Effects of the ventilatory stimulant, doxapram on human TASK-3 (KCNK9, K2P9.1) channels and TASK-1 (KCNK3, K2P3.1) channels.

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.

Identification and Characterization of GAL-021 as a Novel Breathing Control Modulator.

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 KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1 mice lacking the pore-forming α-subunit of the KCa1.1 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 KCa1.1 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.

GAL-021 and GAL-160 are Efficacious in Rat Models of Obstructive and Central Sleep Apnea and Inhibit BKCa in Isolated Rat Carotid Body Glomus Cells.

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.

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist.

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.

1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia.

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.

Inhibition of soluble epoxide hydrolase attenuates endothelial dysfunction in animal models of diabetes, obesity and hypertension.

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.

Discovery of potent, selective, and orally bioavailable 3H-spiro[isobenzofuran-1,4'-piperidine] based melanocortin subtype-4 receptor agonists.

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.

Discovery of a spiroindane based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor agonist.

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.

Characterization of a novel and selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b, in rodents.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

Benzazepinone Nav1.7 blockers: potential treatments for neuropathic pain.

A series of benzazepinones were synthesized and evaluated as hNa(v)1.7 sodium channel blockers. Several compounds from this series displayed good oral bioavailability and exposure and were efficacious in a rat model of neuropathic pain.

Discovery of a novel class of benzazepinone Na(v)1.7 blockers: potential treatments for neuropathic pain.

A series of benzodiazepines and benzazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of the benzazepinone series, compound 47, displayed potent, state-dependent block of hNa(v)1.7, and was orally efficacious in a rat model of neuropathic pain.

Antiobesity efficacy of a novel cannabinoid-1 receptor inverse agonist, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364), in rodents.

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding K(i) of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma C(max) of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30-40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Lead optimization of 5,6-diarylpyridines as CB1 receptor inverse agonists.

Optimization of the biological activity for 5,6-diarylpyridines as CB1 receptor inverse agonists is described. Food intake and pharmacokinetic evaluation of 3f and 15c indicate that these compounds are effective orally active modulators of CB1.

Cyclopentane-based human NK1 antagonists. Part 1: discovery and initial SAR.

An initial investigation of the novel cyclopentane scaffold 6 afforded low nanomolar human NK1 antagonists having enhanced water solubility properties compared to morpholine 1. A synthesis of this cyclopentane scaffold, having three contiguous chiral centers, and the unexpected determination that the 1,2-trans-2,3-trans-ring stereochemistry, as opposed to the cis-ether/phenyl configuration of the known structures 1-5, is optimal for this class of antagonist are described.

Cyclopentane-based human NK1 antagonists. Part 2: development of potent, orally active, water-soluble derivatives.

The synthesis and optimization of a cyclopentane-based hNK1 antagonist scaffold 3, having four chiral centers, will be discussed in the context of its enhanced water solubility properties relative to the marketed anti-emetic hNK1 antagonist EMEND (Aprepitant). Sub-nanomolar hNK1 binding was achieved and oral activity comparable to Aprepitant in two in vivo models will be described.

Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice.

Ghrelin, an acylated peptide secreted from the stomach, acts as a short-term signal of nutrient depletion. Ghrelin is an endogenous ligand for the GH secretagogue receptor 1a, a G protein-coupled receptor expressed in the hypothalamus and pituitary. We used a synthetic oligonucleotide, NOX-B11-2, capable of specific high-affinity binding to bioactive ghrelin to determine whether ghrelin neutralization would alter indices of energy balance in vivo. This novel type of ghrelin-blocking agent, called an RNA Spiegelmer (SPM), is a polyethylene glycol-modified l-RNA oligonucleotide, the nonnatural configuration of which confers in vivo stability. NOX-B11-2 blocked ghrelin mediated activation of GH secretagogue receptor 1a in cell culture (IC50 approximately 5 nm). We explored the effects of acute NOX-B11-2 administration on ghrelin-induced feeding in mice. NOX-B11-2 (66 mg/kg, sc) blocked ghrelin-induced feeding and was without effect on feeding evoked by an orally active nonpeptide ghrelin receptor agonist. We demonstrated that selective ghrelin blockade effectively promoted weight loss in diet-induced obese (DIO) mice. Chronic infusion of NOX-B11-2 (33 mg/kg.d, sc) to DIO mice evoked body weight loss for 13 d and reduced food intake and fat mass relative to control SPM-infused mice. In a 7-d study, DIO mice infused with NOX-B11-2 (33 mg/kg.d, sc) showed body weight loss, compared with animals receiving control SPM. This effect was directly mediated by SPM neutralization of ghrelin because NOX-B11-2 administration to ghrelin-deficient mice resulted in no weight loss. The decreased obesity observed in SPM-treated DIO mice provides validation for ghrelin neutralization as a potential antiobesity therapy.

Synthesis of functionalized 1,8-naphthyridinones and their evaluation as novel, orally active CB1 receptor inverse agonists.

Synthesis, SAR, and binding affinities are described for a new class of 1,8-naphthyridinone CB1 receptor specific inverse agonists. Food intake, knockout mouse, and pharmacokinetic evaluation of 14 indicate that this compound is an effective orally active modulator of CB1.

Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior.

The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels (NaV1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes NaV1.9. Our results confirm that NaV1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of NaV1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

Discovery and activity of (1R,4S,6R)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-2-methyl-2-azabicyclo[2.2.2]octane-6-carboxamide (3, RY764), a potent and selective melanocortin subtype-4 receptor agonist.

A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.