Tino Dyhring

Objective: Preclinical and clinical studies implicate the vascular ATP-sensitive potassium (K_ATP) channel in the signaling cascades underlying headache and migraine. However, attempts to demonstrate that the K_ATP channel inhibitor glibenclamide would attenuate triggered headache in healthy volunteers have proven unsuccessful. It is questionable, however, whether target engagement was achieved in these clinical studies. Methods: Literature data for human glibenclamide pharmacokinetics, plasma protein binding and functional IC₅₀ values were used to predict the K_ATP receptor occupancy (RO) levels obtained after glibenclamide dosing in the published exploratory clinical headache provocation studies. RO vs. time profiles of glibenclamide were simulated for the pancreatic K_ATP channel subtype Kir6.2/SUR1 and the vascular subtype Kir6.1/SUR2B. Results: At the clinical dose of 10 mg of glibenclamide used in the headache provocation studies, predicted maximal occupancy levels of up to 90% and up to 26% were found for Kir6.2/SUR1 and Kir6.1/SUR2B, respectively. Conclusions: The findings of the present study indicate that effective Kir6.1/SUR2B target engagement was not achieved in the clinical headache provocation studies using glibenclamide. Therefore, development of novel selective Kir6.1/SUR2B inhibitors, with good bioavailability and low plasma protein binding, is required to reveal the potential of K_ATP channel inhibition in the treatment of migraine.

Migraine is a highly disabling pain disorder with huge socioeconomic and personal costs. It is genetically heterogenous leading to variability in response to current treatments and frequent lack of response. Thus, new treatment strategies are needed. A combination of preclinical and clinical data indicate that ATP-sensitive potassium (K_ATP) channel inhibitors could be novel and highly effective drugs in the treatment of migraine. The subtype Kir6.1/SUR2B is of particular interest and inhibitors specific for this cranio-vascular K_ATP channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize K_ATP channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different K_ATP subtypes is difficult to assess. Here, we characterize available K_ATP channel activators and inhibitors in fluorescence-based thallium-flux assays using HEK293 cells stably expressing human Kir6.1/SUR2B, Kir6.2/SUR1, and Kir6.2/SUR2A K_ATP channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the K_ATP channel subtype Kir6.1/SUR2B, whereas BMS-191095, NN414, and VU0071306 demonstrated preferred activation of the Kir6.2/SUR1 subtype. In the group of K_ATP channel blockers, only Rosiglitazone and PNU-37783A showed selective inhibition of the Kir6.1/SUR2B subtype. PNU-37783A was stopped in clinical development and Rosiglitazone has a low potency for the vascular K_ATP channel subtype. Therefore, development of novel selective K_ATP channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post‐traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real‐time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, β2, and β4 subunits. Whole‐cell patch‐clamp electrophysiology of chromaffin cells and subtype‐selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA‐5469, was created that is highly selective for α3β2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA‐5469 and the α3β4‐selective α‐conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3β4 nAChRs alone or α3β4 together with the α3β2β4 subtype. Conclusions were derived from observations that acetylcholine‐gated currents in some cells were sensitive to inhibition by PeIA‐5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7‐selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α‐conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3β2β4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.

Essential tremor (ET) is among the most prevalent neurological disorders and the most common cause of abnormal tremors. It is characterized by postural and action tremors ranging from 4 to 12 Hz. The treatments of choice for ET are propranolol and primidone, but their use is associated with adverse effects like hypotension, depression, and cognitive impairments. Benzodiazepines, which nonselectively enhances the effect of GABA at the GABA_A α1/2/3/5 receptors, have been shown to be effective in treating ET. Their use, however, is limited due to sedation, ataxia, tolerance development and memory impairment. Sedation and ataxia are attributed to the activity at the α1 subunit while cognitive impairment is ascribed to the action on the α5 subunit of the GABA_A receptors. It can be hypothesized that subtype selective GABA_A receptor modulators only acting via the α2, and α3 subunits may have an improved side effect profile while retaining the beneficial effects. Here, we have evaluated the effect of subtype selective GABA_A α2/3/5 receptor modulators on harmaline-induced tremors in rats. The tremors were automatically quantified in tremor boxes. We show that the GABA_A α2/3 subtype selective modulator NS16085 significantly and dose-dependently inhibits harmaline-induced tremors in rats, indicating that potentiation of α2- and α3-containing GABA_A receptors is sufficient to ameliorate harmaline-induced tremors. These results provide the first support for a therapeutic role of a subtype selective GABA_A α2/3 modulator in the treatment of ET.

Summary: Aims: Here, we investigate the pharmacology ofNS383, a novel small molecule inhibitor of acid‐sensing ion channels (ASICs).Methods: ASICinhibition byNS383 was characterized in patch‐clamp electrophysiological studies. Analgesic properties were evaluated in four rat behavioral models of pain.Results: NS383 inhibited H⁺‐activated currents recorded from rat homomericASIC1a,ASIC3, and heteromericASIC1a+3 withIC₅₀values ranging from 0.61 to 2.2μM. However,NS383 was completely inactive at homomericASIC2a. Heteromeric receptors containingAISC2a, such asASIC1a+2a andASIC2a+3, were only partially inhibited, presumably as a result of stoichiometry‐dependent binding.NS383 (10–60 mg/kg, i.p.), amiloride (50–200 mg/kg, i.p.), acetaminophen (100–400 mg/kg, i.p.), and morphine (3–10 mg/kg, i.p.) all dose‐dependently attenuated nocifensive behaviors in the rat formalin test, reversed pathological inflammatory hyperalgesia in complete Freund's adjuvant‐inflamed rats, and reversed mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain. However, in contrast to acetaminophen and morphine, motor function was unaffected byNS383 at doses at least 8‐fold greater than those that were effective in pain models, whilst analgesic doses of amiloride were deemed to be toxic.Conclusions: NS383 is a potent and uniquely selective inhibitor of ratASICs containing 1a and/or 3 subunits. It is well tolerated and capable of reversing pathological painlike behaviors, presumably via peripheral actions, but possibly also via actions within central pain circuits.

Large-conductance Ca²⁺-activated K⁺ channels (BK, K_Ca1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader–based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC₅₀ value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K⁺ concentration consistent with a K⁺ channel–mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

The intermediate-conductance Ca²⁺-activated K⁺ channel (K_Ca3.1) has been proposed to play many physiological roles, and modulators of K_Ca3.1 activity are potentially interesting as new drugs. In order to identify new chemical scaffolds, high-throughput screening (HTS) assays are needed. In the current study, we present an HTS assay that has been optimized for the detection of inhibitors as well as activators of K_Ca3.1 in a combined assay. We used HEK293 cells heterologously expressing K_Ca3.1 in a fluorescence-based Tl⁺ influx assay, where the permeability of potassium channels to Tl⁺ is taken advantage of. We found the combined activator-and-inhibitor assay to be robust and insensitive to dimethyl sulfoxide (up to 1%), and conducted an HTS campaign of 217,119 small molecules. In total, 224 confirmed activators and 312 confirmed inhibitors were found, which corresponded to a hit rate of 0.10% and 0.14%, respectively. The confirmed hits were further characterized in a fluorometric imaging plate reader-based concentration-response assay, and selected compounds were subjected to secondary testing in an assay for endogenous K_Ca3.1 activity using human erythrocytes (red blood cell assay). Although the estimated potencies were slightly higher in the RBC assay, there was an overall good correlation across all clusters. The campaign led to the identification of several chemical series of K_Ca3.1 activators and inhibitors, comprising already known pharmacophores and new chemical series. One of these were the benzothiazinones that constitute a new class of highly potent K_Ca3.1 inhibitors, exemplified by 4-{[3-(trifluoromethyl)phenyl]methyl}-2H-1,4-benzothiazin-3(4H)-one (NS6180).