John C. Gordon

The 5-HT3 antagonist tropisetron (ICS 205-930) is a potent and selective α7 nicotinic receptor partial agonist

The 5-HT3 receptor antagonist tropisetron (ICS 205-930) was found to be a potent and selective partial agonist at alpha7 nicotinic receptors. Two other 5-HT3 receptor antagonists, ondansetron and LY-278,584, were found to lack high affinity at the alpha7 nicotinic receptor. Quinuclidine analogues (1 and 2) of tropisetron were also found to be potent and selective partial agonists at alpha7 nicotinic receptors.

Selective α7 nicotinic receptor activation by AZD0328 enhances cortical dopamine release and improves learning and attentional processes

AZD0328, a novel spirofuropyridine neuronal nicotinic receptor partial agonist, was used to investigate the role of alpha7 neuronal nicotinic receptor (NNR) activation in the modulation of midbrain dopamine neuron function, cortical dopamine release and on two behavioral tasks known to be dependent on optimal levels of cortical dopamine. In vivo recordings from area 10 (ventral tegmental area) in rat brain showed an increased firing of putative dopamine neurons in response to low (0.00138 mg/kg) doses of AZD0328. Bursting patterns of dopamine neuron activity remained largely unchanged by application of AZD0328. In vivo microdialysis in awake rats showed an increase in extracellular prefrontal cortical dopamine in response to low doses of AZD0328. Compound-stimulated dopamine release showed an inverted dose effect relation that was maximal at the lowest dose tested (0.00178 mg/kg). Peak extracellular dopamine levels were reached 2h after dosing with AZD0328. Acquisition of operant responding with delayed reinforcement in rats was dose dependently enhanced by AZD0328 with a plateau effect measured at 0.003 mg/kg. This effect was blocked by pre-treatment of animals with the selective alpha7 antagonist methyllycaconitine. AZD0328 improved novel object recognition in mice over a broad range of doses (0.00178-1.78 mg/kg) and the compound effect was found to be absent in homozygous alpha7 KO animals. Together, these data indicate that selective interaction with alpha7 NNRs by AZD0328 selectively enhances midbrain dopaminergic neuronal activity causing an enhancement of cortical dopamine levels; these neurochemical changes likely, underlie the positive behavioral responses observed in two different animal models. Our results suggest selective alpha7 NNR agonists may have significant therapeutic utility in neurologic and psychiatric indications where cognitive deficits and dopamine neuron dysfunction co-exist.

Association of [3H]zacopride with 5-HT3 binding sites.

An assay was developed for [3H]zacopride binding to 5-HT3 specific sites in membranes from rabbit ileum muscularis. The binding was rapid, saturable, reversible, salt-insensitive, unaffected by pH between 6.5 and 9.5, and of high affinity (apparent KD = 0.65 +/- 0.15 nM). ICS 205-930, a potent 5-HT3 antagonist that inhibited competitively, was utilized to define 5-HT3 specific binding. Other 5-HT3 antagonists and agonists, although exhibiting marked differences in potency, were also effective inhibitors; whereas, antagonists of other classes of serotonin receptors, guanyl nucleotides and numerous receptor-specific ligands, including peptide hormones, were inactive. Vagus nerve exhibited the greatest amount of 5-HT3 specific binding amongst rabbit tissues and virtually all of the [3H]zacopride was bound to 5-HT3 binding sites. In rabbit, rat and ferret a fairly uniform distribution of 5-HT3 binding sites was observed along the muscularis of the small bowel. [3H]Zacopride is a high-affinity ligand for detecting 5-HT3 binding sites and rabbit small bowel muscularis membranes are a sensitive system for evaluating the potency of 5-HT3 antagonists or agonists.

Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity.

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications.

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; μ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; μ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo.

SAR development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as kappa opioid receptor antagonists. Part 2

Further structure activity relationship studies on a previously reported 8-azabicyclo[3.2.1]octan-3-yloxy-benzamide series of potent and selective kappa opioid receptor antagonists is discussed. Modification of the pendant N-substitution to include a cyclohexylurea moiety produced analogs with greater in vitro opioid and hERG selectivity such as 12 (kappa IC50=172 nM, mu:kappa ratio=93, delta:kappa ratio=>174, hERG IC50=>33 microM). Changes to the linker conformation and identity as well as to the benzamide ring moiety were also investigated.

Solubilization of a 5-HT3 binding site from rabbit small bowel muscularis membranes

A 5-HT3 binding site, with high affinity for (S-)[3H]zacopride, was solubilized from rabbit small bowel muscularis membranes utilizing 0.5% sodium cholate and 400 mM (NH4)2SO4. Approximately 72% of the (S-)[3H]zacopride binding activity was recovered in a form that retained the high affinity (Kd = 0.7 nM) and specificity for this radioligand that is characteristic of the membrane-bound receptor. ICS 205-930 and other 5-HT3 compounds were effective inhibitors and exhibited the same rank order of potency in the solubilized and membrane-bound preparations. The receptor-detergent complex did not sediment after centrifugation for 1 h at 150,000 x g and eluted between thyroglobulin (MW = 669,000) and apoferritin (MW = 443,000) when fractionated by high-performance liquid chromatography gel filtration. This is the first report of the solubilization of a 5-HT3 binding site.

Synthesis of epibatidine isomers: endo-5- and 6- (6′-chloro-3′-pyridyl-2-azabicyclo[2.2.1]heptanes

Synthesis of the title compounds is described; detailed NMR data are provided in support of the proposed stereostructures. The 5- and 6-endo-compounds show high selectivity for α4β2 versus α7 nAChR subtypes; in contrast, the exo-stereoisomers show comparatively weak affinity at both subtypes.