1521-009X/41/4/791–800$25.00 http://dx.doi.org/10.1124/dmd.112.049569 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 41:791–800, April 2013 Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics Organic Anion Transporter 3 Interacts Selectively with Lipophilic b-Lactam Antibiotics Aaron T. Wolman, Michael R. Gionfriddo, Gregory A. Heindel, Paran Mukhija, Sarah Witkowski, Ajay Bommareddy, and Adam L. VanWert Department of Pharmaceutical Sciences, Nesbitt College of Pharmacy and Nursing, Wilkes University, Wilkes-Barre, Pennsylvania (A.T.W., G.A.H., P.M., S.W., A.B., A.L.V.); and Knowledge and Evaluation Research Unit, Clinical and Translational Sciences, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota (M.R.G.) Received October 9, 2012; accepted January 23, 2013 ABSTRACT Transporters are major determinants of the disposition of xeno- biotics and endogenous chemicals in the body. Organic anion transporter 3 (Oat3) functions in the kidney and brain to remove metabolic waste, toxins, and drugs, and thus transports diverse chemicals. Some b-lactam antibiotics interact with Oat3, and penicillin G exhibits a strong dependence on Oat3 for renal elimination. However, over 80 b-lactams exist, and many have not been assessed for an interaction with Oat3. Moreover, b-lactams continue to receive U.S. Food and Drug Administration approval. This study identified new b-lactam–Oat3 interactions, provided a head-to-head comparison with Oat1, and characterized the physicochemical determinants of affinity for Oat3. Cells expressing mouse Oat3 (mOat3) and Oat1 (mOat1), and human OAT3 (hOAT3) were used to test inhibitors, and high-performance liquid chroma- tography (HPLC) was used to measure transport. Of 26 b-lactams tested, 12 were clear inhibitors of Oat3, and 14 exhibited poor interactions. Inhibitors exhibited a nearly identical rank-order of potency against mOat3 and hOAT3. Oat1 demonstrated a poor interaction with most b-lactams. The majority of Oat3 inhibitors were substrates, and there were clear physicochemical differences between inhibitors and noninhibitors. That is, inhibitors had nearly 40% fewer hydrogen bond donors (P < 0.001), a lower total polar surface area (P < 0.05), and greater lipophilicity (LogP of inhibitors, +1.41; noninhibitors, 21.54; P < 0.001). Pharmacophore mapping revealed a prohibitive hydrogen bond donor group in noninhibitors adjacent to a hydrophobic moiety that was important for binding to Oat3. These findings indicate that Oat3 recognizes lipophilic b-lactams more readily. Moreover, this study has potential implications for designing b-lactams to avoid renal accumulation or brain efflux via Oat3. Introduction Organic anion transporters (OATs) are plasma membrane proteins in the solute carrier family (SLC) (Srimaroeng et al., 2008; VanWert et al., 2010). The role of OATs in physiology and xenobiotic disposition has been under investigation for approximately a century; however, the genetic and amino acid identities of individual members of this group have been revealed only in the past 15 years. OATs mediate drug and toxicant disposition (Sweet, 2005), and their promiscuity lends them a very significant role in pharmacokinetics and pharmacodynamics (Burckhardt and Burckhardt, 2003; VanWert et al., 2010). For example, deletion of renal basolateral Oat1 resulted in a clear reduction in furosemide effectiveness as a result of impaired delivery to its luminal target (Eraly et al., 2006). Likewise, Oat3 knockout mice demonstrated a striking impairment in penicillin G elimination (Vanwert et al., 2007). A brief search of drug interactions using standard databases (e.g., Micromedex) reveals an overwhelming number of warnings related to impaired renal drug secretion. The OATs are considered one major group of transporters central to these untoward renal drug-drug interactions (Burckhardt and Burckhardt, 2011). Oat3 was identified first in mice in a chromosomal region containing the osteosclerosis (oc) mutation (Brady et al., 1999). It was initially named Roct (reduced in oc transporter) because of its downregulation in the kidneys of oc/oc mice. The first functional characterization of Roct (the rat ortholog) resulted in its renaming to Oat3 (Kusuhara et al., 1999). Oat3 protein is expressed (Kojima et al., 2002) and functional (Sweet et al., 2003) in the basolateral membrane of the renal proximal tubule and the blood-brain barrier (Miyajima et al., 2011), and the apical membrane of the choroid plexus (Nagata et al., 2004; Sykes et al., 2004). Thus, its role in drug disposition is multifaceted. In the kidney, Oat3 facilitates the first step of secretion. For some substrates, this precedes apical efflux and excretion; however, for others, such as the b-lactam cephaloridine, renal cells are also the toxicologic target (Sweet, 2005). In addition, Oat3 removes anions from cerebrospinal fluid via the choroid plexus (Nagata et al., 2004; Sykes et al., 2004) and from the brain via the blood-brain barrier (Miyajima et al., 2011). Therefore, although Oat3 function may facilitate toxicity and/or drug pharmacodynamic action in the kidney, Oat3 action has the opposite influence in the brain and the remainder of the body. That is, higher Oat3 function yields a higher elimination rate for its substrates, and lower Oat3 function may yield systemic toxicity, or alternatively, a more pronounced therapeutic effect. For these reasons, achieving a greater understanding of the substrate profile and, perhaps of equal or greater importance, This work was supported by the Mentoring Task Force and a Type I grant at Wilkes University. dx.doi.org/10.1124/dmd.112.049569. ABBREVIATIONS: CHO, Chinese hamster ovary; HEK293, human embryonic kidney 293; hOat, human organic anion transporter; HPLC, high- performance liquid chromatography; mOat, mouse organic anion transporter; Oat3, organic anion transporter 3; oc, osteosclerosis. 791 at Mayo Clinic Library on March 19, 2013 dmd.aspetjournals.org Downloaded from