Evaluation of Proposed In Vivo Probe Substrates and Inhibitors for Phenotyping Transporter Activity in Humans

Transcription

1 Supplement Article Evaluation of Proposed In Vivo Probe Substrates and Inhibitors for Phenotyping Transporter Activity in Humans The Journal of Clinical Pharmacology (2016), 56(S7) S82 S98 C 2016, The American College of Clinical Pharmacology DOI: /jcph.736 Jeremiah D. Momper, PharmD, PhD, Shirley M. Tsunoda, PharmD, and Joseph D. Ma, PharmD, FCP Abstract Drug transporters are present in various tissues and have a significant role in drug absorption, distribution, and elimination. The International Transporter Consortium has identified 7 transporters of increasing importance from evidence of clinically significant transporter-mediated drug drug interactions. The transporters are P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2, organic anion transporters (OAT) 1, and OAT3. Decision trees were created based on in vitro experiments to determine whether an in vivo transporter-mediated drug drug interaction study is needed. Phenotyping is a methodology that evaluates real-time in vivo transporter activity, whereby changes in a probe substrate or probe inhibitor reflect alternations in the activity of the specified transporter. In vivo probe substrates and/or probe inhibitors have been proposed for each aforementioned transporter. In vitro findings and animal models provide the strongest evidence regarding probe specificity. However, such findings have not conclusively correlated with human phenotyping studies. Furthermore, the extent of contribution from multiple transporters in probe disposition complicates the ability to discern if study findings are the result of a specific transporter and thus provide a recommendation for a preferred probe for a drug transporter. Keywords drug drug interaction, transporter, phenotyping, biomarker, drug development Drug transporters are involved in clinically relevant drug drug interactions, thus impacting drug pharmacokinetics (PK), efficacy, toxicity, and dosing. In response to the importance of understanding drug transporters during the drug discovery and development process, the International Transporter Consortium (ITC) was formed. In 2010, the ITC identified 7 transporters of particular importance. The transporters were P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 2, organic anion transporter (OAT) 1, and OAT3. 1 In 2013, the ITC provided an update for these 7 transporters. In addition, based on emerging PK drug drug interaction data and evidence regarding drug interference with transport of endogenous compounds, multidrug and toxin extrusion protein (MATE) 1, MATE2K, multidrug resistance protein (MRP) 2, MRP3, MRP4, and bile salt export pump (BSEP) were also identified as emerging transporters of importance. 2 The goals of the ITC were to establish standards for the in vitro evaluation of transporter-based interactions that may reduce the need for in vivo studies. 2 Decision trees for P-gp, BCRP, OATP, OCT, and OAT substrate and inhibitor interactions were created to determine whether an in vivo clinical study is needed. 1,2 The decision trees are currently included in regulatory draft guidance documents. 3,4 Methods for the evaluation of transporter-based interactions in vitro are detailed, with decision trees providing cutoff values regarding the net flux ratio (eg, P-gp or BCRP substrate), inhibition constant (Ki; eg, P-gp or BCRP inhibitor), and renal clearance (eg, OCT or OAT substrate). 1 If criteria were met that indicated a need to conduct a drug drug interaction study in humans, transporter-specific probe substrate(s) and probe inhibitor(s) were proposed. 1 Transporter-specific probe substrate(s) and probe inhibitor(s) have also been proposed elsewhere. 2,5,6 With the exception of P-gp and BCRP, details on the suitability for use and guidance for the conduct of in vivo transporter-mediated drug drug interaction studies in humans have not been provided. University of California, San Diego, Skaggs School of Pharmacy & Pharmaceutical Sciences, La Jolla, CA, USA Submitted for publication 18 November 2015; accepted 7 March Corresponding Author: Joseph D. Ma, PharmD, FCP, University of California, San Diego, Skaggs School of Pharmacy & Pharmaceutical Sciences, 9500 Gilman Drive, MC 0714, La Jolla, CA joema@ucsd.edu

2 Momper et al S83 Phenotyping is a methodology to evaluate real-time in vivo transporter activity and transporter-mediated drug drug interactions. A drug that is a known substrate or inhibitor of a transporter is used as a probe. For example, coadministration of a probe substrate or inhibitor with an investigational agent that changes the probe PK and/or predefined phenotyping parameter is assumed to reflect alterations in transporter activity. Appropriate selection of a probe substrate, inducer, or inhibitor is vital to accurately interpret study findings. Reviews evaluating probes to phenotype P-gp activity are detailed elsewhere. 7 9 Current US regulatory draft guidance documents have not included decision trees for the evaluation of MATE1-, MATE2K-, MRP2-, MRP3-, MRP4-, and BSEP-mediated drug drug interaction studies and will not be discussed. In vivo probe substrates and/or probe inhibitors have been proposed for BCRP, OATP1B1, OATP1B3, OCT2, OAT1, and OAT3 (Tables 1 and 2). The purpose of this review is to evaluate proposed probe substrates and probe inhibitors for BCRP, OATP1B1, OATP1B3, OCT2, OAT1, and OAT3, as well as highlight advantages and disadvantages for in vivo phenotyping and/or drug drug interaction studies. Evaluation of Proposed In Vivo Transporter Probes Breast Cancer Resistance Protein BCRP is an ATP-binding efflux transporter expressed in numerous tissues, including but not limited to small intestine, liver, blood brain barrier, testis, placenta, and mammary glands. 10,11 Human BCRP is encoded by the ABCG2 gene, located on chromosome 4q22, consisting of 16 exons spanning more than 66 kb. The ABCG2 gene also encodes a 72-kDa membrane protein composed of 655 amino acids. 10 There are at least 20 known ABCG2 polymorphisms, with the 421C>A nonsynonymous single-nucleotide polymorphism (SNP) resulting in decreased activity. 12 BCRP possesses broad substrate and inhibitor specificity, thus transporting endogenous and exogenous substrates that possess diverse physicochemical properties. Several reviews have provided exhaustive lists of compounds that are known BCRP substrates and/or inhibitors based on in vitro data (eg, efflux ratio, K m,k i,ic 50 ). 1,6,13 Rosuvastatin, pitavastatin, and sulfasalazine are proposed in vivo BCRP probe substrates. 1,6,13 Contrasting recommendations exist, as some do not recommend pitavastatinasaninvivobcrpprobe. 6 Current Food and Drug Administration (FDA) guidance documents, as well as the University of California, San Francisco (UCSF) FDA TransPortal have not proposed a BCRP inhibitor probe, whereas review articles have proposed cyclosporine, 1 oral curcumin, and lapatinib. 6 BCRP Substrates Rosuvastatin. Rosuvastatin is a 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitor with an absolute oral bioavailability of 0.2 and limited permeability via passive diffusion. 14,15 Approximately 90% of the dose is recovered in feces, with the majority excreted unchanged, 15 thus suggesting that metabolism is a minor route of clearance. Oral rosuvastatin has been proposed to evaluate intestinal and hepatic BCRP and intravenous rosuvastatin for hepatic BCRP. 6 RosuvastatinistransportedbyBCRPinvitroandinvivo In double-transfected MDCKII cells expressing BCRP and OATP1B1, efflux clearance across the apical membrane was 2.7 times higher compared with OATP1B1 single-transfected cells. 14 Membrane vesicle studies in human BCRP-expressing HEK293 cells reported K m values of ATP-dependent uptake of rosuvastatin of 2.02 ± 1.12 μm. 14 In Bcrp1 (-/-) mice, rosuvastatin total clearance was decreased by 2-fold compared with wild-type mice. 14 A gene-dose effect was observed for the ABCG2 421C>A genetic polymorphism. In healthy Chinese men, rosuvastatin AUC 0-72 (mean ± SD) was 33.8 ± 11.4 and 59.6 ± 22.2 in ABCG2 CC (n = 7) versus CA and AA (n = 7) genotypes. 18 Similar findings are reported elsewhere. 17,19 Human in vivo drug drug interaction and/or phenotyping studies that have used rosuvastatin as a BCRP probe have been conducted in healthy adults who were administered a single oral 10-mg dose. 17,20 25 Exceptions existed as a single 5-mg dose, and 20 mg once daily for 10 days has also been used There is a lack of complete dose proportionality, as rosuvastatin AUC and C max ratios comparing the 20- and 10-mg doses are less than A disadvantage of the use of rosuvastatin is the involvement of other transporters in its disposition. OATP1B1 and OATP1B3 are involved in liver distribution, MRP2 secretes rosuvastatin into bile, and OAT3 and MRP2 are involved in rosuvastatin tubular secretion. 6,14,29 Although intravenous rosuvastatin has been proposed to evaluate hepatic BCRP, this is not readily available. Numerous drug drug interaction studies have been performed to confirm in vitro findings of BCRP-mediated inhibition. The extent of rosuvastatin AUC and C max increases due to BCRP inhibition of precipitant drugs ranges from 1.5- to 7.1- fold and 2- to 10.6-fold, respectively. 20,21,26 28 For a few studies, a lack of agreement between in vitro BCRP inhibition and in vivo findings has been reported, with the precipitant drug showing no statistically or clinically significant change in rosuvastatin PK. 23,24,30

3 S84 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) Table 1. Summary of Advantages and Disadvantages of Proposed Probe Substrates Transporter Probe Substrate Advantages Disadvantages BCRP Rosuvastatin In vivo data demonstrating gene-dose effect with ABCG2 421C>A polymorphism Cotransporter involvement of OATP1B, MRP2, and OAT3 in liver distribution, bile secretion, and renal tubular secretion Substrate for NTCP transporter CYP2C9 polymorphisms do not affect pharmacokinetics Pitavastatin Minimal hepatic (CYP) metabolism Substrate for OATP1B, MRP2, and P-gp Sufficient in vitro evidence of BCRP involvement in biliary In vivo data do not demonstrate gene-dose effect with excretion ABCG2 421C>A polymorphism Sulfasalazine Correlation between AUC in mice with AUC in humans with Substrate for MRP2 and OATP2B1 ABCG2 421C>A polymorphism 20- to 80-Fold interindividual PK variability OATP1B1 Pitavastatin In vitro evidence that 90% of hepatic uptake is mediated by Substrate for BCRP, MRP2, P-gp, and CYP2C9 OATP1B1 Minimal involvement of intestinal transporters Clinical evidence that it is more sensitive and selective versus rosuvastatin Pravastatin Minimal hepatic (CYP) metabolism 50% of total clearance due to OAT3-mediated renal clearance SLCO1B1 521T>C and SLCO1B1*15 associated with OATP inhibition by rifampin minimally affected exposure decreased clearance Minimal involvement of BCRP Rosuvastatin 50% of total hepatic uptake attributed to OATP1B1 NTCP may compensate for impaired OATP activity Knockout mouse model supports role of OATP disposition 25% of total clearance from OAT3-mediated renal elimination Substrate for BCRP, MRP2, NTCP, and P-gp Clinical evidence that it is not as sensitive or selective versus pitavastatin OCT2 Metformin No hepatic metabolism or biliary excretion Also transported by renal MATE1/MATE2 and OCT1 Sufficient in vitro evidence of OCT2 involvement in renal secretion OAT1/OAT3 PAH Sufficient in vitro evidence of OCT1 involvement in renal secretion with limited or no involvement of other renal transporters Requires continuous infusion for optimal assessments, which is technically difficult Can be used to estimate renal plasma flow Zidovudine Elimination is probenecid sensitive Elimination also mediated by UGT2B7, CYP2C9, CYP2E1, CYP3A4, and CYP2A6 Sufficient in vitro evidence of OAT1/OAT3 involvement in renal secretion Tenofovir Minimal hepatic (CYP) metabolism Tubular secretion involves OAT1 and MRP4 Elimination primarily via renal excretion with renal clearance 2 3 times creatinine clearance Ciprofloxacin Elimination is probenecid sensitive Inhibitor of CYP1A2 Sufficient in vitro evidence of OAT3 involvement Minimal OAT1 involvement Acyclovir Sufficient in vitro evidence of OAT1 involvement in renal Substrate for OCT1, MATE1, and MATE2 secretion Elimination is probenecid and cimetidine sensitive Methotrexate Sufficient in vitro evidence of hoat1, hoat3, and hoat4 involvement Significant interindividual PK variability Substrate for BCRP and P-gp AUC, area under the curve; ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; CYP, cytochrome P450; MATE, multidrug and toxin extrusion protein; MRP, multidrug resistance protein; PAH, para-aminohippurate; P-gp, P-glycoprotein; PK, pharmacokinetic; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; NTCP, sodium/taurocholate cotransporting polypeptide; SLCO, solute carrier organic anion transporter; UGT, UDP-glucuronosyltransferase. Pitavastatin. Pitavastatin is another HMG-CoA reductase inhibitor with an absolute oral bioavailability of 0.6 and is considered a class III (low-permeability, high-solubility) drug by Biopharmaceutics Classification System (BCS). 31 Pitavastatin undergoes minimal hepatic metabolism and is eliminated in the bile In membrane vesicle studies in human and mouse BCRP-transfected HEK293 cells, K m values of ATPdependent uptake of pitavastatin were 5.73 ± 1.52 and 4.77 ± 0.5 μm, respectively. 36 BCRP is involved in the

4 Momper et al S85 Table 2. Summary of Advantages and Disadvantages of Proposed Probe Inhibitors Transporter Probe Inhibitor Advantages Disadvantages BCRP Cyclosporine Sufficient in vitro evidence of BCRP inhibition Substrate of P-gp and CYP3A Inhibitor of OATP, P-gp, and MRP2 Moderate CYP3A and weak CYP2C9 inhibitor Curcumin Shown to increase sulfasalazine exposure by >2-fold in humans Evidence of in vivo inhibition in humans is limited to a single study Purity and reproducibility need to be confirmed Lapatinib Most potent TKI to inhibit intestinal and systemic BCRP In vitro P-gp, OATP1B1, and CYP2C8 inhibitor Weak CYP2C8 and CYP3A inhibitor in vivo OATP Cyclosporine Sufficient clinical evidence of OATP inhibition Inhibitor of BCRP, P-gp, and MRP2 Moderate CYP3A and weak CYP2C9 inhibitor Rifampin Sufficient in vitro and in vivo evidence of OATP inhibition CYP3A induction with multiple dosing Clinical evidence that intravenous may be a more selective inhibitor than oral Clinical evidence that it may be a more potent inhibitor than cyclosporine Oral rifampin may affect intestinal transporters and/or metabolic enzymes OCT2 Cimetidine Sufficient in vitro evidence of OCT2 inhibition Nonspecific OCT2 inhibitor Evidence of decreased renal clearance of metformin, an OCT2 substrate Interacts with renal OAT1/OAT3 and CYP1A2, CYP2C19, CYP3A, and CYP2D6 OAT1/OAT3 Probenecid Extensive in vitro and in vivo evidence of OAT1/OAT3 Nonspecific inhibitor that inhibits OAT1 and OAT3 inhibition Used clinically to block tubular secretion of OAT1/OAT3 substrates BCRP,breast cancerresistanceprotein;cyp,cytochrome P450;P-gp,P-glycoprotein;OAT,organic anion transporter;oatp,organic anion transporting polypeptide; OCT, organic cation transporter; TKI, tyrosine kinase inhibitor. biliary excretion of pitavastatin. In Bcrp1 (-/-) mice and wild-type mice, biliary excretion clearance of pitavastatin was 0.11 ± 0.02 and 1.21 ± 0.43 ml/min/kg, respectively. 36 BCRP has a minimal role in the intestinal absorption of pitavastatin. 37 Pitavastatin is also an OATP1B, MRP2, and P-gp substrate, which may not make this an ideal BCRP probe substrate. 26,36 Symmetrical flux of pitavastatin was observed in P-gp- and MRP2-expressing MDCK II monolayers. In double-transfected cells, basal-to-apical flux was 15, 110, and 230 times higher versus apical-tobasal flux in OATP1B1/MRP2, OATP1B1/BCRP, and OATP1B1/P-gp cells, respectively. 36 Although phenotyping studies using pitavastatin as a potential BCRP probe are lacking, pharmacogenetic studies have been published. 26,37,38 In healthy adults (n = 38), there was no difference in pitavastatin AUC based on ABCG2 genotypes. 37 These findings are consistent with another study in healthy Korean men, in which subjects were stratified, in part, by the ABCG2 421C>A genotype. Pitavastatin AUC 0 was ± 41.3, ± 53.6, and ± 30.4 ng h/ml (P =.62) in ABCG2 CC (n = 24), CA(n = 16), and AA (n = 5) genotypes, respectively. 38 Sulfasalazine. Oral sulfasalazine is used to treat inflammatory bowel diseases such as ulcerative colitis and Crohn s disease. Oral sulfasalazine is considered a class IV drug (low-solubility, low-permeability) drug with an oral bioavailability of <15%. 31,39,40 Colonic intestinal flora converts sulfasalazine to sulfapyridine and 5-aminosalicylic acid (5-ASA). The majority of 5-ASA resides in the gut and is N-acetylated via N-acetyltransferase (NAT) 1. Sulfapyridine is readily absorbed and further metabolized to N-acetylsulfapyridine, but by the genetic polymorphic NAT2. 41 In vitro models, BCRPknockout mice, and BCRP inhibitor treated monkeys provide evidence of intestinal BCRP specificity and sensitivity. 42,43 In a mouse everted ileum sac model, steady-state intestinal absorptive clearance increased by 5-fold in the presence of a BCRP inhibitor. 43 In another study, after a single oral dose of 5 mg/kg, sulfasalazine exposure (mean ± SE) was 2550 ± 810 and 511 ± 198 ng h/ml in elacridar (BCRP inhibitor) treated monkeys versus untreated monkeys, respectively (n = 6, P <.01). Similar findings were also observed in BCRPknockout mice, with more than a 100-fold increase compared with wild-type mice. 44 Furthermore, correlations were observed in the animal model AUC changes versus sulfasalazine AUC changes in humans genotyped with the ABCG2 421C>A polymorphism. 42 Conflicting reports regarding a gene-dose effect from the ABCG2 421C>A polymorphism have been reported The lack of a gene-dose effect implies minimal BCRP involvement and/or the involvement of other transporters, such as MRP2, OATP1B1, and/or OATP2B1, which may impact sulfasalazine

5 S86 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) absorption. 43,48,49 Different sulfasalazine doses and formulations have been used. Single oral doses were 100 μg (microdose), 500 mg, 1000 mg, and 2000 mg ,50 Sulfasalazine formulations include intermediate-release (IR), enteric-coated, tablet, and suspension (via crushing of IR tablets into solution). The 100-μg microdose is not recommended, as nonlinear PK were observed and sulfasalazine AUC 0-24 was still 7.4-fold higher after dose normalization. 50 In a study using a single oral 500-mg enteric-coated formulation, a lack of a genedose effect with the ABCG2 421C>A polymorphism and no significant difference in sulfasalazine PK after BCRP inhibition were observed in healthy, Chinese men. 45.In summary, single-dose oral sulfasalazine PK differences are attributed, in part, to dose and formulation. Based on these studies, Lee et al 6 proposed an oral immediate-release 1000-mg sulfasalazine dose to evaluate intestinal BCRP activity. Phenotyping parameters to measure intestinal BCRP activity are primarily sulfasalazine AUC and/or C max. An AUC ratio of parent to metabolite has also been used, but is limited to evaluation in only intermediate- and rapid-acetylator phenotypes. 6,47 Based on an r 2 (coefficient of determination) of 0.93 (P <.0001), there is a suggestion that a single sulfasalazine plasma concentration at 6 hours may be an alternative phenotyping parameter. 46 However, the r 2 is not a measure of predictive performance and can only describe the degree of variability between variables. 51,52 The use of a single sulfasalazine concentration at 6 hours to determine BCRP activity is not recommended. BCRP Inhibitors Cyclosporine. Cyclosporine (cyclosporin A) is an immunosuppressant used for solid organ transplant and for various autoimmune and inflammatory diseases. There is sufficient in vitro evidence to conclude that cyclosporine is a BCRP inhibitor. Cyclosporine EC 50 for inhibition of BCRP-mediated efflux was 4.3 ± 1.9 μm in HEK cells, whereas an IC 50 of 26.1 and 7.3 μm have been reported on evaluating ATPase activities in wild-type BCRP and BCRP R482T mutants. 53,54 There are no published human phenotyping and BCRPspecific drug drug interaction studies on the use of cyclosporine as a BCRP probe inhibitor. In contrast, cyclosporine has been used as an OATP, P- gp, MRP2, and cytochrome P450 (CYP) 3A probe inhibitor. 55 Curcumin. Oral curcumin is a naturally occurring polyphenol and the main curcuminoid of turmeric. Curcumin inhibits ATP-dependent uptake of sulfasalazine by human BCRP in a concentrationdependent manner (IC 50 = 1.6 ± 0.93 μm, K i = 0.7 ± 0.41 μm). BCRP-knockout mice have markedly increased sulfasalazine plasma concentrations with concurrent administration of curcumin. 50 In the same publication, a drug drug interaction study was conducted in healthy Japanese men who were all genotyped as ABCG2 421CC (wild type). Oral curcumin 2000 mg increased sulfasalazine AUC 0 24 by 3.2-fold. 50 However, recent evidence in mice studies suggests that curcumin may also be a MRP2 inhibitor. 56 If these results are confirmed in human studies, this would be a major limitation to the use of curcumin as a BCRP probe inhibitor. Lapatinib. Oral lapatinib is an anticancer tyrosine kinase inhibitor (TKI). In vitro data and extrapolations from clinical data regarding anticancer effects have led a group to propose lapatinib as a BCRP probe inhibitor. A 250-mg dose was proposed, which is intended to be a subtherapeutic dose. Lee et al 6 also suggested that lapatinib is the most potent TKI to evaluate intestinal and systemic BCRP activity based on the rank ordering of inhibitory potency. Lapatinib is not only a BCRP inhibitor, but also a P-gp and OATP1B1 inhibitor in vitro (IC 50 values of μm) and a weak CYP2C8 and CYP3A4 inhibitor in vivo. 57,58 Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 OATPs are a family of 11 transporters encoded by SLCO, which are members of the solute carrier (SLC) family. One physiologic function of OATPs is the transport of bilirubin into hepatocytes, where it undergoes glucuronidation by glucuronosyl transferases (UGTs). Endogenous compounds transported by OATPs include steroid conjugates, thyroid hormones, and prostaglandins. 59 They also transport a wide variety of structurally unrelated substrates: HMG-CoA reductase inhibitors, angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors (ACE-Is), and antidiabetic agents (glinides). OATP1B1 and OATP1B3 are expressed on the basal side of human liver hepatocytes. OATP1B1 is expressed uniformly, whereas OATP1B3 is expressed more in the perivenous section. OATP2B1 is expressed on the basal side of hepatocytes and the apical membrane of intestinal enterocytes, as well as in the heart, 60 brain, 61 and placenta. 62 OATP1A2 is present on vascular endothelial cells in the brain and transports substances across the blood brain barrier. 63 The focus of this section will be on OATP1B1 and OATP1B3, as these have been shown to be important hepatic uptake transporters with involvement in drug drug interactions and alterations in drug response. SLCO1B1 388A>G increases hepatic uptake activity and is present in 40% of whites, 75% of African Americans, and 60% of Asians, whereas SLCO1B1 521T>C decreases hepatic uptake activity and has a

6 Momper et al S87 lower prevalence across ethnic groups (15% of whites, 2% of African Americans, and 15% of Asians) Two haplotypes that decrease uptake activity leading to increased substrate exposure are SLCO1B1*5 (c.388ac.521c) and SLCO1B1*15 (c.388g-c.521c). 68 In general, OATP1B1 and OATP1B3 have overlapping specificity with the compounds that they transport. Many OATP substrates are also substrates for MRP. 69 This is an important consideration when determining the specificity of a compound for OATP. There are also compounds with specificity for OATP1B1 (estrone-3-sulfate) or OATP1B3 (cholecystokinin octapeptide, digoxin, docetaxel, paclitaxel, telmisartan). OATP1B1 and OATP1B3 have been identified as the hepatic transporters that can contribute to drug drug interactions. 1 When the hepatic pathway of an investigational compound is significant (>25% of total clearance), then it should be determined if there is OATP1B1 or OATP1B3 involvement. Proposed probe substrates are pitavastatin, pravastatin, and rosuvastatin. 1,5 The ITC has also provided guidance for determining whether a compound is an OATP inhibitor. 1 Give a compound with a prototypical substrate such as pitavastatin or rosuvastatin and compare it with a positive control with a known OATP inhibitor such as cyclosporine or rifampin. 1 If the IC 50 value is 10 times the unbound C max (unbound C max /IC ), then the compound may need to be further evaluated as an in vivo OATP inhibitor. 1 OATP1B1 Substrates Pitavastatin. Pitavastatin has been proposed as a hepatic OATP1B1 probe substrate. Pitavastatin has minimal hepatic metabolism, minimal involvement of gut transporters, 26 and is excreted unchanged into the bile, and its fraction excreted unchanged by the kidney is less than 2%. Pitavastatin is transported by OATP1B1 and OATP1B3 using HEK293 cells and human cryopreserved hepatocytes, with K m values of 3.0 and 3.3 μm for OATP1B1 and OATP1B3, respectively. 70 Pitavastatin concentrations were influenced by SNPs in SLCO1B1 71 and the haplotype SLCO1B1*15, 38 but not BCRP, which provides evidence of OATP1B1 specificity. 72 Approximately 90% of pitavastatin uptake is accounted for by OATP1B1 in transfected cells and hepatocytes. 70 In a clinical study with oral pitavastatin and single doses of intraveous or oral rifampin, both routes of administration showed a 6- to 8-fold increase in pitavastatin AUC 0, 26 suggesting minimal gut transporter involvement. In addition, this study used a partial AUC from time zero to 4 hours (AUC 0 4 ) to estimate OATP1B activity. 26 The use of a partial AUC is questionable, as validation methods were lacking. The limitations of a partial AUC approach are discussed elsewhere. 7,73 The authors also suggested that pitavastatin is a more sensitive and selective OATP1B1 substrate (and therefore probe) compared with rosuvastatin. 26 There is some evidence that pitavastatin may not be a suitable OATP1B1 probe substrate. The intestinal absorption of pitavastatin may be mediated by OATP facilitating absorption and P-gp mediating efflux. 74,75 In vitro and animal data suggest that both BCRP and MRP may contribute to pitavastatin biliary excretion. In human BCRP membrane-expressing vesicles, pitavastatin uptake had a K m value of 5.73 μm. Knockout bcrp1(-/-) mice had 1/10th the biliary clearance of pitavastatin compared with control. 36 MRP2 is likely involved in the biliary excretion of all statins including pitavastatin, with a K m value of 8.9 μm. 69 In addition, a variant of ABCC2 (encoding for MRP2) together with SLCO1B1*15 accounted for 50% of pitavastatin disposition in a clinical study of healthy Korean men. 38 Finally, individuals with a cytochrome P450 (CYP) 2C9 variant haplotype (CYP2C9*1/*3) had higher pitavastatin AUC 0 and C max compared with the wild-type haplotype. 71 In summary, although there is human clinical data supporting the use of pitavastatin as an in vivo OATP1B probe substrate, caution must be exercised in attributing activity solely to OATP, as BCRP, MRP2, P-gp, and CYP2C9 play a role in its disposition. Pravastatin. Pravastatin is another HMG-CoA reductase inhibitor that is a hydrophilic BCS class III drug 31 with high solubility, low permeability, and low bioavailability of 17%. The low bioavailability may be attributable to chemical degradation in the stomach rather than presystemic metabolism. 76 In addition to being a potential OATP1B1 probe substrate, pravastatin is taken up by OAT3 in kidney proximal tubule cells and undergoes tubular secretion. 77 The SLCO1B1 521T>C and the haplotype SLCO1B1*15 are associated with decreased pravastatin clearance. 68 Conversely, several studies have shown that pravastatin may not be a sensitive probe for OATP1B. Individuals expressing SLCO1B1*15 had a greater decrease in pitavastatin clearance compared with pravastatin. This suggests that pitavastatin may be more sensitive than pravastatin. 78 In addition, the effect of rifampin on pravastatin exposure was minimal (2-fold) 79 compared with the 6- to 8-fold increase in pitavastatin discussed previously. 26 Approximately 50% of pravastatin s total clearance is due to renal elimination partly involving tubular secretion by OAT3. 80 Compared with rosuvastatin and pitavastatin, pravastatin is not a BCRP substrate (Table 1). 81 From all of this taken together, pravastatin does not appear to be an ideal OATP1B probe substrate because of the contribution of OAT3 to its disposition and its lower sensitivity to OATP1B1 activity compared with pitavastatin.

7 S88 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) Rosuvastatin. Rosuvastatin has been proposed as an in vivo OATP probe substrate. Its general properties are summarized in the BCRP section. Rosuvastatin has low passive membrane permeability, limited metabolism, with its disposition dependent on uptake and efflux transporters. Rosuvastatin is a substrate for OATP1B1, OATP1B3, and OATP2B1, 82 but is predominantly transported by OATP1B1. 14 The relative contribution of OATP1B1 was estimated to be 43% 55% of rosuvastatin s total uptake. 14 In vitro and animal studies have shown that rosuvastatin is transported by MRP2 and BCRP into the bile. 14,83 Approximately 72% of absorbed rosuvastatin is eliminated unchanged in bile and 28% in urine. 84 Some data support the role of OATP1B1 and OATP1B3 in rosuvastatin s disposition. In knockout mice, the absence of oatp1a/1b caused an 8-fold increase in systemic exposure, which was attributed to a decrease in the hepatic extraction ratio. 85 In addition, SLCO1B1 521T>C increased rosuvastatin concentrations versus the wild type. 86 However, it has also been shown that sodium/taurocholate cotransporting polypeptide (NTCP) may have the ability to compensate for impaired hepatic uptake by OATP in a knockout mouse model In the absence of oatp1a/1b, systemic concentrations of rosuvastatin were increased; however, hepatic concentrations were not decreased. Whether this is true in humans remains to be investigated. If the role of NTCP in the uptake of rosuvastatin is confirmed in humans, then this would be a limitation to the use of rosuvastatin as an OATP1B1 probe substrate. Hepatic uptake is generally thought to be the rate-limiting step in clearance, so if NTCP can compensate for OATP uptake activity, then measurement of rosuvastatin exposure would not solely reflect OATP. Rosuvastatin C max and AUC are markedly increased in healthy subjects with the ABCG2 421AA genotypesversustheabcg2 421CA and ABCG2 421CC genotypes, suggesting that BCRP plays a major role in the disposition. 90 Approximately 25% of total rosuvastatin clearance is due to renal elimination via OAT3. 91 If rosuvastatin is being used as an OATP1B1 probe, then the renal elimination would need to be accounted for and any changes in OAT3 activity would need to be considered. Finally, in a clinical study, the OATP1B1 inhibitor rifampin was given intravenously and orally together with rosuvastatin. Rifampin increased rosuvastatin C max (6- to 9-fold) and increased AUC (3- to 5-fold), with a greater impact with oral administration. This suggests rifampin s effect on rosuvastatin is primarily presystemic. 92 Therefore, rosuvastatin is not a selective probe for hepatic OATP because of involvement of NTCP, BCNP, MRP2, MPR4, P-gp, and OAT3. OATP Inhibitors Cyclosporine. Cyclosporine is a potent OATP inhibitor with an in vitro K i of 0.24 μm. 93 Cyclosporine also inhibits CYP3A, P-gp, BCRP, and MRP2 in the intestine and the liver. The hypothesis that cyclosporine inhibits uptake transporters in the liver stems from clinical studies. Fluvastatin, pitavastatin, pravastatin, and rosuvastatin are not metabolized to a significant extent by CYP3A, yet cyclosporine increased their AUC by 4- to 20-fold, The mechanism for the interaction is thought to be due in part to cyclosporine inhibition of OATP-mediated uptake of the statins into the liver. In a pig model that measured portal, hepatic, and femoral vein drug concentrations, intravenous cyclosporine increased rosuvastatin AUC and C max 9.1- and 16-fold, 98 respectively, in the hepatic vein. In the portal vein, AUC and C max were only increased by 1.6- and 2.0-fold, respectively. The volume of distribution was decreased by 80%, which suggests that the mechanism of the interaction was inhibition of rosuvastatin uptake from the liver. 98 Cyclosporine may be a useful OATP inhibitor to use in clinical studies to assess whether a compound is an OATP substrate or as a positive control to assess whether a compound is an OATP inhibitor. It is important to recognize that CYP3A, P-gp, and other transporters affect cyclosporine s disposition and, if relevant to the unknown compound, may complicate the assessment of OATP activity. Rifampin. Rifampin is an antibiotic used in the treatment and prevention of various infections including tuberculosis. Rifampin is a potent OATP inhibitor with an in vitro K i of μm. 93, Rifampin IC 50 was 0.27, 0.053, and 0.14 μm for pitavastatin, rosuvastatin, and pravastatin, respectively. 104 A single oral dose of 600 mg rifampin increased plasma concentrations of pitavastatin to a greater extent with oral versus intravenous dosing in cynomolgus monkeys. 105 This suggests that oral rifampin may inhibit gut efflux transporters such as BCRP and MRP2. This possibility was confirmed in a healthy human study that demonstrated a single 600-mg intravenous dose of rifampin was a more selective OATP1B1 inhibitor, as intravenous rifampin caused a 2-fold increase in pitavastatin C max compared with oral rifampin. 92 In contrast, oral rifampin caused a higher rosuvastatin C max, and AUC increased versus intravenous rifampin. In vitro studies showed that oral rifampin also inhibits OATP1B3, BCRP, and MRP2 in the intestine. 92 A single oral dose of rifampin has also been shown to inhibit intestinal P-gp in a clinical study with digoxin. 106 Rifampin activates the nuclear pregnane X receptor, which in turn affects CYP3A, P-gp, and other transporters. In summary, intravenous rifampin appears to be a more

8 Momper et al S89 selective OATP inhibitor compared with oral rifampin and a more potent inhibitor than cyclosporine. Organic Cation Transporter 2 (OCT2) OCT2 (SLC22A2) is an uptake transporter belonging to the solute carrier superfamily. In humans, OCT2 is most strongly expressed in the kidney, where it has the highest mrna expression of all cationic transporters. 107 OCT2 is localized to the basolateral membrane of renal proximal tubules, where it mediates the uptake of exogenous and endogenous small cations, thereby controlling the first step in in the renal secretion of many clinically relevant drugs. 107 The expression of OCT2 in the kidney is under hormonal regulation, and therefore sex and age differences may exist in the functional activity of this pathway Current FDA guidance documents and the ITC recommend determining whether an investigational drug is an OCT2 substrate or inhibitor using cell-based in vitro screening tools. For investigational drugs determined to be in vitro OCT2 inhibitors, the FDA and the ITC recommend a clinical study with metformin as a OCT2 probe substrate. 1,4 OCT2 Substrate Metformin. Metformin is cationic antihyperglycemic drug for type 2 diabetes mellitus. The absolute oral bioavailability is 50% 60%. 111 Metformin is not significantly bound to plasma proteins but does partition into red blood cells. 112 Metformin is excreted unchanged in the urine and is neither significantly metabolized in the liver nor excreted in bile. In subjects with normal renal function, the population mean for renal clearance is 510 ± 120 ml/min, which is approximately 3.5 times greater than creatinine clearance. 113 Therefore, active tubular secretion is the principal route of elimination for metformin. Metformin is a known substrate of human OCT2. In HEK293 cells, metformin uptake is markedly increased in cells transfected with human OCT2, with an apparent K m of 1.38 ± 0.21 mm. 114 The OCT2 inhibitor cimetidine reduced the renal clearance of metformin by an average of 27% (P <.008). 115 Genetic polymorphisms of OCT2 have also been identified and have been shown to partially explain variability in the renal clearance of metformin Despite regulatory recommendations that metformin be used as an OCT2 probe substrate with suspected OCT2 inhibitors, the drug lacks specificity. MATE1 (SLC47A1) and MATE2 (SLC47A2) are also involved in the transport of metformin across the apical membrane of renal tubule cells and thus work in tandem with OCT2 to facilitate renal secretion The role of MATEs in in vivo renal handling of metformin has been established. In a crossover study in 8 healthy volunteers, administration of the MATE inhibitor pyrimethamine reduced metformin renal clearance by 35%. 122 In an in vitro study using HEK293 cells transiently transfected with human OCT1 and OCT2, metformin was shown to be a substrate for both OCT2 and OCT1, although OCT2 had an approximately 10-fold greater capacity on the basis of intrinsic clearance, that is, V max /K m (54.49 ± 4.64 vs 5.09 ± 0.52 μl/min/fmol mrna). 123 Furthermore, in humans, the net metformin clearance from tubular secretion (renal clearance minus creatinine clearance) increases as the number of inactive OCT1 alleles increases. 124 These data suggest that OCT1 contributes to metformin reabsorption in the kidney. Overall, the current understanding of metformin renal handling is that OCT2 contributes to uptake across the basolateral membrane, whereas MATE1 and MATE2 are involved in excretion across the apical membrane and OCT1 is involved in reabsorption across the apical membrane. Therefore, metformin must be used cautiously as an OCT2 probe substrate because altered metformin disposition can be due to changes in the renal OCT2, MATE1/MATE2, and OCT1 transport pathways. A thorough in vitro assessment of the investigational drug of interest (including interactions with MATE1/MATE2 and OCT1) should precede clinical studies with metformin to aid in the interpretation of results. OCT2 Inhibitor Cimetidine. Cimetidine is a proposed OCT2 inhibitor, as IC 50 for cimetidine inhibition of OCT2- mediated transport in HEK293 cells stably expressing human OCT2 was 120 μm. 125 It is now recognized that cimetidine is a nonspecific OCT2 inhibitor that also interacts with multiple anion transporters in the kidney, including OAT1 and OAT Cimetidine is also a known CYP1A2 inhibitor. Organic Anion Transporter 1 (OAT1) and OAT3 OAT1 and OAT3 are highly expressed on the basolateral membrane of the renal proximal tubule and facilitate the active uptake of anionic substrates from blood in exchange for intracellular α-ketoglutarate. 107 The renal anion transport system plays a major role in the disposition of a variety of drugs, including antibiotics, antivirals, ACE-Is, diuretics, and antineoplastics. Few nonsynonymous OAT1 or OAT3 variants have been identified, and none are associated with loss of function. 129 Additional homologues with different substrate specificities have been cloned (OAT2, OAT4, OAT5, OAT6), 130 although their importance is not well understood from a drug interaction perspective. OAT1 and OAT3 often have overlapping substrates and inhibitors, rendering it difficult to quantitate the relative contribution without knocking out individual

9 S90 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) transporters experimentally. The prototypical OAT substrate para-aminohippurate (PAH) as well as the OAT1/OAT3 probe substrates recommended by FDA guidance and the ITC white paper (zidovudine, tenofovir, ciprofloxacin, acyclovir, methotrexate) are reviewed. 1,2,4 OAT1/OAT3 Substrates Para-aminohippurate. PAH is an organic anion with PAH renal clearance used as a diagnostic tool to estimate renal plasma flow. The PAH extraction ratio on a single passage through the kidney is approximately 90% because of (1) extensive secretion by the tubules, (2) free filtration from plasma by the glomerulus, and (3) nonreabsorbtion by the tubules. Human OAT1 efficiently transports PAH. In hoat1-expressing Xenopus laevis oocytes, the uptake of 1 μm PAHwas2.7 ± 0.4 pmol/oocyte after 30 minutes with a K m of 3.1 ± 0.8 μm and 4.0 ± 1.5 μm in the absence and presence of a chloride buffer, respectively. 131 OAT4 and OAT3 show no or limited PAH transport activity relative to OAT1. 131,132 In humans, PAH can be used to measure the effective renal plasma flow and the maximum tubular secretory capacity (difference between the total rate of excretion and quantity filtered by the glomeruli), which is mostly attributable to OAT1 activity. Clearance measurements using a single dose of PAH are generally inaccurate, necessitating continuous intravenous infusions to sustain the plasma PAH concentration. Despite its advantages, PAH is not commonly used in drug drug interaction studies because of the technical and logistical challenges. PAH was used in combination with iohexol to assess the effect of the antiviral drug dolutegravir on renal filtration and effective renal plasma flow in healthy subjects. 133 This study was conducted following in vitro results demonstrating dolutegravir inhibition of OAT1 (IC 50 = 2.12 μm) and OAT3 (IC 50 = 1.97 μm). In the clinical study, for PAH clearance pre- and postdolutegravir treatment after adjusting for placebo, the geometric mean ratios (90%CIs) for PAH clearance were 1.03 ( ) for dolutegravir 50 mg once daily and 0.97 ( ) for dolutegravir 50 mg twice daily. 133 These results suggest that dolutegravir does not significantly affect OAT1 activity in vivo and that clinically relevant drug drug interactions for OAT1 substrates are not expected. Zidovudine. Zidovudine is a nucleoside reverse transcriptase inhibitor indicated in for the treatment of HIV-1 infection. Zidovudine is orally administered, with an absolute oral bioavailability of 64% ± 10% and low plasma protein binding (<38%). The elimination of zidovudine is primarily from hepatic metabolism via UGT2B7-mediated glucuronidation as well as oxidative metabolism by CYP2C9, CYP2E1, CYP3A4, and CYP2A Zidovudine is transported by human OATs with K m values of uptake by hoat1, hoat2, hoat3, and hoat4 of 45.9, 26.8, 145.1, and μm, respectively. 135 Approximately 14% of the parent compound and 74% of the glucuronidated metabolite are eliminated in urine. Renal clearance is 0.34 ± 0.05 L h/kg or approximately 400 ml/min/70 kg, which is 3-fold higher than the glomerular filtration rate (GFR) for a patient with normal renal function, indicating tubular secretion by the aforementioned OAT isoforms. In a small number of subjects (n = 3), probenecid increased the AUC of zidovudine by 106%. Overall, because of the large degree of hepatic metabolism, oral bioavailability variability, and multiple OAT isoforms involved in renal secretion, zidovudine is a poor candidate as an OAT1/OAT3 probe substrate. However, it is widely used in the treatment of HIV-1 infection and therefore may be a suitable opportunistic probe in clinical trials in which zidovudine is coadministered with an investigational drug. Tenofovir. Tenofovir is an antiviral agent indicated for the treatment of HIV-1 infection and chronic hepatitis B. It is administered orally as tenofovir disoproxil fumarate (tenofovir DF) or as tenofovir alafenamide (tenofovir AF), which are both prodrugs converted to tenofovir. Oral bioavailability is approximately 25%, and protein binding is low (<1% to 7.2% over concentrations ranging from 0.01 to mg/l). Neither tenofovir nor tenofovir DF is metabolized by hepatic CYP enzymes. Rather, tenofovir is renally eliminated by glomerular filtration and tubular secretion, with 70% 80% of the administered dose recovered in urine. 136 The renal clearance of tenofovir is 2 to 3 times the creatinine clearance, demonstrating tubular secretion. Tenofovir tubular secretion involves basolateral uptake by OAT1 and apical efflux out into urine by multidrug resistance protein 4 (MRP4) Tenofovir AF results in higher intracellular concentrations of tenofovirdiphosphate (active metabolite), but 90% lower tenofovir plasma concentrations, which is believed to improve the renal and bone safety profile. 140 Tenofovir is most useful as a probe for OAT-mediated secretion when used in an opportunistic fashion in studies of HIV-1-infected patients taking tenofovir as standard of care. For example, a clinical study explored the effect of lopinavir/rinonavir on the renal clearance of tenofovir in HIV-infected patients. 141 After adjusting for renal function, tenofovir renal clearance was 17.5% lower (P <.04) in patients taking lopinavir/ritonavir versus those not taking a protease inhibitor. However, as tenofovir renal secretion involves both OAT1 and MRP4, the specific pathway responsible for

10 Momper et al S91 this marginal renal clearance reduction cannot be conclusively identified. 141 Methodological issues exist with this study, and others have argued that the interaction between protease inhibitors and tenofovir more likely involves intestinal P-glycoprotein and oral absorption of tenofovir DF. 142,143 Ciprofloxacin. Ciprofloxacin is a fluoroquinolone antibiotic indicated for various infections caused by susceptible isolates. Following intravenous administration, approximately 80% of the dose is eliminated in urine as either unchanged parent compound or metabolites. Ciprofloxacin renal clearance is nearly 3 times the GFR and is probenecid sensitive (ie, probenecid reduces renal clearance to a value close to the GFR). In Xenopus laevis oocytes and cell monolayers, OAT3 promoted uptake of ciprofloxacin (moat3; K m = 70 ± 6 μm) with no interaction with either the human or mouse isoforms of OAT This finding was corroborated in Oat3-null mice, in which ciprofloxacin elimination was reduced. 144 Therefore, ciprofloxacin offers an advantage as a more sensitive OAT3 probe substrate than OAT1, although human phenotyping studies are lacking. Acyclovir. Acyclovir is a nucleoside analogue that inhibits the replication of herpes simplex viruses and varicella zoster virus. Approximately 85% of an intravenous dose is eliminated unchanged in the urine. As renal clearance is about 3 times that of GFR, tubular secretion plays a major role in overall clearance. The renal clearance of acyclovir is sensitive to probenecid and cimetidine, signifying the involvement of secretion in addition to filtration. 145,146 Acyclovir is not extensively bound to plasma proteins (<15%). Acyclovir is a substrate of hoat1 and hoct1, with K m values of uptake of and μm, respectively, although hoct1 is mostly expressed in hepatocytes and does not significantly contribute to renal elimination. 147 Acyclovir is not transported by hoat3, hoat2, or hoat4. On the other hand, the apical efflux may be mediated in part by MATE1 and MATE2, although low K m values of 2.64 and 4.32 mm, respectively, bring into question whether these transporters are involved in vivo. 147,148 Clinical phenotyping studies are lacking, but acyclovir has been successfully used in animal studies to evaluate the OAT1 transport pathway. 149,150 Methotrexate. Methotrexate is a folate antimetabolite indicated for various cancers as well as rheumatoid arthritis. Methotrexate clearance involves renal elimination through glomerular filtration and active renal secretion, with limited biliary excretion (<10%). Methotrexate uptake at the tubular basolateral membrane is facilitated by hoat3 and hoat1, and efflux across the apical membrane involves hoat In addition, saturable reabsorption occurs at the apical surface of the distal tubules, which may involve OATP1A2 uptake. 152 Moreover, methotrexate disposition is complex, and interindividual PK variability is high, possibly because of involvement of P-gp and BCRP. 10 Because of the multiple pathways involved in methotrexate elimination as well as saturable reabsorption in the kidney, methotrexate is not an ideal candidate as an OAT1/OAT3 probe substrate. OAT1/OAT3 Inhibitor Probenecid. Probenecid (p-dipropylsulfamoyl benzoic acid) is the classical inhibitor of renal drug secretion, initially developed to decrease the renal excretion of penicillin. 153 Oral probenecid is well absorbed from the gastrointestinal tract and undergoes significant hepatic metabolism, with a plasma half-life of 4 12 hours. 153 Probenecid and its metabolites are renally eliminated, with 5% 10% of the dose appearing in the urine as the parent compound. 153 Probenecid competitively inhibits the active transport (and reduces the renal clearance) of anionic drug molecules in the kidney. Probenecid inhibits OAT1 and OAT3, and therefore a drug drug interaction study with probenecid does not discriminate between these 2 pathways Probenecid inhibition of OAT1/OAT3 generally results in a reduction in renal clearance to a value approaching the GFR. Cidofovir renal clearance, when coadministered with probenecid, is reduced to 87.3 ± 30 ml h/kg (n = 15), which is nearly indistinguishable from creatinine clearance in the same patients (93.4 ± 24.7 ml h/kg; n = 12), indicating near-total inhibition of renal secretion. 157 Future Directions Criteria to evaluate the proposed in vivo BCRP, OATP1B1, OATP1B3, OCT2, OAT1, and OAT3 probes for appropriateness are lacking. The development of criteria is needed and would aid in the design of transporter-mediated drug drug interaction studies as well as provide a starting point to determine the suitability of a probe. Criteria have been proposed for P-gp probes, with an emphasis on specificity, sensitivity, and feasibility. 7,9 Examples of P-gp probe specificity are evidence of no enzyme-mediated metabolism and no involvement of non-p-gp transporters. Examples of P-gp probe sensitivity are the presence of high efflux ratios in bidirectional transport assays and evaluation during inhibitory and induction conditions. Examples of P-gp feasibility criteria are an assessment of probe safety and tolerability, widespread availability for use, and invasiveness of study procedures. 7,9 Some of the proposed P-gp criteria can apply to the transporter probes discussed in this review. However, whether all criteria are applicable remains to be seen. The P-gp criterion of no involvement of non-p-gp transporters