Fast voltage-dependent sodium (NaV ) currents are functionally expressed in mouse corpus cavernosum smooth muscle cells.

BACKGROUND AND PURPOSE: Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. EXPERIMENTAL APPROACH: We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. KEY RESULTS: Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the NaV channel subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7) and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for NaV 1.8 channels. Immunocytochemistry confirmed expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time-dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943. CONCLUSION AND IMPLICATIONS: There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.

Differences in local anaesthetic and antiepileptic binding in the inactivated state of human sodium channel Nav1.4.

Voltage-gated sodium channels play a vital role in nerve and muscle cells, enabling them to encode and transmit electrical signals. Currently, there exist several classes of drugs that aim to inhibit these channels for therapeutic purposes, including local anesthetics, antiepileptics and antiarrhythmics. However, sodium-channel-inhibiting drugs lack subtype specificity; instead, they inhibit all sodium channels in the human body. Improving understanding of the mechanisms of binding of existing nonselective drugs is important in providing insight into how subtype-selective drugs could be developed. This study used molecular dynamics simulations to investigate the binding of the antiepileptics carbamazepine and lamotrigine and the local anesthetic lidocaine in neutral and charged states to the recently resolved human Nav1.4 channel. Replica exchange solute tempering was used to enable greater sampling of each compound within the pore. It was found that all four compounds show similarities in their binding sites within the pore. However, the positions of the carbamazepine and lamotrigine did not occlude the center of the pore but preferentially bound to homologous domain DII and DIII. The charged and neutral forms of lidocaine positioned themselves more centrally in the pore, with more common interactions with DIV. The best localized binding site was for charged lidocaine, whose aromatic moiety interacted with Y1593, whereas the amine projected toward the selectivity filter. Comparisons with our previous simulations and published structures highlight potential differences between tonic and use-dependent block related to conformational changes occurring in the pore.

Ranolazine Improves Glycemic Variability and Endothelial Function in Patients with Diabetes and Chronic Coronary Syndromes: Results from an Experimental Study.

BACKGROUND: Ranolazine is a second-line drug for the management of chronic coronary syndromes (CCS). Glucose-lowering and endothelial effects have also been reported with this agent. However, whether ranolazine may improve short-term glycemic variability (GV), strictly related to the prognosis of patients with type 2 diabetes (T2D), is unknown. Thus, we aimed to explore the effects of adding ranolazine to standard anti-ischemic and glucose-lowering therapy on long- and short-term GV as well as on endothelial function and oxidative stress in patients with T2D and CCS. METHODS: Patients starting ranolazine (n = 16) were evaluated for short-term GV, haemoglobin 1Ac (Hb1Ac) levels, endothelial-dependent flow-mediated vasodilation (FMD), and oxidative stress levels at enrolment and after 3-month follow-up. The same measurements were collected from 16 patients with CCS and T2D that did not receive ranolazine, matched for age, gender, and body mass index. RESULTS: A significant decline in Hb1Ac levels was reported after 3-month ranolazine treatment (mean change -0.60%; 2-way ANOVA p = 0.025). Moreover, among patients receiving ranolazine, short-term GV indexes were significantly improved over time compared with baseline (p = 0.001 for time in range; 2-way ANOVA p = 0.010). Conversely, no significant changes were reported in patients without ranolazine. Finally, greater FMD and lower oxidative stress levels were observed in patients on ranolazine at 3 months. CONCLUSIONS: Ranolazine added to standard anti-ischemic and glucose-lowering therapy demonstrated benefit in improving the glycemic status of patients with T2D and CCS. How this improvement contributes to the overall myocardial benefit of ranolazine requires further studies.

Recombinant production, bioconjugation and membrane binding studies ofPn3a, a selective NaV1.7 inhibitor.

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (NaVs) play a critical role in this process. In particular, the NaV subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a spider venom peptide, µ-TRTX-Pn3a, was found to be a highly selective inhibitor of NaV1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Cytotoxicity of amide-linked local anesthetics on melanoma cells via inhibition of Ras and RhoA signaling independent of sodium channel blockade.

BACKGROUND: Substantial clinical and preclinical evidence have indicated the association between amide-linked local anesthesia and the long-term outcomes of cancer patients. However, the potential effects of local anesthesia on cancer recurrence are inconclusive and the underlying mechanisms remain poorly understood. METHODS: We systematically examined the effects of three commonly used local anesthetics in melanoma cells and analyzed the underlying mechanisms focusing on small GTPases. RESULTS: Ropivacaine and lidocaine but not bupivacaine inhibited migration and proliferation, and induced apoptosis in melanoma cells. In addition, ropivacaine and lidocaine but not bupivacaine significantly augmented the in vitro efficacy of vemurafenib (a B-Raf inhibitor for melanoma with BRAF V600E mutation) and dacarbazine (a chemotherapeutic drug). Mechanistically, ropivacaine but not bupivacaine decreased the activities of Ras superfamily members with the dominant inhibitory effects on RhoA and Ras, independent of sodium channel blockade. Rescue studies using constitutively active Ras and Rho activator calpeptin demonstrated that ropivacaine inhibited migration mainly through RhoA whereas growth and survival were mainly inhibited through Ras in melanoma cells. We further detected a global reduction of downstream signaling of Ras and RhoA in ropivacaine-treated melanoma cells. CONCLUSION: Our study is the first to demonstrate the anti-melanoma activity of ropivacaine and lidocaine but not bupivacaine, via targeting small GTPases. Our findings provide preclinical evidence on how amide-linked local anesthetics could affect melanoma patients.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model.

Experimental evidence regarding the risk of proarrhythmic potential of acehytisine is limited. We assessed its electropharmacological effect together with proarrhythmic potential at intravenous doses of 4 and 10 mg/kg (n = 6) using isoflurane-anesthetized guinea pigs in comparison with that of bepridil at 1 and 3 mg/kg, intravenously (n = 6). Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval, QTc, MAP90(sinus), MAP90(CL300) and MAP90(CL250). At supratherapeutic dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. Quantitative assessment showed that peak changes in P wave duration by acehytisine at 10 mg/kg were 1.7 times longer than bepridil, and in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than bepridil, respectively. Importantly, qualitative assessment indicated that bepridil increased beat-to-beat variability and J-Tpeakc in a dose-related manner, confirming a higher proarrhythmic risk, whereas such dose-related responses were not observed in acehytisine, suggesting a lower proarrhythmic risk. These results suggest that acehytisine exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity compared with bepridil, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.

Identification of CNS-Penetrant Aryl Sulfonamides as Isoform-Selective NaV1.6 Inhibitors with Efficacy in Mouse Models of Epilepsy.

Nonselective antagonists of voltage-gated sodium (NaV) channels have been long used for the treatment of epilepsies. The efficacy of these drugs is thought to be due to the block of sodium channels on excitatory neurons, primarily NaV1.6 and NaV1.2. However, these currently marketed drugs require high drug exposure and suffer from narrow therapeutic indices. Selective inhibition of NaV1.6, while sparing NaV1.1, is anticipated to provide a more effective and better tolerated treatment for epilepsies. In addition, block of NaV1.2 may complement the anticonvulsant activity of NaV1.6 inhibition. We discovered a novel series of aryl sulfonamides as CNS-penetrant, isoform-selective NaV1.6 inhibitors, which also displayed potent block of NaV1.2. Optimization focused on increasing selectivity over NaV1.1, improving metabolic stability, reducing active efflux, and addressing a pregnane X-receptor liability. We obtained compounds 30-32, which produced potent anticonvulsant activity in mouse seizure models, including a direct current maximal electroshock seizure assay.

Challenges and Opportunities for Therapeutics Targeting the Voltage-Gated Sodium Channel Isoform NaV1.7.

Voltage-gated sodium ion channel subtype 1.7 (NaV1.7) is a high interest target for the discovery of non-opioid analgesics. Compelling evidence from human genetic data, particularly the finding that persons lacking functional NaV1.7 are insensitive to pain, has spurred considerable effort to develop selective inhibitors of this Na+ ion channel target as analgesic medicines. Recent clinical setbacks and disappointing performance of preclinical compounds in animal pain models, however, have led to skepticism around the potential of selective NaV1.7 inhibitors as human therapeutics. In this Perspective, we discuss the attributes and limitations of recently disclosed investigational drugs targeting NaV1.7 and review evidence that, by better understanding the requirements for selectivity and target engagement, the opportunity to deliver effective analgesic medicines targeting NaV1.7 endures.

Natural Voltage-Gated Sodium Channel Ligands: Biosynthesis and Biology.

Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage-gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.

New local anesthetics.

Local anesthetics are used for performing various regional anesthesia techniques to provide intraoperative anesthesia and analgesia, as well as for the treatment of acute and chronic pain. Older medications such as lidocaine and bupivacaine as well as newer ones such as mepivacaine and ropivacaine are being used successfully for decades. Routes of administration include neuraxial, perineural, intravenous, various infiltrative approaches, topical, and transdermal. There are new innovations with the use of older local anesthetics in a novel manner, in addition to the development and use of new formulations. This chapter seeks to summarize the pharmacokinetics of local anesthetics and address the role of newer local anesthetics, as well as clinical implications, safety profiles, and the future of local anesthetic research. Finally, some clinical pearls are highlighted.

Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels.

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.

Stimulation of Na+ -K+ -pump currents by epithelial nicotinic receptors in rat colon.

BACKGROUND AND PURPOSE: Acetylcholine-induced epithelial Cl- secretion is generally thought to be mediated by epithelial muscarinic receptors and nicotinic receptors on secretomotor neurons. However, recent data have shown expression of nicotinic receptors by intestinal epithelium and the stimulation of Cl- secretion by nicotine, in the presence of the neurotoxin, tetrodotoxin. Here, we aimed to identify the transporters activated by epithelial nicotinic receptors and to clarify their role in cholinergic regulation of intestinal ion transport. EXPERIMENTAL APPROACH: Ussing chamber experiments were performed, using rat distal colon with intact epithelia. Epithelia were basolaterally depolarized to measure currents across the apical membrane. Apically permeabilized tissue was also used to measure currents across the basolateral membrane in the presence of tetrodotoxin. KEY RESULTS: Nicotine had no effect on currents through Cl- channels in the apical membrane or on currents through K+ channels in the apical or the basolateral membrane. Instead, nicotine stimulated the Na+ -K+ -pump as indicated by Na+ -dependency and sensitivity of the nicotine-induced current across the basolateral membrane to cardiac steroids. Effects of nicotine were inhibited by nicotinic receptor antagonists such as hexamethonium and mimicked by dimethyl-4-phenylpiperazinium, a chemically different nicotinic agonist. Simultaneous stimulation of epithelial muscarinic and nicotinic receptors led to a strong potentiation of transepithelial Cl- secretion. CONCLUSIONS AND IMPLICATIONS: These results suggest a novel concept for the cholinergic regulation of transepithelial ion transport by costimulation of muscarinic and nicotinic epithelial receptors and a unique role of nicotinic receptors controlling the activity of the Na+ -K+ -ATPase.

It takes a team: reflections on insecticide discoveries, toxicological problems and enjoying the unexpected.

Absorption/distribution/metabolism/excretion (ADME)-related studies are mandatory in agrochemical development/registration, but can also play a valuable role in the discovery process. In combination with target-site potency, bioavailability/ADME characteristics determine agrochemical bioactivity and selectivity, and these concerns can dictate the fate of a discovery lead area. Bioavailability/ADME research was critical to the eventual commercialization of three different insecticide chemistries examined in this paper. In one situation, improved systemicity in anthranilic diamides was required to expand pest spectrum. In another, ADME tools were needed to improve the selective toxicity and non-target safety of sodium channel blocker insecticides. Finally, differential ADME characteristics of two classes of hormone agonists dictated differential insecticidal activity, and were useful in optimizing the dibenzoylhydrazine ecdysone agonists. ADME discovery research will help companies to advance novel, efficacious and selective agrochemicals, but organizational patience and a desire to understand lead areas in depth are required. © 2016 Society of Chemical Industry.

Molecular Mechanism of Action and Selectivity of Sodium Ch annel Blocker Insecticides.

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models.

Convergent Evolution of Tetrodotoxin-Resistant Sodium Channels in Predators and Prey.

Convergent evolution of similar adaptive traits may arise from either common or disparate molecular and physiological mechanisms. The forces that determine the degree of underlying mechanistic similarities across convergent phenotypes are highly debated and poorly understood. Some garter snakes are able to consume newts that possess the channel blocking compound tetrodotoxin (TTX). Despite belonging to unrelated lineages, both the predators and prey have independently evolved remarkably similar physiological mechanisms of resistance to TTX that involve chemical and structural changes in voltage-gated sodium channels (NaV). The evolution of TTX resistance in this predator-prey pair constitutes a natural experiment that allows us to explore the causes of molecular convergence. Here, we review broad patterns of convergence at the level of amino acid changes in NaV channels of animals that evolved TTX resistance and make comparisons to known TTX-resistant channels that did not evolve under the selective pressures imposed by TTX. We conclude that convergence likely stems from the interplay of the target specificity of TTX and functional constraints of NaV that are shared among taxa. These and other factors can limit channel evolution to favor a few functionally permissible paths of adaptation, which can explain the observed predictability of changes to channel structure. By studying the functional causes of convergence in NaV channels, we can further our understanding of the role of these important channel proteins at the center of the evolution of the nervous system.

Understanding Sodium Channel Function and Modulation Using Atomistic Simulations of Bacterial Channel Structures.

Sodium channels are chief proteins involved in electrical signaling in the nervous system, enabling critical functions like heartbeat and brain activity. New high-resolution X-ray structures for bacterial sodium channels have created an opportunity to see how these proteins operate at the molecular level. An important challenge to overcome is establishing relationships between the structures and functions of mammalian and bacterial channels. Bacterial sodium channels are known to exhibit the main structural features of their mammalian counterparts, as well as several key functional characteristics, including selective ion conduction, voltage-dependent gating, pore-based inactivation and modulation by local anesthetic, antiarrhythmic and antiepileptic drugs. Simulations have begun to shed light on each of these features in the past few years. Despite deviations in selectivity signatures for bacterial and mammalian channels, simulations have uncovered the nature of the multiion conduction mechanism associated with Na(+) binding to a high-field strength site established by charged glutamate side chains. Simulations demonstrated a surprising level of flexibility of the protein, showing that these side chains are active participants in the permeation process. They have also uncovered changes in protein structure, leading to asymmetrical collapses of the activation gate that have been proposed to correspond to inactivated structures. These observations offer the potential to examine the mechanisms of state-dependent drug activity, focusing on pore-blocking and pore-based slow inactivation in bacterial channels, without the complexities of inactivation on multiple timescales seen in eukaryotic channels. Simulations have provided molecular views of the interactions of drugs, consistent with sites predicted in mammalian channels, as well as a wealth of other sites as potential new drug targets. In this chapter, we survey the new insights into sodium channel function that have emerged from studies of simpler bacterial channels, which provide an excellent learning platform, and promising avenues for mechanistic discovery and pharmacological development.

Recent Trends in the Discovery of Small Molecule Blockers of Sodium Channels.

Voltage-gated sodium channels (VGSC) are responsible for the selective influx of sodium ions in excitable cells. A number of physiological phenomena such as muscle contraction, pain sensation, processing of neuronal information in the brain as well as neuronal regulation of peripheral tissues rely on the activity of these channels. On the other hand, abnormal activity of VGSC are implicated in several pathological processes (e.g., cardiac arrhythmias, epilepsy, and chronic pain) which in some cases (e.g., channelopathies such as myotonias) are linked to specific gene mutations. As a result, VGSC have never stopped attracting the attention of medicinal chemists and the quest for novel drugs to treat these ion channels-associated diseases continues. In this review, VGSC blocking agents reported in the last lustrum are scrutinised with the aim to give a medicinal chemistry perspective on the most interesting compounds classified on the basis of (i) potential therapeutic application, (ii) targeted VGSC isoforms, and (iii) chemical scaffolds. Finally, the clinical potential of selected drug candidates from each chemotype is evaluated by comparing their ligand efficiency metrics. Possible routes for improvement of these preclinical candidates are also discussed.

Modeling interactions between blocking and permeant cations in the NavMs channel.

Mechanisms of sodium channel block by local anesthetics (LAs) are still a matter of intensive studies. In the absence of high-resolution structures of eukaryotic channels, atomic details of LA-channel interactions are analyzed using homology modeling. LAs are predicted to access the closed channel through a sidewalk (fenestration) between the channel repeats, bind in a horizontal orientation, and leave its aromatic moiety in the interface. Recent X-ray structure of a bacterial sodium channel NavMs with a cationic molecule Pl1, which is structurally similar to LAs, has confirmed this theoretical prediction and demonstrated a reduced selectivity filter occupancy by the permeant ions in the Pl1-bound channel. However, the nature of the antagonism between LAs and permeant ions is still unclear. Here we used the NavMs structure and Monte Carlo energy minimizations to model Pl1 binding. Our computations predict that Pl1 can displace permeant ion(s) from the selectivity filter by both steric and electrostatic mechanisms. We hypothesize that the electrostatic mechanism is more general, because it is applicable to many LAs and related drugs, which lack a moiety capable to enter the selectivity filter and sterically displace the permeant ion. The electrostatic mechanism is also consistent with the data that various cationic blockers of potassium channels bind in the inner pore without entering the selectivity filter.

Investigation of Metabolite Profile of YM758, a Novel If Channel Inhibitor.

BACKGROUND: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle. OBJECTIVES: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites. METHODS: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.