A method for assessing and monitoring consistency of nonclinical ECG analysis.

Cardiovascular safety is a key area of concern for new drugs in development, and the collection and analysis of electrocardiograms (ECGs) is a standard and major component of nonclinical testing. Digital data capture technology allows for high-throughput and long-duration ECG collections, resulting in large volumes of data. Consistent analysis of these ECG data is critical for detecting meaningful changes during nonclinical drug development. We developed a method to assess the consistency of nonclinical ECG analysis for a group of analysts over time. Eight­lead ECGs were collected from conscious dogs using Ponemah (v5.2, DSI). Analysts placed Pstart, Qstart, Rpeak, Send, and Tend marks on six waveforms for each animal. The ECG files were randomized and re-marked under blinded conditions 4 to 14 days following initial mark placement. Averages of each parameter measured (RR interval, QRS duration, PR interval, and QT interval) were compiled for each marking session and analyst. A Gage R&R evaluation was completed. Graphical output from the Gage R&R evaluation showed distinct variability on group and individual analyst levels. Differences in inter- and intra-analyst variability (reproducibility and repeatability, respectively) were observed between trained analysts and analysts in training. The Gage R&R method is an effective tool for assessing consistency of digital ECG mark placement at a group level. Furthermore, it is able to identify areas of improvement for individual ECG analysts and to assess ECG analyst consistency during their training period. The assessment results are useful for facilitating discussions on best practices and maintaining consistency of mark placement.

Non-Clinical Safety Pharmacology Evaluations of Trazpiroben (TAK-906), a Novel Dopamine D2/D3 Selective Receptor Antagonist for the Management of Gastroparesis.

BACKGROUND: Gastroparesis is characterized by delayed gastric emptying in the absence of mechanical obstruction. Owing to the potential for serious side effects, current treatments have restrictions on their use and there is a need for novel compounds with favorable safety profiles. Trazpiroben (previously TAK-906) is a peripherally selective dopamine D2/D3 receptor antagonist being developed to treat chronic gastroparesis. Effects of trazpiroben on the central nervous system and pulmonary system in rats and on the cardiovascular system in dogs were assessed. METHODS: Functional observational battery and locomotion assessments were conducted in groups of eight female rats receiving 0 (control), 100, 300, or 1000 mg/kg/day oral trazpiroben for 2 days. Assessments were performed at baseline (pre-dosing) and 0.5 hours post-dose on day 2 of dosing. Pulmonary safety: following administration of the same trazpiroben doses, groups of eight male rats underwent heads-out plethysmography at baseline, through 6 hours post-dose, and approximately 24 hours post-dose on day 1. Four telemetry-instrumented male beagle dogs received 0 (control), 1, 10, or 30 mg/kg of oral trazpiroben in a Latin square crossover design on days 1, 4, 8, and 11. Relevant parameters were continuously measured for approximately 18 hours post-dose. RESULTS: No clinically meaningful effects on central nervous system, pulmonary, or cardiovascular assessments were observed at any trazpiroben dose. Significantly decreased locomotion occurred with increasing dose, including reduced horizontal/vertical ambulation at ≥ 300 mg/kg/day. Small transient decreases in systolic and pulse pressure at ≥10 mg/kg/day were observed, with compensatory increases in heart rate at 30 mg/kg/day. No trazpiroben-related effects on cardiovascular parameters (including QT interval corrected for heart rate) or body temperature were noted. No trazpiroben-related qualitative electrocardiogram abnormalities were observed. DISCUSSION: Our results suggest that trazpiroben has limited central nervous system effects and a favorable cardiac safety profile.

Evaluation of cardiovascular changes in dogs administered three positive controls using jacketed external telemetry-blood pressure (JET-BP).

INTRODUCTION: Nonclinical safety studies are increasingly incorporating cardiac safety endpoints to discover potential cardiovascular liabilities. This trend for more thorough cardiovascular nonclinical safety evaluation is prudent given the high attrition rate of potential therapeutics due to unexpected cardiovascular liabilities discovered in late-stage clinical trials or post-market approval. In particular, the causal relationship of blood pressure changes that lead to risk of major adverse cardiac events suggests hemodynamic changes should be critically evaluated in preclinical studies of novel therapeutics. METHODS: Jacketed external telemetry with an implanted miniature blood pressure transmitter (JET-BP) was used to characterize the tolerability, functionality, and sensitivity of this study design in dogs. Thirty-six male or female beagles (n=6 dogs/sex/group) were administered vehicle control (reverse osmosis water) or etilefrine (1, 10mg/kg), sotalol (3, 30mg/kg), and hydralazine (1, 10mg/kg) on separate days. Telemetry data were evaluated for positive control article-related changes and retrospective power analysis was also completed. Animals were evaluated for instrumentation-related changes in clinical and anatomic pathology endpoints. RESULTS: All three positive controls elicited the expected pharmacologic responses that were statistically different at high and low doses. Retrospective power analysis confirmed this study design was able to statistically differentiate minor (approximately 5 to 15%) changes in electrocardiography and blood pressure values. This study also demonstrated the potential advantages of combining cardiovascular data across sex when the test article exposure and pharmacodynamics were consistent. Data collection using miniature telemetry blood pressure transmitters did not result in anatomic or clinical pathology findings that would prevent their use in general toxicology studies. DISCUSSION: This characterization study indicates that JET-BP in dogs offers a scientifically-robust method to evaluate novel therapeutics for potential cardiovascular liabilities.

Intravenous solid tip ECG lead placement in telemetry implanted dogs: Part 2: High quality telemetry signals yield high sensitivity to drug-induced changes.

INTRODUCTION: Dogs are commonly used in cardiovascular drug safety assessment, and implanted telemetry models include subcutaneous or epicardial electrocardiogram (ECG) electrode placements. The purpose of this study was to determine the sensitivity of a canine telemetry model with intravenous ECG lead placement: the negative ECG lead (solid tip) inserted into the jugular vein and the positive lead sutured to the diaphragm. Reference drugs were administered to test the sensitivity to drug-induced changes. METHODS: Twenty-four dogs were implanted with PCT or PCTP transmitters [Data Sciences International (DSI)]. Three reference drugs were administered: sotalol to eight PCT and milrinone to eight PCTP transmitter-implanted dogs. Twenty-four dogs received moxifloxacin (12 dogs/transmitter type). Telemetry data were collected for 25h and analyzed using double Latin squares for sotalol and milrinone data or a 4×4 or 3×6 parallel design for moxifloxacin data. Evaluated parameters were PR, QT, corrected QT (QTc), QRS, heart rate, left ventricular function, and hemodynamic data. Various correction factors for QTc interval were tested. Retrospective power analysis was performed to detect minimal absolute changes comparing a single to a double Latin square or the two parallel designs. RESULTS: Expected changes on ECG and hemodynamic parameters were observed after administration of all reference drugs. The individual animal corrected QT (QTci) interval provided the optimal correction factor. Retrospective power analysis confirmed detection of smaller differences in double versus single Latin squares. Minimal detectable differences were smaller in both Latin squares compared to parallel designs, with smaller detectable differences in a 3×6 compared to a 4×4 parallel design. DISCUSSION: The solid tip intravenous ECG lead configuration in dogs is a viable radiotelemetry model to detect drug-induced changes with high sensitivity. This model yields comparable signal quality and represents a refinement over epicardial ECG leads and allows for possible reduction in the number of animals if study design and size are selected based on needed assay sensitivity.

Intravenous solid tip lead placement in telemetry implanted dogs. Part 1: Surgical methods, signal quality, and pathological endpoints.

INTRODUCTION: Electrocardiogram (ECG) signals in safety pharmacology studies are generally collected via subcutaneous or epicardial leads. Subcutaneous placement is an easier procedure, but signals often contain artifacts. Epicardial leads offer improved quality but require additional surgical expertise. Signal quality and tolerability of intravenous (IV)/diaphragmatic ECG leads were investigated as a less invasive alternative to the epicardial ECG lead approach for cardiovascular assessment in dogs. METHODS: Twenty-eight beagle dogs were implanted with PCT (n=14) or PCTP (n=14) transmitters with IV (negative)/diaphragmatic (positive) ECG leads arranged in approximate Lead II configuration. Surgical time for previous epicardial and current IV lead placement approaches was compared. The ECG signals were assessed for up to 32 weeks post-surgery. Signal quality was assessed based on good wave/total wave (GW/TW) ratios calculated using ECG PRO (Ponemah Physiology Platform, Version 4.8) and variability in ECG parameter measurements for each surgical model. Clinical pathology was assessed on all animals before surgery and approximately 2 and 12 weeks post-surgery. A specialized necropsy was conducted on four animals (two PCT and two PCTP) to assess the tolerability of telemetry equipment; selected tissues were examined microscopically. RESULTS: Surgical time using the IV lead method was approximately 18% shorter than the epicardial lead method. The GW/TW ratio for IV lead-implanted dogs indicated good durability of signal that was similar to epicardial leads. Intra- and inter-animal variability in ECG parameter measurements was similar between IV lead-implanted and epicardial lead-implanted dogs. Clinical pathology revealed no noteworthy findings, and the IV/diaphragmatic surgical approach had minimal consequences on local vasculature and associated implantation sites. DISCUSSION: Advantages of the IV/diaphragmatic lead model include a less invasive and shorter surgical procedure; high tissue tolerance, ECG signal quality, and durability; and data processing procedures similar to that of epicardial leads. Therefore, the IV/diaphragmatic lead configuration is a viable alternative to more invasive surgical approaches for telemetry device implantation in dogs.

Differential effects of Vitamin D analogs on calcium transport.

It is well known that the efficiency of intestinal active calcium transport is regulated by the Vitamin D receptor pathway and Vitamin D analogs seem to exhibit differential effects on intestinal active calcium transport. To investigate the molecular basis for the difference among Vitamin D analogs, we tested three Vitamin D analogs: 1,25-dihydroxyvitamin D(3), 19-nor-1,25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) ex vivo and in vitro. In 5/6 nephrectomized rat intestinal active calcium transport, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effects on intestinal active calcium transport at all the concentrations tested, while 1alpha-hydroxyvitamin D(2) at 0.33 and 0.67 microg/kg and 1,25-dihydroxyvitamin D(3) at 1microg/kg significantly stimulated calcium transport. In Caco-2 cells, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effect on calcium transport, while 1,25-dihydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(2) (the active form of 1alpha-hydroxyvitamin D(2)) stimulated calcium transport by 934 and 501% at 0.1microM, respectively. 1,25-Dihydroxyvitamin D(2) potently induced the expression of CALB3 and TRPV6 mRNA with an EC(50) of 0.3 and 1.0nM, whereas 19-nor-1,25-dihydroxyvitamin D(2) was 10-fold less potent than 1,25-dihydroxyvitamin D(2) in inducing CALB3 and TRPV6 mRNA. The three Vitamin D analogs had no significant effect on the expression of PMCA1 mRNA. These Vitamin D analogs did not change the expression of Vitamin D receptor (VDR) up to 10nM, but stimulated CYP24A1 expression in a dose-dependent manner with the potency in the order of 1,25-dihydroxyvitamin D(3)>1,25-dihydroxyvitamin D(2)=19-nor-1,25-dihydroxyvitamin D(2). These results suggest that the differential effect of Vitamin D analogs on stimulating intestinal and Caco-2 calcium transport may be in part due to its different effect on stimulating CALB3 and TRPV6 mRNA expression.

Pharmacological MRI in awake rats reveals neural activity in area postrema and nucleus tractus solitarius: relevance as a potential biomarker for detecting drug-induced emesis.

Drug-induced vomiting (emesis) is a major concern in patient care and a significant hurdle in the development of novel therapeutics. With respect to the latter, rodents, such as the rat and mouse, are typically used in efficacy and safety studies; however, drug-induced emesis cannot be readily observed in these species due to the lack of an emetic reflex. It is known that emesis can be triggered by neural activity in brain regions including area postrema (AP) and nucleus tractus solitarius (NTS). In this study, using pharmacological magnetic resonance imaging (phMRI) and a blood-pool contrast agent, we imaged the hemodynamic consequences of brain activity in awake rats initiated by the administration of compounds (apomorphine 0.1, 0.3 micromol/kg i.v. and ABT-594 0.03, 0.1, 0.3 micromol/kg i.v.) that elicit emesis in other species. Regional drug-induced relative cerebral blood volume (rCBV) changes and percent activated area within the AP and NTS were calculated, in which a dose-dependent relationship was evident for both apomorphine and ABT-594. Additionally, to correlate with behavioral readouts, it was found that the activation of AP and NTS was observed at plasma concentrations consistent with those that induced emesis in ferrets for both drugs. Our data thus suggest that phMRI in awake rats may be a useful tool for predicting emetic liability of CNS-acting drugs and may provide insights into depicting the underlying emetic neural pathways in vivo.

Predictive, non-GLP models of secondary pharmacodynamics: putting the best compounds forward.

Secondary pharmacodynamic studies of new chemical entities (NCEs) play a critical role in support of efficient drug discovery. In an era in which speed and efficiency are the norm for pharmaceutical discovery, the need to identify NCEs with greater patient tolerability continues to increase. Early use of secondary pharmacodynamic models (in vivo and in vitro) provides the foundation for critical, early decisions regarding lead molecules. Scientifically robust, non-GLP (good laboratory practices) secondary pharmacodynamic studies can eliminate compounds or structural series with undesirable profiles early, and may prove useful in defining structure-activity relationships (SARs) with regards to off-target effects.

Dopamine D2, but not D4, receptor agonists are emetogenic in ferrets.

Agents that activate the dopamine D2-like family of receptors elicit emesis in humans and other species with a vomiting/emetic reflex; however, the lack of dopamine receptor subtype selective agonists has hampered an understanding of which dopamine D2-like receptor subtype(s) contributes to the emetic response. In this study, stable cell lines expressing the ferret dopamine D2-long (D2L) and D4 receptors were used to characterize known dopamine agonists via radioligand binding and calcium ion flux assays, while emetic activity of these dopamine receptor agonists was determined in male ferrets. Latencies to first emetic event, average number of emetic episodes, and stereotypical behaviors which may be indicative of nausea were also determined. Agonists at dopamine D1-like and D4 receptors had no emetic effect in ferrets. Conversely, stimulation of dopamine D2 and/or D3 receptors resulted in a robust emetic response characterized by a relatively short latency (<15 min) and multiple emetic events. Competitive antagonists of dopamine D2-like receptors (domperidone, haloperidol) dose-dependently blocked the emetic response to PNU95666E, a dopamine D2 receptor selective agonist. Thus, dopamine D2 and/or D3 receptor agonists elicit emesis, while dopamine D1/D5 or D4 receptor-selective agonists are devoid of emetic properties.

ABT-089: pharmacological properties of a neuronal nicotinic acetylcholine receptor agonist for the potential treatment of cognitive disorders.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride salt] is a selective neuronal nicotinic receptor (NNR) modulator with cognitive enhancing properties in animal models of cognitive functioning. Amongst NNR subtypes, ABT-089 shows selectivity for the cytisine binding site on the alpha4beta2 receptor subtype as compared to the alpha-bungarotoxin (alpha-BgT) binding sites on the alpha7 and alpha1beta1deltagamma receptor subtypes. In functional in vitro electrophysiological and cation flux assays, ABT-089 displays differential activity including agonism, partial agonism and antagonism depending upon the NNR subtype and assay. ABT-089 is as potent and efficacious as (-)-nicotine at evoking acetylcholine (ACh) release from hippocampal synaptosomes. Furthermore, ABT-089 is neuroprotective against excitotoxic glutamate insults, with even greater potency seen after chronic treatment. Similarly, ABT-089 is effective in models of cognitive functioning, including enhancement of baseline functioning as well as improvement of impaired cognitive functioning seen following septal lesioning and natural aging. In neuroprotective assays the compound is most potent by chronic administration. In stark contrast to the positive effects in the cognitive models, ABT-089 shows little propensity to induce adverse effects such as ataxia, hypothermia, seizures, cardiovascular or gastrointestinal side effects. Together these data suggest that ABT-089 is a NNR modulator with the potential for treating cognitive disorders with markedly limited adverse cardiovascular and gastrointestinal side effects.

Activation of dopamine D4 receptors by ABT-724 induces penile erection in rats.

Apomorphine, a nonselective dopamine receptor agonist, facilitates penile erection and is effective in patients suffering from erectile dysfunction. The specific dopamine receptor subtype(s) responsible for its erectogenic effect is not known. Here we report that the dopamine D(4) receptor plays a role in the regulation of penile function. ABT-724 is a selective dopamine D(4) receptor agonist that activates human dopamine D(4) receptors with an EC(50) of 12.4 nM and 61% efficacy, with no effect on dopamine D(1), D(2), D(3), or D(5) receptors. ABT-724 dose-dependently facilitates penile erection when given s.c. to conscious rats, an effect that is blocked by haloperidol and clozapine but not by domperidone. A proerectile effect is observed after intracerebroventricular but not intrathecal administration, suggesting a supraspinal site of action. s.c. injections of ABT-724 increase intracavernosal pressure in awake freely moving rats. In the presence of sildenafil, a potentiation of the proerectile effect of ABT-724 is observed in conscious rats. The ability of ABT-724 to facilitate penile erection together with the favorable side-effect profile indicates that ABT-724 could be useful for the treatment of erectile dysfunction.

Emetic liability testing in ferrets.

Evidence of a candidate drug's efficacy and safety is mandatory for successful drug registration by regulatory authorities. However, a third property, tolerability, often determines a drug's acceptance by the patient population. Gastrointestinal events often determine the maximum tolerated dose in Phase I clinical trials. If the plasma concentrations achieved at the maximum tolerable dose are below those required for efficacy, the drug will certainly fail. The identification of a compound's emetic/nauseogenic liability early in the discovery process can be critical to the ultimate success of the drug discovery project. Ferrets are small carnivores (~1 kg) of the Mustelidae family that vomit in response to many pharmacological classes of drugs as well as to cytotoxic chemotherapeutics and radiation. This unit describes a simple method for evaluating the emetic and nauseogenic potential of drug candidates in ferrets.