Role of Biotransformation Studies in Minimizing Metabolism-Related Liabilities in Drug Discovery

Metabolism-related liabilities continue to be a major cause of attrition for drug candidates in clinical development. Such problems may arise from the bioactivation of the parent compound to a reactive metabolite capable of modifying biological materials covalently or engaging in redox-cycling reactions leading to the formation of other toxicants. Alternatively, they may result from the formation of a major metabolite with systemic exposure and adverse pharmacological activity. To avert such problems, biotransformation studies are becoming increasingly important in guiding the refinement of a lead series during drug discovery and in characterizing lead candidates prior to clinical evaluation. This article provides an overview of the methods that are used to uncover metabolism-related liabilities in a pre-clinical setting and offers suggestions for reducing such liabilities via the modification of structural features that are used commonly in drug-like molecules

CYP2C9 protein interactions with cytochrome b5: Effects on the coupling of catalysis

The hemoprotein cytochrome b5 (cyt b5) has been demonstrated to affect the kinetics of drug oxidation by the microsomal cytochromes P450. However, the mechanisms through which cyt b5 exerts these effects are variable and P450 isoform-dependent. While the effects of cyt b5 on the major drug metabolizing enzymes CYP2D6, CYP2E1, and CYP3A4 are well studied, fewer studies conducted over limited ranges of cyt b5 concentrations have been performed on CYP2C9. In the present study with CYP2C9, cyt b5 exerted complex actions upon P450 oxidative reactions by affecting the rate of metabolite formation, the consumption of NADPH by cytochrome P450 reductase, and uncoupling of the reaction cycle to hydrogen peroxide and water. Cytochrome b5 devoid of the heme moiety (apo-b5) exhibited similar effects as native cyt b5. All rates were highly dependent on the cyt b5 to CYP2C9 enzyme ratio suggesting that the amount of cyt b5 present in an in vitro incubation is an important factor that can impact the reliability of extrapolating in vitro generated data to predict the in vivo condition. The major effects of cyt b5 are hypothesized to result from a cyt b5 induced conformational change in CYP2C9 that results in an increased collision frequency between the iron-oxygen species (Cpd I) and the substrate, and a decrease in the oxidase activity. Together, these findings suggest that cyt b5 can alter multiple steps in the P450 catalytic cycle via complex interactions with P450 and P450 reductase

Role for the M₁ Muscarinic Acetylcholine Receptor in Top-Down Cognitive Processing Using a Touchscreen Visual Discrimination Task in Mice

The M₁ muscarinic acetylcholine receptor (mAChR) subtype has been implicated in the underlying mechanisms of learning and memory and represents an important potential pharmacotherapeutic target for the cognitive impairments observed in neuropsychiatric disorders such as schizophrenia. Patients with schizophrenia show impairments in top-down processing involving conflict between sensory-driven and goal-oriented processes that can be modeled in preclinical studies using touchscreen-based cognition tasks. The present studies used a touchscreen visual pairwise discrimination task in which mice discriminated between a less salient and a more salient stimulus to assess the influence of the M₁ mAChR on top-down processing. M₁ mAChR knockout (M₁ KO) mice showed a slower rate of learning, evidenced by slower increases in accuracy over 12 consecutive days, and required more days to acquire (achieve 80% accuracy) this discrimination task compared to wild-type mice. In addition, the M₁ positive allosteric modulator BQCA enhanced the rate of learning this discrimination in wild-type, but not in M₁ KO, mice when BQCA was administered daily prior to testing over 12 consecutive days. Importantly, in discriminations between stimuli of equal salience, M₁ KO mice did not show impaired acquisition and BQCA did not affect the rate of learning or acquisition in wild-type mice. These studies are the first to demonstrate performance deficits in M₁ KO mice using touchscreen cognitive assessments and enhanced rate of learning and acquisition in wild-type mice through M₁ mAChR potentiation when the touchscreen discrimination task involves top-down processing. Taken together, these findings provide further support for M₁ potentiation as a potential treatment for the cognitive symptoms associated with schizophrenia

Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo