Pharmacokinetic Modeling and Monte Carlo Simulation to Predict Interindividual Variability in Human Exposure to Oseltamivir and Its Active Metabolite, Ro 64-0802.

Oseltamivir (Tamiflu®) is a prodrug of Ro 64-0802, a selective inhibitor of influenza virus neuraminidase. There is a possible relationship between oseltamivir treatment and neuropsychiatric adverse events; although this has not been established, close monitoring is recommended on the prescription label. The objective of this study was to predict interindividual variability of human exposure to oseltamivir and its active metabolite Ro 64-0802. By leveraging mathematical models and computations, physiological parameters in virtual subjects were generated with population means and coefficient of variations collected from the literature or produced experimentally. Postulated functional changes caused by genetic mutations in four key molecules, carboxylesterase 1A1, P-glycoprotein, organic anion transporter 3, and multidrug resistance-associated protein 4, were also taken into account. One hundred thousand virtual subjects were generated per simulation, which was iterated 20 times with different random number generator seeds. Even in the most exaggerated case, the systemic areas under the concentration-time curve (AUCs) of oseltamivir and Ro 64-0802 were increased by at most threefold compared with the population mean. By contrast, the brain AUCs of oseltamivir and Ro 64-0802 were increased up to about sevenfold and 40-fold, respectively, compared with the population means. This unexpectedly high exposure to oseltamivir or Ro 64-0802, which occurs extremely rarely, might trigger adverse central nervous system effects in the clinical setting.

Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity.

OBJECTIVE: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance. METHODS: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates. RESULTS: We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. CONCLUSION: OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human kidney drug transporters.

17alpha-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K(m) (Michaelis constant) kinetics (K(m) = 21.1 microM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K(m) model (K(m1) = 1.6 microM; K(m2) = 195 microM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.

Assessment of the renal toxicity of novel anti-inflammatory compounds using cynomolgus monkey and human kidney cells.

PF1, an anti-inflammatory drug candidate, was nephrotoxic in cynomolgus monkeys in a manner that was qualitatively comparable to that observed with the two previous exploratory drug candidates (PF2and PF3). Based on the severity of nephrotoxicity, PF1 ranked between the other two compounds, withPF2 inducing mortality at all doses and PF3 eliciting only mild nephrotoxicity. To further characterize nephrotoxicity in monkeys and enable direct comparisons with humans, primary cultures of proximal tubular (PT) cells from monkey and human kidneys were used as in vitro tools, using lactate dehydrogenase release as the biomarker of cytotoxicity. In both human and monkey PT cells, PF2was by far the most cytotoxic compound of the three drugs. PF1 exhibited modest cytotoxicity at the highest concentration tested in human PT cells but none in monkey kidney cells whereas PF3 exhibited the reverse pattern.Because these drugs are organic anions, mechanistic studies using human organic anion transporters 1 and 3 (hOAT1 andhOAT3) transfected cell lines were pursued to evaluate the potential of these compounds to interact with these transporters. All three drugs exhibited high affinity for hOAT3 (PF1 exhibited the lowest IC50 of 6M) but only weakly interacted with hOAT1 (with no interaction found for PF2). PF2 was a strong hOAT3 (not hOAT1) substrate, whereas PF1 and PF3 were substrates for both hOAT1 and hOAT3.Upon pretreatment of monkeys with the OAT substrate probenecid, PF3 systemic exposure (AUC) and half-life (t1/2) increased approximately 2-fold whereas clearance (CL) and volume of distribution (Vdss) decreased, as compared to naïve monkeys. This indicated that PF3 competed with probenecid for hOAT1 and/or hOAT3mediated elimination of PF3. Thus, hOAT1 and/or hOAT3 may be responsible for the uptake of this series of drugs in renal PT cells, which may directly or indirectly lead to the observed nephrotoxicity in vivo.

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains.

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.