Temporal variability of QT interval and changes in T wave morphology in dogs as markers of the clinical risk of drug-induced proarrhythmia.

INTRODUCTION: The aim of this experiment was to establish the usefulness of assessing temporal variability of the QT interval and changes in the T wave morphology in dogs as markers of pro-arrhythmic risk in humans. For this purpose, the electrocardiographic effects of astemizole and cisapride, two well known pro-arrhythmic drugs in humans, were assessed in dogs. METHODS: Astemizole was administered intravenously at single doses of 1 and 3 mg/kg whilst cisapride was administered orally at doses of 1.5 and 6 mg/kg. Electrocardiograms (ECG) were recorded before and after treatment. From each ECG tracing, QT intervals were recorded over 100 beats for calculation of the mean and standard deviation (SD) of QT and mean corrected QT (QTc). The coefficient of variation of QT (CV=SD/mean) was calculated as an indicator of QT temporal variability. The changes in T wave morphology were assessed in precordial lead CV5RL. RESULTS: Astemizole increased both the QTc interval and the CV of QT. Increases in these parameters also occurred after cisapride, but were less marked than after astemizole. In addition, both compounds produced a notching of the T wave, consisting of the presence of two peaks. DISCUSSION: The effects of astemizole and cisapride on the CV of QT and their propensity to induce of T wave notching are consistent with the blocking of I(Kr) channels and indicate, respectively, an increase in temporal variability of cardiac repolarization and an increased heterogeneity of the repolarization of cardiac cells across the myocardium. These changes are key triggers of arrhythmic events and are thus consistent with the pro-arrhythmic properties of these drugs. This study therefore indicates that the evaluation of CV of QT and T wave morphology in dogs may help in predicting drug-induced pro-arrhythmic risk in humans.

Quantitative relationship between plasma potassium levels and QT interval in beagle dogs.

The aim of the current study was to establish the quantitative relationship between plasma potassium concentrations and the QT interval of the electrocardiogram in dogs. Furosemide, a potent diuretic, was given at increasing doses (5-60 mg/kg) to five male and five female beagle dogs. Electrocardiogram (ECG) was recorded three times each day, simultaneous to blood sampling for measurement of plasma potassium. Furosemide treatment produced a clear hypokalaemia, which was associated with an increase in QT and corrected QT intervals (QTc) duration. On average, the slopes of the negative linear correlation between potassium plasma levels and QT or QTc were steeper in females than in males. These results show that a decrease in potassium plasma level may explain a concomitant increase in QT duration in a toxicity study in dogs, in particular if potassium values are decreased below 3.3 mmol/L. Correction of QT interval for K+ plasma level has, therefore, been established separately for males and females. A global formula correcting QT for K+ and heart rate simultaneously was established. Hypokalaemia was also associated with changes in the morphology of the T wave recorded in CV5RL, in particular, with a flattening and/or a notching of the wave (appearance of a second peak), biphasic aspect or inversion of polarity. These changes are probably related to an increased heterogeneity of repolarization between different populations of cardiomyocytes. In conclusion, hypokalaemia is quantitatively associated with an increase in QT and QTc duration in dogs. The relationship is apparently stronger for females than for males. A formula may be used to correct QT for potassium plasma level.

The electrocardiogram of the Beagle dog: reference values and effect of sex, genetic strain, body position and heart rate.

The aim of the study was to establish a database for electrocardiographic parameters of Beagle dogs used for toxicological studies and to evaluate the influence of supplier, sex, heart rate (HR) and body position for electrocardiogram (ECG) recording on ECG parameters. Peripheral ECG leads were recorded from 934 female and 946 male dogs from Marshall Farms and 27 females and 30 males from Harlan, either standing on a table or restrained in a hammock. HR, RR, PQ and QT intervals, P and QRS duration and P-wave amplitude were measured. There were no major differences between sexes for ECG parameters. The axis of the heart was shifted to the left when the animals were restrained in a hammock compared to when they were standing on a table. The PQ interval was higher (about 9%) in Harlan than in Marshall dogs. HR was negatively correlated with QT (coefficient of linear correlation: r=-0.61 to -0.74), which emphasizes the need for a formula correcting QT interval for HR when interpreting changes in QT interval. HR was also negatively correlated with PQ intervals (r=-0.26 to -0.11), whereas a positive correlation was found between HR and the amplitude of the P wave (r=0.21-0.34). The level of the respiratory sinus arrhythmia (SA) was quantified by calculating the ratio of maximum to minimum RR interval measured over a 10 s period. This ratio was negatively correlated with HR (r =-0.49 to -0.33). Therefore, at high HRs, SA was less marked than at low HRs, but it did not completely disappear. Analysis of beat-to-beat variation indicated that QT and PQ intervals and the amplitude of P wave fluctuated over time and the degree of this variability was positively correlated with the level of SA. In conclusion, we have established reference values for the duration and/or amplitude of some ECG parameters both in terms of means and variability over the recording period, and we have evaluated the influence of body position, genetic strain and HR on the ECG parameters. These data can be used as baseline for the interpretation of the ECG of Beagle dogs.

Time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration.

Adverse drug reactions are a major clinical problem. Drug-induced hepatotoxicity constitutes a large percentage of these reactions. A thorough understanding of the genetic events, specifically, the early "decision-making" processes underlying biological changes caused by drugs and metabolites, is required. To assist in the understanding of these events, we have employed the model hepatotoxin, paracetamol (APAP), and GeneChip technology to investigate global genetic events seen after nontoxic and toxic doses in the mouse. Mice were dosed [vehicle, nontoxic APAP (1 mmol/kg), and toxic APAP (3.5 mmol/kg)], and individual hepatic RNA samples were hybridized to separate chips to determine interanimal variation. Statistical analysis detected 175 CD-1 mouse genes that were significantly regulated (P < 4.1 x 10(-6)), and nonsignificant genes were discarded. For clarity, the significantly regulated genes were then binned into categories according to their major function-antioxidant, glutathione, metabolism, transcription, immune, and apoptosis. There was no hepatic stress observed after dosing 1 mmol/kg APAP, when measured by serum alanine aminotransferase levels. Hepatic toxicity was observed at both 4 and 24 h after a 3.5 mmol/kg dose of APAP. Time course expression profiles for selected genes have been created. These results demonstrate that most active gene expression occurs around 4 h after a toxic dose of APAP. Down-regulation of these genes is observed over 24 h, coinciding with the development of overt toxicity. These data provide a deeper understanding of the in vivo time course of physiological responses of the liver to chemical stress and provide a logical step forward for the investigation of new chemical entities demonstrated positive in chemically reactive metabolite screens. The complete data set can be viewed at http://www.ebi.ac.uk/arrayexpress/. The accession number is E-MEXP-82.

Preclinical safety profile of sildenafil.

Sildenafil citrate, marketed as Viagra, for the treatment of erectile dysfunction, has a proven record of safety in humans as predicted by the results of extensive pharmacological and toxicological testing in animals and in vitro, and confirmed by pharmacokinetic exposure data. The aim of this paper is to review succinctly the main findings resulting from these experiments. Daily doses of sildenafil, within and far beyond the human therapeutic range, were given to dogs and rodents for up to 1 and 2 y, respectively. Plasma analyses were conducted to determine the exposure to sildenafil. We found species-specific effects in dogs (Beagle pain syndrome), mice (marked intestinal dilatation) and rats (adaptive reversible hepatocellular hypertrophy associated with secondary thyroid hypertrophy). All these effects in rodents and dogs have no relevance to humans. Morphometric thickness measurements of the retinal layers carried out in response to clinical observations of visual disturbances in humans indicated no difference between treated and control rats and dogs after up to 24 months of treatment. There was no evidence of histopathologic damage to any structures of the visual pathway. Sildenafil had no effects on fertility, no teratogenic potential, was not genotoxic and has no carcinogenic potential. In rats and dogs, safety ratios were 40:1 and 28:1, respectively, in terms of exposure over 24 h (AUC24 h) and 19:1 and 8:1, respectively, in terms of peak plasma concentration (Cmax). These safety ratios illustrate the separation between exposure to sildenafil of animals at large nontoxic doses and the much smaller human therapeutic exposure. This profile highlights the very low risk of human toxicity for sildenafil. The favourable results of the nonclinical safety evaluation of sildenafil in established animal models have been confirmed by many years of clinical experience during the development and marketing of sildenafil.

Echocardiography, a non-invasive method for the investigation of heart morphology and function in laboratory dogs: 1. Method and reference values for M-mode parameters.

We have set up M-mode echocardiographic (EC) recording in beagles in our laboratory and generated reference values for EC indicators of left ventricle function and morphology. Additionally we assessed the effects of sex, strain and body weight on these parameters and the correlation between parameters. M-mode EC under two-dimensional guidance in longitudinal section was performed on 59 male and 49 female beagles from Marshall (USA) and 13 males and 13 females from Harlan (France). The following parameters were measured or calculated: left ventricle internal diameter in diastole and systole (LVIDd and LVIDs), left ventricle and diastolic and end systolic and stroke volumes (EDV, ESV and SV), cardiac output and index (CO and CI), fractional shortening (FS), ejection fraction (EF), the thickness of the septum and left ventricle posterior wall in diastole (STd and LVPWd) and systole (STs and LVPWs), the percentage of thickening of the septum and left posterior wall (PST and PWT), and the mean and maximal velocities of the left ventricle posterior wall (PWVm and PWVM). Heart rate (HR) was measured by cardiac auscultation. Marshall dogs have higher left ventricle dimensions but lower amplitude and velocity of contraction than Harlan dogs. There were also statistically significant differences between sexes for a number of EC parameters mainly those relating to the size of the left ventricle walls or cavity in diastole. Overall these differences were explained by the correlation between these parameters and body weight. Heart rate correlated only with PWVm and PWVM. There were positive correlations between PST, PWT and EF or FS and between velocities and FS or EF. EDV correlates negatively with EF, FS, PST or PWT.

Echocardiography, a non-invasive method for the investigation of heart morphology and function in laboratory dogs: 2. Effects of minoxidil and quinidine on the left ventricle function.

We used echocardiography to investigate the changes in the cardiac function of dogs treated with minoxidil (a vasodilator, administered at doses which can produce mild lesions in the myocardium of the left ventricle) and quinidine (an antiarrhythmic at doses up to 8 times the upper limit of the therapeutic range in dogs). Groups of three beagles received a single administration of minoxidil at doses of 0.5 or 2 mg/kg. Two groups of two dogs received a single administration of quinidine at doses of 80 or 160 mg/kg. Two groups of three control dogs were treated concurrently with the vehicle alone. M-mode echocardiography was performed under two-dimensional echocardiography guidance on three occasions the day before treatment, immediately before dosing and 1, 3 and 24 h after dosing. We measured or calculated end diastolic, end systolic, and stroke volumes (EDV, ESV and SV), fractional shortening (FS), ejection fraction (EF), the percentage of thickening of the septum and of the left ventricle posterior wall (PST and PWT), and the mean and maximal velocities of the left ventricle posterior wall (PWVm and PWVM). At the same time as echocardiography recording, heart rate was measured by cardiac auscultation. Minoxidil produced a marked tachycardia. Less marked increases in heart rate occurred after quinidine. Both compounds were associated with a decrease in ESV and with marked increases in FS, EF, PWVm and PWVM which, in comparison with data for controls, are indicative of an increase in the amplitude and velocity of cardiac contraction. Both drugs also produced a decrease in EDV and consequently there was no increase in SV despite the increased amplitude of ventricular contraction. Cardiac output increased in proportion to the increase in heart rate. Overall, the effects were dose-related and are consistent with the pharmacological properties of the compounds. However, to date these effects have been demonstrated only by invasive methods. To conclude, we have shown that echocardiography allows a non-invasive investigation of the cardiac effects of suprapharmacological doses of antiarrhythmics and of the changes in heart function induced by vasodilators known to cause left ventricular lesions in dogs.

Binding of drugs to eye melanin is not predictive of ocular toxicity.

Ocular melanin is found in the uveal tract and in the pigmented epithelial layer of the retina. Many structurally and pharmacologically unrelated drugs from different therapeutic classes bind to melanin. Examples include numerous drugs acting on the central nervous system, beta-blockers, beta-agonists, antimalarial drugs, sympathomimetic amines, and antibiotics. The critical factors are the acid/base status and the lipophilicity of the molecule. In all cases, there are no direct consequences of drug-melanin binding. Drug-related toxic effects on the retina described in humans and animals are unrelated to melanin binding: melanin binding and retinal toxicity are two separate entities, the latter being related to the intrinsic toxicity of the compound rather than its ability to bind. Chloroquine and phenothiazines are often used as examples of drugs with retinal toxicity linked to melanin binding. In both cases however, experimental data show that the toxic mechanism is unrelated to binding. Melanin binding has also been found to be protective against the ocular toxicity of some drugs. In conclusion, we believe that potential ocular toxicity of future drugs can be assessed adequately by conducting well-designed toxicology studies, and using nonpigmented rodents in addition to pigmented nonrodent species remains fully justified. Binding of drugs to eye melanin is not predictive of ocular toxicity.

Characterization of the arteritis induced by infusion of rats with UK-61,260, an inodilator, for 24 h. A comparison with the arteritis induced by fenoldopam mesylate.

Administration of fenoldopam mesylate (FM), a dopaminergic agonist, or of cyclic cAMP phosphodiesterase inhibitors (PDE III), for example theophylline and caffeine, induces arteritis in the rat. In this study we characterized the arteritis induced by UK-61,260, an investigational inotropic agent with vasodilatory properties which displays an inhibitory action on cyclic AMP phosphodiesterase, in comparison with lesions induced by FM. The compounds were administered to Sprague-Dawley rats by intravenous infusion over 24 h (FM and UK-61,260), orally or subcutaneously (UK-61,260); the rats were killed and necropsied for pathological examination at various times between 0 h and 28 days post-infusion. Infusion of UK-61,260 at doses of 100, 300 or 400 mg/kg produced arteritis mainly in the mesenteric arteries and occasionally in the renal, pancreatic, gastric and coronary arteries. There were no arterial lesions after infusion of 30 mg/kg, or after administration of 30, 100 or 200 mg/kg per day subcutaneously for 7 days, or after acute administration of 100, 300, 400 or 600 mg/kg orally. Infusion of rats with 72 or 144 mg/kg FM produced arteritis over a wider range of tissues than did UK-61,260. However, the arterial lesions produced by infusion of either drug have the same initial aspect and a similar evolution with time. Immediately after the end of the infusion, minimal necrosis and haemorrhage occurred in the media only, without involvement of the endothelium or the perivascular space. This indicates that the media of the artery is the primary site of injury. The lesions seen 1 and 3 days post-infusion were characterized by severe medial necrosis and haemorrhage with perivascular acute inflammation and appeared macroscopically as haemorrhagic spots on the vessels. On days 7, 14 and 28 post-infusion, no medial necrosis or haemorrhage were present, while perivascular chronic inflammation and moderate smooth muscle hyperplasia were seen. It appeared, therefore, that the lesions underwent repair in 28 days, but footprints of the damage were still present 28 days post-infusion. The similarity between arteritis induced in rats by fenoldopam or by UK-61,260, at doses inducing PDE III inhibition, is consistent with the view that they have a similar pathogenesis. In our view it is probable that these pharmacologically and chemically distinct drugs trigger an increase in intracellular levels of cAMP which in turn triggers vascular damage. The arterial changes observed in the current study after acute administration may explain the increased incidence of polyarteritis nodosa occurring in long term toxicity studies with FM or PDE III inhibitors.

Effects of hydrochlorothiazide and captopril on the survival and heart weight of cardiomyopathic hamsters.

The current study investigated the effect of hydrochlorothiazide (HCTZ), and of captopril, on the survival and heart weight of cardiomyopathic hamsters (CMH). Groups of 51 CMH (about 200 days old) were orally treated with HCTZ (6 mg/kg), captopril (30 mg/kg), a combination of both drugs or vehicle alone (control group), for 217 days. Then survivors were sacrificed and the hearts weighed. The survival of the animals treated with HCTZ alone or in combination with captopril was similar, and was clearly increased when compared to controls (more than 70 days increase in median survival time). A more modest increase in survival occurred in animals treated with captopril alone. The efficacy of the diuretic therapy was consistent with fluid retention being the primary cause of death in CMH and demonstrated the usefulness of the model for the testing of new diuretics. HCTZ or captopril alone had no effect on the heart weight of survivors. However a reduction of cardiac hypertrophy (-18%) was seen when captopril and HCTZ were coadministered. This effect of the ACE inhibitor was considered to result from antagonism of the compensatory activation of the renin-angiotensin system occurring in response to diuretic therapy.

Cardiotoxicity of hydralazine and minoxidil in the rat. Influence of age.

In this study, the cardiotoxicity of isoproterenol, hydralazine and minoxidil was compared between young (1.5 months old) and mature (5 months old) rats. Both age classes were also compared for the effect of hydralazine on blood pressure and heart rate. For the 3 compounds, myocardial necrosis was observed and was more marked in mature than in young rats. The age-related increase in sensitivity to cardiotoxic effects which has already been described for isoproterenol, can therefore be extended to hydralazine and minoxidil. Hydralazine produced hypotension and reflex tachycardia. This latter effect appeared to have a longer duration in mature animals, which may be one of the explanation for their increased sensitivity to the cardiotoxic effect of the compound.

Development of specific antibodies mediating antibody-dependent cell-mediated cytotoxicity following experimental infection of cattle with bovine herpesvirus 1 and subsequent viral reactivation.

Evolution of sensitizing antibodies involved in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-complement cell lysis and complement-facilitated ADCC was followed in bulls after primary infection by a wild strain of infectious bovine rhinotracheitis virus (bovine herpesvirus 1; BHV-1) and after experimental reactivation of the virus. These antibodies were detected between the 4th and the 7th day after primary infection, reached a maximum level after 2 weeks and rose slightly after reactivation of the virus following dexamethasone treatment. The presence of endogenous complement slightly enhanced the ADCC reaction.

[The reaction of antibody-dependent cell-mediated cytotoxicity (ADCC)]. / La réaction de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC).

The antibody-dependent cell-mediated cytotoxicity (ADCC) is a cytotoxic reaction mediated by non immune cells (effector cells) against target cells sensitized by antibodies which are specific of the target cells' surface antigens. Those antibodies belong mostly to the IgG class. Several kinds of leucocytes can play a role in ADCC. All of them bear a receptor for the Fc fragment of the immunoglobulins. The efficacy of each population of effector cells varies according to the animal species and the type of the target cell. Some ADCC effectors are very similar to the natural killers (NK) but they do not belong exactly to the same subpopulation. ADCC can be studied by an indirect method (Cr51 release) or by a direct visual method (single cell assay). Several substances can influence ADCC reactions: corticoids either do not modify or inhibit the reaction. Interferon does not interact with ADCC nor enhances it. Antibiotics do not influence the reaction. Complement enhances the ADCC. Complement reduces the concentration of antibodies and the number of effector cells required for lysis. ADCC plays a role in several biological processes like graft rejection, autoimmune diseases, antitumoral defence, antiparasitical defence, antiviral defence which seems to be its most important role in domestic animals. ADCC can be used to study the evolution of sensitizing antibodies following a viral infection.

Effect of homologous delayed hypersensitivity testing on specific and non-specific lymphoblastic transformation in cattle latently infected with bovine rhinotracheitis virus (bovine herpesvirus 1, BHV 1).

Two cattle latently infected with infectious bovine rhinotracheitis virus (Bovine herpesvirus 1, BHV 1) were intradermally injected with inactivated BHV 1 antigen (delayed hypersensitivity test, DHT, skin test). Another animal, free of BHV 1 infection was similarly treated. Two latent carriers of the virus, intradermally injected with Phosphate-Buffered Saline solution were used as control. The evolution of spontaneous multiplication of lymphocytes and lymphoblastic transformation in vitro induced by three phytomitogens (con A, PHA, PWM) and BHV 1 antigen was followed in all animals. The delayed hypersensitivity test provoked an increase in spontaneous lymphocyte multiplication in latent carriers as well as an increase in mitogen- and antigen-induced blastogenesis on the 2nd and the 9th day following the treatment. A similar increase occurred on the 9th day in the BHV 1-free animal. Therefore, in latent carriers of BHV 1, delayed hypersensitivity testing induces an anamnestic lymphocyte reaction corresponding to the cutaneous one. This first lymphocyte activity is followed, one week later, by a new reaction which occurs both in infected and uninfected animals.