabbit intestinal tissues, a commonly used animal model, have not yet been reported. Kinetic studies revealed that the apical (AP) to basolateral (BL) (i.e. absorptive) transport of etoposide was not apparently mediated by specialized transporters, whereas secretion (i.e. BL to AP transport) by intestinal tissues was concentration dependent and saturable. Half-saturation constant values (K(m), mean+/-standard deviation (S.D.)) ranged from 53.6+/-35.8 microM to 168.7+/-127.3 microM, consistent with previous results from our group in intestinal tissues from other species and Caco-2 cell monolayers. Secretory permeability was greatest in the ileum, whereas values in the upper small intestine and colon were approximately equal, and represented only 50% of the value in the ileum. The ileal secretory transport of etoposide was temperature dependent, with the activation energy (E(a)) >4 kCal/mole at 5 microM, suggesting the involvement of the active, energy dependent mechanism. Etoposide inhibition by verapamil and saquinavir, known inhibitors of intestinal secretion, was characterized as competitive with K(i)'s equal to 193.0+/-164.4 microM and 72.6+/-53.5 microM, respectively. The current results demonstrate that the absorptive transport of etoposide in rabbit tissue was not mediated by specialized carriers, and that secretory transport was regionally dependent, mediated by a transporter or transporters, the K(m)'s were in the micromolar range, and involved the energy dependent mechanism(s). The relatively low k(m) of etoposide compared with its aqueous solubility (0.25-0.34 mM, pH 5-6.5, 25 degrees C) makes it the excellent mixed-mechanism competitive inhibitor for determining the secretory transport properties of putative drug substrates. Understanding the in vitro secretory transport kinetics of etoposide provides a mechanistic basis for ongoing studies exploring the functional role of 'efflux' in vivo.

Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells.

PURPOSE: The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. METHODS: Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 microns to 1000 microns. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4 (100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3 degrees C to 37 degrees C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. RESULTS: Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Ileal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 microns. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Each of the intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. CONCLUSIONS: Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km's were in the micro-molar range, and involved an energy dependent mechanism(s).

Oral absorption of anti-aids nucleoside analogues: 3. Regional absorption and in vivo permeability of 2',3'-dideoxyinosine in an intestinal-vascular access port (IVAP) dog model.

The absolute oral and regional intestinal bioavailabilities (BAs) and pharmacokinetics (PK) of 2',3'-dideoxyinosine (ddI), a nucleoside analog used in the treatment of human immunodeficiency virus (HIV) infection, were investigated in an in vivo intestinal-vascular access port (IVAP) dog model. The mean (+/- SD) absolute regional intestinal BAs of ddI were 49.6 +/- 8.8, 42.7 +/- 7.9, and 13.6 +/- 5.4% after the bolus administration of unbuffered solutions containing 250 mg ddI into the duodenum, ileum, and colon of IVAP beagle dogs, respectively. The BA of the orally administered Videx 250 mg buffered chewable tablets was 44.9 +/- 1.6%. ddI absorption and disposition PK were modeled by simultaneously fitting intravenous, oral, and intestinal plasma level versus time data using a physiologically based PK model. The region-specific apparent absorption rates followed the rank order duodenum > ileum > colon. Apparent regional in vivo intestinal permeabilities correlated well with previously determined regional permeabilities in rats. The intestinal pH was monitored using a radiotelemetric pH monitoring system since ddI is unstable in an acidic environment. While the pH was found to be lower in the duodenum and proximal jejunum (approximately pH 6) than in the ileum or colon (pH > or = 7.0), ddI is reasonably stable across the entire pH range of the dog small intestine. These studies demonstrate that the regional reduction in ddI BA is consistent with a reported distal reduction in intestinal permeability and appears to be a significant contributing factor to the high degree of absorption variability reported for ddI.