Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport.

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17ß-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 µM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 µM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 µM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 µM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17ß-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.

The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1-week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulphated metabolite, 4-methylumbelliferyl sulphate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice.

Identification of diet-derived constituents as potent inhibitors of intestinal glucuronidation.

Drug-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Furanocoumarins in grapefruit juice are cornerstone examples of diet-derived xenobiotics that perpetrate interactions with drugs via mechanism-based inhibition of intestinal CYP3A4. Relative to intestinal CYP3A4-mediated inhibition, alternate mechanisms underlying dietary substance-drug interactions remain understudied. A working systematic framework was applied to a panel of structurally diverse diet-derived constituents/extracts (n = 15) as inhibitors of intestinal UDP-glucuronosyl transferases (UGTs) to identify and characterize additional perpetrators of dietary substance-drug interactions. Using a screening assay involving the nonspecific UGT probe substrate 4-methylumbelliferone, human intestinal microsomes, and human embryonic kidney cell lysates overexpressing gut-relevant UGT1A isoforms, 14 diet-derived constituents/extracts inhibited UGT activity by >50% in at least one enzyme source, prompting IC(50) determination. The IC(50) values of 13 constituents/extracts (≤10 µM with at least one enzyme source) were well below intestinal tissue concentrations or concentrations in relevant juices, suggesting that these diet-derived substances can inhibit intestinal UGTs at clinically achievable concentrations. Evaluation of the effect of inhibitor depletion on IC(50) determination demonstrated substantial impact (up to 2.8-fold shift) using silybin A and silybin B, two key flavonolignans from milk thistle (Silybum marianum) as exemplar inhibitors, highlighting an important consideration for interpretation of UGT inhibition in vitro. Results from this work will help refine a working systematic framework to identify dietary substance-drug interactions that warrant advanced modeling and simulation to inform clinical assessment.

A robust and quantitative method for tracking liposome contents after intravenous administration.

We introduce a method for tracking the rate and extent of delivery of liposome contents in vivo based on encapsulation of 4-methylumbelliferyl phosphate (MU-P), a profluorophore of 4-methylumbelliferone (MU). MU-P is rapidly dephosphorylated by endogenous phosphatases in vivo to form MU after leakage from the liposome. The change in fluorescence spectra when MU-P is converted to MU allows for quantification of entrapped (MU-P) and released (MU) liposome contents by fluorescence or by a sensitive high performance liquid chromatography assay. We define the "cellular availability" of an agent encapsulated in a liposome as the ratio of the amount of released agent in the tissue to the total amount of agent in the tissue; this parameter quantifies the fraction of drug available for therapy. The advantage of this method over existing technologies is the ability to decouple the signals of entrapped and released liposome contents. We validate this method by tracking the circulation and tissue distribution of MU-P loaded liposomes after intravenous administration. We use this assay to compare the cellular availability of liposomes composed of engineered phosphocholine lipids with covalently attached cholesterol, sterol-modified lipids (SML), to liposomes composed of conventional phospholipids and cholesterol. The SML liposomes have similar pharmacokinetic and biodistribution patterns as conventional phospholipid-cholesterol liposomes but a slower rate of contents delivery into the tissue. Thus, MU-P enables the tracking of the rate and extent of liposome contents release in tissues and should facilitate a better understanding of the pharmacodynamics of liposome-encapsulated drugs in animals.

Photoresponsive coumarin-tethered multifunctional magnetic nanoparticles for release of anticancer drug.

Recently, photoresponsive nanoparticles have received significant attention because of their ability to provide spatial and temporal control over the drug release. In the present work, we report for the first time photoresponsive multifunctional magnetic nanoparticles (MNPs) fabricated using coumarin-based phototrigger and Fe/Si MNPs for controlled delivery of anticancer drug chlorambucil. Further, newly fabricated photoresponsive multifunctional MNPs were also explored for cell luminescence imaging. In vitro biological studies revealed that coumarin tethered Fe/Si MNPs of ~9 nm size efficiently delivered the anticancer drug chlorambucil into cancer cells and thereby improving the drug action to kill the cancer cells upon irradiation. Such multifunctional MNPs with strong fluorescence, good biocompatibility and efficient photocontrolled drug release ability will be of great benefit in the construction of light-activated multifunctional nano drug delivery systems.

The chaperone activity and toxicity of ambroxol on Gaucher cells and normal mice.

Gaucher disease (GD), caused by a defect of acid ß-glucosidase (ß-Glu), is one of the most common sphingolipidoses. Recently, ambroxol, an FDA-approved drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified as a chemical chaperone for GD. In the present study, we investigated the chaperone activity and toxicity of ambroxol on both cultured GD patient cells and normal mice. We found that ambroxol treatment significantly increased N370S, F213I, N188S/G193W and R120W mutant ß-Glu activities in GD fibroblasts with low cytotoxicity. Additionally, we measured the ß-Glu activity in the tissues of normal mice which received water containing increasing concentrations of ambroxol ad libitum for one week. No serious adverse effect was observed during this experiment. Ambroxol significantly increased the ß-Glu activity in the spleen, heart and cerebellum of the mice. This result showed its oral availability and wide distribution and chaperone activity in the tissues, including the brain, and its lack of acute toxicity. These characteristics of ambroxol would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations.

Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis.

The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-D-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.

Systematic screening of human ABCC3 polymorphisms and their effects on MRP3 expression and function.

The present study was undertaken to identify genetic polymorphisms of multidrug resistance-associated protein 3 (MRP3, gene name ABCC3), an ATP-binding cassette transporter that mediates the transport of substrates across the basolateral membrane into the blood, and to investigate their effects on ABCC3 expression and MRP3 function. We identified genetic polymorphisms of ABCC3 and evaluated the effects by (1) a luciferase reporter gene assay, (2) measuring mRNA levels, and (3) a human pharmacogenomics study with 4-methylumbelliferone glucuronide (4-MUG). Overall, 61 genetic variants were identified in three ethnic populations; of these variants 17 were novel (7 were non-synonymous: 61Arg>Cys, 132Gln>Stop, 221Trp>Stop, 270His>Gln, 548Leu>Gln, 600Lys>Arg, and 1324Arg>His). However, these mutations occurred at very low frequencies (max. 4.7%). The observed allele frequencies showed considerable inter-ethnic differences. The reporter gene assay indicated a significant reduction of transcriptional activity with the -1767G>A allele compared to the wild-type allele; however, a decreased expression of ABCC3 mRNA was not detected in human liver samples. A human pharmacokinetic study showed that the ABCC3 genotype in the promoter region was not associated with changes in the pharmacokinetics of 4-MUG, a substrate of MRP3. This is the first study to assess the effects of ABCC3 polymorphisms on human pharmacokinetics; however, further investigations are needed to complete the picture.

Differential disposition of intra-renal generated and preformed glucuronides: studies with 4-methylumbelliferone and 4-methylumbelliferyl glucuronide in the filtering and nonfiltering isolated perfused rat kidney.

OBJECTIVES: This study was designed to investigate the renal disposition of 4-methylumbelliferone (4MU) and 4-methylumbelliferyl glucuronide (4MUG) to characterise the contribution of excretion and metabolic clearance to total clearance in the kidney. METHODS: The isolated perfused kidney (IPK) from the male Sprague-Dawley rat was used in filtering and non-filtering mode to study the renal disposition of 4MU, renally generated 4MUG and preformed 4MUG. Perfusate and urine (filtering IPK only) was collected for up to 120 min and 4MU and 4MUG in perfusate and urine were determined by HPLC. Analytes were also measured in kidney tissue collected at 120 min. Non-compartmental analysis was used to derive pharmacokinetic parameters. KEY FINDINGS: The concentration of 4MU in perfusate declined with a terminal half-life of approximately 120 min following administration to the filtering IPK and nonfiltering IPK. There was a corresponding increase in the concentration of 4MUG. Metabolic clearance of 4MU accounted for 92% of total renal clearance. After bolus dosing of preformed 4MUG in the perfusion reservoir of the filtering IPK, the perfusate concentration declined with the terminal half-life of approximately 260 min. The renal excretory clearance of preformed 4MUG accounted for 96% of total renal clearance. 4MU was extensively metabolized by glucuronidation in the filtering and nonfiltering IPK, and the total renal clearance of 4MU was far greater than its renal excretory clearance. This indicated that glucuronidation was the major elimination pathway for 4MU in the kidney. CONCLUSIONS: The data confirmed an important role for the kidney in the metabolic clearance of xenobiotics via glucuronidation and signalled the lack of impact of impaired glomerular filtration on renal drug metabolism.

Functional characterization of multidrug resistance-associated protein 3 (mrp3/abcc3) in the basolateral efflux of glucuronide conjugates in the mouse small intestine.

The intestine expresses metabolic enzymes and transporters and functions as a barrier to orally administered xenobiotics. This study aimed to examine the importance of multidrug resistance-associated protein 3 (Mrp3/Abcc3) in the serosal efflux of glucuronide conjugates formed in the intestine using wild-type and Mrp3(-/-) mice. The everted sacs of the intestine were incubated with 4-methylumbelliferone (4MU), and the efflux rates of intracellularly formed glucuronide conjugate of 4MU (4MUG) into the mucosal and serosal sides were determined. The permeability-surface area product across the serosal membrane (PS(serosal)) of 4MUG in wild-type mice was greatest in the duodenum followed by the jejunum, ileum, and colon. The corresponding parameters were significantly reduced in Mrp3(-/-) mice (approximately 33% of that in wild-type mice) except for the colon where the PS(serosal) of 4MUG was similar between wild-type and Mrp3(-/-) mice. There was no difference in the PS(mucosal) of 4MUG in whole segments of the intestine between wild-type and Mrp3(-/-) mice. In addition to 4MUG, the PS(serosal) of the glucuronide conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) and acetaminophen in the jejunal everted sacs were also significantly reduced in Mrp3(-/-) mice compared with wild-type mice. There was no difference in the mRNA and protein expression of efflux transporters between wild-type and Mrp3(-/-) mice. These results suggest that Mrp3 plays major roles in the efflux transport of various glucuronide conjugates from the enterocytes to the portal blood in the small intestine together with unknown transporter(s), but the contribution of Mrp3 to the basolateral efflux of 4MUG was negligible in the colon.

Silkworm as a model animal to evaluate drug candidate toxicity and metabolism.

To evaluate the feasibility of using the silkworm as a model animal for screening drug candidates, we examined whether the lethal dose of cytotoxic chemicals in silkworm, Bombyx mori, were consistent with those in mammals, and compared the metabolic pathways of these drugs between silkworms and mice. The lethal dose levels of cytotoxic chemicals in silkworms were consistent with those in mammals. We examined the fate of model drugs, 4-methyl umbelliferone, umbelliferone, and 7-ethoxycoumarine, in silkworm larvae. The half-life of 4-methyl umbelliferone in the silkworm larvae hemolymph was 7.0+/-0.1 min, similar to that in mouse blood. In silkworm larvae, 4-methyl umbelliferone was conjugated with glucose, whereas in mammals it is conjugated with glucuronate or sulfate. These results are consistent with a previous report that UDP-glucosyltransferase catalyzes the conjugation of 4-methyl umbelliferone. The glucose-conjugation reaction of 4-methyl umbelliferone was observed in microsomal fractions of fat bodies isolated from silkworms. Furthermore, most umbelliferone and 7-ethoxycoumarine injected into the hemolymph of silkworms was eliminated through the feces in the glucose-conjugated form. These findings suggest that chemicals are metabolized through a pathway common to both mammals and silkworms: reaction with cytochrome P450, conjugation with hydroxylated compounds, and excretion.

The important role of Bcrp (Abcg2) in the biliary excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in mice.

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(-/-), Abcg2(-/-), and Abcb1a(-/-)/Abcb1b(-/-) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also approximately 35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (> or =37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.

Evaluation of the role of multidrug resistance-associated protein (Mrp) 3 and Mrp4 in hepatic basolateral excretion of sulfate and glucuronide metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3-/- and Abcc4-/- mice.

Although glucuronide and sulfate conjugates of many drugs and endogenous compounds undergo appreciable hepatic basolateral excretion into sinusoidal blood, the mechanisms that govern basolateral translocation of these hydrophilic metabolites have not been completely elucidated. In the present study, the involvement in this process of Mrp3 and Mrp4, two basolateral efflux transporters, was evaluated by analyzing the hepatic basolateral excretion of the glucuronide and sulfate metabolites of acetaminophen, 4-methylumbelliferone, and harmol in Abcc3(-/-) and Abcc4(-/-) mice using a cassette dosing approach. In the livers of Abcc3(-/-) and Abcc4(-/-) mice, the basolateral excretory clearance of acetaminophen sulfate was reduced approximately 20 and approximately 20%, 4-methylumbelliferyl sulfate was reduced approximately 50 and approximately 65%, and harmol sulfate was decreased approximately 30 and approximately 45%, respectively. The basolateral excretory clearance of acetaminophen glucuronide, 4-methylumbelliferyl glucuronide, and harmol glucuronide was reduced by approximately 96, approximately 85, and approximately 40%, respectively, in the livers of Abcc3(-/-) mice. In contrast, basolateral excretory clearance of these glucuronide conjugates was unaffected by the absence of Mrp4. These results provide the first direct evidence that Mrp3 and Mrp4 participate in the hepatic basolateral excretion of sulfate conjugates, although additional mechanism(s) are likely involved. In addition, they reveal that Mrp3 mediates the hepatic basolateral excretion of diverse glucuronide conjugates.

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.

Impaired renal excretion of 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) sulfate in breast cancer resistance protein (BCRP1/ABCG2) knockout mice.

Murine breast cancer resistance protein 1 (Bcrp1) is expressed in the brush-border membrane of proximal tubule cells of the kidney. The purpose of the present study is to investigate whether Bcrp1 could be involved in the urinary excretion of the human BCRP substrates, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole sulfate (E3040S) and 4-methylumbelliferone sulfate (4MUS), using Bcrp1(-/-) mice. E3040S and 4MUS were given to the mice by intravenous infusion, and plasma and kidney concentrations and the urinary excretion rate were determined. Knockout of Bcrp1 did not affect the creatinine clearance [7.17 +/- 1.00 and 8.66 +/- 2.02 ml/min/kg for Bcrp1(-/-) and wild-type mice, respectively]. The renal clearance of E3040S was 2.4-fold lower in Bcrp1 (-/-) mice compared with wild-type mice (2.74 +/- 0.41 versus 6.55 +/- 0.52 ml/min/kg). The concentration of E3040S in the kidney was increased in Bcrp1(-/-) mice compared with that in wild-type mice (55.5 +/- 10.5 versus 19.4 +/- 2.7 nmol/g kidney, respectively). In contrast, knockout of Bcrp1 did not affect the pharmacokinetic parameters of 4MUS, although 4MUS was predominantly excreted in the urine. This is to our knowledge the first demonstration of involvement of Bcrp1 in the renal secretion of organic sulfates. However, taking the results of 4MUS into consideration, the renal secretion of organic sulfates cannot be accounted for solely by Bcrp1, and transporters other than Bcrp1 are also involved.

Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid.

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver.

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of drugs. I: studies by in vivo constant infusion.

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

Carrier-mediated entry of 4-methylumbelliferyl sulfate: characterization by the multiple-indicator dilution technique in perfused rat liver.

The hepatocellular entry of 4-methylumbelliferyl sulfate (4MUS) a highly ionized and highly bound anion capable of futile cycling, was examined in the single-pass albumin-free perfused rat liver preparation. Desulfation of 4MUS to 4-methylumbelliferone (4MU) was verified in vitro to be a low-affinity, high-capacity process (Km = 731 micromol/L; Vmax = 414 nmol min(-1) g(-1) liver). With 4MUS given to the perfused rat liver, sulfation of 4MU, the formed metabolite, was attenuated in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a sulfation inhibitor, and when sulfate ion was substituted by chloride ion. 4MU sulfation, being a high-affinity system, was reduced most effectively at the lowest 4MUS concentration (15 micromol/L) used, evidenced by the increased (24%) net hepatic extraction ratio of 4MUS and reduced utilization (72%) of infused tracer 35SO4(2-) by 4MU for 4MU35S formation. Single-pass multiple indicator dilution (MID) studies were thus conducted under identical conditions (DCNP and absence of inorganic sulfate), with injection of [3H]4MUS and a set of noneliminated vascular and cellular reference indicators into the portal vein (prograde) or hepatic vein (retrograde), against varying background bulk concentrations of 4MUS (5 to 900 micromol/L). The steady-state removal rate of 4MUS and formation rates of 4MU and its glucuronide conjugate (4MUG) were not altered with perfusion flow direction, suggesting the presence of even or parallel distributions of 4MUS desulfation and 4MU glucuronidation activities. When the outflow dilution profile of [3H]4MUS was evaluated with the barrier-limited model of Goresky, a slight red cell carriage effect was found for 4MUS. The permeability surface area product for cellular entry for prograde showed a dramatic concentration-dependent decrease (from 0.13 to 0.01 mL sec(-1) g(-1), or 7.4 to 0.56 times the blood perfusate flow rate) and was resolved as saturable and nonsaturable components, while data for retrograde were more scattered, varying from 2.8 to 1 times the blood perfusate flow rate. Efflux (coefficient = 0.0096 +/- 0.0024 and 0.0088 +/- 0.0062 mL sec(-1) g(-1), respectively) was relatively insensitive to concentration and flow direction. The same was observed for the removal capacity for metabolism and excretion (sequestration coefficient: for prograde, 0.0056 +/- 0.0017 mL sec(-1) g(-1); for retrograde, 0.0056 +/- 0.003 mL sec(-1) g(-1)). The decrease in the apparent partition coefficient (ratio of 4MUS concentration estimated in tissue to unbound plasma concentration) and the increase in relative throughput component with concentration further substantiate the claim on the presence of concentrative processes at the sinusoidal membrane.

Uptake of sulfate conjugates by isolated rat hepatocytes.

The uptake of estrone sulfate (E1S; 1 to 400 microM), harmol sulfate (HS; 5 to 900 microM), and 4-methylumbelliferyl sulfate (4MUS; 5 to 1000 microM) was investigated in isolated rat hepatocytes in the presence or absence of inhibitors. Uptake of all of the sulfate conjugates was rapid and exhibited saturation kinetics, best characterized by saturable and nonsaturable (linear transmembrane clearance) transport systems. The KM's were: 16 +/- 6, 123 +/- 28, and 64 +/- 6 microM for E1S, HS, and 4MUS, respectively, with corresponding Vmax's of 0.85 +/- 0.56, 0.48 +/- 14, and 0.42 +/- 0.07 nmol/min/10(6) cells. The nonsaturable uptake clearances, which displayed concentration-independent uptake, were 3 +/- 2, 1 +/- 0.1, 0.5 +/- 0.1 microliter/min/10(6) cells, respectively. Uptake of E1S was inhibited by ouabain (1 mM) and replacement of sodium by choline, whereas HS was insensitive to the addition or substitution. Uptake of both E1S and HS was significantly reduced by metabolic inhibitors (antimycin A, 2.7 microM, rotenone, 30 microM, and KCN, 2 mM) and temperature reduction (from 37 to 27 degrees C). 4,4'-Diisothiocyanostilbene-2-2-'disulfonic acid (2 mM), an inhibitor of anion transport, reduced E1S and HS uptake; E1S uptake was also reduced by HS. HS uptake by both saturable and nonsaturable transport components was depressed by 4MUS (300 microM); the apparent KM was increased by 83% while the Vmax remained unaltered, and the nonsaturable component was decreased by 48%. The data strongly suggest that multiple pathways exist for the uptake of E1S, HS, and 4MUS. E1S uptake is sodium-dependent, requires energy, and is inhibited by anions such as 4,4'diisothiocyanostilbene-2-2'-disulfonic acid and other sulfate conjugates. HS uptake, while being energy dependent, is not sodium dependent, and is inhibited by 4MUS in a competitive fashion. At least one of these pathways is shared.