Pharmacological characterization of A-960656, a histamine H3 receptor antagonist with efficacy in animal models of osteoarthritis and neuropathic pain.

Histamine H(3) receptor antagonists have been widely reported to improve performance in preclinical models of cognition, but more recently efficacy in pain models has also been described. Here, A-960656 ((R)-2-(2-(3-(piperidin-1-yl)pyrrolidin-1-yl)benzo[d]thiazol-6-yl)pyridazin-3(2H)-one) was profiled as a new structural chemotype. A-960656 was potent in vitro in histamine H(3) receptor binding assays (rat K(i)=76 nM, human K(i)=21 nM), and exhibited functional antagonism in blocking agonist-induced [(35)S]GTPγS binding (rat H(3) K(b)=107 nM, human H(3) K(b)=22 nM), and was highly specific for H(3) receptors in broad screens for non-H(3) sites. In a spinal nerve ligation model of neuropathic pain in rat, oral doses of 1 and 3mg/kg were effective 60 min post dosing with an ED(50) of 2.17 mg/kg and a blood EC(50) of 639 ng/ml. In a model of osteoarthritis pain, oral doses of 0.1, 0.3, and 1mg/kg were effective 1h post dosing with an ED(50) of 0.52 mg/kg and a blood EC(50) of 233 ng/ml. The antinociceptive effect of A-960656 in both pain models was maintained after sub-chronic dosing up to 12 days. A-960656 had excellent rat pharmacokinetics (t(1/2)=1.9h, 84% oral bioavailability) with rapid and efficient brain penetration, and was well tolerated in CNS behavioral safety screens. In summary, A-960656 has properties well suited to probe the pharmacology of histamine H(3) receptors in pain. Its potency and efficacy in animal pain models provide support to the notion that histamine H(3) receptor antagonists are effective in attenuating nociceptive processes.

Characterization of A-935142, a hERG enhancer, in the presence and absence of standard hERG blockers.

AIMS: In a previous study we found that A-935142 enhanced hERG current in a concentration-dependent manner by facilitating activation, reducing inactivation, and slowing deactivation (Su et al., 2009). A-935142 also shortened action potential duration (APD90) in canine Purkinje fibers and guinea pig atrial tissue. This study focused on the combined effects of the prototypical hERG enhancer, A-935142 and two hERG current blockers (sotalol and terfenadine). MAIN METHODS: The whole-cell voltage clamp method with HEK 293 cells heterologously expressing the hERG channel (Kv 11.1) was used. KEY FINDINGS: A-935142 did not compete with 3H-dofetilide binding, suggesting that A-935142 does not overlap the binding site of typical hERG blockers. In whole-cell voltage clamp studies we found: 1) 60 µM A-935142 enhanced hERG current in the presence of 150 µM sotalol (57.5±5.8%) to a similar extent as seen with A-935142 alone (55.6±5.1%); 2) 150 µM sotalol blocked hERG current in the presence of 60 µM A-935142 (43.5±1.5%) to a similar extent as that seen with sotalol alone (42.0±3.2%) and 3) during co-application, hERG current enhancement was followed by current blockade. Similar results were obtained with 60 nM terfenadine combined with A-935142. SIGNIFICANCE: These results suggest that the hERG enhancer, A-935142 does not compete with these two known hERG blockers at their binding site within the hERG channel. This selective hERG current enhancement may be useful as a treatment for inherited or acquired LQTS (Casis et al., 2006).

Ventricular rate adaptation: a novel, rapid, cellular-based in-vitro assay to identify proarrhythmic and torsadogenic compounds.

INTRODUCTION: Delayed cardiac repolarization is an established risk factor for proarrhythmia and Torsades-de-Pointes (TdeP) that is typically measured in vitro during slow, regular stimulation. We have developed an alternative, novel, and rapid cellular-based approach for predicting drug-induced proarrhythmia that detects changes in electrical refractoriness based on mechanical responses (measured optically) during increasingly rapid trains of stimulation interspersed with pauses (mimicking the clinically observed short-long-short (SLS) stimulation sequence associated with the TdeP initiation). METHODS: Acutely isolated rabbit ventricular myocytes were superfused and electrically stimulated using an accelerating pacing protocol (APP) consisting of 12 consecutive pacing segments (10 beats per segment) with incrementally faster cycle lengths; trains were separated by pauses to identify loss of stimulus capture as well as to mimic clinically observed SLS sequences. Drug effects were evaluated based on a myocyte's ability to contract during progressively faster pacing segments (rate-adaptation); the earliest rate during which the myocyte fails to respond (longest cycle length with incomplete capture (CLIC)) was used to quantify electrophysiologic effects. RESULTS: Torsadogenic drugs known to delay repolarization during slow stimulation prolonged CLIC and dramatically limited the ability to respond to progressively rapid stimulation. The recognized proarrhythmic compounds E-4031, cisapride, grepafloxacin, and haloperidol rapidly prolonged CLIC at and above therapeutic concentrations in a concentration-dependent manner, while negative controls (captopril, indomethacin, and loratidine) do not affect rate-adaptation. DISCUSSION: Ventricular rate adaptation represents a novel approach for rapidly detecting drugs with torsadogenic risk using rapid rhythms that are typically not employed when evaluating proarrhythmic risk. This method is well suited for detecting and avoiding potential cardiac liabilities early in drug discovery ("frontloading") prior to final selection of candidate drugs.

Negative inotropic effect of a CB2 agonist A-955840 in isolated rabbit ventricular myocytes is independent of CB1 and CB2 receptors.

A-955840, a selective CB2 agonist, has been shown to elicit concentration-dependent decreases in cardiac contractility in the anesthetized dog (decreased maximal velocity of left ventricular pressure development [LV dP/dt max]). However, it is unknown whether this represents a direct effect or a response dependent on other factors (such as autonomic tone and neurohumoral factors) present in vivo. This study examined if A-955840 had a direct effect on contractility of isolated cardiac myocytes, and if so to determine the potential mechanisms. Contractility was assessed in vitro using percent changes in maximal shortening velocity of sarcomeres (dL/dt max) and fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. L-type calcium current in myocytes was recorded using wholecell voltage-clamp techniques. A-955840 reduced dL/dt max and FS in a reversible and concentration-dependent manner with an IC50 of 11.4 µg/mL (based on dL/dt max) which is similar to the estimated IC50 value of 9.8 µg/mL based on the effects of A-955840 on LV dP/dt max in anesthetized dogs. A-955840 (4.1 µg/mL) reduced myocyte contractility (%FS) to a similar extent in the absence and presence of a CB2 antagonist, SR-2 (24.0 ± 3.4 vs 23.1 ± 3.0 %, n=5) or a CB1 antagonist, Rimonabant (18.8 ± 2.3 vs 19.8 ± 2.7 %, n=5). A-955840 (4.1 µg/mL) also reduced L-type calcium current of rabbit ventricular myocytes (1.05 ± 0.11 vs 0.70 ± 0.12 nA, n=5, P < 0.01). These results suggest that A-955840 exerts direct negative inotropic effects on isolated rabbit ventricular myocytes, which is mediated by neither CB2 nor CB1 receptors, and consistent with off-target negative inotropy mediated by inhibition of the cardiac L-type calcium current.

A novel secretagogue increases cardiac contractility by enhancement of L-type Ca2+ current.

N'1-(3,3,6,8-tetramethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yliden)-2-cyanoethanohydrazide (TTYC) increases secretion of glucagon-like peptide-1 and intracellular Ca(2+) concentration in GLUTag cells. The purpose of the present study was to examine if TTYC exerts positive inotropic effects on isolated rabbit ventricular myocytes and in vivo heart in anesthetized rats, and if so to further define the potential mechanism of action. Contractility was assessed in vitro using changes in fractional shortening (FS) of myocyte sarcomere length and in vivo using changes in the velocity of left ventricular pressure. Changes in L-type Ca(2+) current of ventricular myocytes were evaluated using whole-cell voltage-clamp techniques. TTYC increased FS of myocyte sarcomere length in a concentration-dependent manner. The positive inotropic effect was not abrogated by beta-adrenergic blockade (propranolol) or protein kinase A inhibition. TTYC enhanced peak L-type Ca(2+) current in a voltage-dependent manner (current amplitudes increased by 4.0-fold at -10 mV and 1.5-fold at +10 mV). Voltage-dependence of steady-state activation of L-type Ca(2+) current was shifted by 15 mV in the negative direction. Inactivation time course of the L-type Ca(2+) currents at voltages of -10 to 20 mV was significantly slowed by 0.3 microM TTYC. In vivo studies demonstrated that TTYC increased cardiac contractility in a dose-dependent manner. In conclusion, TTYC is a novel L-type Ca(2+) current activator with positive cardiac inotropic effects. Negative shifting of the voltage-dependence of L-type Ca(2+) current activation and reduced inactivation are two mechanisms responsible for the enhanced L-type Ca(2+) current that contribute to the positive inotropic effects.

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.

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.

3-amino-benzo[d]isoxazoles as novel multitargeted inhibitors of receptor tyrosine kinases.

A series of benzoisoxazoles and benzoisothiazoles have been synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs). Structure-activity relationship studies led to the identification of 3-amino benzo[ d]isoxazoles, incorporating a N, N'-diphenyl urea moiety at the 4-position that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor families of RTKs. Within this series, orally bioavailable compounds possessing promising pharmacokinetic profiles were identified, and a number of compounds demonstrated in vivo efficacy in models of VEGF-stimulated vascular permeability and tumor growth. In particular, compound 50 exhibited an ED 50 of 2.0 mg/kg in the VEGF-stimulated uterine edema model and 81% inhibition in the human fibrosarcoma (HT1080) tumor growth model when given orally at a dose of 10 mg/kg/day.

Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

1,4-Dihydroindeno[1,2-c]pyrazoles with acetylenic side chains as novel and potent multitargeted receptor tyrosine kinase inhibitors with low affinity for the hERG ion channel.

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Identification of diamino chromone-2-carboxamides as MCHr1 antagonists with minimal hERG channel activity.

A series of potent 2-carboxychromone-based melanin-concentrating hormone receptor 1 (MCHr1) antagonists were synthesized and evaluated for hERG (human Ether-a-go-go Related Gene) channel affinity and functional blockade. Basic dialkylamine-terminated analogs were found to weakly bind the hERG channel and provided marked improvement in a functional patch-clamp assay versus previously reported antagonists of the series.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

An evaluation of 3,4-methylenedioxy phenyl replacements in the aminopiperidine chromone class of MCHr1 antagonists.

The optimization of potent MCHr1 antagonist 1 with respect to improving its in vitro profile by replacement of the 3,4-methylenedioxy phenyl (piperonyl) moiety led to the discovery of 19, a compound that showed excellent MCHr1 binding and functional potencies in addition to possessing superior hERG separation, CYP3A4 profile, and receptor cross-reactivity profiles.

Constrained 7-fluorocarboxychromone-4-aminopiperidine based Melanin-concentrating hormone receptor 1 antagonists: the effects of chirality on substituted indan-1-ylamines.

The incorporation of constrained tertiary amines into an existing class of N-benzyl-4-aminopiperidinyl chromone-based MCHr1 antagonists led to the identification of a series of chiral racemic compounds that displayed good to excellent functional potency, binding affinity, and selectivity over the hERG channel. Further separation of two distinct chiral racemic compounds into their corresponding pairs of enantiomers revealed a considerable selectivity for MCHr1 for one configuration, in addition to a striking difference in oral exposure between one pair of enantiomers in diet-induced obese mice. Oral administration of the most potent compound in this class in the same animal model led to significant reduction of fat mass in a semi-chronic model for weight loss.

Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: assessment of potency, efficacy, and cardiovascular safety.

Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat, and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular model compared to other series reported by our group. However, this analogue also led to prolongation of the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional blockade of this ion channel.

The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o.

INTRODUCTION: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (IKr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value. METHODS: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K+ channels. The object of these experiments was to (1) compare binding Ki values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K+ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing Ki values from binding studies at 5 and 60 mM [K+]o to functional IC50 values for hERG current block using 56 structurally diverse drugs. RESULTS: We found (a) comparable Ki values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K+]o lowered the Kd and increased the Bmax for [3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding Ki values and functional IC50 values for hERG current block. DISCUSSION: In conclusion, increasing K+ concentrations results in an increase in both [3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between Ki values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K+]o within assays. There is good correlation between binding Ki values and functional (whole-cell patch clamp) IC50 values at both 5 and 60 mM K+ concentrations (R2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K+ channel.

Nonpeptide luteinizing hormone-releasing hormone antagonists derived from erythromycin A: design, synthesis, and biological activity of cladinose replacement analogues.

The design and synthesis of a series of 11,12-cyclic carbamate derivatives of 6-O-methylerythromycin A that are novel, nonpeptide LHRH antagonists, is described. The macrolide antagonist 1, discovered during a screen of our chemical repository, was compared to a macrocyclic peptide antagonist 2 using molecular modeling, thus providing a model for the design of more potent antagonists. Medicinal chemistry efforts to find a replacement for cladinose at position 3 of the erythronolide core provided a series of oxazolidinone carbamates that were equally as active as the cladinose-containing parent macrolides. The descladinose LHRH antagonist 14 has 1-2 nM affinity for both rat and human LHRH receptors and is a potent inhibitor of LH release (pA2 = 8.76) in vitro. In vivo, 14 was found to produce a dose-dependent suppression of LH in male castrate rats via both i.v. and p.o. dosing.

Oral bimoclomol elevates heat shock protein 70 and reduces myocardial infarct size in rats.

Bimoclomol has been shown to increase an inducible member of the heat shock protein 70 family (HSP70) and cytoprotect in vitro. Here, we addressed whether oral pretreatment of rats with bimoclomol could elevate myocardial HSP70 and reduce infarct size in a rat model of ischemia and reperfusion. Rats were pretreated with bimoclomol at 3, 6 or 18 h or with 42 degrees thermal stress 24 h before ischemia. Infarct size was significantly decreased 6 h after oral administration of bimoclomol and 24 h after thermal stress. Left ventricles from a separate group of rats were examined for HSP70 levels. Western blots showed a significant increase in HSP70 6 h after oral administration of bimoclomol and 24 h after thermal stress. There was a significant correlation (P<0.05) between HSP70 induction and infarct size reduction, whether produced by thermal stress or oral administration of bimoclomol. Thus, bimoclomol can increase HSP70 and reduce infarct size in a rat model of ischemia and reperfusion.