Possible involvement of P-glycoprotein in the biliary excretion of grepafloxacin

1. In the present study, we have examined the effects of the quinolones norfloxacin (NFLX), enoxacin (ENX), ofloxacin (OFLX), tosufloxacin (TFLX), lomefloxacin (LFLX), sparfloxacin (SPFX) and grepafloxacin (GPFX) on the efflux of doxorubicin from mouse leukaemia P388/ADR cells expressing P-glycoprotein. The relationship between their partition coefficients (hydrophobicity) and effluxing potencies was also elucidated. 2. Both TFLX and SPFX strongly increased the intracellular accumulation of doxorubicin (5 micromol/L) in P388/ADR cells, but had no effect on P388/S cells not expressing P-glycoprotein. The rank of order of the potency of the quinolones (TFLX > SPFX > GPFX > NFLX) was not related directly to their hydrophobicity. These results suggest that some quinolones can reverse anticancer drug resistance. 3. Because GPFX is more highly excreted into the bile than other known quinolones, the effects of doxorubicin (10 mg/kg) or the well-known inhibitors of P-glycoprotein, namely cyclosporine A (10 mg/kg) and erythromycin (100 mg/kg), on the biliary excretion of GPFX at steady state was studied in rats. 4. Doxorubicin, cyclosporine A and erythromycin significantly decreased the biliary clearance of GPFX. Cyclosporine A and erythromycin had a much stronger inhibitory effect on the biliary excretion of GPFX than doxorubicin. These results suggest the possibility that GPFX is, at least in part, excreted into the bile by a P-glycoprotein-mediated transport mechanism.     

Nonlinear pharmacokinetics of hepatobiliary transport of rose bengal in rats after i.v. bolus administration with varying doses.

To investigate the nonlinear kinetics in the hepatobiliary transport of rose bengal (RB), the time profiles of plasma concentration and biliary excretion rate after its i.v. administration at various doses were measured in rats. The total body clearance decreased remarkably with increased dose. The hepatic uptake clearance also showed a similar dose dependency, and saturation of hepatic uptake at least partly accounts for the dose-dependent change in total body clearance. The peak biliary excretion rate approached the transport maximum (approximately 150 nmol min-1 kg-1) with increased dose. To further clarify which process in RB hepatobiliary transport has nonlinearity, we analysed thus obtained data based on a three-compartment model. The hepatic uptake and sequestration rate constants decreased remarkably with increased dose. The initial hepatic uptake rates assessed from the plasma disappearance rate during the early phase fit well to the Michaelis-Menten equation with a saturable and a nonsaturable component. The maximum uptake velocity and Michaelis constant were 4.7 mumol min-1 kg-1 and 360 microM, respectively. That hepatic uptake has a much higher capacity (about 30 fold) than biliary excretion suggests that biliary excretion can be a rate-determining process in the overall hepatobiliary transport of RB. We conclude that the saturation of both hepatic uptake and biliary excretion could be the main causes for the nonlinear pharmacokinetics of hepatobiliary transport of RB.     

Pharmacokinetics of the hepatic transport of organic anions: Influence of extra- and intracelluar binding on hepatic storage of dibromosulfophthalein and interactions with indocyanine green

The influence of intracellular and extracellular protein binding on the hepatic storage and biliary elimination of dibromosulfophthalein (DBSP) was studied in isolated perfused rat liver. Under first order kinetic conditions the amount of DBSP in the liver at a given plasma concentration (hepatic storage) was determined by extracellular binding to albumin and intracellular binding to the cytosolic Y and Z proteins as well as concentrative membrane transport from plasma into the liver. At higher doses, extensive binding of DBSP to intracellular organelles also occurred while liver cytosol/plasma concentration gradients of unbound DBSP were much lower. Hepatic storage increased with decreasing albumin concentration in the perfusate of isolated perfused rat livers. However, it was shown that this parameter is dose-dependent, and errors can be introduced in its calculation if nonlinearity of sinusoidal and canalicular transport processes as well as nonlinear protein binding are not taken into account. The influence of another organic anion, indocyanine green (ICG) on the hepatic storage, subcellular distribution, and elimination of DBSP was subsequently studied. At equimolar amounts the presence of ICG resulted in a 50% decrease in hepatic clearance and hepatic distribution volume of DBSP. It was inferred that these changes are due to an inhibition of carrier-mediated transport across the sinusoidal and canalicular membrane and preferential displacement from intracellular binding sites. In contrast DBSP in equimolar amount enhanced the initial disappearance rate and biliary excretion of ICG, probably due to increasing its free fraction in plasma. It is concluded that the level and mechanism of interaction of two drugs within the eliminating organ can be characterized by combining clearance studies with data on subcellular and extracellular binding.     

Pharmacokinetics of the hepatic transport of organic anions: influence of extra- and intracellular binding on hepatic storage of dibromosulfophthalein and interactions with indocyanine green

The influence of intracellular and extracellular protein binding on the hepatic storage and biliary elimination of dibromosulfophthalein (DBSP) was studied in isolated perfused rat liver. Under first order kinetic conditions the amount of DBSP in the liver at a given plasma concentration (hepatic storage) was determined by extracellular binding to albumin and intracellular binding to the cytosolic Y and Z proteins as well as concentrative membrane transport from plasma into the liver. At higher doses, extensive binding of DBSP to intracellular organelles also occurred while liver cytosol/plasma concentration gradients of unbound DBSP were much lower. Hepatic storage increased with decreasing albumin concentration in the perfusate of isolated perfused rat livers. However, it was shown that this parameter is dose-dependent, and errors can be introduced in its calculation if nonlinearity of sinusoidal and canalicular transport processes as well as nonlinear protein binding are not taken into account. The influence of another organic anion, indocyanine green (ICG) on the hepatic storage, subcellular distribution, and elimination of DBSP was subsequently studied. At equimolar amounts the presence of ICG resulted in a 50% decrease in hepatic clearance and hepatic distribution volume of DBSP. It was inferred that these changes are due to an inhibition of carrier-mediated transport across the sinusoidal and canalicular membrane and preferential displacement from intracellular binding sites. In contrast DBSP in equimolar amount enhanced the initial disappearance rate and biliary excretion of ICG, probably due to increasing its free fraction in plasma. It is concluded that the level and mechanism of interaction of two drugs within the eliminating organ can be characterized by combining clearance studies with data on subcellular and extracellular binding.     

Phenylbutazone-hydroxycoumarol interactions. Effects on steady state disposition, hepatocellular distribution, and biliary excretion of (S)-acenocoumarol in rats

The effect of phenylbutazone on the disposition of (S)-acenocoumarol in the rat was studied at steady state conditions of distribution and elimination. (S)-Acenocoumarol was administered by constant rate infusions (1 microgram/min). The biliary excretion of 6- and 7-hydroxylated acenocoumarol was followed and the intrahepatic distribution was investigated. Phenylbutazone (50 mg/kg) increased the plasma unbound fraction about 4-fold. (S)-Acenocoumarol plasma clearance was enhanced (2.8 +/- 0.15 vs. 1.54 +/- 0.14 ml/min) but the unbound plasma clearance was reduced by 50% (67 +/- 9 vs. 140 +/- 27 ml/min). Phenylbutazone caused an intrahepatic redistribution of (S)-acenocoumarol, i.e. the drug shifted from the cytosol to the 10,000g pellet. The cytosolic unbound concentration, however, was increased. The (S)-acenocoumarol content in the microsomal fraction was not affected. The biliary excretion rate of total metabolite (free plus conjugated) comprised 50% of the (S)-acenocoumarol infusion rate in controls and was slightly stimulated (+20%) by phenylbutazone. The biliary excretion of free metabolites, however, was greatly increased (62 +/- 7 vs. 22 +/- 6 ng/min for 6-hydroxy-acenocoumarol; 337 +/- 38 vs. 141 +/- 32 ng/min for 7-hydroxy-acenocoumarol). This effect is probably due to stimulation of a hepatic biliary transport system; the rate constant for transport of 7-hydroxy-acenocoumarol was enhanced 5-fold (0.107 +/- 0.03 vs. 0.021 +/- 0.007 min-1).     

Hepatic transport mechanisms for bivalent organic cations. Subcellular distribution and hepato-biliary concentration gradients of some steroidal muscle relaxants

In order to characterize the hepato-biliary transport of bivalent cations in more detail, the subcellular distribution of three steroidal muscle relaxants, that differ physicochemically and kinetically, was studied by differential centrifugation of liver homogenates. Binding of the muscle relaxants to macromolecular compounds was measured in Krebs-albumin solution, in cytosolic fraction of liver homogenate and in bile, to estimate the unbound concentrations in the particular fluids. Cytosol/plasma concentration ratios increased in the order pancuronium less than Org 6368 less than vecuronium, but for all of the compounds did not exceed the value that would be attained by passive equilibration according to the membrane potential. The subcellular distribution patterns of the three substances indicated that the mitochondrial fraction is a major storage compartment in the liver. Yet Org 6368 was bound to the particulate fraction of liver homogenate to a larger extent than pancuronium and vecuronium. The high bile/cytosol concentration ratios indicate that for all of these cations an active transport system is involved in the biliary excretion process. For Org 6368 and vecuronium the bile/cytosol concentration ratios are in the same range (about 30) and substantially higher than for pancuronium (about 6). This suggests that for Org 6368 and vecuronium the transport across the canalicular membrane is more efficient than for pancuronium. The combined data indicate that the extensive binding of Org 6368 to particles within the cell is a major factor in the relative efficient hepatic uptake and the modest biliary excretion of this agent. The limited hepato-biliary transport of pancuronium appears to be due to a relatively small net transport, both at the sinusoidal land at the canalicular membrane.     

Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans.

Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.     

Involvement of multiple efflux transporters in hepatic disposition of fexofenadine

Fexofenadine (FEX) is mainly eliminated from the liver into bile in unchanged form. We demonstrated previously that organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are involved in the hepatic uptake of FEX. However, little is known about the mechanisms controlling the hepatic efflux of FEX from the liver to bile and blood. In the present study, the involvement of hepatic efflux transporters in the pharmacokinetics of FEX was investigated in both in vitro and in vivo studies. Vectorial transport of FEX was observed in OATP1B3/human bile salt export pump (hBSEP) double transfectants but not in OATP1B3/human breast cancer resistance protein double transfectants, which indicates the possible contribution of hBSEP to the biliary excretion of FEX in humans. In multidrug resistance-associated protein 2 (Mrp2)(-/-) mice, the biliary excretion clearance based on the plasma concentration and the liver-to-plasma concentration ratio significantly decreased, whereas the biliary excretion clearance based on the liver concentration decreased only with 20%, suggesting the minimum contribution of Mrp2 to its biliary excretion. ATP-dependent transport of FEX was observed in hMRP3-enriched membrane vesicles but not hMRP4. In Mrp3(-/-) mice, the biliary excretion clearance based on both the plasma and liver concentration and the liver-to-plasma concentration ratio increased, suggesting the significant contribution of Mrp3 to its sinusoidal efflux and the up-regulation of its biliary excretion in Mrp3(-/-) mice. On the other hand, pharmacokinetics of FEX remained unchanged in Mrp4(-/-) mice. This information provides a novel insight into the transporters important for FEX disposition.     

Pharmacokinetics of iodoxamic acid in rhesus monkey: biliary excretion, plasma protein binding, and enterophepatic circulation.

The previously reported steady-state method allowed estimation of the capacity-limited pharmacokinetics of the cholangiographic agent, iodipamide. To circumvent the long time period required to establish each steady-state level, a dynamic method was applied to the study of the rate processes involved in the hepatic uptake and biliary excretion of a new cholangiographic agent, iodoxamic acid, in rhesus monkeys. The dynamic method has the advantage that the pharmacokinetic parameters involved in capacity-limited hepatic uptake or biliary excretion can be obtained from a single infusion experiment. The V max was 1.03 +/- 0.25 mumoles/kg/min (mean +/- SD); Km varied from animal to animal and ranged from 1.5 to 16.4 micrometer. Protein binding was estimated using equilibrium dialysis. The Freundlich isotherm yielded a linear plot when the natural logarithm of unbound iodoxamic acid concentration in plasma was plotted against the natural logarithm of its blood concentration. The plasma protein binding data also could be fitted to the Langmuir isotherm, presuming two independent classes of binding.     

Distribution characteristics of grepafloxacin, a fluoroquinolone antibiotic, in lung epithelial lining fluid and alveolar macrophage

The purpose of this study was to investigate the distribution of Grepafloxacin (GPFX), a new quinolone antimicrobial agent, in the lung epithelial lining fluid (ELF) and the alveolar macrophage (AM) in rats, which are potential infection sites in respiratory tract infections. We also aimed to clarify the mechanism governing the transferability of GPFX into the alveolus compartment from a kinetic point of view. The AUC ratios of ELF/plasma and AM/plasma after the oral administration of GPFX were 5.69 +/- 1.00 and 352 +/- 57, respectively, which were several-fold greater than those of ciprofloxacin (CPFX). Pharmacokinetic analyses of time profiles of GPFX concentrations in ELF and AM revealed that the influx clearance from plasma to ELF across the alveolar barrier is 5-fold greater than the efflux clearance from ELF. In addition, the permeability of GPFX across the cultured AM cell membrane was 7-fold and 11-fold greater than that of levofloxacin (LVFX) and CPFX, respectively. The extent of intracellular binding to AM cells (expressed as a constant (alpha)) was the greatest for GPFX, followed by CPFX and LVFX. There was a significant correlation between the alpha value and the partitioning to the immobilized artificial membrane (IAM) column, which consists of phospholipid residues covalently bound to silica. These results suggest that GPFX is highly distributed in ELF and AM, and that the high transferability of GPFX into ELF may be attributable to the existence of asymmetrical transport across the alveolar barrier. In addition, it was suggested that both rapid permeability across the AM cell membrane and avid binding to the membrane phospholipids may be responsible for the high accumulation of GPFX in AM.     

Mechanism for the tissue distribution of grepafloxacin, a fluoroquinolone antibiotic, in rats.

This study was carried out to investigate the most important factor(s) governing the tissue distribution of grepafloxacin (GPFX), a fluoroquinolone antibiotic, in rats. The tissue-to-blood concentration ratio (K(p)) of GPFX at steady state during constant infusion was highest in the lung, followed by the pancreas, kidney, and spleen. After bolus injection, GPFX was efficiently taken up by most of the organs examined, the uptake clearance other than the lung being almost blood flow-limited. Approximately 10% of the intravenously injected dose was rapidly trapped by the lung, but GPFX distribution rapidly decreased within 30 s due to the washout by the plasma flow. Thus, the higher distribution of GPFX to the lung compared with the other organs cannot be accounted for by a difference in its uptake or efflux. Subcellular fractionation after the infusion indicated that GPFX is primarily distributed to the organelle fractions in most organs, 60% of lung-associated GPFX being recovered in the nucleus and plasma membrane fraction. Such subcellular distribution in the lung was proportional to the phosphatidylserine (PhS) content of each fraction. The steady-state K(p) value in each tissue in vivo also correlated with the tissue content of PhS. GPFX preferentially binds to PhS, compared with other phospholipids, and this binding was inhibited by weakly basic drugs, such as quinidine, imipramine, and propranolol, that have also been reported to bind to PhS. The association of GPFX with PhS synthase transformants of Chinese hamster ovary (CHO-K1) cells depends on the PhS content of each cell line, this association being also inhibited by basic drugs. These results suggest that binding of GPFX to PhS is the major determinant of the high distribution of GPFX to the lung.     

Carrier-mediated uptake of grepafloxacin, a fluoroquinolone antibiotic, by the isolated rat lung cells

Grepafloxacin (GPFX) is a new quinolone antibiotic (NQ) which is highly distributed to the lung and other tissues. In the present study, to characterize the distribution mechanism of GPFX to the lung, the uptake of GPFX by isolated rat lung cells was examined in vitro. GPFX was rapidly taken up by the cells, and the uptake reached a steady-state within 5 min. The cell-to-medium concentration ratio at equilibrium was 56.8+/-1.9 microL/mg protein, which was much higher than the cellular volume. GPFX uptake consisted of a saturable component (Km: 264+/-181 microM, Vmax: 2.94+/-2.33 nmol/min/mg protein) and a nonsaturable component (Pdif: 7.04+/-2.17 microL/min/mg protein). The uptake of GPFX was reduced in the presence of ATP-depletors (FCCP and Rotenone) and by the replacement of sodium with choline in the medium, suggesting that GPFX uptake is at least partially mediated by an Na+- and energy-dependent process. GPFX uptake tended to be reduced in the presence of other NQs such as levofloxacin, lomefloxacin and sparfloxacin, but was only minimally affected by the substrates of several uptake mechanisms already identified in the liver and kidney such as taurocholate, p-aminohippurate, L-carnitine and tetraethylammonium. These results suggested that GPFX is taken up by the lung partially via carrier-mediated transport system(s), distinct from the identified transporters, and such active transport systems may at least partially account for the efficient distribution of GPFX to the lung.     

Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment.

Previous studies have demonstrated that phenobarbital (PB) significantly impairs the biliary excretion of acetaminophen glucuronide (AG) in rats. Studies also suggested that Mrp2 mediates AG biliary excretion, and Mrp3 is involved in AG basolateral export. It was hypothesized that inhibition of Mrp2-mediated AG transport by PB or PB metabolites, and PB induction of Mrp3, may contribute to the impaired biliary excretion of AG by PB. In the present study, the hepatobiliary transport of AG in single-pass isolated perfused Wistar and TR(-) rat livers was investigated. The AG biliary clearance was markedly decreased, and the AG basolateral clearance was significantly increased in TR(-) rat livers. Uptake of AG by Mrp2 and Mrp3, and inhibition of Mrp2- and Mrp3-mediated transport by PB and major PB metabolites, were investigated with rat Mrp2- or Mrp3-expressing Sf9 cell plasma membrane vesicles (Sf9-PMVs). AG was transported by Mrp3 (K(m) approximately 0.91 mM). Net ATP-dependent AG uptake into Mrp2-expressing Sf9-PMVs could not be detected directly. However, AG significantly inhibited Mrp2-mediated 5-(and 6)-carboxy-2',7'-dichlorofluorescein (CDF) transport. p-Hydroxyphenobarbital glucuronide (p-OHPBG), but not PB or p-hydroxyphenobarbital, significantly inhibited Mrp2-mediated CDF transport. The IC(50) values for p-OHPBG inhibition of Mrp2-mediated CDF uptake and Mrp3-mediated AG transport were similar (approximately 0.68 and 0.46 mM, respectively). PB treatment (80 mg/kg/day x 4 days) markedly increased hepatic Mrp3 expression in Wistar rats. In conclusion, inhibition of Mrp2-mediated AG transport by p-OHPBG provided one possible explanation for the impaired biliary excretion of AG after acute PB treatment. However, impaired biliary excretion of AG after PB pretreatment may be attributed primarily to the induction of hepatic Mrp3 by PB.     

Kinetic Analysis of Hepatobiliary Transport for Conjugated Metabolites in the Perfused Liver of Mutant Rats (EHBR) with Hereditary Conjugated Hyperbilirubinemia

Purpose. Previously, we found that the biliary excretion of the 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide is severely impaired in Eisai hyperbilirubinemic rats (EHBR), while that of sulfate remains normal (Takenaka et al., J. Pharmacol. Exp. Then, 274: 1362–1369, 1995). The purpose of the present study is to clarify the mechanisms for impairment of the biliary excretion of E3040 glucuronide in EHBR. Methods. We kinetically analyzed the disposition of the conjugates in the perfused liver at steady state. The uptake of the conjugates into the isolated canalicular membrane vesicles (CMVs) was also examined. Results. At steady state, the bile/liver unbound concentration ratios of the conjugates were 40-400 in both rat strains, indicating a highly concentrated process. The biliary excretion clearance (CL_u,bile) of the glucuronide, defined for the unbound concentration in the liver, was decreased in EHBR to 1/30 of that in normal rats, whereas the CL_u,bile of the sulfate was comparable between the two rat strains. In vitro, the transport of E3040 glucuronide into CMV prepared from SD rats exhibited the ATP dependency, whereas minimal effect of ATP was observed on the uptake of the glucuronide into CMV from EHBR. In contrast, the uptake of E3040 sulfate was comparable between SD rats and EHBR. Furthermore, ATP did not stimulate the uptake of sulfate into the CMVs. Conclusions. It was suggested (1) that the excretion of E3040 glucuronide across the bile canalicular membrane is mediated by the primary active transporter which is defective in EHBR and (2) that the bile canalicular transport system for E3040 sulfate is different from that for the glucuronide in that the former remains normal in EHBR.     

Renal handling of biphosphonate alendronate in rats.

Alendronate is a bisphosphonate that is secreted via a saturable pathway in rat kidney. This study is designed to discover if the rate-determining step in its net renal secretion is uptake into the renal tubule. The tissue uptake clearance of alendronate by the kidney, estimated from an integration plot analysis and normalized with respect to plasma protein binding, was 4.2 times higher at a tracer dose than that of inulin, indicating uptake of alendronate by the renal tubules. The uptake clearance is comparable with the net secretion clearance obtained from an infusion study, indicating that the rate-determining step in the net secretion is uptake under the tracer conditions. When the dose was increased, however, there was no reduction in uptake clearance while the net secretion clearance fell to almost zero. The urinary excretion clearance defined with respect to the steady state concentration in the kidney also fell to almost zero. This result suggests that saturation of the net secretion of alendronate is caused by saturation of membrane transport through the brush-border membrane. Thus, it would seem that there is a transport mechanism for alendronate on the brush-border membrane of kidney epithelial cells.     

Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5]enkephalin.

Rapid and extensive biliary excretion of [D-penicillamine2,5]enkephalin (DPDPE) in rats as the unchanged peptide suggests that multiple transport proteins may be involved in the hepatobiliary disposition of this zwitterionic peptide. Although DPDPE is a P-glycoprotein substrate, the role of other transport proteins in the hepatic clearance of DPDPE has not been established. Furthermore, the ability of various experimental approaches to quantitate the contribution of a specific hepatic uptake or excretion process when multiple transport systems are involved has not been addressed. 3H-DPDPE uptake in suspended Wistar rat hepatocytes was primarily (>95%) due to temperature-dependent transport mechanisms; similar results were obtained in suspended hepatocytes from Mrp2-deficient (TR-) rats. Pharmacokinetic modeling revealed that saturable and linear processes were involved in 3H-DPDPE uptake in hepatocytes. The use of transport modulators suggested that hepatic uptake of 3H-DPDPE was mediated by Oatp1a1, Oatp1a4, and likely Oatp1b2. Accumulation of 3H-DPDPE in sandwich-cultured (SC) hepatocytes was rapid; uptake of 3H-DPDPE in SC rat hepatocytes from control and TR- rats was similar. However, the biliary excretion index and biliary clearance decreased by 83 and 85%, respectively, in TR- SC rat hepatocytes, indicating that DPDPE is an Mrp2 substrate. Rate constants for uptake and excretion of 3H-DPDPE in SC rat hepatocytes were determined by pharmacokinetic modeling; data were consistent with basolateral excretion of 3H-DPDPE from the hepatocyte. These results demonstrate the complexities of hepatobiliary disposition when multiple transport mechanisms are involved for a given substrate and emphasize the necessity of multi-experimental approaches for the comprehensive resolution of these processes.     

Effect of acute renal failure on the disposition and elimination of [3H]N-acetyl procainamide ethobromide in the rat

The effect of glycerol-induced acute renal failure (ARF) on the disposition and elimination of the organic cation [3H]N-acetyl procainamide ethobromide (APAEB) was investigated in the rat. In rats with ARF the plasma clearance, rate constant for the terminal portion of the plasma concentration-time curve and apparent volume of distribution were all decreased (P less than 0.01). Furthermore, the renal clearance of APAEB and the percentage dose excreted in urine were reduced by 85 and 74%, respectively. Decreased renal excretion probably accounted for the altered kinetics of APAEB in ARF because ligation of the renal pedicles of control rats produced changes in the kinetics of APAEB that were similar to those seen in animals with ARF. No change in either the hepatic content of APAEB or its biliary excretion were detected in rats with ARF. Similarly, the hepatic handling of ouabain and taurocholic acid was previously found to be unaltered in ARF; but by contrast, the hepatic uptake and initial biliary excretion of bromosulphophthalein and indocyanine green were decreased (Bowmer & Yates 1984, Br. J. Pharmacol. 83: 773-782). Together these studies indicate that there is a selective impairment of hepato-biliary transport in ARF.     

Canalicular membrane transport is primarily responsible for the difference in hepatobiliary excretion of triethylmethylammonium and tributylmethylammonium in rats.

Two structurally similar quaternary ammonium compounds, triethylmethylammonium (TEMA, M(r) 116) and tributylmethylammonium (TBuMA, M(r) 200) were used as model compounds to identify the unit process of hepatobiliary excretion that is responsible for markedly different biliary excretion of organic cations (OCs). Cumulative biliary excretion (in percentage of dose; i.v., 12 micromol/kg) was 0.17 for TEMA and 34.5 for TBuMA. In vivo uptake clearance into the liver was 0.686 +/- 0.020 ml/min for TEMA and 0.421 +/- 0.028 ml/min for TBuMA. When the uptake clearance was examined in an isolated hepatocyte system, comparable clearance between TEMA and TBuMA was obtained, consistent with the in vivo result. These observations suggest that uptake into the liver is not the major determinant for the difference in biliary excretion of the OCs. Coadministration of colchicine, an inhibitor of microtubule formation, had no effect on biliary excretion of the model compounds, and the primary site of subcellular distribution of the OCs appears to be the cytosol, suggesting that intracellular movement does not play a major role in the markedly different biliary excretion of the OCs. In contrast, in vivo excretion clearance across the canalicular membrane for TBuMA was 180-fold greater than that for TEMA, and in vitro efflux clearance of TBuMA was smaller than that of TEMA (p <.01), indicative of involvement of these processes in the markedly different biliary excretion of the OCs. Therefore, these data indicate that canalicular transport is primarily responsible for the markedly different biliary excretion of TEMA and TBuMA.     

Pharmacokinetics, hepatic biotransformation and biliary and urinary excretion of bromosulfophthalein (BSP) in an experimental liver disease mimicking biliary cirrhosis

We studied the hepatic handling of bromosulfophthalein in healthy rabbits with hepatic coccidiosis 28 days after an experimental infection with sporulated oocysts of Eimeria stiedai, an experimental model of liver disease histopathologically resembling primary biliary cirrhosis in man. A pharmacokinetic study of the results was performed following a multicompartmental model with 7 transfer constants to describe the physiological disposition of the dye. The study showed that the plasma disappearance, distribution volume (Vi), hepatic biotransformation and the biliary and urinary elimination of conjugated (BSPc) and unconjugated (BSPu) bromosulfophthalein were markedly altered. Whereas Vi and urinary excretion of the dye were significantly increased, the hepatic clearance, biotransformation and biliary excretion of BSPc and BSPu were drastically reduced in infected rabbits. Satisfactory agreement was obtained between the experimental and estimated data, particularly those relating to biotransformation clearance and biliary and urinary excretion of the dye. These results demonstrate that severe liver disease in rabbits with histopathological liver alterations resembling several hepatic dysfunctions in man markedly reduce hepatic uptake, metabolism and biliary excretion of a xenobiotic such as BSP.     

Involvement of multiple transport systems in the disposition of an active metabolite of a prodrug-type new quinolone antibiotic, prulifloxacin

Prulifloxacin is a prodrug-type new quinolone. The purpose of this study is to clarify the mechanism of biliary excretion and brain distribution of its active metabolite, UFX. UFX was efficiently excreted into the bile in rats, with its concentration in the bile being 30-60 times higher than that in plasma. The in vivo disposition study revealed that multidrug resistance-associated protein 2 (MRP2) was involved in the biliary excretion of glucuronide metabolite, but not of the unchanged UFX. A transport study using a P-glycoprotein (P-gp) overexpressing cell line, LLC-GA5-COL150, showed that UFX was a substrate of P-gp. Nevertheless, the biliary clearance (CLbile) of UFX in P-gp-gene-deficient mice was not different from that in the normal mice, although the concentration in the liver was slightly higher than that in the normal mice. These observations suggest that multiple transport systems are involved in the biliary excretion of UFX, with minor contribution of P-gp. The distribution of UFX in the rat brain was quite low, and its tissue to plasma concentration ratio (Kp) in the brain was much less than the unity and was increased by cyclosporin A. The Kp in the brain of mdr1a/1b(-/-) mice was higher than that in the normal mice, suggesting that efflux by P-gp played a major role in the limited brain distribution of UFX.