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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.

Introduction Building an understanding of in vivo efficacy based on the evaluation of in vitro affinity or potency is critical in expediting early decision making in drug discovery and can significantly reduce the need for animal studies. The aim of the present study was to understand the translation of in vitro to in vivo endpoints for the cannabinoid receptor 1 (CB1). Methods Using a selection of CB1 agonists we describe an evaluation of in vitro to in vivo translation comparing in vitro receptor affinity or functional potency, using both cAMP and β-arrestin endpoints, to various in vivo CB1 agonist-associated endpoints. Results We demonstrate that in vitro CB1 agonism significantly correlates with the CB1-induced cue in the drug discrimination model in vivo, but not with other purported CB1 agonist-mediated in vivo endpoints, including hypothermia and sedation. Thus, these data challenge common perceptions regarding CB1 agonist-induced tetrad effects in rodents. Discussion This work exemplifies how in vitro profiling of receptor affinity or potency can predict in vivo pharmacodynamic effects, using the CB1 as an example system. The translatability of in vitro activity to in vivo efficacy allows for the ability to rapidly contextualize off-target CB1 in vitro findings, allowing clear and rapid definition of the risk posed by such activity without the need for extensive animal studies. This has significant implications in terms of early decision making in drug discovery and reducing the use of animals in research, while also outlining a template for expanding the approach for additional targets.

A 1,2,4-triazole motif was employed as a bioisostere for the ester commonly used in muscarinic antagonists, and subsequent integrative conjugation to a β2 agonist quinolinone furnished a new class of bifunctional MABAs for the treatment of COPD. Medicinal chemistry optimization using the principles of 'inhalation by design' furnished a clinical candidate with desirable pharmacological, pharmacokinetic and biopharmaceutical properties.

This study describes the analysis of DNA from heat-killed (boilate) isolates of Mycobacterium tuberculosis from two UK outbreaks where DNA was of sub-optimal quality for the standard methodologies routinely used in microbial genomics. An Illumina library construction method developed for sequencing ancient DNA was successfully used to obtain whole genome sequences, allowing analysis of the outbreak by gene-by-gene MLST, SNP mapping and phylogenetic analysis. All cases were spoligotyped to the same Haarlem H1 sub-lineage. This is the first described application of ancient DNA library construction protocols to allow whole genome sequencing of a clinical tuberculosis outbreak. Using this method it is possible to obtain epidemiologically meaningful data even when DNA is of insufficient quality for standard methods.

COPD is a major cause of mortality in the western world. A(2A) agonists are postulated to reduce the lung inflammation that causes COPD. The cardiovascular effects of A(2A) agonists dictate that a compound needs to be delivered by inhalation to be therapeutically useful. A strategy of minimizing side-effect liability by maximizing systemic clearance was followed and pharmacological and pharmacokinetic SAR of a series of inhaled A(2A) agonists described. A sevenfold improvement in potency and 150-fold reduction in side-effect liability over the lead compound CGS-21680, were obtained.

The design and profile of a series of saligenin containing long acting β2-adrenoreceptor agonists is described. Evaluation of these analogues using a guinea-pig tissue model demonstrates that analogues within this series have significantly longer durations of action than salmeterol and have the potential for a once daily profile in human.