bms-690514 br Introduction Nausea and vomiting are among

Introduction Nausea and vomiting are among the most distressing side effects associated with chemotherapy in cancer patients (Billio et al., 2010). Severe emesis can negatively affect a patient's nutritional state, ability to work and motivation, which can, in turn, interfere with the clinical course of the disease and with the patient's acceptance of chemotherapy (Colagiuri et al., 2008). It is therefore important to minimize the risk of nausea and vomiting with each chemotherapy treatment. However, antiemetic drugs also produce adverse effects, so patients should use the lowest dose that provides relief (Marty et al., 1990). The serotonin (5-hydroxytryptamine) type 3 (5-HT3) receptor antagonists are considered the gold standard in treating chemotherapy-induced nausea and vomiting (Billio et al., 2010). 5-HT3 receptors are transmembrane ligand-gated ion channels that are responsible for fast neurotransmission in the nervous systems (Lester et al., 2004). The 5-HT3 receptor complex comprises five subunits surrounding a cation (Na+, Ca2+, K+)-permeable channel pore (Davies et al., 1999, Green et al., 1995, Lummis et al., 2005). The human genome contains five genes encoding different 5-HT3 subunits (5-HT3A–E) (Niesler et al., 2003), and the main subunits involved in the formation of functional receptors are 5-HT3A and 5-HT3B (Niesler et al., 2007). 5-HT3A subunits can form a functional homomeric channel, whereas 5-HT3B subunits cannot (Davies et al., 1999), and instead achieve functionality by forming heteromeric complexes with 5-HT3A subunits, in a proposed stoichiometry of 2A:3B (Barrera et al., 2005, Davies et al., 1999). Some polymorphisms are found in the 5-HT3B subunit. The expression of the subunit may depend on polymorphisms in its promoter region (Tremblay et al., 2003). Moreover, polymorphisms in 5-HT3 receptors are also associated with different risks for chemotherapy-induced vomiting (Sugai et al., 2006). 5-HT3 receptors are located in the gastrointestinal tract, the area postrema of the chemoreceptor trigger zone, and the nucleus of the solitary tract in the emetic complex (Champaneria et al., 1992, Kilpatrick et al., 1989, Pratt and Bowery, 1989). Following exposure to cytotoxic drugs, serotonin is released from enterochromaffin bms-690514 in the mucosa of the small intestine adjacent to vagal afferent neurons expressing 5-HT3 receptors (Andrews et al., 1988). The released 5-HT activates these neurons via the 5-HT3 receptors, leading to a severe emetic response mediated by the medial solitary nucleus (Tyers and Freeman, 1992). Many 5-HT3 receptor antagonists, such as ondansetron, granisetron and palonosetron, are commonly used (Billio et al., 2010), but the efficacy of the 5-HT3 receptor antagonist is not maintained during consecutive cycles (de Wit et al., 1996). We have recently shown that the anticancer drugs irinotecan and topotecan directly modify the 5-HT-mediated 5-HT3 receptor current in vitro experiment (Nakamura et al., 2013, Nakamura et al., 2011). In addition, the modification depends on the subunit and its nonsynonymous polymorphism, which may explain the variation between patients in emetic response to chemotherapy. In the present study, we conducted a global analysis of frequently used anticancer drugs to explore their direct modification of 5-HT3A and 5-HT3AB receptor currents.
Materials and methods
Results The risk of nausea and vomiting after administration of an anticancer drug is graded depending on the proportion of patients who would experience emesis without antiemetic prophylaxis, as follows: high (> 90%), moderate (30–90%), low (10–30%), and minimal (< 10%) using MASCC and ESMO guideline and ASCO guideline for antiemetics in oncology (Hesketh et al., 2017, Roila et al., 2016). We focused here on 35 anticancer drugs across the range of emetic risk (Table 1) (Herrstedt and Dombernowsky, 2007). To examine the action of each drug at the 5-HT3 receptor, human 5-HT3A or 5-HT3A/5-HT3B subunits were expressed in Xenopus laevis oocytes to make homopentameric 5-HT3A or heteropentameric 5-HT3AB receptors, for recording using the two-electrode voltage clamp technique. None of the drugs alone activated the 5-HT3A or 5-HT3AB receptors (data not shown), which showed that they were not agonists for either receptor subtype. We then determined the effect of the drugs on 5-HT-induced receptor current, by applying the drugs in the presence of 2µM 5-HT (the EC20), which enabled measurement of the inhibition and potentiation of 5-HT3 receptor current. In the global analysis, modulation was defined as a > 50% change in current at high drug concentration (100µM). A marked change was observed with 10 of the 35 drugs, including irinotecan and topotecan, which we reported previously (Table 2) (Nakamura et al., 2013, Nakamura et al., 2011). The remaining 25 drugs had no appreciable effect on the 5-HT-induced 5-HT3A or 5-HT3AB receptor currents (Table 1). We then focused on the 10 drugs that modulated the currents. Representative responses and dose-response curves are shown in Fig. 1, Fig. 2, respectively. The 5-HT-induced 5-HT3A receptor current was inhibited by sunitinib, irinotecan, idarubicin, imatinib, doxorubicin, epirubicin, daunorubicin, gefitinib, topotecan and mitoxantrone (Fig. 2 and Table 3), although these effects are weaker than granisetron (IC50 0.0137, LogIC50 7.87 ± 0.25 5-HT3A), one of 5-HT3 receptor specific antagonist. The inhibitory effect of irinotecan on the 5-HT3A receptor was more potent than that on the 5-HT3AB receptor, and the 5-HT-induced 5-HT3AB receptor current showed enhancement by topotecan, confirming previous reports (Fig. 2) (Nakamura et al., 2013, Nakamura et al., 2011). Interestingly, mitoxantrone showed a bell-shaped response curve at the 5-HT3AB receptor, enhancing 5-HT current at lower concentrations and inhibiting it at higher concentrations, although it simply inhibited the 5-HT3A receptor current (Fig. 2B). Focusing on the component of potentiation, mitoxantrone (1µM) itself did not activate the 5-HT3AB receptor (data not shown), suggesting that mitoxantrone does not work as an agonist at the 5-HT3AB receptor. In the presence of a high concentration of 5-HT (10µM), mitoxantrone did not enhance the 5-HT-induced 5-HT3AB receptor current (Fig. 3A, B, E). These data suggested that potentiation of the 5-HT3AB receptor response by 1µM mitoxantrone is completely surmountable by 5-HT. Focusing on the component of inhibition (Figs. 2B and 3C), the currents recorded under 316µM 5-HT in the presence and absence of 100µM mitoxantrone were indistinguishable (Fig. 3D and F), suggesting that mitoxantrone acts as a competitive antagonist at the 5-HT3AB receptor at high concentration. With the exception of irinotecan, topotecan and mitoxantrone, the remaining seven drugs inhibited the 5-HT current with similar efficiency regardless of the receptor composition (Fig. 2, Table 3).