Inhibition of Canonical Transient Receptor Potential Channels 4/5 with Highly Selective and Potent Small-Molecule HC-070 Alleviates Mechanical Hypersensitivity in Rat Models of Visceral and Neuropathic Pain.

Transient receptor potential channels C4/C5 are widely expressed in the pain pathway. Here, we studied the putative analgesic efficacy of the highly selective and potent TRPC4/C5 antagonist HC-070 in rats. Inhibitory potency on human TRPC4 was assessed by using the whole-cell manual patch-clamp technique. Visceral pain sensitivity was assessed by the colonic distension test after intra-colonic trinitrobenzene sulfonic acid injection and partial restraint stress. Mechanical pain sensitivity was assessed by the paw pressure test in the chronic constriction injury (CCI) neuropathic pain model. We confirm that HC-070 is a low nanomolar antagonist. Following single oral doses (3-30 mg/kg in male or female rats), colonic hypersensitivity was significantly and dose-dependently attenuated, even fully reversed to baseline. HC-070 also had a significant anti-hypersensitivity effect in the established phase of the CCI model. HC-070 did not have an effect on the mechanical withdrawal threshold of the non-injured paw, whereas the reference compound morphine significantly increased it. Analgesic effects are observed at unbound brain concentrations near the 50% inhibitory concentration (IC50) recorded in vitro. This suggests that analgesic effects reported here are brought about by TRPC4/C5 blocking in vivo. The results strengthen the idea that TRPC4/C5 antagonism is a novel, safe non-opioid treatment for chronic pain.

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity.

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC50 values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (INa ) and hERG KV 11.1 currents (IhERG ) in a concentration-dependent manner; IC50 values were 23.2 and 10.0 µM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 µM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

1-Sulfonyl-6-Piperazinyl-7-Azaindoles as potent and pseudo-selective 5-HT6 receptor antagonists.

A series of 1-Sulfonyl-6-Piperazinyl-7-Azaindoles, showing strong antagonistic activity to 5-HT6 receptor (5-HT6R) was synthesized and characterized. The series was optimized to reduce activity on D2 receptor. Based on the selectivity against this off-target and the analysis of the ADME-tox profile, compound 1c was selected for in vivo efficacy assessment, which demonstrated procognitive effects as shown in reversal of scopolamine induced amnesia in an elevated plus maze test in mice. Compound 3, the demethylated version of compound 1c, was profiled against a panel of 106 receptors, channels and transporters, indicating only D3 receptor as a major off-target. Compound 3 has been selected for this study over compound 1c because of the higher 5-HT6R/D2R binding ratio. These results have defined a new direction for the design of our pseudo-selective 5-HT6R antagonists.

TRPA1: a transducer and amplifier of pain and inflammation.

The transient receptor potential ankyrin 1 (TRPA1) ion channel on peripheral terminals of nociceptive primary afferent nerve fibres contributes to the transduction of noxious stimuli to electrical signals, while on central endings in the spinal dorsal horn, it amplifies transmission to spinal interneurons and projection neurons. The centrally propagating nociceptive signal that is induced and amplified by TRPA1 not only elicits pain sensation but also contributes to peripheral neurogenic inflammation through a peripheral axon reflex or a centrally mediated back propagating dorsal root reflex that releases vasoactive agents from sensory neurons in the periphery. Endogenous TRPA1 agonists that are generated under various pathophysiological conditions both in the periphery and in the spinal cord have TRPA1-mediated pro-nociceptive and pro-inflammatory effects. Among endogenous TRPA1 agonists that have been shown to play a role in the pathogenesis of pain and inflammatory conditions are, for example, methylglyoxal, 4-hydroxynonenal, 12-lipoxygenase-derived hepoxilin A3, 5,6-epoxyeicosatrienoic acid and reactive oxygen species, while mustard oil and cinnamaldehyde are most commonly used exogenous TRPA1 agonists in experimental studies. Among selective TRPA1 antagonists are HC-030031, A-967079, AP-14 and Chembridge-5861528. Recent evidence indicates that TRPA1 plays a role also in transition of acute to chronic pain. Due to its location on a subpopulation of pain-mediating primary afferent nerve fibres, blocking the TRPA1 channel is expected to have antinociceptive, antiallodynic and anti-inflammatory effects.

Pharmacological characterisation of a structurally novel α2C-adrenoceptor antagonist ORM-10921 and its effects in neuropsychiatric models.

The α2-adrenoceptors (ARs) are important modulators of a wide array of physiological responses. As only a few selective compounds for the three α2-AR subtypes (α2A , α2B and α2C ) have been available, the pharmacological profile of a new α2C-selective AR antagonist ORM-10921 is reported. Standard in vitro receptor assays and antagonism of α2, and α1-AR agonist-evoked responses in vivo were used to demonstrate the α2C-AR selectivity for ORM-10921 which was tested in established behavioural models related to schizophrenia and cognitive dysfunction with an emphasis on pharmacologically induced hypoglutamatergic state by phencyclidine or MK-801. The Kb values of in vitro α2C-AR antagonism for ORM-10921 varied between 0.078-1.2 nM depending on the applied method. The selectivity ratios compared to α2A-AR subtype and other relevant receptors were 10-100 times in vitro. The in vivo experiments supported its potent α2C-antagonism combined with only a weak α2A-antagonism. In the pharmacodynamic microdialysis study, ORM-10921 was found to increase extracellular dopamine levels in prefrontal cortex in the baseline conditions. In the behavioural tests, ORM-10921 displayed potent antidepressant and antipsychotic-like effects in the forced swimming test and prepulse-inhibition models analogously with the previously reported results with structurally different α2C-selective AR antagonist JP-1302. Our new results also indicate that ORM-10921 alleviated the NMDA-antagonist-induced impairments in social behaviour and watermaze navigation. This study extends and further validates the concept that α2C -AR is a potential therapeutic target in CNS disorders such as schizophrenia or Alzheimer's disease and suggests the potential of α2C-antagonism to treat such disorders.

Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy.

Peripheral diabetic neuropathy (PDN) is a devastating complication of diabetes mellitus (DM). Here we test the hypothesis that the transient receptor potential ankyrin 1 (TRPA1) ion channel on primary afferent nerve fibers is involved in the pathogenesis of PDN, due to sustained activation by reactive compounds generated in DM. DM was induced by streptozotocin in rats that were treated daily for 28 days with a TRPA1 channel antagonist (Chembridge-5861528) or vehicle. Laser Doppler flow method was used for assessing axon reflex induced by intraplantar injection of a TRPA1 channel agonist (cinnamaldehyde) and immunohistochemistry to assess substance P-like innervation of the skin. In vitro calcium imaging and patch clamp were used to assess whether endogenous TRPA1 agonists (4-hydroxynonenal and methylglyoxal) generated in DM induce sustained activation of the TRPA1 channel. Axon reflex induced by a TRPA1 channel agonist in the plantar skin was suppressed and the number of substance P-like immunoreactive nerve fibers was decreased 4 weeks after induction of DM. Prolonged treatment with Chembridge-5861528 reduced the DM-induced attenuation of the cutaneous axon reflex and loss of substance P-like immunoreactive nerve fibers. Moreover, in vitro calcium imaging and patch clamp results indicated that reactive compounds generated in DM (4-hydroxynonenal and methylglyoxal) produced sustained activations of the TRPA1 channel, a prerequisite for adverse long-term effects. The results indicate that the TRPA1 channel exerts an important role in the pathogenesis of PDN. Blocking the TRPA1 channel provides a selective disease-modifying treatment of PDN.

Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture.

Long QT syndrome (LQTS) is caused by functional alterations in cardiac ion channels and is associated with prolonged cardiac repolarization time and increased risk of ventricular arrhythmias. Inherited type 2 LQTS (LQT2) and drug-induced LQTS both result from altered function of the hERG channel. We investigated whether the electrophysiological characteristics of LQT2 can be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology. Spontaneously beating cardiomyocytes were differentiated from two iPSC lines derived from an individual with LQT2 carrying the R176W mutation in the KCNH2 (HERG) gene. The individual had been asymptomatic except for occasional palpitations, but his sister and father had died suddenly at an early age. Electrophysiological properties of LQT2-specific cardiomyocytes were studied using microelectrode array and patch-clamp, and were compared with those of cardiomyocytes derived from control cells. The action potential duration of LQT2-specific cardiomyocytes was significantly longer than that of control cardiomyocytes, and the rapid delayed potassium channel (I(Kr)) density of the LQT2 cardiomyocytes was significantly reduced. Additionally, LQT2-derived cardiac cells were more sensitive than controls to potentially arrhythmogenic drugs, including sotalol, and demonstrated arrhythmogenic electrical activity. Consistent with clinical observations, the LQT2 cardiomyocytes demonstrated a more pronounced inverse correlation between the beating rate and repolarization time compared with control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation carrier and arrhythmias can be triggered by a commonly used drug. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as an important platform to study pathophysiological mechanisms and drug sensitivity in LQT2.

Spinal transient receptor potential ankyrin 1 channel contributes to central pain hypersensitivity in various pathophysiological conditions in the rat.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed on nociceptive primary afferent neurons. On the proximal nerve ending within the spinal dorsal horn, TRPA1 regulates transmission to spinal interneurons, and thereby pain hypersensitivity. Here we assessed whether the contribution of the spinal TRPA1 channel to pain hypersensitivity varies with the experimental pain model, properties of test stimulation or the behavioral pain response. The antihypersensitivity effect of intrathecally (i.t.) administered Chembridge-5861528 (CHEM; a selective TRPA1 channel antagonist; 5-10µg) was determined in various experimental models of pain hypersensitivity in the rat. In spinal nerve ligation and rapid eye movement (REM) sleep deprivation models, i.t. CHEM attenuated mechanical hypersensitivity. Capsaicin-induced secondary (central) but not primary (peripheral) mechanical hypersensitivity was also reduced by i.t. administration of CHEM or A-967079, another TRPA1 channel antagonist. Formalin-induced secondary mechanical hypersensitivity, but not spontaneous pain, was suppressed by i.t. CHEM. Moreover, mechanical hypersensitivity induced by cholekystokinin in the rostroventromedial medulla was attenuated by i.t. pretreatment with CHEM. Independent of the model, the antihypersensitivity effect induced by i.t. CHEM was predominant on responses evoked by low-intensity stimuli (⩽6g). CHEM (10µg i.t.) failed to attenuate pain behavior in healthy controls or mechanical hypersensitivities induced by i.t. administrations of a GABA(A) receptor antagonist, or NMDA or 5-HT(3) receptor agonists. Conversely, i.t. administration of a TRPA1 channel agonist, cinnamon aldehyde, induced mechanical hypersensitivity. The results indicate that the spinal TRPA1 channel exerts an important role in secondary (central) pain hypersensitivity to low-intensity mechanical stimulation in various pain hypersensitivity conditions. The spinal TRPA1 channel provides a promising target for the selective attenuation of a central mechanism contributing to pathophysiological pain.

Human embryonic stem cell-derived cardiomyocytes: demonstration of a portion of cardiac cells with fairly mature electrical phenotype.

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESC) provide a promising tool for the pharmaceutical industry. In this study the electrical properties and maturation of hESC-CM derived using two differentiation methods were compared and the suitability of hESC-CMs as a cell model for the assessment of drug-induced repolarization delay was evaluated. CMs were differentiated either in END-2 co-culture or by spontaneous differentiation. Action potentials (APs) were recorded from cells in spontaneously beating areas using the whole-cell patch-clamp technique. The hESC-CMs exhibited predominantly a ventricular-like phenotype with heterogeneous properties. Heterogeneity was indicative of the spectrum of hESC-CM maturation from embryonic-like with AP upstroke velocities <30 V/s and maximum diastolic potential (MDP) of close to -60 mV to more mature with values >150 V/s and -80 mV, respectively. The mean MDP was -70 mV and a significant difference was observed between the two differentiation methods (-66 versus -75 mV, P < 0.001). The age of the CMs did not correlate with phenotype maturation. The addition of the hERG blocker E-4031 and the sodium channel modulator veratridine significantly prolonged the AP duration. Furthermore, proarrhythmic indices were induced. In conclusion, the main observation was the heterogeneity in electrical properties of the hESC-CMs and this was observed with both differentiation methods. One-third of the hESC-CMs exhibited fairly mature electrophysiological properties, suggesting that mature CMs could be obtained from hESCs. However, improved differentiation methods are needed to produce homogeneous mature human CMs for pharmaceutical and toxicological applications.

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol.

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein-dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.

Roles of cutaneous versus spinal TRPA1 channels in mechanical hypersensitivity in the diabetic or mustard oil-treated non-diabetic rat.

Previous results indicate that intaperitoneal administration of a TRPA1 channel antagonist attenuates diabetic hypersensitivity. We studied whether the antihypersensitivity effect induced by a TRPA1 channel antagonist in diabetic animals is explained by action on the TRPA1 channel in the skin, the spinal cord, or both. For comparison, we determined the contribution of cutaneous and spinal TRPA1 channels to development of hypersensitivity induced by topical administration of mustard oil in healthy controls. Diabetes mellitus was induced by streptozotocin in the rat. Hypersensitivity was assessed by the monofilament- and paw pressure-induced limb withdrawal response. Intrathecal (i.t.) administration of Chembridge-5861528 (CHEM, a TRPA1 channel antagonist) at doses 2.5-5.0 microg/rat markedly attenuated diabetic hypersensitivity, whereas 20 microg of CHEM was needed to produce a weak attenuation of diabetic hypersensitivity with intraplantar (i.pl.) administrations. In controls, i.pl. administration of CHEM (20 microg) produced a weak antihypersensitivity effect at the mustard oil-treated site. I.t. administration of CHEM (10 microg) in controls produced a strong antihypersensitivity effect adjacent to the mustard oil-treated area (site of secondary hyperalgesia), while it failed to influence hypersensitivity at the mustard oil-treated area (site of primary hyperalgesia). A reversible antagonism of the rat TRPA1 channel by CHEM was verified using in vitro patch clamp recordings. The results suggest that while cutaneous TRPA1 channels contribute to mechanical hypersensitivity induced by diabetes or topical mustard oil, spinal TRPA1 channels, probably on central terminals of primary afferent nerve fibers, play an important role in maintenance of mechanical hypersensitivity in these conditions.

The action of the novel gastrointestinal prokinetic prucalopride on the HERG K+ channel and the common T897 polymorph.

The human ether-à-go-go related gene (HERG) encodes the alpha-subunit of a delayed rectifier potassium channel important in the repolarisation of the cardiac action potential. Excessive action potential prolongation through HERG channel inhibition is associated with a risk of torsade de pointes arrhythmias and is a major challenge for drug development. The acute effects of the novel prokinetic prucalopride were examined on heterologously expressed HERG channels in human embryonic kidney (HEK) 293 cells using the whole-cell patch-clamp technique. Prucalopride inhibited HERG channels in a concentration-dependent manner with an IC(50) of 4.1 microM. Prucalopride significantly slowed channel deactivation and recovery from inactivation, accelerated and altered the extent of inactivation. Similar concentration-dependency and kinetic changes were observed with the minor T897 polymorphic HERG variant. Prucalopride block was frequency-independent due to rapid state-dependent block, with binding occurring in the open and inactivated states. Though prucalopride blocks HERG channels this is unlikely to be significant at clinically relevant concentrations.

Downregulation of the HERG (KCNH2) K(+) channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation.

The HERG (KCNH2) potassium channel underlies the rapid component of the delayed rectifier current (I(kr)), a current contributing to the repolarisation of the cardiac action potential. Mutations in HERG can cause the hereditary forms of the short-QT and long-QT syndromes, predisposing to ventricular arrhythmias and sudden cardiac death. HERG is expressed mainly in the cell membrane of cardiac myocytes, but has also been identified in cell membranes of a range of other cells, including smooth muscle and neurones. The mechanisms regulating the surface expression have however not yet been elucidated. Here we show, using stable HERG-expressing HEK 293 cells, that ceramide evokes a time-dependent decrease in HERG current which was not attributable to a change in gating properties of the channel. Surface expression of the HERG channel protein was reduced by ceramide as shown by biotinylation of surface proteins, western blotting and immunocytochemistry. The rapid decline in HERG protein after ceramide stimulation was due to protein ubiquitylation and its association with lysosomes. The results demonstrate that the surface expression of HERG is strictly regulated, and that ceramide modifies HERG currents and targets the protein for lysosomal degradation.

Tumor necrosis factor alpha and ceramide depolarise the resting membrane potential of thyroid FRTL-5 cells via a protein kinase Czeta-dependent regulation of K+ channels.

Tumor necrosis factor alpha (TNFalpha) alters the electrophysiological properties of many cell types. In thyroid cells however, the effects have not yet been elucidated. Here, we report the effect of TNFalpha and its second messenger ceramide on the resting membrane potential (RMP) of thyroid FRTL-5 cells. In patch-clamp experiments, we showed that TNFalpha and ceramide depolarise the RMP by inhibiting an acid-sensitive inwardly rectifying potassium current. This depolarisation depended on the activation of protein kinase Czeta (PKCzeta), because it can be blocked by calphostin C, a PKC-inhibitory peptide and a specific inhibitor peptide for PKCzeta. The activation of PKCzeta was confirmed by Western blotting, in which a stimulation with TNFalpha led to the translocation of PKCzeta to the particulate fraction. We conclude that TNFalpha and ceramide depolarise the RMP of thyroid FRTL-5 cells by attenuating a Ba(2+)- and acid-sensitive potassium conductance via activation of PKCzeta.

Functional characterization of the common amino acid 897 polymorphism of the cardiac potassium channel KCNH2 (HERG).

OBJECTIVE: To determine whether the amino acid 897 threonine (T) to lysine (K) polymorphism of the KCNH2 (HERG) potassium channel influences channel performance or patient phenotype. METHODS: The phenotypic effects of this polymorphism were investigated in vitro by electrophysiological experiments in HEK-293 cells and in vivo by exercise electrocardiography in a group of LQTS patients carrying the same genetically proven KCNQ1 mutation. RESULTS: When expressed in HEK-293 cells, the 897T isoform of the KCNH2 channel exhibited changes in inactivation and deactivation properties, and a smaller current density than the more common 897K isoform. Western blot experiments indicated that the decreased current density associated with 897T was caused by reduced channel expression. During a maximal exercise test in 39 LQT1 patients carrying an identical KCNQ1 mutation (G589D) and showing a prolonged QT interval (>440 ms), QT intervals were longer in patients carrying the 897T allele than in those homozygous for the 897K allele. CONCLUSIONS: The K897T variation has an effect on channel function and clinical phenotype. Our data warrant further investigations into the significance of this polymorphism in drug-induced and inherited LQTS.