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SHORT COMMUNICATIONS: Chi-Yuan Wu and Leslie Z. Benet DISPOSITION OF TACROLIMUS IN ISOLATED PERFUSED RAT LIVER: INFLUENCE OF TROLEANDOMYCIN, CYCLOSPORINE, AND GG918 Drug Metab Dispos 2003; 31: 1292-1295 [Abstract] [Full text] [PDF]

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How does P-gp inhibition increase clearance?

Nick Holford   (18 December 2003)

Authors' Response to Comments by Nick Holford

Leslie Z. Benet, Carolyn L. Cummins, Yvonne Y. Lau, Justine E. Lu, and Chi-Yuan Wu   (18 December 2003)

How does P-gp inhibition increase clearance? 18 December 2003
Nick Holford, Associate Professor University of Auckland Send letter to journal: Re: How does P-gp inhibition increase clearance? n.holford{at}auckland.ac.nz Nick Holford	In a recent Short Communication Wu and Benet (1) assert "These results support our hypothesis that the hepatic metabolic clearance of a dual substrate will be increased by inhibiting the efflux transporter". However, I believe their hypothesis about clearance is unreasonable based simply on a mechanism involving P-GP efflux inhibition. They report that the area under the curve of tacrolimus (AUCt) decreased by 25% after a bolus injection to an isolated rat liver when pre -treated with GG918 (a P-gp inhibitor assumed not to affect CYP3A metabolism). Assuming first-order elimination processes this indicates that the total clearance of tacrolimus is increased by 30%. If we assume that P-gp is an elimination clearance mechanism in a well stirred system (as they illustrate in reference (2), Figure 1) for tacrolimus then inhibiting this clearance pathway with GG918 should decrease the total clearance of tacrolimus but exactly the opposite was observed (AUCt decreased with GG918). The authors state "Since P-gp is located on the canalicular membrane of hepatocytes, drug molecules confront CYP3A before P-gp efflux. ... We hypothesize that if P-gp is decreased … the hepatic metabolic clearance of the substrates will be enhanced due to their increased availability to CYP3A." It is reasonable to suppose that hepatocyte concentrations would increase if efflux is inhibited and if this occurred then a greater fraction of tacrolimus might be metabolized by CYP3A but no effect on clearance by CYP3A should occur. This is analogous to a drug being eliminated by renal and non-renal mechanisms. A decrease in renal clearance will increase drug concentration and increase elimination by the non-renal pathway but the non-renal clearance is not affected. Some other results reported by Wu and Benet indicate that the action of GG918 is more complex. They report that the AUC of a metabolite (AUCm) of tacrolimus decreased about 50% with GG918. AUCm reflects the amount of tacrolimus converted to metabolite (metabolite "dose") divided by the clearance of metabolite. By calculating the ratio of AUCm/AUCt the tacrolimus concentration driving the formation of metabolite is normalized. This is equivalent to giving the same dose of tacrolimus if tacrolimus total clearance had remained unchanged. The ratio of AUCm/AUCt decreased by 33% with GG918. A change in this ratio shows how AUCm changes independent of any changes in total tacrolimus clearance. The change could be due to either to a decrease in the formation fraction of tacrolimus eliminated as metabolite leading to a lower metabolite "dose" or to an increase in the clearance of metabolite. Given that the total clearance of tacrolimus was increased with GG918 and inhibiting P-gp efflux, if anything, would increase the formation fraction of the metabolite then it seems plausible that GG918 also increases the clearance of metabolite. It should also be noted that biliary excretion of tacrolimus was not decreased despite a decrease in AUCt with GG918 which argues against P-gp efflux being an important determinant of biliary clearance and even suggests a paradoxical enhancement of excretion by this route with GG918. This same group has made similar assertions about changes in P-gp efflux causing increased CYP3A clearance in women (2). I believe that their simple theory is inadequate and some other mechanism has to be sought to explain the association of decreases in P-gp transporter activity and increases in drug clearance. 1. Wu CY, Benet LZ. Disposition of tacrolimus in isolated perfused rat liver: influence of troleandomycin, cyclosporine, and gg918. Drug Metab Dispos 2003;31(11):1292-5. 2. Cummins CL, Wu CY, Benet LZ. Sex-related differences in the clearance of cytochrome P450 3A4 substrates may be caused by P- glycoprotein. Clin Pharmacol Ther 2002;72(5):474-89.
Authors' Response to Comments by Nick Holford 18 December 2003
Leslie Z. Benet, Professor, Department of Biopharmaceutical Sciences University of California San Francisco, Carolyn L. Cummins, Yvonne Y. Lau, Justine E. Lu, and Chi-Yuan Wu Send letter to journal: Re: Authors' Response to Comments by Nick Holford benet{at}itsa.ucsf.edu Leslie Z. Benet, et al.	We are grateful to Dr. Holford for bringing the apparently contradictory results and interpretation of our recently published isolated perfused rat liver (IPRL) studies (Wu and Benet, 2003) to the attention of the Journal readers. We had previously shown using a cellular system containing both CYP3A4 and P-glycoprotein (P-gp) (Cummins et al., 2002a) and in situ using rat intestinal perfusions (Cummins et al., 2003) that increased extraction ratio and clearance occurred due to P -gp cycling of substrates back into the gut lumen. Suzuki and Sugiyama (2000) and Tam et al. (2003) have noted that present pharmacokinetic theory is not consistent with these results, or at least our interpretation. Similarly, at recent scientific meetings, Professors Yuichi Sugiyama, Sandy Pang and Win Chiou have raised issues similar to those of Dr. Holford when we presented our IPRL results, noting that our interpretation of increased extraction ratio and clearance resulting from inhibition of liver efflux transporters is not possible in terms of current pharmacokinetic theory. We agree that our results are not consistent with present pharmacokinetic theory. However, we continue to publish studies containing these contradictory results. In a second confirmatory IPRL study, Lau et al. (2004, in press but available on the JPET web site) demonstrated that the addition of the P-gp inhibitor quinidine to digoxin perfusions led to decreased areas under the curve (AUCs) for the parent drug and increased AUCs for the primary metabolite, digoxigenin bisdigitoxiside (Dg2). In addition, Lau et al. (2004) showed that adding the oatp2 inhibitor rifampicin to the digoxin IPRL study significantly increased digoxin AUC and decreased Dg2 AUC. We also noted (Cummins et al., 2002b) in P-gp knockout mice that although total clearance of erythromycin, paclitaxel, doxorubicin and vinblastine was lower than in control wild type animals, hepatic extraction (clearance) increased for each of these drugs in knockout animals versus wild type controls. However, as Dr. Holford would point out, each of these results could potentially be explained by suggesting that the increased hepatic elimination fraction (not extraction ratio or clearance) occurs because decreasing one route of elimination allows more drug to be eliminated by the alternative routes (e.g., his renal/non-renal analogy). We counter this argument with our most recent study submitted to this Journal (Lu and Benet, 2004). Here we show that primary rat hepatocytes will more accurately predict in vivo digoxin intrinsic metabolic clearance versus studies in rat hepatic microsomes. Furthermore, we demonstrate that in hepatic microsomes no metabolic interactions occur between digoxin and either rifampin or GG918. However, in hepatocytes, we demonstrate that GG918 will significantly decrease parent drug concentrations while increasing Dg2 levels versus controls; in contrast, addition of rifampin increases digoxin concentrations and significantly decreases Dg2 formation. These results cannot be explained by the alternate elimination route supposition, since no alternate route exists. Similarly, the ratios of metabolite to parent compound in these more recent studies (Lau et al., 2004; Lu and Benet, 2004) are consistent with the changes in clearance that we hypothesize. All of the above studies suggest that the interplay of transporters and enzymes must be considered in defining the intrinsic metabolic clearance of the liver (and other organs such as the intestine and the kidney) and when evaluating potential drug-drug interactions. We believe that the discrepancies between our results and pharmacokinetic theory are due to deficiencies in the current definition of intrinsic clearance. We will describe in a soon to be submitted manuscript the modifications that must be made to accommodate the experimental results showing increased hepatic clearance when hepatic efflux transporters are inhibited. However, we believe that it was critical to first publish the experimental results (Cummins et al., 2002b; Wu and Benet, 2003; Lau et al., 2004; Lu and Benet, 2004) that exposed the deficiencies of the present theory. We thank Dr. Holford for bringing attention to this issue upon the publication of the first of our liver studies (Wu and Benet, 2003). Leslie Z. Benet, Carolyn L. Cummins, Yvonne Y. Lau, Justine E. Lu, and Chi-Yuan Wu Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0466 References Cummins CL, Jacobsen W and Benet LZ (2002a) Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4. J Pharmacol Exp Ther 300: 1036-1045 Cummins CL, Wu CY and Benet LZ (2002b) Sex-related differences in the clearance of cytochrome P450 3A4 substrates may be caused by P- glycoprotein. Clin Pharmacol Ther 72: 474-489 Cummins CL, Salphati L, Reid MJ and Benet LZ (2003) In vivo modulation of intestinal CYP3A metabolism by p-glycoprotein: studies using the rat single-pass intestinal perfusion model. J Pharmacol Exp Ther 305: 306-314 Lau YY, Wu CY, Okochi H and Benet LZ (2004) Ex situ inhibition of hepatic uptake and efflux significantly changes metabolism: hepatic enzyme -transporter interplay. J Pharmacol Exp Ther, in press Lu JE and Benet LZ (2004) Hepatic microsome studies are insufficient to characterize in vivo hepatic metabolic clearance and metabolic drug- drug interactions: studies of digoxin metabolism in primary rat hepatocytes vs. microsomes. Drug Metab Dispos, submitted Suzuki H and Sugiyama Y (2000) Role of metabolic enzymes and efflux transporters in the absorption of drugs from the small intestine. Eur J Pharm Sci 12: 3-11 Tam D, Sun H and Pang KS (2003) Influence of P-glycoprotein, transfer clearances, and drug binding on intestinal metabolism in Caco-2 monolayers or membrane preparations: A theoretical analysis. Drug Metab Dispos 31: 1214-1226 Wu CY and Benet LZ (2003) Disposition of tacrolimus in isolated perfused rat liver: influence of troleandomycin, cyclosporine, and GG918. Drug Metab Dispos 31: 1292-1295