Inhibitory effects of p-aminohippurate and probenecid on the renal clearance of adefovir and benzylpenicillin as probe drugs for organic anion transporter (OAT) 1 and OAT3 in humans
Graphical abstract
Introduction
Drug–drug interactions (DDIs) involving the inhibition of metabolism or excretion increase systemic exposure to victim drugs, leading to their accumulation in the body, and consequently potentiate their pharmacological/adverse effects. To avoid life-threatening events caused by DDIs in the postmarketing stage, US and EU regulatory agencies recently published new draft guidance and guideline on the appropriate risk evaluation of DDIs in the process of drug development (EMA, 2012, FDA, 2012). One of the major revisions in these documents includes the addition of descriptions with decision trees for the risk assessment of transporter-mediated DDIs. These changes were made because various human drug transporters have been characterized extensively and several clinical studies have shown the in vivo significance of these transporters to the effects of coadministered inhibitor drugs and genetic polymorphisms of certain transporters on the pharmacokinetics of clinically used substrate drugs (Konig et al., 2013, Yoshida et al., 2013).
To quantify the DDI risk of an investigational drug as either a victim or perpetrator in humans, it is convenient to use in vivo-selective substrates for, and inhibitors of, metabolic enzymes/transporters in clinical DDI studies. In the field of metabolic enzymes, in vivo probe substrates and inhibitors are well established, and cocktail dosing of probe substrates enables one to determine the in vivo activity of multiple enzymes simultaneously. However, few validated probe drugs that can sensitively detect alterations in the transport activity of a specific transporter have been found.
The urinary excretion mechanism for most anionic drugs comprises both active tubular secretion and glomerular filtration in the kidney (Ito et al., 2013). We have demonstrated previously that the transporters that mediate the uptake process from the blood circulation act as the rate-determining process in the overall tubular secretion of anionic drugs in the kidney (Watanabe et al., 2011, Watanabe et al., 2009). Because of their broad substrate specificities, organic anion transporter 1 (OAT1/SLC22A6) and OAT3/SLC22A8 are considered to be the major transporters in the kidney responsible for the basolateral uptake of various organic anions including drugs and uremic toxins. OAT1 plays a major role in the renal uptake of hydrophilic and small organic anions such as p-aminohippurate (PAH), 2,4-dichlorophenoxyacetate, and acyclic nucleotide phosphonates, whereas OAT3 has a broader substrate specificity than OAT1 and accepts amphipathic and hydrophobic organic anions, and even some organic cations (cimetidine) and zwitterions (fexofenadine) (Burckhardt, 2012, Kusuhara and Sugiyama, 2009). Probenecid has been used to characterize the urinary excretion mechanisms of drugs. Probenecid is a potent inhibitor of both OAT1 and OAT3, and coadministration of probenecid at a therapeutic dose causes a marked inhibition of the tubular secretion of OAT1 and OAT3 substrate drugs (Kusuhara et al., 2013, Shitara et al., 2005). Thus, both the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) selected these transporters as important transporters for drug disposition in their DDI guideline and draft guidance (EMA, 2012, FDA, 2012). However, the selective inhibitors and selective substrates for OAT1 and OAT3 that can be used in clinical situations and the appropriate clinical protocols for these probe drugs have not been established in humans.
The purpose of this study was to understand quantitatively the transport functions of OAT1 and OAT3 by the use of probe substrate drugs and inhibitors in vivo in humans. Based on the published and in house information, we selected adefovir and benzylpenicillin as the probe substrate drugs for OAT1 and OAT3, respectively, because of their selective recognition by each OAT isoform and low renal clearance compared with renal blood flow rate (Cihlar et al., 1999, Nozaki et al., 2007, Vanwert et al., 2007). To evaluate OAT1- and OAT3-mediated transport in a clinical study, PAH and probenecid were selected as inhibitors. PAH is a prototypical OAT1 substrate; because its Michaelis constant (Km) is much larger for OAT3 than for OAT1, it can discriminate OAT1-mediated uptake from the net renal uptake in vitro (Deguchi et al., 2004, Nozaki et al., 2007, Tahara et al., 2005a). Importantly, PAH has been used clinically to measure the capacity of renal secretion clearance by saturating this process (i.e., OAT1-mediated uptake (Vallon et al., 2008)), indicating that PAH can also inhibit OAT1-mediated transport in vivo. By contrast, we found no information about any OAT3-selective inhibitor that would be applicable to a clinical DDI study. Therefore, we decided to use probenecid because it inhibits both OAT1 and OAT3 (Tahara et al., 2005a, Takeda et al., 2001). This study intended to provide a rationale for the use of adefovir and benzylpenicillin as probe drugs for OAT1 and OAT3, respectively, and the use of PAH as an inhibitor to investigate the importance of OAT1 in the renal elimination of test drugs.
Section snippets
Chemicals
[14C]-Benzylpenicillin (52.6 mCi/mmol) and [3H]-adefovir (10 Ci/mmol) were purchased from Moravek Biochemistry, Inc. (Brea, CA). [3H]-p-Aminohippurate (PAH; 4.1 Ci/mmol), [14C]-inulin (8 mCi/mmol), and [3H]-dehydroepiandrosterone sulfate (DHEAS; 74 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Unlabeled adefovir and tenofovir were kindly provided by Gilead Science, Inc. (Forest, CA). Unlabeled PAH and DHEAS were purchased from Sigma–Aldrich (St Louis, MO), and unlabeled
Uptake of adefovir and benzylpenicillin by OAT1 and OAT3, and inhibitory effects of PAH and probenecid on their uptake
The uptake of adefovir and benzylpenicillin was assessed in OAT1- and OAT3-expressing HEK293 cells. Significant uptake of [3H]-adefovir and [14C]-benzylpenicillin (0.1 μM including unlabeled compounds) was observed in OAT1- and OAT3-expressing HEK293 cells, respectively (Fig. 1A and B). PAH preferentially inhibited the OAT1-mediated uptake of [3H]-adefovir compared with the OAT3-mediated uptake of [14C]-benzylpenicillin (Ki values: 6.47 ± 1.51 μM for adefovir and 142 ± 27 μM for benzylpenicillin [mean ±
Discussion
This study aimed to validate human in vivo probe substrates (adefovir and benzylpenicillin) and inhibitors (PAH and probenecid) for studying the renal uptake transporters OAT1 and OAT3 by quantitatively evaluating the results of in vitro inhibition studies and a clinical DDI study.
Consistent with previous reports (Cihlar et al., 1999, Tahara et al., 2005a), we observed that adefovir and benzylpenicillin were specifically transported by OAT1 and OAT3, respectively, into their cDNA-transfected
Conclusions
The present study demonstrated that adefovir and benzylpenicillin can be used as in vivo probe substrate drugs for OAT1 and OAT3, respectively, in clinical studies. Intravenous infusion of PAH at a dosing rate of >120 mg/min/person will show the in vivo role of OAT1 in the renal elimination of drugs, and oral administration of probenecid (1500 mg) will show the net contribution of OAT1 and OAT3 to the overall renal uptake of drugs. These probe substrates for, and inhibitors of, OAT1 and OAT3 will
Conflict of interest
All the authors declare no conflict of interest.
Acknowledgments
This work was partly supported by a Grant-in-Aid for Scientific Research (S) (24229002) (to Y.S. and K.M.) and a Grant-in-Aid for Scientific Research (B) (23390034) (to H.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Japan Research Foundation for Clinical Pharmacology (to K.M.).
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These authors contributed equally to this paper.