Polyspecific organic cation transporters in the liver mediate the elimination of a wide array of endogenous amines and xenobiotics. In contrast to our understanding of the mechanisms of organic cation transport in rat liver, little is known about the mechanisms of organic cation transport in the human liver. We report the cloning, sequencing, and functional characterization of the first human polyspecific organic cation transporter from liver (hOCT1). hOCT1 (554 amino acids) is 78% identical to the previously cloned organic cation transporter from rat, rOCT1 [Nature (Lond.) 372:549-552 (1994)]. In Xenopus laevis oocytes injected with the cRNA of hOCT1, the specific uptake of the organic cation 3H-1-methyl-4-phenylpyridinium (3H-MPP+) was significantly enhanced (8-fold) over that in water-injected oocytes. Uptake of 3H-MPP+ was saturable (K(m) = 14.6 +/- 4.39 microM) and sensitive to membrane potential. Both small monovalent organic cations such as tetraethylammonium and N1-methylnicotinamide and bulkier organic cations (e.g., vecuronium and decynium-22) inhibited the uptake of 3H-MPP+. In addition, the bile acid taurocholate inhibited the uptake of 3H-MPP+ in oocytes expressing hOCT1. Northern analysis demonstrated that the mRNA transcript of hOCT1 is expressed primarily in the human liver, whereas the mRNA transcript of rOCT1 is found in rat kidney, liver, intestine, and colon [Nature (Lond.) 372:549-552 (1994)]. In comparison to rOCT1, hOCT1 exhibits notable differences in its kinetic characteristics and tissue distribution. The functional expression of hOCT1 will provide a powerful tool for elucidation of the mechanisms of organic cation transport in the human liver and understanding of the mechanisms involved in the disposition and hepatotoxicity of drugs.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:We investigated hepatic in vitro intrinsic clearance (CL int,in vitro ) in freshly isolated or cryopreserved hepatocytes and compared with CL int,in vivo by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDPglucuronosyltransferase, sulfotransferase, and/or esterase. CL int,in vitro was determined from substrate disappearance rate at 1 M in hepatocytes. CL int,in vivo was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL int,in vitro in freshly isolated hepatocytes, the rat scaling factor values (CL int,in vivo /CL int,in vitro ) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL int,in vitro values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL int,in vitro were determined by use of cryopreserved hepatocytes. When human CL int,in vitro was regarded as the predicted CL int,in vivo , the observed and predicted CL int,in vivo for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL int,in vitro corrected with the rat scaling factors yielded better predictions of CL int,in vivo that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.Recently, pharmacokinetic investigation has played an increasingly important role in drug discovery. In particular, it is very important to predict human hepatic metabolic clearance because most drugs are eliminated from the body predominantly by hepatic metabolism. For predicting hepatic clearance, theoretical aspects of in vitro/in vivo scaling based on a physiological model and clearance concepts have been developed (Roberts and Rowland, 1986). Application of this method has been successful in predicting in vivo hepatic clearance in rats for many drugs metabolized by P450 1 from in vitro metabolism data using rat liver microsomes and isolated hepatocytes (Sugiyama et al., 1988;Houston and Carlile, 1997). Because human liver samples have become more readily available, it would also be very useful to predict in vivo outcomes from in vitro data in humans. However, there has been relatively limited application of this approach (Hoener, 1994), and there have been some failed attempts at the prediction of human hepatic clearance. For example, Iwatsubo et al. (1997) and Houston and Carlile (1997) reported that CL int,in vitro generally exhibited a positive correlation with CL int,in vivo , but in some cases animal or human clearance values were not well predicted from in vitro studies. To improve the predict...

ABSTRACT:To verify the availability of pharmacokinetic parameters in cynomolgus monkeys, hepatic availability (Fh) and the fraction absorbed multiplied by intestinal availability (FaFg) were evaluated to determine their contributions to absolute bioavailability (F) after intravenous and oral administrations. These results were compared with those for humans using 13 commercial drugs for which human pharmacokinetic parameters have been reported. In addition, in vitro studies of these drugs, including membrane permeability, intrinsic clearance, and p-glycoprotein affinity, were performed to classify the drugs on the basis of their pharmacokinetic properties. In the present study, monkeys had a markedly lower F than humans for 8 of 13 drugs. Although there were no obvious differences in Fh between humans and monkeys, a remarkable species difference in FaFg was observed. Subsequently, we compared the FaFg values for monkeys with the in vitro pharmacokinetic properties of each drug. No obvious FaFg differences were observed between humans and monkeys for drugs that undergo almost no in vivo metabolism. In contrast, low FaFg were observed in monkeys for drugs that undergo relatively high metabolism in monkeys. These results suggest that first-pass intestinal metabolism is greater in cynomolgus monkeys than in humans, and that bioavailability in cynomolgus monkeys after oral administration is unsuitable for predicting pharmacokinetics in humans. In addition, a rough correlation was also observed between in vitro metabolic stability and Fg in humans, possibly indicating the potential for Fg prediction in humans using only in vitro parameters after slight modification of the evaluation system for in vitro intestinal metabolism.Because the development of new drugs is a cost-and labor-intensive task, the selection of candidates with good pharmacokinetic profiles is becoming increasingly common. This practice minimizes the number of drug candidates dropped due to pharmacokinetic problems during the clinical phase (Wishart, 2007).When predicting human pharmacokinetics, the fraction absorbed (Fa), intestinal availability (Fg), and hepatic availability (Fh) are the main factors to consider. Fh prediction has become considerably accurate since several mathematical prediction models have been established, including the physiological model, well stirred model, parallel tube model, and dispersion model (Iwatsubo et al., 1996;Naritomi et al., 2001;De Buck et al., 2007). For FaFg, however, no quantitative prediction method has ever been established, although several qualitative prediction methods using human intestinal microsomes have been reported (Chiba et al., 1997;Shen et al., 1997;Fagerholm, 2007;Fisher and Labissiere, 2007;Yang et al., 2007). For these reasons, we have mainly used animal pharmacokinetic parameters to predict human FaFg in the drug discovery stage.It has been regarded as natural that monkey metabolism is the most similar to that of humans, and cynomolgus monkeys have been widely used in pharmacokinetic or drug-...

We describe the preclinical and clinical pharmacokinetic profiles of FK3453 [6-(2-amino-4-phenylpyrimidin-5-yl)-2-isopropylpyridazin-3(2H)-one] and the mechanism responsible for poor oral exposure of FK3453 in humans. FK3453 showed favourable profiles in preclinical pharmacokinetic studies, including satisfactory absolute bioavailability and total body clearance in animals (30.5%-41.4%, 54.7%-68.2%, and 71.3%-93.4% and 10.8-17.6, 1.9-17.1, and 5.0 mL/min/kg in male rats, female rats, and dogs, respectively), and good metabolic stability in liver microsomes (42.3, 14.5, and 1.1 mL/min/kg in male rats, dogs, and humans, respectively). However, despite these promising preclinical findings, plasma concentrations of FK3453 in humans were extremely low, with the oxidative metabolite of the aminopyrimidine moiety (M4) identified as a major metabolite. Given that aldehyde oxidase (AO) and xanthine oxidase (XO) were presumed to be the enzymes responsible for M4 formation, we investigated the mechanism of M4 formation using human liver subcellular fractions. M4 was detected in the incubation mixture with S9 and cytosol but not with microsomes, and M4 formation was inhibited by AO inhibitors (menadione, isovanillin) but not by cytochrome P-450 inhibitor (1-aminobenzotiazole) or XO inhibitor (allopurinol). These results suggest M4 formation is catalyzed by AO, and therefore, its poor exposure in humans was attributed to extensive AO metabolism.

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1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL(int)) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CL(int) of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL(int) of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL(int) of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

ABSTRACT:This study aimed to establish a practical and convenient method of predicting intestinal availability (F g ) in humans for highly permeable compounds at the drug discovery stage, with a focus on CYP3A4-mediated metabolism. We constructed a "simplified F g model," described using only metabolic parameters, assuming that passive diffusion is dominant when permeability is high and that the effect of transporters in epithelial cells is negligible. Five substrates for CYP3A4 (alprazolam, amlodipine, clonazepam, midazolam, and nifedipine) and four for both CYP3A4 and P-glycoprotein (P-gp) (nicardipine, quinidine, tacrolimus, and verapamil) were used as model compounds. Observed fraction of drug absorbed (F a F g ) values for these compounds were calculated from in vivo pharmacokinetic (PK) parameters, whereas in vitro intestinal intrinsic clearance (CL int,intestine ) was determined using human intestinal microsomes. The CL int,intestine for the model compounds corrected with that of midazolam was defined as CL m,index and incorporated into a simplified F g model with empirical scaling factor. Regardless of whether the compound was a P-gp substrate, the F a F g could be reasonably fitted by the simplified F g model, and the value of the empirical scaling factor was well estimated. These results suggest that the effects of P-gp on F a and F g are substantially minor, at least in the case of highly permeable compounds. Furthermore, liver intrinsic clearance (CL int,liver ) can be used as a surrogate index of intestinal metabolism based on the relationship between CL int,liver and CL m,index . F g can be easily predicted using a simplified F g model with the empirical scaling factor, enabling more confident selection of drug candidates with desirable PK profiles in humans.Given the substantial time and cost associated with drug discovery and development, increasing importance has been placed on the prediction of pharmacokinetics (PK) in humans of drug candidates at the discovery stage to avoid later termination of development because of an undesirable PK profile. Bioavailability (F) of an orally administered drug, which is the fraction of drug reaching systemic blood circulation, is expressed as the product of the fraction of the dose that enters the enterocyte (F a ), intestinal availability (F g ), and hepatic availability (F h ). Therefore, to effectively pass into systemic blood circulation, orally administered drugs must not only have high solubility and permeability in the gastrointestinal tract but also be stable against metabolizing enzymes in the gastrointestinal tract and liver.At the drug discovery stage, compounds selected as drug candidates using high-throughput screening typically are those with high permeability because intestinal permeability is one of the most important factors in determining the F of orally administered drugs. Although P-glycoprotein (P-gp) is highly expressed in intestinal epithelial cells and has the potential to reduce drug absorption, the effect on drug absorption is not qu...

Triple-stage quadrupole (TSQ) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and ion trap ESI-MS/MScan be used to cleave protonated molecules to produce carbocations and neutral molecules in the positive ion mode. Dissociation products which correspond to protonated forms of neutral fragment molecules can also be trapped and detected. These protonated molecules in turn can cleave via carbocation cleavage, ipso cleavage, onium cleavage or McLafferty or related rearrangements. One can elucidate the structures of metabolites from the differences in m/z ratios of the fragments arising from the original drug compound and its metabolite. This strategy for structural elucidation is further facilitated by estimates of the reactivity of drugs with oxygen diradicals involved in cytochrome P-450 cycles.