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Drug-induced cardiac arrhythmia, specifically Torsades de pointes, is associated with QT/QTc interval prolongation, thus prolongation of the QT interval is considered as a biomarker for Torsades de pointes risk (N Engl J Med 350:1013-1022, 2004). Specific inhibition of human ether-a-go-go-related gene (hERG) potassium channels has been recognized as the main mechanism for QT prolongation (Cardiovasc Res 58:32-45, 2003). This mechanism has been demonstrated for a variety of small-molecule agents, which access the inner pore of the hERG channel preferentially from inside the cell. Peptide inhibitors of hERG, such as BeKm-1, interact with the extracellular amino acid residues close to the external pore region of the channel. In this study, the isolated rabbit heart was used to assess whether BeKm-1 could induce QTc prolongation like dofetilide and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (E-4031). Five hearts were perfused with 10 and 100 nM BeKm-1 sequentially. ECG parameters and left ventricular contractility were measured with spontaneously beating hearts. Both concentrations of BeKm-1 prolonged QTc intervals significantly and concentration-dependently (4.7 and 16.3% at 10 and 100 nM, respectively). When evaluated for their inhibitory effect in a hERG functional assay, BeKm-1, dofetilide, and E-4031 caused QTc prolongation at concentrations that caused significant hERG channel inhibition. Lastly, two polyclonal anti-hERG antibodies were also assessed in the hERG channel assay and found to be devoid of any inhibitory effect. These results indicated that the isolated rabbit heart assay can be used to measure QTc changes caused by specific hERG inhibition by peptides that specifically block the external pore region of the channel.

Besipirdine displays potent adrenergic activity in a variety of pharmacological and behavioral tests. Based on this property, we evaluated the effects of besipirdine and its N-despropyl metabolite N-despropyl-besipirdine (P7480) on cardiovascular function in rats and dogs. Besipirdine and P7480 bind alpha-2 adrenoceptors (K(I): 380 and 10 nM, respectively) and facilitate the stimulated release of [3H]norepinephrine from rat cortical slices due to presynaptic autoreceptor blockade. In rat aorta rings and the pithed rat, P7480, but not besipirdine, also behaved as a postsynaptic alpha-1 adrenoceptor agonist. In conscious rats, besipirdine (2-10 mg/kg, p.o.) and P7480 (3-10 mg/kg, p.o.) produced dose-related increases in mean arterial pressure. Inhibition of hepatic cytochrome P-450 enzyme activity blocked the pressor effect of besipirdine, but not of P7480; therefore, P7480 mediated besipirdine's pressor effect. The bradycardia after either agent was unaffected. In conscious dogs, besipirdine (0.1-2 mg/kg, p.o.) also produced dose-related hypertension and bradycardia. The hypertension, but not the bradycardia, were sensitive to prazosin (3 mg/kg, p.o.), but not hexamethonium (10 mg/kg, p.o.). Muscarinic and beta-adrenergic receptor blockade studies in anesthetized dogs demonstrated the bradycardia to be due to withdrawal of cardiac sympathetic tone. These findings suggest that besipirdine's peripheral hypertensive effect is primarily mediated by the pressor metabolite P7480, although facilitated norepinephrine release may contribute. Besipirdine's bradycardic action appears to be centrally mediated, because both compounds lacked direct negative chronotropic activity on spontaneously beating guinea pig atria in vitro.

1-[(3-Fluoro-4-pyridinyl)amino]-3-methyl-1(H)-indol-5-yl methyl carbamate (P10358) is a potent, reversible acetylcholinesterase inhibitor that produces central cholinergic stimulation after oral and parental administration in rats and mice. P10358 is a 2.5 times more potent acetylcholinesterase inhibitor than THA in vitro (IC50 = 0.10 +/- 0.02 microM vs. IC50 = 0.25 +/- 0.03 microM). It also inhibits butyrylcholinesterase activity as potently as THA (IC50 = 0.08 +/- 0.05 microM vs. IC50 = 0.07 +/- 0.01 microM). Ex vivo, P10358 (0.2 - 20 mg/kg, p.o.) produced dose-dependent inhibition of brain acetylcholinesterase activity. At 10 and 20 mg/ kg, it produced profound and long-lasting hypothermia in mice. P10358 enhanced performance in rats in a step-down passive avoidance task (0.62 and 1.25 mg/kg) and in a social recognition paradigm (0.32, 0.64 and 1.25 mg/kg) in mice. It reversed scopolamine-induced deficits in the Morris Water maze in rats (1.25 and 2.5 mg/kg) and a higher dose elevated striatal homovanillic acid levels. These behavioral and biochemical effects are consistent with central cholinergic stimulation. Hemodynamic studies in the rat demonstrated a 16-fold separation between behaviorally active doses (1.25 mg/kg) and those that elevated arterial pressure (20 mg/kg). Lethality in rats occurred at an oral dose of 80 mg/kg, but not at lower doses. Chemically, P10358 is an N-aminoindole and may not have the hepatotoxic liability associated with aminoacridine structure of tacrine. P10358 had weak affinity (>10 microM) at a variety of aminergic and peptidergic receptors and uptake carriers. These properties suggest that P10358 may be a safe and promising symptomatic treatment for Alzheimer's disease.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Previous studies suggest that systemic arterial pressure is tonically regulated by the interaction of peripheral sympathetic nerves with vascular alpha-1A adrenoceptors in vivo. To explore this relationship further, the present study examined the inhibitory effect of selective alpha-1A [5-methylurapidil (5-MU) and nifedipine (NIF)] and alpha-1B [chloroethylclonidine (CEC)] antagonists on the pressor response to electrical stimulation (ES) of the spinal cord in pithed rats. Diastolic pressure changes were measured in the presence of 5-MU or CEC and compared with control responses. Pretreatment with 5-MU (0.5 mg/kg i.v.) significantly suppressed the ES pressor response (50-80% inhibition) at all stimulation frequencies. Likewise, NIF (inhibitor of calcium influx associated with alpha-1A adrenoceptor activation) selectively inhibited the pressor response to ES to the same degree as did 5-MU. CEC (25 mg/kg i.v.) also significantly shifted the ES response curve; however, this effect was mediated by activation of presynaptic alpha-2 receptors on sympathetic terminals because prior administration of idazoxan (5 mg/kg) prevented the inhibitory effect of CEC. Based on the potent inhibitory effects of 5-MU and NIF on the ES pressor response in the pithed rat, it was concluded that vascular alpha-1A adrenoceptors reside in the synaptic region of neurovascular junction where they are primarily activated by neuronal norepinephrine release.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of vascular alpha 1B-adrenergic receptors in the regulation of arterial pressure (MAP) and heart rate (HR) was examined by assessing the effect of i.v. chloroethylclonidine (CEC; irreversible alpha 1B antagonist) in unanesthetized, normotensive Long-Evans rats. MAP, HR and the pressor response to i.v. phenylephrine (PE) were monitored for 24 hr after saline or CEC (15 mg/kg and 25 mg/kg) injection. Neither i.v. saline nor CEC affected MAP or HR throughout the course of the study, yet the PE response was maximally inhibited (> 75% at 15 min) by both doses of CEC. The PE response recovered by 2 hr at the 15-mg/kg dose but remained inhibited up to 4 hr at 25 mg/kg. At 24 hr, all cardiovascular parameters returned to control levels. CEC (100 microM, 30 min) produced irreversible blockade of norepinephrine-induced contractions in rat aortic rings; prazosin (10 nM) and sodium thiosulphate (1 mM, a reagent that inactivates aziridinium ions) reversed CEC's inhibitory effect. Precyclized CEC and its hydrolysis product beta-hydroxyethylclonidine (beta-HEC) poorly antagonized aortic alpha 1B-receptors. Ex vivo analysis of aortic rings from saline and CEC-treated rats showed that PE-induced contractions were shifted to the right and maximally depressed in a dose-dependent manner after 24 hr. These results suggest that 1) CEC produces long lasting blockade of alpha 1B-adrenoceptors in vitro and in vivo via formation of an aziridinium ion intermediate and 2) vascular alpha 1B-adrenoceptors are not coupled to the tonic physiological regulation of MAP in the rat.

Phenylcyclohexyldiethylamine (PCDE) is an analog of phencyclidine with low affinity for the N-methyl-d-aspartate receptor that is metabolized to an active monoethyl derivative, phenylcyclohexylethylamine (PCE). In a pharmacokinetic analysis of the ataxia response of rats to i.p. administered PCDE and PCE, ataxia intensity was determined together with plasma and cerebrospinal fluid concentrations of the drugs. The role of PCE as the active metabolite of PCDE was assessed quantitatively by correlating the response with both the plasma and cerebrospinal fluid drug levels. Increased PCE concentrations in the cerebrospinal fluid and plasma were associated with increased ataxia response when either PCDE or PCE was the administered drug. However, the concentration-response curves did not superimpose and the curve after PCDE was shifted to the left of that after PCE, suggesting that PCDE was contributing an effect not accountable by PCE concentration. This apparent potentiation must involve an interaction at sites other than the N-methyl-daspartate receptor. In the analysis of the behavior responses, PCDE was found to induce a greater backpedalling response which has been attributed to interaction with dopamine or serotonin systems, suggesting that other transmitter systems may contribute to the overall ataxia response.

The relative potencies of the argininolytic agents NG-methyl-L-arginine (L-NMA), NG-nitro-L-arginine (L-NNA) and NG-amino-L-arginine (L-NAA) were assayed by their inhibitory effect on both basal and stimulated release of endothelium-derived NO in vitro and in vivo. Basal NO release was indirectly assessed by the ability of the analogs to contract phenylephrine-preconstricted rat aortic rings and their ability to produce a hypertensive response in awake, unanesthetized normotensive rats. In aortic rings, the three analogs induced vasocontraction and inhibited the vasorelaxation mediated by ACh-stimulated endothelial NO release. In this latter assay, L-NNA was 30 times more potent than either L-NMA or L-NAA. In free-moving rats, the agents caused dose-dependent increases in arterial pressure due to the blockade of endogenous NO formation. Dose-response analysis indicated that L-NNA was 87 and 230 times more potent than L-NMA and L-NAA, respectively. Pretreatment with L-NNA was also found to selectively inhibit, but not abolish, the depressor effects of acetylcholine in unanesthetized and phenylephrine- or vasopressin-infused normotensive-pithed rats. These studies indicate that L-NNA is a potent antagonist of endothelium-derived relaxing factor formation in vitro and in vivo. The contractile and hypertensive effects of the argininolytic agents clearly demonstrates that a continuous basal release of endothelium-derived relaxing factor/NO occurs in both isolated vascular rings and whole animals.

Some tertiary antimuscarinic amines related to oxotremorine were compared with atropine and scopolamine for their ability to block physostigmine-induced hypertension and acetylcholine-induced hypotension in rats. These cardiovascular responses are mediated via muscarinic receptors in the brainstem and on the vasculature, respectively. In urethane-anesthetized rats, physostigmine (77 nmol/kg i.v.) increased mean arterial pressure 40 +/- 5 mm Hg. One hour later, each rat received a single dose of antimuscarinic, then a second bolus of physostigmine 10 min later. Linear regression analysis of antagonist dose-percent inhibition curves showed that all the agents inhibited physostigmine's pressor effect, thus indicating an ability to antagonize brain muscarinic receptors. BM-5 and BoK-1 were estimated to be equipotent with atropine, all having 50% inhibitory doses (ID50) in the 1.5 to 1.8 mumol/kg range. BR-370 and DKJ-21 were 3- and 30-fold less potent, respectively, than atropine in this assay, while scopolamine was the most potent (ID50: 0.039 mumol/kg). In contrast with atropine and scopolamine, the oxotremorine analogs possessed selective central antimuscarinic effects and were completely unable to antagonize the peripheral depressor response. Like atropine, only BM-5 showed significant tachycardiac effects, which suggested that this agent possessed affinity for myocardial muscarinic receptors. These findings support the concept that congeners of oxotremorine behave as centrally selective antimuscarinics at doses which have low peripheral antimuscarinic activity in vivo. This unique pharmacological profile makes them novel probes to study central cholinergic muscarinic mechanisms in cardiovascular regulation.