Evaluation of the translation of multiple cardiovascular regulatory mechanisms in the anesthetized dog.

The strategic and targeted use of an anesthetized canine cardiovascular model early in drug discovery enables a comprehensive cardiovascular and electrophysiological assessment of potential safety liabilities and guides compound selection prior to initiation of chronic toxicological studies. An ideal model would enable exposure-response relationships to guide safety margin calculations, have a low threshold to initiate, and have quick delivery of decision quality data. We have aimed to profile compounds with diverse mechanism of actions (MoAs) of "non-QT" cardiovascular drug effects and evaluate the ability of nonclinical in vivo cardiovascular models to detect clinically reported effects. The hemodynamic effects of 11 drugs (atropine, itraconazole, atenolol, ivabradine, milrinone, enalaprilat, fasudil, amlodipine, prazosin, amiloride, and hydrochlorothiazide) were profiled in an anesthetized dog cardiovascular model. Derived parameters included: heart rate, an index of left ventricular contractility, mean arterial pressure, systemic vascular resistance, and cardiac output. Species specific plasma protein data was generated (human, dog) and utilized to calculate free drug concentrations. Using the anesthetized dog cardiovascular model, 10 of the 11 drugs displayed the predicted changes in CV parameters based on their primary MoAs and corresponding clinically described effects. Interestingly but not unexpected, 1 of 11 failed to display their predicted CV pattern which is likely due to a delay in pharmacodynamic effect that is beyond the duration of the experimental model (hydrochlorothiazide). The analysis from the current study supports the strategic use of the anesthetized dog model early in the drug discovery process for a comprehensive cardiovascular evaluation with good translation to human.

Disconnect between COX-2 selective inhibition and cardiovascular risk in preclinical models.

INTRODUCTION: Secondary pharmacology profiling is routinely applied in pharmaceutical drug discovery to investigate the pharmaceutical effects of a drug at molecular targets distinct from (off-target) the intended therapeutic molecular target (on-target). Data from a randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX, rofecoxib) trial, raised significant concerns about COX-2 inhibition as a primary or secondary target, shaping the screening and decision-making processes of some pharmaceutical companies. COX-2 is often included in off-target screens due to cardiovascular (CV) safety concerns about secondary interactions with this target. Several potential mechanisms of COX-2-mediated myocardial infarctions have been considered including, effects on platelet stickiness/aggregation, vasal tone and blood pressure, and endothelial cell activation. In the present study, we focused on each of these mechanisms as potential effects of COX-2 inhibitors, to find evidence of mechanism using various in vitro and in vivo preclinical models. METHODS: Compounds tested in the study, with a range of COX-2 selectivity, included rofecoxib, celecoxib, etodolac, and meloxicam. Compounds were screened for inhibition of COX-2 vs COX-1 enzymatic activity, ex vivo platelet aggregation (using whole blood from multiple species), ex vivo canine femoral vascular ring model, in vitro human endothelial cell activation (with and without COX-2 induction), and in vivo cardiovascular assessment (anesthetized dog). RESULTS: The COX-2 binding assessment generally confirmed the COX-2 selectivity previously reported. COX-2 inhibitors did not have effects on platelet function (spontaneous aggregation or inhibition of aggregation), cardiovascular parameters (mean arterial pressure, heart rate, and left ventricular contractility), or endothelial cell activation. However, rofecoxib uniquely produced an endothelial mediated constriction response in canine femoral arteries. CONCLUSION: Our data suggest that rofecoxib-related cardiovascular events in humans are not predicted by COX-2 potency or selectivity. In addition, the vascular ring model suggested possible adverse cardiovascular effects by COX-2 inhibitors, although these effects were not seen in vivo studies. These results may also suggest that COX-2 inhibition alone is not responsible for rofecoxib-mediated adverse cardiovascular outcomes.

Automated blood sampling in canine telemetry studies: Enabling enhanced assessments of cardiovascular liabilities and safety margins.

INTRODUCTION: A successful integration of automated blood sampling (ABS) into the telemetry instrumented canine cardiovascular model is presented in this study. This combined model provides an efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in dog while providing a complete Pharmacokinetic/Pharmacodynamic (PK/PD) profile for discovery compounds without handling artifacts, reducing the need for a separate pharmacokinetic study. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points. A series of four use cases utilizing four different test compounds and analytical endpoints are described to illustrate some of the potential applications of the technique. RESULTS: In the four presented use cases, automated blood sampling in telemetry instrumented dogs provides simultaneous cardiovascular (heart rate, arterial blood pressure, and left ventricular pressure), electrophysiological assessment (QTc, PR, and QRS intervals), body temperature, and animal activity, while collecting multiple blood samples for drug analysis. CONCLUSION: The combination of automated blood sampling with cardiovascular telemetry monitoring is a novel capability designed to support safety pharmacology cardiovascular assessment of discovery molecules. By combining telemetry and high-fidelity ABS, the model provides an enhanced PK/PD understanding of drug-induced hemodynamic and electrocardiographic effects of discovery compounds in conscious beagles in the same experimental session. Importantly, the model can reduce the need for a separate pharmacokinetic study (positive reduction 3R impact), reduces compound syntheses requirements, and shorten development timelines. Furthermore, implementation of this approach has also improved animal welfare by reducing the animal handling during a study, thereby reducing stress and associated data artifacts (positive refinement 3R impact).

Automated blood sampling in canine telemetry model: Enhanced assessment of immune liabilities.

INTRODUCTION: This manuscript presents a successful integration of multi-timepoint biomarker blood sampling (e.g., cytokines) in a conscious dog cardiovascular study using automated blood sampling via vascular access ports in telemetry instrumented dogs. In addition to determining plasma exposure of the test compound, the assessment of biomarkers of interest allows for more comprehensive preclinical evaluation on a traditional conscious dog cardiovascular (CV) telemetry study especially for immunology and immune-oncology molecules. This model system provides a rapid and efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in large species that are commonly used for preclinical safety evaluations while collecting multiple blood samples for drug and cytokine analysis. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (ABS) (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points for cytokine analysis. Four beagles received low-dose lipopolysaccharide solution (LPS) (0.1 and 0.5 µg/mL). The following cytokines were measured by Milliplex® map Canine Cytokine Magnetic Bead Panel: Interleukin (IL) 2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, TNF-α, MCP-1, KC-like, GM-CSF, IFN gamma, and IP10. RESULTS: Low dose LPS administration induced a pronounced dose-dependent, transient release of key inflammatory cytokines (IL-2, IL-6, IL-10, TNF-α, MCP-1, and KC-like). Cytokine responses were similar to other canine and human endotoxin models. LPS administration led to an increase in body temperature, heart rate, and mean arterial pressure, as well as a decrease in QTcV interval. CONCLUSION: Successful incorporation of cytokine analysis in telemetry instrumented dogs with vascular access ports allows for translational PK/PD modeling of both efficacy and safety of compounds in the immunology as well as the immune-oncology therapeutic areas designed to modulate the immune system. Remote collection of blood samples simultaneously with CV endpoints is a significant enhancement for assessment of biomarkers that are sensitive to animal handling and excitement associated with room disturbances which are obligatory with manual blood collection. Furthermore, implementing this approach has also refined our animal welfare procedure by reducing the handling during a study and thereby reducing stress (positive refinement 3R impact).

Automated blood sampling in canine telemetry studies: Enabling enhanced assessments of cardiovascular liabilities and safety margins.

INTRODUCTION: A successful integration of automated blood sampling (ABS) into the telemetry instrumented canine cardiovascular model is presented in this study. This combined model provides an efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in dog while providing a complete Pharmacokinetic/Pharmacodynamic (PK/PD) profile for discovery compounds without handling artifacts, reducing the need for a separate pharmacokinetic study. METHODS: Male beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points. A series of four use cases utilizing four different test compounds and analytical endpoints are described to illustrate some of the potential applications of the technique. RESULTS: In the four presented use cases, automated blood sampling in telemetry instrumented dogs provides simultaneous cardiovascular (heart rate, arterial blood pressure, and left ventricular pressure), electrophysiological assessment (QTc, PR, and QRS intervals), body temperature, and animal activity, while collecting multiple blood samples for drug analysis. CONCLUSION: The combination of automated blood sampling with cardiovascular telemetry monitoring is a novel capability designed to support safety pharmacology cardiovascular assessment of discovery molecules. By combining telemetry and high-fidelity ABS, the model provides an enhanced PK/PD understanding of drug-induced hemodynamic and electrocardiographic effects of discovery compounds in conscious beagles in the same experimental session. Importantly, the model can reduce the need for a separate pharmacokinetic study (positive reduction 3R impact), reduces compound syntheses requirements, and shorten development timelines. Furthermore, implementation of this approach has also improved animal welfare by reducing the animal handling during a study, thereby reducing stress and associated data artifacts (positive refinement 3R impact).

Automated Blood Sampling in a Canine Telemetry Cardiovascular Model.

Successful implementation of automated blood sampling (ABS) into a telemetry instrumented canine cardiovascular model provides simultaneous cardiovascular assessment of novel compounds while collecting multiple blood samples for analysis of drug level, cytokines, and biomarkers. Purpose-bred male Beagle dogs (n = 36) were instrumented with a dual-pressure telemetry transmitter and vascular access port. Modifications to acclimation practices, surgical procedures, and housing were required for implementation of ABS in our established cardiovascular canine telemetry colony. These modifications have increased the use and reproducibility of the model by combining early pharmacokinetic and cardiovascular studies, thus achieving both refinement and reduction from a 3R perspective. In addition, the modified model can shorten timelines and reduce the compound requirement in early stages of drug development. This telemetry-ABS model provides an efficient means to quickly identify potential effects on key cardiovascular parameters in a large animal species and to obtain a more complete pharmacokinetic-pharmacodynamic profile for discovery compounds.

Drug-induced QT prolongation: Concordance of preclinical anesthetized canine model in relation to published clinical observations for ten CiPA drugs.

INTRODUCTION: The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative differentiates torsadogenic risk of 28 drugs affecting ventricular repolarization based on multiple in vitro human derived ionic currents. However, a standardized prospective assessment of the electrophysiologic effects of these drugs in an integrated in vivo preclinical cardiovascular model is lacking. This study questioned whether QTc interval prolongation in a preclinical in vivo model could detect clinically reported QTc prolongation and assign torsadogenic risk for ten CiPA drugs. METHODS: An acute intravenous administered ascending dose anesthetized dog cardiovascular model was used to assess QTc prolongation along with other electrocardiographic (PR, QRS intervals) and hemodynamic (heart rate, blood pressures, left ventricular contractility) parameters at plasma concentrations spanning and exceeding clinical exposures. hERG current block potency was characterized using IC50 values from automated patch clamp. RESULTS: All eight drugs eliciting clinical QTc prolongation also delayed repolarization in anesthetized dogs at plasma concentrations within four-fold clinical exposures. In vitro QTc safety margins (defined based on clinical Cmax values/plasma concentrations eliciting statistically significant QTc prolongation in dogs) were lower for high vs intermediate torsadogenic risk drugs. In comparison, hERG IC10 values represented as total drug concentrations were better predictors of preclinical QTc prolongation than hERG IC50 values. CONCLUSION: There was good concordance for QTc prolongation in the anesthetized dog model and clinical torsadogenic risk assignment. QTc assessment in the anesthetized dog remains a valuable part of a more comprehensive preclinical integrated risk assessment for delayed repolarization and torsadogenic risk as part of a global cardiovascular evaluation.

Clinical efficacy of sustained-release buprenorphine with meloxicam for postoperative analgesia in beagle dogs undergoing ovariohysterectomy.

The goal of the current study was to compare the efficacy, adverse effects, and plasma buprenorphine concentrations of sustained-release buprenorphine (SRB) and buprenorphine after subcutaneous administration in dogs undergoing ovariohysterectomy. In a prospective, randomized, blinded design, 20 healthy adult female Beagle dogs underwent routine ovariohysterectomy and received multimodal analgesia consisting of meloxicam and one of two buprenorphine formulations. Dogs were randomly assigned to receive either SRB (0.2 mg/kg SC, once) or buprenorphine (0.02 mg/kg SC every 12 h for 3 d). Blinded observers assessed all dogs by using sedation scores, pain scores, temperature, HR, RR, and general wellbeing. Dogs were provided rescue analgesia with 0.02 mg/kg buprenorphine SC if the postoperative pain score exceeded a prede- termined threshold. Blood samples were collected, and mass spectrometry was used to determine plasma buprenorphine concentrations. Data were analyzed with a linear mixed model and Tukey-Kramer multiple comparison. Age, body weight, anesthetic duration, surgical duration, sevoflurane concentration, and cardiorespiratory variables did not differ significantly between groups. Dogs in both formulation groups had comparable postoperative sedation and pain scores. One dog from each formulation group had breakthrough pain requiring rescue analgesia. Plasma buprenorphine concentrations remained above a hypothesized therapeutic concentration of 0.6 ng/mL for 136.0 ± 11.3 and 10.67 ± 0.84 h for SRB and buprenorphine, respectively. Based on the results of this study, multimodal analgesic regimens consisting of meloxicam and either buprenorphine or SRB are equally efficacious in managing pain associated with an ovariohysterectomy and show comparable side effects.