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The formation of cytotoxic metabolites from the anticonvulsants phenytoin and carbamazepine was investigated in vitro using a hepatic microsomal enzyme system and human mononuclear leucocytes as target cells. Both drugs were metabolised to cytotoxic products. In order to assess the structural requirements for this bioactivation, a series of structurally related compounds was investigated. It was found that molecules which contain either an amide function or an aryl ring may undergo activation in vitro, but only the metabolism-dependent toxicity of the latter is potentiated by pre-treatment of the target cells with an epoxide hydrolase inhibitor. Taken collectively, these data are consistent with the concept that reactive epoxide metabolites of both phenytoin and carbamazepine may produce toxicity in individuals with an inherited deficiency in epoxide hydrolase. [ABSTRACT FROM AUTHOR]

1. The metabolism of the enantiomers of mianserin to stable, chemically reactive and cytotoxic metabolites by human liver microsomes has been investigated in vitro. 2. Both enantiomers were metabolised to three major oxidation products: 8-hydroxymianserin, desmethylmianserin and mianserin 2-oxide. Hydroxylation occurred more readily with the S- enantiomer, whereas desmethylmianserin was always the major metabolite of the R-enantiomer. 3. The generation of chemically reactive metabolites exhibited a marginal degree of stereoselectivity, as assessed by irreversible binding of drug to microsomal protein (S greater than or equal to R; P less than or equal to 0.05). 4. The formation of metabolites which were cytotoxic towards human mononuclear leucocytes was greater (P less than or equal to 0.001] for R(-)- mianserin than for S(+)-mianserin and showed a significant correlation with N-demethylation (r = 0.84, P less than or equal to 0.01). [ABSTRACT FROM AUTHOR]

1. The cytotoxicity of metabolites generated from phenytoin, sorbinil and mianserin by human and mouse liver microsomes was assessed by co- incubation with human mononuclear leucocytes as target cells. Cytotoxicity was determined by trypan blue dye exclusion. 2. Phenytoin and sorbinil were metabolised by NADPH-dependent murine microsomal enzymes to cytotoxic metabolites. Cytotoxicity produced by both drugs was significantly enhanced by the epoxide hydrolase inhibitor trichloropropane oxide (TCPO). No significant cytotoxicity was observed in the presence of human liver microsomes. 3. Mianserin was metabolised by both human and mouse liver microsomes to a cytotoxin. Cytotoxicity was greater in the presence of human liver microsomes (13.7 +/- 2.2%; mean +/- s.d. for four livers, compared with 6.0 +/- 2.4%, mean +/- s.d., n = 4, with mouse liver microsomes), and was unaffected by pretreatment with TCPO. 4. Stable metabolites were quantified by radiometric high performance liquid chromatography. Phenytoin and sorbinil were metabolised to 5-(p-hydroxyphenyl)-5-phenyl-hydantoin (0.3-0.5% of incubated radioactivity) and 2-hydroxysorbinil (0.4-2.7% of incubated radioactivity), respectively, by both human and mouse liver microsomes. 5. Mianserin was metabolised to 8-hydroxymianserin and desmethylmianserin by both human and mouse liver microsomes. Desmethylmianserin was the major product in incubations with human liver microsomes (32.3 +/- 12%, mean +/- s.d. for four livers), whereas 8-hydroxymianserin was the predominant metabolite generated by mouse liver microsomes (25.9 +/- 1.5%, mean +/- s.d., n = 4). 6. Generation of electrophilic metabolites was assessed by determination of the amount of radiolabelled material which became irreversibly bound to protein. Only mouse liver microsomes activated phenytoin to a chemically reactive metabolite, whereas both mouse and human liver microsomes generated reactive metabolites from sorbinil and mianserin. 7. These studies show that drug cytotoxicity can be mediated by low concentrations (circa microM) of metabolites generated by NADPH- dependent hepatic microsomal enzymes; however demonstration of cytotoxicity in vitro has not been established as a means of predicting in vivo toxicity. [ABSTRACT FROM AUTHOR]

Coordinated responses to threats are important for predator evasion in many species. This study examines the effect of developmental social experience on antipredator behavior and group cohesion in a highly gregarious catfish that communicates via tactile interaction, Corydoras aeneus. We reared fish either in a mixed-age group of age-matched peers and adult C. aeneus (mixed-age condition, or MAC), or with age-matched peers only (same-age condition, or SAC). A startle test was conducted with small groups of subadults from either social rearing condition. Prior to any startle events, SAC subadults had increased tactile communication compared to MAC subadults, but SAC individuals were overall less active. SAC fish exhibited a stronger antipredator response to startles, and were more likely to freeze or take refuge in cover in response to a startle than MAC fish. MAC fish tended to respond to startle events by maintaining or decreasing their cohesion, whereas SAC fish tended to maintain or increase their cohesion. These behavioral differences are attributed to MAC fish developing with group protection as a result of shoaling with adults, resulting in reduced antipredator responses when reared with adults. This study underscores how social context during development can be critical in shaping how individuals perceive and respond to potential threats in their environment., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

A variety of fish species have proven instrumental in the investigation of evolution, behavior, ecology, and physiology, among many other fields. Many model systems (e.g., zebrafish, guppies, and three-spined sticklebacks) have been maintained by institutions and have had protocols written with respect to their husbandry. Here we present the protocols we have developed to maintain and breed a variety of Corydoras catfish species, which are native to the tropical Americas. Corydoras species are excellent systems for investigating behavior, ecology, and other topics, and our husbandry protocols would be suitable for nearly every species in the genus. In addition, these protocols are appropriate for a variety of softwater Amazonian species, and we present options for a variety of housing and husbandry conditions. On the whole, we suggest that, in a scientific laboratory setting, the use of remineralized reverse osmosis water is most appropriate and that in context, a single measure, total dissolved solids, can be used to monitor the water chemistry for water introduced to fish enclosures.

Introduction: Transporters are significant in dictating drug pharmacokinetics, thus inhibition of transporter function can alter drug concentrations resulting in drug-drug interactions (DDIs). Because they can impact drug toxicity, transporter DDIs are a regulatory concern for which prediction of clinical effect from in vitro data is critical to understanding risk., Area Covered: The authors propose in vitro strategies to assist mitigating/removing transporter DDI risk during development by frontloading specific studies, or managing patient risk in the clinic. An overview of clinically relevant drug transporters and observed DDIs is provided, alongside presentation of key considerations/recommendations for in vitro study design evaluating drugs as inhibitors or substrates. Guidance on identifying critical co-medications, clinically relevant disposition pathways, and using mechanistic static equations for quantitative prediction of DDI is compiled., Expert Opinion: The strategies provided will facilitate project teams to study the right transporter at the right time to minimize development risks associated with DDIs. To truly alleviate or manage clinical risk, the industry will benefit from moving away from current qualitative basic static equation approaches to transporter DDI hazard assessment towards adopting the use of mechanistic models to enable quantitative DDI prediction, thereby contextualizing risk to ascertain whether a transporter DDI is simply pharmacokinetic or clinically significant requiring intervention.

Animals can gain large benefits from living in groups but must coordinate with their groupmates in order to do so. Social interactions between groupmates drive overall group coordination and are influenced by the characteristics of individual group members. In particular, consistent inter-individual differences in behaviour (e.g. boldness) and familiarity between individuals in groups profoundly affect the individual interactions that mediate group coordination. However, the effects of boldness and familiarity have mostly been studied in isolation. Here we describe how familiarity and boldness interact to affect individual performance, leadership, and group coordination in small shoals of three-spined sticklebacks (Gasterosteus aculeatus) solving a novel foraging task. Groups of higher average boldness were less cohesive, but only when group members were familiar with one another. Familiarity affected shy and bold individuals' foraging performance and leadership tendencies differently depending on group characteristics: the shyest group member experienced declining foraging success and leadership with increased group boldness in familiar groups, but experienced the opposite effect on foraging and no effect on leadership in unfamiliar groups. The boldest group member, in contrast, exhibited the opposite pattern: leading and eating more with increasing group boldness in familiar groups, but eating less with increasing group boldness in unfamiliar groups. These results suggest that both boldness and familiarity are important for establishing group behaviour and coordination, and that consistent inter-individual differences in behaviour may primarily impact group coordination once familiarity has been established., (Copyright © 2022. Published by Elsevier B.V.)

Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.

Early identification of toxicity associated with new chemical entities (NCEs) is critical in preventing late-stage drug development attrition. Liver injury remains a leading cause of drug failures in clinical trials and post-approval withdrawals reflecting the poor translation between traditional preclinical animal models and human clinical outcomes. For this reason, preclinical strategies have evolved over recent years to incorporate more sophisticated human in vitro cell-based models with multi-parametric endpoints. This review aims to highlight the evolution of the strategies adopted to improve human hepatotoxicity prediction in drug discovery and compares/contrasts these with recent activities in our lab. The key role of human exposure and hepatic drug uptake transporters (e.g. OATPs, OAT2) is also elaborated.

To facilitate smoking genetics research we determined whether a screen of mutagenized zebrafish for nicotine preference could predict loci affecting smoking behaviour. From 30 screened F 3 sibling groups, where each was derived from an individual ethyl-nitrosurea mutagenized F 0 fish, two showed increased or decreased nicotine preference. Out of 25 inactivating mutations carried by the F 3 fish, one in the slit3 gene segregated with increased nicotine preference in heterozygous individuals. Focussed SNP analysis of the human SLIT3 locus in cohorts from UK (n=863) and Finland (n=1715) identified two variants associated with cigarette consumption and likelihood of cessation. Characterisation of slit3 mutant larvae and adult fish revealed decreased sensitivity to the dopaminergic and serotonergic antagonist amisulpride, known to affect startle reflex that is correlated with addiction in humans, and increased htr1aa mRNA expression in mutant larvae. No effect on neuronal pathfinding was detected. These findings reveal a role for SLIT3 in development of pathways affecting responses to nicotine in zebrafish and smoking in humans., Competing Interests: JG, AB, MP, RR, DJ, AS, MT, EB, DS, AM, JK, TP, VK, RW, CB No competing interests declared, (© 2020, García-González et al.)

Introduction : Prediction of human absorption, distribution, metabolism, and excretion (ADME) properties, therapeutic dose and exposure has become an integral part of compound optimization in discovery. Incorporation of drug metabolism and pharmacokinetics into discovery projects has largely tempered historical drug failure due to sub-optimal ADME. In the current era, inadequate safety and efficacy are leading culprits for attrition; both of which are dependent upon drug exposure. Therefore, prediction of human pharmacokinetics (PK) and dose are core components of de-risking strategies in discovery. Areas covered : The authors provide an overview of human dose prediction methods and present a toolbox of PK parameter prediction models with a proposed framework for a consensus approach valid throughout the discovery value chain. Mechanistic considerations and indicators for their application are discussed which may impact the dose prediction approach. Examples are provided to illustrate how implementation of the proposed strategy throughout discovery can assist project progression. Expert opinion : Anticipation of human ADME, therapeutic dose and exposure must be deliberated throughout drug discovery from virtual/initial synthesis where key properties are considered and similar molecules ranked, into development where advanced compounds can be subject to prediction with greater mechanistic understanding and data-driven model selection.

Social groups composed of familiar individuals exhibit better coordination than unfamiliar groups; however, the ways familiarity contributes to coordination are poorly understood. Prior social experience probably allows individuals to learn the tendencies of familiar group-mates and respond accordingly. Without prior experience, individuals would benefit from strategies for enhancing coordination with unfamiliar others. We used a social catfish, Corydoras aeneus , that uses discrete, observable tactile interactions to assess whether active interactions could facilitate coordination, and how their role might be mediated by familiarity. We describe this previously understudied physical interaction, 'nudges', and show it to be associated with group coordination and cohesion. Furthermore, we investigated nudging and coordination in familiar/unfamiliar pairs. In all pairs, we found that nudging rates were higher during coordinated movements than when fish were together but not coordinating. We observed no familiarity-based difference in coordination or cohesion. Instead, unfamiliar pairs exhibited significantly higher nudging rates, suggesting that unfamiliar pairs may be able to compensate for unfamiliarity through increased nudging. By contrast, familiar individuals coordinated with comparatively little nudging. Second, we analysed nudging and cohesion within triplets of two familiar and one unfamiliar individual (where familiar individuals had a choice of partner). Although all individuals nudged at similar rates, the unfamiliar group-mate was less cohesive than its familiar group-mates and spent more time alone. Unfamiliar individuals that nudged their group-mates more frequently exhibited higher cohesion, indicating that nudging may facilitate cohesion for the unfamiliar group-mate. Overall, our results suggest that nudges can mitigate unfamiliarity, but that their usage is reduced in the case of familiar individuals, implying a cost is associated with the behaviour., Competing Interests: The authors have no conflicts of interest to declare., (© 2019 The Authors.)

A previous attempt to accurately quantify the increased simvastatin acid exposure due to drug-drug interaction (DDI) with coadministered telithromycin, using a mechanistic static model, substantially underpredicted the magnitude of the area under the plasma concentration-time curve ratio (AUCR) based on reversible inhibition of CYP3A4 and organic anion transporting polypeptide 1B1 (OATP1B1). To reconcile this disconnect between predicted and clinically observed AUCR, telithromycin was evaluated as a time-dependent inhibitor of CYP3A4 in vitro, as well as an inhibitor of OATP1B1. Telithromycin inhibited OATP1B1-mediated [ 3 H]-estradiol 17 β -d-glucuronide (0.02 µ M) transport with a mean IC 50 of 12.0 ± 1.45 µ M and was determined by IC 50 shift and kinetic analyses to be a competitive reversible inhibitor of CYP3A4-mediated midazolam1- hydroxylation with a mean absolute inhibition constant (K i ) value of 3.65 ± 0.531 µ M. The 2.83-fold shift in IC 50 (10.4-3.68 µ M) after a 30-minute metabolic preincubation confirmed telithromycin as a time-dependent inhibitor of CYP3A4; the mean inhibitor concentration that causes half-maximal inactivation of enzyme (K I ) and maximal rate of inactivation of enzyme (k inact ) values determined for inactivation were 1.05 ± 0.226 µ M and 0.02772 ± 0.00272 min -1 , respectively. After the integration of an enzyme time-dependent inhibition component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above, the newly predicted simvastatin acid AUCR (10.8 or 5.4) resulting from perturbation of its critical disposition pathways matched the clinically observed AUCR (10.8 or 4.3) after coadministration, or staggered administration, with telithromycin, respectively. These results indicate the time-dependent inhibition of CYP3A4 by telithromycin as the primary driver underlying its clinical DDI with simvastatin acid., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Progression of new chemical entities is a multiparametric process involving a balance of potency; absorption, distribution, metabolism, and excretion; and safety properties. To accurately predict human pharmacokinetics and estimate human efficacious dose, the use of in vitro measures of clearance is often essential. Low metabolic clearance is often targeted to facilitate in vivo exposure and achieve appropriate half-life. Suspension primary human hepatocytes (PHHs) have been successfully used in predictions of clearance. However, incubation times are limited, hindering the limit of quantification. The aims herein were to evaluate the ability of a novel PHH media supplement, HepExtend, in order to maintain cell function, increase culture times, and define the clearance of stable compounds. Cell activity was analyzed with a range of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) substrates, and the mRNA expression of drug disposition and toxicity marker genes was determined. HepExtend and Geltrex were essential to maintain cell activity and viability for 5 days ( N = 3 donors). In comparison with CM4000 ± Geltrex, HepExtend + Geltrex displayed a higher level of gene expression on day 1, particularly for the P450s, nuclear receptors, and UGTs. The novel medium, HepExtend + Geltrex, was robust and reproducible in generating statistically significant intrinsic clearance values at 0.1 µ l/min/10 6 cells over a 30-hour period ( P < 0.05), which was lower than previously demonstrated. Following regression correction, human hepatic in vivo clearance was predicted within 3-fold for 83% of compounds tested for three human donors, with an average fold error of 2.2. The novel PHH medium, HepExtend, with matrix overlay offers significant improvement in determining compounds with low intrinsic clearance when compared with alternative approaches., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUC inf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUC inf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUC inf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.

Introduction: Drug-drug interactions (DDIs) continue to account for 5% of hospital admissions and therefore remain a major regulatory concern. Effective, quantitative prediction of DDIs will reduce unexpected clinical findings and encourage projects to frontload DDI investigations rather than concentrating on risk management ('manage the baggage') later in drug development. A key challenge in DDI prediction is the discrepancies between reported models. Areas covered: The current synopsis focuses on four recent influential publications on hepatic drug transporter DDIs using static models that tackle interactions with individual transporters and in combination with other drug transporters and metabolising enzymes. These models vary in their assumptions (including input parameters), transparency, reproducibility and complexity. In this review, these facets are compared and contrasted with recommendations made as to their application. Expert opinion: Over the past decade, static models have evolved from simple [I]/k i models to incorporate victim and perpetrator disposition mechanisms including the absorption rate constant, the fraction of the drug metabolised/eliminated and/or clearance concepts. Nonetheless, models that comprise additional parameters and complexity do not necessarily out-perform simpler models with fewer inputs. Further, consideration of the property space to exploit some drug target classes has also highlighted the fine balance required between frontloading and back-loading studies to design out or 'manage the baggage'.

The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08μg/mL at 0.25-0.5h post-dose with an AUC inf of 1.74±0.52μgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08μg/mL at 0.25h post-dose with an AUC inf of 1.94±0.22μgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: Prediction of human pharmacokinetics from in vitro assays and pre-clinical data is an integral part of drug discovery. In vitro stability metabolic studies can provide an estimate of in vivo hepatic intrinsic clearance through inclusion of biological scaling factors. Many labs have personalised stability protocols including marker compounds and have adopted QC criteria and assay limits to ensure data integrity. Contract research organisations (CRO's) provide integrated drug discovery support to academic and pharmaceutical/biotechnology institutions to progress their in-house projects. The majority of these clients have established in-house protocols with associated criteria to ensure data consistency between in-house and external labs., Methods: In this study, numerous assay variables were condensed into one harmonised assay format and a range of compounds with diverse physicochemical properties were evaluated. The protocols were diverse with respect to the following attributes: buffer, microsomal concentration and species strain., Results: Comparison of human lots in vitro CL int between the traditional and consolidated assay formats showed a good correlation with no significant difference. A clear relationship was demonstrated between strains. Interpretation of in vitro intrinsic clearance between the strains for each species was consistent. Using strict classes of in vitro hepatic intrinsic clearance values (<50, 50-100, >150μL/min/mg protein) comparisons across different conditions such as, assay variables, human lots, mouse and rat strains showed >80% agreement., Discussion: A high throughput assay was developed that enables the simultaneous measurement of CL int using mouse, rat and human hepatic microsomes (consolidated assay). By condensing all possible variables into one assay format, consistent data were obtained during head to head tests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The nuclear pregnane X receptor (PXR) regulates the expression of genes involved in the metabolism, hepatobiliary disposition, and toxicity of drugs and endogenous compounds. PXR is a promiscuous nuclear hormone receptor (NHR) with significant ligand and DNA-binding crosstalk with the constitutive androstane receptor (CAR); hence, defining the precise role of PXR in gene regulation is challenging. Here, utilising a novel PXR-knockout (KO) HepaRG cell line, real-time PCR analysis was conducted to determine PXR involvement for a range of inducers. The selective PXR agonist rifampicin, a selective CAR activator, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and dual activators of CAR and PXR including phenobarbital (PB) were analyzed. HepaRG control cells (5F clone) were responsive to prototypical inducers of CYP2B6 and CYP3A4. No response was observed in the PXR-KO cells treated with rifampicin. Induction of CYP3A4 by PB, artemisinin, and phenytoin was also much reduced in PXR-KO cells, while the response to CITCO was maintained. This finding is in agreement with the abolition of functional PXR expression. The apparent EC 50 values for PB were in agreement between the cell lines; however, CITCO was ~threefold (0.3  μ mol/L vs. 1  μ mol/L) lower in the PXR-KO cells compared with the 5F cells for CYP2B6 induction. Results presented support the application of the novel PXR-KO cells in the definitive assignment of PXR-mediated CYP2B6 and CYP3A4 induction. Utilization of such cell lines will allow advancement in composing structure activity relationships rather than relying predominantly on pharmacological manipulations and provide in-depth mechanistic evaluation.

Improving the efficiency of drug discovery remains a major focus for the pharmaceutical industry. Toxicity accounts for 90% of withdrawals and major early-stage terminations relate to suboptimal efficacy and safety. Traditional oral drug space is well defined with respect to physicochemical properties and ADMET risks but increased focus on ligand-lipophilicity efficiency, maximizing enthalpy contributions and new target classes challenge this paradigm. A hybrid space has been identified that combines physical properties and key interactions attributable to drug transporters. A novel algorithm is proposed that incorporates drug-transporter interactions and its utility evaluated against popular ligand efficiency indices. Simply reducing the bulk properties of compounds can exchange one problem for another and creates high-risk areas that challenge the successful delivery from a balanced portfolio., (Copyright © 2015. Published by Elsevier Ltd.)

Introduction: The key role of transporter biology in both the manifestation and treatment of disease is now firmly established. Experiences of sub-optimal drug exposure due to drug-transporter interplay have supported incorporation of studies aimed at understanding the interactions between compounds and drug transporters much earlier in drug discovery. While drug transporters can impact the most pivotal pharmacokinetic parameter with respect to human dose and exposure projections, clearance, at a renal or hepatobiliary level, the latter will form the focus of this perspective., Areas Covered: A synopsis of guidelines on which transporters to study together with an overview of the currently available toolkit is presented. A perspective on when to conduct studies with various hepatic transporters is also provided together with structural "alerts" which should prompt early investigation., Expert Opinion: Great progress has been made in individual laboratories and via consortia to understand the role of drug transporters in disease, drug disposition, drug-drug interactions and toxicity. A systematic analysis of the value posed by the available approaches and an inter-lab comparison now seems warranted. The emerging ability to use physico-chemical properties to guide future screening cascades promises to revolutionise the efficiency of early drug discovery.

Introduction: It is widely accepted that current practice of polypharmacy inevitably increases the incidence of drug-drug interactions (DDIs). Serious DDIs are a major liability for new molecular entities entering the pharmaceutical market. Various strategies are employed to avoid problematic compounds for clinical development. Progress made with reversible CYP DDIs has prompted a switch to study and model time-dependent inhibition and induction interactions., Areas Covered: An overview of popular experimental practices is presented with discussion of techniques and algorithms used to analyse the clinical DDI risk. Emphasis is placed on the transition from early, simple static equations, via more complex net mechanistic, static models to dynamic approaches involving multiple perpetrators and metabolites, simultaneous inhibition and induction., Expert Opinion: Inclusion of the more conservative terms for parameters required for DDI evaluation may eliminate promising chemical space, encourages poor practice and hampers innovation. Breakthroughs have originated from understanding of 'outliers' from such analyses where CYP enzyme-transporter interplay may be involved. The role of key transporters in drug disposition is firmly established as the chemistry required to address new targets deviates from traditional 'drug-like' space. Attempts to model more complex interactions for substrates of both CYP enzymes and drug transporters are still in their infancy and will benefit from dynamic modelling.

1. Optimization of renal clearance is a complex balance between passive and active processes mediated by renal transporters. This work aimed to characterize the interaction of a series of compounds with rat and human organic anion transporters (OATs) and develop quantitative structure-activity relationships (QSARs) to optimize renal clearance. 2. In vitro inhibition assays were established for human OAT1 and rat Oat3 and rat in vivo renal clearance was obtained. Statistically significant quantitative relationships were explored between the compounds' physical properties, their affinity for OAT1 and oat3 and the inter-relationship with unbound renal clearance (URC) in rat. 3. Many of the compounds were actively secreted and in vitro analysis demonstrated that these were ligands for rat and human OAT transporters (IC50 values ranging from <1 to >100 µM). Application of resultant QSAR models reduced renal clearance in the rat from 24 to <0.1 ml/min/kg. Data analysis indicated that the properties associated with increasing affinity at OATs are the same as those associated with reducing URC but orthogonal in nature. 4. This study has demonstrated that OAT inhibition data and QSAR models can be successfully used to optimize rat renal clearance in vivo and provide confidence of translation to humans.

The number of Americans with dementia is expected to increase as the population ages. Developing dementia is feared by many older adults and may result in anxiety in persons with dementia. This article focuses on anxiety, one of the least understood symptoms associated with dementia in community-dwelling older adults, the stigma of dementia, and the relationship between anxiety and stigma in dementia. When undetected and untreated, anxiety and associated stigma can adversely affect quality of life and the ability to age in place., (Copyright © 2014 Elsevier Inc. All rights reserved.)

1. Organic anion transporting polypeptide 1B1 plays a pivotal role in the disposition of many anionic drugs. Significant overlap in substrate specificity between individual OATP isoforms has hampered the identification of the relative importance of individual isoforms for hepatic uptake of xenobiotics. 2. The present study focused on the use of siRNA technology to decrease OATP1B1 selectively in human hepatocytes. Following delivery of siRNA by the novel lipid, AtuFECT01, mRNA expression of OATP1B1 was reduced by 94%-98% with no significant toxicity. Off-target effects were also shown to be minimal as evidenced by the expression of common drug metabolizing enzymes, transporters, nuclear receptors and associated co-regulators. Uptake of estrone-3-sulfate (5 nM) by OATP1B1 was reduced by 82%-95%. This methodology was subsequently used to assess the relative contribution of OATP1B1 uptake in human hepatocytes for olmesartan (42%-62%), valsartan (28%-81%), rosuvastatin (64%-72%), pitavastatin (84%-98%) and lopinavir (64%-89%). These data are consistent with previous values obtained using a relative activity factor approach. 3. The siRNA approach provides a robust and reproducible method for assessing the relative contribution of OATP1B1 to hepatic uptake of new chemical entities. The technique also has potential utility in facilitating detailed characterization of drug-drug interactions involving hepatic drug transporters.

The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A series of dual CCR3/H(1) antagonists based on a bispiperidine scaffold were discovered. Introduction of an acidic group overcame hERG liability. Bioavailability was optimised by modulation of physico-chemical properties and physical form to deliver a compound suitable for clinical evaluation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The pivotal role of organic anion-transporting polypeptide 1B1 (OATP1B1) in drug disposition has become clear over the last decade. Therefore, an OATP1B1 inhibition assay suitable for use within early drug discovery was developed and characterized. IC(50) estimates for 10 literature compounds using pitavastatin and estradiol-17β-glucuronide as substrates were within 2-fold of each other. In addition, the IC(50) estimates using pitavastatin uptake agreed well with literature values (r(2) = 0.92, average fold error = 1.3). However, when estrone-3-sulfate was used, OATP1B1 inhibition was underpredicted by as much as 10-fold. A comparison of uptake in human hepatocytes and OATP1B1 inhibition showed a significant correlation (r(2) = 0.53, P < 0.001) for more than 40 compounds. These data suggest that, for discrete chemical series, OATP1B1 inhibition data may be used as a surrogate for more costly and time-consuming uptake studies in hepatocytes. OATP1B1 inhibition data, determined for over 260 compounds representing both internal AstraZeneca and literature chemistry, were also used to generate a continuous in silico model. The robustness of the model was demonstrated by accurately predicting OATP1B1 inhibition for external test sets using 50 AstraZeneca compounds (root mean square error = 0.45) and 12 literature drugs (RMSE = 0.32). The most important molecular descriptors for the prediction of OATP1B1 inhibition were maximal hydrogen bonding strength followed by cLogP. This study has shown that a well validated OATP1B1 inhibition assay in conjunction with an in silico approaches has the potential to influence significantly the design-make-test cycle and subsequently reduce the propensity of OATP1B1 ligands.

Although the dog is frequently used in pharmacological, pharmacokinetic, and drug safety studies, little is known about canine drug transporters. Dog organic anion-transporting polypeptide (Oatp1b4) has recently been cloned (Comp Biochem Physiol C Toxicol Pharmacol 151:393-399, 2010), but the contribution of Oatp1b4 to hepatic uptake has yet to be clarified. This study compares the transport characteristics of dog Oatp1b4 with those of human OATP1B1/1B3 and demonstrates the importance of Oatp1b4 in the uptake of anionic compounds in dog hepatocytes. Oatp1b4 is the predominant Oatp in dog liver with expression levels double and 30 times those of Oatp2b1 and Oatp1a2, respectively. Uptake of a range of typical OATP substrates by Oatp1b4-expressing HEK293 cells was compared with that in fresh dog hepatocytes. All compounds tested were transported by Oatp1b4 and uptake intrinsic clearance (CL(int, uptake)) in dog hepatocytes in sodium-free buffer was correlated significantly with CL(int, uptake) in Oatp1b4-expressing cells. Dog in vivo clearance for five substrates was predicted more accurately from CL(int, uptake) than from metabolic intrinsic clearance (CL(int, met)), indicating that uptake governs the overall in vivo hepatic clearance of these anionic compounds in dog. The substrate specificities of dog Oatp1b4 appear to be similar to those of human OATP1B1/OATP1B3, whereas the relative uptake clearance of substrates for Oatp1b4 correlate better with OATP1B3 than with the more abundant hepatic analog OATP1B1.

Identifying any extrahepatic excretion phenomenon in preclinical species is crucial for an accurate prediction of the pharmacokinetics in man. This understanding is particularly key for drugs with a small volume of distribution, because they require an especially low total clearance to be suitable for a once-a-day dosing regimen in man. In this study, three animal scaling techniques were applied for the prediction of the human renal clearance of 36 diverse drugs that show active secretion or net reabsorption: 1) direct correlations between renal clearance in man and each of the two main preclinical species (rat and dog); 2) simple allometry; and 3) Mahmood's renal clearance scaling method. The results show clearly that the predictions to man for the methods are improved significantly when corrections are made for species differences in plasma protein binding. Overall, the most accurate predictions were obtained by using a direct correlation with the dog renal clearance after correcting for differences in plasma protein binding and kidney blood flow (r² = 0.84), where predictions, on average, were within 2-fold of the observed renal clearance values in human.

Background: Endothelial function is central to the localization of atherosclerosis. The in vivo endothelial phenotypic footprints of arterial bed identity and site-specific atherosusceptibility are addressed., Methods and Results: Ninety-eight endothelial cell samples from 13 discrete coronary and noncoronary arterial regions of varying susceptibilities to atherosclerosis were isolated from 76 normal swine. Transcript profiles were analyzed to determine the steady-state in vivo endothelial phenotypes. An unsupervised systems biology approach using weighted gene coexpression networks showed highly correlated endothelial genes. Connectivity network analysis identified 19 gene modules, 12 of which showed significant association with circulatory bed classification. Differential expression of 1300 genes between coronary and noncoronary artery endothelium suggested distinct coronary endothelial phenotypes, with highest significance expressed in gene modules enriched for biological functions related to endoplasmic reticulum (ER) stress and unfolded protein binding, regulation of transcription and translation, and redox homeostasis. Furthermore, within coronary arteries, comparison of endothelial transcript profiles of susceptible proximal regions to protected distal regions suggested the presence of ER stress conditions in susceptible sites. Accumulation of reactive oxygen species throughout coronary endothelium was greater than in noncoronary endothelium consistent with coronary artery ER stress and lower endothelial expression of antioxidant genes in coronary arteries., Conclusions: Gene connectivity analyses discriminated between coronary and noncoronary endothelial transcript profiles and identified differential transcript levels associated with increased ER and oxidative stress in coronary arteries consistent with enhanced susceptibility to atherosclerosis.

The method of predicting CYP induction drug-drug interactions (DDIs) from a relative induction score (RIS) calibration has been developed to provide a novel model facilitating predictions for any CYP-inducer substrate combination by inclusion of parameters such as the fraction of hepatic clearance mediated by a specific CYP and fraction of the dose escaping intestinal extraction. In vitro HepaRG CYP3A4 induction data were used as a basis for the approach and a large number of DDIs were well predicted. Primary human hepatocyte data were also used to make predictions, using the HepaRG calibration as a foundation. Similar predictive accuracy suggests that HepaRG and primary hepatocyte data can be used inter-changeably within the same laboratory. A comparison of this 'indirect' calibration method with a direct in vitro-in vivo scaling approach was made and investigations undertaken to define the most appropriate in vivo inducer concentration to use. Additionally, a reasonably effective prediction model based on F(2) (the concentration of inducer taken to increase the CYP mRNA 2-fold above background) was established. An accurate prediction for the CYP1A2-dependent omeprazole-caffeine interaction was also made, demonstrating that the methods are useful for the evaluation of DDIs from induction involving mechanisms other than PXR activation. Finally, a dynamic mechanistic model accounting for the simultaneous influence of CYP induction and reversible and irreversible CYP inhibition in both the liver and intestine was written to provide a prediction of the overall DDI when several interactions occur concurrently. The rationale for using the various models described, alongside commercially available prediction tools, at various stages of the drug discovery process is described.

Atherosclerosis originates as focal arterial lesions having a predictable distribution to regions of bifurcations, branches, and inner curvatures where blood flow characteristics are complex. Distinct endothelial phenotypes correlate with regional hemodynamics. We propose that systemic risk factors modify regional endothelial phenotype to influence focal susceptibility to atherosclerosis. Transcript profiles of freshly isolated endothelial cells from three atherosusceptible and three atheroprotected arterial regions in adult swine were analyzed to determine the initial prelesional effects of hypercholesterolemia on endothelial phenotypes in vivo. Cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) was upregulated at all sites in response to short-term high-fat diet. Proinflammatory and antioxidative endothelial gene expression profiles were induced in atherosusceptible and atheroprotected regions, respectively. However, markers for endoplasmic reticulum stress, a signature of susceptible endothelial phenotype, were not further enhanced by brief hypercholesterolemia. Both region-specific and ubiquitous (ABCA1) phenotype changes were identified as early prelesional responses of the endothelium to hypercholesterolemia.

The ability to predict hepatic metabolic clearance is a key component in the design and selection of small molecule drug candidates within the pharmaceutical industry. The recognition that metabolism-transporter interplay can influence hepatic metabolic clearance has presented new challenges, both in terms of the creation of experimental systems suitable for an industry setting and also in developing an understanding of the pharmacokinetic concepts that underpin them. This paper reviews the pharmacokinetic principles that govern the kinetics of uptake transporter substrates. In addition, new data are presented from a range of test systems for assessing hepatic drug clearance and the impact of drug-drug interactions (DDIs).

Rationale: Endothelial function and dysfunction are central to the focal origin and regional development of atherosclerosis; however, an in vivo endothelial phenotypic footprint of susceptibility to atherosclerosis preceding pathological change remains elusive., Objective: To conduct a comparative multi-site genomics study of arterial endothelial phenotype in atherosusceptible and atheroprotected regions., Methods and Results: Transcript profiles of freshly isolated endothelial cells from 7 discrete arterial regions in normal swine were analyzed to determine the steady state in vivo endothelial phenotypes in regions of varying susceptibilities to atherosclerosis. The most abundant common feature of the endothelium of all atherosusceptible regions was the upregulation of genes associated with endoplasmic reticulum (ER) stress. The unfolded protein response pathway, induced by ER stress, was therefore investigated in detail in endothelium of the atherosusceptible aortic arch and was found to be partially activated. ER transmembrane signal transducers IRE1alpha and ATF6alpha and their downstream effectors, but not PERK, were activated concomitant with a higher transcript expression of protein folding enzymes and chaperones, indicative of ER stress in vivo., Conclusions: The findings demonstrate the prevalence of chronic endothelial ER stress and activated unfolded protein response in vivo at atherosusceptible arterial sites. We propose that chronic localized biological stress is linked to spatial susceptibility of the endothelium to the initiation of atherosclerosis.

Prototypic CYP3A4 inducers were tested in a pregnane X receptor (PXR) reporter gene assay, Fa2N-4 cells, HepaRG cells, and primary human hepatocytes, along with negative controls, using CYP3A4 mRNA and activity endpoints, where appropriate. Over half of the compounds tested (14 of 24) were identified as time-dependent inhibitors of CYP3A4 and high mRNA/activity ratios (>10) were consistent with CYP3A4 time-dependent inhibition for compounds such as troleandomycin, ritonavir, and verapamil. Induction response was compared between two human donors; there was an excellent correlation in the EC(50) estimates (r(2) = 0.89, p < 0.001), and a weak but statistically significant correlation was noted for maximum observed induction at an optimum concentration (E(max)) (r(2) = 0.38, p = 0.001). E(max) and EC(50) estimates determined from the PXR reporter gene assay and Fa2N-4 and HepaRG cells were compared with those from hepatocytes. Overall, EC(50) values generated using hepatocytes agreed with those generated in the PXR reporter gene assay (r(2) = 0.85, p < 0.001) and Fa2N-4 (r(2) = 0.65, p < 0.001) and HepaRG (r(2) = 0.99, p < 0.001) cells. However, E(max) values generated in hepatocytes were only significantly correlated to those determined in Fa2N-4 (r(2) = 0.33, p = 0.005) and HepaRG cells (r(2) = 0.79, p < 0.001). "Gold standard" cytochrome P450 induction data can be generated using primary human hepatocytes, but a restricted, erratic supply and interdonor variability somewhat restrict routine application within a drug discovery setting. HepaRG cells are a valuable recent addition to the armory of in vitro tools for assessing CYP3A4 induction and seem to be an excellent surrogate of primary cells.

The ability to use in vitro human cytochrome P450 (CYP) time-dependent inhibition (TDI) data for in vivo drug-drug interaction (DDI) predictions should be viewed as a prerequisite to generating the data. Important terms in making such predictions are k(inact) and K(I) but first-line screening assays typically involve characterisation of an IC(50) value or a time dependent shift in IC(50). In the work presented here, two key screening methods from the scientific literature were appraised both in terms of practicality and quality of k(inact)/K(I) estimation. The utility of TDI screening data in DDI predictions was investigated and particular reference given to a simple DDI simulation model based on a spreadsheet that calculates the systemic exposure of unbound inhibitor drug following the input of human pharmacokinetic parameters. Using several clinical mechanism-based CYP DDI examples, the effectiveness of the approach was assessed and compared to other widely available approaches (a simple algorithm that employs a single in vivo unbound inhibitor concentration, a seven-compartment physiologically based pharmacokinetic (PBPK) model that defines the extent of interaction as a result of hepatic inhibitor concentrations and the commercially available software SimCYP). All the methods gave predictions that compared favourably with the observed DDIs, but various advantages and disadvantages of each were also given full consideration. The new model facilitates rapid sensitivity analysis (parameters can be easily input and altered to give a visual representation of the impact on the active enzyme concentration) and it was therefore used to derive "rules of thumb" demonstrating the relationship between extent of DDI, time-dependent IC(50) and dose for typical acidic and basic drugs. Additionally, a TDI decision tree linking into reactive metabolite investigations is proposed for use in a Drug Discovery setting.

A series of cytochrome P450 (P450) inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin, and pitavastatin) using hepatocytes and recombinant P450s. The uptake was shown to be temperature-dependent and was inhibited by estrone sulfate, signifying an active component. At the lowest concentrations tested, the inhibitors concentrated up to 1000-fold in rat hepatocytes, but demonstrated only 5-fold greater P450 inhibition relative to recombinant rat P450s, indicating high intracellular binding. Inhibitor accumulation was considerably lower in human hepatocytes and an increase in inhibitory potency relative to recombinant human P450s was not obvious. This study highlights several technical and conceptual issues in the study of P450 inhibition mediated by compounds actively transported across the basolateral hepatocyte membrane. Primarily, the incubation medium concentration once the inhibitor has fully accumulated into the hepatocytes rather than the starting medium concentration, along with the extent of intracellular binding, must be considered as a foundation for in vitro-in vivo extrapolations. Additionally, it is suggested that if the K(m) value for the active uptake process is close to the P450 inhibition K(i), hepatocytes may be used only to establish the free drug accumulation ratio at a clinically relevant drug concentration, and this information, along with the (recombinant P450) K(i) value, may be used to simulate the likely impact of active hepatic uptake on P450 inhibition in vivo.

The disposition of atorvastatin, cerivastatin, and indomethacin, established substrates of rat hepatic basolateral uptake transporters, has been evaluated in suspended rat hepatocytes. Cell and media concentration-time data were simultaneously fitted to a model incorporating active uptake, permeation, binding, and metabolism. Use of the model to estimate the ratio of intracellular to extracellular steady-state free drug concentrations demonstrated the strong influence of active uptake on the kinetics of atorvastatin (18:1) and cerivastatin (8:1), in comparison with indomethacin (3.5:1). Indomethacin, however, was shown to have a higher uptake clearance (599 +/- 101 microl/min/10(6) cells) than atorvastatin (375 +/- 45 microl/min/10(6) cells) and cerivastatin (413 +/- 47 microl/min/10(6) cells). The high passive permeability of indomethacin (237 +/- 63 microl/min/10(6) cells) clearly negated the effect of the active transport on the overall disposition. An analogous physiological model was constructed that allowed prediction of the in vivo pharmacokinetics, including the free intracellular concentration in liver. Hepatic clearance was well predicted by the model, in contrast to predictions based on standard methods. Volume of distribution was well predicted for indomethacin and predicted reasonably for atorvastatin and cerivastatin and higher than might be expected for an acid compound. Furthermore, the terminal half-life predictions for all three compounds were within 2-fold of the observed values. The ability to estimate the free-intracellular hepatic concentration of uptake substrates has major benefits in terms of predicting pharmacokinetics, potential CYP-mediated drug-drug interactions, and efficacy of hepatically targeted therapeutics.

Unbound IC(50) (IC(50,u)) values of 15 drugs were determined in eight recombinantly expressed human cytochromes P450 (P450s) and human hepatocytes, and the data were used to simulate clinical area under the plasma concentration-time curve changes (deltaAUC) on coadministration with prototypic CYP2D6 substrates. Significant differences in IC(50,u) values between enzyme sources were observed for quinidine (0.02 microM in recombinant CYP2D6 versus 0.5 microM in hepatocytes) and propafenone (0.02 versus 4.1 microM). The relative contribution of individual P450s toward the oxidative metabolism of clinical probes desipramine, imipramine, tolterodine, propranolol, and metoprolol was estimated via determinations of intrinsic clearance using recombinant P450s (rP450s). Simulated deltaAUC were compared with those observed in vivo via the ratios of unbound inhibitor concentration at the entrance to the liver to inhibition constants determined against rP450s ([I](in,u)/K(i)) and incorporating parallel substrate elimination pathways. For this dataset, there were 20% false negatives (observed deltaAUC >or= 2, predicted deltaAUC < 2), 77% correct predictions, and 3% false positives. Thus, the [I](in,u)/K(i) approach appears relatively successful at estimating the degree of clinical interactions and can be incorporated into drug discovery strategies. Using a Simcyp ADME (absorption, metabolism, distribution, elimination) simulator (Simcyp Ltd., Sheffield, UK), there were 3% false negatives, 94% correct simulations, and 3% false positives. False-negative predictions were rationalized as a result of mechanism-based inhibition, production of inhibitory metabolites, and/or hepatic uptake. Integrating inhibition and reaction phenotyping data from automated rP450 screens have shown applicability to predict the occurrence and degree of in vivo drug-drug interactions, and such data may identify the clinical consequences for candidate drugs as both "perpetrators" and "victims" of P450-mediated interactions.

A novel series of antagonists of the human P2X7 receptor is described. Modification of substituents enabled identification of compounds selective for the rat P2X7 receptor and provides useful pharmacological tools for evaluation of the role of P2X7 in disease.

Starting from adenosine triphosphate (ATP), the identification of a novel series of P2Y(12) receptor antagonists and exploitation of their SAR is described. Modifications of the acidic side chain and the purine core and investigation of hydrophobic substituents led to a series of neutral molecules. The leading compound, 17 (AZD6140), is currently in a large phase III clinical trial for the treatment of acute coronary syndromes and prevention of thromboembolic clinical sequelae.

The importance of hepatic uptake transporters in drug clearance is well recognized. The subject is reviewed with the intention of providing an overview of the concepts in order to link the increasing knowledge of transporter-mediated uptake into established models of hepatic clearance. In order to understand and quantify their impact, models of hepatic elimination that incorporate permeability barriers are required. Models that include both active and passive uptake into hepatocytes are discussed and simulations of the influence of active uptake and passive diffusion on hepatic clearance are presented. The advantages and weaknesses of a number of in vitro assays of hepatic uptake are described, and their ability to predict hepatic clearance is reviewed.

In this article approaches to predict human pharmacokinetics (PK) are discussed and the capability of the exemplified methodologies to estimate individual PK parameters and therapeutic dose for a set of marketed oral drugs has been assessed. For a set of 63 drugs where the minimum efficacious concentration (MEC) and human PK were known, the clinical dose was shown to be well predicted or in some cases over-estimated using a simple one-compartment oral PK model. For a subset of these drugs, in vitro potency against the primary human targets was gathered, and compared to the observed MEC. When corrected for plasma protein binding, the MEC of the majority of compounds was < or=3 fold over the respective in vitro target potency value. A series of in vitro and in vivo experiments were conducted to predict the human PK parameters. Metabolic clearance was generally predicted well from human hepatocytes. Interestingly, for this compound set, allometry or glomerular filtration rate (GFR) ratio methods appeared to be applicable for renal CL even where CL(renal) > GFR. For approximately 90% of compounds studied, the predicted CL using in vitro-in vivo (IVIV) extrapolation together with a CL(renal) estimate, where appropriate, was within 2-fold of that observed clinically. Encouragingly volume of distribution at steady state (V(ss)) estimated in preclinical species (rat and dog) when corrected for plasma protein binding, predicted human V(ss) successfully on the majority of occasions--73% of compounds within 2-fold. In this laboratory, absorption estimated from oral rat PK studies was lower than the observed human absorption for most drugs, even when solubility and permeability appeared not to be limiting. Preliminary data indicate absorption in the dog may be more representative of human for compounds absorbed via the transcellular pathway. Using predicted PK and MEC values estimated from in vitro potency assays there was a good correlation between predicted and observed dose. This analysis suggests that for oral therapies, human PK parameters and clinical dose can be estimated from a consideration of data obtained from in vitro screens using human derived material and in vivo animal studies. The benefits and limitations of this holistic approach to PK and dose prediction within the drug discovery process are exemplified and discussed.

The wealth of information that has emerged in recent years detailing the substrate specificity of hepatic transporters necessitates an investigation into their potential role in drug elimination. Therefore, an assay in which the loss of parent compound from the incubation medium into hepatocytes ("media loss" assay) was developed to assess the impact of hepatic uptake on unbound drug intrinsic clearance in vivo (CL(int ub in vivo)). Studies using conventional hepatocyte incubations for a subset of 36 AstraZeneca new chemical entities (NCEs) resulted in a poor projection of CL(int ub in vivo) (r2 = 0.25, p = 0.002, average fold error = 57). This significant underestimation of CL(int ub in vivo) suggested that metabolism was not the dominant clearance mechanism for the majority of compounds examined. However, CL(int ub in vivo) was described well for this dataset using an initial compound "disappearance" CL(int) obtained from media loss assays (r2 = 0.72, p = 6.3 x 10(-11), average fold error = 3). Subsequent studies, using this method for the same 36 NCEs, suggested that the active uptake into human hepatocytes was generally slower (3-fold on average) than that observed with rat hepatocytes. The accurate prediction of human CL(int ub in vivo) (within 4-fold) for the marketed drug transporter substrates montelukast, bosentan, atorvastatin, and pravastatin confirmed further the utility of this assay. This work has described a simple method, amenable for use within a drug discovery setting, for predicting the in vivo clearance of drugs with significant hepatic uptake.