Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC 50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC 50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC 50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members.

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.

Although there is considerable variation in the effect of age on drug biotransformation, the metabolism of many drugs is impaired in the elderly. Age-related physiological changes, such as a reduction in liver mass, hepatic metabolising enzyme activity, liver blood flow and alterations in plasma drug binding may account for the decreased elimination of some metabolised drugs in the elderly. It is difficult, however, to separate an effect of aging from a background of marked variation in the rate of metabolism due to factors such as individual metabolic phenotype, environmental influences, concomitant disease states and drug intake. The prevailing data suggest that initial doses of metabolised drugs should be reduced in older patients and then modified according to the clinical response. In most studies the elderly appear as responsive as young individuals to the effects of compounds which induce or inhibit the activity of cytochrome P450 isozymes. Concurrent use of other agents, which induce or inhibit drug metabolism, mandates dose adjustment as in younger patients. Many questions remain unanswered. For instance, limitations of in vitro studies prevent any firm conclusion about changes in hepatic drug metabolising enzyme activity in the elderly. With aging, some pathways of drug metabolism may be selectively affected, but this has not been adequately scrutinised. The possibility that metabolism of stereoisomers may be altered in the elderly has not been adequately tested. The effect of aging on the distribution of polymorphic drug metabolism phenotypes is still not established, despite potential implications for disease susceptibility and survival advantage.