Mechanisms and Modeling of the Intestinal Absorption, Activation, and Systemic Availability of Gemcitabine and a Gemcitabine Prodrug for Oral Administration

Abstract

Gemcitabine (2’-2’-difluoro-deoxycytidine) is an intravenously administered nucleoside analogue used in the treatment of various solid tumor cancers. While oral gemcitabine administration would offer a more patient-friendly, less complex, and less expensive alternative to intravenous administration, the clinical utility of oral gemcitabine administration is hindered by a low oral bioavailability of approximately 10%. This low oral bioavailability was previously believed to be the result of gemcitabine’s low intestinal permeability and oral absorption, followed by significant presystemic metabolism by the enzyme cytidine deaminase (CDA). In one study, we sought to define the mechanisms of gemcitabine intestinal permeability, the potential for saturation of intestinal uptake, and the transporter(s) responsible for mediating the oral absorption of drug using in situ single-pass intestinal perfusions in mice. Concentration-dependent studies were performed for gemcitabine over 0.5 to 2000 µM, along with studies of 5 µM gemcitabine in a sodium-containing buffer +/- thymidine (which can inhibit concentrative (i.e., CNT1 and CNT3) and equilibrative (i.e., ENT1 and ENT2) nucleoside transporters) or dilazep (which can inhibit ENT1 and ENT2), or in a sodium-free buffer (which can inhibit CNT1 and CNT3). Our findings demonstrated that gemcitabine was, in fact, a high-permeability drug in the intestine at low concentrations, that jejunal uptake of gemcitabine was saturable and mediated almost exclusively by nucleoside transporters, and that jejunal flux was mediated by both high-affinity, low-capacity (Km = 27.4 µM, Vmax = 3.6 pmol/cm2/s) and low-affinity, high-capacity (Km = 700 µM, Vmax = 35.9 pmol/cm2/s) transport systems. Thus, CNT(s) and ENT(s) at the apical membrane allow for gemcitabine uptake from the lumen to enterocyte, whereas ENT(s) at the basolateral membrane allow for gemcitabine efflux from the enterocyte to portal venous blood. These results further show that systemic exposure following oral gemcitabine administration is limited by extensive CDA-mediated presystemic metabolism and potentially by saturation of nucleoside transporter-mediated intestinal uptake following oral administration of large doses (i.e., doses which generate saturating gemcitabine concentrations in the intestinal lumen). A subsequent study evaluated the in vivo performance of a peptide transporter 1 (PEPT1)-targeted amino acid ester prodrug of gemcitabine, 5’-L-valyl-gemcitabine (V-Gem), developed to increase gemcitabine’s oral bioavailability. V-Gem was previously shown to be a substrate of the intestinally expressed PEPT1 and stable against CDA-mediated metabolism. However, earlier studies did not evaluate the in vivo oral performance of V-Gem as compared to parent drug. Therefore, we evaluated the pharmacokinetics and in vivo oral absorption of gemcitabine and V-Gem following intravenous and oral administrations in mice. These studies revealed that V-Gem undergoes rapid systemic elimination (half-life < 1 min) and has a low oral bioavailability (< 1%). Most importantly, the systemic exposure of gemcitabine was not different following oral administration of equimolar doses of gemcitabine (gemcitabine bioavailability of 18.3%) and V-Gem (gemcitabine bioavailability of 16.7%). Single-pass intestinal perfusions with portal blood sampling in mice revealed that V-Gem undergoes extensive activation (i.e., conversion to gemcitabine) in intestinal epithelial cells and that gemcitabine undergoes first-pass metabolism in intestinal epithelial cells. Thus, formulation of gemcitabine as the prodrug V-Gem does not increase systemic gemcitabine exposure following oral dosing, due, in part, to the metabolic instability of V-Gem in intestinal epithelial cells. These findings suggest that future development of gemcitabine prodrugs for oral administration should focus on prodrugs with high intestinal permeability, high presystemic stability, and complete in vivo conversion to gemcitabine.