Traditionally, clinical pharmacogenetic studies have begun with identifying a known phenotype (e.g., an aberrant response to a drug) followed by isolating the variant protein and gene responsible for that phenotype. With the sequence of most human genes known, it is possible to carry out pharmacogenetic studies in the reverse manner (i.e., genotype to phenotype). Patients with a known variant of a gene can be studied for their response to certain drugs, which can help elucidate the importance of that gene in drug response. In the proposed study, we plan to use a genotype to phenotype strategy to study the role of OAT3 and related variants in drug response.~Renal elimination of anionic drugs occurs by filtration and active secretion. For secretion, the drug is transported from the blood into the renal tubular cell through the basolateral membrane. This transport occurs against both a concentration and electrochemical gradient, and must therefore be mediated by organic anion transporters (OATs). To date, six human OATs have been identified, OAT 1-5, and OAT7. OAT1 and OAT3 are the dominant OATs found on the basolateral aspect of the renal proximal tubular cells and are hence suspected of playing a significant role in renal anionic drug elimination [3, 4].~Active renal tubular secretion of most beta-lactam antibiotics, including cephalosporins, has long been recognized, with OATs likely playing a major role, though the relative role of each is unclear [5]. In vivo studies have demonstrated that the cephalosporin cefotaxime has a ten-fold higher affinity for OAT3 than OAT1 [6]. Thus, patients with OAT3 variants would be expected to exhibit altered renal secretion of cefotaxime.~Cefotaxime is a common cephalosporin used in the treatment of bacterial infections. Dosed up to 1-2 mg every 6-8 hours, it has been shown to be safe in patients with normal renal function. Cefotaxime is given intravenously, and is metabolized to desacetyl cefotaxime (DACM) which retains antibacterial activity. DACM is further metabolized to two inactive metabolites, M2 and M3; all four forms of cefotaxime are renally eliminated, with active secretion representing a significant percentage of total clearance (44% for cefotaxime, 64% for DACM) [7]. The half-life of cefotaxime and DACM are 1 and 1.5 hours, respectively. Patients with poorly functional OAT3 and related variants might be expected to exhibit reduced tubular secretion of both cefotaxime and its metabolites, and result in higher or prolonged blood levels. Because such variants may reduce the amount of cefotaxime that enters the tubular cells, they may also be associated with a reduced incidence of cephalosporin-induced nephrotoxicity.~The PMT group has a collection of 500 DNA samples from young, healthy adults from four major ethnic groups (125 each from Caucasians, African-Americans, Mexicans, and Chinese). This collection (The Pharmacogenetics of Membrane Transporters), is referred to as SOPHIE (Study of Pharmacogenetics in Ethnically-diverse Populations), and includes only volunteers who have consented to be called back for consideration of enrollment in future studies.~In recent studies, we identified nine non-synonymous OAT3 variants among subjects participating in the SOPHIE study. We cloned and introduced each variant into a heterologous expression system and tested the encoded transporters for their ability to transport estrone sulfate (a documented substrate for OAT3). Four variants were identified that resulted in a complete loss of function: F129L (in one Hispanic subject), R149S (in an Caucasian and an African American subject), Q239stop (in the same African American subject), and R277W (in an Asian subject). In the subject with two non-functional variants, it is unknown whether the variants occur on the same chromosome, or if the variants are on different chromosomes. The latter would potentially make the subject completely OAT3 deficient. An additional variant (I305F) which showed reduced transport ability for some, but not all, OAT3 substrates was identified in three Asian and one Hispanic subject. One variant (A310V) which showed increased transport of estrone sulfate compared to the common allele was identified in a single Caucasian subject.~This study will address the following question: Do individuals who carry altered-function in OAT3 and related variants exhibit differences in the pharmacokinetics of cefotaxime in comparison to individuals who carry the common allele?
In the proposed study, we plan to use a genotype to phenotype strategy to study the role of the organic anion transporter, OAT3, in drug response. More specifically we will examine the contribution of OAT3 to the renal clearance of anionic drugs such as cefotaxime by studying individuals with a non-functional (or poorly-functional) variant of OAT3.