Metabolism and disposition of gemfibrozil in Wistar and multidrug resistance-associated protein 2-deficient TR- rats

1. The roles of multidrug resistance-associated protein (Mrp) 2 deficiency and Mrp3 up-regulation were evaluated on the metabolism and disposition of gemfibrozil. 2. Results from in vitro studies in microsomes showed that the hepatic intrinsic clearance (CLint) for the oxidative metabolism of gemfibrozil was slightly higher (1.5-fold) in male TR- rats, which are deficient in Mrp2, than in wild-type Wistar rats, whereas CLint for glucuronidation was similar in both strains. 3. The biliary excretion of intravenously administered [14C]gemfibrozil was significantly impaired in TR-) rats compared with Wistar rats (22 versus 93% of the dose excreted as the acyl glucuronides over 72 h). Additionally, the extent of urinary excretion of radioactivity was much higher in TR- than in Wistar rats (78 versus 2.6% of the dose). 4. There were complex time-dependent changes in the total radioactivity levels and metabolite profiles in plasma, liver and kidney, some of which appeared to be related to the up-regulation of Mrp3. 5. Overall, it was demonstrated that alterations in the expression of the transporters Mrp2 and Mrp3 significantly affected the excretion as well as the secondary metabolism and distribution of [14C]gemfibrozil.     

Characterization of transport protein expression in multidrug resistance-associated protein (Mrp) 2-deficient rats.

Multidrug resistance-associated protein (Mrp) 2-deficient transport-deficient (TR(-)) rats, together with their transport-competent Wistar counterparts (wild type), have been used to examine the contribution of Mrp2 to drug disposition. However, little is known about potential variation in expression of other transport proteins between TR(-) and wild-type rats or whether these differences are tissue-specific. Sections of liver, kidney, brain, duodenum, jejunum, ileum, and colon were obtained from male TR(-) and wild-type Wistar rats. Samples were homogenized in protease inhibitor cocktail and ultracentrifuged at 100,000g for 30 min to obtain membrane fractions. Mrp2, Mrp3, Mrp4, P-glycoprotein, sodium-dependent taurocholate cotransporting polypeptide, organic anion transporting polypeptides 1a1 and 1a4, bile salt export pump, breast cancer resistance protein, ileal bile acid transporter, UDP-glucuronosyl transferase (UGT1a), glyceraldehyde-3-phosphate dehydrogenase, and beta-actin protein expression were determined by Western blot. Mrp3 was significantly up-regulated in the liver ( approximately 6-fold) and kidney ( approximately 3.5-fold) of TR(-) rats compared with wild-type controls. Likewise, the expression of UGT1a enzymes was increased in the liver and kidney of TR(-) rats by approximately 3.5- and approximately 5.5-fold, respectively. Interestingly, Mrp3 expression was down-regulated in the small intestine of TR(-) rats, but expression was similar to wild type in the colon. Mrp4 was expressed to varying extents along the intestine. Expression of some transport proteins and UGT1a enzymes differ significantly between TR(-) and wild-type rats. Therefore, altered drug disposition in TR(-) rats must be interpreted cautiously because up- or down-regulation of other transport proteins may play compensatory roles in the presence of Mrp2 deficiency.     

Multiple mechanisms are involved in the biliary excretion of acetaminophen sulfate in the rat: role of Mrp2 and Bcrp1.

Previous reports have demonstrated that sulfate metabolites may be excreted into bile by the multidrug resistance-associated protein 2 (Mrp2, Abcc2). Although recombinant human breast cancer resistance protein (BCRP, ABCG2) has affinity for sulfated xenobiotics and endobiotics, its relative importance in biliary excretion of sulfate metabolites in the intact liver is unknown. In the present studies, the potential contribution of Bcrp1 to the biliary excretion of acetaminophen sulfate (AS) was examined following acetaminophen administration (66 micromol, bolus) to isolated perfused livers (IPLs) from wild-type Wistar and Mrp2-deficient (TR(-)) Wistar rats in the presence or absence of the Bcrp1 and P-glycoprotein inhibitor, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide]. Recovery of AS in bile of TR(-) rat livers was approximately 5-fold lower relative to wild-type controls (0.3 +/- 0.1 versus 1.5 +/- 0.3 micromol). In the presence of GF120918, biliary excretion of AS was decreased approximately 2-fold in both TR(-) (0.16 +/- 0.09 micromol) and wild-type (0.8 +/- 0.3 micromol) rat IPLs. These changes were primarily due to alterations in the rate constant governing biliary excretion of AS, which was decreased approximately 90% in TR(-) relative to wild-type rat IPLs (0.02 +/- 0.01 versus 0.2 +/- 0.1 h(-1)) and was further decreased in the presence of GF120918 (0.010 +/- 0.003 and 0.12 +/- 0.05 h(-1); TR(-) and wild-type, respectively). In vitro assays indicated that impaired AS biliary excretion in the presence of GF120918 was due to inhibition of Bcrp1, and not P-glycoprotein. In conclusion, Mrp2 and, to a lesser extent, Bcrp1 mediate biliary excretion of AS in the intact liver.     

Phenobarbital alters hepatic Mrp2 function by direct and indirect interactions

Phenobarbital (PB) treatment impairs the biliary excretion of some organic anions. One mechanism may involve direct competition for biliary excretion by PB and/or a PB metabolite. Alternatively, PB may alter the expression and/or function of hepatic organic anion transport proteins. The role of multidrug resistance-associated protein 2 (Mrp2) in the biliary excretion of PB and metabolites was studied using isolated perfused livers (IPLs) from Wistar and Mrp2-deficient TR- rats. In normal livers, 4.19 +/- 0.53% of the PB dose was recovered in bile as PB metabolites [2.21 +/- 0.69% as 5-ethyl-5-(4-OH phenyl) barbituric acid (PBOH)-glucuronide; 1.98 +/- 0.09% as PBOH-sulfate]. In TR- livers, only PBOH-sulfate was recovered in bile (0.35 +/- 0.16% of dose) during the 2-h perfusion. Mrp2 message was increased (2.3-fold) by PB pretreatment (80 mg/kg i.p. x 4 days) but decreased to control values after a 48-h washout. Mrp2 protein was increased slightly in PB-treated livers and remained slightly elevated after a 24-h washout, but it was decreased significantly to 62 +/-7% of control values after a 48-h washout. The 120-min cumulative biliary excretion of the Mrp2 substrate 5-(and-6)-carboxy-2', 7'-dichlorofluorescein in IPLs from PB-treated rats after a 48-h washout was significantly lower than in vehicle-treated livers (66.3 +/- 9.2% versus 83.4 +/- 2.4% of the dose, respectively). These data support two mechanisms for impaired biliary excretion of some organic anions by PB treatment: 1) PBOH-glucuronide is a substrate for Mrp2 and may compete with other organic anions for biliary excretion and 2) Mrp2 protein expression and functional capacity is decreased 48 h after PB treatment.     

P-glycoprotein plays a major role in the efflux of fexofenadine in the small intestine and blood-brain barrier, but only a limited role in its biliary excretion.

Fexofenadine is a selective, nonsedating H(1)-receptor antagonist approved for symptoms of allergic conditions, which is mainly excreted into feces via biliary excretion. The purpose of this study is to investigate its pharmacokinetics in mice and rats to determine the role of P-glycoprotein (P-gp) in its biliary excretion. In mice, biliary excretion clearance (17 ml/min/kg) accounted for almost 60% of the total body clearance (30 ml/min/kg). Comparing the pharmacokinetics after intravenous and oral administration indicated that the bioavailability of fexofenadine was at most 2% in mice. Knockout of Mdr1a/1b P-gp did not affect the biliary excretion clearance with regard to both plasma and liver concentrations, whereas the absence of P-gp caused a 6-fold increase in the plasma concentration after oral administration. In addition, the steady-state brain-to-plasma concentration ratio of fexofenadine was approximately 3-fold higher in Mdr1a/1b P-gp knockout mice than in wild-type mice. Together, these results show that P-glycoprotein plays an important role in efflux transport in the brain and small intestine but only a limited role in biliary excretion in mice. In addition, there was no difference in the biliary excretion between normal and hereditarily multidrug resistance-associated protein 2 (Mrp2)-deficient mutant rats (Eisai hyperbilirubinemic rats) and between wild-type and breast cancer resistance protein (Bcrp) knockout mice. These results suggest that the biliary excretion of fexofenadine is mediated by unknown transporters distinct from P-gp, Mrp2, and Bcrp.     

Modulation of trabectedin (ET-743) hepatobiliary disposition by multidrug resistance-associated proteins (Mrps) may prevent hepatotoxicity

Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-h continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 microM) exhibited a 2- to 3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2-deficient (TR(-)) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR(-) rats was decreased by approximately 75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR(-) rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or Mrp4.     

Effect of DPC 333 [(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide], a human tumor necrosis factor alpha-converting enzyme inhibitor, on the disposition of methotrexate: a transporter-based drug-drug interaction case study.

DPC 333 [(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] is a potent human tumor necrosis factor alpha-converting enzyme inhibitor with potential therapeutic implications for rheumatoid arthritis. Methotrexate (MTX), a drug for the treatment of rheumatoid arthritis, is eliminated primarily unchanged via renal and biliary excretion in humans as well as in rats and dogs. The objective of the present study was to investigate the potential effect of DPC 333 on the disposition of MTX. In dogs, DPC 333 administered orally at 1.7 mg/kg 15 min before the intravenous administration of [14C]MTX (0.5 mg/kg) did not alter the plasma concentration-time profile of MTX; however, the total amount of radioactivity excreted in urine increased from 58.7% to 92.2% of the dose, and the renal clearance increased from 1.8 ml/min/kg to 2.9 ml/min/kg, suggesting a decrease in MTX disposition via biliary excretion. The biliary excretion of MTX was investigated in isolated perfused livers prepared from wild-type and TR(-) [multidrug resistance-associated protein 2 (Mrp2)-deficient] Wistar rats in the absence and presence of DPC 333. Mrp2-mediated biliary excretion of MTX was confirmed with 95.8% and 5.1% of MTX recovered in the bile of wild-type and TR(-) Wistar rats, respectively. DPC 333 at an initial perfusate concentration of 50 microM completely blocked the biliary excretion of MTX, but not the clearance from perfusate, in both wild-type and TR(-) rats. These results suggest that the enhanced renal elimination of MTX may be due to the potent inhibition of biliary excretion and active renal reabsorption by DPC 333 and/or its metabolites.     

Metabolism and disposition of resveratrol in the isolated perfused rat liver: role of Mrp2 in the biliary excretion of glucuronides

In this study, the hepatic metabolism and transport system for resveratrol was examined in isolated perfused livers from Wistar and Mrp2-deficient TR(-) rats. Based on extensive metabolism to six glucuronides and sulfates (M1-M6), the hepatic extraction ratio and clearance of resveratrol was very high in Wistar and TR(-) rats (E: 0.998 vs. 0.999; Cl: 34.9 mL/min vs. 36.0 mL/min). However, biliary excretion and efflux of conjugates differs greatly in TR(-) rats. While cumulative biliary excretion of the glucuronides M1, M2, M3, and M5 dropped dramatically to 0-6%, their efflux into perfusate increased by 3.6-, 1.8-, 2.5-, and 1.5-fold. In contrast, biliary secretion of the sulfates M4 and M6 was partially maintained in the Mrp2-deficient rats (61% and 39%) with a concomitant decline of their efflux into perfusate by 33.2% and 78.1%. This indicates that Mrp2 exclusively mediates the biliary excretion of resveratrol glucuronides but only partly that of sulfates. Cumulative secretion of unconjugated resveratrol into bile of TR(-) rats was only reduced by 40%, and into perfusate by 19%, suggesting only a minor role of Mrp2 in resveratrol elimination. In summary, resveratrol was dose-dependently metabolized to several conjugates whereby the canalicular transporter Mrp2 selectively mediated the biliary excretion of glucuronides.     

Involvement of Mrp2 (Abcc2) in biliary excretion of moxifloxacin and its metabolites in the isolated perfused rat liver

Moxifloxacin is a novel antibacterial agent that undergoes extensive metabolism in the liver to the glucuronide M1 and the sulfate M2, which are eliminated via the bile. To investigate the role of the multidrug resistance-associated protein (Mrp2) as the hepatic transport system for moxifloxacin and its conjugates, livers of Wistar and Mrp2-deficient TR- rats were perfused with moxifloxacin (10 microM) in a single-pass system. Values for the hepatic extraction ratio (E) and clearance (Cl) were insignificantly higher in TR- rats than Wistar rats (0.193+/-0.050 vs 0.245+/-0.050 for E; 6.85+/-1.96 vs 8.73+/-1.82 mL min(-1) for Cl), whereas biliary excretion and efflux into perfusate over 60 min were significantly lower in the mutant rat strain. Cumulative biliary excretion of M1, M2 and moxifloxacin was significantly reduced to 0.027%, 19.1%, and 29.6% in the TR- rats compared with Wistar rats, indicating that the biliary elimination of M1 is mediated exclusively by Mrp2, whereas that of M2 and moxifloxacin seems to depend mostly on Mrp2 and, to a smaller extent, a further unidentified canalicular transporter. Moxifloxacin stimulates bile flow by up to 11% in Wistar rats, but not in TR- rats, further supporting an efficient transport of this drug and its glucuronidated and sulfated metabolites by Mrp2. Moxifloxacin (10 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bromsulphthalein in Wistar rats by 34%, indicating competition with the elimination of Mrp2-specific substrates. In conclusion, we found that Mrp2 mediates the biliary elimination of moxifloxacin and its glucuronidated and sulfated metabolites in rats. MRP2 may therefore play a key role in the transport of moxifloxacin and its conjugates into bile in humans.     

Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR- rats

Multidrug resistance-associated proteins (Mrps) are a group of ATP-dependent efflux transporters for organic anions. Mrp2 and Mrp4 are co-localized to the apical (brush-border) membrane domain of renal proximal tubules, where they may function together in the urinary excretion of organic anions. Previous reports showed that urinary excretion of some organic anions is not impaired in transport-deficient (TR-) rats, which lack Mrp2, suggesting that up-regulation of other transporter(s) may compensate for the loss of Mrp2 function. The purpose of this study was to determine whether Mrp4 expression in kidney is altered in TR- rats. Mrp4 mRNA expression was quantified using the high-throughput branched DNA signal amplification assay. Mrp4 protein expression was determined by Western blot and immunohistochemical analysis. Mrp4 mRNA in kidney of TR- rats was 100% higher than normal Wistar rats. Western blot analysis showed a 200% increase in Mrp4 protein expression in kidney of the mutant rats compared to normal rats. Immunohistochemical analysis of Mrp4 protein demonstrated apical localization of Mrp4 on renal proximal tubules, and that the immunoreactivity was more intense in kidney sections from TR- rats than those from normal rats. In summary, the results of the present study demonstrate that renal Mrp4 expression is up-regulated in TR- rats, which may explain why urinary excretion of some organic anions remains normal in the mutant rats.     

The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats.

We investigated whether lack of the canalicular multispecific organic anion transporter in transport-deficient (TR-) rats would result in plasma and urinary accumulation of troglitazone or its major metabolites and whether any accumulation would be associated with increased levels of bilirubin or bile acids. Administration of a single oral dose of troglitazone (200 mg/kg) to TR- rats resulted in 2- and 50-fold increases in plasma levels and 30- and 500-fold increases in urinary amounts of troglitazone sulfate and troglitazone glucuronide, respectively, compared with normal rats. No changes were found in the plasma concentrations and urinary amounts of troglitazone or troglitazone-quinone. Accumulation of troglitazone metabolites in plasma was accompanied by a 2-fold increase in the serum level of conjugated bilirubin in TR- rats, whereas no changes were observed in normal animals. Bile acids were detected in the urine of both TR- and normal rats, with an average 3-fold greater level found in the urine of TR- animals. Biliary metabolic profiles revealed a delay in the secretion of troglitazone sulfate and troglitazone glucuronide in TR- rats over the first 2- and 4-h periods, respectively. These results demonstrate the role of multidrug resistant associated protein-2 in biliary secretion of troglitazone glucuronide and troglitazone sulfate and suggest the presence of compensatory mechanisms responsible for transport of troglitazone metabolites and bilirubin-glucuronide at the basolateral and canalicular sites of hepatocytes.     

Increased bioavailability of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in MRP2-deficient rats

MRP2 is an apical transporter expressed in hepatocytes and the epithelial cells of the small intestine and kidney proximal tubule. It extrudes organic anions, conjugated compounds, and some uncharged amphipaths. We studied the transport of an abundant food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in vitro, using an MRP2 transfected epithelial cell line (MDCK II) and intestinal explants from Wistar and MRP2-deficient TR(-) rats in Ussing chambers. In the experiments with the transfected cell line, we could demonstrate more than 3-fold higher transport from basolateral to apical than vice versa, whereas the transport in the parent cell line was equal in both directions. These results were confirmed in studies using isolated pieces of small intestine from Wistar and TR(-) rats in the Ussing chamber. Subsequent in vivo experiments demonstrated that after oral administration, absorption of PhIP was 2-fold higher in the TR(-) rat than in the Wistar rat. Consequently, PhIP tissue levels in several organs (liver, kidney, lung, and colon) were 1.7- to 4-fold higher 48 h after oral administration. MRP2 mediated transport of unchanged PhIP probably involves intracellular GSH, because GSH depletion by BSO-treatment in Wistar rats reduced intestinal secretion in the Ussing chamber to the same level as in TR(-) rats. In accordance, BSO treatment increased oral bioavailability in intact Wistar rats. This study shows for the first time that MRP2-mediated extrusion reduces oral bioavailability of a xenobiotic and protects against an abundant food-derived carcinogen.     

Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides

The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug resistance-associated protein 2 (Mrp2)/Abcc2. To study the possible involvement of Mrp2, we compared intestinal uptake of quercetin-3-glucoside between control and Mrp2-deficient rats, using an in situ intestinal perfusion system. The contribution of Bcrp1 was determined using the specific inhibitor fumitremorgin C (FTC) in Mrp2-deficient rats. Furthermore, vectorial transport of quercetin was studied in Madin-Darby canine kidney (MDCK)II cells transfected with either human MRP2 or murine Bcrp1. In these MDCKII cells, we showed an efficient efflux-directed transport of quercetin by mouse Bcrp1, whereas in control and MRP2-transfected cells no vectorial transport of quercetin was observed. In Mrp2-deficient rats, intestinal uptake of quercetin from quercetin-3-glucoside, efflux of quercetin glucuronides to the gut lumen, and plasma concentration of quercetin were similar to that in control rats. When intestinal Bcrp1 was inhibited by FTC in Mrp2-deficient rats, total plasma concentrations of quercetin and its methylated metabolite isorhamnetin after 30 min of perfusion were more than twice that of controls (12.3 +/- 1.5 versus 5.6 +/- 1.3 muM; p < 0.01), whereas uptake of free quercetin from the intestinal lumen was not affected. Instead, inhibition of Bcrp1 lowered the efflux of quercetin glucuronides into the perfusion fluid by approximately 4-fold. In conclusion, Bcrp1 limits net intestinal absorption of quercetin by pumping quercetin glucuronides back into the lumen.     

Sex differences in the pharmacokinetics, oxidative metabolism and oral bioavailability of oxycodone in the Sprague-Dawley rat

1. The pharmacokinetics and oxidative metabolism of oxycodone were investigated following intravenous and oral administration in male and female Sprague-Dawley (SD) rats. 2. High-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS-MS) was used to quantify plasma concentrations of oxycodone and its oxidative metabolites noroxycodone and oxymorphone following administration of single bolus intravenous (5 mg/kg) and oral (10 mg/kg) doses of oxycodone. 3. The mean (+/-SEM) clearance of intravenous oxycodone was significantly higher in male than female SD rats (4.9 +/- 0.3 vs 3.1 +/- 0.3 L/h per kg, respectively; P < 0.01). Mean areas under the plasma concentration versus time curves (AUC) for oxycodone were significantly higher in female than male SD rats following intravenous (approximately 1.6-fold; P < 0.01) and oral (approximately sevenfold; P < 0.005) administration. 4. The oral bioavailability of oxycodone was low (at 1.2 and 5.0%, respectively) in male and female SD rats, a finding consistent with high first-pass metabolism. Noroxycodone : oxycodone AUC ratios were significantly higher in male than female SD rats after intravenous (approximately 2.4-fold; P < 0.005) and oral (approximately 12-fold; P < 0.005) administration. 5. Circulating oxymorphone concentrations remained very low following both routes of administration. Noroxycodone : oxymorphone AUC ratios were greater in male than female SD rats after intravenous (approximately 13- and fivefold, respectively) and oral (approximately 90- and sixfold, respectively) administration. 6. Sex differences were apparent in the pharmacokinetics, oxidative metabolism and oral bioavailability of oxycodone. Systemic exposure to oxycodone was greater in female compared with male SD rats, whereas systemic exposure to metabolically derived noroxycodone was higher in male than female SD rats. 7. Oral administration of oxycodone to the SD rat is a poor model of the human for the study of the pharmacodynamic effects of oxycodone.     

Induction of hepatic phase II drug-metabolizing enzymes by 1,7-phenanthroline in rats is accompanied by induction of MRP3.

The purpose of the present study was to evaluate the effect of 1,7-phenanthroline (PH), which has been proposed to be a selective phase II enzyme inducer, on the gene expression of xenobiotic transporters, as well as hepatic and renal drug-metabolizing enzymes. After oral administration of PH for 3 days to male Sprague-Dawley rats, mRNA levels in liver (75 and 150 mg/kg doses) and kidney (75 mg/kg dose only) were determined using real-time quantitative polymerase chain reaction. At 150 mg/kg/day, PH treatment resulted in significant increases in hepatic mRNA levels of Mrp3 (36-fold), UGT1A6 (20-fold), UGT2B1 (4-fold), and quinone reductase (QR, 5-fold), compared with the vehicle-treated group. Similar increases in Mrp3 (99-fold), UGT1A6 (17-fold), UGT2B1 (3-fold), and QR (11-fold) mRNA levels were observed in the liver after PH treatment of rats at 75 mg/kg/day. In contrast, the expression levels of CYP2C11 and Oatp2 were decreased by approximately 80 and 50%, respectively. In addition, PH (75 mg/kg/day) elicited statistically significant changes in renal gene expression of CYP3A1, UGT1A6, QR, and Mrp3, but the magnitude of renal Mrp3 induction was less than 2-fold over control. Although PH is known to modulate hepatic glucuronidation in vivo, these data indicated that PH induced mRNA levels of the efflux transporter, Mrp3, which may also affect the disposition of xenobiotics.     

Eisai hyperbilirubinemic rat (EHBR) as an animal model affording high drug-exposure in toxicity studies on organic anions

The Eisai hyperbilirubinemic rat (EHBR) should be a useful animal model for studies on the toxicity of organic anions which are substrates of multidrug resistance-associated protein 2 (Mrp2), since the systemic exposure to these compounds is expected to be increased in EHBR. In this study, we tested the value of EHBR for this purpose, using pravastatin (PV) and methotrexate (MTX) as model compounds. In the case of a single oral dose of PV (200 mg/kg), C(max) in plasma was 4.0-fold higher and AUC(0-infinity) was 3.6-fold larger than those of normal Sprague-Dawley rats (SDR), respectively. When multiple doses of PV were given to EHBR without co-administration of any other compound, drug-induced skeletal muscle toxicity (myopathy/rhabdomyolysis) and increased creatine phosphokinase (CPK) level were observed, whereas a control experiment using SDR did not show any toxic change. When a single dose of MTX (0.6 mg/kg) was given to EHBR orally, C(max) was 1.7-fold higher and AUC(0-infinity) was 1.6-fold larger than those of SDR, respectively. When multiple doses of MTX were given to EHBR, the changes in bone marrow, spleen and intestines were more severe than those in SDR. These findings support the view that EHBR would be a valuable animal model for toxicity studies on organic anion compounds which are substrates of Mrp2.     

Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice

Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs. Hepatic uptake is crucial for the therapeutic effect of pravastatin, a cholesterol-lowering drug and OATP1A/1B substrate. We aimed to gain empirical insight into the relationship between OATPs and pravastatin pharmacokinetics and toxicity. We therefore compared the distribution and toxicity of pravastatin in wild-type and Oatp1a/1b-null mice. Intestinal absorption of pravastatin was not affected by Oatp1a/1b absence, but systemic plasma exposure (AUC) increased up to 30-fold after oral bolus administration. This increased plasma exposure resulted from reduced hepatic uptake, as evident from 10 to 100-fold lower liver-to-plasma concentration ratios. However, the reductions in liver exposure were far smaller (<2-fold) than the increases in plasma exposure. Reduced pravastatin liver uptake in Oatp1a/1b-null mice was more obvious shortly after intravenous administration, with 8-fold lower biliary pravastatin excretion. Although mice chronically exposed to pravastatin for 60 days evinced little muscular toxicity, Oatp1a/1b-null mice displayed 10-fold higher plasma concentrations and 8-fold lower liver concentrations than wild-type mice. Thus, Oatp1a/1b transporters importantly control the hepatic uptake of pravastatin. Activity-reducing human OATP1B polymorphisms may therefore both reduce pravastatin therapeutic efficacy in the liver and increase systemic toxicity risks, thus compromising its therapeutic index in a two-edged way.     

Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 Knockout Rats Using Loperamide, Paclitaxel, Sulfasalazine, and Carboxydichlorofluorescein Pharmacokinetics

Transporter gene knockout rats are practically advantageous over murine models for pharmacokinetic and excretion studies, but their phenotypic characterization is lacking. At present, relevant aspects of pharmacokinetics, metabolism, distribution, and excretion of transporter probes [P-glycoprotein (P-gp): loperamide and paclitaxel; breast cancer resistance protein (Bcrp): sulfasalazine; and multidrug resistance-associated protein 2 (Mrp2): carboxydichlorofluorescein] were studied systematically across SAGE P-gp, Bcrp, and Mrp2 knockout rats. In Mdr1a knockout rats, loperamide and paclitaxel oral bioavailability was 2- and 4-fold increased, respectively, whereas clearance was significantly reduced (40-42%), consistent with the expected 10- to 20-fold reduction in paclitaxel excretion. N-Desmethyl-loperamide pharmacokinetics were not altered in any of the three knockouts after oral loperamide. In rats lacking P-gp, paclitaxel brain partitioning was significantly increased (4-fold). This finding is consistent with observations of loperamide central nervous system opioid pharmacology in Mdr1a knockout rats. Sulfasalazine oral bioavailability was markedly increased 21-fold in Bcrp knockouts and, as expected, was also 2- to 3-fold higher in P-gp and Mrp2 knockout rats. The sulfapyridine metabolite/parent ratio was decreased 10-fold in rats lacking Bcrp after oral, but not intravenous, sulfasalazine administration. Carboxydichlorofluorescein biliary excretion was obliterated in Mrp2 knockout rats, resulting in 25% decreased systemic clearance and 35% increased half-life. In contrast, carboxydichlorofluorescein renal clearance was not impaired in the absence of Mrp2, Bcrp, or P-gp. In conclusion, SAGE Mdr1a, Bcrp, and Mrp2 knockout rats generally demonstrated the expected phenotypes with respect to alterations in pharmacokinetics of relevant probe substrates; therefore, these knockout rats can be used as an alternative to murine models whenever a larger species is practically advantageous or more relevant to the drug discovery/development program.     

Pharmacological studies on triazine derivatives V Sedative and neuroleptic actions of 2-amino-4-[4-(2-hydroxyethyl)-piperazin-1-yl]-6-trifluoromethyl-s-triazine (TR-10).

Pharmacological properties of 2 amino-4-[4-(2-hydroxyethyl)-piperazin-1-yl]-6-trifluoromethyl-s-triazine (TR-10) were investigated in mice and rats. Chlorpromazine served as a reference compound. Tr-10 expressed in general the pharmacological profiles as neuroleptic ascertained by anti-methamphetamine activity, supression of conditioned avoidance response, taming effects, decrease in exploratory behavior and cataleptogenic activity. Among these effects, anti-methamphetamine action was most potent. Different from chlorpromazine, TR-10 showed a similar pharmacological activity pattern in the intraperitoneal and oral routes of administration as depicted from ED50/LD50 values. Although the effects relevant to neuroleptics were less potent than chlorpromazine, such were seen with TR-10 at lower doses than those causing muscle relaxation. TR-10 significantly depressed the spontaneous motor activity but showed no anti-convulsant action in mice. Hypothermic action, potentiating effects of hypnotics and alpha-adrenergic blocking action, characteristic to chlorpromazine, were very weak for TR-10. TR-10 also showed low toxicity in mice (LD50 = 820 mg/kg p.o., 465 mg/kg i.p.) compared with that of chlorpromazine (LD50 = 370 mg/kg p.o., 228 mg/kg i.p.).     

Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion.

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds, including their secretion into the milk. The purpose of this study was to determine whether three widely used fluoroquinolone antibiotics (ciprofloxacin, ofloxacin, and norfloxacin) are substrates of Bcrp1/BCRP and to investigate the possible role of this transporter in the in vivo pharmacokinetic profile of these compounds and their secretion into the milk. Using polarized cell lines, we found that ciprofloxacin, ofloxacin, and norfloxacin are transported by mouse Bcrp1 and human BCRP. In vivo pharmacokinetic studies showed that the ciprofloxacin plasma concentration was more than 2-fold increased in Bcrp1(-/-) compared with wild-type mice (1.77 +/- 0.73 versus 0.85 +/- 0.39 microg/ml, p < 0.01) after oral administration of ciprofloxacin (10 mg/kg). The area under the plasma concentration-time curve in Bcrp1(-/-) mice was 1.5-fold higher than that in wild-type mice (48.63 +/- 5.66 versus 33.10 +/- 4.68 min x microg/ml, p < 0.05) after i.v. administration (10 mg/kg). The milk concentration and milk/plasma ratio of ciprofloxacin were 2-fold higher in wild-type than in Bcrp1(-/-) lactating mice. We conclude that Bcrp1 is one of the determinants for the bioavailability of fluoroquinolones and their secretion into the milk.