Practical permeability-based hepatic clearance classification system (HepCCS) in drug discovery.

BACKGROUND: The use of liver microsomes and hepatocytes to predict total in vivo clearance is standard practice in the pharmaceutical industry; however, metabolic stability data alone cannot always predict in vivo clearance accurately. RESULTS: Apparent permeability generated from Mardin-Darby canine kidney cells and rat hepatocyte uptake for 33 discovery compounds were obtained. CONCLUSION: When there is underprediction of in vivo clearance, compounds with low apparent permeability (less than 3 × 10(-6) cm/s) all exhibited hepatic uptake. A systematic approach in the form of a classification system (hepatic clearance classification system) and decision tree that will help drug discovery scientists understand in vitro-in vivo clearance prediction disconnect early is proposed.

Knockout of a putative ergothioneine transporter in Caenorhabditis elegans decreases lifespan and increases susceptibility to oxidative damage.

In addition to excretion of metabolic waste products, organic ionic transporters facilitate uptake of specific compounds of physiological importance. In animals, the organic cation transporter, OCTN1 was found to enable the specific uptake of the unique amino acid, ergothioneine (EGT). EGT can accumulate in the body at up to millimolar concentrations and is believed to function as a physiological antioxidant. However the main function of EGT and the reasons for its active accumulation in the body remain obscure. Through bioinformatic approaches, we identified an analogous EGT transporter in the nematode, Caenorhabditis elegans. The present study investigated and characterized deletion mutants of this gene, OCT-1, in the nematodes. Gene deletion mutations of the OCT-1 transporter were shown to decrease overall lifespan of the worms and increase oxidative damage. However the absence of impaired EGT uptake and the inability of excess EGT to rescue the debilitating phenotype indicate that EGT transport does not explain the deleterious effects of the gene deletion.

Role of organic cation transporter 1, OCT1 in the pharmacokinetics and toxicity of cis-diammine(pyridine)chloroplatinum(II) and oxaliplatin in mice.

PURPOSE: The goal of this study was to test the hypothesis that by controlling intracellular uptake, organic cation transporter 1, Oct1 is a key determinant of the disposition and toxicity of cis-diammine(pyridine)chloroplatinum(II)(CDPCP) and oxaliplatin. METHODS: Pharmacokinetics, tissue accumulation and toxicity of CDPCP and oxaliplatin were compared between Oct1-/- and wild-type mice. RESULTS: After intravenous administration, hepatic and intestinal accumulation of CDPCP was 2.7-fold and 3.9-fold greater in Oct1 wild-type mice (p < 0.001). Deletion of Oct1 resulted in a significantly decreased clearance (0.444 ± 0.0391 ml/min*kg versus 0.649 ± 0.0807 ml/min*kg in wild-type mice, p < 0.05) and volume distribution (1.90 ± 0.161 L/kg versus 3.37 ± 0.196 L/kg in wild-type mice, p < 0.001). Moreover, Oct1 deletion resulted in more severe off-target toxicities in CDPCP-treated mice. Histologic examination of the liver and measurements of liver function indicated that the level of hepatic toxicity was mild and reversible, but was more apparent in the wild-type mice. In contrast, the effect of Oct1 on the pharmacokinetics and toxicity of oxaliplatin in the mice was minimal. CONCLUSIONS: Our study suggests that Oct1 plays an important role in the pharmacokinetics, tissue distribution and toxicity of CDPCP, but not oxaliplatin.

Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse.

BACKGROUND: It has been proposed that serotonin (5-HT)-mediated constriction of the murine trachea is largely dependent on acetylcholine (ACh) released from the epithelium. We recently demonstrated that ACh can be released from non-neuronal cells by corticosteroid-sensitive polyspecific organic cation transporters (OCTs), which are also expressed by airway epithelial cells. Hence, the hypothesis emerged that 5-HT evokes bronchoconstriction by inducing release of ACh from epithelial cells via OCTs. METHODS: We tested this hypothesis by analysing bronchoconstriction in precision-cut murine lung slices using OCT and muscarinic ACh receptor knockout mouse strains. Epithelial ACh content was measured by HPLC, and the tissue distribution of OCT isoforms was determined by immunohistochemistry. RESULTS: Epithelial ACh content was significantly higher in OCT1/2 double-knockout mice (42 +/- 10 % of the content of the epithelium-denuded trachea, n = 9) than in wild-type mice (16.8 +/- 3.6 %, n = 11). In wild-type mice, 5-HT (1 microM) caused a bronchoconstriction that slightly exceeded that evoked by muscarine (1 microM) in intact bronchi but amounted to only 66% of the response to muscarine after epithelium removal. 5-HT-induced bronchoconstriction was undiminished in M2/M3 muscarinic ACh receptor double-knockout mice which were entirely unresponsive to muscarine. Corticosterone (1 microM) significantly reduced 5-HT-induced bronchoconstriction in wild-type and OCT1/2 double-knockout mice, but not in OCT3 knockout mice. This effect persisted after removal of the bronchial epithelium. Immunohistochemistry localized OCT3 to the bronchial smooth muscle. CONCLUSION: The doubling of airway epithelial ACh content in OCT1/2-/- mice is consistent with the concept that OCT1 and/or 2 mediate ACh release from the respiratory epithelium. This effect, however, does not contribute to 5-HT-induced constriction of murine intrapulmonary bronchi. Instead, this activity involves 1) a non-cholinergic epithelium-dependent component, and 2) direct stimulation of bronchial smooth muscle cells, a response which is partly sensitive to acutely administered corticosterone acting on OCT3. These data provide new insights into the mechanisms involved in 5-HT-induced bronchoconstriction, including novel information about non-genomic, acute effects of corticosteroids on bronchoconstriction.

Renal organic cation and nucleoside transport.

We previously reported that the rat organic cation transporter rOCT1 could transport the nucleoside analog deoxytubercidin (dTub) (Chen R, Nelson JA. Biochem Pharmacol 2000;60:215-9). The cationic form of dTub (dTub(+)) appeared to be the true substrate of rOCT1. We also reported that although rOCT2 is similar to rOCT1, it does not transport dTub at pH 7.4. In this study, we measured the K(m) and V(max) values of dTub(+) uptake at a reduced pH (pH 5.4) for both rOCT1 and rOCT2. The difference in substrate activity appears due, in large part, to a poor affinity of rOCT2 for dTub(+). The transport efficiency estimated by V(max)/K(m) values for rOCT2 was only 6% that of rOCT1. Chimeras constructed between rOCT1 and rOCT2 revealed that the difference in dTub binding lies within transmembrane domains 2-7. To evaluate the potential of OCT1 in the renal secretion of dTub, tissue distribution and urinary excretion of dTub in OCT1 knockout mice were measured. No significant difference was observed in renal elimination, plasma level, and tissue distribution of dTub between the knockout and the wild-type mice. Therefore, dTub is a good substrate for OCT1; however, OCT1 does not appear to be necessary for its renal secretion.

Involvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin.

Metformin, a biguanide, is widely used as an oral hypoglycemic agent for the treatment of type 2 diabetes mellitus. The purpose of the present study was to investigate the role of organic cation transporter 1 (Oct1) in the disposition of metformin. Transfection of rat Oct1 cDNA results in the time-dependent and saturable uptake of metformin by the Chinese hamster ovary cell line with K(m) and V(max) values of 377 microM and 1386 pmol/min/mg of protein, respectively. Buformin and phenformin, two other biguanides, were also transported by rOct1 with a higher affinity than metformin: their K(m) values were 49 and 16 microM, respectively. To investigate the role of Oct1 in the disposition of metformin, the tissue distribution of metformin was determined in Oct1 gene-knockout mice after i.v. administration. Distribution of metformin to the liver in Oct1(-/-) mice was more than 30 times lower than that in Oct1(+/+) mice, and can be accounted for by the extracellular space. Distribution to the small intestine was also decreased in Oct1(-/-) mice, whereas that to the kidney as well as the urinary excretion profile showed only minimal differences. In conclusion, the present findings suggest that Oct1 is responsible for the hepatic uptake as well as playing a role in the intestinal uptake of metformin, whereas the renal distribution and excretion are mainly governed by other transport mechanism(s).