Cardiovascular safety assessments in the conscious telemetered dog: utilisation of super-intervals to enhance statistical power.

INTRODUCTION: ICH S7A and S7B guidelines recommend the use of conscious animals for assessment of non-clinical cardiovascular safety of new chemical entities prior to testing in humans. Protocol design and data analysis techniques can affect the quality of the data produced and can therefore ultimately influence the clinical management of cardiovascular risk. It is therefore essential to have an understanding of the magnitude of changes detectable and the clinical relevance of these changes. This paper describes the utilisation of "super-intervals" to analyse and interpret data obtained from our conscious telemetered dog cardiovascular safety protocol and reports the statistical power achieved to detect changes in various cardiovascular parameters. METHODS: Cardiovascular data from 18 dog telemetry studies were used to calculate the statistical power to detect changes in cardiovascular parameters. Each study followed a test compound versus vehicle cross-over experimental design with 24h monitoring (n=4). 1 min mean raw data from each individual animal was compressed into 15 min mean data for each dose group for visualisation. Larger summary periods, or "super-intervals", were then selected to best represent any observed cardiovascular effects whilst taking into account the pharmacokinetic profile of the drug e.g. intervals of 1 to 6, 7 to 14 and 14 to 22h post-dose. RESULTS: With this methodology and study design we predict, using the median percentile that our studies have 80% power to detect the following changes: HR (+/-10bpm), LV +dP/dt max (+/-375mmHg/s), MBP (+/-5mmHg) and QTc (+/-4ms). DISCUSSION: Super-intervals are a simple way to handle the high degree of natural variability seen with any ambulatory cardiovascular assessment and, in our hands, result in highly statistically powered studies. The ability of this model to detect cardiovascular changes of small, but biologically relevant, magnitude enables confident decision making around the cardiovascular safety of new chemical entities.

Reflex penile erection in anesthetized mice: an exploratory study.

Ejaculatory-like rhythmic contractions of the bulbospongiosus (BS) muscle and penile erection can be elicited in the urethane-anesthetized rat, following spinal cord transection, upon electrical stimulation (ES) of the dorsal penile nerve (DPN). The aim of this work was to investigate this reflex in anesthetized mice. Adult C57BL6 mice were anesthetized with isoflurane. The BS muscle and corpus cavernosum were instrumented to allow quantification of the BS muscle electromyographic activity (BS EMG) and intracavernosal pressure respectively. The femoral artery and jugular vein were catheterized to allow measurement of blood pressure and compound administration. ES of the DPN, via bipolar silver electrodes, reliably evoked erectile responses in mice with intact spinal cords. The overall amplitude of the erectile response was frequency- and pulse duration-dependent. Erectile responses were abolished by bilateral cut of the sensory branch of the pudendal nerve. Transection of the spinal cord potentiated the erectile responses and increased the area under the curve of the BS EMG when compared with those animals with intact spinal cords. However, no coordinated rhythmic contractions of the BS muscle during or after the ES could be observed, with or without spinal transection. Melanotan-II failed to enhance the erectile response induced by ES of the DPN, in mice with intact spinal cords. ES of the DPN in isoflurane-anesthetized mice could be a useful model in which to study the interplay between brain and spinal cord in the control of reflex penile erection, and could take advantage of knockout mice models. However, the lack of efficacy of Melanotan-II suggests that further experiments are necessary to confirm the future utility of this model. In contrast to rats, the expulsion reflex could not be reliably elicited in mice with or without spinal transection. This latter finding suggests the existence of fundamental differences in the organization of the spinal network controlling sexual reflexes between rats and mice.

The roles of nitric oxide in dilating proximal and terminal arterioles of skeletal muscle during systemic hypoxia.

Indirect evidence suggests that dilatation evoked by systemic hypoxia in proximal and terminal arterioles of skeletal muscle is mediated by adenosine acting on A(1) receptors, but that the dilatation of proximal arterioles requires the presence of nitric oxide (NO), whereas that of terminal arterioles is mediated by NO. In the present study, we showed that primary and terminal arterioles of spinotrapezius muscle (diameters: 45 +/- 4 and 9 +/- 1 microm, respectively) in anaesthetised rats dilated by 15 +/- 7 and 48 +/- 24%, respectively, during hypoxia (breathing 12% O(2)). Inhibition of NO synthesis with intravenous nitro-L-arginine methyl ester attenuated these responses (to -2 +/- 3 and -4 +/- 4%, respectively). However, when a tonic level of NO was subsequently restored by infusion of NO donor, hypoxia evoked dilatation of primary arterioles (+24 +/- 8%) but not terminal arterioles (0 +/- 4%). The adenosine A(1) receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (intravenous) attenuated this 'restored' primary arteriolar vasodilatation (to +6 +/- 3%). Similar results were obtained with 8% O(2). We propose that (1) proximal arteriolar dilatation evoked by systemic hypoxia depends on a background level of NO, which facilitates the action of adenosine on A(1) receptors, and (2) hypoxia-evoked dilatation of terminal arterioles is mediated by NO synthesised via A(1) receptor stimulation.

Oxygen delivery and oxygen consumption in rat hindlimb during systemic hypoxia: role of adenosine.

1. In anaesthetised rats, the increase in femoral vascular conductance (FVC) evoked by moderate systemic hypoxia is mediated by adenosine acting on A(1) receptors. It is also nitric oxide (NO) dependent: it is attenuated by NO synthase (NOS) inhibition, but restored when baseline FVC is restored by sodium nitroprusside (SNP), a NO donor. However, under these conditions there was in increase in the critical O(2) delivery (D(O2,crit)) at which hindlimb O(2) consumption (V(O2)) becomes directly dependent upon O(2) delivery (D(O2)), indicating that V(O2) is regulated by newly synthesised NO. 2. In the present study, after NOS inhibition, when baseline FVC was restored with SNP infusion, the increases in FVC evoked by breathing 12 and 8 % O(2) were reduced by the A(1) receptor antagonist DPCPX, by 60 and 40 %, respectively (n = 8). The A(2A) receptor antagonist ZM241385 reduced the FVC increase evoked by 12 % O(2) (by 45 %, n = 8), but did not alter that evoked by 8 % O(2). 3. DPCPX also reduced the increases in FVC evoked by graded systemic hypoxia, breathing 14-6 % O(2) and increased D(O2,crit), from 0.64 +/- 0.06 to 0.95 +/- 0.07 ml O(2) min(-1) kg(-1) (control vs. DPCPX). However, ZM241385 (n = 8) had no effect on the FVC increases or on D(O2,crit) (0.70 +/- 0.02 ml O(2) min(-1) kg(-1), n = 8). 4. Thus, the increases in FVC evoked by mild to severe systemic hypoxia are mediated by A(1) receptors. These responses, which are attributable to proximal arteriolar dilatation, help maintain D(O2). Even after NOS inhibition, adenosine still increases FVC via A(2A) (moderate hypoxia only) and A(1) receptors, providing baseline levels of NO are present. Furthermore, adenosine, acting via A(1) receptors, is important in determining D(O2,crit) and therefore in maintaining V(O2). We propose that this is achieved by A(1)-evoked dilatation of terminal arterioles and is mediated by increased synthesis of NO.

Vasodilatation, oxygen delivery and oxygen consumption in rat hindlimb during systemic hypoxia: roles of nitric oxide.

We have investigated the relationship between O2 delivery (DO2) and O2 consumption (VO2) in hindlimb muscle of anaesthetised rats during progressive systemic hypoxia. Since muscle vasodilatation that occurs during hypoxia is nitric oxide (NO) dependent, we examined the effects of the NO synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME). In control rats (n = 8), femoral vascular conductance (FVC) increased at each level of hypoxia. Hindlimb DO2 decreased with the severity of hypoxia, but muscle VO2 was maintained until the critical DO2 value (DO2,crit) was reached at 0.64 +/- 0.06 ml O2 min-1 kg-1; below this VO2 declined linearly with DO2. This is a novel finding for the rat but is comparable to the biphasic relationship seen in the dog. In another group of rats (n = 6), L-NAME caused hindlimb vasoconstriction and attenuated the hypoxia-evoked increases in FVC DO2 was so low after L-NAME administration that VO2 was dependent on DO2 at all levels of hypoxia. In a further group (n = 8), femoral blood flow and DO2 were restored after L-NAME by infusion of the NO donor sodium nitroprusside (20 g x kg(-1) x min(-1). Thereafter, hypoxia-evoked increases in FVC were fully restored. Nevertheless, DO2,crit was increased relative to control (0.96 +/- 0.07 ml O2 min(-1) x kg(-1), P < 0.01). As NOS inhibition limited the ability of muscle to maintain VO2 during hypoxia, we propose that hypoxia-induced dilatation of terminal arterioles, which improves tissue O2 distribution, is mediated by NO. However, since the hypoxia-evoked increase in FVC was blocked by L-NAME but restored by the NO donor, we propose that the dilatation of proximal arterioles is dependent on tonic levels of NO, rather than mediated by NO.

Effects of tumour necrosis factor-alpha on left ventricular function in the rat isolated perfused heart: possible mechanisms for a decline in cardiac function.

1. The cardiac depressant actions of TNF were investigated in the isolated perfused rat heart under constant flow (10 ml min(-1)) and constant pressure (70 mmHg) conditions, using a recirculating (50 ml) mode of perfusion. 2. Under constant flow conditions TNF (20 ng ml(-1)) caused an early (< 25 min) decrease in left ventricular developed pressure (LVDP), which was maintained for 90 min (LVDP after 90 min: control vs TNF; 110 +/- 4 vs 82 +/- 10 mmHg, P < 0.01). 3. The depression in cardiac function seen with TNF under constant flow conditions, was blocked by the ceramidase inhibitor N-oleoylethanolamine (NOE), 1 microM, (LVDP after 90 min: TNF vs TNF with NOE; 82 +/- 10 vs 11 +/- 5 mmHg, P < 0.05). 4. In hearts perfused at constant pressure, TNF caused a decrease in coronary flow rate (change in flow 20 min after TNF: control vs TNF; -3.0 +/- 0.9 vs -8.7 +/- 1.2 ml min(-1), P < 0.01). This was paralleled by a negative inotropic effect (change in LVDP 20 min after TNF: control vs TNF; -17 +/- 7 vs -46 +/- 6 mmHg, P < 0.01). The decline in function was more rapid and more severe than that seen under conditions of constant flow. 5. These data indicate that cardiac function can be disrupted by TNF on two levels, firstly via a direct, ceramidase dependant negative inotropic effect, and secondly via an indirect coronary vasoconstriction.

Effects of tumour necrosis factor-alpha on the coronary circulation of the rat isolated perfused heart: a potential role for thromboxane A2 and sphingosine.

1. The actions of tumour necrosis factor-alpha (TNF-alpha) on the coronary circulation were investigated in the rat isolated heart, perfused under constant flow, recirculating conditions. 2. An early increase in coronary perfusion pressure (CPP) was observed upon treatment with TNF-alpha (increase in CPP 10 min after TNF-alpha treatment: 45+/-12 mmHg vs control: 15+/-4 mmHg, P<0.05). The role of sphingosine, prostanoids and endothelins, in this coronary constrictor action, was investigated with the use of pharmacological inhibitors and antagonists. 3. The TNF-alpha induced increase in coronary tone was blocked by indomethacin, 10 microM (increase in CPP after 10 min: 13+/-4 mmHg vs TNF-alpha alone, P<0.05). 4. The thromboxane receptor antagonist GR32191, 10 microM, attenuated the TNF-alpha induced coronary constriction (12+/-2 mmHg vs TNF-alpha alone, P<0.05), as did the joint thromboxane A2 synthesis inhibitor and receptor antagonist ZD1542, 10 microM (8+/-1 mmHg vs TNF-alpha alone, P<0.05). 5. The ceramidase inhibitor N-oleoylethanolamine (NOE), 1 microM, also blocked the TNF-alpha induced response (8+/-4 mmHg vs TNF-alpha alone, P<0.05). 6. In contrast, the coronary constrictor action of TNF-alpha was not inhibited by the endothelinA/B receptor antagonist bosentan, 3 microM (38+/-9 mmHg vs TNF-alpha, P=NS). 7. These data indicated that the early coronary vasoconstriction induced by TNF-alpha was mediated by both thromboxane A2 and sphingosine, suggesting an interaction between both the sphingomyelinase and phospholipase A2 metabolic pathways.

Selective inhibition of pyrimidine biosynthesis and effect on proliferative growth of colonic cancer cells.

A highly selective inhibition of de novo pyrimidine synthesis in the intact cell has been demonstrated by the action of N-(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of the reaction catalyzed by asparate transcarbamylase. The effect of pyrimidine deprivation induced by this agent on the viability and survival of human normal (WI-38) and colonic cancer cells (HT-29) was examined. The PALA-treated, pyrimidine-deprived cells failed to grow but demonstrated a normal rate of glucose utilization with impaired glycogen synthesis. Pyrimidine deprivation and lack of cell growth were maintained long after PALA removal. Growth inhibition of HT-29 cells by PALA was found to reflect an apparent steady state between newly formed and dying cells induced by limited pyrimidine availability. The highly selective nature of PALA action was confirmed by the ability of an exogenous source of pyrimidine to restore the normal growth pattern of the cell. Significant antitumor activity of PALA was found against a transplantable colonic tumor (line 26) carried in mice.