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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.

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.

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 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.

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.

The synthesis and the in vitro properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors is described. Despite bearing structural similarity to rolipram, several of these compounds have much reduced affinity for the [3H]rolipram binding site.

The synthesis and biological properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors are described. These catechol benzimidazoles were designed from rolipram and initial compounds reflected a similarly high affinity for the [ 3 H]rolipram b binding site (500 to 1000X greater affinity for the [ 3 H]rolipram binding site over the PDE IV inhibitory site). However, SAR studies on the 3-alkoxy position revealed that this [ 3 H]rolipram binding site affinity could be attenuated, while potentiating the PDE IV inhibitory activity. This resulted in the 2-indanyl analog 13 which is a potent, selective PDE IV inhibitor with a 15X differential in favor of PDE IV binding.

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.