ESI+ MS/MS confirmation of canine ivermectin toxicity.

Ivermectin is a semisynthetic macrocyclic lactone anthelmintic of the avermectin family derived from Streptomyces fermentation products. Avermectins are used as antiparasitic agents in domestic animals; although considered relatively safe, one must consider animal species, breed, weight, and age in dosage determinations.In January 2006, two canines were presented to the UK Livestock Disease Diagnostic Center after dying from suspected ivermectin overdoses [30-50 mg/kg body weight]. To confirm this clinical diagnosis we developed a rapid, sensitive semiquantitative ElectroSpray Ionization-Mass Spectrometry (ESI/MS) method for ivermectin in canine tissue samples. Pharmaceutical ivermectin contains two ivermectins differing by a single methyl group, and each compound forms interpretation-confounding adducts with tissue Na(+) and K(+) ions. We now report that ivermectin administration was clearly confirmed by comparison with standard and dosage forms of ivermectin, and simple proportionalities based on mass spectral intensity of respective molecular ions allowed semiquantitative estimates of injection site tissue concentrations of 20 and 40 microg/g tissue (wet weight) in these animals, consistent with the history of ivermectin administration and the clinical signs observed.There is a distinct need for both rapid detection and confirmation of toxic exposures in veterinary diagnostics, whether for interpretation of clinical cases antemortem or for forensic reasons postmortem. It is vital that interpreters of analytical results have appropriate guidance in the scientific literature and elsewhere so as to enable clear-cut answers. The method presented here is suitable for routine diagnostic work in that it allows rapid extraction of ivermectin from tissue samples, avoids the need for high-performance liquid chromatography and allows ready interpretation of the multiple ivermectin species seen by ESI(+) MS/MS in samples originating from veterinary dosage forms.

Predicting QT prolongation in humans during early drug development using hERG inhibition and an anaesthetized guinea-pig model.

BACKGROUND AND PURPOSE: Drug-induced prolongation of the QT interval can lead to torsade de pointes, a life-threatening ventricular arrhythmia. Finding appropriate assays from among the plethora of options available to predict reliably this serious adverse effect in humans remains a challenging issue for the discovery and development of drugs. The purpose of the present study was to develop and verify a reliable and relatively simple approach for assessing, during preclinical development, the propensity of drugs to prolong the QT interval in humans. EXPERIMENTAL APPROACH: Sixteen marketed drugs from various pharmacological classes with a known incidence -- or lack thereof -- of QT prolongation in humans were examined in hERG (human ether a-go-go-related gene) patch-clamp assay and an anaesthetized guinea-pig assay for QT prolongation using specific protocols. Drug concentrations in perfusates from hERG assays and plasma samples from guinea-pigs were determined using liquid chromatography-mass spectrometry. KEY RESULTS: Various pharmacological agents that inhibit hERG currents prolong the QT interval in anaesthetized guinea-pigs in a manner similar to that seen in humans and at comparable drug exposures. Several compounds not associated with QT prolongation in humans failed to prolong the QT interval in this model. CONCLUSIONS AND IMPLICATIONS: Analysis of hERG inhibitory potency in conjunction with drug exposures and QT interval measurements in anaesthetized guinea-pigs can reliably predict, during preclinical drug development, the risk of human QT prolongation. A strategy is proposed for mitigating the risk of QT prolongation of new chemical entities during early lead optimization.

Rapid determination of ethylene glycol and glycolic acid in biological fluids.

Ethylene glycol poisoning of companion animals is a common occurrence and is sometimes involved in human intoxication. Ethylene glycol is of limited toxicity, but the metabolites including glycolic acid are responsible for poisoning. Conventional treatment has employed substances to prevent alcohol dehydrogenase from metabolizing the ethylene glycol, but to be effective, therapy must begin within hours of ethylene glycol consumption. We describe a rapid (10 min) analysis of biological fluids for ethylene glycol and glycolic acid using isocratic HPLC, a refractive index detector, and a Waters fast fruit juice analytical column.

Terfenadine alters action potentials in isolated canine Purkinje fibers more than acrivastine.

Acrivastine and terfenadine are second-generation antihistamines with similar pharmacologic profiles and comparable clinical efficacies for allergic rhinitis. However, terfenadine therapy has been associated with cardiovascular side effects that include prolonged QT interval, torsades de pointes, and ventricular fibrillation (VF). We examined the adverse effects induced by terfenadine on evoked action potentials (APs) in isolated canine cardiac Purkinje fibers and determined whether acrivastine causes similar disturbances in this preparation. Terfenadine produced a statistically significant decrease in the maximal rate of increase in the AP (dV/dt) at 10(-7) M, which corresponds to the highest plasma concentration observed clinically. The IC50 (mean +/- SEM) value for terfenadine-induced inhibition of dV/dt was 1.3 +/- 0.3 x 10(-6) M. The decrease in dV/dt caused by terfenadine became more pronounced with faster rates of stimulation. Acrivastine at a concentration of 10(-5) M, a value 10 times higher than plasma concentrations observed in clinical studies, caused no significant changes in AP duration (APD) or dV/dt. The IC50 (mean +/- SEM) value for the acrivastine-induced inhibition of dV/dt was estimated to be 8.0 +/- 3.7 x 10(-3) M. Terfenadine blocked the evoked AP at 3 x 10(-6) M, whereas no block was observed with acrivastine at 10(-3) M. The effective serum concentration of acrivastine is approximately 100 times higher than that of terfenadine. Because the IC50 value for inhibition of dV/dt for acrivastine is approximately 6,000 times greater than that for terfenadine, we estimate that acrivastine is approximately 60-fold less likely to cause disturbances in cardiac conduction than terfenadine.

Lamotrigine, phenytoin and carbamazepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells.

Lamotrigine is a chemically novel anticonvulsant drug that has been reported to inhibit veratrine-induced neurotransmitter release from cortical slices in vitro. To characterize further the mechanism of action of lamotrigine, we have investigated the effects of this drug together with the anticonvulsant drugs phenytoin and carbamazepine on voltage-sensitive sodium channels present in N4TG1 mouse neuroblastoma clonal cells. Lamotrigine, phenytoin and carbamazepine produced a tonic inhibition of sodium channels with IC50 values of 91, 58 and 140 microM, respectively. At a concentration of 100 microM, all compounds shifted the voltage-dependency of steady-state inactivation toward more negative potentials by 7 to 15 mV, slowed the rate of recovery from inactivation and produced a use-dependent inhibition of sodium channels. Our data show that lamotrigine inhibits sodium channels in a manner that is similar to that produced by phenytoin and carbamazepine. This inhibition of neuronal activity is consistent with the reduction of glutamate release that was previously reported in neurochemical studies, and it expands our understanding of the mechanism of action of this anticonvulsant drug.

Tetraethylammonium ion sensitivity of a 35-pS CA2(+)-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone.

Single Ca2(+)-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K+, respectively) Ca2(+)-activated K+ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca2(+)-activated K+ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca2+ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca2+ is low; at positive membrane potentials, this channel requires Ca2+ concentrations greater than 10 microM for activation. However, 35-pS channels have a much higher sensitivity to Ca2+ in the first minute after excision (activated by 1 microM Ca2+ at -50 mV). Therefore, it is possible that the Ca2+ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca2+ concentration and by Ca2+ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (Kd = 17 mM) and is relatively resistant to inhibition by apamin.

An electrophysiological comparison of solitary type I and type II vestibular hair cells.

We have studied the electrophysiological properties of enzymatically dissociated adult pigeon semicircular canal type I (chalice shaped) and type II (cylindrical shaped) hair cells using whole cell patch clamp techniques. Under current clamp conditions, type I hair cells often exhibit more hyperpolarized resting potentials than type II hair cells, and type I hair cells also have higher input conductances (measured with negative current steps) than type II hair cells. Under voltage clamp conditions, type I hair cells showed large, persistent outward currents over the range of about -70 to -50 mV, whereas, type II hair cells showed little or no current over this potential range. The persistent outward current of type I hair cells was not inactivated at a holding potential of -30mV, a potential that inactivated the rapidly inactivating, IA, and delayed rectifier, IK, currents of type II hair cells. This current probably contributes to both the resting potential and input conductance of type I hair cells.

Tetraethylammonium blockade of apamin-sensitive and insensitive Ca2(+)-activated K+ channels in a pituitary cell line.

1. The pharmacological sensitivities and physiological contributions of two types of Ca2(+)-activated K+ channels (BK and SK) in GH3 cells were examined by the outside-out, whole-cell and cell-attached modes of the patch-clamp technique. 2. BK channels (250-300 pS in symmetrical 150 mM-K+) in outside-out patches were blocked by external tetraethylammonium (TEA) and by 50 nM-charybdotoxin (CTX), but were not blocked by apamin. 3. SK channels (9-14 pS in symmetrical 150 mM-K+) in outside-out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM-CTX. 4. The dissociation constant (Kd) for TEA block of SK channels (3.1 +/- 0.37 mM) was 12-fold greater than the Kd for the BK channels (260 +/- 21 microM). The TEA blockade of both channels was not strongly voltage dependent: for both channels the TEA binding site sensed less than 20% of the membrane electric field. 5. Application of blockers of the BK channels (1 mM-TEA and 50 nM-CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after-hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30% of the spontaneously pacing cells. 6. It is suggested that BK channels contribute to action potential repolarization: whereas SK channels contribute to the regulation of action potential firing rate.

Studies of solitary semicircular canal hair cells in the adult pigeon. II. Voltage-dependent ionic conductances.

1. The ionic conductances present in putative type II hair cells enzymatically dissociated from the anterior, posterior, and lateral semicircular canal cristae of the white king pigeon (Columba livia) vestibule were studied under whole cell voltage clamp. 2. Two classes of voltage-dependent potassium conductances were distinguishable on the basis of the time course of activation and inactivation and pharmacologic sensitivity. The rapid potassium conductance, IA, as inhibited by 6 mM 4-aminopyridine (4-AP), whereas the slow potassium conductance, IK, was inhibited by 50 mM tetraethylammonium (TEA). These conductances were not affected by extracellular calcium removal. IA was quite similar to the rapidly-inactivating A-current of molluscan soma, whereas IK was more like the delayed rectifier of molluscan soma. 3. The steady-state inactivation of IA occurred over a potential range from -100 to -40 mV. The threshold for activation of IA occurred between -60 and -50 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 13.7 nS when the conditioning pulse was -100 mV, and we estimate it to be approximately 1-2 nS from the resting membrane potential of -56 mV. 4. The steady-state inactivation of IK was approximately 60% at -40 mV and was completely removed at -80 mV. The threshold for activation of IK was between -50 and -40 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 10.5 nS when the conditioning pulse was -80 mV, and we estimate it to be approximately 6-7 nS from the resting potential of -56 mV. 5. At -56 mV (the average resting membrane potential of putative type II semicircular canal hair cells), approximately 10-14% of IA channels and approximately 57-70% of IK channels were not inactivated: thus IA and IK can contribute to the outward current during small depolarizations from rest. 6. A small calcium-dependent outward current, IK(Ca), could be elicited during step depolarizations from a holding potential of -40 mV. This calcium-dependent current was active over the range of -20 to +40 mV. 7. Inward currents could not be detected when the cells were exposed to normal physiological solutions. However, when the outward currents were blocked with internal cesium and the external solution contained 20 mM barium, sustained inward currents with rapid activation kinetics could be detected. The threshold for activation of the inward current occurred at -40 mV, and the I-V relationship peaked at -10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of solitary semicircular canal hair cells in the adult pigeon. I. Frequency- and time-domain analysis of active and passive membrane properties.

1. Hair cells were enzymatically dissociated from the neuroepithelium (cristae ampullares) of the semicircular canals of white king pigeons (Columba livia). Those hair cells determined to be type II by an anatomic criterion, the ratio of the minimum width of the neck to the width of the cuticular plate, were studied with the use of the whole cell patch-clamp technique. 2. The mean +/- SD zero-current membrane potential, Vz, was found to be -54 +/- 12 mV for anterior crista hair cells (n = 71), -62 +/- 14 mV for posterior crista hair cells (n = 14), and -55 +/- 12 mV for lateral (horizontal) crista hair cells (n = 18). The mean +/- SD value of Vz for hair cells from all cristae (n = 103) was -56 +/- 13 mV. 3. Active and passive membrane properties were calculated in the time domain, in voltage- or current-clamp mode, from responses to voltage or current pulses and, in the frequency domain, by fitting a membrane model to admittance magnitude and phase data resulting from current responses to sum-of-sines voltages at different d.c. levels of voltage-clamp membrane potential. 4. The average value +/- SE of input resistance (Rin), over the range from -100 to -60 mV, was found to 1.5 +/- 0.3 G omega from a mean-voltage-as-a-function-of-current plot, V-I, (n = 7) and a mean of 1.4 +/- 0.3 G omega from individual (n = 15) current-as-a-function-of-voltage plots, I-V. A lower mean value 0.8 +/- 0.4 G omega was obtained for the input resistance from frequency-domain calculations for a different set of cells (n = 21). Also, in two different sets of cells, average input capacitance (Cin) was determined to be 12 +/- 3 pF (n = 7) from time-domain estimates and 14 +/- 3 pF (n = 21) from frequency-domain estimates. The (Rin)(Cin) product was 11 ms based on frequency-domain estimates and 17 ms from time-domain estimates. 5. I-V curves for hair cells voltage clamped at -60 mV showed some anomalous rectification for hyperpolarizations between -60 and -120 mV but no detectable N-shape for depolarizations between -50 and 90 mV. The I-V relation showed increasing slope with depolarization through the resting potential (Vz) and increased linearly between -40 and 80 mV; the best-fit straight-line maximum slope conductance for six cells over this range was 17.4 +/- 0.3 nS.(ABSTRACT TRUNCATED AT 400 WORDS)

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line.

Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250-300 pS and 9-14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at-50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at -50 mV, and its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.

A light and transmission electron microscope study of the neural processes within the pigeon anterior semicircular canal neuroepithelium.

This report addresses two questions. First, what is the incidence and distribution of multiple type I hair cells within a single NC in the pigeon's anterior semicircular canal crista? Second, are the synaptic structures found in the avian anterior crista similar to those found in the mammalian crista and if so are they different for single and multiple hair cell calyxes? Three pigeon anterior cristae were studied using interference LM and one pigeon anterior crista was studied using TEM. The light microscope studies showed that about 28% of the NCs studied contained a single type I hair cell; 64% contained 2-5 type I hair cells; and about 8% contained 6-12 type I hair cells. It was noted that the largest number of type I hair cells/unit area was located on the lower slopes of the crista while the smallest number of type I hair cells/unit area was located on the upper slopes and apex of the crista. The TEM studies showed that of 51 hair cells studied, 35% were type II hair cells and 65% were type I hair cells. These studies also showed that synaptic structures described in mammals are also seen in the pigeon. So far, in our preliminary studies we have been unable to demonstrate any difference in synaptic structures associated with single calyxes and those associated with multiple calyxes.