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 nitric oxide on the sinusoidal uptake of organic cations and anions by isolated hepatocytes

The issue of whether or not the presence NOx (NO and oxidized metabolites) in the hepatocytes at pathological levels affects the functional activity of transport systems within the sinusoidal membrane was investigated. For this purpose, the effect of the pretreatment of isolated hepatocytes with sodium nitroprusside (SNP), a spontaneous NO donor, on the sinusoidal uptake of tributylmethylammonium (TBuMA) and triethylmethyl ammonium (TEMA), representative substrates of the organic cation transporter (OCT), and taurocholate, a representative substrate of the Na+/taurocholate cotransporting polypeptide (NTCP), was measured. The uptake of TBuMA and TEMA was not affected by the pretreatment, as demonstrated by the nearly identical kinetic parameters for the uptake (i.e., Vmax, Km and CL(linear)). The uptake of mannitol into hepatocytes was not affected, demonstrating that the membrane integrity remained constant, irregardless of the SNP pretreatment. On the contrary, the uptake of taurocholate was significantly inhibited by the pretreatment, resulting in a significant decrease in Vmax, thus providing a clear demonstration that NOx preferentially affects the function of NTCP rather than OCT on the sinusoidal membrane. A direct interaction between NOx and NTCP or a decrease in Na+/K+ ATPase activity as the result of SNP pretreatment might be responsible for this selective effect of NOx.     

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.     

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.     

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.     

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)     

Functional Impairment of Sinusoidal Membrane Transport of Organic Cations in Rats with CCl4-Induced Hepatic Failure

Purpose. The effect of CCl₄-induced experimental hepatic failure(EHF) on the sequential hepatobiliary transport of model organiccations (OCs), triethylmethylammonium (TEMA), and tributylmethylammonium (TBuMA), was investigated in rats. Methods. EHF was induced by an i.p. injection of CCl₄ at a dose of1 ml/kg 24 hr prior to the transport study. The cumulative in vivobiliary excretion, in vitro hepatic uptake by isolated hepatocytes,in vitro efflux (i.e., release) from hepatocytes, andin vivo hepatobiliary excretion clearance were measured for normal and CCl₄-EHF rats. Results. The CCl₄-EHF decreased the apparentin vivo biliary clearance (CL_o) and the in vitromaximum uptake rate (V_{max, uptake}) of TBuMA by 66 and 48%,respectively. The CCl₄-EHF had no effect on the CL_oof TEMA, but decreased both the V_{max, uptake} (59%) and thein vitromaximum hepatic efflux rate (Vmax, efflux) of TEMA (80%). On thecontrary, the CCl₄-EHF had no influence on the in vivohepatobiliaryexcretion clearance (CL_exc) of both OCs. Conclusions. Transport systems for the OCs on the sinusoidal membrane(uptake and/or efflux), rather than those on the bile canalicularmembrane (excretion) appear to be prone to damage by the CCl₄-EHF.     

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).     

A physiologically based model of hepatic ICG clearance: interplay between sinusoidal uptake and biliary excretion

Although indocyanine green (ICG) has long been used for the assessment of liver function, the respective roles of sinusoidal uptake and canalicular excretion in determining hepatic ICG clearance remain unclear. Here this issue was addressed by incorporating a liver model into a minimal physiological model of ICG disposition that accounts of the early distribution phase after bolus injection. Arterial ICG concentration-time data from awake dogs under control conditions and from the same dogs while anesthetized with 3.5% isoflurane were subjected to population analysis. The results suggest that ICG elimination in dogs is uptake limited since it depends on hepatocellular uptake capacity and on biliary excretion but not on hepatic blood flow. Isoflurane caused a 63% reduction in cardiac output and a 33% decrease in the ICG biliary excretion rate constant (resulting in a 26% reduction in elimination clearance) while leaving unchanged the sinusoidal uptake rate. The terminal slope of the concentration-time curve, K, correlated significantly with elimination clearance. The model could be useful for assessing the functions of sinusoidal and canalicular ICG transporters.     

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.     

Differences in the hepatobiliary transport of two quinolone antibiotics, grepafloxacin and lomefloxacin, in the rat

The biliary excretion of grepafloxacin (GPFX) was compared with that of lomefloxacin (LFLX) in rats. The biliary clearances (Cl(plasma)(bile)) of GPFX was 2.9 times greater than LFLX based on the plasma concentration reached during constant intravenous (i.v.) infusion. The liver-plasma unbound concentration ratio, K(pu), of GPFX (1.7) was also higher than that of LFLX (0.7). The hepatic uptake clearance, assessed from an integration plot analysis, of GPFX was comparable with the hepatic blood flow rate, and 1.5 times that of LFLX, indicating that membrane transport in the uptake process is more efficient for GPFX. This was also supported by the difference between the uptake clearance of GPFX and LFLX in isolated rat hepatocytes. The bile-liver unbound concentration ratio of GPFX and LFLX was approximately 6 and 3, respectively, and the biliary clearance based on the unbound liver concentration of GPFX was 1.8 times that of LFLX. These results suggest that the concentrative transport of GPFX also across the canalicular membrane was more efficient than that of LFLX. Thus, the membrane transport activity via both sinusoidal and canalicular membranes determines the net excretion of each compound.     

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--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)     

Loading-washout studies of the stereoselective sinusoidal uptake of (R)- and (s)-2-phenylpropionyl acyl glucuronide

The vectorial movement of glucuronide conjugates from blood into bile can be an important elimination route for many drug metabolites, however the intrinsic hydrophilicity of those conjugates may conceptually act to reduce the overall efficiency of that process by limiting the flux of such conjugates across the sinusoidal membrane domain of hepatocytes. In this investigation, the hepatic disposition of the diastereomeric glucuronides of (R)- and (S)-2-phenylpropionic acid (a model "profen" compound) have been studied using the isolated perfused rat liver to establish whether a permeability barrier at the sinusoidal membrane domain (demonstrated previously for those conjugates) is of a sufficient magnitude to impact on the overall biliary excretion of these conjugates. Livers were perfused (30 mL/min) with perfusate containing either (R)-PPA, (S)-PPA, (R)-PPA-Glucuronide or (S)-PPA-Glucuronide in order to determine the dispositional profile of each glucuronide administered to the liver as both a preformed and an hepatically-generated metabolite. Once an apparent steady-state condition had been reached, infusion of test compound was ceased in order to establish the kinetics of the hepatic washout. The extent of biliary excretion of each glucuronide was dependent upon whether the glucuronide was presented to the liver as a preformed or hepatically-generated metabolite, and those differences, when analysed using a physiologically-based pharmacokinetic model, were consistent with the sinusoidal membrane acting as a barrier to the cellular entry of the glucuronides. Furthermore, that barrier was more pronounced for (R)-PPAG than it was for (S)-PPAG, suggesting that the hepatocellular uptake of the two diastereomers is stereoselective.     

Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats.

The relationship between biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats was examined. The biliary excretion of seven model substrates in 96-h sandwich-cultured rat hepatocytes was determined by differential cumulative uptake of substrate in the monolayers preincubated in standard buffer (intact bile canaliculi) and Ca2+-free buffer (disrupted bile canaliculi). Biliary excretion in vivo was quantitated in bile duct-cannulated rats. The biliary excretion index of model substrates, equivalent to the percentage of retained substrate in the canalicular networks, was consistent with the percentage of the dose excreted in bile from in vivo experiments. The in vitro biliary clearance of inulin, salicylate, methotrexate, [D-pen2,5]enkephalin, and taurocholate, calculated as the ratio of the amount excreted into the bile canalicular networks and the area under the incubation medium concentration-time profile ( approximately 0, approximately 0, 4.1 +/- 1.0, 12.6 +/- 2.2, and 56. 2 +/- 6.0 ml/min/kg, respectively), correlated with their intrinsic in vivo biliary clearance (0.04, 0, 17.3, 34.4, and 116.9 ml/min/kg, respectively; r2 = 0.99). The model compound 264W94 was not excreted in bile either in vivo or in vitro. The glucuronide conjugate of 2169W94, the O-demethylated metabolite of 264W94, was excreted into bile in vitro when 2169W94, but not 264W94, was incubated with the monolayers; 2169W94 glucuronide undergoes extensive biliary excretion after administration of 264W94 or 2169W94 in vivo. Biliary excretion in long-term sandwich-cultured rat hepatocytes correlates with in vivo biliary excretion. The study of biliary excretion of metabolites in the hepatocyte monolayers requires consideration of the status of metabolic activities.     

Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport.

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.     

Mirex exposure inhibits the uptake of estradiol-17 beta(beta-D-glucuronide), taurocholate, and L-alanine into isolated rat hepatocytes.

The insecticides mirex and chlordecone have previously been found to suppress the biliary excretion of a wide variety of compounds. In the present studies, the effects of mirex, chlordecone, and phenobarbital on the uptake of two endogenous organic anions, estradiol-17 beta(beta-D-glucuronide) (E217G), an estrogen metabolite, taurocholate (TC), a common bile acid, and an essential amino acid, L-alanine (L-Ala) (0.5 mM), into isolated rat hepatocytes was investigated. Female Sprague-Dawley rats were orally dosed with mirex (12.5, 25, and 50 mg/kg) or chlordecone (6.25, 12.5, and 18.75 mg/kg) dissolved in corn oil for 3 days and isolated rat hepatocytes were prepared 2 days later. Rats were also dosed orally with phenobarbital (50 mg/kg on the first day and 80 mg/kg for the next 4 days) dissolved in distilled deionized water, and isolated hepatocytes were prepared on the sixth day. Mirex significantly reduced the uptake of both organic anions (0.5, 10, and 50 microM E2 17G; 10 microM TC) into hepatocytes by 40-70%, whereas chlordecone had no effect on their uptake. Mirex at 50 mg/kg significantly reduced the Vmax for the low- and high-affinity E217G uptake sites by 70% and decreased the Km for the low affinity uptake site by 60%. Mirex also significantly decreased the Vmax for TC uptake from 1.11 to 0.82 nmol/min/mg protein but had no effect on its Km (23.2 vs 22.9 microM). Mirex at 50 mg/kg was also found to reduce the uptake of 0.5 mM L-Ala by nearly 40%. Phenobarbital had no effect on the uptake of E217G (0.5 microM), TC (10 microM), or L-Ala (0.5 mM). Mirex treatment had no effect on hepatic plasma membrane Na+,K(+)- or Mg2(+)-ATPase activity. Neither mirex nor chlordecone at 50-100 microM had any effect on the uptake of 10 microM TC when added directly to hepatocytes from naive rats. These results indicate that mirex decreases the transport of organic anions and L-Ala across the basolateral domain of the hepatocyte in addition to its inhibitory effects on biliary excretion.     

Novel In Vitro-In Vivo Extrapolation (IVIVE) Method to Predict Hepatic Organ Clearance in Rat

Purpose  

Drug elimination in the liver consists of uptake, metabolism, biliary excretion, and sinusoidal efflux from the hepatocytes to the blood. We aimed to establish an accurate prediction method for liver clearance in rats, considering these four elimination processes. In vitro assays were combined to achieve improved predictions.     

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.     

Organ distribution and cellular uptake of methyl mercury in the rat as influenced by the intra- and extracellular glutathione concentration

Intravenous administration of CH₃HgCl (4 μmole/kg) premixed with glutathione or cysteine (8 μmole/kg) to female rats caused a rapid uptake of mercury in the kidney and a depressed content in the liver and blood as compared to CH₃HgCl given alone. GSH depletion in the tissues, produced by injection of diethylmaleate, DEM (3.9 mmole/kg) did not influence the kidney uptake of mercury from administered CH₃Hg⁺-GSH, whereas the uptake of injected CH₃HgCl was depressed. Both GSH and cysteine (8 μmole/kg) promoted the biliary excretion of methyl mercury. In suspensions of rat erythrocytes and isolated hepatocytes, additions of GSH reduced the cellular uptake of CH₃Hg⁺ from the medium, whereas this was increased in the hepatocytes by adding cysteine or methionine. Cysteine addition slightly reduced the uptake of CH₃Hg⁺ in the erythrocytes. GSH-depletion as obtained by DEM pretreatment of the cells, reduced the CH₃Hg⁺ uptake into hepatocytes by 40%, in contrast to only a negligible effect on the erythrocytes. Our results support previous reports that a physiological CH₃Hg⁺-GSH-complexation takes place intracellularly, at least in liver cells. Our results are furthermore consistent with the assumption that biliary excreted CH₃Hg⁺-GSH, which can be reabsorbed, only to a limited extent is taken up by the liver, whereas this GSH-complexation and reabsorption is of importance for the CH₃Hg⁺-uptake in the kidneys.