Ruth L. Martin

Preclinical Characterization of A‐582941: A Novel α7 Neuronal Nicotinic Receptor Agonist with Broad Spectrum Cognition‐Enhancing Properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A‐582941. A‐582941 was found to exhibit high‐affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A‐582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A‐582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short‐term recognition memory, memory consolidation, and sensory gating deficit. A‐582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Utility of hERG Assays as Surrogate Markers of Delayed Cardiac Repolarization and QT Safety

HERG (human-ether-a-go-go-related gene) encodes for a cardiac potassium channel that plays a critical role in defining ventricular repolarization. Noncardiovascular drugs associated with a rare but potentially lethal ventricular arrhythmia (Torsades de Pointes) have been linked to delayed cardiac repolarization and block of hERG current. This brief overview will discuss the role of hERG current in cardiac electrophysiology, its involvement in drug-induced delayed repolarization, and approaches used to define drug effects on hERG current. In addition, examples of hERG blocking drugs acting differently (i.e., overt and covert hERG blockade due to multichannel block) together with the utility and limitations of hERG assays as tools to predict the risk of delayed repolarization and proarrhythmia are discussed.

Protein kinase-dependent Cl- currents in feline ventricular myocytes.

A Cl- current (ICl) induced by isoproterenol (ISO) has been identified in isolated guinea pig ventricular myocytes. This ISO-induced ICl can be inhibited by propranolol and mimicked by forskolin (FSK), suggesting that beta-receptors, cAMP, and protein kinase A (PKA) are involved in regulating the involved Cl- channel. Because activation of protein kinase C (PKC) mediated via alpha-adrenergic receptor stimulation is also known to regulate several ion channels, the idea that activation of PKC also can induce ICl was investigated by using isolated feline ventricular myocytes and the whole-cell patch-clamp technique. We found that extracellularly applied phorbol 12-myristate 13-acetate (PMA) could activate ICl in feline ventricular cells. Control experiments indicated that in the absence of PMA or other interventions, the steady-state current-voltage relation of patches maintained for more than 40 minutes was unchanged over a voltage range from -100 to +80 mV. This suggests that the present findings are not complicated by the development over time after patching of a steady-state ICl, similar to the findings reported for canine atrial myocytes. When induced by PMA, ICl was noninactivating and outwardly rectifying; it reversed polarity at approximately the equilibrium potential for Cl- and was sensitive to the Cl- channel blocker 9-anthracene carboxylic acid. In contrast, PMA failed to induce ICl when either staurosporine or calphostin C was added to the patch pipette solution used to internally dialyze the myocytes. The kinetic properties of PMA- and FSK-induced ICl were similar. When supramaximal concentrations of both ISO (1 mumol/L) and PMA (6 mumol/L) were applied simultaneously, the size of the induced ICl was the same as that induced by the same concentrations of either agonist applied alone. In addition, maximal induction of ICl with PMA (6 mumol/L) prevented the effects of FSK (1 mumol/L, the concentration causing approximately 40% of the maximal response [approximately EC40]), yet the effects of simultaneously applied submaximal concentrations (eg, approximately EC25 to approximately EC40) of both 0.5 mumol/L PMA and 1 mumol/L FSK were roughly additive. The results suggest that (1) both PMA and ISO or FSK can induce ICl with approximately equal efficacy, (2) the PMA- and ISO- or FSK-induced ICls are similar, and (3) they all flow through the same set of Cl- channels, implying that channel phosphorylation via either PKA or PKC can activate this feline cardiac ICl.

Electrophysiologic characterization of a novel hERG channel activator

Activators of the human ether-a-go-go-related gene (hERG) K(+) channel have been reported recently to enhance hERG current amplitude (five synthetic small molecules and one naturally occurring substance). Here, we characterize the effects of a novel compound A-935142 ({4-[4-(5-trifluoromethyl-1H-pyrazol-3-yl)-phenyl]-cyclohexyl}-acetic acid) on guinea-pig atrial and canine ventricular action potentials (microelectrode techniques) and hERG channels expressed in HEK-293 cells (whole-cell patch clamp techniques). A-935142 shortened cardiac action potentials and enhanced the amplitude of the hERG current in a concentration- and voltage-dependent manner. The fully activated current-voltage relationship revealed that this compound (60 microM) increased both outward and inward K(+) current as well as the slope conductance of the linear portion of the fully activated I-V relation. A-935142 significantly reduced the time constants (tau) of hERG channel activation at two example voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05) and shifted the voltage-dependence for hERG activation in the hyperpolarizing direction by 9 mV. The time course of hERG channel deactivation was slowed at multiple potentials (-120 to -70 mV). A-935142 also reduced the rate of inactivation and shifted the voltage-dependence of inactivation in the depolarizing direction by 15 mV. Recovery of hERG channel from inactivation was not affected by A-935142. In conclusion, A-935142 enhances hERG current in a complex manner by facilitation of activation, reduction of inactivation, and slowing of deactivation, and abbreviates atrial and ventricular repolarization.

In vitro preclinical cardiac assessment of tolterodine and terodiline: multiple factors predict the clinical experience.

Terodiline and tolterodine are drugs used to treat urinary incontinence. Terodiline was removed from the market in 1991 for proarrhythmia, whereas tolterodine has a generally benign clinical cardiac profile. To assess differences in the electrophysiologic actions of these drugs, we evaluated their effects on hERG current (HEK cells) and cardiac Purkinje fiber repolarization. The IC50 for hERG block (37°C) by tolterodine was 9.6 nM and by terodiline was 375 nM, values near or below clinical concentrations. Tolterodine elicited concentration-dependent prolongation of the action potential duration (APD90). In contrast, terodiline depressed the action potential plateau and induced triangulation without affecting APD90. The triangulation ratios (normalized ratio of APD50 over APD90) for terodiline were 0.94 and 0.59 for 1.0 and 10 μM and for tolterodine, were 0.99 and 0.97 at 7 and 70 nM. In summary, tolterodine, a potent hERG blocker, has a benign clinical cardiac profile at therapeutic concentrations that may be due to its lack of triangulation, as well as extensive plasma protein binding. However, at supratherapeutic concentrations, preclinical data predict risk of QT prolongation. These data suggest that hERG block and triangulation are among multiple factors that must be considered in preclinical cardiac safety assessments.

Comparison of the effects of internal [Mg2+] on IK1 in cat and guinea-pig cardiac ventricular myocytes

The effects of internal Mg2+ (Mg2+i) on processes underlying the inward rectification of the cardiac IK1 in enzymatically isolated cardiac ventricular myocytes (CVM) obtained from cat, guinea pig and rabbit were compared using the whole-cell and excised inside-out patch configurations of the voltage-clamp technique. In confirmation of the findings of other investigators, Mg(2+)i-sensitive outward IK1 currents could be elicited from guinea pig CVM at 15 degrees C when Mg2+i was reduced from 1 mM to less than 1 microM, suggesting that Mg2+i has an important role in the inward rectification of IK1 in guinea pig CVM at unphysiologically low temperatures. However, as temperature was raised to more physiological levels (e.g., 30 degrees C), Mg(2+)i-sensitive outward IK1 currents could no longer be evoked. In contrast with the results obtained with guinea pig CVM, IK1 remained inwardly rectifying in cat and rabbit CVM independent of the Mg2+i concentration ([Mg2+]i) at 37 degrees, 15 degrees, 10 degrees, and 5 degrees C. Reduced [Mg2+]i at low temperature (15 degrees C), shifted the voltage dependence of guinea pig IK1 activation in the depolarizing direction; this resulted in an apparent linearization of IK1 conductance. When the range of the membrane potential examined was expanded to include voltages up to +80 mV, the guinea pig IK1 was found to exhibit inward rectification with the inflection in the I-V relationship that is found at approximately EK at normal temperature, also shifted 80 mV or more to much less negative voltages at low temperatures. In contrast, irrespective of [Mg2+]i or temperature, the voltage dependencies of the IK1 activation curves for both cat and rabbit myocytes were not changed from that defined when [Mg2+]i was 1 mM and temperature was 37 degrees C. We propose that Mg2+i affects inward rectification of IK1 in cold guinea pig CVM by altering the voltage dependence of activation.

Hydroxypropyl ??-Cyclodextrins: A Misleading Vehicle for the In Vitro hERG Current Assay

Delayed cardiac repolarization and fatal proarrhythmia have been linked to block of the repolarizing current, Ikr or hERG (human ether-a-go-go related gene) current. Thus, determining the potency of hERG block is critical in evaluating cardiac safety during preclinical development. Hydroxypropyl β-cyclodextrins (HβC) are cyclic oligosaccharides used to enhance drug solubility. To evaluate the utility of HβC to enhance drug solubility in hERG screening assays, we studied the effect of HβC on hERG current and the sensitivity of the hERG assay to 3 structurally different hERG blocking drugs using whole-cell voltage clamp technique and HEK-293 cells expressing the hERG channel. HβC inhibited hERG activation and tail current and accelerated current deactivation in a concentration-dependent manner. HβC (6%) reduced the apparent potency of block by terfenadine (IC50 12000 nM vs 45 nM), cisapride (IC50 281 nM vs 28 nM), and E-4031 (163 nM vs 26 nM). Reduced potency of block was consistent with loss of activity as a result of complexation with HβC by terfenadine and cisapride (demonstrated in solubility studies) and interactions with HβC by E-4031 (demonstrated in absorbance studies). These results demonstrate that HβC is an unsuitable agent for enhancing compound solubility in the in vitro hERG current assay and may mask drug effects, allowing potentially dangerous drugs to advance into clinical development.

In vitro studies on a class of quinoline containing histamine H3 antagonists.

A series of quinoline containing histamine H(3) antagonists is reported herein. These analogs were synthesized via the Friedlander quinoline synthesis between an aminoaldehyde intermediate and a methyl ketone allowing for a wide diversity of substituents at the 2-position of the quinoline ring.

Altering extracellular potassium concentration does not modulate drug block of human ether-a-go-go-related gene (hERG) channels

1 Drug‐induced block of the rapidly activating delayed rectifier K+ current (IKr), encoded by human ether‐a‐go‐go‐related gene (hERG), has been linked to acquired long QT syndrome (aLQTS). Hypokalaemia is a recognized risk factor in aLQTS. To further understand why hypokalaemia is a risk factor in aLQTS, we examined the effect of [K+]o on drug block of the hERG potassium channel stably expressed in human embryonic kidney (HEK‐293) cells using whole‐cell voltage‐clamp techniques. 2 The effects of selected [K+]o (1–20 mmol/L) on hERG block with four structurally diverse compounds (dofetilide, mesoridazine, quinidine and terfenadine) from different therapeutic classes were evaluated. Reducing [K+]o from 20 to 1 mmol/L had little effect on IC50 values for hERG current block for all four compounds. For example, evaluating quinidine in external potassium concentrations of 20, 10, 5 and 1 mmol/L resulted in IC50 values of 1.82 ± 0.33, 2.04 ± 0.28, 1.57 ± 0.52 and 1.14 ± 0.21 mmol/L, respectively. No statistically significant difference (P > 0.35, anova) was observed between drug block of hERG in different external potassium concentrations. These data are in contrast with previously reported results examining hERG channel modulation expressed in AT‐1 cells under similar experimental conditions. 3 These results demonstrate that [K+]o does not directly modulate drug block of hERG channels expressed in an HEK‐293 cell line. The enhanced risk of Torsades de Pointes associated with hypokalaemia in aLQTS may be due to reduction of other (non‐hERG) potassium currents, further reducing the repolarization reserve, and not due to direct modulation of hERG block by [K+]o.

Discovery of N-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/mivebresib), a Potent and Orally Available Bromodomain and Extraterminal domain (BET) Family Bromodomain Inhibitor

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.