Conscious Dogs Research Articles

A cross-species validation of single-beat metrics of cardiac contractility.

The assessment of left ventricular (LV) contractility in animal models is useful in various experimental paradigms, yet obtaining such measures is inherently challenging and surgically invasive. In a cross-species study using small and large animals, we comprehensively tested the agreement and validity of multiple single-beat surrogate metrics of LV contractility against the field-standard metrics derived from inferior vena cava occlusion (IVCO). Fifty-six rats, 27 minipigs and 11 conscious dogs underwent LV and arterial catheterization and were assessed for a range of single-beat metrics of LV contractility. All single-beat metrics were tested for the various underlying assumptions required to be considered a valid metric of cardiac contractility, including load-independency, sensitivity to inotropic stimulation, and ability to diagnose contractile dysfunction in cardiac disease. Of all examined single-beat metrics, only LV maximal pressure normalized to end-diastolic volume (EDV), end-systolic pressure normalized to EDV, and the maximal rate of rise of the LV pressure normalized to EDV showed a moderate-to-excellent agreement with their IVCO-derived reference measure and met all the underlying assumptions required to be considered as a valid cardiac contractile metric in both rodents and large-animal models. Our findings demonstrate that single-beat metrics can be used as a valid, reliable method to quantify cardiac contractile function in basic/preclinical experiments utilizing small- and large-animal models KEY POINTS: Validating and comparing indices of cardiac contractility that avoid caval occlusion would offer considerable advantages for the field of cardiovascular physiology. We comprehensively test the underlying assumptions of multiple single-beat indices of cardiac contractility in rodents and translate these findings to pigs and conscious dogs. We show that when performing caval occlusion is unfeasible, single-beat metrics can be utilized to accurately quantify cardiac inotropic function in basic and preclinical research employing various small and large animal species. We report that maximal left-ventricular(LV)-pressure normalized to end-diastolic volume (EDV), LV end-systolic pressure normalized to EDV and the maximal rate of rise of the LV pressure waveform normalized to EDV are the best three single-beat metrics to measure cardiac inotropic function in both small- and large-animal models.

Effects of recording device, body position, electrode placement, and sedation on electrocardiogram intervals in dogs

Selected electrocardiograph (ECG) intervals may be useful when incorporated into prediction models for cardiac risk assessment in dogs. Standard recommendations for ECG acquisition may not be adhered to in practice. Study objectives were to compare duration of P, PR, QRS, QT, and R wave peak time intervals in: (1) lead II ECGs vs. single lead precordial ECGs in conscious dogs; (2) lead II ECGs with electrodes placed in proximal limb (PL) vs. distal limb (DL) positions with dogs in right lateral (RL) recumbency, left lateral (LL) recumbency and standing positions; (3) single lead precordial ECGs from dogs in RL, LL and standing positions; and (4) before and after sedation with butorphanol in lead II ECGs obtained in RL recumbency. All intervals could be measured in all dogs (conscious and sedated) from a RL lead II ECG with both PL and DL electrode positioning. This was reduced to 98% for lead II ECGs with dogs in LL and standing positions. Intervals that were not different regardless of recording device, dog position, electrode limb position or sedation included P, QRS and P + QRS, suggesting that these intervals have the greatest clinical utility across a variety of recording conditions. The main impact of positioning in healthy dogs was the lack of ability to consistently measure all intervals in standing dogs, particularly P wave duration. Further investigation is needed to determine if this is applicable to dogs with cardiac disease.

An investigation into the detection of the pulse in conscious and anaesthetized dogs

ObjectivesTo record the success rate of veterinary professionals and students at identifying the pulse in conscious and anaesthetized dogs. To explore the influence of clinical experience, pulse location, anaesthesia and likely confounding variables on the success of pulse palpation. Study designProspective, observational, randomized study. AnimalsA total of 54 client-owned dogs scheduled for general anaesthesia. MethodsFor each dog, three participants (senior anaesthetist, anaesthesia resident/nurse, veterinary student/animal care assistant) attempted pulse palpation at three locations (femoral, radial and dorsal pedal pulse) in conscious and anaesthetized dogs. The time to pulse palpation was measured with a stopwatch for each attempt and data were modelled using a multivariate Cox regression survival analysis (significance p < 0.05). ResultsThe overall success rate of pulse palpation was 77%, with a median time of 10.91 seconds (interquartile range 9.09 seconds). Success rate was lower in conscious dogs (67%) than in anaesthetized dogs (87%). There was a 77% lower likelihood of success at the radial than at the femoral pulse [hazard ratio (HR) 0.23, 95% confidence interval (CI) 0.38–0.69, p < 0.001]. Veterinary students/animal care assistants had a 71% lower likelihood of success than senior anaesthetists (HR 0.29, 95% CI 0.22–0.39, p < 0.001). Age, weight and American Society of Anesthesiologists physical status had no significant influence. Premedication/anaesthetic drugs, heart rate or mean arterial pressure had no significant influence on the time to pulse palpation in anaesthetized dogs. The median time to palpation was less than 10 seconds for all experience groups at the femoral location. ConclusionsPalpation of the femoral location had the greatest likelihood of success with the least amount of time. Monitoring the femoral pulse during induction of anaesthesia is suggested as a method for confirming spontaneous circulation. Pulse palpation improves with clinical experience.

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. Eight‑lead 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.

Use of a human wrist blood pressure monitor for arterial blood pressure measurements in normotensive conscious dogs in comparison to veterinary high-definition oscillometry.

BackgroundThe gold standard in canine blood pressure (BP) measurement is the invasive method; however, non‐invasive blood pressure measurement techniques (NIBP) are more commonly used. The lack of small, lightweight, cheap, fast and portable NIBP still remains a point to improve in the emergency setting.Key FindingsA human wrist blood pressure (WBP) device was evaluated in comparison with the veterinary high‐definition oscillometry (HDO) in conscious normotensive dogs. Systolic and diastolic BPs were evaluated in two groups of dogs. The bias, the limits of agreement and correlation between variables were calculated. Twenty‐five and 36 dogs were, respectively, included in Study No. 1 (dogs weighting ≥10 kg) and 2 (dogs weighting ≥20 kg). In both studies, correlation between the two devices was moderate. A better agreement was displayed for diastolic pressure and poor precision for both systolic and diastolic pressures measurements (as determined by wide limits of agreement). The WBP method underestimated both the systolic and diastolic BP with respect to the HDO method. The inclusion of bigger dogs (Study No. 2) with limb circumference more like to human limb dimension did not improve the agreement between considered methods.SignificanceThe human WBP monitor considered is not suitable to replace the commonly used veterinary HDO for in‐clinic BP monitoring in normotensive conscious dogs.

Evaluation of Physicochemical Properties, Pharmacokinetics, Biodistribution, Toxicity, and Contrast-Enhanced Cancer MRI of a Cancer-Targeting Contrast Agent, MT218.

ObjectivesPreclinical assessments were performed according to the US Food and Drug Administration guidelines to determine the physicochemical properties, pharmacokinetics, clearance, safety, and tumor-specific magnetic resonance (MR) imaging of MT218, a peptidic gadolinium-based MR imaging agent targeting to extradomain B fibronectin for MR molecular imaging of aggressive tumors.Materials and MethodsRelaxivity, chelation stability, binding affinity, safety-related target profiling, and effects on CYP450 enzymes and transporters were evaluated in vitro. Magnetic resonance imaging was performed with rats bearing prostate cancer xenografts, immunocompetent mice bearing murine pancreatic cancer allografts, and mice bearing lung cancer xenografts at different doses of MT218. Pharmacological effects on cardiovascular, respiratory, and central nervous systems were determined in rats and conscious beagle dogs. Pharmacokinetics were tested in rats and dogs. Biodistribution and excretion were studied in rats. Single and repeated dosing toxicity was evaluated in rats and dogs. In vitro and in vivo genotoxicity, in vitro hemolysis, and anaphylactic reactivity were also performed.ResultsAt 1.4 T, the r1 and r2 relaxivities of MT218 were 5.43 and 7.40 mM−1 s−1 in pure water, 6.58 and 8.87 mM−1 s−1 in phosphate-buffered saline, and 6.54 and 8.70 mM−1 s−1 in aqueous solution of human serum albumin, respectively. The binding affinity of MT218 to extradomain B fragment is 3.45 μM. MT218 exhibited no dissociation of the Gd(III) chelates under physiological conditions. The peptide degradation half-life (t1/2) of MT218 was 1.63, 5.85, and 2.63 hours in rat, dog, and human plasma, respectively. It had little effect on CYP450 enzymes and transporters. MT218 produced up to 7-fold increase of contrast-to-noise ratios in the extradomain B fibronectin–rich tumors with a dose of 0.04 mmol/kg for at least 30 minutes. MT218 had little pharmacological effect on central nervous, cardiovascular, or respiratory systems. MT218 had a mean plasma elimination half-life (t1/2) of 0.31 and 0.89 hours in rats and dogs at 0.1 mmol/kg, respectively. No detectable Gd deposition was observed in the brain at 6 hours postinjection of MT218 at 0.1 mmol/kg in rats. MT218 was not mutagenic and had no mortality or morbidity in the rats or dogs up to 1.39 and 0.70 mmol/kg/d, respectively. The no observed adverse effect level of MT218 in Sprague-Dawley rats was 1.39 mmol/kg for single dosing and 0.46 mmol/kg/d for repeated dosing. The no observed adverse effect level in dogs was 0.07 mmol/kg/d. MT218 exhibited no genotoxicity, hemolysis, and anaphylactic reactivity.ConclusionThe preclinical assessments showed that the targeted contrast agent MT218 has high r1 and r2 relaxivities, satisfactory physicochemical properties, pharmacokinetic, and safety profiles and produces effective tumor enhancement in multiple cancer types in rats and mice at reduced doses.

A novel cardiovascular systems model to quantify drugs effects on the inter-relationship between contractility and other hemodynamic variables.

The use of systems‐based pharmacological modeling approaches to characterize mode‐of‐action and concentration‐effect relationships for drugs on specific hemodynamic variables has been demonstrated. Here, we (i) expand a previously developed hemodynamic system model through integration of cardiac output (CO) with contractility (CTR) using pressure‐volume loop theory, and (ii) evaluate the contribution of CO data for identification of system‐specific parameters, using atenolol as proof‐of‐concept drug. Previously collected experimental data was used to develop the systems model, and included measurements for heart rate (HR), CO, mean arterial pressure (MAP), and CTR after administration of atenolol (0.3–30 mg/kg) from three in vivo telemetry studies in conscious Beagle dogs. The developed cardiovascular (CVS)‐contractility systems model adequately described the effect of atenolol on HR, CO, dP/dtmax, and MAP dynamics and allowed identification of both system‐ and drug‐specific parameters with good precision. Model parameters were structurally identifiable, and the true mode of action can be identified properly. Omission of CO data did not lead to a significant change in parameter estimates compared to a model that included CO data. The newly developed CVS‐contractility systems model characterizes short‐term drug effects on CTR, CO, and other hemodynamic variables in an integrated and quantitative manner. When the baseline value of total peripheral resistance is predefined, CO data was not required to identify drug‐ and system‐specific parameters. Confirmation of the consistency of system‐specific parameters via inclusion of data for additional drugs and species is warranted. Ultimately, the developed model has the potential to be of relevance to support translational CVS safety studies.

Influence offentanyl, ketamine orlidocaine infusion onthe intraocular pressure and pupil size inconscious dogs.

Fifty-five healthy conscious dogs were included inaprospective randomised double-blinded clinical study. The dogs allocated toone offour groups received intravenous bolus followed byinfusion offentanyl (FEN-group), orketamine (KET-group), orlidocaine (LID-group), orsaline (SAL-group). The intraocular pressure (IOP), pupil size (PS), heart rate (HR) and mean arterial pressure (MAP) were measured prior toand at2, 5, 10, 20 and 30min after initiation ofthe drug administration. The data were analysed using ananalysis ofvariance and theSteel-Dwass test. Nosignificant difference inthe IOP within orbetween the groups was detected. Inthe FEN-group, the PS decreased significantly atall the measured times. Inthe KET-group, the PS increased significantly at2, 5and 10minutes. The PS was significantly smaller inthe FEN-group compared tothe KET-group at2, 5, 10 and 20min, compared tothe SAL-group at5, 10, 20 and 30minutes. Inthe FEN-group the HR significantly decreased compared tothe baseline and was significantly lower compared tothe KET-group and LID-group. Fentanyl, ketamine orlidocaine administered atthe doses studied asabolus followed bya30-min infusion seem tocause noeffect onthe IOP inhealthy conscious non-painful dogs without ocular abnormalities. Fentanyl decreased and ketamine transiently increased the PS.

Threshold for Anaphylactoid Reaction to Polysorbate 80 in Canines.

Polysorbate 80 (PS80) functions as a dispersing agent or solubilizer in many pharmaceuticals, and as a stabilizer in biopharmaceuticals. Topical or parenteral administration of low doses of PS80 in biopharmaceuticals has been associated with mild allergic reactions, including local injection site reactions in humans. High doses of PS80, such as levels found in traditional Chinese herbal parenteral medicines, have been linked to systemic effects consistent with anaphylactoid-type reactions, which are characterized by the direct release of histamine from mast cells (degranulation). Nonclinical safety assessments of PS80 in vivo have mainly focused on canine model systems, a species established to be particularly sensitive to PS80. However, there is conflicting data about the dose and route of administration of PS80 required to elicit an anaphylactoid-type reaction in this model system. Therefore, studies using multiple dosing regimens in anesthetized and conscious dogs including a combination of cardiovascular data, clinical signs, and biomarkers of mast cell degranulation were conducted. An intravenous (IV) bolus of 1mg/kg PS80 (0.25% w/v) elicited a positive anaphylactoid reaction including increased heart rate, hypotension, and clinical signs associated with anaphylactoid reactions (e.g., reddened muzzle). However, a full reaction was not observed with a subcutaneous (SC) injection of PS80 (0.25% w/v) up to 20mg/kg and IV bolus or IV infusions up to 0.5mg/kg. These data establish a threshold dose for eliciting an anaphylactoid reaction in canine which varies depending on the route of administration as well as the rate of PS80 infusion.

Comparison between the Parks Medical Doppler and the Mano Médical Vet BP Doppler for recording indirect systemic blood pressure in conscious dogs.

Background:The acquisition of systemic blood pressure (SBP) provides valuable information regarding cardiovascular function and tissue perfusion in human and veterinary species.Aim:To evaluate the agreement between Parks Medical Doppler (PMD) and Mano Médical Vet BP (MMVBP) Doppler for assessing SBP in conscious dogs.Methods:40 client-owned dogs were prospectively enrolled; SBP measurements were acquired by a single operator using the PMD and then the MMVBP. The mean of five consecutive measurements for each device was classified according to target organ damage (TOD) risk scores (1: <140 mmHg; 2: 140–160 mmHg; 3: 160–180 mmHg; 4: ≥ 180 mmHg).Results:Total mean SBP for the devices was not statistically different (p = 0.77). However, the Bland–Altman analysis revealed wide limits of agreement (LoA), with MMVBP slightly underestimating SBP compared to PMD (bias = −0.6 mmHg, 95% LoA: −26.3 to 25.09). Both devices correlated well ( r = 0.8269; p < 0.0001) and had identically acceptable intra-observer repeatability (coefficients of variation = 4.09% for MMVBP and 3.86% for PMD). Four dogs (10%) had a TOD score of 3 by one device but scored <3 with the other.Conclusion:A good agreement and correlation was observed between the PMD and the MMVBP, suggesting that both devices can be used interchangeably for assessment of SBP in conscious dogs. The wide LoA observed between both devices was most likely associated with intraindividual variability in SBP over time.

Effects of auriculopalpebral nerve block on ocular parameters in dogs

The objective of this study was to compare the effects of two local anesthetics used on auriculopalpebral block on eyelid akinesia, tear production, intraocular pressure (IOP) and tear break-up time (TBUT) in conscious dogs. A blind, randomized, prospective study was conducted to determine the effects of auriculopalpebral block using ropivacaine 0.75% and bupivacaine 0.5% in 12 healthy non-brachycephalic dogs (24 eyes). Threat response and eyelid reflex tests, Schirmer tear test (STT), IOP and tear break-up time were conducted before blockage and at 30, 60, 120, 240 and 360 minutes after application. A difference was observed between the values found at 30, 60, 120 and 240 minutes compared to baseline for threat response and eyelid reflex tests in the two groups evaluated, proving eyelid akinesia after blockages. No difference was found for STT, IOP and TBUT between baseline values and post-anesthesia times or between groups. It was possible to conclude that ropivacaine and bupivacaine on auriculopalpebral block in conscious dogs promoted eyelid akinesia for at least 240 minutes, not altering ocular physiological parameters of tear production, intraocular pressure, and tear break-up time after blockages.

Influence of Fentanyl, Ketamine, and Lidocaine on Tear Production in Healthy Conscious Dogs

Tear production is an important factor in maintaining proper function of the cornea and conjunctiva. The purpose of this study was to determine the effects of bolus followed by infusion of fentanyl, lidocaine, and ketamine on tear production as measured by the Schirmer I Tear Test (STT-I) in dogs. A prospective, randomized, “double-blind” study was performed. A total of 55 healthy conscious client-owned dogs were included in the study. Dogs were randomly allocated to one of four groups and given intravenous fentanyl 0.005 mg kg-1 followed by 0.005 mg kg-1 hour−1 (FEN-group), ketamine 0.6 mg kg−1 followed by 0.6 mg kg−1 hour−1 (KET-group), lidocaine 1 mg kg-1 followed by 1 mg kg−1 hour−1 (LID-group), or saline 0.3 mL kg−1 followed by 2 mL kg−1 hour−1 (SAL-group). The STT-I was performed prior to (baseline) and again 30 minutes (T30) after initiation of drug administration. Data were expressed as the median (minimum – maximum) and analyzed by Wilcoxon and Steel-Dwass tests (P < .05). The STT-I values increased little but were statistically significant in the KET-group from 18 (14-23) to 19 (14-25) (P = .039) and in the LID-group from 21 (14-25) to 20 (17-29) (P = .027). At 30 minutes, STT-I values were significantly higher in LID-group 20 (17-29) than in FEN-group 18 (12-22) (P = .006). Fentanyl, ketamine, and lidocaine administered at the studied doses as a bolus and then followed by an infusion within 30 minutes in healthy conscious dogs demonstrated a clinically insignificant effect on tear production as measured by STT-I.

Lack of pro-arrhythmic potential for the novel soluble guanylate cyclase stimulator vericiguat: results from preclinical studies

Abstract Introduction Vericiguat is an orally-administered soluble guanylate cyclase stimulator, developed for the treatment of symptomatic chronic heart failure (HF) in adult patients who have had a previous decompensation event. At the maximum therapeutic dose of 10 mg once daily in patients with HF, the protein-unbound plasma concentrations of vericiguat and its major pharmacologically inactive N-glucuronide metabolite M-1 are approximately 18 nmol/l and 43 nmol/l, respectively. As part of an integrated risk assessment, vericiguat and its M-1 metabolite were characterised electrophysiologically in vivo and in vitro. This was performed according to the International Council for Harmonisation standard S7B guideline and to recent related “best practice” revisions (draft ICH E14/S7B Q&amp;A), being adopted as a result of the Comprehensive In Vitro Proarrhythmia Assay (CIPA) initiative. Purpose To assess the potential for a proarrhythmic risk from vericiguat and its M-1 metabolite in a series of preclinical studies. Methods The potential for proarrhythmic risk was investigated in conscious telemetered dogs and in a series of in vitro electrophysiological studies, including mechanistic ion channel studies, using both generally accepted and CIPA voltage-clamp protocols under conditions simulating normal and diseased physiological states. The ion channels studied were hERG, hNav1.5, hCav1.2, hKvLQT1/minK and hKv4.3. Transfected human embryonic kidney cell lines were used for the hERG, hNav1.5 and hKvLQT1/minK studies; transfected Chinese hamster ovary cell lines were used for the hCav1.2 and hKv4.3 studies. Results In dogs, administration of vericiguat as single oral doses was associated with dose-dependent decreases in arterial blood pressure (consistent with its mode of action) and compensatory increases in heart rate (Table 1). Heart rate-corrected QT (QTc) intervals were not prolonged by vericiguat to a clinically meaningful extent. Neither vericiguat nor its M-1 metabolite inhibited cardiac ion channels (hERG, hNav1.5, hCav1.2, hKvLQT1/minK and hKv4.3) at exposure multiples of &amp;gt;150-fold (Table 2). Conclusion There was no preclinical evidence of proarrhythmic risk from the in vitro (simulating normal and diseased physiological states) and in vivo assessment of vericiguat or its major N-glucuronide metabolite M-1. This integrated risk assessment of non-clinical data supports the conclusion that administration of vericiguat 10 mg once daily in humans is not associated with meaningful QTc prolongation. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Funding for this research was provided by Bayer AG, Berlin, Germany and Merck Sharp &amp; Dohme Corp., a subsidiary of Merck &amp; Co., Inc., Kenilworth, NJ, USA.

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

IntroductionThis 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. MethodsMale 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. ResultsLow 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. ConclusionSuccessful 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).

Impact of region-of-interest size and location on quantitative contrast-enhanced ultrasound of canine splenic perfusion

BackgroundDuring contrast enhanced ultrasound (CEUS), the features of the regions of interest (ROI) can affect the value of the perfusion-related parameters obtained from a time intensity curve (TIC). In veterinary medicine, conflicting have been reported on the influence of ROI size and location on renal CEUS. There are some disagreeing evidences regarding the optimal method for selecting ROI in quantitative analysis of renal perfusion using CEUS.The aim of this study was to evaluate the effect of the size and location of ROIs in the spleen of conscious dogs on perfusion variables determined using sulphur hexafluoride contrast-enhanced ultrasounds.ResultsA prospective observational study on 15 client-owned mixed-breed adult dogs was performed using a system equipped with contrast-tuned imaging technology. Qualitative and quantitative assessments of the spleen enhancement pattern were carried out. Three square ROIs (0.05 cm2) were manually drawn in a row and spaced 1 mm apart, placing adjacent ROIs at three different depths. Three medium rectangular ROIs (0.3 cm2) include the 3 smallest ROIs in each row, indicated by the letters A, B and C, and a single large square ROI (1 cm2) was drawn containing all previous ROIs. Software analysis of time-intensity curves generated within each ROI allowed us to calculate the perfusion-related parameters: peak enhancement, time to peak, regional blood flow, mean transit time and regional blood volume.The coefficient of variation for all blood-related parameters was always lower in the larger ROI than in the other smaller ROIs. ROI A and B, positioned proximally and medially, levels respectively, showed similar coefficients of variation to the largest ROI. The analysis of variance model exhibited a significant effect of location and size of the ROIs in the quantitative analysis of canine spleen perfusion, with a reduction of perfusion-related parameters in the distal ROI.ConclusionsThe recommendation for a quantitative CEUS examination of a dog’s spleen is to analyze splenic perfusion by drawing a sufficiently large ROI proximal to the ultrasound beam on the splenic parenchyma. This may be of clinical relevance in the diagnosis of splenic diseases.

Effects of fadolmidine, an α2 -adrenoceptor agonist, as an adjuvant to spinal bupivacaine on antinociception and motor function in rats and dogs.

α2‐Adrenoceptor agonists such as clonidine and dexmedetomidine are used as adjuvants to local anesthetics in regional anesthesia. Fadolmidine is an α2‐adrenoceptor agonist developed especially as a spinal analgesic. The current studies investigate the effects of intrathecally administered fadolmidine with a local anesthetic, bupivacaine, on antinociception and motor block in conscious rats and dogs. The antinociceptive effects of intrathecal fadolmidine and bupivacaine alone or in combination were tested in the rat tail‐flick and the dog's skin twitch models. The durations of motor block in rats and in dogs were also assessed. In addition, the effects on sedation, mean arterial blood pressure, heart rate, respiratory rate and body temperature were evaluated in telemetrized dogs. Concentrations of fadolmidine in plasma and spinal cord were determined after intrathecal and intravenous administration in rats. Co‐administration of intrathecal fadolmidine with bupivacaine increased the magnitude and duration of the antinociceptive effects and prolonged motor block without hypotension. The interaction of the antinociceptive effect was synergistic in its nature in rats. Concentration of fadolmidine in plasma was very low after intrathecal dosing. Taken together, these studies show that fadolmidine as an adjuvant to intrathecal bupivacaine provides enhanced sensory‐motor block and enables a reduction of the doses of both drugs. The results indicate that co‐administration of fadolmidine with intrathecal bupivacaine was able to achieve an enhanced antinociceptive effect without hypotension and could thus represent a suitable combination for spinal anesthesia.

Evaluation of moxifloxacin in canine and non-human primate telemetry assays: Comparison of QTc interval prolongation by timepoint and concentration-QTc analysis.

The in vivo correct QT (QTc) assay is used by the pharmaceutical industry to characterize the potential for delayed ventricular repolarization and is a core safety assay mentioned in International Conference on Harmonization (ICH) S7B guideline. The typical telemetry study involves a dose‐response analysis of QTc intervals over time using a crossover (CO) design. This method has proven utility but does not include direct integration of pharmacokinetic (PK) data. An alternative approach has been validated and is used routinely in the clinical setting that pairs pharmacodynamic (PD) responses with PK exposure (e.g., concentration‐QTc (C‐QTc) analysis. The goal of our paper was to compare the QTc sensitivity of two experimental approaches in the conscious dog and non‐human primate (NHP) QTc assays. For timepoint analysis, a conventional design using eight animals (8 × 4 CO) to detect moxifloxacin‐induced QTc prolongation was compared to a PK/PD design in a subset (N = 4) of the same animals. The findings demonstrate that both approaches are equally sensitive in detecting threshold QTc prolongation on the order of 10 ms. Both QTc models demonstrated linearity in the QTc prolongation response to moxifloxacin dose escalation (6 to 46 ms). Further, comparison with human QTc findings with moxifloxacin showed agreement and consistent translation across the three species: C‐QTc slope values were 0.7‐ (dog) and 1.2‐ (NHP) fold of the composite human value. In conclusion, our data show that dog and NHP QTc telemetry with an integrated PK arm (C‐QTc) has the potential to supplement clinical evaluation and improve integrated QTc risk assessment.

Genetic Toxicology and Safety Pharmacological Evaluation of Forsythin.

Introduction Forsythin is the main ingredient of Forsythia suspensa and is widely used in treatment of fever, viral cold, gonorrhea, and ulcers clinically. This study aimed to evaluate the potential genetic toxicity of forsythin and its safety for human use. Methods Based on the Good Laboratory Practice regulations and test guidelines, the genetic toxicity of forsythin was assessed by the Ames test, chromosome aberration (CA) test, and bone marrow micronucleus (MN) test in vivo. In the Ames test, five strains of Salmonella typhimurium were exposed to different concentrations of forsythin in the presence or absence of the S9 mixture, and then, the number of His + revertant colonies was counted. In the CA test, Chinese hamster lung (CHL) fibroblast cells were treated with different concentrations of forsythin, mitomycin C, or cyclophosphamide in the presence or absence of the S9 mixture, and the chromosomal aberrations were determined. In the MN test, bone marrow was isolated from the mice with different treatments, and the ratios of polychromatic erythrocytes (PCE) and erythrocytes (PCE/(PCE + NCE)) were measured. Finally, beagle dogs were divided into four groups (negative control, low dose, medium dose, and high dose groups), and then, a telemetry system was used to evaluate the safe use of forsythin. Results Ames test results showed that the number of colonies in all test strains with different treatments showed no significantly dose-dependent increase in the presence or absence of the S9 mixture (p > 0.05). In the CA test, the number of cells with aberrations in the CHL fibroblast cells treated with low, medium, and high doses of forsythin for 24/48 h in the absence of the S9 mixture was, respectively, 5.0/2.5, 4.5/1.5, and 5.0/5.0, and in the presence of the S9 mixture, the number was, respectively, 5.0, 5.0, and 4.5. These results showed that there was no significant difference compared to the negative control group either in the presence (2.0) or in the absence (4.0/2.5 for 24/48 h) of the S9 mixture (p > 0.05). The MN test showed that the values of PCE/(PCE + NCE) in the negative, positive controls, and forsythin treatment groups were all more than 20%, which indicated that forsythin had no cytotoxicity. Additionally, no significant toxicological effects of forsythin on blood pressure, respiration, temperature, electrocardiogram, and other physiological indicators in the conscious beagle dogs of different groups were observed by the telemetry method. Conclusion Our findings showed that forsythin has low probability of genetic toxicity and no significant toxicological effects, which implied that forsythin is suitable for further development and potential application.

Thyroid hormone activated upper gastrointestinal motility without mediating gastrointestinal hormones in conscious dogs

This study was conducted to clarify the relationship between thyroid function and gastrointestinal motility. We established an experimental configuration in which the feedback of thyroid function was completely removed using conscious dogs. With hypothyroidism, time of phase I of interdigestive migrating contractions (IMC) was longer, time of phase II and phase III was significantly shortened, and both the continuous time of strong tetanic contraction at antrum and 10-h frequency of phase III counted from the first IMC after meal significantly decreased. Whereas, hyperthyroidism caused the opposite events to those with hypothyroidism. Furthermore, We found giant migrating contractions (GMC) occurred from the upper gastrointestinal tract when we administrated high dose of thyroid hormone. One GMC occurred from anal sides propagated to cardiac, and this propagation was similar to the emesis-like interdigestive motor activity, the other GMC occurred from oral sides propagated to anal sides and this was similar to the diarrhea-like interdigestive motor activity. We examined the relationship between thyroid function and gastrointestinal hormones including of ghrelin, GLP-1, and cholecystokinin (CCK). However, we could not find significant differences under different thyroid hormone status. This is the first report that thyroid hormone activated upper gastrointestinal motility without mediating gastrointestinal hormones.

The in vivo QTc core assay: An evaluation of QTc variability, detection sensitivity and implications for the improvement of conscious dog and non-human primate telemetry studies

The ICH S7B guideline describes the requirement to conduct an in vitro IKr (hERG) and in vivo QTc assay for human risk assessment of new drug products, but the guidance is devoid of recommendations on study execution or quality. In the absence of standard practice, multiple study designs and experimental approaches have been utilized, especially with the nonclinical QTc assay. Since 2009, our approach to the in vivo QTc assay has been consistent for small molecules and yields reproducible and sensitive levels for QTc signal detection. Our database and experience indicate that nonrodent telemetry studies can achieve high sensitivity and a calculated metric of study power can be used to indicate study quality. Using a retrospective statistical power analysis of multiple studies (n = 14 dog; n = 6 NHP), the detection sensitivity for a specific study design (N = 8; double Latin square cross-over) was determined. The output of the power analysis is the minimal detectable effect at 80% power and a 95% probability level. The design provided an average sensitivity to detect a 4.7 (2.0%) and 6.5 (1.9%) msec QTcI change in dog and NHP, respectively. These findings suggest that this experimental approach has a consistent and reproducible sensitivity to enable a robust QTcI risk evaluation and can be used confidently to support an integrated nonclinical-clinical pro-arrhythmia risk assessment. The inclusion of power analysis (i.e., QTc sensitivity) data in a regulatory submission provides key information to critical stakeholders about the quality of the in vivo QTc assessment and its value for human safety testing.