Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted

1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (−/−) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow.
2. ³H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine.
3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdr1a/1b (−/−) mice compared to wild-type.
4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdr1a/1b (−/−) mice compared to wild-type mice. The absence of the mdr1-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice.
5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdr1a/1b (−/−) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expresion of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function.
6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdr1-type P-glycoproteins.

     

Different Activity of ATP Dependent Transport Across the Canalicular Membrane for Tributylmethyl-ammonium and Triethylmethyl-ammonium as a Potential Mechanism of the Preferential Biliary Excretion for Tributylmethylammonium in the Rat

Purpose. The mechanism(s) responsible for the significantly higher biliary excretion of tributyl methyl ammonium (TBuMA) than of tri-ethyl methyl ammonium (TEMA) was investigated in canalicular liver plasma membrane vesicles (cLPM). Methods. The uptake of [³H]TBuMA and [³H]TEMA into cLPM in the presence of a pH gradient or ATP was measured by a rapid filtration technique. Results. The uptake of substrates into the vesicle was significantly increased by an outwardly directed pH gradient. The pH dependent uptake was saturable and cross-inhibited by the other organic cation, indicating that TEMA and TBuMA share a common transport mechanism. Kinetic analysis revealed the two compounds show similar characteristics for the pH-gradient dependent uptake. Thus, the organic cation/H⁺ exchange mechanism does not appear to explain the significant difference in biliary excretion of the organic cations. In the presence of ATP, however, uptake into cLPM was readily observed for TBuMA while TEMA uptake was negligible. Inhibition studies with typical P-glycoprotein substrates indicated the uptake may be mediated by the P-glycoprotein. Conclusions. Differences between TBuMA and TEMA in reactivity for an ATP dependent transport process, rather than for an organic cation/H⁺ exchanger, may be responsible for the markedly different biliary excretion of TBuMA and TEMA.     

Hepatic uptake and biliary excretion of organic cations--I. Characterization of three new model compounds

Three quaternary ammonium compounds (QACs) with different lipophilicity, triethylmethyl ammonium iodide (TEMA), tripropylmethyl ammonium iodide (TPMA) and tri-n-butylmethyl ammonium iodide (TBuMA) were given as a bolus injection of 10 mumole and 1 mumole in an isolated perfused liver. TPMA and TBuMA exhibited saturation kinetics at a dose of 10 mumole, but not when 1 mumole of the agents was given. Biliary clearance of TEMA was equal to the bile flow (0.010 ml/min), whereas for TPMA and TBuMA much higher values of 0.8 ml/min and 2.2 ml/min were found respectively. Partition coefficients of TEMA, TPMA and TBuMA between n-octanol and Krebsbicarbonate solution were 0.0013, 0.013 and 0.14 respectively. Liver-to-plasma concentration ratios were 4, 16 and 30 in the post-distribution phase, whereas bile-to-liver ratios were calculated to be 0.1, 1.3 and 14 respectively. The latter parameter varied roughly proportionally to the lipophilicity of the compounds. The liver/plasma concentration ratios corrected for intracellular binding exceeded a value of 12 indicating that accumulation in the liver of these agents cannot soley be explained by passive equilibration according to the membrane potential. Transport from liver into the bile of TPMA and TBuMA presumably also occurred against an electrochemical gradient. It was inferred that the small molecular weight compounds such as TEMA, can be transported from plasma into bile paracellularly by a passive process. Rapid uptake into the liver of such compounds may not lead to an appreciable biliary output and can even reduce the rate of biliary excretion. QACs with intermediate or high lipophilicity are transported by carrier mediated processes both at the level of hepatocyte uptake and bile canalicular transport. The influence of choleresis on hepato-biliary transport of the three QACs was investigated by giving sodium taurocholate (Tc) by constant infusion of 60 mumole/hr, increasing bile flow from 9 to 16 microliter/min. The biliary output of TEMA appeared to be basically unaffected, whereas the biliary excretion of TPMA and TBuMA was clearly elevated when the bile flow was increased. The stimulatory influence of taurocholate on the biliary output of the latter organic cations is explained by an increased net uptake of these agents into the liver and an increased net canalicular transport. This effect is proposed to be due to a reduced reabsorption from the biliary tree as a consequence of the higher bile flow and/or biliary micelle binding. Taurocholate increased liver-to-plasma ratios.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Contribution of Ion-Pair Complexation with Bile Salts to the Transport of Organic Cations Across LLC-PK1 Cell Monolayers

Purpose. To examine the effect of ion-pair complexation with endogenous bile salts on the transport of organic cations (OCs) across LLC-PK1 cell monolayers. Methods. The transport of tributylmethyl-ammonium (TBuMA) and triethylmethylammonium (TEMA) across the cell monolayer was measured in the presence of taurodeoxycholate (TDC), an endogenous organic anion that forms an ion-pair complex with TBuMA, but not with TEMA. Results. Under proton gradient conditions (i.e., pH 6.0 apical/pH 7.4 basal), the above OCs exhibited similar transport charactersistics, consistent with the well-established OC/H⁺ antiporter, and the presence of TDC had no measurable effect on the transport of these OCs. Under pH-equilibrated conditions (i.e., pH 7.4 apical/pH 7.4 basal); however, basal to apical transport of TBuMA, not that of TEMA, was increased in the presence of TDC, probably as a result of the formation of a lipophilic ion-pair complex between TBuMA and TDC. The transport and efflux of the TBuMA-TDC complex across the apical membrane of the cell was inhibited by representative substrates of the P-glycoprotein (P-gp), indicating the involvement of P-gp in this process. The increased affinity of the ion-pair complex to P-gp is consistent with a mechanism involving increased transport. Conclusion. In cases where there is no proton gradient between the plasma and urine, the formation of lipophilic ion-pair complexes in the kidney with endogenous bile salts might be involved in the in vivo urinary excretion of large Mw OCs, such as TBuMA.     

Hepatic uptake and biliary excretion of organic cations--II. The influence of ion pair formation

The influence of an anorganic anion iodide (I-) and an organic anion tetraphenylborate (TPB-) on the hepatic uptake and biliary excretion of three organic cations, triethylmethyl ammonium (TEMA), tripropylmethyl ammonium (TPMA) and tri-n-butylmethyl ammonium (TBuMA) was studied. The compounds were injected as a bolus (D = 1 mumole) and studied in isolated perfused livers. In the perfusion medium 25% of the amount of NaCl (3 mmole) was replaced by NaI, whereas in two other experiments TPB- was added to the medium in two concentrations (2 microM and 200 microM). NaI did not affect the biliary output of the three quaternary ammonium compounds (QACs) although an increased net rate of hepatic uptake was found for all compounds, most likely due to a decreased liver to plasma transport. Liver to plasma concentration ratios were increased, while the ratios between bile to liver and bile to plasma were not affected. TPB- in catalytic amounts added to the medium (2 microM) decreased the biliary output of TEMA and TBuMA, whereas the kinetic profile of TPMA was unchanged. The decreased biliary excretion rate of TEMA was explained by a decreased plasma level (due to the increased liver uptake) assuming that the small molecular weight compounds can enter the bile directly from plasma via the junctional complexes between the cells. The bile to plasma (B/P) ratio was not affected. In contrast, the bile to plasma (B/P) ratio and the bile to liver (B/L) ratio of TBuMA were decreased, compared with the control, probably due to an increased reabsorption from the bile, whereas the back transport from the liver into the plasma was also decreased. A large amount of TPB- (200 microM), added to the perfusion medium, dramatically changed the kinetic profile of the three QACs. Ion pair formation between the QACs and TPB- was supposed to be responsible for this effect. Plasma levels dropped more rapidly as a result of an increased rate of liver uptake. The biliary excretion of all compounds was greatly reduced (the excretion rates were 0.022, 0.19 and 0.18 nmole/min, compared with 0.047, 0.71 and 7.5 nmole/min for the controls). It is concluded that ion pair formation may play a role in the hepatobiliary transport. The rate of liver uptake of the QACs is enhanced in the presence of an anion, which is due to an increase in plasma to liver transport (k12) and a reduced liver to plasma transport (k21).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of organic anions and vinblastine on biliary excretion of erythromycin in rats.

Since little is known about the mechanism of biliary excretion of cationic drugs, biliary excretion of erythromycin was studied in rats. Infusion of sulfobromophthalein and taurocholate significantly decreased biliary erythromycin excretion, whereas infusion of dibromosulfophthalein, cefpiramide, ursodeoxycholate-3-O-glucuronide and taurolithocholate-3-sulfate had no effect on biliary excretion of erythromycin. Vinblastine significantly inhibited biliary erythromycin excretion. Phenothiazine treatment significantly increased biliary erythromycin excretion. However, erythromycin infusion did not affect biliary vinblastine excretion. These findings indicate a multiplicity of biliary excretory pathways for organic cations; at least one additonal pathway may exist for organic cations apart from P-glycoprotein.     

Effects of butylated hydroxytoluene (BHT) on biliary excretion of xenobiotics and bile flow in rats

A significant enhancement in the biliary excretion of iv injected sulfobromophthalein (BSP), phenol- 3,6 -dibromphthalein disulfonate (DBSP), procaine amide ethobromide (PAEB) and ouabain was observed in rats maintained on diets containing 0.25% BHT for periods of 10 days. The enhanced biliary excretion of these drugs in BHT treated rats appears to be correlated with the increase in bile flow produced by BHT. The increased bile flow was due to an increase in canalicular bile production rather than a change in net ductular secretion or reabsorption of fluid since bile to plasma concentration ratios of erythritol were unchanged and no permeability change in the biliary tree was observed when mannitol was administered by retrograde intrabiliary injection. The increase in bile flow was not due to an enhanced excretion of bile salts into bile, because both the biliary bile acid concentration and total biliary excretion of bile acids were lower in BHT-treated rats than in control rats. It appears that the increase in bile flow produced by BHT is due to the osmotic choleresis related to the secretion of BHT and its metabolites into bile.     

Decreased biliary excretion of tributylmethyl ammonium in cholestyramine pretreated rats due to reduced formation of ion-pair complexes with hepatic bile salts

The hypothesis that higher molecular weight (MW) quaternary ammoniums (QAs) form lipophilic ion-pair complexes with bile salts in the liver, and are subsequently excreted into bile via a canalicular transporter, P-gp, was re-examined in the present study for its validity. The biliary excretion of tributylmethyl ammonium (TBuMA), a QA with a MW of 200, in bile salt-depleted rats was determined. Depletion was induced by a daily oral administration of a resin, cholestyramine, at a dose of 0.5 g/kg for 2 consecutive weeks, which decreased the concentration of total bile salts in the liver by 38%. When TBuMA was administered intravenously (12 micromol/kg) to these rats, the plasma level, area under the plasma concentration-time curve (AUC), systemic clearance (CL) and volume of distribution (V(ss)) of the compound remained unchanged, whereas bile flow (23.03 vs 16.94 microl/min, p<0.05) and biliary clearance (CL(bile), 12.75 vs 5.34 ml/min/kg, p<0.01) were decreased significantly. These results implied the biliary clearance of TBuMA in rats with bile salt depletion was significantly decreased as a result of decreased ion-pair complexation of TBuMA. The above results are consistent with our hypothesis and the existence of a MW threshold (i.e. 200+/-50 for rats) for the biliary excretion of QAs.     

Mechanism of the stationary canalicular excretion of tributylmethyl ammonium in rats with a CCl4-induced acute hepatic injury

The in vivo canalicular excretion clearance of tributylmethyl ammonium (TBuMA), a P-glycoprotein (P-gp) substrate, was previously reported to be unaffected by the induction of an experimental hepatic injury (EHI) by CCl(4) despite the increased expression of P-gp in the EHI liver. The objective of this study, therefore, was to elucidate the mechanism for the unchanged canalicular excretion clearance of TBuMA in EHI rats. TBuMA uptake was increased in cLPM vesicles from EHI rats compared with that from control rats. The total bile salt concentration in EHI liver was significantly reduced compared with that in a control liver. Because, in our previous studies, the uptake of TBuMA by cLPM vesicles was found to be significantly enhanced in the presence of bile salts, the reduction in bile salt levels in the EHI liver may be related to the unaltered TBuMA clearance. Despite the fact that the uptake of TBuMA by cLPM vesicles was increased by the addition of an EHI liver extract, the extent of the increase was comparatively less compared to the addition of a control liver extract. The in vivo excretion clearance of TBuMA was increased in a taurodeoxycholate dose-dependent manner in EHI rats. These observations suggest, therefore, that despite the induction of P-gp expression by the EHI, the in vivo canalicular excretion clearance of TBuMA remains unaltered as the result of an offset by reduced levels of bile salt(s).     

Recent Advances in Carrier-mediated Hepatic Uptake and Biliary Excretion of Xenobiotics

Purpose. Besides renal excretion, hepatic metabolism and biliary excretion are the major pathways involved in the removal of xenobiotics. Recently, for many endogenous and exogenous compounds (including drugs), it has been reported that carrier-mediated transport contributes to hepatic uptake and/ or biliary excretion. In particular, primary active transport mechanisms have been shown to be responsible for the biliary excretion of anticancer drugs, endogenous bile acids and organic anions including glutathione and glucuronic acid conjugates. Primary active excretion into bile means the positive removal of xenobiotics from the body, and this elimination process is now designated as Phase III (T. Ishikawa, Trends Biochem. Sci., 17, 1992) in the detoxification mechanisms for xenobiotics in addition to Phase I by P-450 and Phase II by conjugation. Methods. The transporters, which have been called P-glycoprotein (MDR), multidrug resistance related protein (MRP) and GS-X pump and which are believed to be involved in the primary active pumping of xenobiotics from the cells, are now known as the ATP-binding cassette (ABC) transporters. In this review, we first describe the HMG-CoA reductase inhibitor, pravastatin, as a typical case of a carrier-mediated active transport system that contributes to the liver-specific distribution in the body. Results. Regarding biliary excretion, we have summarized recent results suggesting the possible contribution of the ABC transporters to the biliary excretion of xenobiotics. We also focus on the multiplicities in both hepatic uptake and biliary excretion mechanisms. Analyzing these multiplicities in transport is necessary not only from a biochemical point of view, but also for our understanding of the physiological adaptability of the living body in terms of the removal (detoxification) of xenobiotics. Conclusions. Clarification of these transport mechanism may provide important information for studying the pharmacokinetics of new therapeutic drugs and furthermore, leads to the development of the drug delivery systems.     

Investigations on the hepatic uptake systems for organic cations with a photoaffinity probe of procainamide ethobromide.

Azido procainamide methoiodide (APM), a photolabile derivative of the transport model compound procainamide ethobromide (PAEB), shows a close resemblance to PAEB from a physicochemical point of view. Like PAEB it is effectively taken up by the liver and excreted into bile. Kinetics of the uptake of APM in isolated hepatocytes revealed that in addition to a non-saturable process, two saturable uptake systems are involved (Km1 = 3 microM, Vmax1) = 80 pmol/min/10(6) cells, Km2 = 100 microM, Vmax2 = 130 pmol/min x 10(6) cells). The uptake rate of APM was inhibited markedly in the presence of other organic cations. Organic anions and uncharged compounds generally had no inhibitory effect on the APM uptake. These results support the theory that there is a separate hepatic uptake system for organic cations like APM. Photoaffinity labeling of intact hepatocytes as well as plasma membrane sub-fractions enriched with sinusoidal domains disclosed two major binding polypeptides with apparent M(r) of 48,000 and 72,000. Such labeling patterns were not observed in membranes from hepatoma cells that are deficient in organic solute uptake. Differential photoaffinity labeling with other cationic compounds such as tributylmethyl ammonium and d-tubocurarine reduced the incorporation of APM in these polypeptides. The 48- and 72-kDa proteins might be involved in carrier-mediated transport of type I organic cations at the hepatic uptake level.     

Altered pharmacokinetics and hepatic uptake of TBuMA in ethynylestradio-induced cholestasis

The objective of this study was to examine the pharmacokinetics of organic cations in intrahepatic cholestatic rats. A pretreatment with 17alpha-ethynylestradiol was used to induce intrahepatic cholestasis, and tributylmethylammonium (TBuMA) was used as a representative model organic cation. When [3H]TBuMA was intravenously administered1 the AUC value for TBuMA was significantly increased by 79% in cholestasis, and its total systemic clearance was consequently decreased by 46%. In addition, the in vivo hepatic uptake clearance of TBuMA from the plasma to the liver was decreased by 50% in cholestasis. The concentration of bile salts in plasma was increased by 2.1 fold in cholestatic rats. Since TBuMA forms ion-pair complexes with anionic components such as bile salts, the decreased hepatic uptake of TBuMA in cholestasis may be due to a change in endogenous components, e.g., bile salts in the plasma. In isolated normal hepatocytes, the uptake clearance for TBuMA in the presence of cholestatic plasma was decreased by 20% compared with normal plasma. Therefore, we conclude that the inhibition of the hepatic uptake process by the cholestasis may be in part due to the increased formation of ion-pair complexes of TBuMA with bile salts in the plasma.     

Effect of a new hepatoprotective agent, YH-439, on the hepatobiliary transport of organic cations (OCS): Selective inhibition of sinusoidal OCs uptake without influencing glucose uptake and canalicular OCs excretion

The effect of a new hepatoprotective agent, YH-439, on the hepatobiliary transport of a model organic cation (OC), TBuMA (tributylmethylammonium), was investigated. The area under the plasma concentration-time curve (AUC) from time zero to 4 h following iv administration of TBuMA (6.6 micromol/kg) was increased significantly when YH-439 in corn oil (300 mg/kg) was orally administered to rats 24 h prior to the experiment. Nevertheless, the cumulative biliary excretion of TBuMA remained unchanged. As a consequence, the apparent biliary clearance (CLb) of TBuMA was decreased significantly as a result of YH-439 pretreatment, consistent with the fact that the in vivo excretion clearance of TBuMA across the canalicular membrane (CLexc) was not changed by the pretreatment. The in vitro uptake of TBuMA into isolated hepatocytes was decreased by one half by the pretreatment, owing to a decrease in the apparent Vmax and CLlinear, but the Km for the process remained constant. Most interestingly, however, the sinusoidal uptake of glucose, a nutrient, into hepatocytes was not influenced by the pretreatment, suggesting the YH-439 pretreatment specifically impaired the sinusoidal uptake of OCs. Thus, the OC-specific inhibition of hepatic uptake, without influencing the uptake of glucose, a nutrient, appeared to be associated with the hepatoprotective activity of YH-439.     

Hepatobiliary excretion of tributylmethylamonium in rats with lipopolysaccharide-induced acute inflammation

The alteration in the pharmacokinetic behaviors of organic cations (OCs) in rats during acute inflammation (Al) was investigated. Al was induced by an intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg) 24 hr prior to the start of pharmacokinetic studies. Tributylmethylammonium (TBuMA) was selected as a model OC since it is largely excreted into bile, and is neither metabolized nor binds to proteins in the body. When TBuMA was administered intravenously to Al rats at a dose of 6.6 micromole/kg, the AUC was increased, while biliary excretion (i.e., cumulative amount and apparent clearance) was decreased compared to normal rats. When TBuMA was administered intravenously to Al rats at a constant rate (i.e., a bolus injection at a dose of 1.5 micromole/kg followed by a constant infusion at a rate of 1.5 micromole/kg/hr for 165 min), steady-state concentrations of plasma and liver concentrations of TBuMA were increased significantly, while in vivo hepatic uptake (amount) and canalicular excretion (clearance) were decreased. These results are consistent with a hypothesis in which both the sinusoidal uptake of TBuMA into hepatocytes via the OCT1 and the canalicular excretion of the compound from hepatocytes via the P-gp are decreased by LPS-induced Al.     

Effect of pregnane X receptor ligand on pharmacokinetics of substrates of organic cation transporter Oct1 in rats.

Because rat organic cation transporter 1 (Oct1, SLC22a1) is expressed mainly in the liver and mediates drug transport, its activity may determine the hepatic handling of cationic drugs. Here, we studied the regulation mechanism of the expression of Oct1, focusing on the nuclear receptors. In vitro studies using cultured hepatocytes indicated that expression of Oct1 was up-regulated by treatment with pregnenolone-16 alpha-carbonitrile (PCN) and by overexpression of rat pregnane X receptor (PXR). In addition, isolated rat hepatocytes exhibited an increase of 1-methyl-4-phenylpyridinium (MPP(+)) uptake on treatment with PCN. When rats were subcutaneously administered PCN, an increase of biliary excretion clearance and distribution volume was observed for drugs such as MPP(+), metformin, and tetraethylammonium, although the effects on pharmacokinetic parameters were variable among the tested drugs. In addition, the expression of Oct2 in kidney was increased by treatment with PCN. Thus, PXR ligands appear to regulate the expression of organic cation transporters in rats and thereby to influence the pharmacokinetic properties of cationic drugs. Because PXR ligands include various clinically used drugs, alterations of hepatic drug handling may arise from interactions between cationic drugs that are substrates of Oct1 and ligands of PXR.     

Low‐dose probenecid selectively inhibits urinary excretion of phenolsulfonphthalein in rats without affecting biliary excretion

Renal organic anion transport systems play an important role in the excretion of anionic drugs and toxic compounds. Probenecid has been used as a potent inhibitor of urinary and biliary excretion of anionic compounds mediated by transporters such as organic anion transporters and multidrug resistance‐associated protein 2 (Mrp2). The purpose of this study was to optimize the dose of probenecid required for selective inhibition of urinary excretion of anionic compounds in rats, without inhibition of biliary excretion. Phenolsulfonphthalein (PSP), a model anionic compound that is excreted in urine and bile, was intravenously administered to rats after intraperitoneal injection of different doses of probenecid (0, 0.2, 2, 10, 100, 200 and 400 mg kg^?1). Treatment with 100, 200 or 400 mg kg^?1 probenecid decreased both renal clearance (CL_r) and biliary clearance (CL_b) of PSP, whereas 0.2 mg kg^?1 probenecid did not have any effect. Probenecid administered at doses of 2 and 10 mg kg^?1 decreased only CL_r. The median effective doses of probenecid for inhibiting CL_r and CL_b were 0.925 and 23.9 mg kg^?1, respectively. These data suggest that a low dose of probenecid selectively inhibits urinary excretion of PSP that may be mediated by organic anion transporters, without affecting biliary excretion that may be mediated by Mrp2. Copyright © 2011 John Wiley & Sons, Ltd.     

The rate-limiting step in the biliary elimination of some substrates of uridine diphosphate glucuronyltransferase in the rat

Phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline mutually inhibit each others enzymatic conjugation by microsomal UDP glucuronyltransferase (EC 2.4.1.17; acceptor unspecific) in vitro. After intravenous injection these UDP glucuronyltransferase substrates are excreted in the rat in bile as β-d-glucuronides. When these glucuronides were injected i.v. they were excreted partially in the bile and also in the urine. Ligation of the kidneys caused an increased biliary excretion of the i.v. injected glucuronides. To determine if these UDP glucuronyltransferase substrates also inhibited each others glucuronidation in vivo, two compounds were injected i.v. simultaneously into rats and the mutual effects on the biliary excretion of these compounds were studied. Phenolphthalein inhibited the biliary excretion of 4-methylumbelliferone in the form of its glucuronide conjugate in bile. This inhibition was not due to substrate competition for UDP glucuronyltransferase but to inhibition of excretion of the formed glucuronides from the liver cell into bile. From further experiments in which the mutual effects of the i.v. injected glucuronides on each others biliary excretion were studied it could be concluded that the rate-limiting step in the biliary elimination of phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline as glucuronides in the rat was the excretion of the products of UDP glucuronyltransferase activity in vivo into the lumen of the bile canaliculus and not conversion by UDP glucuronyltransferase.     

Possible involvement of P-glycoprotein in biliary excretion of CPT-11 in rats.

In our previous work, we found that the biliary excretion of the carboxylate form of irinotecan, CPT-11, on rat bile canalicular membrane consists of two components, the low-affinity one being canalicular multispecific organic anion transporter (cMOAT). In the present study, we have investigated the high-affinity component by studying the uptake in canalicular membrane vesicles. The ATP-dependent uptake of the carboxylate form of CPT-11 was inhibited significantly by several substrates and/or modulators of P-glycoprotein, including PSC-833, verapamil, and cyclosporin A, at a substrate concentration of 5 microM, at which the high-affinity component is involved predominantly in CPT-11 transport. When the concentration of the carboxylate form of CPT-11 was 250 microM, at which the low-affinity component (cMOAT) is involved predominantly in its transport, the inhibitory effect of the above compounds was reduced greatly. Similarly, there was also much lower inhibition of the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione, a substrate of cMOAT, by the above compounds. Taurocholic acid, a substrate of canalicular bile acid transporter, failed to inhibit the uptake of CPT-11 at the substrate concentration of both 5 and 250 microM. These results suggest that P-glycoprotein may act as the high-affinity component in the biliary excretion of the carboxylate form of CPT-11 in rats.