Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.