Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2

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.

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells

“…The K m values of hENT1 and hENT2 were similar in MIA‐PaCa2 and As‐PC1 cells. These results are similar to our previous results for normal hepatocytes (Shimada et al, ).…”

“…We have already reported that the hepatic extraction of GEM is mediated by hENT1 and hENT2, in addition to a non‐saturable component, in normal human hepatocytes, and we showed that at clinically relevant dose levels in GEM‐HAI therapy, hENT2‐mediated uptake would not be completely saturated, although hENT1 was saturated (Shimada et al, ). However, GEM uptake kinetics and the roles of transporters in metastatic tumors have not been clarified.…”

“…GEM is transported by equilibrative nucleoside transporters (ENTs; SLC29 family) and concentrative nucleoside transporters (CNTs; SLC28 family) (Mackey et al, , ; Ritzel et al, 2001). Also, we previously reported that the hepatic extraction of GEM was mediated by human ENT1 (hENT1; SLC29A1) and hENT2 (SLC29A2), in addition to a non‐saturable process, and we concluded that at a clinically relevant dose of GEM‐HAI, hENT2‐mediated uptake would not be completely saturated (Shimada et al, ). In addition, we have reported that the administration of 400 mg GEM/body for 30 min by HAI did not lead to severe adverse events in patients with postoperative liver metastases from pancreatic cancer (Tajima et al, ), and we confirmed the safety of GEM‐HAI therapy (Tajima et al, , , ).…”

“…The uptake study was performed as described previously, with some modifications (Shimada et al, ). Briefly, a mixture of aliquots of cell suspension (110 μl) and incubation medium containing 59 nM [ 3 H]uridine or 62 nM [ 3 H]GEM (37 kBq/ml) in HBRS buffer (final concentration) was incubated at 37°C for designated times with or without non‐labeled GEM; then, the cells were collected by centrifugation for 1 min at 10,000× g and transferred to 3 M KOH.…”

Application of Quantitative Targeted Absolute Proteomics to Profile Protein Expression Changes of Hepatic Transporters and Metabolizing Enzymes During Cholic Acid-Promoted Liver Regeneration

“…In addition, human ENT1 mediates portal blood-to-hepatic extraction of gemcitabine, which is used for the treatment of hepatic metastasis of pancreatic cancer. 40 A knockout study in mice has shown that ent1 is responsible for hepatic uptake of ribavilin, which is used for the treatment of hepatitis C virus infection. 41 Iikura et al 42 have also reported that ENT1 is a possible determinant of the antiviral effectiveness of ribavirin by using OR6 cells as a hepatitis C virus replication system.…”