Probenecid-impaired biliary excretion of acetaminophen glucuronide and sulfate in the rat.

Acetaminophen (APAP; 100 mg/kg iv) and probenecid (50 mg/kg bolus + 11.4 mg/hr/kg infusion) were administered to male Sprague-Dawley rats to examine the disposition of APAP, and its glucuronide (AG) and sulfate (AS) conjugates in plasma, bile, and urine. Probenecid significantly decreased the formation clearance of AG from 3.65 +/- 0.434 to 1.94 +/- 0.441 ml/min/kg and the renal clearance of AS from 9.32 +/- 2.26 to 3.15 +/- 1.21 ml/min/kg. The biliary excretion of AG was reduced approximately 3- to 4-fold by probenecid, from 6.54 to 1.87% of the APAP dose, and the AG biliary excretion rate was decreased 4- to 5-fold during probenecid treatment. The more extensive impairment of AG biliary excretion relative to AG formation suggests that probenecid may inhibit the hepatobiliary transport of AG. The significant reduction in the biliary excretion rate at early time points for AS suggests that probenecid may inhibit hepatic AS transport. The study results indicate that probenecid impairs AG and AS formation, AS renal secretion, and AG and AS biliary excretion.     

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.     

Role of breast cancer resistance protein (Bcrp1/Abcg2) in the extrusion of glucuronide and sulfate conjugates from enterocytes to intestinal lumen

The purpose of this study is to examine the significance of efflux transporters in the small intestine to extrude glucuronide (G) and sulfate (S) conjugates into the intestinal lumen. From this standpoint, we performed in situ intestinal perfusion experiments by using Eisai hyperbilirubinemic rats (EHBRs) in which the multidrug resistance protein 2 (Mrp2/Abcc2) is hereditarily defective and breast cancer resistance protein (Bcrp1/Abcg2) knockout mice. The intestinal lumen of EHBRs and Bcrp1 (-/-) mice was perfused with medium containing 4-methylumbelliferone (4MU) and E3040 [6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridilmethyl) benzothiazole] to determine the efflux of metabolites into the outflow. The efflux of E3040-glucuronide (G) in EHBRs was significantly lower compared with that in normal rats. However, no significant difference was observed for the efflux of 4MU-G, 4MU-sulfate (S), and E3040-S between EHBRs and normal rats. In contrast, the efflux of intracellularly formed 4MU-G, 4MU-S, and E3040-G in Bcrp1 (-/-) mice was significantly lower than that in normal mice. Therefore, Bcrp1 has an important role in extruding glucuronide and sulfate conjugates formed in enterocytes into the intestinal lumen, whereas Mrp2 is responsible for the efflux of some glucuronide conjugates.     

Altered hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein in Abcg2 (Bcrp1) and Abcc2 (Mrp2) knockout mice.

This study characterized the hepatobiliary disposition of 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF), a model Abcc2/Mrp2 (canalicular) and Abcc3/Mrp3 (basolateral) substrate, in perfused livers from male C57BL/6 wild-type, Abcg2-/-, and Abcc2-/- mice. After single-pass liver perfusion with 1 muM CDF diacetate for 30 min and an additional 30-min perfusion with CDF-free buffer, cumulative biliary excretion of CDF in Abcg2-/- mice was significantly higher than in wild-type mice (65 +/- 6 and 47 +/- 15% of dose, respectively, p < 0.05), whereas CDF recovery in bile of Abcc2-/- mice was negligible. Cumulative recovery of CDF in perfusate was significantly higher in Abcc2-/- (90 +/- 8% of dose) relative to wild-type (35 +/- 11% of dose) mice. Compartmental pharmacokinetic analysis revealed that the rate constant for CDF biliary excretion was significantly increased in Abcg2-/- (0.061 +/- 0.005 min(-1)) compared with wild-type (0.039 +/- 0.011 min(-1)) mice. The rate constant governing the basolateral excretion of CDF was approximately 4-fold higher in Abcc2-/- (0.12 +/- 0.02 min(-1)) relative to wild-type (0.030 +/- 0.011 min(-1)) mice but was not altered in Abcg2-/- (0.031 +/- 0.004 min(-1)) mice. Hepatic Abcc3 protein levels, determined by immunoblot analysis, were approximately 60% higher in Abcc2-/- mice than in wild-type mice. In contrast, neither Abcc3 protein levels nor Abcc2 mRNA levels were altered in Abcg2-/- relative to wild-type mice. These data in knockout mouse models demonstrate that loss of expression and function of one canalicular transport protein may change the route and/or extent of excretion into bile or perfusate because of alterations in the function of other basolateral or canalicular transport proteins.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. iii: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate

1.?To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions.

2.?The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL_eff) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate.

3.?Under Cl^?-depleted conditions, the CL_eff of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl^? might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL_eff of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl^? is involved in the sinusoidal efflux of 4MUS.

4.?The effect of glutathione was examined. CL_eff of 4MUG was not affected by the additional glutathione, but CL_eff of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG.

5.?Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. III: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.     

Biliary and urinary excretion of drug conjugates: effect of diuresis and choleresis on excretion of harmol sulphate and harmol glucuronide in the rat.

1. Harmol (7-hydroxy-1-methyl-9H-pyrido [3,4b] indole) is converted to harmol sulphate and harmol glucuronide in the rat in vivo. Harmol sulphate is excreted mainly in urine, while harmol glucuronide is excreted about equally in bile and urine, when harmol is given intravenously. 2. In rats with ligated kidneys, only choleresis produced by glycodehydrocholate and dehydrocholate caused enhanced biliary excretion of harmol sulphate; harmol glucuronide was unaffected. Several other bile salts had no effect. 3. Mannitol diuresis markedly increased urinary excretion of harmol sulphate, and decreased its biliary excretion. Harmol glucuronide was much less affected. 4. Nafenopin pretreatment increased liver weight and bile flow, and enhanced biliary excretion of harmol sulphate at the expense of its urinary excretion. 5. For harmol sulphate, urine and bile are compensatory pathways of elimination that can be influenced by urine and bile flow changes through diuresis and choleresis.     

Effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of phytoestrogens

The effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of the phytoestrogens daidzein, genistein, and coumestrol was investigated using Bcrp(-/-) mice. Expression of the genes for either mouse Bcrp or human BCRP in MDCK II cells induced apically directed transport of the three phytoestrogens, whereas their transcellular transport was identical in mock and LLC-PK1 cells expressing mouse Mdr1a. After oral administration, the plasma levels of daidzein and genistein were increased in Bcrp(-/-) mice, but only a minimal change was observed for coumestrol. At steady state, tissue-to-plasma concentration ratios of the three phytoestrogens in the brain and testis of wild-type mice were very small and similar to those of [(14)C]inulin, whereas those were significantly increased in the brain and testis of Bcrp(-/-) mice. The largest increases were observed with genistein (9.2- and 5.8-fold in the brain and testis, respectively). The distributions of genistein in the epididymis and fetus, but not the ovary, were also increased in Bcrp(-/-) mice. The Bcrp protein was localized in the luminal membrane of the endothelial cells in the testis and the body of the epididymis and in both the luminal and abluminal side of ducts in the head of the epididymis. These results suggest that Bcrp limits the oral availability and distribution into the brain and testis, epididymis, and fetus of phytoestrogens.     

The effects of multidrug resistance protein 2 (Mrp2) and breast cancer resistance protein (Bcrp) on biliary excretion of cefditoren in rats

Objective Cefditoren,a third-generation cephalosporin antibiotics,has been used in clinic extensively.Whether Mrp2 or other canalicular transporters such as Bcrp and P-gp are involved in the biliary excretion of cefditoren is unknown.This study is performed to investigate the role of the canalicular transporters in the biliary excretion of cefditoren and the effect of cefditoren on expression levels of some hepatic transporters.Methods We examined the hepatobiliary disposition of cefditoren using probenecid,novobiocin and verapamil as the inhibitors of Mrp2,Bcrp and P-gp respectively in perfused rat livers.The concentration of cefditoren in the perfusate and bile were determined by RP-HPLC with ultraviolet detection at 295nm using a mobile phase composed of 0.1% ammonium acetate-methanol(65∶35).We also investigated the effects of cefditoren on expression of hepatic transporters.The change in mRNA of main canalicular transporters was investigated by RT-PCR and Western blot after administration of cefditoren.Results The values for the hepatic extraction ratio did not change,whereas cumulative biliary excretion rates of cefditoren were significantly reduced to 43.78% and 79.52% over 25 min in the perfused probenecid and novobiocin rats,respectively.After oral administration of cefditoren,the expression levels of Mrp2,Bcrp,Oat2 mRNA were markedly up-regulated,while Mdr1a and Oct1 mRNA were down-regulated by RT-PCR.In concordance with RT-PCR results,Mrp2 expression level was up-regulated by Western blot.Conclusions Mrp2 and Bcrp mediated the biliary excretion of cefditoren,whereas P-gp had no contribution to the transportation of cefditoren into bile.The expression levels of Mrp2,Bcrp and Oat2 mRNA were up-regulated and the expression levels of Mdr1a and Oct1 mRNA were down-regulated by cefditoren.These results provide important data for drug-drug interaction.     

Udp glucuronyltransferase and phenolsulfotransferase from rat liver in vivo and in vitro—III: The effect of phenolphthalein and its sulfate and glucuronide conjugate on conjugation and biliary excretion of harmol

Harmol (7-hydroxy-l-methyl-9H-pyrido-(3,4b) indol) is excreted in the rat in bile and urine after intravenous (i.v.) injection, in the form of harmol-sulfate and harmol-glucuronide. The effect of phenolphthalein, phenolphthalein-β-d-glucuronide and phenolphthalein-sulfate on both harmol conjugations in vitro and in vivo has been studied. Phenolphthalein and phenolphthalein-glucuronide inhibited strongly glucuronidation of harmol in vitro by a Triton X-100 activated post-nuclear supernatant. Phenolphthalein-sulfate was less inhibitory. Sulfation of harmol was only inhibited at higher concentrations of phenolphthalein-glucuronide and phenolphthalein-sulfate. When phenolphthalein and its conjugates were injected simultaneously with harmol, a decrease of biliary excretion of harmol-sulfate and harmol-glucuronide was found and a great increase in urinary excretion of these conjugates. The total amount of harmol-sulfate synthesized in vivo increased and the amount of harmol glucuronide decreased. These results can be explained by (1) inhibition of glucuronidation of harmol by the phenolphthalein derivatives and (2) an inhibition of biliary excretion of harmol-sulfate and harmol-glucuronide by the phenolphthalein derivatives, probably due to competition of harmol and phenolphthalein conjugates for the same transport-carrier from liver cell to bile. This carrier should transport both sulfate and glucuronide conjugates.     

Evaluation of the role of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in the urinary excretion of sulfate and glucuronide metabolites of edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one).

Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. Edaravone is mainly excreted into the urine after conjugation to glucuronide or sulfate. Previous studies have demonstrated that edaravone sulfate is a good substrate of human organic anion transporter (OAT) 1 (SLC22A6) and human OAT3 (SLC22A8). In this study, we examined the involvement of breast cancer resistance protein [BCRP (ABCG2)] and [multidrug resistance-associated protein 4 MRP4 (ABCC4)] in the luminal efflux in the kidney. Increased ATP-dependent uptake of edaravone sulfate but not edaravone glucuronide was observed in BCRP-expressing membrane vesicles compared with control vesicles (Km = 16.5 microM). In contrast, edaravone glucuronide, but not edaravone sulfate, exhibited greater ATP-dependent uptake in MRP4-expressing membrane vesicles than that in control vesicles (Km = 9.85 microM). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of edaravone glucuronide by MRP4. The functional importance of BCRP and MRP4 in the urinary excretion of edaravone sulfate and edaravone glucuronide, respectively, was investigated using Bcrp and Mrp4 knockout mice. The renal clearance with respect to the kidney concentration of edaravone sulfate was reduced significantly but not abolished in Bcrp knockout mice compared with wild-type mice (3.62 versus 4.85 ml/min/kg b.wt.). The renal clearance of edaravone glucuronide was lower in Mrp4 knockout mice than wild-type mice (2.01 versus 5.06 ml/min/kg BW). Our results suggest that Bcrp and Mrp4 are partly involved in the luminal efflux of edaravone sulfate and edaravone glucuronide, respectively.     

Role of breast cancer resistance protein (Bcrp/Abcg2) in fetal protection during gestation in rat

Breast cancer resistance protein (BCRP/ABCG2) is an ABC family drug efflux transporter expressed in a number of physiological tissues including placenta. Here we investigated the expression and function of Bcrp in the rat placenta and fetus during pregnancy. We show that the expression of Bcrp mRNA in placenta peaks on gestation day (gd) 15 and declines significantly to one third up to term. In fetal body tissue, 6.9 and 7.4-fold Bcrp mRNA increase was detected on gds 15 and 18, respectively, compared to the early gd 12. The expression of Bcrp mRNA in fetal organs on gds 18 and 21 is also demonstrated. Additionally, the function of placental and fetal Bcrp during pregnancy was studied by fetal exposure to cimetidine infused to the maternal circulation. The relative amount of drug that penetrated to fetus was highest on gd 12 and decreased to one tenth thereafter. Studies on cimetidine distribution in fetus revealed 2- and 4.4-times lower penetration to the brain on gds 18 and 21, respectively, compared to the whole fetal tissue. Our results indicate that the rat fetus is protected by Bcrp against potentially detrimental substances from gd 15 onwards. Moreover, we propose that the protection of fetus by placental Bcrp is further strengthened by fetal Bcrp.     

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1β, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac’s mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.     

Urinary excretion of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA), including the sulfate and glucuronide of 4-hydroxy-3-methoxymethamphetamine (HMMA), in humans and rats

The urinary concentrations of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), specifically 4-hydroxy-3-methoxymethamphetamine sulfate (HMMA-Sul) and 4-hydroxy-3-methoxymethamphetamine glucuronide (HMMA-Glu), have been directly measured in both MDMA users and rats by an established liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) procedure. The concentrations of these conjugates in urine from MDMA users (n = 25) ranged from 6.5 to 202 µM (from 1.8 to 55.6 µg ml^?1) for HMMA-Sul and from 1.3 to 87.0 µM (from 0.5 to 32.3 µg ml^?1) for HMMA-Glu, and the ratio of HMMA-Sul to HMMA-Glu ranged from 1.6 to 9.9 (3.1 ± 1.8). These results demonstrate that the sulfation is quantitatively more significant than the glucuronidation for HMMA in humans. In rats, in contrast, almost all the conjugated HMMA (>99%) was excreted as the glucuronide. These findings indicate that hydrolysis should be carefully made in urine analysis by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) by using either an acid or an enzyme possessing both sulfatase and β-glucuronidase activities. It is concluded that a considerable interspecies variation exists in the conjugation of HMMA between humans and rats.     

Urinary excretion of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA), including the sulfate and glucuronide of 4-hydroxy-3-methoxymethamphetamine (HMMA), in humans and rats

The urinary concentrations of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), specifically 4-hydroxy-3-methoxymethamphetamine sulfate (HMMA-Sul) and 4-hydroxy-3-methoxymethamphetamine glucuronide (HMMA-Glu), have been directly measured in both MDMA users and rats by an established liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) procedure. The concentrations of these conjugates in urine from MDMA users (n = 25) ranged from 6.5 to 202 microM (from 1.8 to 55.6 microg ml(-1)) for HMMA-Sul and from 1.3 to 87.0 microM (from 0.5 to 32.3 microg ml(-1)) for HMMA-Glu, and the ratio of HMMA-Sul to HMMA-Glu ranged from 1.6 to 9.9 (3.1 +/- 1.8). These results demonstrate that the sulfation is quantitatively more significant than the glucuronidation for HMMA in humans. In rats, in contrast, almost all the conjugated HMMA (>99%) was excreted as the glucuronide. These findings indicate that hydrolysis should be carefully made in urine analysis by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) by using either an acid or an enzyme possessing both sulfatase and beta-glucuronidase activities. It is concluded that a considerable interspecies variation exists in the conjugation of HMMA between humans and rats.     

Pharmacokinetics and metabolism of an alpha,beta-blocker, amosulalol hydrochloride, in mice: biliary excretion of carbamoyl glucuronide

The pharmacokinetics and metabolism of an alpha,beta-blocker, amosulalol hydrochloride, were investigated in mice. After intravenous administration (10 mg/kg), the plasma concentration of the unchanged drug declined biphasically, with a terminal half-life of 1.1 h. The maximum plasma concentrations were reached at 0.25 h after oral administration, and then declined with apparent half-lives of 0.8-1.3 h. The systemic bioavailability of a 10-mg/kg dose was 38.7%. The area under the plasma concentration curve increased more than proportionally to the dose, which suggests metabolic saturation. After oral and intravenous administrations of (14)C-labelled amosulalol hydrochloride, 64.7% and 81.0% of the radioactivity were recovered, respectively, in the urine within 48 h. HPLC-UV and LC/MS analyses demonstrated that the major urinary metabolite was the glucuronide of M-2 (desmethyl metabolite at the o-methoxyphenoxy group) followed by M-5, the M-3 glucuronide, and the M-4 glucuronide, in that order. In the bile sample, amosulalol carbamoyl glucuronide was found as a new metabolite of this drug.     

The biliary excretion and enterohepatic circulation of benzo(a)pyrene and its metabolites in the rat

The enterohepatic circulation of benzo(a)pyrene (BP) has been investigated in the rat with a view to determining the availability of potentially toxic metabolities to tissues within this cycle. Some 60% of the dose of [¹⁴C]-BP (3 μmoles kg^?1, i.v.) is excreted in bile in 6 hr, with less than 3% in urine. The biliary metabolities are mainly polar conjugates; only 8% of the ¹⁴C in 2 hr bile samples can be directly extracted into ethyl acetate. However, following hydrolysis by β-glucuronidase some 40% of the ¹⁴C is extractable at pH 7. The extract consisted of polar metabolites (polyhydroxylated and/or conjugated; 37.5%), BP 4,5-diol (16.8%), BP 3,6-quinone (5.9%). 9-hydroxy BP (5.4%) and 3-hydroxy BP (5.3%) as indicated by co-chromatography with authentic standards on reversed phase HPLC, together with several unidentified metabolites. The proximate carcinogen BP 7, 8-diol was not detected. Biliary metabolites of [¹⁴C]-BP undergo enterohepatic recirculation in the rat; following the intraduodenal infusion of bile containing metabolites of [¹⁴C]-BP into bile duct cannulated rats, approximately 20% of the dose is absorbed and excreted in bile in 30 hr, with only 1% in urine. The pattern of metabolites in this bile is very similar to that in bile from rats administered [¹⁴C]-BP i.v. Following a single i.v. dose of [¹⁴C]-BP (3 μmoles kg^?1) to rats with re-entrant bile duct cannulae, which allowed intermittent collection of bile over a period of several days with minimal interference to the enterohepatic circulation, the proximate carcinogen BP 7, 8-diol was detected in recirculating bile. Biliary metabolites of BP, which have recently been shown to be mutagenic, can thus traverse the intestine to undergo enterohepatic circulation in the rat.     

Integration of hepatic drug transporters and phase II metabolizing enzymes: Mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites

The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.     

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.     

The formation and toxicity of catechol metabolites of acetaminophen in mice

Acetaminophen is metabolized to a catechol, 3'-hydroxy-4'-hydroxyacetanilide (3-hydroxyacetaminophen), by mouse liver microsomes, and to both catechol and methylated catechol metabolites by the mouse in vivo. Although 3-hydroxyacetaminophen is less hepatotoxic in mice than acetaminophen itself, 3-methoxyacetaminophen is as hepatotoxic as acetaminophen and is subject to a glutathione threshold effect. However, neither metabolite is formed in sufficient amounts to account for the hepatotoxicity caused by acetaminophen in the mouse. Mouse liver microsomes catalyze the oxidation of acetaminophen to the catechol in an apparent cytochrome P-450-mediated reaction that is induced by phenobarbital and inhibited by piperonyl butoxide, but is surprisingly not altered by cobaltous chloride. Lineweaver-Burk analysis of the oxidation carried out by liver microsomes from control animals gave curvilinear plots that may indicate catalysis by two enzyme sites with apparent Km values of 0.011 and 0.271 mM, and apparent Vmax values of 87 and 162 pmol/mg/min, respectively. Neither an isotope effect nor an NIH shift were measurable in the microsomal metabolism of selectively deuterated analogs of acetaminophen to 3-hydroxyacetaminophen. These results, coupled with results of previous investigations with 18O2 and epoxide hydrolase, indicate that a mechanism different from either direct insertion or epoxidation is involved in the formation of the catechol metabolite of acetaminophen.