Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HHEP) are two common in vitro systems used in metabolic stability and inhibition studies. The comparison between the assays using the two systems can provide mechanistic insights on the interplay of metabolism, passive permeability and transporters. This study investigated the critical factors impacting the unbound intrinsic clearance (CLint,u) and IC50 of CYP3A inhibition between HLM and HHEP. The HLM/HHEP CLint,u ratio and HHEP/HLM IC50 ratio are inversely correlated to passive permeability, but have no correlation with P-gp efflux ratio. Cofactor-supplemented permeabilized HHEP (MetMax™) collapses the IC50 differences between HHEP and HLM. P-gp inhibitor, encequidar, shows minimal impact on CLint,u and IC50 in HHEP. This is the first study that is able to separately investigate the effects of passive permeability and efflux transport. These data collectively show that passive permeability plays a critical role in metabolism and enzyme inhibition in HHEP, while P-gp efflux has a minor role. This may be due to low functional P-gp activity in suspension HHEP under the assay conditions. Low passive permeability may limit metabolism and enzyme inhibition in HHEP, leading to lower CLint,u and higher IC50 in HHEP compared to HLM. When liver microsomes give higher CLint,u than hepatocytes, microsomes are more predictive of in vivo clearance than hepatocytes.

Quantitative Contribution of Six Major Transporters to the Hepatic Uptake of Drugs: "SLC-Phenotyping" Using Primary Human Hepatocytes.

Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using single-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 µM) inhibits three OATPs, while rifampicin (5 µM) inhibits OATP1B1/1B3 only. Further, hepatitis B virus myristoylated-preS1 peptide (0.1 µM), quinidine (100 µM), and ketoprofen (100-300 µM) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (ft ) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 1B/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/1B3 contribution, respectively. OCT1-mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro ft values were corrected using quantitative proteomics data to obtain "scaled ft " Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/1B3 ft was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting.

In Vitro-In Vivo Extrapolation of Key Transporter Activity at the Blood-Brain Barrier.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.

Development of a high-speed, multiplexed sample-delivery instrument for LC-MS/MS bioanalysis.

BACKGROUND: The number of new chemical entities and types of in vitro and in vivo samples that require bioanalysis in drug discovery is large and diverse. In addition, method development time is limited as data turnaround is the highest priority. These circumstances require that a well-defined set of bioanalysis options be available in short timeframes to triage samples for analysis. METHOD: The Apricot Designs Dual Arm (ADDA) instrument is an LC-MS/MS sample delivery system that features a flexible hardware design coupled with software automation to enhance throughput in LC-MS/MS bioanalysis drug discovery. The instrument can perform high-throughput LC-MS/MS (8-10 s/sample) for screening and in vitro bioanalysis, as well as multiplexed LC for traditional gradient or isocratic LC approaches. The instrument control software is designed to integrate with DiscoveryQuant™ software (AB Sciex) and a global database of MS/MS conditions. CONCLUSION: Development of the sample delivery platform and its application in high-throughput and gradient LC will be described.

Impact of recovery on fraction unbound using equilibrium dialysis.

Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.

Development of a new permeability assay using low-efflux MDCKII cells.

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

Species independence in brain tissue binding using brain homogenates.

Species independence of brain tissue binding was assessed with a large number of structurally diverse compounds using equilibrium dialysis with brain homogenates of seven species and strains (Wistar Han rat, Sprague-Dawley rat, CD-1 mouse, Hartley guinea pig, beagle dog, cynomolgus monkey, and human). The results showed that the fractions unbound of the seven species and strains were strongly correlated with correlation coefficients ranging from 0.93 to 0.99. The cross-species/strain correlations were not significantly different from the interassay correlation with the same species. The linear correlation between Wistar Han and other species had a slope close to 1 and an intercept near 0. Based on orthogonal statistical analysis, no correction is needed for extrapolation of fraction unbound from Wistar Han rat to the other species or strains. Hence, brain tissue binding of Wistar Han rat can be used to obtain binding of other species and strains in drug discovery.

Pharmacokinetic-pharmacodynamic modelling of the effect of Moxifloxacin on QTc prolongation in telemetered cynomolgus monkeys.

INTRODUCTION: Delayed ventricular repolarisation is manifested electrocardiographically in a prolongation of the QT interval. Such prolongation can lead to potentially fatal Torsades de Pointes. Moxifloxacin is a fluoroquinolone antibiotic which has been associated with QT prolongation and, as a result, is recommended by the regulatory authorities as a positive control in thorough QT studies performed to evaluate the potential of new chemical entities to induce QT prolongation in humans. The sensitivity of the cynomolgus monkey as a quantitative preclinical predictor of the PK-QTc relationship is discussed. METHODS: Cardiovascular monitoring was performed in the telemetered cynomolgus monkey for 22 h following oral administration of Moxifloxacin (10, 30 and 90 mg/kg) or placebo. QTc was derived using an individual animal correction factor (ICAF): RR-I = QT-I--(RR-550)* (IACF). A PKPD analysis was performed to quantify the increase in placebo-adjusted QTc) elicited by administration of Moxifloxacin. In addition, the rate of onset of hERG channel blockade of Moxifloxacin was compared to Dofetilide by whole cell patch clamp technique in HEK-293 cells stably expressing the hERG channels. RESULTS: Moxifloxacin induced a dose dependent increase in QTc). A maximum increase of 28 ms was observed following administration of 90 mg/kg Moxifloxacin. The corresponding maximum free systemic exposure was 18µM. Interrogation of the PK-QTc relationship indicated a direct relationship between the systemic exposure of Moxifloxacin and increased QTc. A linear PKPD model was found to describe this relationship whereby a 1.5 ms increase in QTc was observed for every 1 µM increase in free systemic exposure. DISCUSSION: The exposure dependent increases in QTc observed following oral administration of Moxifloxacin to the cynomolgus monkey are in close agreement with those previously reported in human subjects. A direct effect linear relationship was found to be conserved in both species. As a result of the quantitative agreement in both species, the utility of the telemetered cynomolgus monkey as a preclinical predictor of QTc) prolongation is exemplified. Furthermore, the rate of onset of hERG channel blockade observed in patch clamp offers a mechanistic insight into the relative rates of channel blockade observed in vivo with both Moxifloxacin and Dofetilide.

Strategic use of plasma and microsome binding to exploit in vitro clearance in early drug discovery.

Apparent intrinsic clearance (CLia) determined from microsomal stability assays is a cornerstone in drug discovery. Categorical bins are routinely applied to this end point to facilitate analysis. However, such bins ignore the interdependent nature of apparent intrinsic microsome clearance on several ADME parameters. Considering CLia as a determinant for both metabolic stability and potential dose is more appropriate. In this context with proper accounting for nonspecific binding to microsomes and plasma, consideration of compounds with higher CLia may be warranted. The underlying benefit is the potential increase in the number of hits or chemical diversity for evaluation during the early stages of programs.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

A new chemical tool for exploring the role of the PDE4D isozyme in leukocyte function.

Nicotinamide (2) is a potent and selective inhibitor of the PDE4D isozyme and as a chemical tool selectively blocks eosinophil mediator release and chemotaxis thus linking the role of PDE4D to eosinophil function.

Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase.

Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states.