Search

Introduction: Assessment of the propensity of novel drugs to cause proarrhythmia is essential in the drug development process. It is increasingly recognized, however, that QT prolongation alone is an imperfect surrogate marker for Torsades de Pointes (TdP) arrhythmia prediction. In the present study we investigated the behavior of a novel surrogate marker for TdP, the electro-mechanical (E-M) window, prior to triggering of TdP episodes with sympathetic stimulation after administration of a number of reference compounds., Methods: Experiments were carried out in closed chest pentobarbital anesthetized guinea pigs. Test compounds were administered intravenously together with a specific I(Ks) blocker (JNJ303; 0.2 mgkg(-1)min(-1) for 3 min) and adrenaline (0.06 mgkg(-1)min(-1) for 2 min) was applied to trigger TdP. ECG, blood- and left ventricular pressure signals were measured continuously throughout the experiments. The E-M window i.e. the duration of the mechanical systole (QLVP(end) interval) minus the duration of the electrical activity (QT interval) was assessed for individual beats., Results: Drugs with documented TdP liability (quinidine, haloperidol, domperidone, terfenadine, moxifloxacin, ciprofloxacin and dofetilide) produced TdP in the protocol after adrenaline infusion, whereas negative control compounds (verapamil, ranolazine, amiodarone and saline) did not cause TdP arrhythmia, even though increases in repolarization times were observed. TdP were typically preceded by large (greater than -50 ms) negative electro-mechanical windows and were accompanied by aftercontractions., Discussion: The present study in anesthetized guinea pigs indicates that negative E-M windows are a prerequisite for sympathetically-driven TdP induction after the administration of various agents with known proarrhythmic potential. These data are a first step in the validation of this novel protocol; however we believe that this proarrhythmia model in small animals might be a valuable additional tool in the prediction of TdP risk of new chemical entities at the early stages of drug discovery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Evaluation of drug-related effects on cardiovascular function is part of the core battery described in the ICH S7A guideline. Anesthetized guinea-pigs are excellent models for the evaluation of drug-induced prolongation of ventricular repolarization; however less information is available regarding other cardio-hemodynamic parameters in this model. The current study aimed to document cardio-hemodynamic responses in anesthetized guinea-pigs after administration of a number of reference drugs with known pharmacological actions., Methods: Experiments were carried out in closed chest pentobarbital anesthetized female guinea-pigs. Compounds were administered intravenously while arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were measured continuously. The rate of LVP contraction (LV dP/dt(max)) was used to evaluate cardiac performance; and was compared to the QA interval; which has previously been proposed as an indirect measurement of cardiac function., Results: Baseline values for heart rate and blood pressure were lower in anesthetized animals compared to literature data of conscious guinea-pigs. Heart rate increased after administration of adrenaline, isoprenaline and salbutamol, but not after L-phenylephrine. Verapamil and amiodarone decreased heart rate and blood pressure. Zatebradine infusion led to a decrease in heart rate with minimal effects on blood pressure. Sodium nitroprusside (SNP) caused a reduction in mean blood pressure at higher doses followed by reflex tachycardia. Both adrenaline and L-phenylephrine increased arterial blood pressure. Furthermore, adrenaline, isoprenaline and salbutamol increased LV dP/dt(max) and decreased the QA interval. L-phenylephrine increased LV dP/dt(max), but transiently prolonged the QA interval. Both verapamil and amiodarone decreased LV dP/dt(max) and prolonged the QA interval, whereas zatebradine did not affect this parameter., Discussion: In addition to its utility for the assessment of test compounds on ventricular repolarization the pentobarbital anesthetized guinea-pig model shows promise for early stage cardio-hemodynamic screening. Furthermore, the QA interval shows potential for prediction of adverse effects on cardiac contractility., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The potential of biosolid colloids to transport metals associated with organic-waste amendments through subsurface soil environments was investigated with leaching experiments involving undisturbed soil monoliths. The monoliths (25 cm in height and 18 cm in diameter) were carved from the upper solum of an Alfisol, a Mollisol, and an Entisol in the Bluegrass region of Kentucky. Biosolid colloids were fractionated from two municipal wastes (lime-stabilized/LSB, and aerobically-digested/ADB), and a poultry-manure (PMB) organic waste and applied onto the monoliths at a rate of approximately 0.7 cm/h. Eluents were monitored for soluble and sorbed Cd, Cr, and Mo concentrations over 16-24 pore volumes of leaching. Colloid-free solutions with metal concentrations similar to those of the biosolid colloids were used as controls. The results indicated significantly (P<0.05) higher (up to 4 orders of magnitude) cationic and anionic metal elutions in association with the biosolid colloids in both, total and soluble fractions, over the control treatments. The elution of significant soluble metal loads in association with the biosolid colloids is attributed to increasing organic-metal complexation and exclusion processes, and emphasizes their importance as contaminant carriers and facilitators. Eluted metal loads varied with metal, colloid, and soil type, following the sequences Mo>Cd>Cr for the metals, and ADB>PMB>LSB (Cd and Cr) or ADB>LSB>PMB (Mo) for the colloids. Metal elution was generally enhanced by soil macroporosity and increasing OM content, while pH and Fe-Al oxides had significant, but opposite effects for the elution of cationic and anionic metal forms. Colloid and metal breakthrough curves were correlated well, being mostly asymmetrical with several maxima and minima caused by multiple clogging and flushing cycles. Soil- and colloid-metal sorption affinities were not reliable predictors of metal attenuation/elution loads, underscoring the dynamic nature of transport processes. The findings demonstrate the important role of biosolid colloids as contaminant carriers and the significant risk they pose, if unaccounted, for soil and ground water contamination in areas receiving heavy applications of biosolid waste amendments.

Heat-pain threshold and stimulus response characteristics can be evaluated with graduated heating pulses from a radiant heat source or a contact thermode. Results may be used to: (1) evaluate differences in sensation among anatomical sites, sides of the body, and with development and aging; and (2) provide an end-point for the study of the efficacy of drugs; or to follow the course of sensory alteration in disease (medical practice, epidemiologic studies, and controlled clinical trials). Because there is great variability in how tests of this kind are performed and scored, comparisons of results among medical centers are difficult. To meet this need, we have developed, and here describe, a standardized and validated test of heat-pain. We use both pyramidal and trapezoid-shaped stimuli. The range of stimulus magnitudes we recommend is sufficient to test heat-pain at a sensitive region (the face) of young people and an insensitive region (the foot) of healthy old people. From tests on healthy subjects and patients, we find that neither our previously published forced-choice or 4, 2, and 1 stepping algorithms are suitable for testing heat-pain sensation. We, therefore, introduce the Non-Repeating Ascending with Null Stimuli (NRA-NS) algorithm which performs satisfactorily. The graphed data points of responses to increasingly stronger heat pulses were made up of two components-the no pain (0) response line and the heat-pain response line (> or = 1 numerical scaling of the pain responses graded from 1 [least] to 10 [greatest]). For the pain responses, we found that usually a curve could be fit using a quadratic equation. Using this equation, or interpolation where necessary, it is possible to compute the heat-pain detection threshold (HPDT or HP:0.5), an intermediate heat-pain response (HP:5.0), and the difference between the two (HP:5.0-0.5). Our studies show that a certain time is needed between successive stimuli and tests to minimize changing basal skin temperature or threshold. We also demonstrated that low or high baseline skin temperatures can affect heat-pain responses, therefore, we advocate specific testing conditions. Based on a study of 25 healthy subjects, the reproducibility of the test falls within +/-1 stimulus steps 88% of the time for HP:5.0 and 76% of the time for HP:0.5. The precise approaches employed to make the test standard and reproducible are described. We illustrate that the algorithm and testing system is able to document altered pain threshold with skin abrasion, with intradermal injection of nerve growth factor, and with diabetic polyneuropathy.

During endochondral bone formation, chondrocytes in the cartilaginous anlage of long bones progress through a spatially and temporally regulated differentiation program before being replaced by bone. To understand this process, we have characterized the differentiation program and analyzed the relationship between chondrocytes and their extracellular environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of proliferating chondrocytes is not contiguous with the zone of hypertrophic chondrocytes identified by the transcription of the type X collagen gene. We find that the postproliferative chondrocytes which make up the zone between the zones of proliferation and hypertrophy specifically transcribe the gene for cartilage matrix protein (CMP). This zone has been termed the zone of maturation. The identification of this unique population of chondrocytes demonstrates that the chondrocyte differentiation program consists of at least three stages. CMP translation products are present in the matrix surrounding the nonproliferative chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic chondrocytes. As a result of the changes in gene expression during the differentiation program, chondrocytes in each zone reside in an extracellular matrix with a unique macromolecular composition. Chondrocytes in primary cell culture can proceed through the same differentiation program as they do in the cartilaginous rudiments. In culture, a wave of differentiation begins in the center of a colony and spreads to its periphery. The cessation of proliferation coincides with the appearance of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches at the proliferative-maturation transition and at the maturation-hypertrophy transition during chondrocyte differentiation and indicate that chondrocytes synthesize new matrix molecules and thus modify their preexisting microenvironment as differentiation progresses. However, when "terminally" differentiated hypertrophic chondrocytes are released from their surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen. Thus they are capable of recapitulating all three stages of the differentiation program in vitro. The data suggest a high degree of plasticity in the chondrocyte differentiation program and demonstrate that the progression and maintenance of this program is regulated, at least in part, by the extracellular environment which surrounds a differentiating chondrocyte during endochondral bone formation.

A sensitive and specific analytical method was developed to determine the enantiomeric purity of naproxen. A simple derivatization of naproxen with (S)-(+)-2-octanol proceeded quantitatively and gave a mixture of diastereomeric esters displaying baseline separation on liquid chromatography. The ratio of these esters was the same as the ratio of enantiomers present in naproxen samples assayed. Reproducibility of the method was excellent.

The degradation of the prostaglandin fenprostalene (III) was studied in aqueous solution. The reaction was both specific acid and base catalyzed. The only reaction found to occur was hydrolysis of the methyl ester at C-1. Activation energies for the acid- and base-catalyzed reactions were determined and are nearly identical to that for the hydrolysis of ethyl acetate, a model ester. A competing acid-catalyzed reaction of the C-1 free acid of III was found to be approximately 10 times slower than the hydrolysis of III.

The kinetics of degradation of fenprostalene (I) in polyethylene glycol 400 solution was examined using HPLC. The degradation of I at 80 degrees C was shown to depend on the presence of oxygen and a large number of polar products were produced, as evidenced by using 3H-labeled I. Evidence that autoxidation of the polyethylene glycol 400 was concurrent with degradation of I was found from a drop in the apparent pH. Antioxidants were very effective in retarding the rate of degradation in the presence of oxygen. Degradation of I in polyethylene glycol 400 appears to arise from a reaction between the drug and reactive peroxide intermediates formed through air-oxidation of polyethylene glycol 400. This is supported by the finding that I reacts exclusively by a slow transesterification reaction in diethylene glycol, a solvent that is stable to autoxidation.