Involvement of Multidrug Resistance Associated Protein 1 (Mrp1) in the Efflux Transport of 17β Estradiol-D-17β-Glucuronide (E217βG) Across the Blood-Brain Barrier

Purpose.The purpose of present study is to investigate the involvement of multidrug resistance-associated protein 1 (Mrp1), Mrp2, and P-glycoprotein (Mdr1a) in the efflux transport of 17—estradiol-D-17-glucuronide (E₂17G) across the blood—brain barrier (BBB). Methods. The expression of Mrp1 and Mrp2 at the BBB was investigated by RT-PCR and Western blot analyses. The time profiles of the remaining radioactivity of [³H]E₂17G in the brain were compared in wild-type, Mdr1a/Mdr1b and Mrp1 knockout mice and normal and Mrp2-deficient mutant rats [Sprague-Dawley and Eisai hyperbilirubinemic rats (EHBR), respectively] after intracerebral microinjection. Results. RT-PCR and Western blot analyses revealed the expression of Mrp1 in isolated rat brain capillary; however, RT-PCR was unable to detect any expression of Mrp2. Significant elimination of E₂17G was observed in wild-type mice at a rate constant of 0.007 min^–1, which was significantly decreased (0.004 min^–1) in Mrp1 knockout mice. In contrast, there was no difference in the efflux of E₂17G from the brain in wild-type and Mdr1a/Mdr1b knockout mice and in normal and EHBR. No significant difference was observed in the accumulation of E₂17G by brain slices prepared from wild-type and Mrp1 knockout mice. Conclusion. Mrp1, but not Mrp2, is involved in the excretion of E₂17G at the BBB and provides a barrier function by extruding conjugated metabolites into the blood.     

Impact of Mrp2 on the Biliary Excretion and Intestinal Absorption of Furosemide,Probenecid, and Methotrexate Using Eisai Hyperbilirubinemic Rats

Purpose. This study assesses the impact of rat multidrug resistance-associated protein 2 (Mrp2) on the biliary excretion and oral absorption of furosemide, probenecid, and methotrexate using Eisai hyperbilirubinemic rats (EHBR). Methods. To assess Mrp2-mediated biliary excretion, rats received a 2-h intravenous infusion of furosemide, probenecid, or methotrexate. Blood and bile samples were collected at specified intervals. To assess Mrp2's impact on oral absorption, rats received furosemide, probenecid, or methotrexate orally at 5 mg/kg. Jugular and portal blood samples were obtained at timed intervals. All samples were analyzed by LC-MS/MS. Pharmacokinetic parameters were estimated using WinNonlin and standard pharmacokinetic equations. Results. Thirty seven- and 39-fold reductions in biliary clearance were observed in EHBR as compared to control rats for probenecid and methotrexate, respectively. Biliary clearance was comparable between EHBR and control rats for furosemide. In all cases, no significant difference in absorption was observed between EHBR and control rats. Conclusions. This study provides the first evidence that Mrp2 mediates the biliary excretion of probenecid but not furosemide. Additionally, Mrp2 apparently has a less profound impact on intestinal absorption than biliary excretion of its substrates. Furthermore, alteration in systemic clearance in EHBR indicates that a potential compensatory mechanism may occur in EHBR.     

Determination of the Rate-Limiting Step in the Hepatic Elimination of YM796 by Isolated Rat Hepatocytes

Purpose. The membrane permeability clearance and intrinsic metabolic clearance of a drug in the liver were estimated using isolated rat hepatocytes, and the rate-limiting step in the overall intrinsic clearance of the drug in vivo was investigated. For this purpose, an anti-dementia drug, (S)-(-)-2,8-dimethyl-3-methylene-l-oxa-8-azaspiro [4,5] de-cane-L-tartarate monohydrate (YM796) was used as a model drug. Methods. The parent drug and its metabolites in both medium and cells were separated by thin-layer chromatography (TLC). The total amount of drug taken up by hepatocytes and the total amount of metabolites were plotted against the AUC of YM796 in the medium or cells to obtain the kinetic parameters. Results. While the influx clearance (PS_int) through the sinusoidal membrane defined in terms of YM796 concentration in the medium was almost constant, irrespective of the concentration of YM796 in the medium, the intrinsic metabolic clearance (CL_int) and the efflux clearance (PS_eff) defined in terms of the total concentration of YM796 in the cells markedly decreased and increased, respectively, as the concentration of YM796 increased. The overall intrinsic metabolic clearance (Cl_int,all), defined in terms of the YM796 concentration in the medium, corresponding to the hepatic intrinsic clearance obtained from thein vivo pharmacokinetic data on the drug, was comparable with PS_inf at low concentrations of YM796. As the YM796 concentration increased, however, Cl_int,all fell markedly approaching CL_int. Conclusions. While, at low concentrations of YM796, CL_int,all was predominantly affected by membrane permeability clearance, at high concentrations it was completely rate-determined by the intrinsic metabolic clearance because of the marked reduction in CL_int resulting from the saturation of YM796 metabolism.     

Study on Brain Interstitial Fluid Distribution and Blood-Brain Barrier Transport of Baclofen in Rats by Microdialysis

Purpose. This study was performed to examine the distribution in the brain interstitial fluid (ISF) and the blood-brain barrier (BBB) transport of baclofen in rats by a microdialysis technique. Methods. Following an i.v. bolus administration and/or the constant i.v. infusion of baclofen to the microdialysis cannula-bearing anesthetized rats, the concentrations of baclofen in the hippocampal ISF, whole brain tissue, cerebrospinal fluid (CSF), and plasma were determined by high-performance liquid chromatography (HPLC). Data were kinetically analyzed to estimate the transport parameters, i.e., the influx clearance (CL_in) from plasma to brain and the efflux rate constant (k_eff) from brain to plasma, and the steady-state volume of distribution in the brain (V_d). Results. The concentrations of baclofen in ISF, whole brain tissue, and CSF at the pseudo-steady state were almost 30-fold lower than the plasma unbound concentration, suggesting the restricted distribution of baclofen in the brain. The estimated values of CL_in and k_eff were 0.00157 ± 0.00076 ml/min/g of brain and 0.0872 ± 0.0252 min^–1, respectively. The efflux clearance (CL_out) calculated by multiplying k_eff by V_d (0.816 ± 0.559 ml/g of brain) was 0.0712 ± 0.0529 ml/min/g of brain, and it was significantly 40-fold greater than the CL_in value and fully greater than the convective flow in ISF. Furthermore, no significant concentration gradient was observed between ISF and CSF. These results suggest that the CL_out value mainly reflects the efflux clearance through the BBB. Additionally, the hippocampal ISF/plasma concentration ratio of baclofen was markedly increased by both systemic administration of probenecid and its direct instillation into ISF. Conclusions. The restricted distribution of baclofen in the brain ISF may be ascribed to the efficient efflux from the brain through the BBB which is regulated possibly by a probenecid-sensitive organic anion transport system.     

Kinetics of hepatic transport of 4-methylumbelliferone in rats. Analysis by multiple indicator dilution method

Hepatic elimination of 4-methylumbelliferone (4MU), which has been used as a model compound for conjugative metabolism, was studied by means of a multiple indicator dilution (MID) method in the isolated perfused rat liver. Using this method, three intrinsic hepatic clearances, CL _int,inf , CL _{int,eff, and} CL _{int,seq, which represent the influx, efflux, and sequestration processes, respectively, were obtained. When the dose was increased from a low dose (50 g/rat liver) to a high dose (3000 g/rat liver), the hepatic availability of 4MU increased from 0.11 to 0.73. With increasing dose, the} CL _{int,eff value increased approximately two times, while the} CL _{int,seq value decreased to approximately one-third. The remarkable dose dependence of hepatic availability was due to nonlinearity in both} CL _{int,eff and} CL _{int,seq values. However, the} CL_int,inf value was almost independent of dose. The dose-dependent change in CL_int,seq might be explained by the saturation of conjugative metabolism of 4-MU, while the increase in the CL _{int,eff value with increasing dose might be partly explained by the nonlinear tissue binding of 4-MU, since the tissue unbound fraction determined by an ultrafiltration method using liver homogenate increased approximately 1.5 times at higher concentration of 4-MU compared to that at lower concentrations. In addition, based on a comparison of the individual intrinsic clearances, i.e.}, CL _int,inf , CL _{int,eff, and} CL _{int,seq, the major determining process of the apparent hepatic intrinsic clearance of 4MU is thought to be the sequestration process at the high dose. However, at the low dose, the membrane transport process (influx and efflux processes) as well as the sequestration process also determine the apparent hepatic intrinsic clearance}.     

Basolateral Efflux Mediated by Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3) Facilitates Intestinal Absorption of Folates in Mouse

Purpose

This study investigated the role of an ABC transporter, Mrp3/Abcc3 in intestinal folate absorption.

Methods

Plasma concentrations of folic acid and leucovorin, given orally, were determined in wild-type and Mrp3 ^?/? mice. Mucosal-to-serosal transport was determined in the everted intestinal sacs. The plasma concentrations of endogenous 5-methyltetrahydrofolic acid, homocysteine and vitamin B₁₂, and mRNA levels of hepatic and intestinal folate metabolizing enzymes were compared between wild-type and Mrp3 ^?/? mice.

Results

C _max and area-under plasma concentration–time curve of folic acid were 3.0- and 2.3-fold lower in Mrp3 ^?/? mice compared with wild-type mice, whereas the total body clearance was unchanged. Absorption of leucovorin was significantly delayed in Mrp3 ^?/? mice. Mucosal-to-serosal transport of folic acid and leucovorin was significantly decreased in the duodenum of Mrp3 ^?/? mice, where their PS _serosal was decreased to 6.3 and 22% of that in wild-type mice, respectively. PS _serosal of 5-methyltetrahydrofolic acid was moderately decreased in Mrp3 ^?/? mice. There was no obvious abnormality in folate homeostasis in Mrp3 ^?/? mice.

Conclusions

Mrp3 accounts for the serosal efflux of folic acid and leucovorin, while it makes a moderate contribution to the serosal efflux of 5-methyltetrahydrofolic acid in mice. Mrp3 dysfunction does not disrupt folate homeostasis in mouse.     

Improved lung delivery from a passive dry powder inhaler using an Engineered PulmoSphere powder

Purpose. To assess the pulmonary deposition and pharmacokinetics of an engineered PulmoSphere® powder relative to standard micronized drug when delivered from passive dry powder inhalers (DPIs). Methods. Budesonide PulmoSphere (PS_bud) powder was manufactured using an emulsion-based spray-drying process. Eight healthy subjects completed 3 treatments in crossover fashion: 370 g budesonide PulmoSphere inhaled from Eclipse® DPI at target PIF of 25 L·min^-1 (PS_bud25), and 50 L·min^-1 (PS_bud50), and 800 g of pelletized budesonide from Pulmicort® Turbuhaler® at 60 L·min^-1(TH_bud60). PS_bud powder was radiolabeled with ^99mTc and lung deposition determined scintigraphically. Plasma budesonide concentrations were measured for 12 h after inhalation. Results. Pulmonary deposition (mean ± sd) of PS_bud was 57 ± 7% and 58 ± 8% of the nominal dose at 25 and 50 L·min^-1, respectively. Mean peak plasma budesonide levels were 4.7 (PS_bud25), 4.0 (PS_bud50), and 2.2 ng·ml^-1 (TH_bud60). Median t_max was 5 min after both PS_bud inhalations compared to 20 min for Turbuhaler (P < 0.05). Mean AUCs were comparable after all inhalations, 5.1 (PS_bud25), 5.9 (PS_bud50), and 6.0 (TH_bud60) ng·h·ml^-1. The engineered PS_bud powder delivered at both flow rates from the Eclipse® DPI was twice as efficiently deposited as pelletized budesonide delivered at 60 L·min^-1 from the Turbuhaler. Intersubject variability was also dramatically decreased for PS_bud relative to TH_bud. Conclusion. Delivery of an engineered PulmoSphere formulation is more efficient and reproducible than delivery of micronized drug from passive DPIs.     

Stereoselective hepatic disposition of a diastereomeric pair of αvβ3 antagonists in rat

1.?The study investigated mechanisms underlying the stereoselective hepatic disposition observed in rats of a zwitterionic diastereomeric pair ((3S)-3-{(3R or 3S)-2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]pyrrolidin-1-yl}-3-quinolin-3-ylpropanoic acid) with different lipophilicities.

2.?In a recirculating isolated rat liver system, the more hydrophilic diastereomer II possessed biliary clearance, CL_b, and bile-to-liver concentration ratio higher (about 10–30-fold) than the lipophilic zwitterion I, whereas both I and II exhibited comparably high concentration ratios between liver and perfusate. Although MK-571, a known multidrug resistance protein (MRP) inhibitor, significantly inhibited the CL_b of both compounds, it did not inhibit their canalicular transport, as evident by unchanged concentration ratios between bile and liver of either I or II.

3.?Following an intravenous infusion of I or II to Sprague–Dawley rats, the biliary clearance calculated either based on plasma (CL_b,p) or liver concentration (CL_b,l), of II was much higher than that of I (about 5–50-fold). In rats lacking multidrug resistance protein 2 (Mrp2) (Eisai hyperbilirubinemic rat, EHBR), the biliary excretion rate and CL_b,p of II were also higher than the corresponding values for I. However, both CL_b,p or CL_b,l of either I or II were not reduced in EHBR, as compared with control SD rats.

4.?In the in vitro rat canalicular membrane vesicle study, I and II exhibited no differences in their inhibitory effect on the Mrp2 mediated ATP-dependent [³H]DNP-SG initial uptake (no inhibition at 10?μM and only about 40% inhibition at 100?μM).

5.?Collectively, these results suggested that (1) the difference in the hepatic disposition between the two isomers was due primarily to the difference in their transport mechanism across the canalicular membrane and (2) Mrp2 did not play a major role in the observed differences in the biliary excretion of the diastereomers I and II in rats.     

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

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

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

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

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

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

Transport of HIV-protease inhibitors across 1 alpha,25di-hydroxy vitamin D3-treated Calu-3 cell monolayers: modulation of P-glycoprotein activity

Purpose. The presence of P-glycoprotein (P-gp) within the lipid bilayers of the absorptive cells greatly influences drug entry into the HIV-infected sanctuary sites. The objective of this study was to access the potential role of pulmonary cells expressing high levels of P-gp in the efflux of potent anti-HIV drugs such as protease inhibitors. Methods. Human airway epithelium-derived Calu-3 cells grown in the presence of 0.025 mM 1,25di-hydroxy Vitamin D₃ (di-OH vit D₃) were used as a model to evaluate the effects of p-glycoprotein efflux of HIV protease inhibitors. Cells used as controls were not treated with di-OH vit D₃. The anti-HIV agents ³H Ritonavir and Saquinavir (50 M) were used as model compounds for influx and efflux studies. Results. Di-OH vit D₃ treatment enhanced the differentiation of Calu-3 cells indicated by more cilia and mucus secretion. It also caused elevated P-gp expression as demonstrated by Western Blot analysis and enhanced basal to apical transport of cyclosporine as compared with untreated cells. The amount of Saquinavir transported, after 3 h, across untreated Calu-3 cells (A-B) was 3-fold higher (1.62 g; Papp = 2.4 (± 0.79) × 10^–6 cm/s) than di-OH vit D₃-treated cells (0.57 g with the Papp = 5.02 (± 0.62) × 10^–7 cm/s). Similar transport profiles were obtained for ³H ritonavir and a significant increase (p < 0.05) in the A-B transport (2.5-fold) of ³H ritonavir was observed when the cell monolayers were preincubated with testosterone prior to transport studies. However, transport of AZT remained unaltered in di-OH vit D₃ treated monolayers. Conclusion. Modulation of P-gp activity may be necessary to increase the therapeutic efficacy of protease inhibitors against HIV-1 reservoirs across alveolar lining cells and fluids.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

Altered Disposition and Effect of Lerisetron in Rats with Elevated Alpha1-acid Glycoprotein Levels

Purpose. To examine the effect of changes in plasma 1-acid glycoprotein (AAG) levels on the pharmacokinetics (PK) and pharmacodynamics (PD) of lerisetron, a novel serotonin 5-HT₃ receptor antagonist, in the rat. Methods. After subcutaneous administration of turpentine oil, AAG was significantly elevated compared with controls. The PK of unchanged lerisetron (UL; high-performance liquid chromatography with radioactivity monitoring) and total lerisetron (TL; unchanged + changed, scintillation counting) was characterized post intravenous (i.v.) ¹⁴C lerisetron (50 g/kg) in control and turpentine oil pretreated rats. The PK (0 – 180 min) was described by a two-compartmental model. Protein binding of lerisetron in vitro was measured using an ultrafiltration technique. The effect of lerisetron (5 g/kg, i.v.) over 180 min was measured in anesthetized rats (control and pretreated) with the Bezold-Jarisch reflex (inhibition of bradycardia after 16 g/kg serotonin i.v.) as the endpoint. PD parameters were estimated by sigmoid E_max models. Results. The unbound fraction was significantly diminished in pretreated rats (mean ± SEM) (6.60 ± 1.23% vs. control 14.4 ± 1.40%, P < 0.05). Volume of distribution (V) and clearance for UL and TL were significantly decreased when compared to the controls (P < 0.0001 for UL and P < 0.05 for TL). Plasma clearance based on unbound concentration for UL did not differ between groups but the unbound V and steady-state unbound V remained decreased (P < 0.05 and P < 0.0001). Pretreated rats showed a significantly diminished drug effect: the area under the E-t curve over 180 min was (mean ± SEM) 5189 ± 657.7 in control animals vs. 3486 ± 464.4 in the pretreated group (P < 0.05). The EC₅₀ (concentration at half maximum effect) for UL and TL were increased in pretreated rats and were not compensated when the unbound concentration was used. Conclusions. An increase in AAG causes alterations in the PK and PD of lerisetron, and because this is not compensated with the unbound concentration, we suggest that mechanisms not linked to protein binding may be involved.     

Surface Activity and Concentration Dependent Intestinal Permeability in the Rat

Purpose. To investigate the relation between intestinal effective permeability (P_eff) and surface activity of fluvastatin and verapamil. Methods. P_eff values were determined for fluvastatin, antipyrine and D-glucose following colon perfusions in the rat in situ. The perfusion solutions differed regarding concentrations of fluvastatin (0-2500 M) and surface tension (58.9-43.7 mN/m). A cellulose derivative, ethyl-(hydroxyethyl) cellulose (EHEC), was added to lower the surface tension of one of the perfusion solutions. The surface tension of perfusion solutions containing R/S-verapamil (8-814 M) and R/S-verapamil + chlorpromazine (814 M + 10 mM) were related to the corresponding P_eff values from the literature. Results. The P_eff of fluvastatin correlated inversely (r² = 0.985, p < 0.05) with the surface tension of the perfusion solutions below the critical micelle concentration (CMC, 1 mM). Decreasing the surface tension with EHEC increased the P_eff of fluvastatin by 36% (p < 0.001), but not to the extent anticipated from the correlation between the P_eff and the surface tension. EHEC also increased the P_eff of antipyrine by 49% (p < 0.01) but not for D-glucose. The P_eff of R/S-verapamil correlated inversely with the surface tension (r² = 0.980, p < 0.001). Conclusions. The ability of fluvastatin to decrease the surface tension at the membrane surface can partly explain the concentration dependent colonic P_eff of fluvastatin. This study shows that the surface activity of the drug molecule itself is an important physicochemical factor that should be taken into consideration when evaluating drug absorption studies performed in vitro or in situ.     

Are MDCK Cells Transfected with the Human MRP2 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MRP2 gene (MDCK-MRP2) are a good model of the human intestinal mucosa. Methods. MRP2 expression in Caco-2 cells was compared with the expression of this efflux transporter in MDCK-wild type (MDCK-WT) and MDCK-MRP2 cells using Western blotting methods. The polarized efflux activities of MRP2 in the MDCK-MRP2, MDCK-WT, MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and Caco-2 cells were compared using vinblastine as a substrate. Apparent Michaelis-Menten constants (K_M, Vmax) for the efflux of vinblastine in Caco-2 and MDCK-MRP2 cells were determined in the presence of GF120918 (2 M), which inhibits P-glycoprotein but does not affect MRP2. Apparent inhibitory constants (K_I) of known substrates/inhibitors of MRP2 were determined by measuring their effects on the efflux of vinblastine in these cell lines. Results. MDCK-MRP2 cells expressed higher levels of MRP2 than MDCK-WT and Caco-2 cells as measured by Western blotting technique. Two isoforms of MRP2 expressed in Caco-2 and MDCK cells migrated at molecular weights of 150 kD and 190 kD. In MDCK-MRP2 cells, the 150 kD isoform appeared to be overexpressed. MDCK-MRP2 cell monolayers exhibited higher polarized efflux of vinblastine than Caco-2 and MDCK-WT cell monolayers. K_M values for vinblastine in Caco-2 and MDCK-MRP2 cells were determined to be 71.8 ± 11.6 and 137.3 ± 33.6 M, respectively, and V_max values were determined to be 0.54 ± 0.03 and 2.45 ± 0.31 pmolcm^–2s^–1, respectively. Known substrates/inhibitors of MRP2 showed differences in their ability to inhibit vinblastine efflux in Caco-2 cells as compared to MDCK-MRP2 cells Conclusions. These data suggest that MDCK-MRP2 cells overexpress only the 150 kD isoform of MRP2. The 190 kD isoform, which was also found in Caco-2 cells and MDCK-WT cells, was present in MDCK-MRP2 cells but not over expressed. The apparent kinetics constants and affinities of some MRP2 substrates were different in Caco-2 cells and MDCK-MRP2 cells. These differences in substrate activity could result from differences in the relative expression levels of the MRP2 isoforms present in Caco-2 cells and MDCK-MRP2 cells and/or differences in the partitioning of substrates in these two cell membrane bilayers.     

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.     

Pharmacokinetic Study of the Hepatobiliary Transport of Indomethacin

Purpose. The biliary excreted amount of indomethacin and itsglucuronide is related to the intestinal toxicity of this drug. In the presentstudy, we investigated the hepatobiliary transport of indomethacin.Methods. The uptake of indomethacin into primary cultured rathepatocytes and COS-7 cells transfected with cDNA encoding sodiumtauro-cholate co-transporting polypeptide or organic anion transportingpolypeptide 1 was examined. Moreover, we compared the biliaryexcretion of indomethacin and its glucuronide between Sprague-Dawley(SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective.Results. The uptake of indomethacin into rat hepatocytes was mediatedby Na⁺-dependent and independent active transport systems. Neithertransfectant stimulated the uptake of indomethacin. After intravenousinfusion of indomethacin to SD rats, the biliary excretion ofindomethacin glucuronide exceeded that of indomethacin. The indomethacintransport clearance across the bile canalicular membrane wascomparable between SD rats and EHBR, whereas the corresponding value forindomethacin glucuronide in EHBR was approximately 50% that inSD rats.Conclusions. These results indicate that another transporter(s) isinvolved in the hepatic uptake of indomethacin and the canaliculartransport of indomethacin glucuronide is mediated by cMOAT/MRP2whereas that of indomethacin is not mediated by cMOAT/MRP2.     

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

1.?To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation.

2.?Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CL_eff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CL_b) of glucuronides were approximately two times larger than those of sulfates.

3.?The ratios of CL_eff to CL_b were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated.

4.?For both compounds, CL_b of glucuronide was comparable with CL_b of sulfate, meaning that CL_b is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates.

5.?Higher concentration of glucuronides in the liver, partly caused by much lower CL_eff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CL_eff than CL_b.

6.?In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.     

Altered Pharmacokinetics of Paclitaxel in Experimental Hepatic or Renal Failure

No HeadingPurpose. The aim of this study was to investigate the effect of hepatic or renal insufficiency on the pharmacokinetics of paclitaxel in rats.Methods. Rats were treated with carbon tetrachloride (CCl₄; 0.5 ml/kg) to induce hepatic failure or were subjected to 5/6 nephrectomy (5/6 Nx) to induce renal failure. Paclitaxel (3 mg/kg) was administered intravenously or intraportally. Testosterone 6-hydroxylase activity, which is a marker of CYP3A activity, was measured in rat liver microsomes from CCl₄-treated or 5/6 Nx rats.Results. After paclitaxel was administered intravenously, total body clearance was significantly reduced by 73% and 34% relative to each control value in CCl₄-treated and 5/6 Nx rats, respectively (control, 1.82 ± 0.42 vs. CCl₄-treated, 0.49 ± 0.11; sham, 1.54 ± 0.07 vs. 5/6 Nx, 1.01 ± 0.12 L h^–1 kg^–1; mean ± SE, n = 5 to 6). Testosterone 6-hydroxylase activity was reduced by 92% and 59% relative to each control value in rat liver microsomes from CCl₄-treated and 5/6 Nx rats, respectively. After the intraportal administration of paclitaxel, apparent clearance was reduced by 85% relative to control value in rats with hepatic failure, while that in rats with renal failure was the same as the reduction in systemic clearance.Conclusions. These results suggested that not only hepatic failure but also renal failure could modify the pharmacokinetics of paclitaxel in vivo.     

Sex Specificity in Methadone Analgesia in the Rat: A Population Pharmacokinetic and Pharmacodynamic Approach

Purpose. To quantify the extent to which a sex-specific dichotomy in the temporal evolution of the analgesic effect, after intravenous (i.v.) methadone injection in the rat, relates to the pharmacokinetics (PK) and pharmacodynamics (PD) that mediate the dose-to-effect pathway. Methods. Tail-flick analgesia was measured after i.v. methadone injection (0.35 mg/kg) in female (n = 16) and male (n = 16) Sprague-Dawley rats. The PK were evaluated in separate female (n = 56) and male (n = 56) rats after they had received the same dose of methadone i.v. (0.35 mg/kg). A bicompartmental model described the kinetics and a sigmoid E_max model-related drug effect vs. simulated concentrations (pharmacodynamics) at the times of effect measurement. All model parameters as well as interanimal and assay variabilities were estimated with a mixed-effects population method using the program NONMEM. Results. The area under the effect-time curve (AUCE_0-120) was (mean ± interanimal SD) 1859 ± 346 min in the females, which was significantly lower than the 4871 ± 393 min in the males (P < 0.0001). On the contrary, the profiles of concentration vs. time were higher in females and, therefore, corresponded inversely to the effect vs. time-relative magnitudes. The central volume of distribution, V1, was 1.94 ± 0.37 l/kg for female rats and 3.01 ± 0.33 l/kg for male rats. Also, the central clearance was 0.077 ± 0.006 l/min/kg and 0.102 ± 0.005 l/min/kg, respectively, for female and male rats. Both parameters differed significantly between sexes (P < 0.0001). The pharmacodynamic maximum observed effect parameter (E_max) was 37% ± 29% in female rats and 85% ± 16% in male rats, and these values were significantly different (P < 0.0001). The parameter for the concentration eliciting half of E_max (EC₅₀) was 24.1 ± 7.5 g/l in female rats and 20.3 ± 2.9 g/l in male rats, and the Hill-related exponent, , was 6.3 ± 3.9 in female rats and 5.5 ± 4.1 in male rats. These parameters did not differ significantly (at the P < 0.05 level). Conclusions. A sex-specific dichotomy in the methadone antinociceptive effect, in the rat, was not proportionally related to plasma concentrations. Each sex corresponded to a distinct subpopulation of the PK parameters and one of the pharmacodynamic parameters (E_max). When the course of a drug involves PK or PD subpopulations, PK/PD modeling can afford the safest prediction of the effect-time evolution for a particular dose.     

Effect of GF120918, a Potent P-glycoprotein Inhibitor,on Morphine Pharmacokinetics and Pharmacodynamics in the Rat

Purpose. The objective of this study was to evaluate the effect of a potent P-gp inhibitor, GF120918, on the systemic pharmacokinetics and antinociceptive pharmacodynamics of a single intravenous dose of morphine in rats. Methods. Male Sprague-Dawley rats received either 500 mg base/kg/d GF120918 or vehicle for 4 days by gavage, or no pretreatment. On day 4, morphine was administered as a 1- or 2-mg/kg i.v. bolus. Antinociception, expressed as percent of maximum possible response (%MPR), was evaluated over 300 min after morphine administration. Serial blood samples were collected and analyzed for morphine and morphine-3-glucuronide (M3G) by HPLC. Results. Morphine clearance and distribution volume were not altered significantly by GF120918. M3G AUC in the GF120918-treated rats was approximately 2-fold higher than in vehicle-treated rats. For both morphine doses, %MPR and the area under the effect-time curve at 300 min were significantly higher in the GF120918-treated rats. A pharmacokinetic/pharmacodynamic effect model accurately described the effect-concentration data for the rats that received 1-mg/kg morphine; k_e0 was significantly smaller for GF 120918- vs. vehicle-treated and control rats (0.060 ± 0.028 vs. 0.228 ± 0.101 vs. 0.274 ± 0.026 min^–1, p=0.0023). EC₅₀ and were similar between treatment groups. Conclusions. Pretreatment with GF 120918 enhanced morphine antinociception, as assessed by the hot-lamp tail-flick assay, and elevated systemic M3G concentrations in rats. The differential pharmacologic response to morphine in the GF120918-treated animals could not be attributed to alterations in systemic morphine pharmacokinetics.