Suitability of Digoxin as a P Glycoprotein Probe: Implications of Other Transporters on Sensitivity and Specificity

Transcription

1 Review Suitability of Digoxin as a P Glycoprotein Probe: Implications of Other Transporters on Sensitivity and Specificity The Journal of Clinical Pharmacology XX(XX) , The American College of Clinical Pharmacology DOI: /jcph.200 Ahmed M. Nader, PhD 1 and David R. Foster, PharmD, FCCP 2,3 Abstract The study of transporter mediated drug drug interactions (DDI) requires use of appropriate probes to reflect transporter function. Digoxin is often used as a probe in DDI studies involving P glycoprotein (P gp) and is recommended by FDA for this purpose, despite several lingering questions regarding suitability of digoxin as P gp probe. This review aims to critically evaluate use of digoxin as a probe for P gp mediated clinical DDI studies, with focus on sensitivity and specificity of digoxin for P gp. Although previous reviews have evaluated digoxin transport by P gp, the purpose of the current review is to critically evaluate such literature in light of newly evolving literature suggesting digoxin transport by non P gp transporters. Keywords digoxin, P glycoprotein, drug drug interaction, transporter, OATP The study of transporter mediated drug drug interactions (DDI) requires use of appropriate probes to reflect transporter function. Digoxin is often used as a probe in DDI studies involving P glycoprotein (P gp) and is recommended by FDA for this purpose, despite several lingering questions regarding suitability of digoxin as P gp probe. This review aims to critically evaluate use of digoxin as a probe for P gp mediated clinical DDI studies, with focus on sensitivity and specificity of digoxin for P gp. Although previous reviews have evaluated digoxin transport by P gp, the purpose of the current review is to critically evaluate such literature in light of newly evolving literature suggesting digoxin transport by non P gp transporters. Role of P Glycoprotein in Drug Disposition and Drug Drug Interactions P glycoprotein is a member of the ATP binding cassette family of transporters that plays a protective role through facilitating renal and biliary drug excretion as well as limiting intestinal drug absorption or penetration into brain or placental tissues. 1 Examples of P gp substrates include vincristine, paclitaxel, ritonavir, talinolol, loperamide, cyclosporine, fexofenadine, digoxin, and verapamil. 2 There is a wide overlap between P gp and Cytochrome P450 (CYP) 3A substrates 3 which adds to the complexity of studies on P gp substrates or inhibitors. Numerous examples of P gp mediated DDI exist, many of which are clinically important. Examples include drug interactions between loperamide and quinidine and between digoxin and verapamil, clarithromycin, cyclosporine, ritonavir, or grapefruit juice. 4,5 These, and other similar examples, have led to the recommendation by FDA, in 2006, for all drugs under development to be tested in vitro and (possibly) in vivo for their effects (inhibition and induction) on P gp function. 6 P glycoprotein mediated drug transport may be influenced by genetic polymorphisms in the MDR1 gene coding for P gp. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene have been identified as being associated with changes in P gp function. The most common of these are the C3435T and G2677T SNPs. A meta analysis of the effects of MDR1 C3435T polymorphism on digoxin pharmacokinetics concluded that the SNP has no effect on digoxin area under the curve (AUC) 1 Pharmaceutical Sciences Section, College of Pharmacy, Qatar University, Doha, Qatar 2 Department of Pharmacy Practice, College of Pharmacy, Purdue University, Indianapolis and West Lafayette, IN, USA 3 Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN, USA Submitted for publication 28 July 2013; accepted 30 September Corresponding Author: David R. Foster, PharmD, FCCP, Department of Pharmacy Practice, College of Pharmacy, Purdue University, FTB Faculty Office Building, 640 Eskenazi Avenue, Indianapolis, IN drfoster@purdue.edu Author contributions: Ahmed M. Nader conducted search, analyzed data, and wrote manuscript. David R. Foster Analyzed data and wrote manuscript.

2 2 The Journal of Clinical Pharmacology / Vol XX No XX (2013) and is not associated with MDR1 mrna expression. 7 In contrast, Japanese individuals with both SNPs were found to have higher digoxin bioavailability. 8 A recent study conducted in Xenopus laevis oocytes showed that digoxin efflux was similar between oocytes expressing wild type P gp and those expressing triple variant (1236 C>T, 2677 G>T, and 3435 C>T) P gp. 9 These results indicate that the studied SNPs may have no functional role in digoxin efflux by P gp. Although the effects of such SNPs on P gp transport function are generally controversial, most studies support a decrease in P gp function with these mutations. 10 Possible presence of other unknown mutations in linkage disequilibrium with the known MDR1 SNPs may make it harder to consistently link changes in digoxin or other P gp substrate pharmacokinetics to specific MDR1 SNPs. Characterization of drug effects on P gp function requires the use of a suitable probe that reflects clinically important changes in P gp transport function. Several criteria have been suggested for evaluation of P gp substrates for use as in vivo probes. 11,12 These criteria include specificity (no or minimal involvement of other transporters or metabolizing enzymes in the probe disposition), sensitivity (in vivo P gp activity correlates with P gp content and with activity of other validated P gp probes, net flux ratio of Basal Apical to Apical Basal transport is >2), feasibility (available, inexpensive, and safe for use in humans), and a permeability rate limited absorption (in case of evaluation of intestinal effects). 11,12 Fullfillment of such criteria is of critical importance in order to ensure that conclusions obtained from such DDI studies can be mainly attributed to modulation of P gp activity. In addition to digoxin, several drugs have been suggested for use as in vivo P gp probes, including fexofenadine, talinolol, and quinidine. 11 With talinolol and fexofendine being substrates for organic anion transporting polypeptide (OATP) and multi drug associated resistance protein (MRP2) transporters and quinidine being metabolized by CYP3A, none of these drugs has presented itself as an appropriate in vivo P gp probe. 11,51 Such involvement of enzymes and non P gp transporters in the disposition of those drugs highlights the need for further evaluation of such involvement in digoxin disposition. Digoxin as a Probe for P Glycoprotein Function Digoxin is classified as a Biopharmaceutics Classification System (BCS) class II drug with low solubility and high intestinal permeability. 13 Digoxin is rapidly absorbed after oral administration with an average oral bioavailability of 70% for tablets and 80 90% for capsules. 14 Elimination occurs primarily through renal excretion of the unchanged drug (approx. 75% of the dose) by both glomerular filtration and active tubular secretion. 15 The remaining digoxin dose that is not renally eliminated is subject to metabolism by gut flora, hepatic metabolism, as well as biliary excretion. 1 Digoxin is an established substrate for P gp transport in vitro. 16,17 P gp has also been shown to play a role in the in vivo absorption of digoxin in addition to influencing renal and biliary excretion Such involvement of P gp in digoxin disposition and the minimal involvement of metabolizing enzymes in digoxin elimination has lead to the widespread use of digoxin as a probe for P gp function. Digoxin has the advantages of minimal metabolism, availability as both oral and intravenous dosage forms, low cost, and relative safety at moderate doses (0.5 1 mg). However, the suitability of digoxin as a P gp probe has been questioned due to possible lack of specifcity or reduced sensitivity to P gp activity. In 2006, the U.S. FDA provided a guidance for recommended P gp probes to be used for in vitro and in vivo studies evaluating the effects of investigational drugs on P gp function (Table 1). 6,21 Choice of probe substrates is based on their directional permeabilities in cell systems expressing P gp, where higher apparent permeabilities (P app ) in the basal to apical (B A) direction indicates efflux by P gp in cell systems. The guidance identifies digoxin as one of the most suitable substrates for use for in vitro and in vivo studies involving P gp (Table 1). Since Table 1. P Glycoprotein Substrates Deemed Acceptable by the U.S. Food and Drug Administration Drug Conc. used in vitro (mm) Caco 2 P app, B A /P app, A B a In vitro cell line MDR1 MDCK b MDR1 LLCPK b Digoxin Loperamide Quinidine Vinblastine c >9 d 3 Talinolol Adapted from: DevelopmentResources/DrugInteractionsLabeling/ucm htm#pgptransport. a P app,b A /P app,a B ;P app ¼ apparent permeability, A B ¼ Apical to Basal, B A ¼ Basal to Apical. b Data for MDR1 MDCK and MDR1 LLCPK are the ratio observed in transfected cells relative to the ratio observed in respective wild type cells. c Vinblastine is also a substrate for MRP2 that is constitutively expressed in Caco 2, and wild type MDCK and LL CPK1 cells. d Data are derived from net B to A flux in the absence of GF120918, a potent P gp inhibitor, relative to that observed in the presence of GF MDR1 MDCK: Multidrug resistance gene transfected Madin Darby Canine Kidney cells. MDR1 LLCPK: Multidrug resistance gene transfected renal epithelial cell lines from porcine kidney.

3 Nader and Foster 3 the release of this guidance, many other transporters have been shown to play important roles in disposition of several drugs including digoxin. A newer draft guidance released in February 2012 did not include any updates on the use of digoxin as a P gp probe. 6 Consequently, we believe that it is necessary to re evaluate the use of digoxin as a P gp probe in light of the emerging evidence of role of other transporters in its disposition. Suitability of Digoxin as an In Vivo P gp Probe Suitability of digoxin for use as a probe for P gp function requires sufficient sensitivity and specificity to P gp. Studies relating digoxin disposition or transport to P gp function help establish its sensitivity to P gp activity, whereas specificity can be evaluated in presence of in vitro, animal, or clinical studies that suggest roles for other transporters in digoxin disposition or transport. An analysis of such studies is provided below. Evidence Supporting the Use of Digoxin as a P gp Probe Evidence to support the use of digoxin as a P gp probe includes in vitro, animal, and clinical studies that evaluate the effects of changing P gp function on digoxin transport or disposition. This approach helps build an association between digoxin transport or pharmacokinetics and P gp function which in turn would support its use as a P gp probe. Table 2 lists studies (in vitro, animal, and clinical) that suggest a clear association between digoxin transport and/or disposition and P gp function. In Vitro Evidence of Digoxin Transport by P gp. In vitro investigation of P gp mediated digoxin transport generally either involves the use of MDR1 transfected cells or cells normally expressing P gp (e.g., Caco 2 cells). The former may yield more definitive results since P gp expression is ensured through the transfection process. Digoxin transport is then studied in wild type and transfected cells or in the presence and absence of known P gp inhibitors. Comparison of digoxin transport in wild type and transfected cells can highlight the role of P gp in substrate digoxin movement; however, it still does not evaluate or exclude the involvement of other transporters normally expressed by those cells. In other words, such studies may indicate the sensitivity of digoxin to P gp function but not its specificity to P gp transport. In addition, selective inhibition of P gp is not necessarily guaranteed. Possible inhibition of other unknown transporters frequently cannot be excluded and hence full attribution of the results to P gp is not always correct. Animal Studies Relating Digoxin Pharmacokinetics to P gp Function. Animal studies that evaluate P gp involvement in digoxin disposition often involve the use of mdr1 knockout mice models. Such studies involving gene knockout can be useful for testing digoxin sensitivity to P gp function and potentially providing data regarding the magnitude of a given effect on digoxin PK. The degree of inhibition of P gp shown in animal studies is often lower than what would be expected based on in vitro studies (four to eightfold change). For example, only 1.4 to 2 fold increases in digoxin plasma and tissue concentrations were observed in mice after coadministration with quinidine as compared to digoxin administration alone. 23 Similar to in vitro studies, animal studies cannot be used to discern the specificity of digoxin to P gp transport as multiple transporters are present in vivo. For example, in the studies conducted by Fromm et al. and Mayer et al. (Table 2), quinidine and valspodar increased digoxin concentrations in madr1a and mdr1a/b knockout mice, respectively. 23,24 This is unexpected given the minimal contribution of metabolizing enzymes to digoxin elimination and suggests that other transporters may be involved in digoxin disposition and are also affected by coadministration of quinidine and valspodar. Ultimately, the involvement of other non P gp transporters in digoxin disposition cannot be excluded from in vitro or animal studies unless specific inhibitors of such transporters are used or genes coding for those transporters are knocked out. Clinical Trials Relating Changes in P gp Function to Digoxin Pharmacokinetics. Clinical trials to evaluate the role of P gp in digoxin disposition usually involve the oral and/or intravenous (IV) administration of the drug and study of its pharmacokinetics. Effects of P gp on digoxin disposition can be illustrated through the coadministration of a P gp inhibitor or inducer and comparing digoxin pharmacokinetics in presence and absence of the interacting drug. Alternatively, digoxin pharmacokinetics can be compared between individuals carrying different P gp allelic variants. Results from human trials are generally regarded as the most informative since this approach simulates actual drug administration in patients. However, neither of the clinical approaches identified above are free from limitations. The use of inhibitors requires the choice of a selective inhibitor that only inhibits P gp and is safe for use in humans. Such selectivity cannot be confirmed for most of the currently available P gp inhibitors. For example, a commonly used P gp inhibitor, quinidine, may not be a selective inhibitor for only P gp (as described above, quinidine affects digoxin PK in mdr1a knockout mice). 23 Conversely, studying digoxin pharmacokinetics in different P gp genotypes is only informative if the effects of each of those genotypes on P gp function are clearly known and lead to quantifiable changes in P gp function and digoxin disposition. However, these assumptions are largely unfounded, particularly for the most studied SNP in the MDR1 gene, the C3435T SNP, where results have indicated increased, decreased, and similar P gp activity between different genotypes. 7

4 4 The Journal of Clinical Pharmacology / Vol XX No XX (2013) Table 2. Studies Supporting the Use of Digoxin as a P Glycoprotein Probe Type of study Design/system Findings Comments Refs. In vitro, inhibition MDR1 transfected LLC PK1 cells B A transport eightfold higher than A B Transport inhibited by quinidine and verapamil In vitro, inhibition Caco 2 cells B A transport higher than A B Transport inhibited by vinblastine and verapamil In vitro, inhibition MDR1 transfected MDCKII cells B A transport fivefold higher than A B Ratio decreased to twofold with verapamil Animal MDR1 knockout mice Digoxin brain concentrations 35 fold higher in knockout mice compared to wild type mice Animal MDR1 knockout mice 11% of IV digoxin dose excreted in intestinal lumen in wild type mice as compared to 2.5% in knockout mice Animal MDR1 knockout mice AUC 0 24 increased threefold and renal and biliary clearances decreased 70% in knockout mice Animal, inhibition Wild type mice Coadministration of quinidine led to increased plasma, liver, kidney, and brain concentrations by 1.4 to 2 fold Clinical, inhibition Digoxin verapamil DDI study in atrial Verapamil resulted in fibrillation decrease in digoxin biliary clearance, increase in steady state plasma concentrations, and no change in digoxin renal clearance Clinical Clinical, inhibition Digoxin PK and intestinal MDR1 expression in healthy volunteers Digoxin PK with quinidine using multilumen perfusion catheters Changes in digoxin AUC correlated with intestinal P gp expression Quinidine increased digoxin absorption and increased AUC and C max by fourfold Clinical, induction Digoxin rifampin DDI study Rifampin decreased digoxin AUC and bioavailability Clinical, inhibition Digoxin ritonavir DDI study Ritonavir increased digoxin AUC and decreased digoxin renal and nonrenal clearance Ensured P gp involvement due to use of transfected cells Does not indicate specificity for P gp since only MDR1 transfection was used Use of inhibitors indicates involvement of P gp Involvement of other transporters expressed by Caco 2 cells not studied Ensured P gp involvement due to use of transfected cells Use of inhibitor suggests involvement of P gp Specific to P gp due to use of selectively knockout mice Specific to P gp due to use of knockout mice Indicates P gp involvement in limiting digoxin absorption Specific to P gp due to use of knockout mice Indicates P gp involvement in digoxin renal and biliary excretion Effects of P gp inhibitors have a smaller in vivo magnitude than would be expected from in vitro studies Role of P gp in digoxin PK were not known at the time the study was conducted Unexpected results on renal clearance Illustrates the involvement of P gp in digoxin absorption Results cannot be solely attributed to P gp Illustrates the involvement of P gp in digoxin Quinidine may not be a selective inhibitor of P gp Induction of intestinal P gp reduces digoxin absorption Suggests involvement of P gp in digoxin renal and biliary excretion Tanigawara et al. 16 Cavet et al. 17 Taub et al. 22 Schinkel et al. 25 Mayer et al. 18 Kawahara et al. 20 Fromm et al. 23 Hedman et al. 29 Hoffmeyer et al. 19 Igel et al. 28 Greiner et al. 27 Ding et al. 30 (Continued)

5 Nader and Foster 5 Table 2. Continued Type of study Design/system Findings Comments Refs. Clinical, inhibition Digoxin PK with clarithromycin and MDR1 genotype in healthy volunteers Clarithromycin increased digoxin bioavailability G2677T and C3435T SNPs were associated with increased digoxin bioavailability Clinical, induction Digoxin rifampin DDI study Rifampin increased nonrenal clearance and decreased renal excretion of digoxin Clarithromycin did not affect digoxin total or renal clearance Unexpected results if P gp is involved in renal and biliary excretion Unexpected effects of rifampin on renal excretion Might suggest involvement of other unknown transporters Kurata et al. 8 Drescher et al. 31 MDR1, multi drug resistance gene; LLC PK1, renal epithelial cells from porcine kidney; B A, basal to apical; MDCK, Madine Darby Canine kidney; AUC, area under the curve; PK, pharmacokinetics; SNP, single nucleotide polymorphism; DDI, drug drug interaction. Sensitivity of Digoxin to Changes in P gp Function Most DDI studies between digoxin and P gp inducers/ inhibitors show only modest effects on digoxin pharmacokinetics (20 30% changes in digoxin AUC or C max ). 26,27 In part, this may be a result of the high absolute bioavailability (70 80%) of digoxin from oral capsules. This means that even if P gp is the only transporter involved in digoxin absorption and if we assume that it is completely inhibited by an inhibitor, the maximum change in digoxin bioavailability would only be 1.25 to 1.4 fold. The relatively low sensitivity of digoxin to P gp function in vivo was observed in a study of the effects of MDR1 C3435T polymorphism on digoxin plasma concentrations. Individuals homozygous for the T allele had more than twofold lower intestinal MDR1 expression; however, the increase in digoxin C max observed in those individuals was only 30 40% as compared to individuals homozygous for the C allele (Table 2). 19 The relatively small changes in digoxin pharmacokinetics with P gp inhibitors suggest that digoxin absorption may not be highly dependent on P gp in vivo; other processes such as passive diffusion or uptake by non P gp transporters may play a more important role in digoxin absorption. Digoxin is s a BCS class II drug with low solubility and high permeability which suggests that it is either highly passively diffused or is dependent on uptake transporters to cross the intestinal wall. Sensitivity of digoxin to P gp function is also dependent on the site of interaction. Interactions between digoxin and P gp inhibitors in the kidneys are often of lower magnitude in vivo than would be expected from in vitro results of P gp inhibition. This may be explained by the fact that, for a significant alteration of P gp in renal tubules to occur, the inhibitor concentration in renal tubules should be much higher than the IC 50 for P gp inhibition. This of course is dependent on the dose of the inhibitor used, route of administration, whether single or multiple doses are given, and whether or not the inhibitor reaches renal tissues in high concentrations. For example, verapamil reduces digoxin biliary clearance by 43% but does not affect its renal clearance (although this comparison was made under steady state conditions). 29 This is probably due to lack of sufficiently high verapamil concentrations in renal tissues to inhibit P gp. Similarly, induction of P gp by rifampin increases digoxin AUC to a much lesser extent after intravenous digoxin administration compared to oral digoxin administration. 27 Further, in this study, digoxin renal clearance and half life were not changed, indicating that the interaction was mainly at the level of the intestine and not the kidney. 27 Appropriate choice of the pharmacokinetic parameter to evaluate when examining P gp mediated DDI is essential. Use of an inaccurate marker of digoxin disposition within a specific study can result in false positive or false negative results. Choice of the pharmacokinetic parameter to evaluate will primarily depend on the anticipated time course and site of interaction. For example, interactions involving renal P gp (especially with IV drug administration) should be evaluated using digoxin total and renal clearances, urinary excretion rate, and half life. On the other hand, interactions following oral administration (involving intestinal, biliary, and renal P gp) are better evaluated using AUC 0 1 and C max.itis important to note that evaluation of partial AUC (e.g., AUC 0 4 ) is not necessarily reflective of intestinal P gp activity alone. Digoxin elimination starts early enough to have some effect on partial AUC as well as C max. In order to avoid false interpretation of DDI study results, a study design that enables an analysis of digoxin full pharmacokinetic profile (C max,t max, renal clearance, total clearance, partial AUC, AUC 0 1, and urinary excretion rate) should be used. In that aspect, a regulatory consensus is needed on the most appropriate and most sensitive pharmacokinetic

6 6 The Journal of Clinical Pharmacology / Vol XX No XX (2013) Table 3. Studies Suggesting the Involvement of Non P Glycoprotein Transporters in Digoxin Disposition Type of study Design/system Findings Comments Reference In vitro Uptake into oatp2 expressing Xenopus laevis oocytes Digoxin uptake was 11 fold higher in oatp2 transfected oocytes compared to control In vitro Uptake into human and rat hepatocytes Digoxin uptake into human hepatocytes at 4 C was 5% of that at 37 C In vitro Uptake into OATP transfected Xenopus laevis oocytes Digoxin uptake was inhibited by quinidine only in rate hepatocytes Digoxin uptake was >2 fold higher in OATP8 expressing oocytes as compared to control Suggests the affinity of digoxin for rat oatp2 (analogue to human OATP1B1) Indicates the possible involvement of a carrier mediated process in digoxin hepatic uptake Human OATP8 is also known as OATP1B3 Indicates digoxin might be a substrate for OATP1B3 Uptake by oocytes expressing OATP1B1, OATP1A2, or OATP2B1 was similar to control In vitro, inhibition Uptake into isolated rat hepatocytes Amiodarone decreased digoxin uptake into hepatocytes Indicates the possible role of transporters in digoxin hepatic uptake but does not suggest a specific transporter In vitro, inhibition Uptake into oatp2 expressing Xenopus laevis oocytes In vitro Uptake into MDCK cells transfected with human OATP4C1 gene In vitro Transport in rat intestinal brush border membrane vesicles (BBMV) In vitro, inhibition Transport in rat intestinal tissue in Ussing chambers In vitro, inhibition Transport in Caco 2 and MRP2 transfected MDCKII cells Amiodarone decreased digoxin uptake into oatp2 transfected oocytes Digoxin uptake was more than twofold higher in transfected cells compared to control Digoxin uptake into BBMV was inhibited by DIDS (anionic transporter inhibitor) Digoxin intestinal absorptive permeability decreased in presence of DIDS (anionic transporter inhibitor) and BSP (OATP inhibitor) MK571 (selective MRP inhibitor) and DIDS reduced digoxin intestinal exsorptive permeability MK571 (MRP2 inhibitor) reduced digoxin secretion from Caco 2 cells In vitro Transport into MDCKII hmdr1 cells Kinetic modeling of digoxin transport suggests that both basolateral and apical uptake transporters are required in addition to P gp and passive diffusion to describe digoxin transport In vitro Uptake in sandwich cultured human hepatocytes Radiolabeled digoxin uptake was inhibited by non labeled digoxin but not by OATP1B1, OATP1B3, OATP2B1, More specific results than above. Suggests the role of rat oatp2 (analogue to human OATP1B1) in digoxin hepatic uptake Results are specific to OATP4C1 due to use of transfected gene OATP4C1 may not be present in liver or intestine but these results still indicate the affinity of digoxin to OATP transporters Suggests the involvement of an intestinal anionic transporter in digoxin intestinal uptake Suggest the involvement of an anionic uptake transporter (may be OATP) in digoxin intestinal absorption Also, suggests a role for a non P gp efflux transporter in digoxin intestinal secretion Digoxin secretion in MRP2 transfected cells was only 1.2 fold higher than controls Might suggest involvement of an MK571 sensitive efflux transporter other than MRP2 Indirect data that suggests digoxin might be a substrate for uptake transporters Results are from mathematical modeling of transport data rather than actual studies with specific uptake transporters Indicates that digoxin might not be a substrate for OATP mediated hepatic uptake in humans OAT2, OCT1, or MCT8 inhibitors Uptake into the liver was 50% by passive diffusion and 50% through a carrier mediated process with an unknown transporter Noé et al. 32 Olinga et al. 42 Kullak Ublick et al. 39 Kodawara et al. 33 Kodawara et al. 33 Mikkaichi et al. 43 Yao et al. 37 Yao et al. 37 Lowes et al. 48 Acharya et al. 38 Kimoto et al. 41 (Continued)

7 Nader and Foster 7 Table 3. Continued Type of study Design/system Findings Comments Reference In vitro Uptake in OATP expressing HEK293, MDCK, and CHO cells In vitro, inhibition Digoxin uptake in cryopreserved human hepatocytes (CHH) Ex situ, inhibition Digoxin rifampicin coadministration in isolated perfused rat liver Ex situ, inhibition Digoxin rifampicin coadministration in isolated perfused rat liver Digoxin uptake in cells transfected with OATP1B1, OATP1B3, OATP1A2, and OATP2B1 was similar to controls Digoxin uptake was sodium dependent in nontransfected HEK293 cells Digoxin uptake into hepatocytes was not significantly inhibited by rifampin (OATP inhibitor) Rifampicin (OATP inhibitor) decreased the uptake of digoxin into the liver and increased AUC in liver perfusate Rifampicin (OATP1B1 inhibitor) decreased the digoxin uptake into the liver Ex situ Uptake in isolated perfused rat liver Digoxin uptake was mediated by two different uptake systems Digoxin inhibited uptake of OATP1B1 and OATP1B3 substrates Suggests that digoxin may be a substrate for a sodium dependent transporter Taub et al. 40 Suggests that digoxin may not be an OATP substrate De Bruyn et al. 49 Suggests a role for hepatic sinusoidal uptake transporters (esp. OATP) in digoxin entrance into liver Hepatocellular uptake of digoxin was concentration dependant indicating the involvement of a carrier mediated process Rifampicin increased Km for the uptake process, potentially due to inhibition of oatp2 A high capacity low affinity system and a low capacity high affinity system were involved in digoxin uptake Lau et al. 34 Weiss et al. 35 Weiss et al. 36 No specific transporters were identified Animal Amiodarone digoxin DDI study in rats IV amiodarone increased digoxin AUC by >2 fold and Amiodarone is an oatp2 inhibitor Funakoshi et al. 50 reduced digoxin renal and biliary excretion, total clearance, and liver distribution Animal mdr1a knockout mice Quinidine increased digoxin plasma and tissue levels by % in mdr1a knockout mice Animal mdr1a/b double knockout mice Valspodar increased brain to plasma ratio and reduced fecal excretion of digoxin in mdr1a/b double knockout mice Clinical Digoxin PK and SLCO1B3 genetic polymorphism in hemodialysis patients Clinical Digoxin PK after coadministration with grapefruit juice Concentration to dose ratio was higher in patients with deletion Polymorphisms in the OATP1B3 gene Grapefruit juice reduced digoxin absorption rate constant and increased lag time Indicates the possible role of rat oatp2 in digoxin hepatic uptake Reduction of renal excretion suggests inhibition of oatp4c1 as well Decreased liver distribution confirms hepatic uptake involvement Suggests inhibition of non P gp transporters by quinidine and/or contribution of mdr1b to digoxin efflux by P gp Complete knockout of P gp genes Suggests inhibition of non P gp transporters by valspodar Patients with lower OATP1B3 expression have higher digoxin concentration to dose ratio (possible due to lower hepatic uptake) Might indicate the role of OATP1B3 in digoxin hepatic uptake Results suggest possible inhibition of an intestinal uptake rather than an efflux transporter Fromm et al. 23 Mayer et al. 24 Tsujimoto et al. 51 Parker et al. 47 MDR1, multi drug resistance gene; LLC PK1, renal epithelial cells from porcine kidney; B A, basal to apical; MDCK, Madin Darby Canine Kidney; AUC, area under the curve; PK, pharmacokinetics; SNP, single nucleotide polymorphism; oatp, rat organic anion transporting polypeptide; OATP, human organic anion transporting polypeptide; SLCO, solute carrier gene; DIDS, 4,4 0 diisothiocyanatostilbene 2,2 0 disulfonate; BSP, bromosulfophthalein; MRP, mutli drug resistance protein; MDCK hmdr1, MDCK cells overexpressing human MDR1 gene; HEK293, human embryonic kidney cells; CHO, Chinese hamster ovary cells.

8 8 The Journal of Clinical Pharmacology / Vol XX No XX (2013) parameter to use when conducting P gp mediated digoxin DDI studies. Specificity of Digoxin as a P gp Probe Selection criteria for best in vivo P gp probes include adequate sensitivity and specificity of the probe substrate to P gp. Specificity to P gp means that changes in probe pharmacokinetics (absorption, distribution, and elimination) are almost exclusively affected by P gp (in addition to passive diffusion) with minimal involvement of other transporters or metabolizing enzymes. If this condition is not fulfilled, then the observed effects of coadministered inhibitors or inducers on the probe pharmacokinetics in DDI studies cannot be exclusively attributed to changes in P gp function. This is frequently the case since specificin vivo P gp inhibitors or inducers are generally unavailable. Involvement of non P gp transporters in digoxin disposition has been the focus of numerous studies and will be addressed below. Role of Enzymes and Non P gp Transporters in Digoxin Transport and Disposition. Metabolizing enzymes play a minor role in digoxin elimination and are generally not considered a confounding factor in digoxin DDI studies with P gp inhibitors or inducers. However, the contribution of non P gp transporters cannot be similarly excluded. Several in vitro studies have been conducted to evaluate the role of transporters other than P gp in digoxin pharmacokinetics (Table 3). Those studies usually involve evaluation of digoxin uptake or transport in cells transfected with specific transporters. Alternatively, digoxin transport in intestinal cells or tissues and in hepatocytes can be evaluated to investigate the role of transporters normally expressed in these cells. Most of the recent in vitro studies of digoxin transport have focused on the role of organic anion transporting polypeptides (OATPs). Other studies have also focused on non P pg efflux transporters as well as sodium dependent uptake transporters. Digoxin Uptake by Organic Anion Transporting Polypeptides (OATP) (Table 3). One of the earliest in vitro studies to suggest the potential affinity of digoxin to OATPs was conducted in oatp2 transfected X. laevis oocytes. Digoxin uptake was 11 fold higher in oocytes transfected with the rat oatp2 gene compared to non transfected cells. 32 The rat oatp2 gene is the analog to the human liver specific transporter OATP1B1. Similar results were observed in another study where amiodarone (an OATP inhibitor) inhibited digoxin uptake in rat oatp2 transfected oocytes. 33 Ex situ experiments in isolated perfused rat livers also demonstrate that digoxin hepatic uptake is reduced by the OATP inhibitor rifampicin. 34,35 Digoxin hepatocellular uptake occurs in a concentration dependent manner, indicating the likely involvement of carrier mediated processes. 35 Two distinct uptake systems (a high capacity low affinity system and a low capacity high affinity system) are responsible for digoxin uptake into rat liver but neither has been specifically identified. 36 Other studies suggest that digoxin may be a substrate for uptake by other, yet unknown, transporter(s). 37,38 Kinetic modeling of digoxin transport in MDCKII hmdr1 cells suggests the involvement of both basolateral and apical uptake transporters in digoxin disposition. 38 Also, digoxin uptake in intestinal brush border membrane vesicles is inhibited by anion transporter inhibitors. 37 It is important to note that the above mentioned studies were conducted using rat OATP genes or rat livers. Different results are obtained when the human OATP genes are used. Specifically, digoxin uptake in oocytes, HEK293, MDCK, and CHO cells transfected with human OATP1B1, OATP2B1, or OATP1A2 genes is similar to uptake in non transfected cells/oocytes. 39,40 In two studies conducted in OATP1B3 transfected oocytes, digoxin uptake was more than twofold higher than control in only one of the studies. 39,40 In a recent study of digoxin transport by OATP transporters conducted in sandwichcultured human hepatocytes, digoxin hepatic uptake was mediated equally by passive diffusion and active carriermediated uptake. Use of specific inhibitors in hepatocytes and transfected HEK293 cells indicate that digoxin is not a substrate for uptake by OATP1B1, OATP1B3, OATP2B1, OATP1A2, OAT2, OCT1, or MCT8. 41 The results of in vitro studies of digoxin transport by OATPs indicate that digoxin might have different affinities for human and rat OATP transporters. Indeed, digoxin uptake into hepatocytes is inhibited by quinidine only in rat and not in human hepatocytes. 42 This is important when results from studies done using rat genes coding for OATP transporters are used to predict the effects of OATP on digoxin pharmacokinetics in humans. Studies conducted using human genes or human hepatocytes have consistently demonstrated that digoxin is not transported by OATPs. Evaluation of the involvement of uptake transporters in digoxin renal excretion is very limited. In a single study conducted in OATP4C1 transfected MDCKII cells, digoxin uptake was found to be higher than controls. 43 Clinical significance of OATP4C1 mediated digoxin uptake in renal tubules has not been studied due to the lack of specific inhibitors. Even in the presence of an OATP4C1 inhibitor, digoxin renal excretion may not be significantly affected, because inhibitor concentrations at the renal tubules need to be high enough to sufficiently exceed IC 50 for inhibition of OATP4C1. Hence, involvement of non P gp transporters in digoxin renal excretion is not likely to be clinically relevant. Possible Role for Other Uptake Transporters in Digoxin Disposition (Table 3). Although studies conducted in human hepatocytes do not indicate OATP mediated digoxin uptake, these studies still suggest that an active uptake process is involved in digoxin entry into the liver. 41

9 Nader and Foster 9 Digoxin inhibits active hepatic uptake of other drugs, which might indicate an affinity of digoxin to hepatic sinusoidal uptake transporters. 40,44 To this end, digoxin uptake in non transfected HEK293 cells is sodiumdependent (although this study did not evaluate whether this process is concentration dependent). 40 This might suggest that digoxin is a substrate of one of the sodiumdependent transporters expressed in HEK293 cells such as the sodium dependent multivitamin transporter (SMVT) or the apical sodium bile acid transporter (ASBT). Further studies in cells specifically transfected with genes coding for each of those transporters are needed to confirm this observation. Both SMVT and ASBT are expressed on the apical membrane of enterocytes and hence may play a role in digoxin absorption if digoxin is indeed one of their substrates. 45 The involvement of a sodium dependent uptake process in digoxin disposition was also demonstrated in a study with ribosomal protein L3 (RPL3). X. laevis oocytes transfected with the RPL3 gene showed a fourfold increase in digoxin uptake compared to controls and uptake was sodium dependent. 46 Although the design of this study does not definitively establish RPL3 as a digoxin transporter, the results, in conjunction with those above, suggest that one or more sodium dependent uptake transporters might be involved in digoxin uptake in vivo, although the identity and the clinical significance of such transporters are currently unknown. Involvement of uptake transporters in digoxin disposition has not yet been evaluated in clinical studies In a clinical DDI study between grapefruit juice and digoxin, grapefruit juice reduced digoxin absorption rate constant and increased absorption lag time. 47 Although grapefruit juice was hypothesized to be a P gp inhibitor, reduction in absorption rate is more consistent with inhibition of an uptake rather than an efflux transporter. The fact that this interaction is not consistent with P gp further emphasizes the lack of specificity of digoxin as a P gp probe. The effects of grapefruit juice on sodium dependent transporters such as ASBT or SMVT are currently unknown. Summary and Conclusions Digoxin remains a commonly used in vitro and in vivo probe for P gp function. Despite its widespread use as a P gp probe, however, questions remain regarding the sensitivity and specificity of digoxin to P gp activity. Sensitivity of digoxin to in vivo P gp activity can be reduced by the relatively high bioavailability of digoxin, dissolution limited absorption (rather than permeability limited absorption), and the lack of sufficiently high inhibitor concentrations at sites other than the intestine. Related to specificity, results from in vitro studies of digoxin transport are controversial. Most studies conducted using human OATP genes or human hepatocytes suggest that digoxin is not transported by most human OATP transporters (OATP1B1, OATP1B3, OATP1A2, and OATP2B1). In contrast, in a clinical trial relating digoxin pharmacokinetics to OATP transport, there was an association between OATP1B3 genotype and digoxin pharmacokinetics in patients with renal failure. 51 Whether this is a true association or the result of other unidentified factors or genetic variations needs to be confirmed in additional prospective clinical trials. Based on currently available data, we believe that digoxin should not be considered an OATP substrate in humans. 40,41,49 Digoxin uptake by non OATP transporters requires further investigation. Several in vitro studies have suggested that digoxin might be a substrate for sodiumdependant uptake transporters or other unknown intestinal or hepatic sinusoidal uptake transporters ,40 Definitive in vitro studies of digoxin uptake into cells/oocytes transfected with ASBT, SMVT, or other uptake transporters, and/or using specific inhibitors of these transporters are warranted to confirm such observations. Knockout mice models, and potentially, clinical studies would ultimately be required to confirm in vitro observations, as it is possible that sodium dependant transporters affect digoxin in vitro and in animals but still have a minor role in digoxin disposition in humans. Notably, clinical evaluation would require the use of specific and clinically safe inhibitors of ASBT and/or SMVT transporters. It is also possible that further in vitro and animal studies lead to identification of other not currently identified transporters involved in digoxin pharmacokinetics. Involvement of uptake transporters in digoxin disposition, if confirmed, may pose two concerns. The first relates to the relative contribution of P gp and non P gp transporters to digoxin disposition. This will determine both the clinical importance of non P gp transporters in digoxin disposition as well as the sensitivity of digoxin to changes in P gp activity. The second concern is that drugs being tested as P gp inhibitors/inducers might also affect non P gp transporters involved in digoxin disposition. Despite the uncertainty regarding the involvement of other non P gp transporters in digoxin disposition, an argument can be made that digoxin is still be the best available (if not, the most practical) P gp probe. Other currently available probes for P gp activity include loperamide, vinblastine, talinolol, quinidine, and fexofenadine. 21 However, none of those substrates can be considered an ideal P gp probe due to a number of limitations (for example: safety for loperamide and vinblastine, OATP transport for talinolol and fexofenadine, and CYP3A metabolism for quinidine). 52 In contrast, digoxin is not significantly metabolized and is relatively safe at doses used in DDI studies. Given the limitations of other P gp probes, and the fact that other transport proteins have not conclusively been established as influencing

10 10 The Journal of Clinical Pharmacology / Vol XX No XX (2013) digoxin disposition, digoxin remains one of the only feasible P gp probes in humans. This presents researchers and regulatory agencies alike with a substantial dilemma. On one hand, the lack of a reliable P gp probe does not necessarily justify the recommendation of an unreliable probe by regulatory agencies (examples of the perils of this approach are abundant in the drug transporter literature). Alternatively, it can be argued that the need for premarket clinical testing for P gp mediated effects justifies the use of the best available probe, even if it is imperfect, and by providing a recommendation, regulatory agencies at least foster a standardized approach to evaluation. There is a definite need for clarity and transparency regarding use of digoxin as a P gp probe. Guidances should be clear that results from studies using digoxin should be interpreted with the caveat that is an imperfect P gp probe. 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