Reevaluating safety pharmacology respiratory studies within the ICH S7A core battery: A multi-company evaluation of preclinical utility and clinical translation.

Optimization of ICH safety guideline studies for inclusion into regulatory submissions is critical for resource conservation, animal use reduction, and efficient drug development. The ICH S7A guidance for Safety Pharmacology (SP) studies adopted in 2001 identified the core battery of studies to evaluate the acute safety of putative pharmaceutical molecules prior to First in Human (FIH) trials. To assess the utility of respiratory studies in predicting clinical AE's, seven pharmaceutical companies pooled preclinical and clinical respiratory findings. A large database of novel molecules included all relevant data from standard S7A respiratory (n = 459) and FIH studies (n = 309). The data were analyzed with respect to the progression of these molecules, clinical adverse event reporting of these same molecules, and achieved exposures. These S7A respiratory assay findings had no impact on compound progression, and only 12 of 309 drug candidates were 'positive' preclinically and reported a respiratory-related AE in clinical trials (i.e. cough, dyspnea, etc.), an overall incidence rate of 3.9%. Contingency tables/statistics support a lack of concordance of these preclinical assays. Overall, our extensive analysis clearly indicated that the preclinical respiratory assay fails to provide any prognostic value for detecting clinically relevant respiratory adverse events.

Adrenalectomy reduces neuropeptide Y-induced insulin release and NPY receptor expression in the rat ventromedial hypothalamus.

Chronic central administration of neuropeptide Y (NPY) causes hyperphagia, hyperinsulinemia, and obesity, a response that is prevented by prior adrenalectomy (ADX) in rats. The basis of NPY's effect and how the acute responses to this peptide are affected by ADX remain unknown. This study investigates the role of glucocorticoids in acute NPY-stimulated food intake, acute NPY-induced insulin release, and hypothalamic NPY-receptor mRNA expression levels. NPY-induced food intake was similar in ADX and control rats after acute intracerebroventricular injection of NPY. Injection of NPY caused a significant increase in plasma insulin in control rats, but this effect was completely absent in ADX rats in which basal plasma insulin levels were also lower than controls. In addition, ADX significantly reduced the number of neurons expressing NPY receptor Y(1) and Y(5) mRNAs in the ventromedial hypothalamus (VMH), without affecting Y(1)- or Y(5)-mRNA expression in the paraventricular hypothalamus or the arcuate nucleus. These data indicate that glucocorticoids are necessary for acute NPY-mediated insulin release and suggest that the mechanisms involve glucocorticoid regulation of Y(1) and Y(5) receptors specifically within the VMH nucleus.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

2-36[K4,RYYSA(19-23)]PP a novel Y5-receptor preferring ligand with strong stimulatory effect on food intake.

Members of the neuropeptide Y (NPY) family regulate many physiological processes via interaction with at least four functional, pharmacologically distinct Y-receptors. However, selective antagonists developed for several subtypes have not been useful in defining particular Y-receptor functions in vivo. To identify critical residues within members of the NPY family required for Y-receptor subtype-selectivity we have determined the contribution of each residue within NPY to receptor binding by replacing them with L-alanine. In a second study, chimeric peptides where single or stretches of residues were interchanged between members of the NPY family were generated and tested in radioligand binding studies. Overall, substituted alanine analogues exhibited similar orders of affinities at each Y-receptor subtype with no obvious subtype-selectivity. Residues of particular interest are Leu30 which exhibited selectivity for the Y4-receptor, whereas Asp16 does not appear to play any role in ligand binding. Several chimeric peptides, e.g., [K4]pancreatic polypeptide ([K4]PP) and [RYYSA(19-23)]PP clearly showed higher affinity at the Y4 and Y5 subtypes compared to the Y1 and Y2 subtypes. In addition, the transfer of a proline residue from position 14 to 13 in peptide YY decreases its affinity at the Y1-, Y4- and Y5-receptors but is unchanged at the Y2 subtype. Combining these results, and with the help of molecular modelling, second generation chimeras were designed. The most significant improvement was achieved in chimera 2-36[K4,RYYSA(19-23)]PP where the affinity for the Y5 subtype increased by ninefold over that from NPY. Several of these compounds were also tested for their ability to stimulate food intake in a rat model. Interestingly, again 2-36[K4,RYYSA(19-23)]PP showed the most dramatic effect with a major increase on food intake over a range of doses compared to NPY suggesting a possible synergistic effect of several Y-receptors on feeding behaviour.

Effect of hypercapnic acidemia on anisotropic propagation in the canine ventricle.

BACKGROUND: Impulse propagation in the ventricle depends on both sodium channel availability and cell-to-cell coupling through gap junctions. Sodium channel block has been shown to depress conduction velocity (theta) more longitudinal (LONG) to than transverse (TRANS) to fiber orientation. Because exposure to CO2 produces intracellular acidosis and decreased gap junction conductance in vitro, we tested the hypothesis that increased PCO2 would result in preferential depression of transverse conduction in vivo. METHODS AND RESULTS: In anesthetized dogs, when atrial pH was reduced to 6.70 +/- 0.04 by increasing the fraction of inhaled CO2 to 40%, theta TRANS fell from 0.23 +/- 0.04 to 0.19 +/- 0.02 m/s (-16 +/- 8%, P < .03), while theta LONG was unchanged (-3 +/- 7%, P = NS). In contrast, with the same degree of acidemia produced by HCl infusion, only theta LONG fell (-8 +/- 7%), coincident with a rise in serum K+. CONCLUSIONS: The observed effect of CO2 on propagation in the intact heart is consistent with its previously described in vitro actions to uncouple cell-to-cell communication and may provide a model to study the role of cell-to-cell coupling in normal and abnormal propagation.

Virtual cathode effects during stimulation of cardiac muscle. Two-dimensional in vivo experiments.

We have found that when suprathreshold cathodal stimuli were applied to the epicardium of canine ventricle, impulse propagation originated at a "virtual cathode" with dimensions greater than those of the physical cathode. We report the two-dimensional geometry of the virtual cathode as a function of stimulus strength; the results are compared with the predictions of an anisotropic, bidomain model of cardiac conduction recently developed in our laboratories. Data were collected in six pentobarbital-anesthetized dogs by using a small plaque electrode sewn to the left ventricular epicardium. Arrival times at closely spaced bipolar electrodes oriented radially around a central cathode were obtained as a function of stimulus strength and fiber orientation. The dimensions of the virtual cathode were determined by linear back-extrapolation of arrival times to the time of stimulation. The directional dependence of the conduction velocity was consistent with previous reports: at 1 mA, longitudinal (0 degree) and transverse (90 degrees) velocities were 0.60 +/- 0.03 and 0.29 +/- 0.02 m/sec, respectively. At 7 mA, the longitudinal velocity was 0.75 +/- 0.05 m/sec, whereas there was no significant change in the transverse velocity. In contrast to conduction velocity, the virtual cathode was smallest in the longitudinal orientation and largest between 45 degrees and 60 degrees. Virtual cathode size was dependent on both orientation and stimulus strength: at 0 degree, the virtual cathode was small (approximately 1 mm) and relatively constant over the range of 1-7 mA; at oblique orientations (45 degrees-90 degrees), it displayed a roughly logarithmic dependence on stimulus strength, approximately 1 mm at 1 mA and approximately 3 mm at 7 mA. The bidomain, anisotropic model reproduced both the stimulus strength and the fiber-orientation dependence of the virtual cathode geometry when the intracellular and extracellular anisotropies were 10:1 and 4:1, respectively, but not when the two anisotropies were equal. We suggest that the virtual cathode provides a direct measure of the determinants of cardiac activation; its complex geometry appears to reflect the bidomain, anisotropic nature of cardiac muscle.

Suppression of longitudinal versus transverse conduction by sodium channel block. Effects of sodium bolus.

BACKGROUND: Arrhythmias resulting from treatment with sodium channel-blocking antiarrhythmic drugs have been successfully treated with sodium infusion, although the mechanism underlying this effect is uncertain. METHODS AND RESULTS: In this study, we used a multielectrode array to examine the effects of O-desmethyl encainide (ODE), a potent sodium channel-blocking metabolite of encainide, on conduction in canine ventricle. ODE depressed both longitudinal and transverse conduction velocities in a plasma concentration-related fashion (r = -0.74, -0.60; p less than 0.001). At ODE concentrations less than or equal to 300 ng/ml (n = 34), conduction velocity was depressed to the same extent in the longitudinal (-21.9 +/- 8.4%, SD) and transverse orientations (-22.0 +/- 8.8%). However, at concentrations greater than 300 ng/ml (n = 17), conduction was significantly more impaired longitudinally than transversely (-44.5 +/- 11.7% versus -34.4 +/- 13.7%, p less than 0.02). In 12 animals with high concentrations (mean, 432 +/- 32 ng/ml), a 5-meq/kg bolus of sodium chloride over 1 minute immediately increased conduction velocity; this effect was significantly greater and longer lasting in the longitudinal orientation. In two animals, conduction block in the longitudinal orientation was documented at high plasma ODE and was immediately reversed by sodium bolus. CONCLUSIONS: We conclude that the major effect of sodium in animals with excess sodium channel block is improvement of longitudinal propagation; this effect may underlie the antiarrhythmic action of sodium in the analogous clinical setting.

Phase shifting of circadian rhythms and depression of neuronal activity in the rat suprachiasmatic nucleus by neuropeptide Y: mediation by different receptor subtypes.

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slices in vitro (n = 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5-8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration-response relationships and very different phase-response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAA antagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13-36 and (Cys2,8-aminooctanoic acid5,24,D-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [D-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.