Ayşe Ufuk, Norikazu Matsunaga, David Hallifax, Aleksandra Galetin
Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom
Drug-drug interactions (DDIs) associated with lysosomal sequestration have been postulated in addition to potential therapeutic or adverse consequences of this process. Despite lysosomotropic properties of both CYP2D substrates and inhibitors, there are currently no data on the role lysosomal sequestration in metabolic CYP2D6 DDIs. Therefore, the current study investigated the importance of lysosomal sequestration on cellular disposition of the basic drug desipramine (CYP2D6 substrate) in the absence and presence of quinine, quinidine and fluoxetine (CYP2D inhibitors) and ammonium chloride (NH4Cl) (abolishes lysosome-cytosol pH gradient) in rat and human hepatocytes (both poor (PM) and extensive (EM) CYP2D6 metabolisers). Desipramine cell-to-medium ratio (Kp) was measured over a concentration range (0.1-300 µM) in plated rat and human hepatocytes over 45 and 90 minutes, respectively in the absence and presence of agents. Concentrations of desipramine and its major metabolite 2-OH desipramine were monitored by LC‑MS/MS. In rat hepatocytes, desipramine Kp increased over low concentration range to reach its maximum (Kp,max) of 300 at 3 µM and declined to ~13 (Kp,min, attributed to its membrane partitioning) at the highest drug concentration. However, in the presence of quinine, desipramine Kp was maximal (400) at 0.1 µM, highlighting the impact of desipramine metabolic elimination in determining its Kp at low drug concentrations. Increase in lysosomal pH by NH4Cl (20 mM) did not affect either Kp or metabolic clearance (CLmet) of desipramine at low concentrations, but reduced its Kp,max by 2-fold.
In contrast to rat hepatocytes, desipramine cellular disposition was different in EM human hepatocytes, as metabolism by CYP2D6 did not affect desipramine Kp at low concentrations (Kp,max of 386 achieved at 0.1 µM). This finding is supported by 6.5-fold lower desipramine CLmet in EM human hepatocytes compared with rat hepatocytes (15.9 vs. 103 µL/min/mg protein). Among CYP2D inhibitors investigated, fluoxetine caused the most pronounced reduction in desipramine Kp,max in both rat and human hepatocytes (up to 55%), in agreement with its greater membrane partitioning than quinine and quinidine. Rank order in the observed reduction of desipramine Kp,max by the agents investigated did not differ between CYP2D6 EM and PM human hepatocytes. The findings of this study suggest that lysosomal sequestration of CYP2D inhibitors has minimal influence on desipramine metabolic DDIs associated with this enzyme. However, it highlights important species differences in the interplay between metabolism and lysosomal sequestration and subsequently desipramine cellular disposition which need to be considered when evaluating CYP2D6 DDI potential.