Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.

In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Aberrant signaling by transforming growth factor-b (TGF-b) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-b signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-b signaling via ALK5 plays a critical role in maintaining heart valve integrity.

Indole-3-carbinol (I3C) and 3,3-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr 1 ), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr 1 , and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr 1 were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.

ABSTRACT:3,3-Diindolylmethane (DIM) is a naturally occurring indole, which is currently under investigation as a potential chemopreventive agent. The concentrations of DIM in plasma, liver, kidney, lung, heart, and brain tissues were determined following oral administration of two different formulations to mice (250 mg/kg). Mice were sacrificed periodically from 0 to 24 h after administration of either a crystalline or an absorption-enhanced formulation (BioResponse-DIM; Indolplex) of DIM, and plasma and tissue concentrations were determined by high-performance liquid chromatography (UV detection, 280 nm). A physiologically based pharmacokinetic (PBPK) model was developed to characterize the pharmacokinetic properties of the two different formulations. The final model included parameters reflecting linear firstorder absorption, systemic clearance, and distributional clearance in the remainder compartment, which were considered independent of formulation. All pharmacokinetic profiles from the two formulations were fitted simultaneously to estimate unknown model parameters. Plasma and tissue concentrationtime profiles exhibited a rapid rise to peak values at 0.5 to 1 h, followed by a polyexponential decline with an extended terminal phase. These profiles were well described by the final model and unknown parameters were estimated with relatively low coefficients of variation. Relative drug exposure and absorption parameters suggest that BioResponse-DIM exhibited approximately 50% higher bioavailability than the crystalline formulation. Clearance of DIM was estimated as 7.18 ml/h. This is the first study to characterize the pharmacokinetics of DIM in mice, and the established PBPK model should prove useful in the design and analysis of future preclinical studies aimed at evaluating the in vivo pharmacological effects of DIM.

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.

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