Rate limiting steps in hepatic drug clearance: comparison of hepatocellular uptake and metabolism... more Rate limiting steps in hepatic drug clearance: comparison of hepatocellular uptake and metabolism with microsomal metabolism of saquinavir, nelfinavir and ritonavir
Drug metabolism and disposition: the biological fate of chemicals, Jul 1, 2018
This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic... more This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good...
Drug metabolism and disposition: the biological fate of chemicals, 2018
Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, i... more Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using sapo...
Drug metabolism and disposition: the biological fate of chemicals, 2018
Hepatocyte drug depletion-time assays are well established for determination of metabolic clearan... more Hepatocyte drug depletion-time assays are well established for determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, an adaptation of the conventional depletion assay involving centrifugation of hepatocytes prior to sampling, allowing estimation of uptake in addition to metabolism. Using experimental procedures consistent with a high throughput, a selection of 12 compounds with a range of uptake and metabolism characteristics (atorvastatin, cerivastatin, clarithromycin, erythromycin, indinavir, pitavastatin, repaglinide, rosuvastatin, saquinavir, and valsartan, with two control compounds-midazolam and tolbutamide) were investigated in the presence and absence of the cytochrome P450 inhibitor 1-aminobenzotriazole and organic anion transporter protein inhibitor rifamycin SV in rat hepatocytes. Data were generated simultaneously for a given drug, and provided, through the use of a mechanistic cell mo...
Journal of Pharmacology and Experimental Therapeutics, 2004
Introduction: 628 Discussion: 1200 d) List of non-standard abbreviations: %act, percent activatio... more Introduction: 628 Discussion: 1200 d) List of non-standard abbreviations: %act, percent activation caused by 100µM effector and 100µM carbamazepine; carbamazepinediol, carbamazepine-trans-diol (trans-10,11-dihydro-10,11-dihydroxycarbamazepine); carbamazepine-ep, carbamazepine-10,11-epoxide; Css CBZ , Css CBZ-ep , plasma steady state concentration of carbamazepine and of carbamazepine-10,11-epoxide respectively; CYP, cytochrome P450; DHEA, dehydroepiandrosterone; HLM, human liver microsomes; v 150% , concentration of heteroactivator giving 150% of reaction velocity.
The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-s... more The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B-subfamily, we previously demonstrated that genetic ablation causes reduced hepatic clearance capacity for substrates. In this report we focused on the physiological function of the hepatic OATP1B transporters. First we studied the influence of the Oatp1b2 deletion on bile acid metabolism showing that lack of the transporter results in a significantly reduced expression of Cyp7a1 the key enzyme of bile acid synthesis, resulting in elevated cholesterol levels after high dietary fat challenge. Furthermore, Slco1b2 −/− mice exhibited delayed clearance after oral glucose challenge resulting from reduced hepatic glucose uptake. In addition to increased hepatic glycogen content, Slco1b2 −/− exhibited reduced glucose output after pyruvate challenge. This is in accordance with reduced hepatic expression of PEPCK in knockout mice. We show this phenotype is due to the loss of liver-specific Oatp1b2-mediated hepatocellular thyroid hormone entry, which then leads to reduced transcriptional activation of target genes of hepatic thyroid hormone receptor (TR) including the prior mentioned Cyp7a1 and Pepck, but also Dio1 and Glut2. Importantly, we assessed human relevance using a cohort of archived human livers where OATP1B1 expression was noted to be highly associated with TR target genes, especially for GLUT2. Furthermore, GLUT2 expression was significantly decreased in livers harboring a common genetic polymorphism in SLCO1B1.
CL active , clearance by active uptake; CL met , clearance by metabolism; CL obs , observed in vi... more CL active , clearance by active uptake; CL met , clearance by metabolism; CL obs , observed in vitro clearance (either by uptake or metabolism); CL uptake , total uptake clearance by active and passive processes; fu cell , fraction of unbound drug in the hepatocyte; k dep , initial depletion rate constant; Kp total , tissue-to-medium total drug concentration ratio; Kp u , hepatocyte-to-medium unbound drug concentration ratio; OATP, organic anion transporter polypeptide; P diff , passive uptake clearance.
With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug di... more With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic aniontransporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under-and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.
Interindividual variability in activity of uptake transporters is evident in vivo, yet limited da... more Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic aniontransporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CL active, u), bidirectional passive diffusion (P diff), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CL active, u was estimated at a single concentration. The unbound affinity constant (K m, u) and P diff values were consistent across donors, whereas V max was on average up to 2.8-fold greater in donor HU4122. Consistency in K m, u values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CL active across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake K m, u and CL active, u were on average 4.3-and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed. This work was supported by GlaxoSmithKline, Ware, UK (Ph.D. studentship to K.M.) and the Biotechnology and Biological Sciences Research Council, UK. Parts of this work were previously presented at the following conference: Mé nochet K, Kenworthy KE, Houston JB, and Galetin A (2010) Contribution of active uptake to the hepatic clearance of seven OATP substrates in rat and human plated hepatocytes.
A progress curve method for assessing time-dependent inhibition of CYP3A4 is based on simultaneou... more A progress curve method for assessing time-dependent inhibition of CYP3A4 is based on simultaneous quantification of probe substrate metabolite and inhibitor concentrations during the experiment. Therefore, it may overcome some of the issues associated with the traditional two-step method and estimation of inactivation rate (k inact) and irreversible inhibition (K I) constants. In the current study, seven time-dependent inhibitors were investigated using a progress curve method and recombinant CYP3A4. A novel mechanistic modeling approach was applied in order to determine This article has not been copyedited and formatted. The final version may differ from this version.
Previous studies have shown the importance of the addition of albumin for characterization of hep... more Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL int, UGT) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL int, UGT on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL int, UGT in different tissues. Although BSA increased CL int, UGT in all tissues, the extent was tissue-and drug-dependent. Scaled CL int, UGT in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml ⅐ min ؊1 ⅐ g tissue ؊1 in liver, kidney, and intestinal microsomes. Renal CL int, UGT (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL int, UGT for UGT2B7 substrates represented approximately onethird of hepatic estimates. Scaled renal CL int, UGT (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL int, UGT is particularly important for UGT1A9 substrates. The work was funded by a consortium of pharmaceutical companies (Glaxo-SmithKline, Lilly, Pfizer, and Servier) within the Centre for Applied Pharmacokinetic Research at the University of Manchester. K.L.G. is a recipient of a Ph.D. studentship from Biotechnology and Biological Sciences Research Council. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
Predictions of intrinsic clearance (CL int) from human liver microsomes often underestimate in vi... more Predictions of intrinsic clearance (CL int) from human liver microsomes often underestimate in vivo observations. In this study, a series of five benzodiazepines were used as prototypic CYP3A4 substrates in order to investigate the prediction of clearance from the less studied alternative in vitro system, cryopreserved human hepatocytes. Formation of the two major metabolites from midazolam, triazolam, diazepam, flunitrazepam and alprazolam was measured in cryopreserved human hepatocytes from five donors; the kinetics were characterised and CL int values determined and scaled to predict CL int in vivo. At least one of the two major pathways of metabolic clearance of each benzodiazepine was characterised by autoactivation in hepatocytes; the extent to which this occurred varied depending on substrate and liver (up to 8-fold). Heteroactivation by testosterone of these pathways was also observed (up to 6-fold). The maximum autoactivated clearance was used to predict in vivo CL int (1.6-138ml/min/kg) which closely agreed with values previously obtained using human liver microsomes. Comparison with in vivo CL int indicates that cryopreserved human hepatocytes systematically under-predict CL int. When three previous studies (documenting CL int for substrates of various enzymes in cryopreserved human hepatocytes using drug depletiontime profiles) were considered as well, the combined data show a consistent underprediction of 5.6-fold. Collectively it is demonstrated that the predicted hepatic intrinsic clearances from cryopreserved hepatocytes show an excellent rank order with in vivo findings but are systematically under-predicting the in vivo value.
The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important ... more The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important drug-drug interaction involving cytochrome P450 inhibition. The availability of an in vivo K i value (unbound K i , 21 M) obtained from a series of steady-state inhibitor infusion studies allowed assessment of several in vitroderived predictions of this inhibition interaction. Studies monitoring substrate depletion with time were used to obtain in vitro K i values that were evaluated against the more traditional metabolite formation approach using microsomes and hepatocytes. OMP inhibited the metabolism of DZ to its primary metabolites 4-hydroxydiazepam, 3-hydroxydiazepam, and nordiazepam to different extents over a range of concentrations (0.3-150 M), and a competitive inhibition model best fitted the data. The K i values observed using the substrate depletion approach (16 ؎ 3 M and 7 ؎ 2 M in microsomes and hepatocytes, respectively) were in good agreement with the overall weighted K i values obtained using the standard metabolite formation approach (12 ؎ 2 M and 16 ؎ 5 M in microsomes and hepatocytes, respectively). In vitro binding and cell uptake studies as well as human serum albumin studies in hepatocytes confirmed the importance of both intracellular and extracellular unbound concentrations of inhibitor when considering inhibition predictions. Both kinetic approaches and both in vitro systems predicted the in vivo interaction well and provide a good example of the ability of in vitro inhibition studies to quantitatively predict an in vivo drug-drug interaction successfully.
The hepatic uptake of imipramine and propranolol was investigated following incubation with isola... more The hepatic uptake of imipramine and propranolol was investigated following incubation with isolated rat hepatocytes over a wide concentration range (0.04-400 µM). The cell-tomedium concentration ratio (K p) was concentration-dependent and could be described using a two-site binding model incorporating a high affinity/low capacity site and a linear component for a site which was apparently not saturated. Maximum (at 0.04 µM) and minimum K p values (at 400 µM) were 360 and 280 and 110 and 70 for imipramine and propranolol, respectively. During these incubations, metabolism was inhibited using aminobenzotriazole (an irreversible inhibitor of P450). Pre-treatment of cells either by freeze-thawing or with saponin (which permeabilises the plasma membrane) eliminated the saturable process. The saturable uptake process of imipramine was also inhibited by 18 other lipophilic amine drugs (including propranolol). This uptake component may involve membrane transporter(s) whereas the non-saturable component probably represents partition into the phospholipid component of membranes. Propranolol was further investigated to determine the impact of high Kp values on hepatocellular clearance. The area under the curve for propranolol concentrations in the total incubate (medium including the cells) from the depletion-time profile was substantially greater than the corresponding area under the curve for the drug concentration in the extracellular medium and this difference approximated to the non-saturable uptake component. It is concluded that the clearance of propranolol in isolated hepatocytes is not rate limited by hepatic uptake and is directly proportional to unbound drug concentration being independent of the higher K p value.
The current study assesses hepatic and intestinal glucuronidation, sulfation and P450 metabolism ... more The current study assesses hepatic and intestinal glucuronidation, sulfation and P450 metabolism of raloxifene, quercetin, salbutamol and troglitazone using different in vitro systems. Fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine and importance of multiple metabolic pathways on accuracy of clearance prediction assessed. In vitro intrinsic sulfation clearance (CL int,SULT) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL int,UGT) and P450 clearance (CL int,CYP) expressed per gram tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL int,SULT ranged between 0.7-11.4ml/min/g liver and 0.1-3.3ml/min/g intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with high extent of sulfation (51% and 28% of total CL int for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared to predictions based only on primary pathway (22-36%). The assumption of no intestinal first-pass resulted in under-prediction of oral clearance for raloxifene, troglitazone and quercetin (3-22% of observed, respectively). Accounting for intestinal contribution to oral clearance via estimated intestinal availability, improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation of metabolic clearance.
A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance co... more A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance compounds) was performed in CYP3A4, CYP3A5 and CYP2C19 baculovirus-expressed recombinant systems. The data obtained in the expression systems were scaled and compared to human liver microsomal predicted clearance and observed in vivo values, using either CYP relative activity factors (RAF) or the relative abundance approach. Inter-individual variability, both in content (CYP3A4, CYP3A5) and activity (CYP3A4, CYP3A5 and CYP2C19), were incorporated in the clearance prediction by bootstrap analysis. These re-sampling Monte Carlo based simulations were performed in order to justify any distribution assumptions in the generated range of the predicted clearance due to a limited sample size. Therefore, this approach allowed extrapolation of the recombinant clearance data to specific population groups and investigation of the role of 'minor' forms like CYP3A5 and CYP2C19 in comparison to the most prolific CYP3A4. The use of quinidine 3hydroxylation and alprazolam 1'-hydroxylation, as RAF markers for CYP3A4 and CYP3A5 activity, respectively, and the incorporation of variability improved the clearance prediction of the selected benzodiazepines (apart from flunitrazepam) to within the 2-fold of the in vivo value. Clearance estimates from the immunoquantified protein levels was approximately 8-fold lower in comparison to the RAF approach. The differences observed in the benzodiazepines metabolite pathway ratios between CYP3A4 and CYP3A5, particularly for 1'-to 4-hydroxymidazolam and alprazolam provided useful measure of inter-individual differences within the CYP3A family.
The hepatic uptake of quinine, fluvoxamine and fluoxetine (0.1-10 µM) was investigated with fresh... more The hepatic uptake of quinine, fluvoxamine and fluoxetine (0.1-10 µM) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K p) were concentration-dependent: the mean maximum K p (at 0.1 µM) were 150 (quinine), 500 (fluvoxamine) and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K p (at 10 µM) were 30 (quinine), 200 (fluvoxamine) and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to FCCP (ATP inhibitor). The difference between the maximum and minimum K p for these three amine drugs, as well as for dextromethorphan, propranolol and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes which actively maintain their low internal pH (~5) using ATP and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K p correlated strongly with the observed maximum K p. Thus at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs this can be readily estimated assuming a common degree of uptake into lysosomes.
sulphate; HLMs, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry;... more sulphate; HLMs, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under plasma concentration time curve; EC 200 , concentration of effector required to produce 200% control activation; CL max , maximal clearance; fu inc , fraction unbound in the incubation; CYP, cytochrome P450; fm CYP3A4, fraction of a substrate drug metabolized by the heteroactivated pathway via CYP3A4.
The substrate depletion method is a popular approach used for the measurement of in vitro intrins... more The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL int). However, the incubation conditions used in these studies can vary, the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam, and triazolam. Subsequent predictions of in vivo CL int (ranging from 3 to 200 ml/min) and hepatic clearance (CL H) (ranging from 0.3 to 15 ml/min) demonstrated that the general predictive ability of this approach was similar to that of the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL int estimates indicated that the concentration of enzyme used is of particular importance. The metabolism of triazolam, clonazepam, and diazepam was monoexponential at all cell densities using hepatocytes; however, with microsomes, biphasic depletion was apparent, particularly at higher microsomal protein concentrations (2-5 mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8 to 65% activity after a 1-h incubation) compared with the hepatocyte system (approximately 100% activity after a 1-h incubation). For clonazepam (a low clearance substrate), these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL int and CL H when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.
Rate limiting steps in hepatic drug clearance: comparison of hepatocellular uptake and metabolism... more Rate limiting steps in hepatic drug clearance: comparison of hepatocellular uptake and metabolism with microsomal metabolism of saquinavir, nelfinavir and ritonavir
Drug metabolism and disposition: the biological fate of chemicals, Jul 1, 2018
This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic... more This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good...
Drug metabolism and disposition: the biological fate of chemicals, 2018
Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, i... more Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using sapo...
Drug metabolism and disposition: the biological fate of chemicals, 2018
Hepatocyte drug depletion-time assays are well established for determination of metabolic clearan... more Hepatocyte drug depletion-time assays are well established for determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, an adaptation of the conventional depletion assay involving centrifugation of hepatocytes prior to sampling, allowing estimation of uptake in addition to metabolism. Using experimental procedures consistent with a high throughput, a selection of 12 compounds with a range of uptake and metabolism characteristics (atorvastatin, cerivastatin, clarithromycin, erythromycin, indinavir, pitavastatin, repaglinide, rosuvastatin, saquinavir, and valsartan, with two control compounds-midazolam and tolbutamide) were investigated in the presence and absence of the cytochrome P450 inhibitor 1-aminobenzotriazole and organic anion transporter protein inhibitor rifamycin SV in rat hepatocytes. Data were generated simultaneously for a given drug, and provided, through the use of a mechanistic cell mo...
Journal of Pharmacology and Experimental Therapeutics, 2004
Introduction: 628 Discussion: 1200 d) List of non-standard abbreviations: %act, percent activatio... more Introduction: 628 Discussion: 1200 d) List of non-standard abbreviations: %act, percent activation caused by 100µM effector and 100µM carbamazepine; carbamazepinediol, carbamazepine-trans-diol (trans-10,11-dihydro-10,11-dihydroxycarbamazepine); carbamazepine-ep, carbamazepine-10,11-epoxide; Css CBZ , Css CBZ-ep , plasma steady state concentration of carbamazepine and of carbamazepine-10,11-epoxide respectively; CYP, cytochrome P450; DHEA, dehydroepiandrosterone; HLM, human liver microsomes; v 150% , concentration of heteroactivator giving 150% of reaction velocity.
The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-s... more The role of Organic Anion Transporting Polypeptides (OATPs), particularly the members of OATP1B-subfamily, in hepatocellular handling of endogenous and exogenous compounds is an important and emerging area of research. Using a mouse model lacking Slco1b2, the murine ortholog of the OATP1B-subfamily, we previously demonstrated that genetic ablation causes reduced hepatic clearance capacity for substrates. In this report we focused on the physiological function of the hepatic OATP1B transporters. First we studied the influence of the Oatp1b2 deletion on bile acid metabolism showing that lack of the transporter results in a significantly reduced expression of Cyp7a1 the key enzyme of bile acid synthesis, resulting in elevated cholesterol levels after high dietary fat challenge. Furthermore, Slco1b2 −/− mice exhibited delayed clearance after oral glucose challenge resulting from reduced hepatic glucose uptake. In addition to increased hepatic glycogen content, Slco1b2 −/− exhibited reduced glucose output after pyruvate challenge. This is in accordance with reduced hepatic expression of PEPCK in knockout mice. We show this phenotype is due to the loss of liver-specific Oatp1b2-mediated hepatocellular thyroid hormone entry, which then leads to reduced transcriptional activation of target genes of hepatic thyroid hormone receptor (TR) including the prior mentioned Cyp7a1 and Pepck, but also Dio1 and Glut2. Importantly, we assessed human relevance using a cohort of archived human livers where OATP1B1 expression was noted to be highly associated with TR target genes, especially for GLUT2. Furthermore, GLUT2 expression was significantly decreased in livers harboring a common genetic polymorphism in SLCO1B1.
CL active , clearance by active uptake; CL met , clearance by metabolism; CL obs , observed in vi... more CL active , clearance by active uptake; CL met , clearance by metabolism; CL obs , observed in vitro clearance (either by uptake or metabolism); CL uptake , total uptake clearance by active and passive processes; fu cell , fraction of unbound drug in the hepatocyte; k dep , initial depletion rate constant; Kp total , tissue-to-medium total drug concentration ratio; Kp u , hepatocyte-to-medium unbound drug concentration ratio; OATP, organic anion transporter polypeptide; P diff , passive uptake clearance.
With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug di... more With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic aniontransporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under-and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.
Interindividual variability in activity of uptake transporters is evident in vivo, yet limited da... more Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic aniontransporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CL active, u), bidirectional passive diffusion (P diff), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CL active, u was estimated at a single concentration. The unbound affinity constant (K m, u) and P diff values were consistent across donors, whereas V max was on average up to 2.8-fold greater in donor HU4122. Consistency in K m, u values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CL active across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake K m, u and CL active, u were on average 4.3-and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed. This work was supported by GlaxoSmithKline, Ware, UK (Ph.D. studentship to K.M.) and the Biotechnology and Biological Sciences Research Council, UK. Parts of this work were previously presented at the following conference: Mé nochet K, Kenworthy KE, Houston JB, and Galetin A (2010) Contribution of active uptake to the hepatic clearance of seven OATP substrates in rat and human plated hepatocytes.
A progress curve method for assessing time-dependent inhibition of CYP3A4 is based on simultaneou... more A progress curve method for assessing time-dependent inhibition of CYP3A4 is based on simultaneous quantification of probe substrate metabolite and inhibitor concentrations during the experiment. Therefore, it may overcome some of the issues associated with the traditional two-step method and estimation of inactivation rate (k inact) and irreversible inhibition (K I) constants. In the current study, seven time-dependent inhibitors were investigated using a progress curve method and recombinant CYP3A4. A novel mechanistic modeling approach was applied in order to determine This article has not been copyedited and formatted. The final version may differ from this version.
Previous studies have shown the importance of the addition of albumin for characterization of hep... more Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL int, UGT) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL int, UGT on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL int, UGT in different tissues. Although BSA increased CL int, UGT in all tissues, the extent was tissue-and drug-dependent. Scaled CL int, UGT in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml ⅐ min ؊1 ⅐ g tissue ؊1 in liver, kidney, and intestinal microsomes. Renal CL int, UGT (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL int, UGT for UGT2B7 substrates represented approximately onethird of hepatic estimates. Scaled renal CL int, UGT (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL int, UGT is particularly important for UGT1A9 substrates. The work was funded by a consortium of pharmaceutical companies (Glaxo-SmithKline, Lilly, Pfizer, and Servier) within the Centre for Applied Pharmacokinetic Research at the University of Manchester. K.L.G. is a recipient of a Ph.D. studentship from Biotechnology and Biological Sciences Research Council. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
Predictions of intrinsic clearance (CL int) from human liver microsomes often underestimate in vi... more Predictions of intrinsic clearance (CL int) from human liver microsomes often underestimate in vivo observations. In this study, a series of five benzodiazepines were used as prototypic CYP3A4 substrates in order to investigate the prediction of clearance from the less studied alternative in vitro system, cryopreserved human hepatocytes. Formation of the two major metabolites from midazolam, triazolam, diazepam, flunitrazepam and alprazolam was measured in cryopreserved human hepatocytes from five donors; the kinetics were characterised and CL int values determined and scaled to predict CL int in vivo. At least one of the two major pathways of metabolic clearance of each benzodiazepine was characterised by autoactivation in hepatocytes; the extent to which this occurred varied depending on substrate and liver (up to 8-fold). Heteroactivation by testosterone of these pathways was also observed (up to 6-fold). The maximum autoactivated clearance was used to predict in vivo CL int (1.6-138ml/min/kg) which closely agreed with values previously obtained using human liver microsomes. Comparison with in vivo CL int indicates that cryopreserved human hepatocytes systematically under-predict CL int. When three previous studies (documenting CL int for substrates of various enzymes in cryopreserved human hepatocytes using drug depletiontime profiles) were considered as well, the combined data show a consistent underprediction of 5.6-fold. Collectively it is demonstrated that the predicted hepatic intrinsic clearances from cryopreserved hepatocytes show an excellent rank order with in vivo findings but are systematically under-predicting the in vivo value.
The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important ... more The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important drug-drug interaction involving cytochrome P450 inhibition. The availability of an in vivo K i value (unbound K i , 21 M) obtained from a series of steady-state inhibitor infusion studies allowed assessment of several in vitroderived predictions of this inhibition interaction. Studies monitoring substrate depletion with time were used to obtain in vitro K i values that were evaluated against the more traditional metabolite formation approach using microsomes and hepatocytes. OMP inhibited the metabolism of DZ to its primary metabolites 4-hydroxydiazepam, 3-hydroxydiazepam, and nordiazepam to different extents over a range of concentrations (0.3-150 M), and a competitive inhibition model best fitted the data. The K i values observed using the substrate depletion approach (16 ؎ 3 M and 7 ؎ 2 M in microsomes and hepatocytes, respectively) were in good agreement with the overall weighted K i values obtained using the standard metabolite formation approach (12 ؎ 2 M and 16 ؎ 5 M in microsomes and hepatocytes, respectively). In vitro binding and cell uptake studies as well as human serum albumin studies in hepatocytes confirmed the importance of both intracellular and extracellular unbound concentrations of inhibitor when considering inhibition predictions. Both kinetic approaches and both in vitro systems predicted the in vivo interaction well and provide a good example of the ability of in vitro inhibition studies to quantitatively predict an in vivo drug-drug interaction successfully.
The hepatic uptake of imipramine and propranolol was investigated following incubation with isola... more The hepatic uptake of imipramine and propranolol was investigated following incubation with isolated rat hepatocytes over a wide concentration range (0.04-400 µM). The cell-tomedium concentration ratio (K p) was concentration-dependent and could be described using a two-site binding model incorporating a high affinity/low capacity site and a linear component for a site which was apparently not saturated. Maximum (at 0.04 µM) and minimum K p values (at 400 µM) were 360 and 280 and 110 and 70 for imipramine and propranolol, respectively. During these incubations, metabolism was inhibited using aminobenzotriazole (an irreversible inhibitor of P450). Pre-treatment of cells either by freeze-thawing or with saponin (which permeabilises the plasma membrane) eliminated the saturable process. The saturable uptake process of imipramine was also inhibited by 18 other lipophilic amine drugs (including propranolol). This uptake component may involve membrane transporter(s) whereas the non-saturable component probably represents partition into the phospholipid component of membranes. Propranolol was further investigated to determine the impact of high Kp values on hepatocellular clearance. The area under the curve for propranolol concentrations in the total incubate (medium including the cells) from the depletion-time profile was substantially greater than the corresponding area under the curve for the drug concentration in the extracellular medium and this difference approximated to the non-saturable uptake component. It is concluded that the clearance of propranolol in isolated hepatocytes is not rate limited by hepatic uptake and is directly proportional to unbound drug concentration being independent of the higher K p value.
The current study assesses hepatic and intestinal glucuronidation, sulfation and P450 metabolism ... more The current study assesses hepatic and intestinal glucuronidation, sulfation and P450 metabolism of raloxifene, quercetin, salbutamol and troglitazone using different in vitro systems. Fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine and importance of multiple metabolic pathways on accuracy of clearance prediction assessed. In vitro intrinsic sulfation clearance (CL int,SULT) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL int,UGT) and P450 clearance (CL int,CYP) expressed per gram tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL int,SULT ranged between 0.7-11.4ml/min/g liver and 0.1-3.3ml/min/g intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with high extent of sulfation (51% and 28% of total CL int for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared to predictions based only on primary pathway (22-36%). The assumption of no intestinal first-pass resulted in under-prediction of oral clearance for raloxifene, troglitazone and quercetin (3-22% of observed, respectively). Accounting for intestinal contribution to oral clearance via estimated intestinal availability, improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation of metabolic clearance.
A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance co... more A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance compounds) was performed in CYP3A4, CYP3A5 and CYP2C19 baculovirus-expressed recombinant systems. The data obtained in the expression systems were scaled and compared to human liver microsomal predicted clearance and observed in vivo values, using either CYP relative activity factors (RAF) or the relative abundance approach. Inter-individual variability, both in content (CYP3A4, CYP3A5) and activity (CYP3A4, CYP3A5 and CYP2C19), were incorporated in the clearance prediction by bootstrap analysis. These re-sampling Monte Carlo based simulations were performed in order to justify any distribution assumptions in the generated range of the predicted clearance due to a limited sample size. Therefore, this approach allowed extrapolation of the recombinant clearance data to specific population groups and investigation of the role of 'minor' forms like CYP3A5 and CYP2C19 in comparison to the most prolific CYP3A4. The use of quinidine 3hydroxylation and alprazolam 1'-hydroxylation, as RAF markers for CYP3A4 and CYP3A5 activity, respectively, and the incorporation of variability improved the clearance prediction of the selected benzodiazepines (apart from flunitrazepam) to within the 2-fold of the in vivo value. Clearance estimates from the immunoquantified protein levels was approximately 8-fold lower in comparison to the RAF approach. The differences observed in the benzodiazepines metabolite pathway ratios between CYP3A4 and CYP3A5, particularly for 1'-to 4-hydroxymidazolam and alprazolam provided useful measure of inter-individual differences within the CYP3A family.
The hepatic uptake of quinine, fluvoxamine and fluoxetine (0.1-10 µM) was investigated with fresh... more The hepatic uptake of quinine, fluvoxamine and fluoxetine (0.1-10 µM) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K p) were concentration-dependent: the mean maximum K p (at 0.1 µM) were 150 (quinine), 500 (fluvoxamine) and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K p (at 10 µM) were 30 (quinine), 200 (fluvoxamine) and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to FCCP (ATP inhibitor). The difference between the maximum and minimum K p for these three amine drugs, as well as for dextromethorphan, propranolol and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes which actively maintain their low internal pH (~5) using ATP and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K p correlated strongly with the observed maximum K p. Thus at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs this can be readily estimated assuming a common degree of uptake into lysosomes.
sulphate; HLMs, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry;... more sulphate; HLMs, human liver microsomes; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under plasma concentration time curve; EC 200 , concentration of effector required to produce 200% control activation; CL max , maximal clearance; fu inc , fraction unbound in the incubation; CYP, cytochrome P450; fm CYP3A4, fraction of a substrate drug metabolized by the heteroactivated pathway via CYP3A4.
The substrate depletion method is a popular approach used for the measurement of in vitro intrins... more The substrate depletion method is a popular approach used for the measurement of in vitro intrinsic clearance (CL int). However, the incubation conditions used in these studies can vary, the consequences of which have not been systematically explored. Initial substrate depletion incubations using rat microsomes and hepatocytes were performed for eight benzodiazepines: alprazolam, clobazam, clonazepam, chlordiazepoxide, diazepam, flunitrazepam, midazolam, and triazolam. Subsequent predictions of in vivo CL int (ranging from 3 to 200 ml/min) and hepatic clearance (CL H) (ranging from 0.3 to 15 ml/min) demonstrated that the general predictive ability of this approach was similar to that of the traditional metabolite formation method. A more detailed study of the substrate depletion profiles and CL int estimates indicated that the concentration of enzyme used is of particular importance. The metabolism of triazolam, clonazepam, and diazepam was monoexponential at all cell densities using hepatocytes; however, with microsomes, biphasic depletion was apparent, particularly at higher microsomal protein concentrations (2-5 mg/ml). Enzyme activity studies indicated that enzyme loss was more pronounced in the microsomal system (ranged from 8 to 65% activity after a 1-h incubation) compared with the hepatocyte system (approximately 100% activity after a 1-h incubation). For clonazepam (a low clearance substrate), these biphasic profiles could be explained by loss of enzyme activity. To ensure accurate predictions of in vivo CL int and CL H when using the substrate depletion approach, based on the results obtained for this class of drugs, it is recommended that low enzyme concentrations and short incubation times are used whenever possible.
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