Vasodilation activity of dipfluzine metabolites in isolated rat basilar arteries and their underlying mechanisms

Highlights

• Among the 5 metabolites of Dip, only M1, like its parent compound, had vasodilation activity.

• Mechanisms of M1 are related to blocking two Ca²⁺ channel and NO & EDHF release.

• Dip only opens ATP-sensitive K⁺ channel, but M1 also opens voltage-sensitive K⁺ channels.

• The latter may partially explain why M1 has stronger vasodilatory effects than Dip.

Abstract

Identifying the metabolites of a drug has become an indispensable task in the development of new drugs. Dipfluzine (Dip) is a promising candidate for the treatment of cerebral vascular diseases and has 5 metabolites (M1∼M5) in rat urine and liver microsomes, but their biological activity is still unknown. Because selective cerebral vasodilation is a main role of Dip, we investigated the vasodilation of Dip and its 5 metabolites in isolated Sprague-Dawley (SD) male rat basilar arteries preconstricted with high-K⁺ or 5-HT. The results showed that only M1 possessed concentration-dependent inhibitory activity on the vasoconstriction of arteries with or without the endothelium, and M1 has a more potent vasodilatory effect than Dip on both contraction models. Like Dip, the vasodilatory mechanisms of M1 may be not only related to receptor-operated and voltage-dependent calcium ion channels of smooth muscle cells but also to the release of NO and EDHF from endothelial cells and the opening of Ca²⁺-activated K⁺ channels and ATP-sensitive potassium ion channels. Unlike Dip, the vasodilation mechanism of M1 is also related to the opening of voltage-sensitive K⁺ channel. Together with more selectivity to non-VDCC than Dip, this may partially explain why M1 has stronger vasodilatory effects than Dip. The mechanisms of vasodilation of Dip and M1 may result from the combined action of these or other factors, especially blocking non-endothelium dependent non-VDCC and endothelium dependent IK_Ca channels. These results point to the possibility that M1 provides synergism for the clinical use of Dip, which may inform the synthesis of new drugs.

Introduction

Since the introduction of metabolites in the safety testing (MIST) guidance by the Food and Drug Administration in 2008, drug discovery efforts have increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions. There have been increased attempts to obtain a comprehensive and quantitative profile of human metabolites at the earliest stage of drug development (Luffer-Atlas and Atrakchi, 2017; Schadt et al., 2018). After a drug enters the body, it interacts with the body and undergo a series of metabolic changes. In these metabolic processes, some drugs lose their pharmacological activity and are eventually excreted out of the body with urine and faeces, while others produce active metabolites (AMs) with stronger pharmacological activity than their parent drugs (Hecker et al., 1994; Murata et al., 2016; Wang et al., 2012; Wang and LeCluyse, 2003). These AMs may play an important role in drug therapy. Accordingly, identifying the metabolites of a drug has become a more urgent, valuable, and indispensable task in the development stage of new drugs. Identifying AMs is of great significance to ensure the safety and effectiveness of clinical drugs and to inform better drug design.

Dipfluzine (Dip) is a novel diphenylpiperazine Ca²⁺ channel blocker designed and synthesized by the College of Pharmacy, Hebei Medical University. Previous studies have demonstrated that Dip is a highly selective cerebral vasodilator that exerts protective effects against focal or global cerebral ischaemic injury, inhibiting platelet aggregation in vitro and thrombus formation in vivo via multiple mechanisms (Wang and He, 1993; Bai and Wang, 2002; Zhang et al., 2005; Wang and He, 1994a). Evidence from in vivo and in vitro experiments has revealed that the pharmacological activity of Dip is more potent than that of its analogues cinnarizine or flunarizine (Wang and He, 1993, 1994b; Zhu et al., 1996). These results suggest that Dip is a promising drug candidate for the treatment of cerebral vascular diseases.

In a previous study, we evaluated the metabolic pathway of Dip in rats and identified its metabolites in rat urine by liquid chromatography/diode array detection/mass spectrometry (LC/DAD/MS) methods and in rat liver microsomes by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS) analysis (Liu et al., 2005; Guo et al., 2012, 2014). These results indicate that Dip was extensively transformed into 5 metabolites, 1-(4-fluorophenyl)-4-piperazinyl- butanone (M1), 4-hydroxy-benzophenone (M2), 4-fluoro-γ-hydroxy-benzene- butanoic acid (M3), diphenylmethanol (M4), and benzophenone (M5), in rats via multiple pathways, including N-dealkylation at positions 1 and 4 of the piperazine ring (Fig. 1). However, the biological activities of active metabolites have not been studied.

Dip shows a higher selectivity for cerebral vasodilation and blocker characteristics for the voltage-dependent Ca²⁺ channel (VDCC) and receptor-operated Ca²⁺ channel (ROCC) (also called non-voltage- dependent Ca²⁺ channel (non-VDCC)) in inhibiting contractile responses in vitro in the rabbit aortic artery, porcine basilar, coronary and radial arteries evoked by KCl, rabbit aortic artery by NE, and porcine basilar artery by 5-HT, and it decreases the vascular resistance of vertebral, coronary and femoral arteries in anaesthetized dogs (Wang et al., 1990; Wang and He, 1993; Zhu et al., 1996). Therefore, the present study aimed to compare the vasodilatory effects of Dip and its 5 metabolites on the vasoconstriction of isolated SD male rat basilar arteries induced by high-K⁺ depolarization and 5-HT receptor activation to screen the AMs of Dip and to observe whether the AMs, such as Dip, also possess characteristics of a Ca²⁺ channel blocker.

Vasodilation includes two pathways. One is the non-endothelium dependent pathway by blocking Ca²⁺ channels of vascular smooth muscle, and this is mainly related to the inhibition of extracellular calcium influx and intracellular calcium release to reduce intracellular calcium concentration (Lizuka et al., 2006). The other is the endothelium-dependent pathway which occurs by releasing endothelium-derived relaxing factor (EDRF) from the vascular endothelium in addition to nitric oxide (NO), prostacyclin (PGI₂), and this EDRF is mainly related to the secretion of endothelium-dependent hyperpolarizing factor (EDHF), a third endothelium- dependent vasodilatory factor in both cerebral and peripheral circulations (Cohen, 2005; Jin et al., 2011). This EDHF-mediated vasodilation is distinct from NO- and PGI2-mediated mechanisms and is further characterized by its dependence on an intact endothelium and the activation of calcium-sensitive potassium channels (K_Ca) to produce a smooth muscle hyperpolarization and subsequent vasodilatation (Quilley et al., 1997). K_Ca are important to vascular endothelial function. There are 5 members in the Kca channel family, characterized by their unitary conductance: large (BK_Ca), intermediater (IK_Ca), and 3 subtypes of small (SK_Ca): SK_Ca1, SK_Ca2 and SK_Ca3. IK_Ca and SK_Ca channels are thought to be the predominant K_Ca channels expressed in systemic vascular endothelia, which opening is the mechanism that underlies the classical EDHF pathway (Edwards et al., 1999). However, the EDHF response in cerebral blood vessels is quite different from that in peripheral blood vessels. For example, in many peripheral blood vessels, EDHF can activate IK_Ca and SK_Ca, while in cerebral blood vessels, EDHF only involves the activation of IK_Ca. In addition, EDHF was inhibited by NO in normal peripheral blood vessels, but not in cerebral vessels (Zygmunt and Högestätt, 1996; Marrelli et al., 2003). EDHF-mediated vasodilatation appears to result from a far more complicated process requiring specific interactions between smooth muscle and the endothelium, in which there are a number of unifying hallmarks common among EDHF-mediated dilations. One in particular is the essential involvement of K_Ca (Feletou and Vanhoutte, 1999). Although it was initially assumed that the K_Ca channels critical to EDHF-mediated dilations were BK_Ca located on the smooth muscle (Quilley et al., 1997), it is now generally believed that the K_Ca involved include IK_Ca alone or in combination with SK_Ca located on the endothelium (Eichler et al., 2003). The mechanism by which endothelial K_Ca activation translates into smooth muscle hyperpolarization is by the stimulation of smooth muscle inwardly rectifying K channels (K_IR) (Edwards et al., 1999).

Because the mechanism that underlies the classical EDHF pathway is opening of K_Ca channels on the plasma membrane of endothelial cells (Edwards et al., 2010), and to confirm one or more metabolites of Dip as AMs that block VDCC and non-VDCC as their main mechanism of vasodilation, the present study aims to further explore the endothelium-dependent mechanisms and potassium channel mechanism of Dip and its AMs at relaxing rat basilar arteries with or without the endothelium. This study applies various EDRF inhibitors and K⁺ channel blockers, especially EDHF-mediated vasodilatation mechanisms related to the opening of K_Ca channels.