Ruth A. Duffy

The gerbil elevated plus-maze II: anxiolytic-like effects of selective neurokinin NK1 receptor antagonists.

Neurokinin NK1 receptor antagonists may have therapeutic potential in the treatment of anxiety and depression. Species variants in the NK1 receptor result in reduced affinity of NK1 receptor antagonists at rat and mouse NK1 receptors, making it difficult to test NK1 antagonists in traditional preclinical models of anxiety and depression. Gerbil NK1 receptors are similar in homology to the human NK1 receptor. In a companion article, we described the anxiety-like behavioral profile of gerbils on an adapted elevated plus-maze, and the ability of anxiolytic drugs to produce anti-anxiety effects in the gerbil elevated plus-maze. The aim of the present study was to determine whether oral (p.o.) administration of the NK1 receptor antagonists MK-869, L-742,694, L-733,060, CP-99,994, and CP-122,721 produced anxiolytic-like effects in the gerbil elevated plus-maze. Upon testing, all five NK1 antagonists produced anxiolytic-like effects. MK-869 (0.01–3 mg/kg) was the most potent NK1 antagonist, producing anxiolytic-like effects on percentage of open arm time, percentage of open arm entries, stretch-attend postures, and head dips at 0.03–0.3 mg/kg doses. L-742,694 (1–30 mg/kg) and L-733,060 (1–10 mg/kg) produced anxiolytic-like effects on percentage of open arm time and stretch-attend postures at 3–10 mg/kg doses. CP-99,994 (3–30 mg/kg) only produced an anxiolytic-like effect on stretch-attend postures. CP-122,721 (3–30 mg/kg) produced an anxiolytic-like effect on percentage of open arm time at 30 mg/kg. The order of potency of the NK1 antagonists to increase percentage of open arm time was very similar to their potency to block NK1 agonist-induced foot-tapping. These studies demonstrate that neurokinin NK1 receptor antagonists produce anxiolytic-like effects in a novel gerbil elevated plus-maze, and suggest that this is an appropriate model to test NK1 antagonists for preclinical anxiolytic activity.

Design and synthesis of piperidinyl piperidine analogues as potent and selective M2 muscarinic receptor antagonists.

Identification of a number of highly potent M2 receptor antagonists with >100-fold selectivity against the M1 and M3 receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain acetylcholine release after oral administration.

Serotonergic component of SCH 23390: in vitro and in vivo binding analyses

A series of benzazepines related to SCH 23390 were tested for binding to the 5HT-2 receptor. The compounds tested inhibited the binding of 3H-ketanserin with KI values generally greater than those observed for the D-1 receptor, but less than those for the D-2 receptor. When this serotonergic activity was correlated to the D-1 activity, the resulting coefficient was 0.84, indicating a strong correlation between the two activities. Conversely, the 5HT-2 activity did not show a good correlation with the D-2 activity. To further test the significance of the 5HT-2 binding of the SCH 23390, in vivo binding studies were performed using 125I-SCH 38840 in the frontal cortex, an area containing both D-1 and 5HT-2 receptors. The in vivo binding of 125I-SCH 38840 to frontal cortex exhibited peak levels one hour following subcutaneous administration, similar to the time course previously observed in striatum. The binding was both D-1 and tissue specific. Competition studies with selected standards demonstrated that inhibition of the binding to frontal cortex, in contrast to the inhibition observed in the striatum, exhibited a Hill coefficient less than unity, implying interaction at more than one receptor subtype. When SCH 23390 and ketanserin were administered simultaneously, the inhibition of the in vivo binding of 125I-SCH 38840 to striatum was not different than that observed with SCH 23390, alone. However, the inhibition of binding to frontal cortex was significantly greater than that demonstrated with either SCH 23390 or ketanserin, alone, suggesting that 125I-SCH 38840 was binding to both D-1 and 5HT-2 receptors, in vivo.

Muscarinic agonists and antagonists in the treatment of Alzheimer's disease☆

Alzheimers disease (AD) is a neurodegenerative disease characterized by cognitive impairment and personality changes. The development of drugs for the treatment of the cognitive deficits of AD has focused on agents which counteract loss in cholinergic activity. Although symptoms of AD have been successfully treated with acetylcholinesterase inhibitors (tacrine, donepezil. rivastigmine, galanthamine), limited success has been achieved with direct M1 agonists, probably due to their lack of selectivity versus other muscarinic receptor subtypes. Muscarinic M2 antagonists have been reported to increase synaptic levels of acetylcholine after oral administration to rats (e.g. BIBN-99, SCH-57790), but their selectivity versus other muscarinic receptor subtypes is modest. Exploration of a series of piperidinylpiperidines has yielded the potent and selective M2 antagonist SCH-217443. This antagonist has excellent bioavailability in rats and dogs and shows activity in a rat model of cognition.

Cloning, expression, and pharmacological characterization of the GPR120 free fatty acid receptor from cynomolgus monkey: Comparison with human GPR120 splice variants

Activation of the GPCR GPR120 by free fatty acids has been reported to cause GLP-1 release in rodent intestine. One genetic sequence was reported for rodents, while two sequences were reported for human GPR120, BC101175 and NM_181745. A 1086 base pair sequence cloned from cynomolgus monkey colon cDNA has 85.1% and 83.4% homology with the mouse and rat GPR120 sequences, and 97.5% homology with the human BC101175 sequence. No splice variants of the cynomolgus monkey GPR120 receptor were found. Eight non-synonymous cSNPs were discovered with frequencies less than 4% in monkey samples tested. Real-time PCR demonstrated that, like the human, the highest GPR120 expression in cynomolgus monkey is in lung and colon. Studies measuring intracellular calcium release produced by free fatty acids and the small molecule GPR120 agonist GW9508 in cells expressing the cynomolgus monkey GPR120 receptor were compared to those expressing the human BC101175 splice variant. Long-chain free fatty acids produced the greatest response in cynomolgus monkey GPR120-expressing cells. GW9508 had similar efficacy at the cynomolgus monkey and at the BC101175 human GPR120 receptors. The cynomolgus monkey and the human GPR120 (BC101175) receptors have similar sequences and pharmacology. The possible significance of the alternate splice variant in human is discussed.

Design and synthesis of ether analogues as potent and selective M2 muscarinic receptor antagonists

Novel, selective M2 muscarinic antagonists, which replace the metabolically labile styrenyl moiety of the prototypical M2 antagonist 1 with an ether linkage, were synthesized. A detailed SAR study in this class of compounds has yielded highly active compounds that showed M2 Ki values of < 1.0 nM and >100-fold selectivity against M1, M3, and M5 receptors.

Identification of novel cannabinoid CB1 receptor antagonists by using virtual screening with a pharmacophore model.

CB1 receptor antagonists have proven to be clinically effective in treating obesity and related disorders. We report here the identification of a novel class of azetidinone CB1 antagonists by using virtual screening methods. For this purpose, we developed a pharmacophore model based on known representative CB1 antagonists and employed it to screen a database of about a half million Schering-Plough compounds. We applied a stepwise filtering protocol based on molecular weight, compound availability, and a modified rule-of-five to reduce the number of hits. We then combined Bayesian modeling and clustering techniques to select a final set of 420 compounds for in vitro testing. Five compounds were found to have >50% inhibition at 100 nM in a CB1 competitive binding assay and were further characterized by using both CB1 and CB2 assays. The most potent compound has a CB1 K i of 53 nM and >5-fold selectivity against the CB2 receptor.

Facilitation of acetylcholine release and improvement in cognition by a selective M2 muscarinic antagonist, SCH 72788.

Current treatment of Alzheimers Disease (AD) requires acetylcholinesterase inhibition to increase acetylcholine (ACh) concentrations in the synaptic cleft. Another mechanism by which ACh levels can be increased is blockade of presynaptic M2 muscarinic autoreceptors that regulate ACh release. An antagonist designed for this purpose must be highly selective for M2 receptors to avoid blocking postsynaptic M1 receptors, which mediate the cognitive effects of ACh. Structure-activity studies of substituted methylpiperadines led to the synthesis of 4-[4-[1(S)-[4-[(1,3-benzodioxol-5-yl)sulfonyl]phenyl]ethyl]-3(R)-methyl-1-piperazinyl]-4-methyl-1-(propylsulfonyl)piperidine. This compound, SCH 72788, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 0.5 nM, and its affinity at M1 receptors is 84-fold lower. SCH 72788 is a functional M2 antagonist that competitively inhibits the ability of the agonist oxotremorine-M to inhibit adenylyl cyclase activity. In an in vivo microdialysis paradigm, SCH 72788 increases ACh release from the striatum of conscious rats. The compound is also active in a rodent model of cognition, the young rat passive avoidance response paradigm. The effects of SCH 72788 suggest that M2 receptor antagonists may be useful for treating the cognitive decline observed in AD and other dementias.

[3H]SCH 39166, A New D1‐Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 {(‐)‐trans‐6,7,7a,8,9,13b‐hexahydro‐3‐chloro‐2‐hydroxy‐N‐ methyl‐5H ‐ benzo ‐[d]naphtho[2,1b]azepine} has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable. high‐affinity fashion, with a KDof 0.79 nM. In competition studies, D1‐selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further. when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.

SCH 57790: A novel M2 receptor selective antagonist

As a decrease in cholinergic neurons has been observed in Alzheimers Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.