Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

Veterinary Anaesthesia and Analgesia 2017, xxx, 1e11

http://dx.doi.org/10.1016/j.vaa.2016.12.058

RESEARCH PAPER

Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum Q5

Q4

alveolar concentration of sevoflurane in dogs Rachel C Hectora, Marlis L Rezendea, Khursheed R Mamaa, Eugene P Steffeyb, Heather K Knychb, Ann M Hessc, Juhana M Honkavaarad, Marja R Raekalliod & Outi M Vainiod a

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences,

Colorado State University, Fort Collins, CO, USA b Kenneth L Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of CaliforniaeDavis, Davis, CA, USA c

Department of Statistics, College of Natural Sciences, Colorado State University, Fort Collins, CO,

USA d

Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of

Helsinki, Helsinki, Finland Correspondence: Marlis L Rezende, Department of Clinical Sciences, Colorado State University, Fort Collins, CO, 80523-1678, USA. Email: [email protected] Present address: JM Honkavaara, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of CaliforniaeDavis, Davis, CA, USA.

Q1

Abstract Objective To determine the effects of low and high dose infusions of dexmedetomidine and a peripheral a2-adrenoceptor antagonist, MK-467, on sevoflurane minimum alveolar concentration (MAC) in dogs. Study design Crossover experimental study. Animals Six healthy, adult Beagle dogs weighing 12.6 ± 0.9 kg (mean ± standard deviation). Methods Dogs were anesthetized with sevoflurane in oxygen. After a 60-minute instrumentation and equilibration period, the MAC of sevoflurane was determined in triplicate using the tail clamp technique. PaCO2 and temperature were maintained at 40 ± 5 mmHg (5.3 ± 0.7 kPa) and 38 ± 0.5 ºC, respectively. After baseline MAC determination, dogs were administered two incremental loading and infusion doses of either dexmedetomidine (1.5 mg kg
1 then 1.5 mg kg
1 hour
1 and 4.5 mg kg
1 then 4.5 mg kg
1 hour
1) or MK-467 (90 mg kg
1 then 90 mg kg
1 hour
1 and 180 mg kg
1 then 180 mg kg
1 hour
1); loading doses were administered over 10 minutes. MAC was redetermined in duplicate starting 30 minutes

after the start of drug administration at each dose. End-tidal sevoflurane concentrations were corrected for calibration and adjusted to sea level. A repeated-measures analysis was performed and comparisons between doses were conducted using Tukey's method. Statistical significance was considered at p < 0.05. Results Sevoflurane MAC decreased significantly from 1.86 ± 0.3% to 1.04 ± 0.1% and 0.57 ± 0.1% with incremental doses of dexmedetomidine. Sevoflurane MAC significantly increased with high dose MK-467, from 1.93 ± 0.3% to 2.29 ± 0.5%. Conclusions and clinical relevance Dexmedetomidine caused a dose-dependent decrease in sevoflurane MAC, whereas MK-467 caused an increase in MAC at the higher infusion dose. Further studies evaluating the combined effects of dexmedetomidine and MK-467 on MAC and cardiovascular function may elucidate the potential benefits of the addition of a peripheral a2-adrenergic antagonist to inhalation anesthesia in dogs. Keywords adrenergic a2-receptor antagonist, dexmedetomidine, dog, minimum alveolar concentration, MK-467.

1

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

Introduction Dexmedetomidine is an a2-adrenoreceptor agonist used in dogs for procedural sedation, as a premedicant prior to general anesthesia, and as an adjunct during inhalation anesthesia. In addition to providing reliable sedation, medetomidine (and its active isomer, dexmedetomidine) are reported to have potent antinociceptive effects (Murrell & Hellebrekers 2005; van Oostrom et al. 2011; Bennett et al. 2016). It has also been shown that dexmedetomidine significantly reduces the minimum alveolar concentration (MAC) of isoflurane in dogs when used either as a single bolus (Bloor et al. 1992) or as a constant rate infusion (CRI; Pascoe et al. 2006). Similar MAC sparing effects of dexmedetomidine have been reported for sevoflurane (Moran-Mu~ noz et al. 2014). The administration of dexmedetomidine is, however, associated with some adverse cardiovascular effects. After intravenous (IV) administration, typically systemic vascular resistance and mean arterial pressure (MAP) increase. This, in turn, results in reflex bradycardia and decreases in cardiac output and oxygen delivery (Bloor et al. 1992; Pascoe 2015). These predominantly peripheral effects have been shown to be largely dose independent (Pypendop & Verstegen 1998). Hence, despite the beneficial effects, side effects may limit its use in debilitated or cardiovascularly compromized animals. When dexmedetomidine is administered to provide sedation, an antagonist can be administered if the cardiovascular effects are adversely affecting the animal. The a2-adrenoreceptor antagonist, atipamezole, has been reported to reverse both central and peripheral effects of dexmedetomidine (V€ah€a-Vahe 1990; Bloor et al. 1992). Thus, not only the undesired cardiovascular effects may be reversed, but also the sedation and antinociception. MK-467 is an a2adrenoreceptor antagonist under investigation that poorly penetrates the bloodebrain barrier (Clineschmidt et al. 1988). This provides an avenue whereby peripheral effects of a2-adrenoreceptor agonists may be reversed with little to no impact on central effects (Pagel et al. 1998; Honkavaara et al. 2008; Restitutti et al. 2011). In dogs sedated with medetomidine and butorphanol, MK-467 attenuated the cardiovascular changes associated with medetomidine with little effect on the degree of sedation (Salla et al. 2014a). Similarly, MK-467 ameliorated the cardiovascular effects of medetomidine when both drugs were administered prior to isoflurane

2

anesthesia in dogs (Salla et al. 2014b), and when medetomidine and MK-467 were administered concurrently as CRIs in anesthetized dogs (Kaartinen et al. 2014). Reversal of peripheral cardiovascular effects of dexmedetomidine was demonstrated in cats (Pypendop et al. 2016) and of detomidine and romifidine in horses (Vainionp€ a€ a et al. 2013; Pakkanen et al. 2015; de Vries et al. 2016) with MK-467 administration. MK-467, therefore, shows promise for use in patients that would poorly tolerate the cardiovascular effects but could benefit from the central sedation and analgesia provided by the a2adrenoreceptor agonists. Although MK-467 is believed to have largely peripheral actions, its influence on the MAC of inhalation anesthetics has not been evaluated. In an effort to systematically evaluate the potential influence of MK-467 during anesthesia management, the effect of two doses of MK-467 on the MAC of sevoflurane in dogs was investigated. As part of a larger study designed to ultimately evaluate the combined effects of MK-467 and dexmedetomidine on sevoflurane MAC and cardiovascular variables, the effect of two doses of dexmedetomidine on the MAC of sevoflurane in dogs was also evaluated. We hypothesized that MK-467 would result in no change in sevoflurane MAC, and dexmedetomidine would dose-dependently decrease sevoflurane MAC. Materials and methods Dogs Six healthy, adult (age, 2.0 ± 0.1 years) purpose-bred Beagle dogs (three males and three females, weighing 12.6 ± 0.9 kg) [mean ± standard deviation (SD)] were used in this study, which was approved by the Colorado State University Institutional Animal Care and Use Committee. Sample size was determined based on the results of similar studies evaluating the effect of dexmedetomidine on MAC (Pascoe et al. 2006; Moran-Mu~ noz et al. 2014). Physical examination, complete blood count, and serum chemistry values were assessed as normal for each dog before the start of the study. Dogs were group-housed, with ad libitum fresh water and commercial dry dog food. They were socialized by students in the behavioral studies program on a daily basis and familiarized with the study environment and personnel prior to the study. Food, but not water, was withheld for approximately 12 hours before anesthesia.

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

Anesthesia and instrumentation Each dog was anesthetized on two occasions, separated by 1 week. Anesthesia was induced by sevoflurane (vaporizer setting 5%) in oxygen (5 L minute
1) delivered by a mask from a circle system (DRE Moduflex Optimax; DRE Medical Inc., KY, USA). The time from the first breath of sevoflurane to orotracheal intubation with a cuffed tube and connection to the breathing circuit was recorded. Induction quality was scored by a single observer (MLR) on a scale of 1 to 5, with 5 as the highest quality (Appendix 1). During instrumentation, anesthesia was maintained with sevoflurane (vaporizer 2.5e3.5%) in oxygen (1 L minute
1). The dog was positioned in right lateral recumbency for the first treatment and left lateral recumbency for the second treatment. Treatments were assigned using a crossover design as described later. A 20 gauge, 48 mm catheter (BD Insyte; Becton Dickinson Infusion Therapy Systems Inc., UT, USA) was placed percutaneously in a cephalic vein for drug and fluid delivery. Immediately, infusion of lactated Ringer's solution (Hospira Inc., IL, USA) was started and continued at 2e4 mL kg
1 hour
1 for the duration of anesthesia. A 22 gauge, 25 mm catheter (BD Insyte; Becton Dickinson Infusion Therapy Systems Inc.) was placed percutaneously in a dorsal pedal or medial metacarpal artery and connected to a pressure transducer (Argo Trans e Model 2/Macro; Argon Medical Devices Inc., TX, USA) for continuous monitoring of systolic (SAP), MAP, and diastolic (DAP) arterial pressures, and to facilitate blood sampling for pH, arterial partial pressure of carbon dioxide (PaCO2) and arterial partial pressure of oxygen (PaO2), plasma drug concentrations, packed cell volume (PCV) and total protein (TP) measurements. The blood-gas analyzer (800ABL Flex; Radiometer America Inc., CA, USA) was calibrated every 4 hours with electrolyte and metabolite solutions and gas mixtures of known concentrations. The pressure transducer was calibrated against a mercury column within the range of anticipated working pressures (0e200 mmHg) and zeroed at sternal midline to approximate the level of the right atrium. All dogs were ventilated (Bird Mark 7 Respirator; CareFusion, CA, USA) to maintain PaCO2 between 35 and 45 mmHg (4.7e6.0 kPa). As sampling of blood for PaCO2 measurements was intermittent, end-tidal partial pressure of carbon dioxide (PE0 CO2) was used to guide ventilation. Continuous sampling of respiratory gases, including both CO2 and sevoflurane,

was facilitated by placement of a red rubber catheter in the lumen of the endotracheal tube, with the distal tip of the catheter positioned in the thoracic trachea. This catheter was connected to an anesthetic gas analyzer (Datex-Ohmeda Cardiocap 5; GE Healthcare, WI, USA; sampling rate, 200 ± 20 mL minute
1). Just prior to MAC determination time points, manual sampling of end-tidal expired gas was performed through this catheter using a glass syringe to ensure the accuracy of the continuously sampled end-tidal sevoflurane (FE0 Sevo) measurements. The gas analyzer was calibrated daily before the study and the calibration checked intermittently and after the conclusion of the study with oxygen alone (zero) and two known CO2 and five known sevoflurane concentration standards (compressed gas, N.O.S 1.5, 2.5, 3.0, 3.5, 4.0 and 5.0% sevoflurane, balance nitrogen; Air Liquide Healthcare, Air Liquide Healthcare America Corporation, PA, USA) spanning the endtidal concentrations measured in the study. A calibration curve was generated and the measured FE0 Sevo was adjusted based on this using a regression line. These values were then corrected for barometric pressure using the following equation: (barometric pressure of location in mmHg/760)  (MAC value). Barometric pressure, which was measured using a barometer within the blood-gas analyzer on each study day, ranged from 632 to 640 mmHg (84.3e85.3 kPa). A lead II electrocardiogram was used to continuously monitor both heart rate (HR) and rhythm (Prism; Medical Data Electronics Inc., CA, USA). Arterial hemoglobin oxygen saturation percentage (SpO2) was measured using a pulse oximeter probe (Nellcor; Medtronic, MN, USA) placed on the tongue. Body temperature was monitored via a thermistor (calibrated using water baths and a certified Bureau of Standards thermometer) placed in the thoracic esophagus. A heat lamp (Model PUL-300-HD; Fostoria Industries Inc., OH, USA), circulating water blanket (Gaymar T/Pump; Gaymar Industries Inc., NY, USA) and forced warm air (Bair Hugger Warming Unit Model 585; Arizant Healthcare Inc., MN, USA) were used alone or in combination to maintain a target of 37.5e38.5  C throughout the study. A urinary catheter (8 Fr 300 mm Foley Catheter; SurgiVet, MN, USA) was inserted aseptically to maintain an empty bladder and quantify urine output. After catheter placement, the bladder was fully evacuated of urine, after which urine was allowed to accumulate in a 1-L collection bag for periodic measurement.

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

3

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

Experimental design An instrumentation and equilibration period of a minimum of 60 minutes was followed by maintenance of constant FE0 Sevo for 15 minutes prior to initiating baseline sevoflurane MAC determination using the tail clamp method. This technique involved a single observer (RCH) blinded to treatments, vaporizer settings, blood-gas values and monitored hemodynamic variables, applying constant pressure with Carmalt forceps with 6 cm tips covered in plastic tubing to the tail for 60 seconds or until a positive response was observed. The forceps were not locked but always closed up to the first ratchet. The tail was moved throughout stimulus application. Only a purposeful movement (e.g. raising of the head) was considered a positive response. Other changes, such as increased respiratory rate (fR) and effort or swallowing, were not considered positive responses. If there was no purposeful movement within the 60 seconds, the response was considered negative and the stimulus discontinued. If a positive response to the stimulus was observed, the FE0 Sevo was increased by approximately 10%, and the stimulus was reapplied after a 15-minute anesthetic re-equilibration period. If a negative response was observed, the FE0 Sevo was decreased in a similar fashion. MAC was determined as the average of a positive and negative response. Baseline MAC was determined in triplicate for sevoflurane for both treatments. A different location on the tail within the middle third of the tail was randomly selected at each stimulus to avoid tissue damage at one site. After the determination of baseline MAC, dogs were administered their preassigned treatment of either dexmedetomidine (SevoDex) or MK-467 (SevoMK). Treatments were assigned using a crossover design, where three dogs were administered SevoDex during the first anesthesia and three dogs were administered SevoMK. The other treatment was administered during the second anesthesia. The order in which the dogs were anesthetized was selected by drawing slips of paper containing the dogs' names. A minimum of 1 week (range, 7e9 days) elapsed between anesthetic episodes for each dog. During SevoDex, the dog was administered an IV loading dose of dexmedetomidine (1.5 mg kg
1; Dexdomitor; Zoetis Inc., NJ, USA) over 10 minutes followed by a CRI (1.5 mg kg
1 hour
1). After MAC determination, a second IV loading dose of dexmedetomidine (4.5 mg kg
1) was administered over 10 minutes followed by a CRI (4.5 mg kg
1 hour
1). For SevoMK treatment, the dog 4

was administered an IV loading dose of MK-467 (90 mg kg
1) over 10 minutes followed by a CRI (90 mg kg
1 hour
1) and then a second loading dose (180 mg kg
1) over 10 minutes followed by a CRI (180 mg kg
1 hour
1). The drugs were delivered by a syringe infusion pump (Medfusion 3500; Smiths Medical, OH, USA) that had been verified for accuracy over the rates being administered. Sevoflurane MAC was determined in duplicate at each low and high dose infusion rate for each treatment, starting 20 minutes after the beginning of each CRI. Arterial blood was collected for measurement of plasma drug concentrations at fixed time points during MAC determination (as described below). Measurements Body weight, HR, fR, and rectal temperature (RT) were recorded for each dog before each anesthesia. Esophageal temperature, HR, fR, FE0 Sevo, PE0 CO2, SpO2, SAP, MAP and DAP were monitored throughout anesthesia and recorded just prior to each MAC determination. These values from the positive and negative responses that were used to calculate MAC at each MAC determination (Sevo, high and low dose Dex and MK-467) were averaged. Arterial blood was anaerobically collected before and after each MAC determination for measurement of pH, PaCO2, PaO2, bicarbonate (HCOe 3 ), base excess (BE), glucose, lactate and creatinine concentrations. PCV and TP were also measured from the same sample at each of these time points. Arterial blood was also sampled at the end of each dexmedetomidine and MK-467 loading dose, then at 30 minute intervals during drug infusion and at the end of the infusion when MAC determination was completed, for analysis of plasma dexmedetomidine and MK-467 concentrations. Urine volume, urine glucose and urine specific gravity (USG) were measured at the end of each MAC determination. Urine volume was quantified using a graduated cylinder (Tekk 100 mL; Kimble Chase Life Science and Research Products LLC, TN, USA), urine glucose was measured with reagent strips (KetoDiastix; Bayer HealthCare LLC, IN, USA) and USG measured with a refractometer. Urine output (mL kg
1 hour
1) was calculated for each dose and treatment. Recovery At the end of anesthesia, sevoflurane and positive pressure ventilation were discontinued and the dog

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

was allowed to recover under close observation. Total anesthesia time (from intubation to discontinuation of sevoflurane), and time from discontinuation of sevoflurane to extubation and standing were recorded. Overall recovery quality was scored by a single observer (MLR) (Appendix 1). Carprofen (4 mg kg
1; Rimadyl; Zoetis Inc., MI, USA) was administered subcutaneously and cefazolin (22 mg kg
1; Hikma Farmaceutica, Portugal) IV over 10 minutes during the recovery period. After complete recovery, when the dog was able to ambulate and interact normally, the dog was returned to group housing and examined twice daily for any complications or abnormal behaviors. Dexmedetomidine and MK-467 analyses Arterial blood samples for drug plasma concentration analysis (3 mL) were collected into tubes containing sodium heparin (BD Vacutainer; Becton Dickinson, NJ, USA) and stored on ice. At the end of each study day, samples were centrifuged and the plasma was harvested and stored at
80 ºC until analysis. Calibrator and quality control (QC) stock solutions of dexmedetomidine (Toronto Research Chemicals, ON, Canada) were prepared in methanol, and MK467 (Vetcare Ltd, Finland) stock solutions were prepared in water and acetonitrile (ACN) at 1 mg mL
1 free base. For analysis, dexmedetomidine and MK467 were combined into one working solution. Calibration curves and negative control samples were prepared fresh for each quantitative assay. In addition, QC samples (plasma fortified with analyte at three concentrations within the standard curve) were included with each sample set as an additional check of accuracy. Prior to sample processing and analysis, all plasma samples were diluted with 100 mL of water containing the internal standards antipyrine (AP; Sigma Aldrich, MO, USA) and 2-(1-hydroxyethyl) promazine sulfoxide (HEPS; Frontier BioPharm, KY, USA) at 100 ng mL
1. The injection volume was 30 mL for the liquid chromatography tandem-mass spectrometry (LC-MS/MS) system. The concentrations of dexmedetomidine and MK467 were measured in plasma by LC-MS/MS using positive heated electrospray ionization [HESI(þ)] (Thermo Scientific, CA, USA). Quantitative analysis was performed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) coupled with a turbulent flow chromatography system (TFC TLX2; Thermo Scientific) having LC-10ADvp liquid

chromatography systems (Shimadzu, Japan) and operated in laminar flow mode. Product masses and collision energies of each analyte were optimized by infusing the analytes into the mass spectrometer. Chromatography used an ACE 3 C18 10 cm  2.1 mm 3 mm column (Mac-Mod Analytical, PA, USA) and a linear gradient of ACN in water with a constant 0.2% formic acid at a flow rate of 0.35 mL minute
1. The initial ACN concentration was held at 10% for 0.50 minutes, ramped to 90% over 5 minutes, held at that concentration for 0.33 minutes, before re-equilibrating for 4.17 minutes at initial conditions. Detection and quantification was conducted using selective reaction monitoring of initial precursor ion for dexmedetomidine [mass/charge ratio (m/z) 201.16], MK-467 [(m/z) 419.18], internal standards AP [(m/z) 189.14] and HEPS [(m/z) 345.15]. The response for the product ions for dexmedetomidine (m/z 41.2, 68.2, 95.1), MK-467 (m/z 199.9, 237.9, 281), AP (m/z 77.1, 131.1, 147.2) and HEPS (m/z 58.1, 86, 242.9) were plotted and peaks at the proper retention time integrated using Quanbrowser software (Thermo Scientific). Quanbrowser software was used to generate calibration curves and quantitate dexmedetomidine and MK-467 in all samples. A weighting factor of 1/X was used for all calibration curves. The response for dexmedetomidine and MK-467 was linear and gave a coefficient of determination of 0.99. Accuracy was reported as percent nominal concentration, and precision was reported as percent relative standard deviation. The technique was optimized to provide a limit of quantitation of 0.1 ng mL
1 for both dexmedetomidine and MK-467 in plasma. The limit of detection was approximately 0.02 ng mL
1 for both dexmedetomidine and MK467. Statistical analysis Statistical analyses were performed using SAS Version 9.4 (SAS Institute Inc., NC, USA). For baseline physiologic values (HR, fR, RT), time to intubation, to extubation, to standing, the total anesthesia time and total fluid volume administered, data were summarized as mean ± SD. A paired t test was used to test for differences comparing the two treatments (SevoDex versus SevoMK). Induction and recovery quality scores were summarized as median (range), and the Wilcoxon's matched-pairs signed rank test was used to compare these between the treatments.

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

5

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

For all other variables (MAC, HR, fR, SAP, DAP, MAP, esophageal temperature, SpO2, PE0 CO2, pH, PaCO2, PaO2, BE, HCO3, creatinine, lactate, glucose, PCV, TP, urine output and plasma drug concentrations), data were summarized using mean ± SD. A repeated-measures analysis of variance was performed for each response variable separately using SAS Proc Mixed. Specifically, treatment (SevoDex or SevoMK) and dose (baseline sevoflurane, low dose infusion, high dose infusion) and treatment  dose interaction were treated as fixed effects. In order to account for the crossover design, dog and dog  treatment were included in the model as random effects. For each treatment, comparisons between doses were performed using Tukey's method. For all analyses, p < 0.05 was considered significant. Results Preanesthetic HR, fR and RT were not significantly different between treatments. Induction time, induction quality, time to extubation, total anesthesia time and total fluid volumes were also not different between treatments, but time to standing was significantly longer (p ¼ 0.02) and the recovery quality score significantly higher (p ¼ 0.03) in SevoDex (Table 1). Three dogs defecated, two dogs vomited and one dog vomited and defecated within 30 minutes of extubation in SevoMK. No postanesthesia vomiting or defecation occurred in SevoDex. Baseline sevoflurane MAC was not different between treatments (Table 2). Within SevoDex, sevoflurane MAC was significantly reduced with both low

Table 1 Comparison of induction time (start of sevoflurane to intubation), extubation time (from discontinuation of sevoflurane and infusion to extubation) and standing times (from discontinuation of sevoflurane and infusion to standing), total anesthesia time (duration of sevoflurane), and induction and recovery quality in six healthy Beagle dogs administered sevoflurane and intravenous infusion of either dexmedetomidine (SevoDex) or MK-467 (SevoMK). Timed values are means ± standard deviations. Qualitative assessments are median (range)

Q2

Variable

SevoDex

SevoMK

Total anesthesia time (minutes) Induction time (minutes) Time to extubation (minutes) Time to standing (minutes)* Induction quality Recovery quality*

341 ± 66 8.7 ± 2.7 17 ± 10 36 ± 17 4 (3e5) 5 (5)

340 ± 53 6.7 ± 1.6 7±2 15 ± 9 4 (2e5) 3 (2e4)

*Significant difference between treatments (p < 0.05).

6

and high dose infusions compared with baseline (p < 0.001). Dogs administered high dose dexmedetomidine had significantly lower MAC than dogs administered low dose dexmedetomidine (p ¼ 0.001). Within SevoMK, sevoflurane MAC during high dose MK-467 infusion was significantly higher compared with baseline (p ¼ 0.007; Table 2). No differences in MAC were observed between the high and low infusion doses or between baseline and low dose MK-467. Dogs administered low and high doses of dexmedetomidine had significantly lower HR than at baseline (p < 0.001), with a further decrease occurring during the high dose compared with low dose infusion (p < 0.001; Table 2). Within SevoMK, dogs administered low and high dose infusions had significantly higher HR compared with baseline (p < 0.001) with no difference in HR between infusion doses. Within SevoDex, dogs administered low and high dose infusions had significantly higher SAP (p < 0.001), DAP (p ¼ 0.003 and 0.03, respectively) and MAP (p ¼ 0.002 and 0.001, respectively) compared with baseline. Significantly lower SAP (p ¼ 0.03), DAP (p ¼ 0.003) and MAP (p ¼ 0.01) were seen in the high dose SevoMK as compared with baseline; no differences were noted with the low dose. PaCO2 was not significantly different between infusion doses of either drug, and pH, PaO2, HCOe 3 and BE were within clinically accepted ranges throughout the study (Table 2). While esophageal temperature was maintained in the targeted range of 37.5e38.5  C, dogs administered high dose dexmedetomidine (p ¼ 0.001) were significantly warmer than during baseline (Table 2). Compared with baseline, blood glucose concentrations were significantly higher with both low (p ¼ 0.02) and high (p ¼ 0.01) dose dexmedetomidine infusions and significantly lower with the high dose MK-467 infusion (p ¼ 0.01; Table 2). No dogs developed glucosuria. Urine output was significantly higher in SevoDex than in SevoMK for both low (p ¼ 0.04) and high (p ¼ 0.002) doses. Within SevoDex, PCV was significantly increased from baseline in low (p < 0.001) and high (p < 0.001) dose infusions, and between doses (p ¼ 0.01). PCV was unchanged in SevoMK. Plasma concentrations of dexmedetomidine and MK-467 did not change significantly within treatments during the infusion period for each dose. Significantly higher plasma concentrations were found when comparing concentrations at the end of the loading dose to plasma concentrations at later

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al. Table 2 Minimum alveolar concentration (MAC), cardiopulmonary and other physiological measurements during sevoflurane MAC determination in six healthy Beagle dogs administered low and high dose intravenous infusions of dexmedetomidine (SevoDex) or MK-467 (SevoMK). Data are means ± standard deviations Variable

MAC (%) % change in MAC Temperature ( C) HR (beats minute
1) fR (breaths minute
1) SAP (mmHg) MAP (mmHg) DAP (mmHg) pH PaCO2 (mmHg) PaCO2 (kPa) PaO2 (mmHg) PaO2 (kPa)
1 HCOe 3 (mmol L ) BE (mmol L
1) PCV (%) TP (g L
1) Glucose (mmol L
1) Lactate (mmol L
1) Creatinine (mmol L
1) Urine output (mL kg
1 hour
1)

SevoDex

SevoMK ¡1

Baseline

1.5 mg kg hour¡1

1.86 ± 0.3* N/A 37.8 ± 0.1* 111 ± 6* 10 ± 3* 110 ± 20* 96 ± 15* 87 ± 14* 7.35 ± 0.02 36 ± 2 4.8 ± 0.3 435 ± 29 58.0 ± 3.9 19.4 ± 0.8 e5.0 ± 1.0 36 ± 6* 50 ± 4 6.2 ± 0.4* 2.5 ± 0.4 50.4 ± 8.8* 1.7 ± 1.1

1.04 ± 0.1y e44 ± 10 38.0 ± 0.2*,y 61 ± 7y 14 ± 6y 128 ± 17y 112 ± 11y 101 ± 10y 7.37 ± 0.06 34 ± 5 4.5 ± 0.7 429 ± 27 57.2 ± 3.6 19.2 ± 1.3 e4.8 ± 1.5 43 ± 4y 50 ± 3 6.9 ± 0.7y 2.3 ± 0.2 46 ± 8.8y 2.7 ± 2.2

4.5 mg kg hour¡1

¡1

0.57 ± 0.1z e69 ± 7 38.1 ± 0.1y 43 ± 6z 9 ± 3*,y 131 ± 17y 113 ± 12y 98 ± 11y 7.35 ± 0.04 36 ± 5 4.8 ± 0.7 423 ± 36 56.4 ± 4.8 19.1 ± 1.8 e5.4 ± 1.6 48 ± 4z 52 ± 3 6.9 ± 0.9y 2.1 ± 0.3 45.1 ± 8.8y 3.7 ± 2.6

Baseline

90 mg kg¡1 hour¡1

180 mg kg¡1 hour¡1

1.93 ± 0.3* N/A 38.0 ± 0.1 114 ± 8* 8±1 114* ± 14 101 ± 13* 92 ± 12* 7.33 ± 0.04 39 ± 5 5.2 ± 0.7 440 ± 28 58.7 ± 3.7 20.1 ± 1.2 e4.7 ± 1.0 39 ± 7 51 ± 3* 6.2 ± 0.6* 2.5 ± 0.7 51.3 ± 8.8 2.3 ± 1.8

2.12 ± 0.3*,y þ10 ± 5 38.1 ± 0.1 140 ± 14y 8±2 111*,y ± 13 95 ± 11*,y 85 ± 11*,y 7.34 ± 0.02 39 ± 3 5.2 ± 0.4 437 ± 24 58.3 ± 3.2 20.4 ± 1.4 e4.5 ± 1.5 37 ± 8 50 ± 3*,y 5.6 ± 0.3y 2.5 ± 1.2 52.2 ± 8.8 0.7 ± 0.4

2.29 ± 0.5y þ 19 ± 15 38.1 ± 0.1 140 ± 14y 8±1 103y ± 11 89 ± 12y 77 ± 10y 7.35 ± 0.01 38 ± 2 5.1 ± 0.3 416 ± 23 55.5 ± 3.1 20.7 ± 1.1 e3.9 ± 1.1 36 ± 6 48 ± 4y 5.5 ± 0.3y 2.0 ± 0.5 48.6 ± 8.8 0.4 ± 0.2

*,y,z

Significant difference between time points within a treatment (p < 0.05). BE, base excess; DAP, diastolic arterial pressure; fR, respiratory rate; HCOe 3, bicarbonate; HR, heart rate; MAP, mean arterial pressure; PCV, packed cell volume; SAP, systolic arterial pressure; TP, total protein.

1.9 ± 0.2% and 1.82 ± 0.06% (Wilson et al. 2008; Moran-Mu~ noz et al. 2014) or slightly lower than 2.36 ± 0.46% and 2.1 ± 0.3% reported in other studies (Kazama & Ikeda 1988; Greene et al. 2002; Mahidol et al. 2015). These discrepancies could be related to a number of factors, including MAC determination site and technique, varying interpretations of what constitutes a positive or negative response, or individual variation among dogs.

time points. Drug plasma concentrations are not available for all dogs at 60 minutes, as MAC determination was completed and the infusion discontinued before this time in some dogs (Table 3). Discussion Sevoflurane MAC values (corrected to a barometric pressure of 760 mmHg) for dogs in the current study are similar to previously reported values of

Table 3 Plasma drug concentrations for dexmedetomidine and MK-467 measured at the end of the loading dose and at fixed time points during infusion in six healthy Beagle dogs. n ¼ 6 except where indicated. Data are means ± standard deviations Treatment [drug (infusion rate)]

Dexmedetomidine (1.5 mg kg
1 hour
1) Dexmedetomidine (4.5 mg kg
1 hour
1) MK-467 (90 mg kg
1 hour
1) MK-467 (180 mg kg
1 hour
1) Q3

Plasma drug concentrations (ng mL¡1) End of loading dose

30 minutes

60 minutes

End of infusion

5.0 ± 0.9* 19.0 ± 5.1* 726.6 ± 109.3* 1906.7 ± 317.5*

1.9 ± 0.4y 6.8 ± 0.9y 552.6 ± 128.5y 1388.2 ± 269.3y

1.6 ± 0.4y (n ¼ 4) 6.2 ± 1.3y 636.8 ± 242.8*,y (n ¼ 3) 1586.4 ± 503.1y (n ¼ 4)

1.7 ± 0.4y 6.0 ± 1.0y 613.1 ± 159.8y 1472.9 ± 371.4y

*,y

Significant difference between time points within a treatment (p < 0.05).

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

7

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

Factors known to decrease MAC such as variations in body temperature, severe hypotension, hypoxemia and hypercapnia (Steffey et al. 2015) were not observed during the study and so were presumed to have no influence on results. The dose-dependent reduction in sevoflurane MAC with dexmedetomidine was anticipated. Previously, dexmedetomidine doses of 0.5, 2.0 and 3.0 mg kg
1 hour
1 were shown to reduce sevoflurane or isoflurane MAC by approximately 18, 44 and 59%, respectively (Pascoe et al. 2006; MoranMu~ noz et al. 2014). The present study used different doses (1.5 and 4.5 mg kg
1 hour
1) to expand on available information regarding anesthesia-sparing effects of dexmedetomidine, and to explore the potential for increased magnitude of sparing effects at the high dose. A MAC reduction of 44% was measured at the lower dose (similar to that observed by Pascoe et al. 2006 with 2 mg kg
1 hour
1) with a further reduction of 69% with administration of 4.5 mg kg
1 hour
1. It is possible that further reduction in MAC would be seen at higher dosage rates. Physiologic changes attributed to the a2-adrenergic agonist effects of dexmedetomidine such as decreases in HR, increases in blood glucose and increases in arterial pressure (Lin et al. 2008; Pascoe 2015) were also noted in this study. Hemodynamic changes were clinically significant. Increases in blood glucose, although statistically significant, were of limited magnitude and clinical importance. As has been previously reported in dogs (Pascoe et al. 2006; Lin et al. 2008; Pascoe 2015), a significant increase in PCV was observed during dexmedetomidine infusion. The cause of this change is unknown. Although it has been speculated to be a result of a2-adrenergic-induced red blood cell release from the spleen (Pascoe 2015), changes in splenic volume (e.g. splenic contraction) in response to dexmedetomidine were not seen with computed tomography in healthy dogs (Baldo et al. 2012). The increase in PCV could also be a result of hemoconcentration from dexmedetomidine-induced diuresis. This is plausible, as dogs in this study received a constant fluid rate in the face of increasing urine output in the SevoDex treatment. Changes in TP, however, were not seen, suggesting this is unlikely to be the sole mechanism. No changes in PCV were detected with MK-467 administration. MK-467 caused a significant overall increase (19%) in sevoflurane MAC at the high dose infusion 8

rate with marked individual variability observed. Three dogs in the high dose treatment had increases in MAC > 20%, with one reaching 43%. The MAC increase in the three remaining dogs was within 10% of baseline. Additionally, regardless of dose, the MAC in dogs administered MK-467 was never lower than their baseline sevoflurane MAC. Interestingly, the increase in MAC did not consistently parallel plasma MK-467 concentrations. MK-467 doses used in the study were selected based on prior work (Kaartinen et al. 2014) and in consultation with coinvestigators (OMV, MRR) in an effort to assess suitability for subsequent use with dexmedetomidine. MK-467 has previously been reported to increase the clearance of a2-adrenergic agonists, probably via improvements in cardiac output and hepatic perfusion, thus decreasing the level of sedation (Honkavaara et al. 2012; Vainionp€ a€ a et al. 2013). In this study, MK-467 was used in dogs that were not administered dexmedetomidine, so the effect is likely to be related to the drug's action on a2-adrenergic receptors. This is particularly interesting as MK-467 is reported to be a peripherally acting antagonist based on measured brain concentrations in marmosets, being 6% or less when compared with plasma drug concentrations (Clineschmidt et al. 1988). Spinal cord penetration was not reported in that study. MAC is reportedly mediated primarily through effects at the level of the spinal cord (Eger et al. 2002), and there is evidence in other species that permeability of the bloodebrain barrier versus the blood spinal cord barrier differs for some molecules (Pan et al. 1997). Penetration of MK-467 into the central nervous system (spinal cord and brain) is not known for dogs, but based on these results it is plausible that penetration is occurring, if only to a limited degree, to cause the observed increase in MAC. However, the a2-adrenoceptor antagonist atipamezole, which has the ability to reverse both central and peripheral affects, has been shown to have no effect on isoflurane MAC in dogs and rats (Ewing et al. 1993; Eger et al. 2003). One could hypothesize that these differences are a result of differential brain versus spinal cord penetration or that MK-467 has some other unknown central stimulatory or additional receptor effects. Further study is necessary to verify and explore potential mechanisms. Hemodynamic and physiologic changes typical of antagonist activity at a2-adrenoreceptors such as increases in HR, decreases in arterial pressure and decreases in blood glucose concentrations were seen

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

in dogs administered MK-467. These changes have been previously reported (Honkavaara et al. 2011; Restitutti et al. 2012). Reduction in blood glucose was mild and clinically insignificant, which is consistent with a prior report showing MK-467 to be a poor antihyperglycemic drug in humans (Schafers et al. 1992). Clinically significant hypotension was not observed in the present study when MK-467 was administered in combination with MAC concentrations of sevoflurane. This may be related to the sevoflurane dose or because a2-antagonist-induced decreases in systemic vascular resistance were offset by increases in HR, which would be expected to improve cardiac output (Honkavaara et al. 2011). Recovery quality was poorer and with wider variability for MK-467 than for dexmedetomidine, suggesting that the sedative effects of dexmedetomidine are beneficial during recovery from sevoflurane. Gastrointestinal side effects occurred in three MK467-treated dogs in the early recovery period; no additional adverse effects were noted. In horses, MK467 increased defecation and occasionally caused abdominal discomfort, presumed to be due to enhanced intestinal motility (de Vries et al. 2016). In humans, MK-467 has been reported to cause nausea (Schafers et al. 1992), and some dogs in this treatment subjectively appeared nauseous (e.g. drooling with occasional vomiting) for a short time after anesthetic recovery. However, these dogs had the smallest increases in MAC associated with MK-467 administration. In the current study, MAC determinations were performed over several hours, and a fixed dosing order (low to high) was used for each treatment, introducing time as a potential confounder. Randomizing the dosing order or using one dose for each anesthetic episode are possible alternatives. A limitation of randomizing dosing order is the unknown time required for plasma concentrations to return to baseline between doses. Increasing the number of anesthetic episodes increases the use of each research dog (which is discouraged by the Institutional Animal Care and Use Committee in the absence of significant added justification of value) and can be cost prohibitive. When compared with a prior MAC study (Pascoe et al. 2006) in which a single dose of dexmedetomidine was used per anesthetic (and where MAC determination was performed in triplicate versus duplicate), the overall study time was similar,

indicating that by using the current study design, anesthetic time was not increased significantly. Although a discussion of time is included, it is of value to note that the MAC of an inhalation anesthetic does not change over time. Hence, the relevance of time in this study is on the potential to influence plasma concentration of the infused drug, which in turn may influence independent MAC point and alter the average MAC value. Plasma drug concentrations were therefore measured during each infusion, and no significant differences over time were noted. Temporal effects on cerebral and cardiovascular variables have been reported during constant inhalation anesthesia (Turner et al. 1984; Steffey et al. 2015) and could have influenced the cardiovascular variables reported in this study. Even though the cardiovascular effects of dexmedetomidine and MK-467 were not the focus of this study, other studies have reported similar results for HR and arterial blood pressure (Lin et al. 2008; Honkavaara et al. 2011; Restitutti et al. 2012; Pascoe 2015), indicating that the effects of time, if any, were minimal. Efforts to ensure that the investigator applying the tail clamp was unaware of the drug treatment included masking the vaporizer settings, gas analyzer and multiparametric monitor and limiting access only to the tail and tail clamp. Mucous membranes (of the anus and vulva) were not specifically shielded from view and the investigator was aware of the study design that incorporated a low to high dose sequence. Nonetheless, the anesthetic sparing effects of dexmedetomidine described in the current study are in agreement with previous reports (Pascoe et al. 2006; Moran-Mu~ noz et al. 2014). This study determined the effects of two infusion rates of dexmedetomidine and of MK-467 on sevoflurane MAC in dogs. Consistent with previous studies, dexmedetomidine reduced sevoflurane MAC in a clinically significant and dose-dependent manner. Concomitant decreases in HR and increases in arterial pressure, blood glucose and PCV are similar to observations from previous reports. Dogs administered dexmedetomidine infusions had an excellent recovery from anesthesia. An unexpected, significant increase in sevoflurane MAC with the high MK-467 infusion dose was recorded. Clinically insignificant increased HR and decreased arterial pressures and blood glucose occurred during MK467 administration.

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

9

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al.

Acknowledgements Funding for this study was provided by Vetcare Ltd, Finland. The authors thank Dr Nadhapat Bunnag, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, for assistance with data collection. Authors' contributions RCH: data collection and interpretation, preparation of the manuscript; MLR, KRM, EPS: study design, data collection and interpretation, preparation of the manuscript; JMH, present for some data collection, reviewed the manuscript; HKK: sample preparation and analysis; AMH: statistical analysis and interpretation; MRR, OMV: contributed to study design (dosing), reviewed the manuscript. All authors approved the manuscript prior to submission. Conflict of interest statement Authors declare no conflict of interest. References Baldo CF, Garcia-Pereira FL, Nelson NC et al. (2012) Effects of anesthetic drugs on canine splenic volume determined via computed tomography. Am J Vet Res 73, 1715e1719. Bennett RC, Salla KM, Raekallio MR et al. (2016) Effects of MK-467 on the antinociceptive and sedative actions and pharmacokinetics of medetomidine in dogs. J Vet Pharmacol Ther 39, 336e343. Bloor BC, Frankland M, Alper G et al. (1992) Hemodynamic and sedative effects of dexmedetomidine in dog. J Pharmacol Exp Ther 263, 690e697. Clineschmidt BV, Pettibone DJ, Lotti VJ et al. (1988) A peripherally acting alpha-2 adrenoceptor antagonist: L-659,066. J Pharmacol Exp Ther 245, 32e40. Eger EI, Eisenkraft JB, Weiskopf RB (2002) The pharmacology of inhaled anesthetics. Baxter Healthcare Corporation, USA. Eger 2nd E.I., Xing Y, Laster MJ, Sonner JM (2003) a-2 adrenoreceptors probably do not mediate the immobility produced by inhaled anesthetics. Anesth Analg 96, 1661e1664. Ewing KK, Mohammed HO, Scarlett JM, Short CE (1993) Reduction of isoflurane anesthetic requirement by medetomidine and its restoration by atipamezole in dogs. Am J Vet Res 54, 294e299. Greene SA, Benson GJ, Tranquilli WJ, Grimm KA (2002) Relationship of canine bispectral index to multiples of sevoflurane minimal alveolar concentration, using patch or subdermal electrodes. Comp Med 52, 424e428.

10

Honkavaara JM, Raekallio MR, Kuusela EK et al. (2008) The effects of L-659,066, a peripheral a2-adrenoceptor antagonist, on dexmedetomidine-induced sedation and bradycardia in dogs. Vet Anaesth Analg 35, 409e413. Honkavaara JM, Restitutti F, Raekallio MR et al. (2011) The effects of increasing doses of MK-467, a peripheral alpha(2)-adrenergic receptor antagonist, on the cardiopulmonary effects of intravenous dexmedetomidine in conscious dogs. J Vet Pharmacol Ther 34, 332e337. Honkavaara J, Restitutti F, Raekallio M et al. (2012) Influence of MK-467, a peripherally acting a2adrenoceptor antagonist on the disposition of intravenous dexmedetomidine in dogs. Drug Metab Dispos 40, 445e449. Kaartinen J, del Castillo JR, Salla K et al. (2014) Haemodynamic interactions of medetomidine and the peripheral alpha-2 antagonist MK-467 during step infusions in isoflurane-anaesthetised dogs. Vet J 202, 353e360. Kazama T, Ikeda K (1988) Comparison of MAC and the rate of rise of alveolar concentration of sevoflurane with halothane and isoflurane in the dog. Anesthesiology 68, 435e437. Lin GY, Robben JH, Murrell JC et al. (2008) Dexmedetomidine constant rate infusion for 24 hours during and after propofol or isoflurane anaesthesia in dogs. Vet Anaesth Analg 35, 141e153. Mahidol C, Niyom S, Thitiyanaporn C et al. (2015) Effects of continuous intravenous infusion of morphine and morphineetramadol on the minimum alveolar concentration of sevoflurane and electroencephalographic entropy indices in dogs. Vet Anaesth Analg 42, 182e186. Moran-Mu~ noz R, Ibancovichi JA, Gutierrez-Blanco E et al. (2014) Effects of lidocaine, dexmedetomidine or their combination on the minimum alveolar concentration of sevoflurane in dogs. J Vet Med Sci 76, 847e853. Murrell JC, Hellebrekers LJ (2005) Medetomidine and dexmedetomidine: a review of cardiovascular effects and antinociceptive properties in the dog. Vet Anaesth Analg 32, 117e127. van Oostrom H, Doornenbal A, Schot A et al. (2011) Neurophysiological assessment of the sedative and analgesic effects of a constant rate infusion of dexmedetomidine in the dog. Vet J 190, 338e344. Pagel PS, Proctor LT, Devcic A et al. (1998) A novel alpha 2-adrenoceptor antagonist attenuates the early, but preserves the late cardiovascular effects of intravenous dexmedetomidine in conscious dogs. J Cardiothorac Vasc Anesth 12, 429e434. Pakkanen SAE, Raekallio MR, Mykk€ anen AK et al. (2015) Detomidine and the combination of detomidine and MK-467, a peripheral alpha-2 adrenoceptor

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Sevofluraneedexmedetomidine or MK-467 in dog RC Hector et al. antagonist, as premedication in horses anaesthetized with isoflurane. Vet Anaesth Analg 42, 527e536. Pan W, Banks WA, Kastin AJ (1997) Bloodebrain barrier permeability to ebiratide and TNF in acute spinal cord injury. Exp Neurol 146, 367e373. Pascoe PJ (2015) The cardiopulmonary effects of dexmedetomidine infusions in dogs during isoflurane anesthesia. Vet Anaesth Analg 42, 360e368. Pascoe PJ, Raekallio M, Kuusela E et al. (2006) Changes in the minimum alveolar concentration of isoflurane and some cardiopulmonary measurements during three continuous infusion rates of dexmedetomidine in dogs. Vet Anaesth Analg 33, 97e103. Pypendop BH, Verstegen JP (1998) Hemodynamic effects of medetomidine in the dog: a dose titration study. Vet Surg 27, 612e622. Pypendop BH, Honkavaara J, Ilkiw JE (2016) Cardiovascular effects of dexmedetomidine, with or without MK-467, following intravenous administration in cats. Vet Anaesth Analg. http://dx.doi.org/10.1111/ vaa.12397. Restitutti F, Honkavaara JM, Raekallio MR et al. (2011) Effects of different doses of L-659'066 on the bispectral index and clinical sedation in dogs treated with dexmedetomidine. Vet Anaesth Analg 38, 415e422. Restitutti F, Raekallio M, Vainionp€ a€ a M et al. (2012) Plasma glucose, insulin, free fatty acids, lactate and cortisol concentrations in dexmedetomidine-sedated dogs with or without MK-467: a peripheral a-2 adrenoceptor antagonist. Vet J 193, 481e485. Salla K, Restitutti F, Vainionp€ a€ a M et al. (2014a) The cardiopulmonary effects of a peripheral alpha-2adrenoceptor antagonist, MK-467, in dogs sedated with a combination of medetomidine and butorphanol. Vet Anaesth Analg 41, 567e574. Salla K, Bennett RC, Restitutti F et al. (2014b) A comparison in dogs of medetomidine, with or without MK-467, and the combination acepromazineebutorphanol as premedication prior to anaesthesia induced by propofol and maintained with isoflurane. Vet Anaesth Analg 41, 163e173. Schafers RF, Elliott HL, Howie CA, Reid JL (1992) A preliminary, clinical pharmacological assessment of L-659,066, a novel alpha 2-adrenoceptor antagonist. Br J Clin Pharmacol 34, 521e526. Steffey EP, Mama KR, Brosnan RJ (2015) Inhalation anesthetics Lumb & Jones' Veterinary Anesthesia and Analgesia (5th edn). Grimm KA, Lamont LA, Tranquilli WJ et al. (eds.). Wiley Blackwell Publishing, USA. pp. 297e331.

Turner DM, Kassell NF, Sasaki T et al. (1984) Timedependent changes in cerebral and cardiovascular parameters in isoflurane-nitrous oxide-anesthetized dogs. Neurosurgery 14, 135e141. V€ ah€ a-Vahe AT (1990) The clinical effectiveness of atipamezole as a medetomidine antagonist in the dog. J Vet Pharmacol Ther 13, 198e205. Vainionp€ a€ a MH, Raekallio MR, Pakkanen SA et al. (2013) Plasma drug concentrations and clinical effects of a peripheral alpha-2-adrenoceptor antagonist, MK467, in horses sedated with detomidine. Vet Anaesth Analg 40, 257e264. de Vries A, Pakkanen SA, Raekallio MR et al. (2016) Clinical effects and plasma drug concentrations of romifidine and the peripheral a2-adrenoceptor antagonist MK-467 in horses. Vet Anaesth Analg 43, 599e610. Wilson J, Doherty TJ, Egger CM et al. (2008) Effects of intravenous lidocaine, ketamine, and the combination on the minimum alveolar concentration of sevoflurane in dogs. Vet Anaesth Analg 35, 289e296. Received 26 August 2016; accepted 26 December 2016. Available online xxx

Appendix 1. Scoring system used to assess induction and recovery quality. Score

Induction

Recovery

5

Accepts mask easily, calm with no excitement or struggling Quiet and smooth except for brief excitement, no struggling Mild struggling or paddling, brief excitement Moderate struggling and excitement

Extubates calmly, quiet and smooth with normal mentation

4

3

2

1

Resists mask, struggling and vocalization throughout

Predominantly smooth with minor restlessness resolving quickly Generally quiet with some restlessness or excitement Some paddling, brief thrashing or disorientation Emergence delirium, thrashing and vocalizing

© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e11

11

Please cite this article in press as: Hector RC, Rezende ML, Mama KR et al. Effects of constant rate infusions of dexmedetomidine or MK-467 on the minimum alveolar concentration of sevoflurane in dogs, Veterinary Anaesthesia and Analgesia (2017), http://dx.doi.org/10.1016/j.vaa.2016.12.058

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52