1 Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M 2 subtype whereas the remaining minority belong to the M 3 subtype. The objective of the present study was to assess the functional role of M 2 and M 3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2 In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC 50 =6.3) which were unaected by tetrodotoxin (0.1 mM). These contractions were antagonized by muscarinic antagonists with the following rank order of anity (pA 2 ) estimates: atropine (9.1) 4 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) 4 darifenacin (8.5) 4 para¯uoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) 4 pirenzepine (6.8) 4 methoctramine (5.9). These pA 2 estimates correlated most favourably (r=0.99, P50.001) with the binding anity (pK i ) estimates of these compounds at human recombinant muscarinic m 3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically de®ned M 3 receptor. 3 As M 2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M 2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M 3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 mM) to protect M 2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC 50 =5.8) but had marginal eects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA 2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M 2 and M 3 receptors and were consistent with the involvement of both of these subtypes. 4 In urethane-anaesthetized rats, the cholinergic component (*55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID 35%inh , nmol kg 71 , i.v.): 4-DAMP (8.1) 4 atropine (20.7) 4 methoctramine (119.9) 4 darifenacin (283.3) 4 pirenzepine (369.1) 4 p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pK i estimates of these compounds at human recombinant muscarinic m 2 receptors. This is consistent with a major contribution of M 2 receptors in the generation of volumeinduced bladder contractions, although the modest potency of darifenacin does not exclude a role of M 3 receptors. Pretreatment with propranolol (1 mg kg 71 , i.v.) increased the ID 35%inh of methoctramine signi®cantly from 95.9 to 404.5 nmol kg 71 but had...

No abstract

No abstract

CB1 cannabinoid receptor antagonists have therapeutic utility in CNS as well as metabolic disorders. We identified multiple novel structural classes of CB1 antagonists and characterized a compound called AC‐462 in detail. AC‐462 is structurally distinct from rimonabant and other biarylpyrazoles, utilizing a dibenzothiazepine structure as its scaffold. The binding Ki's of AC‐462 to human and rat CB1 receptors are 0.2 and 2 nM, respectively. AC‐462 is highly selective for CB1 receptors over CB2, and over 60 other GPCRs, transporters and ion channels. AC‐462 has pro‐cognitive actions in radial arm maze and novel object recognition paradigms and augments apomorphine‐induced rotations in the 6‐hydroxy dopamine lesion model of Parkinson's disease. AC‐462 reverses CP 55,940 induced analgesia and hypothermia in mice, and is orally active as an appetite suppressant, reducing food intake in fasted Sprague‐Dawley rats. Once daily dosing of AC‐462 to Zucker rats over 14‐days significantly decreased body weight compared to vehicle‐treated animals. A large number of analogs of AC‐462 with similar in vitro and in vivo activities were produced. One of these analogs called AC‐999 produced robust decreases in body weight of diet‐induced obese (DIO) mice dosed orally qd over 14‐days. These results demonstrate that AC‐462 and its analogs have therapeutic potential for a wide array of indications.

The atypical antipsychotics share the common attributes of working on serotonin and dopamine receptors. However, a comprehensive mechanism of action of these agents is unknown. In order to better elucidate the mechanism by which these drugs produce their therapeutic and the undesired effects, a robust method for measuring in vivo receptor binding is needed. The authors developed a liquid chromatography/triple quad mass spectral detectors (LC/MS/MS) method to measure the brain distribution of receptor occupancy using tracers targeting dopamine D2, serotonin 5‐HT2A, 5‐HT2C and muscarinic M1 receptors. The tracers are raclopride, MDL‐100907, SB‐221284 and pirenzepine, respectively. All four non‐radiolabeled tracers were detectable in discrete rat brain areas after intravenous administration. These tracers demonstrated a differential brain distribution corresponding to the regional differences in their respective receptor densities. Oral pretreatment of various antipsychotic agents that occupy these receptors decreased this differential distribution in a dose‐dependent manner. Our results demonstrate the utility of LC/MS/MS to quantify the in vivo receptor occupancy of antipsychotic and other agents acting in the CNS.