br Methods br Results br Discussion
Discussion Skin biopsy and microneurography have both been described as useful tools in the diagnosis of PAN, directly identifying peripheral sympathetic fibers without the mediation of specific bcl-2 family organs (Donadio et al., 2005, Donadio et al., 2012a, Donadio and Liguori, 2015, Giannoccaro et al., 2011). In the majority of PAN patients, microneurography failed to disclose sympathetic outflow in either skin or muscle branches, whereas skin biopsy disclosed adrenergic and cholinergic abnormalities supporting a non-selective and widescale peripheral autonomic denervation (Donadio et al., 2012a, Donadio and Liguori, 2015) as described in our control PAN control patient. Furthermore, autonomic adrenergic fibers showed a poor but preserved staining even when PAN was associated with a prevalent adrenergic failure (Guaraldi et al., 2011). By contrast, in our patient with chronic OH microneurography showed increased MSNA, as previously described (Thompson et al., 1995), suggesting structurally preserved sympathetic adrenergic postganglionic fibers and the presence of a possible compensatory mechanism counteracting the lack of adrenergic function underlying OH whereas sympathetic sudomotor outflow to skin was in the normal range. Accordingly, skin biopsy displayed richly innervated SG and preserved staining of adrenergic fibers around the APM using a pan-neuronal marker (i.e. PGP) suggesting the structural integrity of these fibers, but DβH staining was absent supporting a selective adrenergic dysfunction. Although we have not used a specific marker of APM adrenergic fibers (i.e. TH) the PGP staining likely expressed adrenergic fibers since: 1) the nearly totality of APM fibers are usually adrenergic (Donadio et al., 2006); 2) fibers stained by PGP showed the classical wavy pattern typical of adrenergic fibers (Fig. 1B). A careful analysis of APM innervation identified VIP positive fibers in muscles of the proband with a similar rate of control and control patient. These data suggested that VIP pilomotor fibers are expressed in a subset of pilomotor fibers and are functionally independent form DbH pathway as previously reported (Nolano et al., 2010) although their exact role remains to be defined.
Conflicts of interest
Introduction P-glycoprotein (P-gp; ABCB1) and Breast Cancer Resistance Protein (BCRP; ABCG2) are efflux transporters from the ATP-binding cassette family with broad and partly overlapping substrate specificity (Xia et al., 2005). Their widespread and privileged location in several tissues greatly influences the absorption, distribution and elimination of endogenous compounds and structurally diverse drugs (Chen et al., 2016). At the blood-brain barrier (BBB) of rodents and humans, P-gp and BCRP cooperatively contribute to efflux xenobiotics from cerebral endothelial cells, and when one is inhibited, the other compensates its function (Agarwal et al., 2011; Bauer et al., 2016; Kodaira et al., 2010). Hence, identifying P-gp and BCRP substrates and/or inhibitors is not only important to comprehend brain exposure, but also to investigate the occurrence of drug-drug interactions (DDIs) that may compromise clinical efficacy and safety. Thus, in vitro assays are recommended by the Food and Drug Administration and European Medicines Agency (EMA) to evaluate P-gp and BCRP-mediated efflux (European Medicines Agency, 2012; Food and Drug Administration, 2012). In this context, the main objective of the present work was to investigate the interaction of three dopamine β-hydroxylase (DBH) inhibitors with P-gp and BCRP. DBH catalyses the conversion of dopamine into noradrenaline in the catecholamine biosynthetic pathway and its inhibitors have been developed for the treatment of resistant hypertension, congestive heart failure and pulmonary arterial hypertension, associated with an overactivity of the sympathetic nervous system (Bonifácio et al., 2015; Igreja et al., 2017). First and second generation DBH inhibitors did not reach the market due to low potency, poor selectivity and severe adverse effects (Bonifácio et al., 2015). Despite having demonstrated greater potency, nepicastat, a third-generation inhibitor, altered noradrenaline and dopamine levels in the cerebral cortex, indicating that it crosses the BBB, which could lead to undesired central effects (Beliaev et al., 2009). Thus, peripheral selectivity is an important clinical requirement for the development of DBH inhibitors, as the decrease of noradrenaline levels should occur at the periphery in sympathetically enervated tissues, thereby avoiding potentially serious side-effects in the CNS (Almeida et al., 2013). In contrast, etamicastat is a reversible DBH inhibitor with limited access to the brain. It decreases peripheral noradrenaline levels in sympathetically innervated tissues, without affecting noradrenaline levels in parietal and frontal cortex (Beliaev et al., 2006). Similarly, zamicastat also reversibly inhibits DBH without effect in brain tissue (Igreja et al., 2012).