Aims The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK-1 and TASK-1/TASK-3 heterodimer channels, than TASK-3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK-1 and TASK-3. Methods Whole-cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK-1, TASK-3 and TASK-2 channels. Mutations of amino acids in the pore-lining region of TASK-3 channels were introduced using site-directed mutagenesis. Results Doxapram was an equipotent inhibitor of human TASK-1 and TASK-3 channels, compared with mouse channel variants, where it was more selective for TASK-1 and heterodimers of TASK-1 and TASK-3. The effect of doxapram could be attenuated by either the removal of the C-terminus of human TASK-3 channels or mutations of particular hydrophobic residues in the pore-lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK-3, zinc. The positive enantiomer of doxapram, GAL-054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL-053, had little inhibitory effect. Conclusion These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK-1 and TASK-3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents.

GAL-021 and GAL-160 are alkylamino triazine analogues, which stimulate ventilation in rodents, non-human primates and (for GAL-021) in humans. To probe the site and mechanism of action of GAL-021 and GAL-160 we utilized spirometry in urethane anesthetized rats subjected to acute bilateral carotid sinus nerve transection (CSNTX) or sham surgery. In addition, using patch clamp electrophysiology we evaluated ionic currents in carotid body glomus cells isolated from neonatal rats. Acute CSNTX markedly attenuated and in some instances abolished the ventilatory stimulant effects of GAL-021 and GAL-160 (0.3 mg/kg IV), suggesting the carotid body is a/the major locus of action. Electrophysiology studies, in isolated Type I cells, established that GAL-021 (30 μM) and GAL-160 (30 μM) inhibited the BK(Ca) current without affecting the delayed rectifier K(+), leak K(+) or inward Ca(2+) currents. At a higher concentration of GAL-160 (100 μM), inhibition of the delayed rectifier K(+) current and leak K(+) current were observed. These data are consistent with the concept that GAL-021 and GAL-160 influence breathing control by acting as peripheral chemoreceptor modulators predominantly by inhibiting BK(Ca) mediated currents in glomus cells of the carotid body.

Background: The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator.Methods: The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve-transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays.Results: GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited K(Ca)1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1(-/-) mice lacking the pore-forming -subunit of the K(Ca)1.1 channel.Conclusions: GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting K(Ca)1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

Doxapram is a clinical respiratory stimulant but is limited by analeptic and cardiovascular adverse events (AEs). We hypothesized that the enantiomers of doxapram may diverge with respect to efficacy and tolerability. In the 1st series of experiments, we administered vehicle, doxapram (4, 20, & 40 mg/kg, IV) and equivalent doses of GAL‐054 and GAL‐053 (each at 2, 10, & 20 mg/kg, IV) to conscious rats. Doxapram and GAL‐ 054 dose‐dependently increased tidal volume, respiratory frequency, and minute ventilation (Vmin) compared to vehicle, whereas GAL‐053 did not. The magnitudes of the effect of 20 mg/kg doxapram and 10 mg/kg GAL‐054 on Vmin were equivalent. All rats that received 40 mg/kg doxapram seized and died, whereas 2 of 6 rats that received 20 mg/kg GAL‐053 seized and one died. In the 2nd series of experiments, all 3 compounds (each at 1 – 30 mg/kg, IV) were administered to anesthetized rats. The ED50 values for doxapram and GAL‐054 for increasing Vmin were 6.4 and 1.8 mg/kg, respectively, but GAL‐053 was essentially inactive. Dysrhythmias were observed with doxapram and GAL‐ 053. These data indicate that the (+) enantiomer, GAL‐054, retains all of the respiratory stimulant properties of doxapram, whereas the AEs (dysrhythmias, seizures, and death) are restricted to the (−) enantiomer, GAL‐053. Thus, of the enantiomers of doxapram, GAL‐054, is the eutomer, and displays a markedly higher therapeutic index than GAL‐053.

Opioid-induced respiratory depression (OIRD) is a common side effect of opioid analgesics and can be life-threatening, especially during the perioperative period and sleep. Current therapy for OIRD includes administering opioid antagonists and/or decreasing subsequent opioid doses, both of which can compromise analgesia. Here, we describe a novel respiratory stimulant, GAL-021, that increases minute ventilation (Vmin) and reverses OIRD without diminishing analgesia. GAL-021 administered as an IV bolus (0.01 – 3 mg/kg) to anesthetized rats or by infusion (0.01 – 0.4 mg/kg/min × 60-min) to conscious rats increased Vmin, tidal volume (VT), and respiratory frequency (f). In anesthetized rats, the ED50 for increasing Vmin was 140 μg/kg. In conscious rats, OIRD was induced with morphine (10 mg/kg, IV), which increased PaCO2 and decreased Vmin, VT, f, pH, and PaO2 for up to 2 hours. Subsequent infusion of GAL-021 (0.03 – 0.4 mg/kg/min, IV) resulted in dose-dependent diminution of the evoked respiratory depression for the duration of the infusion (20 min). The increase in Vmin induced by GAL-021 was due to an increase in VT but not f. Termination of GAL-021 infusion restored OIRD. In contrast, GAL-021 did not diminish morphine-induced analgesia assessed by the tail-flick assay. These data demonstrate that GAL-021 stimulates breathing in conscious and anesthetized rats and reverses OIRD without attenuating analgesia.

GAL‐021 is a novel respiratory stimulant that increases minute ventilation in rats, mice, and Cynomolgus monkeys. We hypothesized that GAL‐021 increases minute ventilation via mechanisms at the level of the carotid body. Using in vivo electrophysiological techniques, we examined the dose‐dependence of the effects of GAL‐021 (0.01 – 3 mg/kg, IV) on carotid sinus nerve (CSN) activity and phrenic motoneuron activity in mechanically ventilated, vagotomized, and neuromuscularly paralyzed adult rats, and on tidal volume (VT) in anesthetized spontaneously breathing rats. GAL‐021 dose‐dependently increased CSN (ED50 0.13 mg/kg) and phrenic nerve neurogram amplitude (ED50 0.08 mg/kg), values in reasonable accord with effects on VT (ED50 0.04 mg/kg). Thus, GAL‐021 is virtually equipotent in stimulating the afferent, efferent and effector components of the ventilatory response. When administered to conscious rats 7 days after either bilateral CSN transection or sham operation, the ventilatory effects (VT and frequency) of GAL‐021 (0.2 mg/kg, IV) were markedly diminished in the CSN transected rats compared to sham‐operated controls. Collectively, these data suggest that GAL‐021 stimulates breathing, at least in part, by effects on the carotid body resulting in increased carotid sinus nerve activity.

GAL‐021 is a novel respiratory stimulant that increases minute ventilation (Vmin) in rats and Cynomolgus monkeys. Transecting the carotid sinus nerve decreases this effect in rats, suggesting that part of the evoked stimulation is mediated by effects at the level of the carotid body. BK (or Maxi K) channels, which consist of a pore‐forming α‐subunit (slo1) and various â regulatory subunits, are present in the carotid body and have been implicated in oxygen sensing. Hypoxia reportedly decreases BK potassium current and increases carotid sinus nerve activity. In preliminary studies using GH3 cells we demonstrated that GAL‐021 exerts modest inhibition of BK channels (56%I at 10 μM). To test the hypothesis that GAL‐021 may stimulate breathing via inhibition of BK channels, we administered GAL‐021 (0.03 ‐ 3 mg/kg, IV) as bolus injections to isoflurane anesthetized, wild‐type and BK á‐subunit knockout (Slo1−/−) mice. In wild‐type mice, GAL‐021 dose‐dependently increased Vmin (maximum response 143 ± 34 % above baseline; ED50 0.5 mg/kg) consistent with prior studies in rats and monkeys. In Slo1−/− mice the effects of GAL‐021 on Vmin (maximum response 64 ± 24 % above baseline; ED50 2.1 mg/kg) were significantly attenuated (but not abolished) in comparison to the wild‐type. These data suggest that BK channels contribute to the effects of GAL‐021 on ventilation but that non‐BK‐dependent mechanisms are also involved.

We have demonstrated that the (+) enantiomer of doxapram, GAL‐ 054, is the respiratory stimulant eutomer in drug naïve rats and that its tolerability profile is superior to the (−) enantiomer (or distomer), GAL‐053. Given that doxapram is used clinically to reverse drug‐induced respiratory depression, we hypothesized that GAL‐054 would also be superior to GAL‐053 at reversing opioid‐induced respiratory depression (OIRD). Tidal volume (VT), respiratory frequency (f), minute ventilation (Vmin), and arterial pH and blood gases were measured from conscious rats using whole‐body plethysmography and blood gas analysis before and after bolus morphine injection and then during subsequent doxapram, GAL‐054, and GAL‐053 infusion. Morphine (10 mg/kg, IV) decreased Vmin, by decreasing both VT, and f. GAL‐ 054 (1.0 and 3.0 mg/kg/min) infusion reversed OIRD in a dose‐dependent manner by increasing VT, but not f. In contrast, GAL‐ 053 infusion had minimal effects on breathing in morphine‐treated rats. As expected, morphine increased PaCO2 and decreased pH and PaO2. Doxapram (3 mg/kg/min) and GAL‐054 (3 mg/kg/min) diminished these effects, whereas GAL‐053 infusion (3 mg/kg/min) did not. No adverse events were observed for doxapram or either enantiomer during this study. Collectively, these data support the classification of GAL‐054 as the respiratory stimulant eutomer and GAL‐053 as the distomer of racemic doxapram.

Opioid‐induced respiratory depression (OIRD) is a common side effect of opioid‐induced analgesia and can be life‐threatening, especially during the perioperative period and sleep. Current therapy for OIRD includes administering opioid antagonists and/or decreasing subsequent opioid doses, both of which can compromise analgesia. We have demonstrated that GAL‐021, a novel respiratory stimulant, increases minute ventilation (Vmin) and reverses OIRD in rats, without diminishing opioid‐induced analgesia. Here, we describe the effects of GAL‐021 in Cynomolgus monkeys. In isoflurane‐anesthetized opioid‐naïve monkeys, GAL‐021 administered by IV bolus (0.01 – 0.3 mg/kg) or infusion (0.02 – 0.1 mg/kg/min) increased Vmin, tidal volume, and respiratory frequency. In conscious animals, OIRD was induced by morphine (2.4 – 3.2 mg/kg, slow IV bolus), titrated in each animal to decrease Vmin by 20 to 30% and increase ETCO2 by 10 mmHg. Administration of GAL‐021 15‐min post‐morphine diminished OIRD in a dose‐dependent manner. Loading GAL‐021 at 0.1 mg/kg/min × 5 min and maintaining infusion at 0.05 mg/kg/min × 10 min reduced OIRD by 75%. In contrast, loading at 0.2 mg/kg/min × 5 min and maintaining infusion at 0.1 mg/kg/min × 10 min returned ETCO2 to pre‐morphine levels. These data demonstrate that GAL‐021 stimulates respiration in conscious and anesthetized monkeys and is capable of reversing OIRD in non‐human primates.

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.

Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension. It has been well established that epoxyeicosatrienoic acids (EETs) act as an endothelial derived hyperpolarization factor (EDHF). Soluble epoxide hydrolase (s-EH) rapidly hydrolyses certain epoxylipids (e.g. EETs) to less bioactive diols (DHETs), thereby attenuating the evoked vasodilator effects. The aim of the present study was to examine if inhibition of s-EH can restore impaired endothelial function in three animal models of cardiometabolic diseases. Isolated vessel rings of the aorta and/or mesenteric artery from mice or rats were pre-contracted using phenylephrine or U46619. Endothelium-dependent and independent vasorelaxation to acetylcholine and sodium nitroprusside (SNP) were measured using wire myography in vessels isolated from db/db or diet-induced obesity (DIO) mice, and angiotensin II-induced hypertensive rats treated chronically with s-EH inhibitors AR9281 or AR9276 or with vehicle. Vasorelaxation to acetylcholine, but not to SNP was severely impaired in all three animal models. Oral administration of AR9281 or AR9276 abolished whole blood s-EH activity, elevated epoxy/diol lipid ratio, and abrogated endothelial dysfunction in all three models. Incubating the mesenteric artery of db/db mice with L-NAME and indomethacin to block nitric oxide (NO) and prostacyclin formation did not affect AR9821-induced improvement of endothelial function. These data indicate that inhibition of s-EH ameliorates endothelial dysfunction and that effects in the db/db model are independent of the presence of NO and cyclooxygenase derived prostanoids. Thus, preserving vasodilator EETs by inhibition of s-EH may be of therapeutic benefit by improving endothelial function in cardiometabolic diseases.

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Improved methods for non-invasive in vivo assessment are needed to guide development of animal models of atherosclerosis and to evaluate target engagement and in vivo efficacy of new drugs. Using novel 3D-micro-ultrasound technology, we developed and validated a novel protocol for 3D acquisition and analysis of imaging to follow lesion progression in atherosclerotic mice. The carotid arteries of ApoE receptor knockout mice and normal control mice were imaged within the proximal 2mm from the aortic branch point. Plaque volume along that length was quantified using a semi-automated 3D segmentation algorithm. Volumes derived by this method were compared to those calculated using 3-D histology post-mortem. Bland-Altman comparison revealed close correlation between these two measures of plaque volume. Furthermore, using a segmentation technique that captures early positive and 33 week negative remodeling, we found evidence that plaque volume increases linearly over time. Each animal and each plaque served as its own control, allowing accurate comparison. The high fidelity anatomical registration of this protocol provides increased spatial resolution and therefore greater sensitivity for measurement of plaque wall size, an advance over 2-dimensional measures of intimal-medial-thickening. Further, 3-dimensional analysis ensures a point of registration that captures functional markers in addition to the standard structural markers that characterize experimental atherosclerosis. In conclusion, this novel imaging protocol provides a non-invasive, accurate surrogate marker for experimental atherosclerosis over the life of the entire lesion.