Conscious Dogs Research Articles

A "one-step" approach to heart rate correction and statistical analysis applied to conscious dog QTc studies.

A "one-step" method which combined the heart rate correction and statistical analysis for conscious nonhuman primate (NHP) QTc assessment was recently published. The principles of this method are applicable to other species. In the current analysis, we demonstrate the utility of the technique in conscious dog QTc studies. Two studies in male dogs (n = 8 and n = 7) implanted with telemetry devices were used. In both studies, treatments were randomized and all animals received all treatments. In the primary study, the effect on QTc of moxifloxacin was compared with vehicle. Each treatment (vehicle and moxifloxacin) was given on two separate occasions. In the second study, dogs were given vehicle or dofetilide. Conventional QTc analysis was compared with the "one-step" method. The effect on QTc relative to vehicle was determined along with the median minimal detectable difference. As expected, both moxifloxacin and dofetilide gave QTc increases with a maximum of ~ 20 ms. There was a significant increase in the sensitivity to detect a QTc effect when using the "one-step" method. The minimal detectable difference was 1.6 ms for the "one-step" method compared with 6.2 ms for the conventional method. These analyses are consistent with the increased sensitivity described for the "one-step" method applied to studies in NHP. The increased sensitivity should enhance the ability to support an integrated assessment of the QTc prolongation liability for new drugs.

Antinociceptive and anesthetic-sparing effects of levomethadone/fenpipramide cannot be enhanced by coadministration of metamizole in awake and anesthetized Beagles.

To determine the effect of levomethadone/fenpipramide and metamizole alone and in combination on acute nociception. 8 healthy, adult Beagles were used in 2 separate randomized, complete crossover, experimental trials (threshold testing and determination of minimal alveolar concentration [MAC]) with masked observers. In both trials, treatments were 0.2 mg·kg-1 levomethadone/fenpipramide (L), 75 mg·kg-1 metamizole (M), or their combination (LM). In conscious dogs, mechanical thresholds were determined using constantly rising force. Thermal thresholds were measured via ramped contact heat. The MAC of sevoflurane was determined using the bracketing method with electrical stimulus (50 V, 50 Hz, 10 ms) before and 1 and 4 hours after treatment. Mechanical thresholds in L and LM were significantly increased above baseline (BL) for 165 minutes and above M for 135 minutes. Percent thermal threshold excursion significantly increased above BL in L for 75 minutes and in LM for 135 minutes. In L and LM, the percent thermal threshold excursion was significantly higher than in M from 15 to 75 or 135 minutes, respectively. In L and LM, the MAC of sevoflurane was significantly reduced at 1 hour compared to BL and M. Duration but not the magnitude of thermal antinociception of levomethadone/fenpipramide was increased by metamizole. Mechanical antinociception in awake dogs and anesthetic-sparing effects of levomethadone/fenpipramide were not altered. Coadministration of levomethadone/fenpipramide and metamizole to increase antinociception is not justified.

Real-world diagnostic potential of bacterial biomarkers of canine periodontitis.

The objective of this study was to investigate the diagnostic potential of bacterial biomarkers by comparing the performance of molecular detection assays with clinical assessments of dog's oral health performed by veterinarians. Supragingival and subgingival plaque samples were collected from 127 client-owned dogs, pre-booked for procedures under general anesthesia, visiting veterinary practices in the United States. DNA was extracted and bacterial biomarkers quantified using quantitative polymerase chain reaction. Gingivitis and periodontitis were recorded by a trained clinician using the Weighted Gingivitis Periodontitis Score which involved assessing the buccal surfaces of 18 teeth while under general anesthesia. Intraoral dental radiographs of the left and right mandibular first molar teeth were also obtained. These data were then used to establish the diagnostic performance of the molecular assay to detect periodontitis. An initial conscious, visual oral examination performed by the veterinarian identified 67.7% of dogs as having periodontitis, but examination under general anesthesia indicated a higher proportion (86.6%). Analysis of supragingival plaque samples collected by veterinarians from conscious and unconscious dogs demonstrated the assay had an accuracy of 77.7 to 80.9%, a sensitivity of 77.6 to 81.0%, and a specificity of 80.0%. Use of this molecular screening tool in conscious dogs has the potential to improve early periodontal disease detection and support veterinary decision making, ultimately improving the oral health of dogs and consequently their quality of life.

Effect of Butorphanol-Medetomidine and Butorphanol-Dexmedetomidine on Echocardiographic Parameters during Propofol Anaesthesia in Dogs.

This study compared the effects of butorphanol-medetomidine and butorphanol-dexmedetomidine combinations on echocardiographic parameters during propofol anaesthesia in dogs. The dogs were randomly divided into two groups. In the butorphanol-medetomidine (BM) group, butorphanol (0.2 mg/kg) and medetomidine (15 μg/kg) were intravenously administered; in the butorphanol-dexmedetomidine (BD) group, butorphanol (0.2 mg/kg) and dexmedetomidine (7.5 μg/kg) was used. Anaesthesia was induced with propofol and maintained with a constant-rate infusion of propofol (0.2 mg/kg/min). The echocardiographic parameters were assessed in conscious dogs (T0). Echocardiography was conducted again at 10 min post premedication (T1), followed by assessments at 30 (T2), 60 (T3), and 90 (T4) mins. The dogs were subjected to diagnostic procedures (radiography, computed tomography) under anaesthesia. A significant reduction in heart rate and cardiac output was noted in both groups at T1. There was no significant difference in the stroke volume between the BM and BD groups. The application of butorphanol-dexmedetomidine caused a significant increase in the left ventricular internal diameter in diastole and the diameter of the left atrium compared to that caused by butorphanol-medetomidine. This study documented that butorphanol-medetomidine and butorphanol-dexmedetomidine combinations caused similar reductions in heart rate and cardiac output in both groups. 'New´ valvular regurgitation occurred following their administration.

AM Insulinization of the Liver Is an Important Driver of PM Hepatic Glucose Uptake

Glucose tolerance is significantly improved in response to consuming a second, identical meal later in the day. It is known that physiologic (portal vein) exposure to insulin in the morning primes the liver for increased hepatic glucose uptake and glycogen storage in the afternoon. Although this phenomenon has been observed in both healthy and diabetic individuals, it has not yet been established whether or not morning peripheral insulin delivery could bring about the same response. Thus, the aim of this study was to determine how the route of morning insulin delivery impacts the liver’s ability to extract and store glucose later in the day. To explore this aim, we delivered insulin into the portal vein (Po Ins) or a leg vein (Pe Ins) in the morning (AM) and subsequently challenged conscious dogs to a hyperinsulinemic-hyperglycemic (HIHG) clamp in the afternoon (PM). We then assessed the impact that each route of AM insulin delivery had on hepatic glucose uptake (HGU), non-HGU, and glycogen storage in the PM. The insulin infusion rates used in the Po Ins group were selected to mimic the rise in endogenous insulin secretion previously observed during a 4hr AM duodenal glucose infusion (2.1 mU/kg/min [0-30 min], 2.4 mU/kg/min [30-60 min], and 1.5 mU/kg/min [60-240 min]). In efforts to expose both groups to the same amount of insulin in the periphery while creating a difference at the liver, these rates were halved in the Pe Ins group. In the PM clamp, all groups received 4x basal insulin, 2x basal glycemia, and portal glucose infusion to simulate a second meal. During the 2.5hr PM clamp, the mean HGU was 1.8-fold higher in the Po Ins group vs. the Pe Ins group (HGU of 6.28±0.62 vs. 3.49±0.31 mg/kg/min; net AUC of 827±82 vs. 474±40 mg/kg/2.5hr, p<0.002), respectively, with no significant difference in non-HGU (net AUC of 819±118 mg/kg/2.5hr in Po Ins vs. 684±83 mg/kg/2.5hr in Pe Ins). Additionally, mean PM hepatic glycogen content was 47% larger in Po Ins vs. Pe Ins (23.6±1.1 vs. 12.5±1.5 mg/g liver, p<0.001), respectively. When compared to a group from an earlier study that received morning saline and a HIHG clamp in the afternoon (Sal Ctrl), there was no significant difference between Pe Ins and Sal Ctrl for any parameter measured. Therefore, physiologic hepatic insulin exposure in the morning is critical for increased hepatic glucose disposition and glycogen storage later in the day. 5T32DK007563 5R01DK131082-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Integration of metabolic flux with hepatic glucagon signaling and gene expression profiles in the conscious dog.

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

Use of video-electroencephalography as a first-line examination in veterinary neurology: development and standardization of electroencephalography in unsedated dogs and cats.

To assess the feasibility and validate the use of video-electroencephalography (EEG) in conscious dogs and cats and to propose guidelines of routine EEG in veterinary clinical practice. Prospective clinical study. One hundred and fifty EEG recordings were carried out to validate the clinical adding-value, reproducibility, and guidelines on 140 owned animals. One hundred and one EEGs were performed on dogs and 49 on cats. We compared recordings performed with 8 EEG unwired stud Ag/AgCl electrodes held by elastic straps and 8 EEG wired cup Ag electrodes held by a tailor-made manufactured headset combined with a wired video-EEG device. Electrodes placement was determined according to previously published animal EEG protocols. Physiological sensors, such as electrocardiography, electromyography, and respiratory sensors were added. Stimulation protocols were tested. Quality and interpretability were evaluated. Headsets and recording procedures appeared suitable for all skull shapes and sizes. Video-EEG recordings were successfully performed without tranquilization or anesthesia except for 9 animals. Median EEG recordings time was 40 min. Impedance remained below 20 kΩ in 99% of dog EEGs and 98% of cat EEGs. Isosynchrony was reported in 6% of the channels. Seventy-five percent of dog EEGs and 83% of cat EEGs were readable for more than 50% (to 100%) of their duration. Successful discrimination of vigilance states from rhythm analysis (wakefulness, drowsiness, and sleepiness) was possible in 99% of dog EEGs and 91% of cat EEGs. Photic driving responses during photic stimulations were observed in 11% of dog EEGs and 85% of cat EEGs. Electroencephalography recordings were directly informative in 32% of the examinations: in 25% EEG abnormalities were associated with clinical signs and 7% concerned EEG abnormalities without clinical symptoms during recording. Thirteen percent of dogs subjected to photic stimulation exhibited epileptic anomalies. Among 9 EEGs with other history-based stimulations, three displayed epileptic graphoelements. We have developed a standardized unanesthetized video-EEG procedure easily performed and reproducible in dogs and cats. Qualitative and quantitative technical and medical criteria were evaluated and were in accordance with human EEG recommendations. Moreover, we have demonstrated its relevance and accuracy for diagnostic purposes, providing further arguments for the use of EEG as a first-line neurological functional exploration test.

Validity and Repeatability Characteristics of a Non-Invasive, Infrared-Based Method Estimating Plasma Indocyanine Green Decay in Healthy Dogs.

Plasma clearance of indocyanine green (ICG-CL) is an invasive method to evaluate liver dysfunction. We aimed to investigate the practicality of a noninvasive, transcutaneous, infrared-based method estimating the disappearance rate of indocyanine green (ICG-PDR). In a randomized, cross-over study, both ICG-CL and ICG-PDR were determined in eight healthy dogs while conscious and when sedated with medetomidine and medetomidine-vatinoxan. ICG-PDR was further repeated in six of the dogs to assess its repeatability. Differences were tested with repeated-measures analysis of variance and post hoc t-tests with Bonferroni corrections, while associations were evaluated by both Spearman and Pearson correlation analyses. Furthermore, repeatability was assessed by examining calculated coefficients of variation (CV). A significant decrease in ICG-CL was observed in dogs sedated with medetomidine, while no difference between conscious and sedated states was detected with ICG-PDR. Overall, correlations between ICG-CL and ICG-PDR were poor, as was the intrasubject repeatability of ICG-PDR in conscious dogs with CV consistently above 20%. While some of the results may be explained by poor signal quality for the non-invasive method, we conclude that in healthy dogs ICG-PDR performed poorly.

Systolic blood pressure measurements with Doppler ultrasonic flow detector and high-definition oscillometry are comparable on population level but show large intra-individual differences in apparently healthy elderly dogs.

Agreement of systolic blood pressure measurements (SBP) between noninvasive blood pressure devices in conscious dogs is poorly studied. Situational hypertension is expected in clinics, but studies are lacking. This study aimed to compare SBP measurements obtained with Doppler ultrasonic flow detector (Doppler) versus high-definition oscillometry (HDO) in clinics and at home and to estimate the prevalence of situational hypertension in conscious, apparently healthy elderly dogs. 122 apparently healthy elderly or geriatric dogs were prospectively recruited. Systolic blood pressure was obtained consecutively with Doppler and HDO techniques in a randomized order per dog, following a standardized protocol. An at-home measurement was advised for in-clinic hypertensive dogs (SBP ≥ 160 mmHg), also using both devices. Dual measurements were available in 102 dogs. Median SBP was 147.3 mmHg (range, 105 to 239 mmHg) for Doppler and 152.3 mmHg (range, 113 to 221 mmHg) for HDO. Forty-six percent (56/122) were hypertensive, of which 9% (11/122) were hypertensive with both methods. No significant difference was found between the 2 devices in the global study population or within the group of hypertensive dogs. Repeated at-home measurements were performed in 20/56 (35.7%) hypertensive dogs, resulting in a 10 and 26 mmHg lower median SBP value for Doppler and HDO, respectively (P > .05). In-clinic situational hypertension was presumed in 8/20 (40%) dogs. The choice of the noninvasive blood pressure device did not significantly impact SBP results, but large interindividual differences in SBP between techniques occurred. Situational hypertension was frequently observed in clinic.

1600-P: Duration of Morning Hyperinsulinemia Is Key to the Enhancement of Hepatic Glucose Uptake Later in the Day

The second meal phenomenon, which occurs in normal and diabetic individuals, refers to the improvement in glucose tolerance seen following a second identical meal. We previously showed that 4h of morning (AM) hyperinsulinemia, but not hyperglycemia, enhanced hepatic glucose uptake (HGU) and glycogen storage during a PM hyperinsulinemic hyperglycemic clamp (HIHG) later that day. Our aim was to determine if the duration of morning hyperinsulinemia is important for the PM response to a HIHG clamp. To determine this, we administered the same amount of insulin over 2h in the first half of the morning (Ins2h-A), over 2h in the 2nd half of the morning (Ins2h-B), or over the entire 4h (Ins4h) of the morning. We then assessed the impact of the AM insulin duration on HGU during a PM HIHG clamp in conscious dogs. The dogs underwent an AM hyperinsulinemic euglycemic clamp from 0-120 min, 120-240 min, or 0-240 min (Ins2h-A, Ins2h-B, or Ins4h; n=6/group). The insulin infusion rates used in the Ins4h were selected to mimic the rise in endogenous insulin secretion previously observed during a 4h AM duodenal glucose infusion (2.1 mU/kg/min (0-30 min), 2.4 mU/kg/min (30-60 min), and 1.5 mU/kg/min (60-240 min)). These rates were doubled in both 2h groups to match the total amount of insulin being infused in the 4h AM clamp. In the PM, all groups had 4x basal insulin, 2x basal glycemia, and portal glucose infusion to simulate a 2nd meal. During the 4h PM clamp, there were no significant differences between the two 2h groups. However, there was a marked effect on the mean HGU in the Ins4h group compared to the Ins2h-A and Ins2h-B groups (HGU of 6.3±0.9 vs 3.8±0.3 and 4.4±0.4 mg/kg/min; AUC of 1393±199 vs 822±68 and 971±93 mg/kg/4hr, P<0.05), respectively, despite matched hepatic glucose loads. The longer duration (Ins4h) of AM hyperinsulinemia allowed for a 41% and 66% greater uptake of glucose in the PM clamp (vs Ins2hA + B). Thus, the duration of AM hyperinsulinemia is an important determinant of hepatic metabolism later in the day. Disclosure H.L.Waterman: None. M.S.Smith: None. B.Farmer: None. G.Kraft: None. M.Scott: None. D.S.Edgerton: None. A.D.Cherrington: Advisory Panel; Metavention, vTv Therapeutics, Diakard, Sekkei Bio, Sensulin Labs, LLC, Other Relationship; Fractyl Health, Inc., Novo Nordisk, Abvance Therapeutics, Research Support; Cellular Longevity, Inc, dba Loyal, Senda Biosciences. M.C.Moore: None. Funding National Institutes of Health (5T32DK007563-34, 5R01DK131082-02)

1560-P: Combining Glucagon with Insulin Can Lessen the Hypoglycemic Potential of Insulin

We showed previously in people with T1D that by infusing glucagon along with insulin can buffer insulin’s hypoglycemic effect without worsening its ability to limit hyperglycemia. Our aim was to determine in the conscious dog the mechanisms by which glucagon limits the insulin driven fall in glucose. Somatostatin was infused to disable the endocrine pancreas and insulin and glucagon were infused into a leg vein at basal rates. After a 140 min control period, the insulin infusion rate was increased 4-7-fold for 3h to induce hypoglycemia. Glucagon was also infused into a leg vein, then was either increased proportionately to insulin (constant I/G molar ratio, HiGGN), or in a second study on the same dog, it was kept at its basal rate (Ba GGN). Hepatic sinusoidal insulin levels rose from 11±2 to 65±17 μU/ml regardless of the glucagon level, which remained basal in BaGGN (50±10pg/mL) but rose from 52±8 pg/ml to 238±50pg/ml in HiGGN. Plasma glucose fell over the first hour, reaching a nadir of 45±2 mg/dl when glucagon was basal, and fell more slowly when glucagon was elevated (nadir of 50±2 mg/dl). This difference was due to a greater increase in net hepatic glucose output in the HiGGN group (1.4 ± 0.2 vs 2.4 ± 0.2 mg/kg/min in BaGGN and HiGGN respectively). Interestingly the rise in plasma cortisol and epinephrine were reduced by 33 and 39%, respectively, in the group with elevated glucagon. Further, this lessening in the counterregulatory response was associated with reductions in lipolysis and lactate production by muscle. Thus, by maintaining a fixed I/G molar ratio the hypoglycemic potential of insulin is buffered. This could allow more aggressive insulin treatment which would in turn increase time in range for glucose. Disclosure G.Kraft: None. M.Scott: None. M.S.Smith: None. B.Farmer: None. D.S.Edgerton: None. A.D.Cherrington: Advisory Panel; Metavention, vTv Therapeutics, Diakard, Sekkei Bio, Sensulin Labs, LLC, Other Relationship; Fractyl Health, Inc., Novo Nordisk, Abvance Therapeutics, Research Support; Cellular Longevity, Inc, dba Loyal, Senda Biosciences.

Electropharmacological Characterization of Licorice Using the Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Sheets and the Chronic Atrioventricular Block Dogs.

Licorice has been traditionally prescribed for palpitation, whereas its overdose has caused lethal arrhythmias including torsade de pointes. Licorice contains glycyrrhizic acid of ≥ 2% (w/w), which is hydrolyzed to glycyrrhetinic acid (GRA) in the intestine. Since their cardiac electropharmacological properties are not fully understood, we assessed them to ask mechanism of licorice-induced torsade de pointes. GRA at 0.1, 1 and 10μg/mL was cumulatively applied to the human induced pluripotent stem cell-derived cardiomyocytes sheets (n = 6). GRA shortened spontaneous activation interval and repolarization period, and decreased maximum contraction velocity, indicating Ca2+ channel blockade. It prolonged effective refractory period and post-repolarization refractoriness with a steep frequency-dependency, whereas it delayed conduction with a modest use-dependency, resembling lidocaine in the mode of Na+ channel-blocking action. Meanwhile, Kanzoto containing a decoction of licorice alone in a dose of 2 or 6g/body/day was orally administered to the conscious chronic atrioventricular block dogs for 3days (n = 4). Kanzoto prolonged QT interval with increasing its temporal dispersion, suggesting K+ channel suppression, and slightly decreased the plasma K+ concentration without inducing torsade de pointes. Moreover, it significantly suppressed atrial and idioventricular rates, leading to sinus arrest along with the onset of ventricular fibrillation in one animal, possibly due to Na+ channel blockade. These results indicate that electropharmacological profile of licorice can be explained by Na+, Ca2+ and K+ channels blockade, which may be associated with low torsadogenic risk, but might contribute to the onset of other types of lethal ventricular arrhythmias.

Altered cardiac repolarization associated with cellular electrophysiological remodeling following chronic testosterone undecanoate administration in a canine model

Abstract Funding Acknowledgements Type of funding sources: None. Background The influence of testosterone on ventricular ion channels is widely investigated. Despite the legally stipulated availability of different androgen anabolic steroids (AAS), these agents are very popular among young adults, however, the direct effects of supra-physiological testosterone level are still unclear. Supposedly, the chronic use of AASs may result in structural and functional remodeling of the heart. Purpose The aim of our study was to investigate the potential electrophysiological effects of chronic administration of testosterone-undecanoate in a canine model in in vivo and in vitro studies. Methods Eight male beagle dogs were randomized into control (’Cont’) and treated (’Tr’) groups (n = 4; n = 4). The latter group received 15 mg/kg of long-lasting testosterone-undecanoate intramuscular injections weekly for 3 months. Blood samples were taken for monitoring testosterone levels. To investigate the altered repolarization in conscious dogs electrocardiography studies were performed. Ventricular myocytes were enzymatically dissociated via retrograde perfusion. The transmembrane ionic currents were recorded using the whole-cell configuration of the patch-clamp technique and the action potential duration (APD) was measured by the perforated patch-clamp technique. Results Testosterone level was significantly higher in the ‘Tr’ group compared to the ‘Cont’ group (47.02 nm/L vs. 15.23 nm/L; p=0.0002). The chronic treatment did not affect the heart rate between the examined groups (93.7±23.5 vs.108.8±21.4 beats/min), however, it resulted in significantly shortened QT (226±49 vs. 244.9±26.6 ms; p<0.05) and QTc (26.06±2.5 vs. 29.6±3.01 ms, p<0.05) intervals. ECG recordings also presented prolonged PQ (112.1±15.5 vs. 61.65±12.7ms; p<0.05), QRS (72.37±15.4 vs. 61.65±12.7 ms; p<0.05), and Tp-Te (32.95±7.45 vs. 53.46±16.6 ms; p<0.05) intervals in the ‘Tr’ group. Additionally, the APD of isolated left ventricular myocytes significantly shortened in the ‘Tr’ group compared to the ‘Con’ group (235.2±26.7 vs. 283.6±28.5 ms; p<0.05). Patch-clamp experiments revealed increased magnitude of transient outward potassium current (Ito), the inward rectifier potassium current (Ik1), and the slowed delayed rectifier potassium current (Iks) in the ‘Tr’ group. Conclusion The repolarization of the canine ventricular myocardium was significantly modified by constantly high level of testosterone. Many beneficial effects are attributed to physiological testosterone levels; however, the supra-physiological testosterone level may lead to potentially harmful alternations in the cardiac repolarization that may promote arrhythmogenesis, especially in the presence of various heart diseases. Presumably, the constantly high testosterone level induced electrophysiological changes may contribute to the development of life-threatening arrhythmias under certain circumstances.

Nonclinical Cardiovascular Assessment of the Soluble Guanylate Cyclase Stimulator Vericiguat.

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action-related dose-dependent changes in systolic arterial blood pressure (up to -20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (-6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1. SIGNIFICANCE STATEMENT: There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.

Use of pupillometry for the evaluation of analgesia in dogs hospitalized in intensive care: A prospective study

A prospective study was performed on hospitalized conscious dogs. The objectives were: 1) to evaluate the feasibility and reliability of portable infrared pupillometry using a measure of photo light reflexes, 2) to identify parameters influencing measures, and 3) to compare parameters before and after the administration of analgesia.Twenty-nine dogs were included. Pupillometry was feasible by a single person. There was an excellent reliability for the evaluation of pupil diameter. There was poor to good reliability for the other parameters. There was an association between weight and mean pupil diameter (estimate = 0.1 mm, CI95 = [0.0; 0.1], P = 0.02), mean diameter variation (estimate = 0.2%, CI95 = [0.0; 0.4], P = 0.01), and mean velocity (estimate = 0.08 ms, CI95 = [0.03; 0.13], P = 0.002). Male dogs had a significantly larger mean pupil diameter than females (estimate = 1.3 mm, CI95 = [0.1; 2.5], P = 0.03). Independently of weight and sex, there was an association between mean pupil diameter and pain score: the higher the pain score, the lower the mean pupil diameter. There was a correlation between mean pupil diameter and morphine dose (ρ = −0.78, P = 0.0004). There was no significant difference in pupil diameter, velocity, and latency before and after analgesic administration.In conclusion, results do not support the use of PLR measurement by pupillometry as a pain assessment technique in hospitalized conscious dogs.

The effect of eye stimulation using graded external weights on the oculocardiac reflex in beagle dogs

The objective of this study was to determine to what extent the oculocardiac reflex (OCR) progressively rises with increasing external pressures over the superior eyelid. Controlled external weights were applied to the left eyelid of ten conscious healthy adult beagle dogs, using an electronic von Frey aesthesiometer with a modified probe. Four different weights (200 g, 300 g, 400 g and 800 g) were applied on four different non-consecutive days (with one-week intervals). Pressure was applied by pushing the device against the superior eyelid. The baseline heart rate was primarily recorded using an electrocardiograph (ECG). Then, indices of heart rate variability such as R-R intervals and vasovagal tonus index (VVTI) were calculated from the ECG tracings using twenty consecutive QRS complexes from each dog, before and after a selected set of graded weights were applied to the eye. Median R-R interval significantly increased when an external weight of 200 g was applied and peaked at 300 g. VVTI values peaked at 400 g. With higher pressures, the proportional increase was smaller in both R-R intervals and VVTI. We concluded that external eye pressure using a weight of 200 g or more is sufficient to elicit an OCR response in conscious dogs. Pressures above 400 g started to activate compensatory mechanisms that counteracted with the OCR, probably linked to an adrenergic response induced by the animal’s discomfort. Changes in R-R intervals might be used as an early OCR marker, while VVTI apparently was less influenced by potential sympathetic responses.

Testing the nonclinical Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm with an established anti-seizure medication: Levetiracetam case study.

Levetiracetam (LEV), a well-established anti-seizure medication (ASM), was launched before the original ICH S7B nonclinical guidance assessing QT prolongation potential and the introduction of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm. No information was available on its effects on cardiac channels. The goal of this work was to "pressure test" the CiPA approach with LEV and check the concordance of nonclinical core and follow-up S7B assays with clinical and post-marketing data. The following experiments were conducted with LEV (0.25-7.5 mM): patch clamp assays on hERG (acute or trafficking effects), NaV 1.5, CaV 1.2, Kir 2.1, KV 7.1/mink, KV 1.5, KV 4.3, and HCN4; in silico electrophysiology modeling (Virtual Assay® software) in control, large-variability, and high-risk human ventricular cell populations; electrophysiology measurements in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and dog Purkinje fibers; ECG measurements in conscious telemetered dogs after single oral administration (150, 300, and 600 mg/kg). Except a slight inhibition (<10%) of hERG and KV 7.1/mink at 7.5 mM, that is, 30-fold the free therapeutic plasma concentration (FTPC) at 1500 mg, LEV did not affect any other cardiac channels or hERG trafficking. In both virtual and real human cardiomyocytes, and in dog Purkinje fibers, LEV induced no relevant changes in electrophysiological parameters or arrhythmia. No QTc prolongation was noted up to 2.7 mM unbound plasma levels in conscious dogs, corresponding to 10-fold the FTPC. Nonclinical assessment integrating CiPA assays shows the absence of QT prolongation and proarrhythmic risk of LEV up to at least 10-fold the FTPC and the good concordance with clinical and postmarketing data, although this does not exclude very rare occurrence of QT prolongation cases in patients with underlying risk factors.

Colorectal and bladder prokinetic activity of [Lys5, MeLeu9, Nle10]-NKA(4-10) after intranasal or sublingual delivery in dogs.

The feasibility of eliciting defecation and urination after intranasal (IN) or sublingual (SL) delivery of a small peptide NK2 receptor agonist, [Lys5, MeLeu9, Nle10]-NKA(4-10), was examined using prototype formulations in dogs. In anesthetized animals, administration of 100 or 300 µg/kg IN or 2.0-6.7 mg/kg SL increased colorectal peak pressure and area under the curve. Peak bladder pressure was also increased at the same doses, and this was accompanied by highly efficient voiding at normal physiological bladder pressure. The onset of these effects was rapid (≤2.5 min), and the primary contractions lasted ∼25 min, returning to baseline in <60 min. Slight hypotension lasting a few minutes and causing<10% change from baseline was detected after higher doses and was statistically significant after only 100 µg/kg IN. In conscious dogs, there was a dose-related increase in voiding responses and reduction in the latency to urinate and defecate after 300 and 1000 µg/kg IN; emesis was also observed at these doses. SL administration of 6.7 mg/kg induced urination within 10 min, but not defecation or emesis. These findings support the feasibility of developing a convenient dosage form of small peptide NK2 receptor agonists as on-demand defecation or urination therapies.

Abstract 9515: Cariporide, a Selective Na+-H+ Exchange Inhibitor, Enhances Cardiac Positive Inotropic and Lusitropic Responses to Dobutamine in Heart Failure: Assessment by Pressure-Volume Analysis

Background: Inability of failing hearts to respond to positive inotropic agents such as dobutamine (DOB) represents a major clinical problem. We have shown previously that in addition to intracellular acidosis, various signaling pathways such as endothelin-1 and angiotensin II also activate cardiac Na + -H + exchange (NHE). In heart failure (HF), increased stimulation of NHE decreases myocardial calcium sensitivity. We hypothesized that blocking NHE with cariporide (CAR), a NHE-1-selective inhibitor, may improve β-adrenergic inotropic responsiveness in HF and enhance left ventricle (LV) positive inotropic and lusitropic responses of DOB. Methods: We compared the effect of clinically relevant doses of CAR (3.5 μg/kg, iv) on LV functional performance and LV contractile response to DOB (6 μg/kg/min, iv, 10 min) in 6 instrumented, conscious dogs after pacing-induced HF. LV contractile performance was measured by pressure (P)-volume (V) analysis. Animals received DOB first. After stopping DOB for 30 minutes, both DOB and CAR were infused. Results: After HF, DOB caused a 17% increase in E ES (5.3 ±1.5 vs 4.4±0.8 mmHg/ml), a16% increase in M SW (53.8±4.9 vs 45.3±3.7 mmHg), and an 11% decrease in the time constant of LV relaxation (τ) (40.4±2.6 vs 45.8±2.5 ms). DOB followed by CAR resulted in significantly greater effects with a 42% increase in E ES (6.1±2.3 vs 4.3±1.9 mmHg/ml) and a 37% increase in M SW (61.4±5.9 vs 45.1±3.2 mmHg), and a 23 % decrease in τ (34.1±3.6 vs 44.5±2.4 ms). Compared with DOB alone, DOB plus CAR caused no significant changes in heart rate (126±11 vs 130±12 bpm) and LV end-systolic P (110±8.6 vs 112±6.5 mmHg), but significantly decreased mean left atrial P (27.2±5.0 vs 32.9±5.4 mmHg), and increased stroke volume (32%, 13.3±1.7 vs 10.1±2.7 ml). DOB plus CAR also produced about 28% increased LV mechanical efficiency, measured as the ratio of stroke work to total P-V area (0.59±0.15 vs 0.46±0.17) ( p &lt;0.05). Conclusions: In pacing-induced HF, Na + -H + exchange inhibition by cariporide enhances LV positive inotropic and lusitropic responses to DOB. This suggests that combination therapy with intravenous cariporide and DOB may be appropriate in HF, thus providing a potential method of enhancing β-adrenergic responsiveness of the failing myocardium.

Influence of Methadone on Intraocular Pressure, Pupil Size, and Aqueous Tear Production in Healthy Dogs

Intraocular pressure (IOP), pupil size (PS), and tear production are variables important in maintaining eye homeostasis. The purpose of this study was to determine the effects of methadone on IOP, PS, and tear production measured by Schirmer I tear test (STT-I) in healthy nonpainful dogs. A prospective, randomized, “double-blind” clinical study was performed. A total of 40 healthy conscious client-owned dogs were included in the study. Dogs were allocated randomly to 1 of 3 groups and given intravenous methadone 0.3 mg/kg (Met-IV, n = 15), intramuscular methadone 0.3 mg/kg (Met-IM, n = 15), or saline 0.3 mL/kg (SAL, n = 10). IOP, PS, STT-I, heart rate (HR), and mean arterial pressure (MAP) were measured prior to (baseline) and at 2, 5, 10, 20, and 30 minutes after drug administration. Data were analyzed using 1-way and 2-way repeated measures ANOVA or their nonparametric equivalents (P < .05). No significant differences in IOP and PS within or between the groups were detected. In the Met-IV group, the STT-I decreased significantly after 30 minutes (P = .025), however, the values remained within the physiological ranges. In Met-IV group, HR decreased significantly at 5, 10, 20, and 30 minutes, respectively. No other significant differences were observed. Methadone administered at a dose of 0.3 mg/kg intravenously or intramuscularly seems to cause within 30 minutes no clinically important effect on IOP, PS, and STT-I in healthy conscious nonpainful dogs without ocular abnormalities.