The intestinal permeability (Peff
) of ribavirin was unaffected by the absence of mEnt1 at any of the ribavirin perfusate concentrations examined (), indicating that mEnt1 does not mediate the absorption of ribavirin from the intestinal lumen in mice. In contrast, the large reduction of ribavirin transport in the absence of sodium as well as the saturability of the process with increasing ribavirin perfusate concentration indicate that the absorption of ribavirin from the intestinal lumen in mice is an active, sodium-dependent process, most likely mediated by the concentrative nucleoside transporters (Cnts) (). The data obtained after co-perfusion with 500 μM formycin B (Cnt2 inhibitor) or 250 μM thymidine (Cnt3 inhibitor) confirmed that this transport was dominated by mCnt2 (). The Ki
value of formycin B for hCNT2 is ~200 μM 19
. Assuming competitive inhibition, we would expect 64% inhibition of mCnt2 by formycin B at 500 μM - we obtained 61% inhibition. The dominant role of mCnt2 is supported by the similarity in the Km
of ribavirin loss from the intestinal lumen (44.5 ± 7.6 μM) with the in vitro
of ribavirin uptake into MDCK cells over-expressing mCnt2 (29.2 ± 2.9 μM).
Although others have suggested that Ent1 may contribute to the uptake of nucleosides across the enterocyte brush border membrane 25
, we found no such involvement of mEnt1 in the absorption of ribavirin into the enterocytes in the mouse (). This observation is consistent with our functional and expression data where we found minimal hENT1 activity/expression at the brush border membrane of the human enterocytes 12–13, 14
, but found high expression at the membranes of the crypt cells 26
and lesser expression at the basolateral membrane of the enterocytes. Our results demonstrate that mEnt1 does not play a role in the absorption of ribavirin into the enterocytes, therefore Ent1 expression in the enterocytes is either minimal or basolateral. If the same holds true in humans, this differential localization of hCNT2 on the brush border membrane of the enterocytes and hENT1 on the basolateral membrane of the enterocytes and the crypt cells suggest that these nucleoside transporters work in tandem to vectorially transport substrates from the lumen of the intestine into the mesenteric circulation. We tested this hypothesis by analyzing the ribavirin concentration in the intestinal tissue from wild-type and Ent1(−/−) mice after each of the ribavirin perfusions. Consistent with our hypothesis, after the 20 μM ribavirin perfusion, there was significantly greater accumulation (8.0-fold) of ribavirin in the intestinal tissue of Ent1(−/−) mice than in wild-type mice (). That is, when mEnt1 was absent in the intestine, ribavirin’s vectorial transport into the mesenteric circulation was compromised.
Surprisingly, the difference in intestinal accumulation of ribavirin between wild-type and Ent1(−/−) after 20 μM perfusions disappeared after the 200 and 5000 μM ribavirin perfusions. After the 200 μM perfusions, ribavirin’s accumulation in the wild-type and Ent1(−/−) mice intestine increased more than proportionally compared to that observed after the 20 μM perfusion (; note that the values plotted are dose-normalized to 20 μM perfusions). This increase is surprising because if mEnt1 transport was the rate-limiting step in the egress of ribavirin from the intestinal tissue, the dose-normalized intestinal tissue ribavirin concentration in the Ent1(−/−) mice should not have increased with increasing ribavirin perfusate concentration. In addition, in the wild-type mice, when the intestinal ribavirin perfusate concentration was increased, the intestinal ribavirin tissue concentration should have increased until ribavirin’s mEnt1-mediated egress from the intestine was saturated. Consequently, the dose-normalized accumulation of ribavirin in the intestinal tissue of wild-type mice, at the 200 and 5000 μM intestinal perfusate concentrations, should not exceed that in the Ent1(−/−) mice at 20 μM. The fact that it did suggests the presence of additional processes involved in the elimination of ribavirin from the intestinal tissue, which are saturated as the perfusate ribavirin concentration was increased from 20 to 200 μM and 5000 μM. This conclusion is supported upon examination of the total radioactivity accumulated in the intestinal tissue (). In both Ent1(−/−) and wild-type mice, the ratio of total intestinal radioactivity to radioactivity attributable to ribavirin (plus phosphorylated metabolites) decreased as the perfusate concentration increased (). This indicates that non-phosphorylation metabolism of ribavirin (e.g. deribosylation) was most likely saturated. However, simultaneous saturation of transporters other than mEnt1 (e.g. nucleobase transporters or perhaps MRP727
) cannot be discounted. At the higher perfusate ribavirin concentrations (200 and 5000 μM), the intestinal tissue concentrations of ribavirin increased in proportion to the dose (i.e. they are not significantly different when dose normalized). These data suggest that during 200 and 5000 μM perfusions, although the ribavirin concentrations were above the Km
for mCnt2 and most likely saturating, the remaining concentrative transport plus diffusion into the enterocytes was of such magnitude that all processes responsible for egress (including mEnt1) or metabolism (other than phosphorylation) of ribavirin were saturated and ribavirin egress from the intestine became a diffusion-limited process. At first sight, saturation of mEnt1 does not appear to be consistent with the concentrations of ribavirin observed in the intestinal tissue (~10 – 250 μM), which is lower than the Km
of ribavirin transport by mEnt1 (~321 μM) 17
. However, it is important to note that the measured ribavirin concentration was that in the entire intestine rather than only where mEnt1 is expressed. Therefore, the ribavirin concentrations within the regions expressing mEnt1 may have been much greater than the Km
of ribavirin transport by mEnt1.
Consistent with the egress of ribavirin from the intestine being rate-limited by mEnt1 after the 20 μM ribavirin perfusions, the dose-normalized ribavirin plasma concentrations (at 33 minutes) in wild-type mice were 3-fold greater than in the Ent1(−/−) mice (). At first sight, the greater than proportional increase in absolute plasma ribavirin concentrations in wild-type and Ent1(−/−) mice after the 200 and 5000 μM perfusions compared to the 20 μM perfusions appears contradictory to our conclusion that egress from the intestine is rate-limiting at the 20 μM perfusate concentrations. However, these data can potentially be explained by saturable first-pass hepatic extraction during the 200 and 5000 μM ribavirin perfusions, which we have demonstrated28
In summary, we have presented an in situ
perfusion study in wild-type and Ent1(−/−) mice which allowed us to elucidate the contribution of the nucleoside transporters to the intestinal absorption of ribavirin. The perfusion data presented here show that ribavirin’s bioavailability after oral administration to mice is rate-limited by several processes represented in . First, the absorption of ribavirin into the enterocytes from the intestine lumen is a saturable, sodium-dependent process, largely mediated by mCnt2. However, once in the enterocytes, the vectorial transport of ribavirin into the mesenteric blood is mediated by mEnt1 plus other unknown processes (metabolism and transport) which are also saturable. These observations in mice are largely consistent with observations made (where possible) in humans and suggest that a more optimal dosing strategy for ribavirin should be considered which takes these complex intestinal transport processes into consideration. Since the liver is the target of ribavirin therapy it is important to determine the in vivo
role of nucleoside transporters in the hepatic distribution of ribavirin. In vitro
studies in our laboratory with human hepatocytes suggest that hENT1 plays a dominant role in the hepatic distribution of ribavirin 26
. However, studies need to be conducted in vivo,
in both Ent1(−/−) mice as well as in humans, to gain a better understanding of factors that govern the in vivo
bioavailability (intestinal absorption and hepatic bioavailability) of ribavirin. Such studies could help guide development of therapeutic strategies that maximize the efficacy and reduce the toxicity of ribavirin in patients with hepatitis C virus infection.
Figure 4 A graphical illustration describing the proposed mechanisms of intestinal absorption of 20 or 200 and 5000 μM ribavirin in mice. The absorption of ribavirin into the enterocytes from the intestinal lumen is a saturable, sodium-dependent process, (more ...)