RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2

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ABSTRACT

Hepatic arterial infusion (HAI) chemotherapy with gemcitabine (GEM) is expected to be more effective and safer method to treat hepatic metastasis of pancreatic cancer compared with intravenous administration, because it affords higher tumor exposure with lower systemic exposure. Thus, a key issue for dose selection is the saturability of hepatic uptake of GEM. Therefore, we investigated GEM uptake in rat and human isolated hepatocytes. Hepatic GEM uptake involved high- and low-affinity saturable components with Km values of micromolar and millimolar order, respectively. The uptake was inhibited concentration dependently by S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and was sodium-ion-independent, suggesting a contribution of equilibrative nucleoside transporters (ENTs). The concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low-affinity binding site. Therefore, the high- and low-affinity sites correspond to ENT1 and ENT2, respectively. Our results indicate hepatic extraction of GEM is predominantly mediated by the low-affinity site (hENT2), and at clinically relevant hepatic concentrations of GEM, hENT2-mediated uptake would not be completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over intravenous administration in terms of severe adverse events. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3162–3169, 2015

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INTRODUCTION

Pancreatic cancer is a major cause of cancer-related death globally, and has a 5-year survival rate of only about 5% for all stages combined.1 Surgical treatment offers the best possibility for cure, but pancreatic cancer is likely to become metastatic,1 especially to the liver (60%).2 Even patients who receive surgery experience a 50%–80% local recurrence rate and a 25%–50% risk of developing distant metastases,3 and the 5-year survival rate after recurrence is less than 30%.4., 5. Therefore,

Chemicals

[3H]GEM (16.3 Ci/mmol) and [14C]inulin (16 mCi/mmol) were purchased from Moravek Biochemicals, Inc. (Brea, California) and American Radiolabeled Chemicals, Inc. (St. Louis, Missouri), respectively. Nonlabeled GEM was purchased from Tokyo Chemical Industry Company, Ltd. (Tokyo, Japan). Collagenase Type I from Clostridium histolyticum was obtained from Sigma–Aldrich (St. Louis, Missouri). All other chemicals were commercial products of analytical or cell-culture grade. S

Time Course of [3H]GEM Uptake in Rat Hepatocytes

[3H]GEM uptake by rat hepatocytes increased time-dependently up to 30 min. Initial uptake (cell-to-medium ratio) was approximately linear up to 5 min and the uptake clearance at 5 min was 18 μ L/mg protein (Fig. 1). [3H]GEM uptake was markedly inhibited by 77% of initial uptake at 30 min in the presence of 10 mM nonlabeled GEM (Fig. 1). Therefore, the uptake time was fixed at 5 min for subsequent studies.

Kinetic Analysis of [3H]GEM Uptake in Rat and Human Hepatocytes

Initial uptake rate of GEM in rat hepatocytes followed Michaelis–Menten kinetics (Fig. 2a). The

DISCUSSION

We have confirmed the effectiveness and safety of GEM HAI in a previous phase I clinical study,18., 19., 20., 21. but nevertheless, dose selection remains a critical issue, because saturation of hepatic extraction in HAI would lead to increased systemic exposure, resulting in potentially severe toxicity. Therefore, in this study, we examined the saturability of hepatic extraction of GEM at clinically relevant concentrations in normal hepatocytes to provide a rational basis for dose selection

CONCLUSION

Our results indicate that hepatic extraction of GEM is predominantly mediated by hENT2, and at clinically relevant dose levels, hENT2-mediated uptake is not completely saturated. This is critical for HAI, because saturation of hepatic uptake would result in a marked increase of GEM concentration in the peripheral circulation, abrogating the advantage of HAI over i.v. administration in terms of the likelihood of severe adverse events. Thus, expression of ENT2 in hepatic tissue may be a key

ACKNOWLEDGMENT

This study was supported in part by JSPS KAKENHI grant number 23590173.

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