Joseph J. Salata

Cardiac Electrophysiological Actions of the Histamine H1-Receptor Antagonists Astemizole and Terfenadine Compared With Chlorpheniramine and Pyrilamine

We compared the cardiac electrophysiological actions of two types of H1-receptor antagonists--the piperidines, astemizole and terfenadine, and the nonpiperidines, chlorpheniramine and pyrilamine-in vitro in guinea pig ventricular myocytes and in vivo in chloralose-anesthetized dogs. Astemizole and terfenadine significantly increased action potential duration of guinea pig myocytes. This concentration-dependent prolongation of action potential duration was reverse frequency dependent and led to development of early afterdepolarizations, which occurred more frequently at higher concentrations and slower pacing frequencies. Astemizole and terfenadine potently blocked the rapidly activating component of the delayed rectifier, IKr, with IC50 values of 1.5 and 50 nmol/L, respectively. At 10 mumol/L, terfenadine but not astemizole blocked the slowly activating component of the delayed rectifier, IKs (58.4 +/- 3.1%), and the inward rectifier, IK1 (20.5 +/- 3.4%). Chlorpheniramine and pyrilamine blocked IKr relatively weakly (IC50 = 1.6 and 1.1 mumol/L, respectively) and IKs and IK1 less than 20% at 10 mumol/L. Astemizole and terfenadine (1.0 to 3.0 mg/kg IV) significantly prolonged the QTc interval and ventricular effective refractory period in vivo. Chlorpheniramine and pyrilamine (< or = 3.0 mg/kg) did not significantly affect these parameters. Block of repolarizing K+ currents, particularly IK1, by astemizole and terfenadine produces reverse rate-dependent prolongation of action potential duration and development of early afterdepolarizations, delays ventricular repolarization, and may underlie the development of torsade de pointes ventricular arrhythmias observed with the use and abuse of these agents.

Saxitoxin is a gating modifier of hERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent “agonist-like” effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics.

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.

Left Ventricular Hypertrophy Decreases Slowly but Not Rapidly Activating Delayed Rectifier Potassium Currents of Epicardial and Endocardial Myocytes in Rabbits

BackgroundDelayed rectifier K+ currents are critical to action potential (AP) repolarization. The present study examines the effects of left ventricular hypertrophy (LVH) on delayed rectifier K+ currents and their contribution to AP repolarization in both epicardial (Epi) and endocardial (Endo) myocytes. Methods and ResultsLVH was induced in rabbits by a 1-kidney removal, 1-kidney vascular clamping method. Slowly (IKs) and rapidly (IKr) activating delayed rectifier K+ currents were recorded by the whole-cell patch-clamp technique, and APs were recorded by the microelectrode technique. In normal rabbit left ventricular myocytes, IKs densities were larger in Epi than in Endo (1.1±0.1 versus 0.43±0.07 pA/pF), whereas IKr density was similar between Epi and Endo (0.31±0.05 versus 0.36±0.07 pA/pF) at 20 mV. LVH reduced IKs density to a similar extent (≈40%) in both Epi and Endo but had no significant effect on IKr in either Epi or Endo. Consequently, IKr was expected to contribute more to AP repolarization in LVH than in control. This was confirmed by specific IKr block with dofetilide, which prolonged AP significantly more in LVH than in control (31±3% versus 18±2% in Epi; 53±6% versus 32±4% in Endo at 2 Hz). In contrast, L-768,673 (a specific IKs blocker) prolonged AP less in LVH than in control. The very small IKs density in Endo with LVH is consistent with the greater incidence of early afterdepolarizations induced in this region by dofetilide. ConclusionsLVH induces a decrease in IKs density and increases the propensity to develop early afterdepolarizations, especially in Endo.

Species variants of the IsK protein: differences in kinetics, voltage dependence, and La3+ block of the currents expressed in Xenopus oocytes

We have compared the slowly activating K+ currents (IsK) resulting from the expression of the human, mouse, or rat IsK proteins in Xenopus oocytes, utilizing natural, species-dependent sequence variations to initiate structure-function studies of this channel. Differences were found between the human and rodent currents in their voltage dependence, kinetics, and sensitivity to external La3+. The current/voltage relationships of the human and rat IsK currents differed significantly, with greater depolarizations required for activation of the human channel. The first 30 s of activation during depolarizations to potentials between −10 and +40 mV was best described by a triexponential function for each of the three species variants. The activation rates were, however, significantly faster for the human current than for either of the rodent forms. Similarly, deactivation kinetics were best described as a biexponential decay for each of the species variants but the human currents deactivated more rapidly than the rodent currents. The human and the rodent forms of IsK were also differentially affected by external La3+. Low concentrations (10, 50 μM) rapidly and reversibly reduced the magnitude of the mouse and rat currents during a test depolarization and increased the deactivation rates of the tail currents. In contrast, the magnitude and deactivation rates of the human IsK currents were unaffected by 50 μM La3+.

Assessing use-dependent inhibition of the cardiac Na± current (INa) in the PatchXpress automated patch clamp

INTRODUCTIONnThe cardiac Na+ current (I(Na)) underlies the rapid depolarization of the cardiac myocyte, and block of the current slows cardiac conduction and increases the risk of ventricular arrhythmia. A feature of Na+ channel block termed use-dependence is important to the assessment of blocking potency. We developed a robust automated patch clamp assay to rapidly and routinely assess the use-dependent block of I(Na) by drug candidates. The assay clarifies whether drug candidates block more potently at increased heart rates and provides a quantitative score of use-dependence.nnnMETHODSnA use-dependent cardiac I(Na) assay was implemented on the PatchXpress 7000A, an automated whole-cell patch clamp device, using a HEK cell line stably expressing the human cardiac Na+ channel, Na(V)1.5. Stable recordings lasting up to 30 minutes were achieved by selection of holding potential (-100 mV) as well as an appropriate osmotic gradient to prevent time-dependent loss of cell capacitance and current. The final protocol allows evaluation of I(Na) inhibition at three pulsing rates at three test concentrations for each recorded cell.nnnRESULTSnIC(50) values obtained for three standard I(Na) blockers lidocaine, mexiletine, and flecainide, at pulsing frequencies of 0.2 Hz, 1 Hz, and 3 Hz, were compared to IC(50) values obtained with conventional pipette patch clamp of the Na(V)1.5 cell line and of guinea pig cardiac myocytes using matched voltage protocols and pulsing rates. Absolute potencies were well correlated only under conditions of matched holding potential and fell within an approximately three-fold window. While absolute potencies could vary widely with holding potential, the fold increases in potency with increases in pulsing rates were less prone to variation of the holding potential.nnnDISCUSSIONnUse-dependence of cardiac Na+ channel block can be rapidly assessed in the PatchXpress platform and quantified at early stages of drug development to guide lead optimization.

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones as potent and selective IKs-blocking class III antiarrhythmic agents

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones having various N-l substituents were identified as potent and selective blockers of the slowly activating cardiac delayed rectifier potassium current (I(Ks)). Compound 11 is the most potent I(Ks) channel blocker reported to date.

WITHDRAWN: Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics

Amgen, Inc, Department of Investigative Toxicology, Thousand Oaks, CA, 91320, USA Schering-Plough Research Institute, Kenilworth, NJ, 07033, USA F. Hoffmann-La Roche Ltd., Non-Clinical Safety, Basel, Switzerland Wyeth Research, Chazy, New York, 12921, USA Abbott Laboratories, Department of Integrative Pharmacology, Abbott Park, IL 60064, USA AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10-4TG, U.K. GlaxoSmithKline, Safety Assessment, The Frythe, Welwyn, Herts, AL6 9AR, U.K. Novartis Pharmaceutic Corp., One Health Plaza, East Hanover, NJ, 07936, USA Merck Research Laboratories, Safety Assessment, West Point, PA, 19486, USA Lilly Research Laboratories, Global Safety Pharmacology, Greenfield, IN 46140, USA Johnson & Johnson PR&D, Global Preclinical Toxicology/Pathology, Raritan, NJ, 00869, USA

Patent Update: Patent Trends in Antiarrhythmic Agents during 1991–1992

SummaryAs a consequence of the widespread use during the past decade of sophisticated voltage clamp techniques that allow identification of specific types of ion channels, there have been a host of potassium channel subtypes identified in cardiac cells [10]. Numerous medicinal chemicals that modulate the various potassium channel subtypes, either through blockade or activation, have been synthesized and studied for their cardiovascular effects and have provided potential therapies for a number of cardiovascular disorders, including cardiac arrhythmias. The pharmacologic control and modulation of cardiac arrhythmias will continue to expand as new chemicals and new channels are discovered and explored.