Chronobiological perspectives on myocardial electrophysiological parameters under three types of general anaesthesia in a rat model

The specific aim of the present study, with respect to dependence on the light–dark (LD) cycle under in vivo conditions in spontaneously breathing rats was to review initial state in electrophysiological parameters that may predict the development of heart rhythm disorders in pentobarbital (40 mg/kg), ketamine–xylazine (100 + 15 mg/kg) and zoletil (30 mg/kg) anaesthetized animals. The study was performed using female Wistar rats that were adaptated to an LD cycle (12 h:12 h). Heart rate, PQ and QT intervals were evaluated for their dependence on the LD cycle. The longest PQ interval duration is under zoletil anaesthesia in the light period and the longest QT interval duration is under ketamine–xylazine anaesthesia in both light periods. We concluded that the most significant predisposition toward the development of ventricular arrhythmias originating from disorders of impulse production and conduction occurred under zoletil anaesthesia in the light period; those resulting from disorders in the dispersion of refractory periods occurred under ketamine–xylazine anaesthesia in both the light periods.     

Pharmacometabolomic Approach to Predict QT Prolongation in Guinea Pigs

Drug-induced torsades de pointes (TdP), a life-threatening arrhythmia associated with prolongation of the QT interval, has been a significant reason for withdrawal of several medicines from the market. Prolongation of the QT interval is considered as the best biomarker for predicting the torsadogenic risk of a new chemical entity. Because of the difficulty assessing the risk for TdP during drug development, we evaluated the metabolic phenotype for predicting QT prolongation induced by sparfloxacin, and elucidated the metabolic pathway related to the QT prolongation. We performed electrocardiography analysis and liquid chromatography–mass spectroscopy-based metabolic profiling of plasma samples obtained from 15 guinea pigs after administration of sparfloxacin at doses of 33.3, 100, and 300 mg/kg. Principal component analysis and partial least squares modelling were conducted to select the metabolites that substantially contributed to the prediction of QT prolongation. QTc increased significantly with increasing dose (r = 0.93). From the PLS analysis, the key metabolites that showed the highest variable importance in the projection values (>1.5) were selected, identified, and used to determine the metabolic network. In particular, cytidine-5′-diphosphate (CDP), deoxycorticosterone, L-aspartic acid and stearic acid were found to be final metabolomic phenotypes for the prediction of QT prolongation. Metabolomic phenotypes for predicting drug-induced QT prolongation of sparfloxacin were developed and can be applied to cardiac toxicity screening of other drugs. In addition, this integrative pharmacometabolomic approach would serve as a good tool for predicting pharmacodynamic or toxicological effects caused by changes in dose.     

Lethal effects of cocaine are reduced by the dopamine-1 receptor antagonist SCH 23390 but not by haloperidol

The prevalence of cocaine abuse has been associated with a host of medical complications and deaths. We investigated the effects of two dopamine antagonists with different affinities for dopamine-1 and dopamine-2 receptor subtypes on cocaine-induced lethality. Male Fischer-344 rats were given cocaine HCl (i.p.) and observed for lethality at 24 hrs. Cocaine was not lethal at 50 mg/kg and produced a steep dose-effect function from 60 to 100 mg/kg. Lethality was 88.9% at 100 mg/kg and the LD 50 was 79.7 mg/kg (95% CL: 74.8-84.9). Doses as high as 180 mg/kg failed to kill all rats. Lethality was often but not invariably associated with convulsions. Haloperidol (0.3-3 mg/kg i.p.) given 30 min prior to cocaine did not alter the lethal effects of cocaine but did reduce the lethality of methamphetamine. SCH 23390 (0.1-1 mg/kg i.p., 30 min prior) shifted the cocaine dose-effect function to the right at 0.3 mg/kg. Maximum protection was conferred by 0.3 mg/kg SCH 23390 where the LD 50 was increased to 100.1 mg/kg (95% CL: 91.5-109.5). Comparable protection was not observed if SCH 23390 was given 5 min after cocaine. These results suggest that dopamine receptors may play a role in the lethal effects of cocaine and that the D1 dopamine receptor subtype appears to be more relevant to lethality than the D2 subtype.     

Some toxicological characteristics of three venomous soft corals from the Red Sea

Three common Red Sea soft corals (Cnidaria: Anthozoa), Nephthea sp, Dendronephthya sp and Heteroxenia fuscescens sting humans. Nematocyst venoms of each animal are lethal to mice and hemolytic to human erythrocytes. However, these hemolysins are partially inhibited by known anti-hemolytic agents. Venoms and their gel chromatography-separated fractions have different dermonecrosis and vasopermeability potency in mouse skin. The venom of Heteroxenia fuscescens (Hf) was more lethal (LD50: 0.7 mg/kg), with one prominent 97-kDa protein fraction (LD50: 0.55 mg/kg). Hf venom was more hemolytic, more dermonecrotic, and had more vasopermeable factors than that of the two other species. SDS polyacrylamide gel electrophoresis of soft coral whole venoms and fractions showed different protein molecular masses ranging from 200 to less than 6 kDa. High IgG titers were assayed from venom-sensitized mice blood sera. Enzyme-linked immunosorbent assays (ELISA) marked significant immunological cross-reaction between the studied soft coral venoms and their bioactive fractions.     

The effect of NADP+ on cardiomyocyte electrophysiological properties of C57BL/6 and mdx mice

We have studied the influence of NADP⁺ on routine electrocardiography (ECG) in 6-month-old C57BL/6 and mdx mice. The animals were anesthetized by ether before ECG recording. ECG registration was carried out at a speed of 100 mm/s. The first ECG recording was made before intraperitoneal NADP⁺ injection in a dose of 13 or 80 mg/kg. The second ECG recording was made 10 min after NADP⁺ injection. Anesthesia was then terminated. The mice were occasionally anesthetized 45–60 min later, and the third ECG was recorded 1 h after injection of NADP⁺. ECG recording was carried out at a speed of 100 mm/s in standard leads I, II, and III and unipolar leads AvR, AvL, and AvF. Values of standard ECG characteristics, such as the P wave and the intervals PQ, QT, RR, and the QRS complex, were measured in milliseconds in standard lead II. We did not observe any differences between ECG magnitudes of 2- to 3-month-old C57BL/6 and mdx mice during trial experiments. Mice of both strains had a sinus rhythm in their heart rate. The QRS complex in mdx mice had a tendency to be larger than in C57BL/6 mice. Heart rates fluctuated between 722 ± 22 and 681 ± 21 beats per minute. The effect of NADP⁺ was studied in 6-month-old male mice. The increase in the RR interval and the decline in heart rate from 697 ± 21 to 461 ± 23 and 491 ± 28 beats per min for C57BL/6 mice (p < 0.01) and from 722 ± 28 beats per minute to 454 ± 31 beats per min for mdx mice were registered 10 min after NADP⁺ injection at a dose of 80 mg/kg. The increase in the RR interval can be explained by an increase in the QT interval. A statistically significant reduction in the QT interval leading to a diminished RR interval was observed in mdx mice 1 h after NADP⁺ injection. NADP⁺ at a dose of 13 mg/kg did not significantly change the ECG properties in mdx mice. ECG of mdx mice was characterized by negative repolarization of the T wave in 37% of all leads. The amount of leads with negative T-wave repolarization decreased up to 3% 1 h after NADP⁺ injection in dose of 80 mg/kg. The results have shown that cytomembranes of ventricular cardiac myocytes and the degree of oxidative stress are the main targets of the action of NADP⁺ in C57BL/6 and mdx mouse hearts.     

Antitumor activity of 3,4-dihydroquinazoline dihydrochloride in A549 xenograft nude mice

In the previous article we have reported that 3,4-dihydroquinazoline 1 is a potent and selective T-type calcium channel blocker that exhibited strong anti-cancer activity in vitro. Compound 1·2HCl was further in vivo evaluated against A549 xenograft in BALB/c nude mice, which exhibited 49% tumor-weight inhibition through intravenous administration of 2 mg/kg of body weight and was more potent than doxorubicin. Moreover, compound 1·2HCl has an oral bioavailability of 98% with LD(50) values of 693 mg/kg (p.o. route) and 40.0 mg/kg (i.v. route) of body weight. In addition, its efficient scale-up synthetic method was developed.     

Issues in QT interval measurement

The QT interval, apart from clinical implications is crucial for safety assessment of new drugs under development. A QTc prolongation of even 10 msec in a study group is a warning signal for a new drug. There are various issues involved in the measurement of the QT interval especially regarding the ending of the T wave and different morphological pattern of T-U complex. The other issue is significant spontaneous variability in the QT interval, resulting in spurious QT prolongation and unnecessary concern.To minimize all these confounding factors, all clinical trials for assessing QT interval prolongation should be randomized and double blinded with appropriate control groups including placebo. ECG measurements should be done by trained readers with electronic calipers at ECG core Lab. ECGs should be compared with multiple baseline values with multiple, time-matched on-treatment values.     

Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.     

Conventional QT Variability Measurement vs. Template Matching Techniques: Comparison of Performance Using Simulated and Real ECG

Increased beat-to-beat variability in the QT interval (QTV) of ECG has been associated with increased risk for sudden cardiac death, but its measurement is technically challenging and currently not standardized. The aim of this study was to investigate the performance of commonly used beat-to-beat QT interval measurement algorithms. Three different methods (conventional, template stretching and template time shifting) were subjected to simulated data featuring typical ECG recording issues (broadband noise, baseline wander, amplitude modulation) and real short-term ECG of patients before and after infusion of sotalol, a QT interval prolonging drug. Among the three algorithms, the conventional algorithm was most susceptible to noise whereas the template time shifting algorithm showed superior overall performance on simulated and real ECG. None of the algorithms was able to detect increased beat-to-beat QT interval variability after sotalol infusion despite marked prolongation of the average QT interval. The QTV estimates of all three algorithms were inversely correlated with the amplitude of the T wave. In conclusion, template matching algorithms, in particular the time shifting algorithm, are recommended for beat-to-beat variability measurement of QT interval in body surface ECG. Recording noise, T wave amplitude and the beat-rejection strategy are important factors of QTV measurement and require further investigation.     

小鼠心肌肥厚发展过程中心电图动态变化

目的：探讨小鼠心肌肥厚发展过程中心电图的动态变化。方法：复制小鼠压力超负荷性心肌肥厚模型,连续动态监测小鼠从心肌肥厚早期至心力衰竭发展过程中的不同阶段体表心电图改变。结果：①对照组和模型组术后2周内小鼠未见自发性心律失常,而模型组术后5周、9周和13周小鼠出现自发性心律失常,主要表现为频发的室性早搏以及阵发性室性心动过速,心律失常发生率分别为15％、28％和63％。②与同期对照相比,术后2周、5周、9周和13周组动物伽间期以及帆间期明显延长,分别延长20．4％、32．7％、49．7％、61．0％和27．1％、32．1％、43．9％、59．1％（P〈0．01）。③心肌肥厚小鼠心电图的另一个特征为J波变化。所有对照组动物心电图均为正向J波,而模型组动物从2周开始J波正向值下降,5周逐渐变平,到13周时完全翻转。④与同期对照相比,模型组的PR间期没有改变,但术后2周RR问期轻微缩短。结论：心肌肥厚小鼠自发性心律失常发生率逐渐增加,QT间期进行性延长,J波幅值逐渐降低,表明随着疾病的进展心室复极化异常逐渐加重。     

Electrocardiography in two models of isoproterenol-induced left ventricular remodeling

We studied the ability of the ECG to detect pathological changes in isoproterenol-induced remodeling of rat heart. Myocardial hypertrophy in rats was induced by repeated injections of isoproterenol (5 mg/kg s.c. 7 days, Iso5, n=7). Single overdose of isoproterenol (150 mg/kg s.c., Iso150, n=7) evoked myocardial infarction followed with ventricular remodeling. The electrocardiograms were recorded in anesthetized animals (thiopenthal 45 mg/kg i.p.) and myocardial contractile performance was analyzed in isolated hearts perfused according to Langendorff. The hypertrophic hearts were characterized by increased heart and left ventricular (LV) weight as well as by thicker LV free wall and interventricular septum. Mean values of LV contraction did not significantly differ from controls. Longer QT interval, QRS complex, negative Q and S waves, higher R amplitude were typical characteristics for Iso5 rats. Iso150 animals showed tendency to decreased systolic blood pressure and heart frequency. Decrease in the thickness of LV compared to Iso5 as well as impaired LV function were related to the dilated left ventricle. Iso150 ECG showed longer QRS and QT, deepened negativity of S wave and mild decrease of R(II) compared to Iso5. Voltage criteria showed that Sokolow-Lyon index is a good predictor of left ventricular hypertrophy in isoproterenol-induced cardiac remodeling without systemic hypertension.     

Age and anesthetic effects on murine electrocardiography

Murine models offer potential insights regarding human cardiac disease, but efficient and reliable methods for phenotype evaluation are necessary. We employed non-invasive electrocardiography (ECG) in mice, investigating statistical reliability of these parameters with respect to anesthetic and animal age. Mice (C57BL/6, 8 or 48 weeks) were anesthetized by ketamine/xylazine (K/X, 80/10 mg/kg ip) or by inhalation anesthetic (halothane, HAL; sevoflurane, SEV) and 6 lead ECGs were recorded. P wave duration and QT interval was significantly prolonged with K/X compared to HAL and SEV, indicating slowed atrial and ventricular conduction. P-R interval (atrio-ventricular conduction) was significantly increased in aged mice under all anesthetics. Heart rate was inversely correlated to QT interval and P wave duration. We also detected significant age effects with respect to optimal approaches for QT interval corrections. Power analysis showed 4-fold higher number of mice/group, were required for K/X, to achieve identical statistical sensitivity. These data demonstrate the importance of anesthetic selection for relevant and reliable ECG analysis in mice and illustrate the selective influences of anesthetics and age on cardiac conductance in this species.     

Anti-ulcer compound from Voacanga africana with possible histamine H2 receptor blocking activity.

Voacanga africana is used in Cameroonian ethnomedicine for the treatment of peptic ulcers. We have tested the cytoprotective, anti-secretory and ulcer healing actions of an alkaloid (TN) obtained from the fruit extract. Oral administration of TN (50-100 mg/kg) dose-dependently prevented ulcer formation by HCl/ethanol (36-75%), absolute ethanol (43-75%), HCl-ethanol/indomethacin (58-84%), Pylorus ligation (31-100%), cold restraint stress (68-100%) and histamine (49-100%). The inhibitory effect at 50 and 100 mg/kg against HCl/ethanol was not suppressed by pre-treatment with indomethacin (20 mg/kg, i.p.). TN reduced Shay-ligated gastric acid secretion from 77 mEq/l in the controls to 46 and 25 mEq/l for the 50 and 100 mg/kg doses. Augmented histamine-induced gastric acid secretion was reduced from 84 mEq/l in the controls to 45 and 21 mEq/l for the two doses of TN, with total inhibition of gastric and duodenal ulcers by the 50 mg/kg dose. Healing rate of chronic acetic acid-induced ulcers was 62 and 83%, respectively, for the dose of 50 and 100 mg/kg of TN compared with the controls. TN has gastric anti-secretory effects similar to histamine receptor blockers. Its cytoprotective and ulcer healing properties are related to its ability to strengthen gastric mucosal defenses through enhanced gastric mucus production.     

Effect of bile and lipids on the stereoselective metabolism of halofantrine by rat everted‐intestinal sacs

The everted rat intestinal‐sac model was utilized to assess the effect of post‐prandial conditions on the stereoselective intestinal metabolism of halofantrine to its active metabolite desbutylhalofantrine. Everted intestinal sacs were incubated with (±)‐halofantrine HCl in the presence of simulated bile solution (containing lecithin, lipase and cholesterol) and lipids to mimic post‐prandial conditions in the small intestine. The halofantrine enantiomer concentrations in intestinal sacs were relatively constant in the presence of bile, but decreased significantly on addition of lipids to the incubation media. Formation of desbutylhalofantrine enantiomers was inversely proportional to bile concentration whereas addition of lipids in the presence of bile caused a significant decrease in desbutylhalofantrine:halofantrine ratio of (?) enantiomers. Pre‐treatment of rats with peanut oil had no significant effect on desbutylhalofantrine formation in the incubated sacs or microsomal preparations, nor did it affect the expression of intestinal cytochrome P450. Addition of extra cholesterol to the bile incubations caused a significant increase in tissue halofantrine and desbutylhalofantrine concentrations, which as for lower cholesterol, were diminished on addition of other lipids. The results were consistent with previous in vivo evaluations showing that the desbutylhalofantrine to halofantrine ratio was decreased by the ingestion of a high fat meal. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of chromosome aberrations, micronucleus formation and sperm abnormalities in mouse following carbofuran exposure

Carbofuran was tested to study in vivo cytogenetic effects in mouse bone marrow cells and morphological alterations in sperms. The acute oral and intraperitoneal (i.p.) LD(50) of carbofuran was determined to be 9.5 or 2.0 mg/kg b.w. in mice, respectively. The animals were orally administered 1.9, 3.8 or 5.7 mg/kg b.w. (20, 40 and 60% of LD(50)) of carbofuran for 24 h or 1.9 mg/kg b.w. for 4 consecutive days (cumulative 7.6 mg/kg or 80% of LD(50)) to analyse chromosome aberrations (CAs). For micronucleus test (MT) animals were orally exposed to 5.7 mg/kg b.w. for 24 and 48 h or 1.9 mg/kg b.w. for 4 consecutive days. For reference mice were exposed to peanut oil (negative control) and cyclophosphamide (20 mg/kg) or ethyl methanesulfonate (EMS: 100 mg/kg) positive control for CAs and MT respectively. To analyse the effect on sperm morphology mice were exposed to single i.p. dose of 1 and 2 mg/kg b.w. of carbofuran and repeatedly to 0.5 mg/kg for 5 consecutive days. Cytogenetic analysis revealed that all the test doses induced mitotic inhibition, CAs, micronucleus (MN) formation and sperm abnormalities in a dose dependent manner. Present observations concurrent with earlier reports substantiate the genotoxic potential of carbofuran and possible risk to human beings.     

The Effect of Phenothiazines on the Electrocardiogram

Thioridazine, chlorpromazine and trifluoperazine were administered to six psychiatric patients. Each was used in four dosage levels (thioridazine and chlorpromazine: 200, 400, 800 and 1200 mg. daily; trifluoperazine: 8, 16, 32, 64 mg. daily); and each increase in dosage was effected after four days of drug administration.Before the trial, twice during each drug period and before commencement of the next dose regimen, an electrocardiogram (ECG) was recorded. The findings indicated that thioridazine modifies the terminal portion (S-T segment, T and U waves) of the human ECG. A similar change occurred in three of six subjects while taking chlorpromazine and in one of six while taking trifluoperazine. Thioridazine induced changes in all six subjects studied, viz., blunting and notching of T waves with or without prolongation of QT interval. In some the notching produced a doublehump appearance in which a T wave of reduced voltage formed the proximal hump and a positive U wave of increased voltage formed the distal hump.Thioridazine-induced alterations in the ECG have been described as resembling those caused by quinidine; they also resemble changes associated with hypokalemia.     

Vectorcardiography analysis of the repolarization response to pharmacologically induced autonomic nervous system modulation in healthy subjects

Autonomic nervous system activity is essential for regulation of ventricular repolarization (VR) and plays an important role in several arrhythmogenic conditions. This study in 31 healthy adult subjects (16 men, 15 women) evaluated the VR response to pharmacologically modulated autonomic nervous system activity applying vectorcardiography (VCG) analysis. During continuous VCG recording, 0.01-0.1 μg·kg(-1)·min(-1) isoprenaline (Iso) was infused at an increasing flow rate until three targeted heart rates (HR) were reached. After Iso washout, one intravenous bolus of 0.04 mg/kg atropine was given followed by an intravenous bolus of 0.2 mg/kg propranolol. A 5-min steady-state VCG recording was analyzed for each of the seven phases (including baseline 1 and 2). Furthermore, during the first 4 min following atropine, six periods of 10-s VCG were selected for subanalysis to evaluate the time course of change. The analysis included QRS, QT, and T-peak to T-end intervals, measures of the QRS and T vectors and their relation, as well as T-loop morphology parameters. By increasing HR, Iso infusion decreased HR dependent parameters reflecting total heterogeneity of VR (T area) and action potential morphology (ventricular gradient). In contrast, Iso prolonged QT HR corrected according to Bazett and increased the T-peak to T-end-to-QT ratio to levels observed in arrhythmogenic conditions. HR acceleration after atropine was accompanied by a transient paradoxical QT prolongation and delayed HR adaptation of T area and ventricular gradient. In addition to the expected HR adaptation, the VR response to β-adrenoceptor stimulation with Iso and to muscarinic receptor blockade with atropine thus included alterations previously observed in congenital and acquired long QT syndromes, demonstrating substantial overlap between physiological and pathophysiological electrophysiology.     

The role of dopamine and serotonin in the prolongation of post-decapitation convulsions in mice.

L-DOPA (320 mg/kg, i.p.) increased the duration of the clonic phase of post-decapitation convulsions (PDC) by 60% in mice pretreated with the peripheral decarboxylase inhibitor, Ro 4-4602 (50 mg/kg, i.p.). Assays of brains at the time of decapitation showed a 300% increase in dopamine (DM), an 80% reduction in serotonin (5-HT) and no change in norepinephrine (NE) levels. The effect of L-DOPA on PDC was not blocked by haloperidol (0.5 – 5.0 mg/kg), a blocker of DM receptors, nor by diethyldithiocarbamate (400 mg/kg) an inhibitor of NE synthesis. Parachlorophenylalanine (300 mg/kg × 3 days) produced an 80% reduction in 5-HT and a prolongation of PDC similar to that observed after L-DOPA. Prolongation of PDC was also seen after the 5-HT antagonists methysergide (5 mg/kg) and cinanserin (10 mg/kg), but not after cyproheptadine (10 mg/kg). The 5-HT precursor, 5-hydroxytryptophan (100 mg/kg), produced no change in PDC when used alone but inhibited L-DOPA's prolongation of PDC. The results suggest that L-DOPA acts by depleting 5-HT in bulbospinal pathways and thus enhancing reflex activity in the spinal cord.     

Plasmodium yoelii nigeriensis (MDR)-efficacy of oral pyronaridine against multidrug-resistant malaria in Swiss mice

A multidrug-resistant strain of Plasmodium yoelii nigeriensis (MDR) showing a wide spectrum of resistance to chloroquine, amodiaquine, mepacrine, mefloquine, halofantrine, quinine, and quinidine was used in this study for in vivo evaluation of the blood schizontocidal activity of pyronaridine, a topoisomerase II inhibitor, in Swiss mice. The parasite produces 100% lethal infection in mice. The drug was administered orally once a day from day 0 onward. The initial studies showed that low doses of pyronaridine (0.625 to 5.0 mg base/kg x9 days) did not completely control blood-induced P. yoelii nigeriensis infection. Finally a series of doses of pyronaridine ranging from 1.25 to 30.0 mg/kg administered orally for 7 consecutive days were evaluated and in spite of high level of resistance to standard antimalarials, the parasite P. yoelii nigeriensis has shown complete susceptibility to pyronaridine (15 mg/kg dose x7 days). The present paper also compares the merits of a single MDR strain vs a battery of different resistant lines for quick antimalarials screening.