In vitro assessment of the interactions of dopamine β-hydroxylase inhibitors with human P-glycoprotein and Breast Cancer Resistance Protein

Inhibition of the biosynthesis of noradrenaline is a currently explored strategy for the treatment of hypertension, congestive heart failure and pulmonary arterial hypertension. While some Dopamine β-hydroxylase (DBH) inhibitors cross the blood-brain barrier (BBB) and cause central as well as peripheral effects (nepicastat), Others have limited access to the brain (etamicastat, zamicastat). In this context, peripheral selectivity is clinically advantageous, in order to prevent alterations of noradrenaline levels in the CNS and the occurrence of adverse central effects. A limited brain exposure results from the combination of several factors, such as a reduced passive permeability or affinity for efflux transporters, but efflux liabilities may also lead to unwanted drug-drug interactions (DDIs) in the presence of co-administered substrates or inhibitors. Thus, the purpose of the study herein presented was to explore the interaction of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), the two major efflux transporters of the BBB that hamper the entry of several drugs to the brain, with the DBH inhibitors, etamicastat, nepicastat and zamicastat. Madin-Darby canine kidney cells (MDCK II) and transfected lines with human MDR1 (MDCK-MDR1) and ABCG2 (MDCK-BCRP) genes were used as a BBB surrogate model. P-gp and BCRP substrates and/or inhibitors were identified through intracellular accumulation and bidirectional permeability assays. The obtained data revealed that zamicastat is a concentration-dependent dual P-gp and BCRP Inhibitor with IC₅₀ values of 73.8 ± 7.2 μM and 17.0 ± 2.7 μM, while etamicastat and nepicastat inhibited BCRP to greater extent than P-gp, with IC₅₀ values of 47.7 ± 1.8 μM and 59.2 ± 9.4 μM, respectively. Additionally, etamicastat was identified as P-gp and BCRP dual substrate, as demonstrated by net flux ratios of 5.84 and 3.87 and decreased >50% by verapamil and Ko143. Conversely, nepicastat revealed to be a P-gp-only substrate, with a net flux ratio of 2.01, reduced to 0.92 in the presence of verapamil. Furthermore, nepicastat displayed a consistently higher apparent permeability (>8.49 × 10^-6 cm s^-1) than etamicastat (<0.58 × 10^-6 cm s^-1). The identification of etamicastat as a dual efflux substrate suggests that P-gp and BCRP may be partially responsible for the limited central exposure of this compound, in association with its low passive permeability. Moreover, the weak efflux inhibitory potencies of etamicastat and nepicastat revealed a low DDI risk, while the dual P-gp/BCRP inhibition of zamicastat could be studied in the future with synergically effluxed compounds, for which BBB penetration is severely impaired.