Whole-Cell Configuration of the Patch-Clamp Technique in the hERG Channel Assay.

In vitro electrophysiological safety studies have become an integral part of the drug development process because, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or their native current, the rapidly activating delayed rectifier potassium current (IKr ). Therefore, according to the ICH S7B guideline, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong the QT interval prior to first-in-human testing. The protocols described in this article are designed to assess the effects of acute or long-term exposure to new chemical entities on the amplitude of IKr in HEK293 cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with moxifloxacin, terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. © 2024 Wiley Periodicals LLC. Basic Protocol: Measurement of the acute effects of test items in the hERG channel test Alternate Protocol: Measurement of the long-term effects of test items in the hERG channel test.

Electrophysiological characteristics and pharmacological sensitivity of two lines of human induced pluripotent stem cell derived cardiomyocytes coming from two different suppliers.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as preclinical tool for predicting drug-induced QT prolongation and arrhythmias. This study was conducted to assess the electrophysiological characteristics and the pharmacological sensitivity of two commercialized hiPSC-CMs. The baseline electrophysiological characteristics measured with a multi-electrode array (MEA) technology differ between Cor.4U and iCell2: higher beat rate (+32bpm) and shorter field potential duration (FPD, -201ms) for Cor.4U. The FPD lengthening after cisapride (100nM: +65% versus +18%), quinidine (10µM: +65% versus +31%), sotalol (30µM: +90% versus +47%) or flecainide (3µM: +76% versus +22%) application appeared earlier in iCell2 as compared to Cor.4U. Arrhythmia occurrence also appeared earlier in iCell2 as compared to Cor.4U for the 3 substances mentioned above. The FPD shortening recorded after verapamil or nifedipine application was similar in both hiPSC-CMs. In conclusion, Cor.4U and iCell2 hiPSC-CMs are both sensitive enough to detect drug-induced delayed or shortened repolarization and arrhythmia and can provide useful predictive cardiac electrophysiology data. Arrhythmias occurred at concentrations higher than clinical free maximum plasma concentrations with an overestimation of the risk with cisapride. However, quantitative differences of baseline electrophysiological characteristics or pharmacological sensitivity of both cell types have to be considered with caution during the interpretation of data. The new chemical entities included within a given drug development program should be evaluated in hiPSC-CMs coming from a single supplier.

Proarrhythmic risk assessment using conventional and new in vitro assays.

Drug-induced QT prolongation is a major safety issue in the drug discovery process. This study was conducted to assess the electrophysiological responses of four substances using established preclinical assays usually used in regulatory studies (hERG channel or Purkinje fiber action potential) and a new assay (human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)-field potential). After acute exposure, moxifloxacin and dofetilide concentration-dependently decreased IKr amplitude (IC50 values: 102 µM and 40 nM, respectively) and lengthened action potential (100 µM moxifloxacin: +23% and 10 nM dofetilide: +18%) and field potential (300 µM moxifloxacin: +76% and 10 nM dofetilide: +38%) durations. Dofetilide starting from 30 nM induced arrhythmia in hiPSC-CMs. Overnight application of pentamidine (10 and 100 µM) and arsenic (1 and 10 µM) decreased IKr, whereas they were devoid of effects after acute application. Long-term pentamidine incubation showed a time- and concentration-dependent effect on field potential duration. In conclusion, our data suggest that hiPSC-CMs represent a fully functional cellular electrophysiology model which may significantly improve the predictive validity of in vitro safety studies. Thereafter, lead candidates may be further investigated in patch-clamp assays for mechanistic studies on individual ionic channels or in a multicellular Purkinje fiber preparation for confirmatory studies on cardiac conduction.

Complementarity of in vitro and in vivo models for the evaluation of gastro-protective effects of pharmacological substances.

Gastric mucosa is frequently exposed to various gastric irritants, and there is a continuing requirement to develop new gastro-protective agents. This study compares the effects of three such agents, sucralfate, rebamipide, and cimetidine in both in vivo and in vitro indomethacin-induced gastric damage models. For the in vivo approach, rats were orally administered sucralfate, rebamipide, and cimetidine at 300 mg/kg before an acute dose of indomethacin (30 mg/kg). Gastric lesions were then macroscopically examined. For the in vitro approach, gastric mucosal cells were incubated with sucralfate (3 and 5 mg/mL), rebamipide (0.3 and 1 mm), and cimetidine (10 and 50 µg/mL) before exposure to indomethacin (3.8 mm). The release of lactate dehydrogenase (LDH) and mitochondrial function were then measured. Sucralfate, rebamipide, and cimetidine displayed gastro-protective effects in vivo (decreased number of gastric ulcers: -50% P < 0.05, -22% NS, and -69% P < 0.05, respectively, and reduced length of gastric lesions: -62% P < 0.05, -29% NS, and -70% P < 0.001, respectively). Cell damage induced by indomethacin in vitro was inhibited by sucralfate (LDH release) and by rebamipide and cimetidine (mitochondrial function and LDH release). In contrast, sucralfate accentuated the indomethacin-induced decrease in mitochondrial function. Although cultured gastric cells offer a promising tool for evaluating the cytotoxic or protective effects of test compounds, data from in vivo models are still needed to confirm in vitro data. Using both approaches provides more comprehensive insight into the effects of test compounds on the gastric mucosa.

Comparison of three preclinical models for nausea and vomiting assessment.

INTRODUCTION: Nausea is a subjective sensation often preceding emesis in humans. Drug-induced nausea remains difficult to predict in preclinical tests. The aim of this study was to compare the effects of emetic agents in rats (pica behavior), ferrets (acute and delayed phases of emesis) or dogs (emesis and cardiovascular endpoints). METHODS: Rats and ferrets were administered cisplatin (±aprepitant/ondansetron or aprepitant) or apomorphine (±domperidone). Telemetered dogs were administered apomorphine (±domperidone). Food and kaolin intake was measured in rats whereas the number of emetic events was counted in ferrets and dogs. Cardiovascular changes were also monitored in dogs. RESULTS: In rats, cisplatin (6mg/kg, i.p.) increased kaolin intake (+2257%, p<0.001). The cisplatin effects were not reversed by the combination of aprepitant/ondansetron (2mg/kg, p.o./2mg/kg, i.p.) or by aprepitant (30mg/kg, p.o.). Apomorphine (10mg/kg, i.p.) did not induce pica behavior. In ferrets, cisplatin (8mg/kg, i.p.) induced acute and delayed emesis (371.8±47.8 emetic events over 72h) which was antagonized by aprepitant (1mg/kg, p.o.). Apomorphine (0.25mg/kg, s.c.) induced acute emesis (38.8±8.7 emetic events over 2h) which was abolished by domperidone (0.1mg/kg, s.c.). In dogs, apomorphine (100µg/kg, s.c.) induced emesis and tachycardia which were decreased by domperidone (0.2mg/kg, i.v.). CONCLUSIONS: The assessment of emesis in the ferret or in the dog displays a strong predictive value. In contrast, assessing nausea remains challenging in all animal species and the use of pica behavior remains questionable in the context of antiemetic drug development.

Comparison of the effects of clonidine, loperamide and metoclopramide in two models of gastric emptying in the rat.

Several methods are used to evaluate gastric emptying (GE) in rats, which is an important endpoint in preclinical drug development. Although phenol red model or monitoring of plasma acetaminophen levels are well-established procedures for GE assessment, their capacity to detect the effects of pharmacological agents has rarely been compared. This study was therefore designed to evaluate clonidine with loperamide and metoclopramide in the two test models. Rats were administered phenol red or acetaminophen test meals. The remaining amount of phenol red in the stomach or the time course of plasma acetaminophen levels was then measured. In the phenol red test, loperamide (8 mg/kg, p.o.) and clonidine (100 µg/kg, s.c.) decreased GE (-88 and -42%, P < 0.001 and P < 0.01, respectively). Metoclopramide (10 mg/kg, s.c.) accelerated GE (+42%, P < 0.01). Loperamide reduced acetaminophen plasma levels (-45% at T15 min, P < 0.05), suggesting a delayed GE. Clonidine and metoclopramide increased acetaminophen plasma levels (+115 and +152% at T15 min, P < 0.05 and P < 0.001, respectively), suggesting an accelerated GE. The three substances did not affect plasma acetaminophen levels when acetaminophen was subcutaneously injected, thereby suggesting that acetaminophen metabolism/excretion was not modified. Whereas the phenol red test allows the evaluation of GE at a single time point, the measurement of plasma acetaminophen levels over the time would appear more informative. Nevertheless, the fact that clonidine, in contrast to expectation, increased plasma acetaminophen levels, suggests that data obtained with the acetaminophen method should be interpreted with caution for new chemical entities susceptible to modify absorption of acetaminophen from the small intestine.

Automated analysis of delayed emesis in the telemetered ferret: detection of synergistic effects of aprepitant and ondansetron.

Nausea and vomiting are common side effects of cancer chemotherapy. We have previously described a model in the ferret where delayed emesis can be measured automatically using telemetry. This study was designed to examine the sensitivity of this automated emesis model for detecting moderate and/or additive pharmacological effects by investigating low-dose effects of aprepitant alone or in combination with ondansetron. Ferrets implanted with telemetry devices (Data Sciences International) were orally treated with aprepitant (0.03 mg/kg) and/or ondansetron (0.3 mg/kg) and then challenged with cisplatin (8 mg/kg, i.p.). Abdominal pressure was recorded in unrestrained animals from 18 to 72 h post-challenge, and the pressure signals were automatically analyzed using adapted software (Emka Technologies). Ondansetron administered alone 1 h before cisplatin challenge had no significant effects on the delayed emesis phase. Once-daily treatment with aprepitant (2 h before cisplatin and then 24 and 48 h after cisplatin challenge) slightly reduced the total number of emetic events (-32%, NS). When administered together, aprepitant and ondansetron exhibited synergistic effects on delayed-phase emesis. The combined treatment markedly and significantly decreased the mean number of emetic events recorded between 24 and 54 h after cisplatin dosing (-75%, P < 0.05) and the total number of emetic events (-56%, p < 0.05). Our results demonstrate that the automated cisplatin-induced emesis model in the ferret is sensitive enough to detect the synergistic effects of aprepitant and ondansetron in combination, creating new and important perspectives for the evaluation of combined therapy in the reduction of side effects of cancer chemotherapy.

In vitro safety cardiovascular pharmacology studies: impact of formulation preparation and analysis.

Collection of formulation samples is required for GLP in vitro studies to check the exposure of the test system and allow reliable determinations of safety margins. In vitro studies conducted in-house were investigated to evaluate problems of solubility, stability and adsorption of the formulations. Terfenadine was used as reference substance to illustrate the purpose. Lowered target concentrations of test substances in in vitro studies can be attributed to the solubility limitation in the superfusion medium, the low stability under frozen conditions (24% of the final solutions stable at -20 °C) and/or the adsorption on the superfusion tubing (30% of the studies). Terfenadine also showed a limited solubility (measured concentrations ranging from 0.597 µM to 0.833 µM instead of 1 µM) and a loss of substance through the superfusion tubing from -30.2% to -39.2% with dimethylsulfoxide, ethanol or methanol. Terfenadine solubility was improved with 2-hydroxypropyl-ß-cyclodextrin, no adsorption was observed, but its capacity to block the hERG channel was decreased. It is recommended to determine the substance solubility in appropriate buffers, to evaluate possible adsorption during method validation (formulation samples collected after superfusion), and to prepare fresh formulation each testing day with immediate analysis in absence of stability data. This strategy clearly favors single-site as opposed to multi-site studies.

An overview of QT interval assessment in safety pharmacology.

Medicinal products that prolong cardiac repolarization, as assessed in terms of prolongation of the QT interval of the electrocardiogram, may trigger torsade de pointe, a potentially fatal arrhythmia. The lethality of this risk necessitates a detailed preclinical evaluation before initiating clinical trials. The strategy for assessing the potential of new chemical entities to cause QT interval prolongation involves two complementary approaches. An in vivo test provides information on the potential of the agent to prolong the QT interval under near-physiological conditions. The results are mostly descriptive, providing little insight into the mechanisms of action. In vitro experiments provide more mechanistic data, although the test procedure is far removed from the clinical situation. While both approaches have reasonable predictive value, the results may depend largely on the experimental conditions employed. Discussed in this unit are experimental issues that should be considered when testing agents for their potential to cause arrhythmias, as well as general strategies for understanding the problems associated with this cardiovascular risk.

Using telemetry to automate the detection of emesis in the ferret: new vistas for delayed emesis assessment.

INTRODUCTION: Nausea and vomiting are common side effects of cancer chemotherapy. The ferret is a highly appropriate animal species to evaluate both early and delayed emetic events occurring hours and days after administration, respectively. If early emesis can be easily investigated in ferrets by direct observation, alternative methods are required to quantify delayed emesis. This study was designed to validate a new method of automated detection of abdominal pressure changes related to retches or vomits induced by a cytotoxic substance in the ferret. METHODS: Five ferrets implanted with telemetry devices (Data Sciences International) were challenged with cisplatin (8 mg/kg, i.p.) and abdominal pressure was recorded in unrestrained animals for 72 h. The pressure signals were analyzed both manually and automatically using an adapted version of ecgAUTO software (Emka Technologies). Over the first 3 h, the emetic response was also quantified via direct observation of the animals. The data produced by the 3 methods of detection were compared using a Spearman's rank correlation coefficient. RESULTS: Visual, manual and automated detections of early emetic events over the first 3-hour recording period were well correlated when compared per 30-, 15- or 5-minute epoch: correlation coefficients ranging from 0.8640 to 0.9289, p<0.0001 for all comparisons. Manual and automated detections of early and delayed emetic events over the 72-hour recording period were also well correlated when compared per 3-hour epoch: correlation coefficient=0.9190, p<0.0001. DISCUSSION: These findings demonstrate that automated detection of abdominal pressure changes with adapted software is a reliable method for measuring emetic events in the ferret. The results obtained open major possibilities for the rapid, comprehensive and objective analysis of delayed emesis. They should thereby facilitate the development of novel chemotherapeutic agents and anti-emetic therapies.

Overview of safety pharmacology.

Safety pharmacology entails the assessment of the potential risks of novel pharmaceuticals for human use. As detailed in the ICH S7A guidelines, safety pharmacology for drug discovery involves a core battery of studies on three vital systems: central nervous (CNS), cardiovascular (CV), and respiratory. Primary CNS studies are aimed at defining compound effects on general behavior, locomotion, neuromuscular coordination, seizure threshold, and vigilance. The primary CV test battery includes an evaluation of proarrhythmic risk using in vitro tests (hERG channel and Purkinje fiber assays) and in vivo measurements in conscious animals via telemetry. Comprehensive cardiac risk assessment also includes full hemodynamic evaluation in a large, anesthetized animal. Basic respiratory function can be examined in conscious animals using whole-body plethysmography. This allows for an assessment of whether the sensitivity to respiratory-depressant effects can be enhanced by exposure to increased CO2 . Other safety pharmacology topics detailed in this unit are the timing of such studies, ethical and animal welfare issues, and statistical evaluation.

Whole-cell configuration of the patch-clamp technique in the hERG channel assay to predict the ability of a compound to prolong QT interval.

In vitro electrophysiological safety studies have become an integral part of the drug development process since, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or its native current, the rapidly activating delayed rectifier potassium current (I(Kr)). Therefore, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong QT interval. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long-term exposure on the amplitude of I(Kr) in human embryonic kidney 293 (HEK293) cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes.

The comparative sensitivity of three in vitro safety pharmacology models for the detection of lidocaine-induced cardiac effects.

INTRODUCTION: In the current ICH S7B guideline, in vitro evaluation of proarrhythmic liability is limited to the risk of QT interval prolongation, whilst the effect of new chemical entities on cardiac conductivity is often overlooked. The aim of this work was to compare the effects of the sodium channel blocker, lidocaine in three in vitro safety pharmacology models: hNa(v)1.5 channel test, atrial action potential (AP) and Purkinje fiber AP and to identify the most sensitive model for detecting cardiac conduction slowing. METHODS: Whole-cell patch-clamp methods were used to record the sodium current (I(Na)) encoded by hNa(v)1.5 in stably transfected HEK293 cells at ambient temperature. Transmembrane APs were recorded in rabbit Purkinje fibers and rabbit and guinea-pig left stimulated atria at physiological temperature. Parameters involved in depolarization or repolarization were reported. RESULTS: Lidocaine (from 10 to 1000 µM) decreased the amplitude of I(Na) (IC(50): 256±37 µM) in a concentration-dependent manner. In the Purkinje fiber assay, lidocaine (10, 30 and 100 µM) had no effects on maximal upstroke velocity (Vmax), but shortened AP duration at 90% repolarization (APD(90)). At 30 and 100 µM, lidocaine also increased AP triangulation. In guinea-pig atria, lidocaine decreased Vmax starting from 30 µM and conduction velocity (CV) at 100 µM, but had no effects on other parameters. In rabbit atria, lidocaine decreased Vmax and CV at 100 µM without affecting APD(90). The effects of 100 µM lidocaine on Vmax and CV were more marked in rabbit than in guinea-pig atria. CONCLUSION: Rabbit atria are more sensitive than rabbit Purkinje fibers or guinea-pig atria for detecting lidocaine-induced cardiac conduction slowing. These data suggest that isolated rabbit atria in addition to the hNa(v)1.5 channel assay could be relevant models to predict drug-induced conduction slowing.

Supplemental studies for cardiovascular risk assessment in safety pharmacology: a critical overview.

Safety Pharmacology studies for the cardiovascular risk assessment, as described in the ICH S7A and S7B guidelines, appear as being far from sufficient. The fact that almost all medicines withdrawn from the market because of life-threatening tachyarrhythmias (torsades-de-pointes) were shown as hERG blockers and QT interval delayers led the authorities to focus mainly on these markers. However, other surrogate biomarkers, e.g., TRIaD (triangulation, reverse-use-dependence, instability and dispersion of ventricular repolarization), have been identified to more accurately estimate the drug-related torsadogenic risk. In addition, more attention should be paid to other arrhythmias, not related to long QT and nevertheless severe and/or not self-extinguishing, e.g., atrial or ventricular fibrillation, resulting from altered electrical conduction or heterogeneous shortening of cardiac repolarization. Moreover, despite numerous clinical cases of drug-induced pulmonary hypertension, orthostatic hypotension, or heart valvular failure, few safety investigations are still conducted on drug interaction with cardiac and regional hemodynamics other than changes in aortic blood pressure evaluated in conscious large animals during the core battery mandatory studies. This critical review aims at discussing the usefulness, relevance, advantages, and limitations of some preclinical in vivo, in vitro, and in silico models, with high predictive values and currently used in supplemental safety studies.

Ventilatory function assessment in safety pharmacology: optimization of rodent studies using normocapnic or hypercapnic conditions.

Although the whole body plethysmography for unrestrained animals is the most widely used method to assess the respiratory risk of new drugs in safety pharmacology, non-appropriate experimental conditions may mask deleterious side effects of some substances. If stimulant or bronchodilatory effects can be easily evidenced in rodents under standard experimental conditions, i.e. normal air breathing and diurnal phase, drug-induced respiratory depression remains more difficult to detect. This study was aimed at comparing the responsiveness of Wistar rats, Duncan Hartley guinea-pigs or BALB/c mice to the respiratory properties of theophylline (50 or 100 mg/kg p.o.) or morphine (30 mg/kg i.p.) under varying conditions (100% air versus 5% CO2-enriched air, light versus dark day phase), in order to select the most appropriate experimental conditions to each species for safety airway investigations. Our results showed that under normocapnia the ventilatory depressant effects of morphine can be easily evidenced in mice, slightly observed in guinea-pigs and not detected in rats in any day phase. Slight hypercapnic conditions enhanced the responsiveness of rats to morphine but not that of guinea-pigs and importantly they did not blunt the airway responsiveness of rats to the stimulation and bronchodilation evoked by theophylline, the most widely used reference agent in safety pharmacology studies. In conclusion, hypercapnic conditions associated with the non-invasive whole body plethysmography should be considered for optimizing the assessment of both the ventilatory depressant potential of morphine-like substances or the respiratory stimulant effects of new drugs in the rat, the most extensively used species in rodent safety and toxicological investigations.

Cardiomyopathic Syrian hamster as a model of congestive heart failure.

Cardiomyopathic Syrian hamsters (Bio TO-2 dilated strain) constitute an animal model of congestive heart failure, which progressively develops an alteration of cardiac function leading to decreased arterial blood pressure and musculo-cutaneous blood flow associated with a complex process of cardiac remodeling including left ventricle dilation, wall thinning, and greater collagen density. The protocols described in this unit are designed to assess the pharmacological effects of new therapeutic strategies on cardiac and systemic hemodynamics, morphometry (body and target organs weight), cardiac remodeling (left ventricle dilation and collagen density), and survival in this model of dilated cardiomyopathy. Examples of results obtained with enalapril, an angiotensin I converting enzyme inhibitor, are provided for illustrative purposes.

The action potential of the Purkinje fiber: an in vitro model for evaluation of the proarrhythmic potential of cardiac and noncardiac drugs.

The proarrhythmic potential of new chemical entities can be investigated using in vitro electrophysiological techniques measuring the cardiac action potential in isolated Purkinje fibers. Different types of arrhythmias may occur as early afterdepolarizations (EADs), which are favored by action potential duration lengthening and bradycardia, or as delayed afterdepolarizations (DADs), which are facilitated by tachycardia. The effects of a test compound on the occurrence of these arrhythmias, thought to be responsible for the development of torsades de pointes in the clinic can be studied using the experimental protocols described in this unit.

Combined effects of enalapril and spironolactone in hamsters with dilated cardiomyopathy.

Chronic angiotensin I-converting enzyme inhibition can be associated with aldosterone escape. We investigated the effects of enalapril, spironolactone, and their combination on hemodynamics and cardiac remodeling in cardiomyopathic hamsters to determine whether these drugs could exert additive effects. Cardiomyopathic hamsters, Bio TO-2 dilated strain, were orally treated with enalapril (20 mg. kg. day ) and/or spironolactone (20 mg. kg. day ) according to a 2 x 2 factorial design from 120 days of age. Animals were investigated at 180 (10 animals per group) and 240 (16 animals per group) days of age. Compared with corresponding untreated groups, enalapril significantly decreased mean blood pressure (-18%); enalapril and spironolactone significantly increased cardiac output (+28%, +11%) and femoral blood flow (+10%, +12%) and significantly decreased systemic (-38%, -17%) and femoral (-26%, -13%) vascular resistances. Enalapril and spironolactone significantly decreased left ventricle cavity area (-21%, -26%) and left (-34%, -47%) and right (-37%, -48%) ventricle collagen density. Spironolactone significantly increased left ventricle wall thickness (+4%). There were significant enalapril x spironolactone interactions for most variables (compared with control group, +52%, +36%, +45% for cardiac output; +26%, +28%, +26% for femoral blood flow; -50%, -30%, -45% for systemic vascular resistance; -33%, -20%, -35% for femoral vascular resistance; -27%, -31%, -40% for left ventricle cavity area; and -46%, -58%, -60% for left and -39%, -50%, -66% for right ventricle collagen density in enalapril, spironolactone, and enalapril + spironolactone groups, respectively). In cardiomyopathic hamsters, enalapril and spironolactone in combination did not improve hemodynamics more than enalapril alone but induced stronger effects than each drug alone on cardiac remodeling.

Combined effects of metoprolol and spironolactone in dilated cardiomyopathic hamsters.

The use of beta-blockers reduces angiotensin II levels, but could not adequately suppress aldosterone production. Thus, the combination of a beta-blocker with an aldosterone receptor antagonist could exert additive effects. The effects of metoprolol and spironolactone and their combination on hemodynamics and cardiac remodeling in cardiomyopathic hamsters (CMH) were investigated. The Bio TO-2 dilated strain of CMH was treated orally with metoprolol (10 mg/kg/day), spironolactone (20 mg/kg/day), or both according to a 2 x 2 factorial design (24 animals per group) from 120 days of age and during 120 days. As compared to corresponding untreated groups, metoprolol significantly decreased mean blood pressure (-7%), and metoprolol and spironolactone significantly increased cardiac output (18% and 19%, respectively), mesenteric blood flow (11% and 14%), and femoral blood flow (13% and 17%), and significantly decreased systemic (-24% and -15%), mesenteric (-14% and -13%) and femoral (-19% and -10%) vascular resistances. Metoprolol significantly increased renal blood flow (22%) and significantly decreased renal vascular resistance (-23%). Metoprolol and spironolactone significantly decreased the cavity area of the left ventricle (-21% and -32%, respectively) and the collagen density of the left (-36% and -39%) and right (-38% and -43%) ventricles. Although the combination did not induce stronger effects than each drug alone on the systemic and most regional hemodynamic variables, it did have a stronger effect on the cardiac remodeling (compared to control group: -24%, -34%, and -46% for the left ventricle cavity area, -33%, -35%, and -62% for collagen density in the left ventricle, and -52%, -57%, and -59% for collagen density in the right ventricle, respectively, in the metoprolol, spironolactone, and metoprolol + spironolactone groups). In CMH, metoprolol and spironolactone combined did not improve hemodynamics more than each drug alone, but did exert additive effects on cardiac remodeling.

Systemic and regional hemodynamic and cardiac remodeling effects of candesartan in dilated cardiomyopathic hamsters with advanced congestive heart failure.

The effects of the selective angiotensin II type 1 receptor antagonist candesartan on cardiac, systemic, and regional hemodynamics and on cardiac, pulmonary, and hepatic histomorphometry were investigated in cardiomyopathic hamsters (CMHs), Bio TO-2 dilated strain, with advanced congestive heart failure (CHF). Two groups were treated orally with candesartan cilexetil at 22 or 50 mg/kg/d from 190 days of age and compared with a control group (38 animals/group). Investigations were performed at 225, 255, and 285 days of age. Left ventricle (LV) and systemic blood pressures and cardiac output and mesenteric and femoral blood flows were measured in anesthetized animals. LV cavity area, LV and right ventricle (RV) wall thickness and collagen density, and pulmonary and hepatic congestion were assessed. Compared with the control group, candesartan did not modify cardiac hemodynamics but significantly and dose-dependently decreased systemic vascular resistances (on average: -23 and -32% after 22 and 50 mg/kg, respectively) and increased stroke volume (+32 and +42%) and cardiac output (+27 and +34%). Candesartan did not modify mesenteric vascular resistances and blood flow but significantly and dose-dependently decreased femoral vascular resistances (-19 and -33%) and increased femoral blood flow (+33 and +43%). Candesartan significantly decreased LV cavity area (-14 and -8%) and LV (-15 and -31%) and RV (-16 and -24%) collagen density but did not modify LV and RV wall thickness. Candesartan decreased pulmonary congestion at 255 and 285 days of age but did not modify hepatic congestion. In CMHs with advanced CHF, candesartan cilexetil improves systemic and femoral hemodynamics, partly reverses cardiac remodeling, and decreases pulmonary congestion.