DISCOVERY AND DEVELOPMENT OF VETERINARY PHARMACEUTICALS IN TELEMETERED ANIMALS

R. J. G. Zwijnenberg

2010

ISBN: 978‐90‐39354742

DISCOVERY AND DEVELOPMENT OF VETERINARY PHARMACEUTICALS IN TELEMETERED ANIMALS

Ontdekking en ontwikkeling van veterinaire farmaceutische produkten in van telemeters voorziene dieren

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 14 december 2010 des middags te 2.30 uur

door:

Raphaёl Johannes Gerhardus Zwijnenberg geboren op 22 december 1961 te Winterswijk

Promotor: Prof. dr. J. Kirpensteijn

Colophon

My parents own a house in Palma de Gandia, Valencia, Spain. It is a typically Spanish house with many terraces and retaining walls. All these retaining walls have been built by hand by my parents. As they were building the retaining walls in their garden, my parents lovingly named each one of these walls by a different name. E.g. the longest wall was called “la Langedocienne” after a piece of the French autoroute that is long and doesn’t seem to end. The one wall I tried to help construct my parents named the “klaagmuur” in Dutch. The translation in English means “the wall of complaints” as the Wailing Wall is called in Dutch. I must have complained a lot during construction, or at least my parents thought so. Also doing a PhD by publication, as is the custom in the Netherlands, is a long process of writing and re‐ writing articles, sign‐off procedures, satisfying both scientific and company standards and procedures. When submitting my fifth article for sign off, some people within the company were clearly showing signs of fatigue for being confronted again by a “nagging” and demanding author. Over the centuries the Wailing Wall has been a source of conflict but also a source of inspiration, a symbol of hope and freedom. Reaching my PhD I will regard as a personal freedom from a desire I had since I graduated as a veterinarian in 1987. I herewith would like to express my thanks to Guy Rosner (Tel Aviv, Israel) who as a personal friend and a great artist agreed to make this painting for the cover of my thesis.

Raphael Zwijnenberg. Sydney, 1 November 2010.

Contents

Chapter 1 Introduction 1

Chapter 2 Effects of perzinfotel on the minimum alveolar concentration of in dogs when given as a pre‐anesthetic IV, IM or SQ and in combination with 9

Chapter 3 Effects of perzinfotel and PLA‐ 695 on kinetic gait and subjective lameness scores in a sodium urate‐induced synovitis model in dogs 25

Chapter 4 Effects of perzinfotel, butorphanol and a butorphanol‐ perzinfotel combination on the minimum alveolar concentration of isoflurane in cats 39

Chapter 5 Evaluation of oscillometric and vascular‐access‐port derived arterial blood pressure measurement techniques in anesthetized cats: comparative performance versus implanted telemetry 55

Chapter 6 Evaluation of the potential for interaction between a metaflumizone‐amitraz combination and dexmedetomidine hydrochloride in dogs 69

Chapter 7 Discussion 79

Chapter 8 Conclusion 89

Samenvatting 91 Acknowledgements 93 Curriculum Vitae 95 List of publications 97

CHAPTER 1

Introduction CHAPTER 1

Pharmaceutical Drug Discovery

Pharmaceutical drug discovery is the process by which drugs are discovered and/or designed and commonly takes place in scientific institutions and pharmaceutical companies. In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery. A new approach has been to understand how disease and infection are controlled at the molecular and physiological level and to target specific entities based on this knowledge1. This field of science is typified by great discoveries of novel actives through active research programs into specific classes of chemistry, endless screening of libraries of potential actives as well as (still) pure serendipity. Pharmaceutical drug discovery can also be disappointing, e.g. when promising actives fail safety and/or efficacy criteria. Despite significant advances in understanding of disease on a molecular and physiological level, drug discovery is still a lengthy, expensive and inefficient process with a relatively low rate of success2. It is estimated that the discovery and development of a human pharmaceutical takes on average 13 years at an average cost of US$1.8 billion dollars for each new molecular entity3. Discovery studies such as proof of concept, efficacy, safety, dose titration etc., are typically done in a limited number of animals (n=6‐8) per treatment group. The statistical power and the level of significance (P < 0.05) of effects observed are used as considerations to move projects into development. Ethical considerations are an important factor to limit the number of animals used in discovery studies. Typically, after establishing a satisfactory efficacy and safety profile (including toxicology), a marketing need, formulation, reasonable manufacturing costs etc., drugs can move into the process of drug development.

1.2 Pharmaceutical Drug Development Pharmaceutical drug development is often an interaction between a pharmaceutical company and a regulatory body to identify the necessary trials to compose a dossier that would be acceptable for approval. In the veterinary field, these would typically include field trials in client owned animals as well as residue and environmental studies (especially in case of production animals). Due to more rigorous regulation the number of truly new medicines approved by the United States Food and Drug Administration (FDA) between 2003 and 2008 has decreased by 50% compared to the previous 5 years4.

2 INTRODUCTION

1.3 Perzinfotel, the drug In the Wyeth Central Nervous System (CNS) facility in Princeton, New Jersey (NJ), a novel potent N‐methyl‐D‐aspartate (NMDA) antagonist (EAA‐090, later named perzinfotel) was designed and synthesized for pharmaceutical research5. It was thought that perzinfotel, being a unique NMDA antagonist with a favourable preclinical profile, may offer advantages over existing NMDA antagonists for the treatment of neurological disorders such as and head trauma. Later research confirmed that both perzinfotel and another NMDA antagonist (EAB‐318) protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate‐ and NMDA‐induced neurotoxicity. Both antagonists preferentially blocked NMDA‐elicited currents mediated by N‐methyl‐d‐aspartate receptor (NR)1 splice variants containing the N‐terminal insertion. They also favoured NR2A‐versus NR2B‐ or NR2C‐ containing NMDA receptors, with perzinfotel showing the greatest selectivity. Perzinfotel was 10 times more potent at blocking NR2A‐versus NR2B‐ or NR2C‐containing NMDA receptors6. NMDA receptors (NMDAR’s) are glutamate receptors that control synaptic plasticity and memory function and are located in the hippocampus and the cortex. The NMDAR is a heterotetramer consisting of two obligatory NR1 subunits and two of four possible NR2 subunits: NR2A, NR2B, NR2C, and NR2D. The NR2 subunits in the adult hippocampus and cortex are usually NR2A and NR2B subunits7. NR2A and NR2B have different roles in mediating cell death and cell survival. This was observed in an in vivo rat model of focal ischemic stroke. It was found that blocking NR2B‐mediated cell death was effective in reducing infarct volume only when the receptor antagonist was given before the onset of stroke and within 4.5 hours (h) after stroke. In great contrast, activation of NR2A‐mediated cell survival with administration of either alone or in the presence of NR2B antagonist significantly attenuated ischaemic brain damage even when delivered 4.5 h after stroke onset. Hence perzinfotel, with a greater selectivity for the NR2A subunit, was considered as a potential drug candidate against stroke. Some NMDA antagonists like and have effects. A study in rats showed that perzinfotel lacked antinociceptive effects but dose‐ and time‐dependently (10‐100 mg/kg) blocked prostaglandin E(2) (PGE(2))‐ and capsaicin‐induced thermal hypersensitivity in a warm‐water tail‐withdrawal assay in rats8. Perzinfotel was also considered for neuropathic pain in humans. Compared to current drugs on the market perzinfotel did not outperform. Because other Wyeth drugs were also considered for stroke, perzinfotel was offered for consideration to the Animal Health Division of Wyeth, Fort Dodge Animal Health, in 2006.

1.4 The veterinary discovery process of perzinfotel in canines After Fort Dodge Animal Health was given the opportunity to assess perzinfotel for veterinary use, an initial study was done in beagles to look at the clinical effects of perzinfotel with dosages ranging between 10‐60 mg/kg9. Perzinfotel, given at a dose of ≥30 mg/kg produced

3 CHAPTER 1

increasing muscular rigidity. This rigidity was transient and all dogs recovered uneventfully. All doses of perzinfotel resulted in pupil dilatation. Also the pupil dilation was a temporary side‐ effect from which all dogs recovered within 2‐8 hours. These observed side effects, however mild in nature, are the reason that in subsequent studies only doses up to 30 mg/kg were used. Perzinfotel alone did not produce anaesthesia at the studied doses. Although veterinary anaesthesia has become safer over the years through safer anaesthetics, improved monitoring and better quality of equipment, safety of anaesthesia is a constant concern for practising veterinarians. In the recent years the anaesthetic sparing effect of different drugs has been studied and debated as a means to reduce the amount of anaesthetic gases given to animals during anaesthesia10, 11. It was theorised that perzinfotel might have an anaesthetic sparing effect similar or greater to ketamine. The first studies during which perzinfotel was given IV after propofol induction and at the time of isoflurane administration, demonstrated that perzinfotel, indeed, produced a significant anaesthetic sparing effect that was greater than the anaesthetic sparing effect produced by ketamine12, 13. Subsequent to these findings it was proposed to include perzinfotel in a pre‐anaesthetic protocol and do the necessary study to demonstrate the anaesthetic sparing effects of perzinfotel as a pre‐anaesthetic alone and in combination with butorphanol (Chapter 2).

1.5 Telemetry Through past experience in development of a new anaesthetic compound in close alignment with FDA requirements14 and through scientific advice it was clear that reliable measurement of blood pressure would be essential for an acceptable dossier of perzinfotel for approval by the FDA. The only acceptable, standardised and validated method of measuring blood pressure is through telemetry. Telemetry is a technology that allows remote measurement and reporting of information. This technology has applications in the field of motor racing (e.g. Engine Control Unit (ECU), tire temperatures), agriculture (e.g. air temperature, relative humidity, precipitation, leaf wetness, soil moisture), water management (e.g. automatic meter reading (AMR), groundwater monitoring, leak detection in distribution pipelines and equipment surveillance), defense, space and resource exploration systems (e.g. development phase of missiles, satellites and aircraft), rocketry (monitoring progress of rocket launch), enemy intelligence (e.g. encrypting and decrypting telemetry signals for missile launches etc.), energy monitoring (sophisticated air‐condition units), resource distribution (channeling), medicine (biotelemetry, e.g. constant monitoring of patients with a risk of abnormal heart activity)15. In this case the telemetry devices were implanted in dogs and cats under general anaesthesia with isoflurane. The telemetry devices provided signals for body temperature, electrocardiogram (ECG) and arterial blood pressure through an anti‐thrombotic gel‐filled catheter and a 2‐electrode lead ECG. Each device was subcutaneously secured to the superficial abdominal musculature of the left flank via a ventral skin incision. The arterial pressure catheter was tunneled to the left

4 INTRODUCTION

hind limb, and introduced/placed in the abdominal aorta via the left femoral artery. Two ECG electrodes were tunneled subcutaneously and secured to the superficial muscles of the left and right sides of the chest wall using a lead II configuration. The digital telemetry signals were picked up by a receiver and manually recorded as well as printed out through a conventional printer. The telemeters were battery operated and could be switched on and off externally. Although telemetry allows 24 hour observation of blood pressure and temperature during the studies described in this thesis, telemetry was only used to measure blood pressure just before, during and after anaesthesia.

1.6 Possible analgesic effects of perzinfotel in dogs Peri‐ and post operative pain control is an integral part of veterinary surgery. Although rat models showed that the analgesic effect of perzinfotel is variable8, no data were available regarding the analgesic effect of perzinfotel in dogs. If perzinfotel would be administered as a part of a pre‐anaesthetic protocol, an analgesic effect would be advantageous. The analgesic properties of perzinfotel were studied in an induced urate synovitis model and compared with the analgesic properties of a proprietary PLA‐inhibitor (PLA‐695) and a positive control (Carprofen, Pfizer Animal Health) (Chapter 3).

1.7 The effect of perzinfotel in felines and more telemetry After studying the anaesthetic sparing effect of perzinfotel in dogs, the question arose what the anaesthetic sparing effect of perzinfotel in cats would be. A preliminary study showed that cats seem to be more sensitive to perzinfotel than dogs17. Therefore a dose titration to study the effect on cats was included as the first part of the study. After it was established perzinfotel had only minor transient side effects like immobilisation and pupil dilatation, appropriate doses were selected for the second part of the study (Chapter 4). As in the previously discussed study in canines, this feline study also needed to be done in telemetered animals. Looking forward though, the development of perzinfotel would need field studies in client owned animals that would not be telemetered. Therefore the decision was made to do a study to validate three different methods of measuring blood pressure: internal telemetry, external telemetry with a vascular access port (VAP) and oscillometry with a tail cuff. External telemetry is technology that uses a femoral artery indwelling catheter. This catheter was implanted by performing a cut‐down over the right femoral artery, and introducing a 3F catheter into the abdominal aorta. This catheter was subcutaneously tunneled to a subcutaneous pocket on the animal’s right hip, where it was attached to the VAP and secured to the underlying fascia. The VAP was used in conjunction with a wearable telemetry transmitter to provide a less invasive telemetric arterial pressure signal. In contrast with internal telemetry, no major body cavity was opened/accessed during the VAP

5 CHAPTER 1

implantation procedures. After surgery the animals were allowed to recover for ≥ 30 days during which they were observed daily for signs distress while the wound sites were monitored for signs of infections. No signs of infection, pain or distress related to the surgery could be observed at any time. Depending on the results in this study, the less invasive and less expensive VAP technology could replace the internal telemetry for future in house studies. Non‐invasive oscillometry was evaluated for field studies that would be required for the completion of a dossier that would comply with regulatory requirements (Chapter 5).

1.8 Additional study with telemetered dogs Although telemetry is the accepted, validated method to measure blood pressure in cats and dogs, it is invasive and expensive. The conclusion of the perzinfotel dog study (Chapter 3) coincided with the launch of a new Fort Dodge Animal Health product, Promeris for Dogs®. During and just after the launch of Promeris, some clinicians in the USA and Europe asked the question whether Promeris, with amitraz as an active ingredient, and dexmedetomidine hydrochloride (Dexdomitor®, Pfizer Animal Health) could interact as it was theorised that amitraz, when administered systemically to mammals, would bind with α2‐receptors 16 producing sedative effects that may exacerbate the effects of α2‐sedatives like Dexdomitor. This study was designed to investigate the cardiovascular effects, if any, of a topical metaflumizone‐amitraz combination administered cutaneously to telemetered dogs that were subsequently sedated with dexmedetomidine (IV) (Chapter 6).

1.9 Research hypotheses Perzinfotel or a combination of perzinfotel and butorphanol will have a significant effect on minimum alveolar concentration of isoflurane and on haemodynamic parameters like medium, diastolic and systolic blood pressure in dogs compared to control dogs not receiving perzinfotel. To prove this hypothesis, six healthy sexually intact telemetered Beagles were induced with propofol after the administration of a placebo, perzinfotel or a perzinfotel/butorphanol combination and anaesthesia was maintained with a mixture of isoflurane in oxygen. During anaesthesia the ECG, heart rate (HR), blood pressure, end‐tidal (ET)iso, ETco2, PO2 and body temperature was constantly monitored. Results were recorded, statistically analysed and interpreted.

Perzinfotel and PLA‐695 will have an effect on kinetic gait and subjective lameness scores in a sodium urate‐induced synovitis model in dogs. To prove this hypothesis, eight adult mixed‐breed dogs were with either treated with perzinfotel, PLA‐695, carprofen (positive control) or not treated (negative control) in a blinded four way crossover study using an induced urate synovitis model. Ground reaction forces

6 INTRODUCTION

were measured by using two force plates while experienced veterinarians were recording subjective lameness data. Results were recorded, statistically analysed and interpreted. Perzinfotel or a combination of perzinfotel and butorphanol will have a significant effect on minimum alveolar concentration of isoflurane and on haemodynamic parameters like medium, diastolic and systolic blood pressure in cats compared to control cats. To prove this hypothesis, six healthy sexually intact telemetered domestic shorthair cats were induced with propofol and anaesthesia was maintained with a mixture of isoflurane in oxygen, after the administration of a placebo, perzinfotel or a perzinfotel/butorphanol combination. During anaesthesia the ECG, HR, blood pressure, ETiso, ETco2, PO2 and body temperature was constantly monitored. Results were recorded, analysed and interpreted.

There is no significant difference in measurement of blood pressure during anaesthesia between oscillometric methods (tail cuff), vascular‐access port (VAP) derived arterial blood pressure and implanted telemetry in cats. To prove this hypothesis, six healthy sexually intact cats were impanted with invasive telemetry and semi‐invasive VAP technology. A tail cuff was administered during anaesthesia with isoflurane. Arterial blood pressure was recorded using all three techniques at normotension and hypertension. Results were recorded, analysed and interpreted.

Metaflumizone/amitraz does not interact in terms of heart rate and/or haemodynamic parameters with a compound that has α2‐adrenergic agonist activity like dexmedetomidine. Six healthy sexually intact telemetered Beagles were sedated with dexmedetomidine first without any pre‐treatment and then after pre‐treatment with metaflumizone and amitraz.

During anesthaesia the ECG, HR, blood pressure, PO2 and body temperature were constantly monitored. Results were recorded, analysed and interpreted.

References

1. Drug discovery. http://en.wikipedia.org/wiki/Drug_discovery Retrieved 5 June 2010. 2. Anson, Blake D.; Ma, Junyi; He, Jia‐Qiang (1 May 2009), "Identifying Cardiotoxic Compounds", Genetic Engineering & Biotechnology News, TechNote (Mary Ann Liebert) 29 (9): 34–35 3. Steven M. Paul, Daniel S. Mytelka, Christopher T. Dunwiddie, Charles C. Persinger, Bernard H. Munos, Stacy R. Lindborg & Aaron L. Schacht (2010). "How to improve R&D productivity: the pharmaceutical industry's grand challenge". Nature Reviews Drug Discovery 9 (3): 203–214. 4. Mathieu, M. P., ed. Parexel's Bio/Pharmaceutical R&D Statistical Sourcebook 2008/2009. (Parexel International Corporation, Waltham, 2008). 5. Kinney WA, Abou‐Gharbia M, Garrison DT, Schmid J, Kowal DM, Bramlett DR, Miller TL, Tasse RP, Zaleska MM, Moyer JA. Design and synthesis of [2‐(8,9‐dioxo‐2,6‐diazabicyclo[5.2.0]non‐ 1(7)‐en‐2‐yl)‐ethyl]phosphonic acid (EAA‐090), a potent N‐methyl‐D‐aspartate antagonist, via the use of 3‐cyclobutene‐1,2‐dione as an achiral ‐amino acid bioisostere. Journal of Medicinal Chemistry. 1998 Jan 15;41(2):236‐46.

7 CHAPTER 1

6. Sun L, Chiu D, Kowal D, Simon R, Smeyne M, Zukin RS, Olney J, Baudy R, Lin S. Characterization of two novel N‐methyl‐D‐aspartate antagonists: EAA‐090 (2‐[8,9‐dioxo‐2,6‐diazabicyclo [5.2.0]non‐1(7)‐en2‐yl]ethylphosphonic acid) and EAB‐318 (R‐alpha‐amino‐5‐chloro‐1‐ (phosphonomethyl)‐1H‐benzimidazole‐2‐propanoic acid hydrochloride). Journal of Pharmacology and Experimental Therapeutics. 2004 Aug;310(2):563‐70 7. Fei Li and Joe Z. Tsien, Memory and the NMDA receptors. N Engl J Med, 361:302, July 16, 2009 8. Brandt MR, Cummons TA, Potestio L, Sukoff SJ, Rosenzweig‐Lipson S. Effects of the N‐methyl‐ D‐aspartate receptor antagonist perzinfotel [EAA‐090; [2‐(8,9‐dioxo‐2,6‐ diazabicyclo[5.2.0]non‐1(7)‐en‐2‐yl)‐ethyl]phosphonic acid] on chemically induced thermal hypersensitivity. Journal of Pharmacology and Experimental Therapeutics. 2005 Jun;313(3):1379‐86. 9. Fort Dodge Animal Health data on file, study report GASR 13‐13.00 10. Sérgio S Souza*, Tatiana R Intelisano, Christianni P De Biaggi*, Cláudio A Moura†, André L Selmi†, Ricardo A Dias‡ & Sílvia R G Cortopassi. Cardiopulmonary and isoflurane‐sparing effects of epidural or intravenous infusion of dexmedetomidine in cats undergoing surgery with epidural lidocaine. Veterinary anaesthesia and analgesia. 2009. Volume 37, issue 2, pages 106‐115. 11. Muir WW III, Wiese AJ, March PA. Effects of , lidocaine, ketamine, and morphine‐ lidocaine‐ketamine drug combination on minimum alveolar concentration in dogs anesthetized with isoflurane. Am J Vet Res 2003; 64: 1155 – 1160. 12. Kushiro T, Wiese AJ, Eppler MC et al. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2007; 68: 12: 1294 – 1299. 13. Ueyama Y, Lerche P, Eppler M, Muir WWIII. Effects of Perzinfotel, Fentanyl, and a Combination of Perzinfotel and Fentanyl on the Minimum Alveolar Concentration of Isoflurane in Dogs. Am J Vet Res 2009; 70: 12: 1459‐1464. 14. Cleale RM, Muir WW, Waselau AC, Lehmann MW, Amodie DM. Pharmacokinetic and pharmacodynamic evaluation of propofol administered to cats in a novel, aqueous, nano‐ droplet formulation or as an oil‐in‐water macroemulsion. J Vet Pharm Ther 2009; 2 5: 436 – 445. 15. Telemetry. http://en.wikipedia.org/wiki/Telemetry Retrieved 6 June 2010. 16. Queiroz‐ Neto A, Zamur A, Gonçalves SC et al. Characterization of the antinociceptive and sedative effect of amitraz in horses. J. vet. Pharmacol. Therap. 1998; 21: 400 – 405. 17. Fort Dodge Animal Health Data on file, study report GASR 13‐12.00

8

CHAPTER 2

Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs when given as a pre‐anesthetic IV, IM or SQ and in combination with butorphanol

Raphael J Zwijnenberg DVM, MVPHMgt* Carlos L del Rio DVM, PhD** Robert A Pollet, PhD* William W Muir DVM, PhD**

*Fort Dodge Animal Health, PO Box 5366, Princeton, NJ 08543 **QTest Labs, PO Box 12381, Columbus, OH 43212

Am J Vet Research 2010; 71 (6): 604‐609 CHAPTER 2

Objective – To compare the effects of perzinfotel administered alone IM, IV, SQ or administered in combination with butorphanol on minimal alveolar concentration (MAC) of isoflurane and hemodynamic parameters in dogs versus baseline values without perzinfotel or butorphanol alone.

Animals ‐ Six healthy sexually intact Beagles, 4 males and 2 females, 18.5 ‐ 31 months of age and weighing 9.8 ‐ 12.4 kg.

Procedures – After administration of placebo, perzinfotel or a perzinfotel/butorphanol combination, dogs were induced with propofol and anesthesia was maintained with a mixture of isoflurane in oxygen. The ECG, HR (beats/min), systolic (SAP), diastolic (DAP) and mean

(MAP) arterial pressure (mm Hg), ETISO (%), ETco2 (mm Hg), PO2 (mm Hg) by pulse oximetry (%), body temperature (ºC), inspiration and expiration concentrations of isoflurane were continuously monitored. A noxious stimulation protocol was used and MAC was determined twice during anesthesia.

Results ‐ IV, SQ and IM administration of perzinfotel (10‐30 mg/kg) decreased the mean isoflurane MAC values by 32 – 44%. The greatest MAC reduction (59%) was observed with a combination of 20 mg/kg perzinfotel and 0.2 mg/kg butorphanol, while the administration of butorphanol alone gave a 15% reduction of the isoflurane MAC.

Conclusions and Clinical Relevance ‐ All doses of perzinfotel resulted in a significant reduction of MAC as well as significantly higher BIS (Bispectral Index) values. A dose of 30 mg/kg resulted in significantly higher HR and DAP. SQ, IM or IV administration of perzinfotel as a pre‐ anesthetic treatment prior to isoflurane anesthesia improves anesthetic safety by reducing anesthetic requirements. N‐methyl‐D‐aspartate (NMDA) receptors (NMDARs) are a class of glutamate‐gated ion channels that regulate Na and Ca transmembrane flux. NMDA receptors have received particular attention from both scientists and clinicians because of their crucial roles in excitatory synaptic transmission, plasticity, and prevention of neurodegeneration in the central nervous system (CNS)1,2. NMDARs contain a variety of sites at which endogenous ligands and subunit selective drugs modulate receptor activity. They are comprised of NR1 (eight splice variants) and NR2 (A, B, C, and D subtypes) subunits with further variation possibly provided by the recently discovered NR3 (A and B) subunits. These subunits represent a class of structurally different binding sites with different affinities for agonists and antagonists. Recent evidence suggests that activation of NR2B and NR3A subtypes play important roles in perception of pain and neuronal injury, respectively.

10 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

There is considerable evidence that pain associated with peripheral tissue or nerve injury involves NMDA receptor activation3. NMDA receptors have been identified on myelinated and unmyelinated axons in peripheral somatic tissues4,5. Local injections of glutamate or NMDA result in nociceptive behaviors in rats that can be attenuated by the peripheral administration of NMDA receptor antagonists6,7,8,9. NMDA receptor antagonists have demonstrated effective alleviation of pain in both animal models and clinical situations10,11. Although the effect of NMDA receptor antagonists has been well documented, their use as may be limited by side effects such as memory impairment, effects, ataxia and motor incoordination. Antinociceptive selective antagonists of NR2B‐containing NMDA receptors (e.g. ifenprofil) have a much lower side‐effect profile compared with some other NMDA receptor antagonists11. Perzinfotel (EAA‐090) is a potent NMDA receptor antagonist12. A single bolus dose of perzinfotel, administered IV, following permanent occlusion of the middle cerebral artery in the rat, reduced infarct size by 57%. In vivo characterization showed that perzinfotel was 10 times more potent at blocking NR2A‐ versus NR2B‐ or NR2C‐containing NMDA receptors, and protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate‐ and NMDA‐induced neurotoxicity13. When compared with uncompetitive channel blockers (e.g., memantin, , and ketamine), a NR2B selective antagonist (e.g., ) and other glutamate antagonists (e.g., , CCP, CGP‐39653), perzinfotel had superior therapeutic ratios for effectiveness in treating pain versus adverse behavioral effects14. NMDA receptor antagonists may reduce the amount of inhaled anesthetic needed to maintain anesthesia (anesthetic sparing) in addition to producing analgesic effects. The minimum alveolar concentration (MAC) of an inhalant anesthetic is defined as the amount of inhaled anesthetic required to prevent gross purposeful movement to a noxious stimulus in 50% of the subjects15. The MAC is a measure of anesthetic potency and provides a guide to the concentration of inhaled anesthetic needed to induce unconsciousness and immobility. Apart from an analgesic effect, currently available NMDA antagonists like ketamine reduce the MAC of isoflurane needed to maintain dogs under anesthesia by up to 25%16,17. Recently, perzinfotel has been shown to have similar or greater anesthetic‐sparing effects in dogs18. The synthetic derivative butorphanol is a mixed agonist‐antagonist analgesic frequently administered alone or in conjunction with other sedatives as preanesthetic prior to general anesthesia in dogs19,20. Current opinion suggests that butorphanol acts as a partial mu (u) agonist, pure kappa (κ) agonist and delta (δ) antagonist although species differences have been noted21. Studies investigating the inhalant anesthetic (MAC) sparing effects of butorphanol in dogs have demonstrated variable effects and minimal (8‐19%) inhalant anesthetic sparing effects19,22,23,24,25,26. Several of these same studies suggested that butorphanols’ anesthetic sparing effect was enhanced and potentially additive when administered in combination with nonsteriodal anti‐inflammatory drugs although appropriate experiments for this conclusion were not performed22,23,27.

11 CHAPTER 2

Bispectral Index (BIS) processing is a proprietary method for analyzing the degree of sedation and hypnosis28,29. Bispectral analysis examines the harmonic and phase relation of EEG signals and quantifies the amount of synchronization in the EEG. The BIS is a numeric value derived from the EEG and provides a reasonably accurate index of anesthetic depth and the presence or absence of consciousness30. Values <70 generally are associated with pronounced sedation, and values < 60 indicate unconsciousness from which the animal cannot be aroused. Changes in BIS values are used to indicate a return to consciousness during inhalant anesthesia and to help identify differences between drug induced analgesic and hypnotic effects. The purpose of this study was to determine the anesthetic sparing effects of perzinfotel when given as a pre‐anesthetic via IV, IM or SQ routes, and in combination with butorphanol. Alteration of MAC and BIS values was examined and measurements of hemodynamic parameters like heart rate (HR) and blood pressure were conducted. In addition, time to sternal recumbency after anesthesia was measured.

Materials and Methods

Animal care and instrumentation: The study was approved by the Institutional Animal Care and Use Committee. Six healthy sexually intact Beagles, 4 males and 2 females, 18.5 to 31 months of age and weighing 9.8 to 12.4 kg, were the subjects of this study. Each dog was equipped with a telemetry device that had been surgically implanted a minimum of 2 weeks before beginning the study. The telemetry device permitted the simultaneous and continuous monitoring of respiration, ECG, arterial (femoral artery) blood pressure, and body temperature.

Experimental design: Each dog was submitted to eight different treatments (Table 1). Apart from the first and the last treatment (MACo and G, respectively) all treatments were done in a Latin square cross‐over design (Table 2). All treatments were separated by a minimum washout period of 7 days. The first treatment, saline control, allowed the determination of the baseline MAC and other parameters in all dogs. During subsequent treatments, perzinfotel and/or butorphanol were administered as a treatment 30 minutes before induction. Isoflurane MAC was determined twice during each treatment at approximately 30 minutes after anesthesia onset and 2 hrs later (MAC1 and MAC2, respectively). During the last treatment (Treatment G), the MAC of isoflurane was re‐determined under control conditions

(MACo) in all dogs in order to evaluate any possible confounding effects (i.e., lessening of anesthetic requirements) resulting from habituation to the laboratory environment, the noxious stimulation protocol, and/or the repeated anesthesia (i.e., temporal factors).

Following the MACo determination, the independent anesthetic sparing effect of butorphanol was determined.

12 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

Table 1: Description of treatments

Treatment Dosing Rate

MACo Determination of control MAC1 & MAC2, no perzinfotel A 20 mg/kg IV B 20 mg/kg SQ C 20 mg/kg IM D 10 mg/kg IM E 30 mg/kg IM F 20 mg/kg IM + 0.2 mg/kg butorphanol IM G Saline followed (after MAC1 determination) by 0.2 mg/kg butorphanol IM (i.e., for MAC2)

Table 2: Experimental design

Dog number 1‐4978137 2‐5103720 3‐5017521 4‐5288762 5‐5271932 6‐5104505

MACo MACo MACo MACo MACo MACo A F E D C B B A F E D C C B A F E D D C B A F E E D C B A F Treatment* F E D C B A G G G G G G * All treatments were administered at an interval of approximately 7 days (10.0 ± 0.8 days; median: 7 days; 25th‐75th percentile: 7 ‐ 13 days).

Experimental procedures: Dogs were fasted for 12 hours and water was withheld for 2 hours prior to the administration of treatment (pre‐medication) with either saline, perzinfotel, perzinfotel + butorphanol or butorphanol on the day of each experiment. The degree of sedation after administration of perzinfotel was scored. A cephalic vein was catheterized and propofola was administered at a dose of 4‐6 mg/kg (to effect). The dogs were orotracheally intubated and positioned in right lateral recumbency. Isofluraneb in oxygen was used to maintain anesthesia through an out‐of‐circle, agent‐specific vaporizerc in a semi‐closed d anesthetic circle rebreathing system . The end‐tidal carbon dioxide concentration (ETco2; mm Hg) was maintained between 35 and 45 mm Hge by means of controlled breathing. The ECG, HR (beats/min), systolic (SAP), diastolic (DAP) and mean (MAP) arterial pressure (mm Hg),

ETISO (%), ETco2 (mm Hg), PO2 (mm Hg) by pulse oximetry (%), body temperature (°C), inspiration and expiration concentrations of isoflurane were continuously monitoredf,g.

13 CHAPTER 2

Heating padsh and hot water blankets were used during anesthesia to maintain body temperature between 37.5 and 38.5°C.

Determination of isoflurane MAC: Isoflurane MAC was determined by delivering a noxious supramaximal electrical stimulus to the buccal mucosa15. Two 24‐gauge, 10‐mm insulated i stimulating electrodes were inserted 1 cm apart into the buccal mucosa at a location dorsal and caudal to the incisors. The opposite ends of the electrodes were connected to an electrical stimulatorj that delivered a predetermined stimulus of 50 V, 5 Hz, and 10 ms duration. Stimulation continued for 1 min unless the dog showed gross purposeful movement before completion of the 1‐min stimulation. Lifting of the head and repeated movement of the limbs were considered gross purposeful movement. Slight paw movement, arching of the back, chewing, swallowing, blinking, opening of the eyes, and nystagmus were not considered gross purposeful movement (negative response). The ETISO was initially set at

1.5% during each dog’s first MAC0 determination and at 1.2 times each dogs control MAC value during subsequent days when experimental treatments were administered. If there was a negative response to the stimulus, the ETISO was decreased by 20% and allowed to equilibrate for at least 15 minutes before applying the stimulus. This process was continued until the dog responded with gross purposeful movement. The ETISO was then increased by increments of 10% until the dog failed to demonstrate gross purposeful movement. The MAC was considered to be the average of the lowest ETISO value that did not produce gross purposeful movement and the highest ETISO value that produced gross purposeful movement15.

Determination of BIS: The BIS value was derived by continuously monitoring EEG activity. The EEG was obtained from platinum subdermal needle electrodes using a 3‐lead referential montage, arranged in a bifrontal configuration with the reference electrode positioned on the midline of the head rostral to the medial canthus of the eyes. The ground electrode was positioned on the midline in the atlantooccipital region15, 30. The EEG and BIS values were continuously acquired and displayed by use of a proprietary BIS monitork with the high‐ frequency filter set at 70 Hz and the low‐frequency filter set at 2 Hz. The BIS number was automatically calculated and digitally displayed every 5 sec and represented the EEG activity during the previous 60 seconds. Eight BIS values were recorded during a 2 min period before and after buccal mucosal stimulation. It has been previously demonstrated that perzinfotel does not change BIS values in isoflurane anesthetized dogs31.

Time to sternal recumbency: Time to sternal recumbency (min:sec) is the time between extubation (laryngeal cough reflex) and maintenance of sternal recumbency. Time was measured with a digital clock.

Statistical analysis: For each animal, average values for all MAC determinations at a given

14 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

treatment were calculated. Group data for each treatment are reported as the mean ± SD. Responses of all groups treated with either experimental compounds or saline were compared. Comparisons of hemodynamic parameters and BIS were made at MAC level. These comparisons were made by ANOVA, with LS Means of groups compared to each other by two‐ sided Student's t‐test at the 5% level of significance l.

Results

The control (MACo) MAC values for isoflurane were 1.13 ± 0.12% (CTRL‐MAC1) and 1.20 ± 0.10% (CTRL‐ MAC2) when determined approximately 30 min and 2 hrs later after the onset of anesthesia. The CTRL MAC remained stable since MAC determined at the end of the study was 1.12 ± 0.05%. The IV, SQ and IM administration of perzinfotel 30 minutes before induction to anesthesia significantly decreased the mean isoflurane MAC values by 32 – 44% for all doses of perzinfotel (Table 3). The decrease after administration of perzinfotel, 30 mg/kg IM, was also significant compared to 20 mg/kg kg IV and 10 mg/kg IM doses and as such demonstrated mild dose dependency. The greatest MAC reduction (59%, significant compared to all other treatments) was observed when the combination of 20 mg/kg perzinfotel and 0.2 mg/kg butorphanol was administered IM, while the administration of butorphanol alone gave a much smaller reduction of the isoflurane MAC (15%). The administration of perzinfotel alone produced little sedative effect during the 30 min prior to induction. However, the combination of perzinfotel and butorphanol produced moderate sedation. The administration of perzinfotel produced dose dependent statistically significant increases in the time required to sternal recovery following extubation (Table 5). Control dogs required 0:58 ± 0:53 minutes to reach a sternal position after anesthesia with propofol and isoflurane; pre‐medication with 20 mg/kg perzinfotel IV increased this time to 11:36 ± 5:48 minutes, while doses of 10, 20 and 30 mg/kg perzinfotel IM produced sternal recovery times of 3:10 ± 2:39, 9:03 ± 3:51 and 16:33 ± 9:26 minutes respectively. The combination of 20 mg/kg perzinfotel IM and 0.2 mg/kg butorphanol IM resulted in a sternal recovery time of 13:03 ± 11:50 minutes, compared with 3:29 ± 2:24 minutes in dogs that received 0.2 mg/kg butorphanol. Apart from the recovery times after treatment with 10 mg/kg perzinfotel IM alone and 0.2 mg/kg butorphanol IM alone, all other recovery times were significantly longer compared to control (Table 5). No adverse reactions were observed during anesthesia or during recovery. The BIS values significantly increased with all doses of perzinfotel administered (Table 3). Also, when perzinfotel was given in combination with butorphanol BIS increased significantly, while administration of butorphanol alone had no effect on BIS. These BIS values were measured at MAC level.

15 CHAPTER 2

Table 3: MAC, HR and BIS; changes in these parameters relative to saline controls for six dogs anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel and or butorphanol. HR and BIS were measured at MAC level.

MAC Heart rate Bispectral Index 1 Treatment N Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline control3 18 1.14±0.11a ‐ 92.0±20.1b,c ‐ 73.1±8.6c ‐ 20 mg 12 0.74±0.10c,d ‐35% 91.1±16.8 b,c ‐1% 82.5±0.8a 13% perzinfotel/kg IV 20 mg 12 0.77±0.08c ‐32% 108.7±14.1a,b 18% 84.8±4.8a 14% perzinfotel/kg SQ 10 mg 12 0.78±0.11c ‐32% 105.8±26.8 15% 80.9±4.9a,b 11% perzinfotel/kg a,b IM 20 mg 12 0.72±0.13c,d ‐37% 108.9±18.5 18% 79.8±3.4a,b 9% perzinfotel/kg a,b IM 30 mg 12 0.64±0.11d ‐44% 117.9±17.7a 28% 83.6±4.9a 14% perzinfotel/kg IM 20 mg 12 0.47±0.05e ‐59% 83.5±11.7c ‐9% 85.6±7.4a 17% perzinfotel/kg IM + 0.2 mg butorphanol/kg IM 0.2 mg 6 0.97±0.12b ‐15% 74.7±16.3c ‐19% 74.8±5.1b,c 2% butorphanol/kg IM 1N = Number of measurements. 2Mean ± Standard Deviation. 3Two saline control measurements at the beginning and one at the end of the study for each dog. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05).

Heart rate decreased (non‐significantly) compared to no treatment (control) heart rate in anesthetized dogs after treatment with 20 mg/kg perzinfotel IM with 0.2 mg/kg butorphanol IM (9%) and with 0.2 mg/kg butorphanol IM alone (19%) (Table 3). Heart rate increased (≥ 15%) compared to the control heart rate of anesthetized dogs for all other doses of perzinfotel administered. Only treatment with 30 mg/kg perzinfotel significantly increased the

16 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

heart rate compared to the control heart rate of anesthetized dogs. These HR values were measured at MAC level.

Table 4: DAP, SAP and MAP; changes in these parameters relative to saline controls for six dogs anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel and or butorphanol. All these parameters were measured at MAC level.

Diastolic arterial blood Systolic arterial blood Mean arterial blood Treatment N1 pressure pressure pressure Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline control3 18 66.5±19.9b,c ‐ 115.0±23.9a,b ‐ 84.6±21.8a,b ‐ 20 mg 12 79.6±19.6a,b 20% 134.3±20.3a 17% 98.0±18.8a 16% perzinfotel/kg IV 20 mg 12 82.8±16.4a,b 25% 134.6±23.1a 17% 101.6±18.3a 21% perzinfotel/kg SQ 10 mg 12 79.1±17.5a,b 19% 127.5±22.3a 11% 98.1±16.7a 16% perzinfotel/kg IM 20 mg 12 81.1±15.9a,b 22% 130.3±19.8a 13% 99.1±17.4a 17% perzinfotel/kg IM 30 mg 12 85.2±12.0a 28% 134.2±16.3a 17% 101.2±13.2a 20% perzinfotel/kg IM 20 mg 12 65.4±12.4b,c ‐2% 123.8±11.9a,b 8% 84.3±11.7a,b 0% perzinfotel/kg IM + 0.2 mg butorphanol/kg IM 0.2 mg 6 52.8±13. ‐21% 100.5±16.1b ‐13% 69.0±14.0b ‐18% butorphanol/kg IM 1N= Number of measurements. 2Mean ± Standard Deviation. 3Two saline control measurements at the beginning and one at the end of the study for each dog. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05).

17 CHAPTER 2

Treatment with perzinfotel resulted in a significant increase of DAP at a dose of 30 mg/kg. Other increases of MAP, SAP and DAP (Table 4) during isoflurane anesthesia were not statistically significant. The combination of 20 mg/kg perzinfotel IM with 0.2 mg/kg butorphanol IM had little effect on blood pressure, while the administration of butorphanol alone decreased DAP, SAP and MAP by an average of 13‐21% (non‐significant). These blood pressure values were measured at MAC level.

Table 5: Time from extubation to sternal recumbency

Treatment Time (min:sec)

line control3 0:58 ± 0:53× 20 mg perzinfotel/kg IV 11:36 ± 5:48*,¤ 20 mg perzinfotel/kg SQ 11:53 ± 4:05*,¤ 10 mg perzinfotel/kg IM 3:10 ± 2:39× 20 mg perzinfotel/kg IM 9:03 ± 3:51* 30 mg perzinfotel/kg IM 16:33 ± 9:26*,¤ 20 mg perzinfotel/kg IM + 0.2 mg 13:03 ± 11:50*,¤ butorphanol/kg IM 0.2 mg butorphanol/kg IM 3:29 ± 2:24× *Significantly (P<0.05) different compared to control. ¤,×Significantly (P<0.05) different compared to other doses.

Discussion

These results support and extend previous studies investigating the anesthetic sparing effects of perzinfotel in dogs. All doses of perzinfotel investigated decreased isoflurane MAC values regardless of route (IV, IM, SQ) of administration or combination with the opioid agonist butorphanol. Escalating IM doses of perzinfotel demonstrated mild dose dependent reductions in isoflurane MAC values which were augmented when perzinfotel was combined with butorphanol. The 0.2 mg/kg IM dose of butorphanol produced a smaller, but also statistically significant reduction in isoflurane MAC. Collectively, these studies provide evidence that perzinfotel decreases inhalant anesthetic requirements and does not negatively impact the anesthetic sparing effects of butorphanol in dogs.

18 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

The MAC of an inhaled anesthetic required to prevent gross purposeful movement in 50% of the subjects in response to a supramaximal noxious stimulus is used as a clinical index of drug potency and a guide to selection of the inhalant anesthetic concentration required for general anesthesia15. The repeatability and stability over time of the control MAC values reported in this study indicate that the measured decrease in isoflurane MAC values is scientifically valid. The decrease in MAC values after pretreatment with perzinfotel was greater than the relatively small decrease in MAC after pretreatment with butorphanol. Furthermore, isoflurane MAC reductions displayed mild dose dependency following IM administration of perzinfotel. The decrease in isoflurane MAC values are associated with statistically significant increases in BIS values (increase in consciousness). These increases in BIS suggest a reduction in CNS and anesthetic associated depression. The lack of change in BIS and hemodynamic values, among all treatment groups, when isoflurane concentration was held constant at 1.5% suggests that changes in the isoflurane concentration was the main factor responsible for the changes observed. Butorphanol produced comparatively minimal effects on isoflurane MAC and BIS values, although heart rate and arterial blood pressure tended to be decreased from control values. The combination of perzinfotel and butorphanol produced the greatest decreases in isoflurane MAC (59%). These results support previous studies suggesting that butorphanol produces minimal inhalant anesthetic sparing effects in isoflurane anesthetized dogs32. We did not perform the types of experiments required to determine if this drug interaction relative to isoflurane MAC reduction was additive or synergistic but did not observe an antagonistic effect30. The combination of perzinfotel and butorphanol decreased HR in anesthetized dogs compared to control (‐9%), but increased HR compared to butorphanol alone (+12%) although not significantly. The DAP, MAP and SAP in these anesthetized dogs were increased compared to untreated controls for all five doses of perzinfotel that were administered. This increase was only statistically significant for DAP for the highest dose of perzinfotel (30 mg/kg) compared to the untreated controls. The increases were statistically significant for all doses of perzinfotel administered alone compared to the administration of butorphanol alone. The combination of perzinfotel and butorphanol increased the DAP, MAP and SAP compared to butorphanol alone, an effect that was most likely due to the greater decrease in isoflurane concentration; this increase, however, was not statistically significant and the values were similar to control. Given its anesthetic sparing effect, perzinfotel pre‐treatment tended to limit the decrease in arterial pressure normally associated with isoflurane anesthesia at MAC level. In contrast, butorphanol alone tended to result in a further lowering of blood pressure (MAP) by about 18% relative to control (non‐significant). Butorphanols’ effects upon heart rate and blood pressure when administered in combination with perzinfotel or alone suggest that it possesses mild cardiovascular depressant activity in isoflurane anesthetized dogs33. Further studies are required, however, to determine the dose dependent cardiovascular

19 CHAPTER 2

effects of perzinfotel alone and in combination with other opioid agonists in isoflurane anesthetized dogs31. Recovery was longer when dogs were pretreated with perzinfotel. These data suggest that perzinfotel may produce some immobilizing activity when combined with inhalant anesthetics and supports studies suggesting that NMDA receptor inhibition contributes part of the immobilizing activity of aromatic volatile anesthetics34. These longer recovery times would be of little or no clinical significance in a clinical setting (e.g. 9:03 ± 3:51 min at a dose of 20 mg/kg IM). Some of the results previously discussed were not statistically significant compared to controls (see Tables 3,4,5). The authors are of the opinion that as increases in Heart Rate, DAP, MAP and SAP were a general and consistent finding throughout the study and were significant for the highest dose of perzinfotel used (Heart Rate and DAP), it was of clinical significance to mention and discuss these data. Results that are statistically significant are not necessarily biologically or clinically important (e.g. recovery times) and vice versa35,36. In conclusion, pretreatment with perzinfotel (IM, IV, SQ) produced significant, dose dependant decreases in isoflurane MAC values which were associated with improvement in BIS and hemodynamic values in sexually intact male and female dogs. The isoflurane MAC reduction was augmented by the concomitant use of butorphanol and did not produce any adverse effects. The SQ, IM or IV administration of perzinfotel as a pre‐anesthetic treatment prior to isoflurane anesthesia improves anesthetic safety by reducing inhalant anesthetic requirements.

Footnotes: a Propo Flo, Abbott Laboratories, North Chigago, ILL b IsoFlo. Abbott Laboratories, North Chicago, ILL c Isotec 3, Ohmeda, Madison, Wis d LEI Medical, Boring, Ore e Veterinary Anesthesia Ventilateor Modle 2KIE, Hallowell Engineering and Manufacturing Corp, Pittsfield, Mass f DSI Physio Tel D70‐PCT transmitter, Data Sciences International, Saint Paul, Minn g Passport 2, Datascope, Montvale, NJ h T/Pump, Gaymar Industries Inc., Orchard Park, NY i Genuine grass platinum subdermal needle electrodes, Astro‐Med, Inc., West

Warwick, RI j Grass SD9 Stimulator, Grass Medical Instruments, Quincy, MA k A‐1000 EEG Monitor, Aspect Medical Systems, Inc., Newton, MA l SAS version 8.2, SAS Institute, Inc. (Cary, NC)

20 EFFECTS OF PERZINFOTEL ON THE MINIMUM ALVEOLAR CONCENTRATION OF ISOFLURANE IN DOGS

Acknowledgements

The authors would like to thank Dr. Mark Eppler PhD for scientific support and Deborah Amodie BS for statistical analysis. This study was funded by Fort Dodge Animal Health.

References

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16. Solano AM, Pyendop BH, Boscan PL et al. Effect of intravenous administration of ketamine on the minimum alveolar concentration of isoflurane in anesthetized dogs. Am J Vet Res 2006; 67:21‐25. 17. Muir WW III, Wiese AJ, March PA. Effects of morphine, lidocaine, ketamine, and morphine‐ lidocaine‐ketamine drug combination on minimum alveolar concentration in dogs anesthetized with isoflurane. Am J Vet Res 2003; 64: 1155 – 1160. 18. Kushiro T, Wiese AJ, Eppler MC et al. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2007; 68: 12: 1294 – 1299. 19. Monteiro ER, Junior AR, Assis HM et al. Comparative study on the sedative effects of morphine, methadone, butorphanol or , in combination with acepromazine, in dogs. Vet Anaesth Analg. 2009; 36(1):25‐33. 20. Sano T, Nishimura R, Mochizuki M, Sasaki N. Effects of midazolam‐butorphanol, acepromazine‐ butorphanol and medetomidine on an induction dose of propofol and their compatibility in dogs. J Vet Med Sci. 2003 Oct;65(10):1141‐3. 21. Commiskey S, Fan LW, Ho IK, Rockhold RW. Butorphanol: effects of a prototypical agonist‐ antagonist analgesic on kappa‐opioid receptors. J Pharmacol Sci. 2005; 98(2):109‐16. 22. Yamashita K, Okano Y, Yamashita M et al. Effects of carprofen and meloxicam with or without butorphanol on the minimum alveolar concentration of in dogs. J Vet Med Sci. 2008; 70(1):29‐35. 23. Ko JC, Lange DN, Mandsager RE et al. Effects of butorphanol and carprofen on the minimal alveolar concentration of isoflurane in dogs. J Am Vet Med Assoc. 2000; 217(7):1025‐8. 24. Grimm KA, Tranquilli WJ, Thurmon JC, Benson GJ. Duration of nonresponse to noxious stimulation after intramuscular administration of butorphanol, medetomidine, or a butorphanol‐ medetomidine combination during isoflurane administration in dogs. Am J Vet Res. 2000; 61(1):42‐7. 25. Quandt JE, Raffe MR, Robinson EP. Butorphanol does not reduce the minimum alveolar concentration of in dogs. Vet Surg. 1994; 23(2):156‐9. 26. Murphy MR, Hug CC Jr. The sparing effect of morphine, butorphanol, and . Anesthesiology. 1982; 57(6):489‐92 27. Shafer SL, Hendrickx JF, Flood P, Sonner J, Eger EI 2nd. Additivity versus synergy: a theoretical analysis of implications for anesthetic mechanisms. Anesth Analg. 2008; 107(2):507‐24. 28. Kissin I. Depth of anesthesia and bispectral index monitoring. Anesth Analg 2000; 90: 1114 – 1117. 29. Johansen JW. Update on Bispectral Index monitoring. Best Practice & Research Clinical Anaesthesiology. 2006; 20: 81‐99. 30. March PA, Muir WW III. Bispectral analysis of the electroencephalogram: a review of its development and use in anesthesia. Vet Anesth Analg 2005; 32: 241 – 255. 31. Ueyama Y, Lerche P, Eppler M, Muir WWIII. Effects of Perzinfotel, Fentanyl, and a Combination of Perzinfotel with Fentanyl on the Minimum Alveolar Concentration of Isoflurane in Dogs. (accepted: AJVR‐08‐10‐0355.R2) 32. Ko JC, Lange DN, Mandsager RE, Payton ME et al. Effects of butorphanol and carprofen on the minimal alveolar concentration of isoflurane in dogs. J Am Vet Med Assoc. 2000; 217(7):1025‐8. 33. Tyner CL, Greene SA, Hartsfield SM. Cardiovascular effects of butorphanol administration in isoflurane‐O2 anesthetized healthy dogs. Am J Vet Res. 1989; 50(8):1340‐2. 34. Sewell JC, Raines DE, Eger EI 2nd et al. A comparison of the molecular bases for N‐methyl‐D‐ aspartate‐receptor inhibition versus immobilizing activities of volatile aromatic anesthetics. Anesth Analg. 2009; 108(1):168 – 75. 35. Petrie A, Watson P. The distinction between statistical and biological difference. In: Statistics for Veterinary and Animal Science. 1rst ed. Oxford: Blackwell Science Ltd, 2003; 74.

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36. Thrusfield M. Statistical versus clinical (biological) significance. In: Veterinary Epidemiology.2nd ed. Oxford: Blackwell Science Ltd, 2001; 202.

23

CHAPTER 3

Effects of perzinfotel and PLA‐ 695 on kinetic gait and subjective lameness scores in a sodium urate‐induced synovitis model in dogs

Steven Budsberg DVM,MS, Bryan Torres DVM, Raphael Zwijnenberg DVM, MVSc, James Clark PhD, Mark Eppler PhD, Curtis Cathcart DVM, Lisa Reynolds BS, Sami Al‐Nadaf BS.

From the Department of Small Animal Medicine and Surgery (Budsberg, Torres, Cathcart, Reynolds, Al‐Nadaf), College of Veterinary Medicine, University of Georgia, Athens, GA 30602. From Pfizer (Zwijnenberg, Eppler), Kalamazoo, MI 49001. From Pfizer Inflammation and Immunology (Clark), Cambridge, MA 02140.

Am J Vet Research, accepted for publication March 2010

CHAPTER 3

Abstract

Objective: To investigate the ability of perzinfotel (a NMDA receptor antagonist), and a proprietary PLA2 inhibitor (PLA‐695) to attenuate lameness in dogs created by an induced urate synovitis model.

Animals: Eight adult purpose‐bred mixed‐breed dogs were used. Procedures: A blinded four way crossover study was performed. Treatment groups were randomly assigned between perzinfotel (10mg/kg), PLA‐695 (10mg/kg), carprofen (4.4 mg/kg‐ positive control); and no treatment (negative control). Synovitis was induced on the fourth day of treatment by intra‐articular injection of sodium urate (SU) one hour prior to the last treatment dose. Measurements were taken at baseline and 2, 4, 6, 8, 12, and 25 hours after SU injection. Ground reaction forces and clinical lameness evaluations were collected at each timepoint. All data were analyzed with a repeated measures ANOVA. All hypotheses tested were 2‐sided with P<0.05.

Results: Compared to baseline values, peak vertical force (PVF) and vertical impulse (VI) for both the negative control and perzinfotel groups had significantly lower forces at 2 and 4 hours. No differences from baseline values for PVF and VI were seen in the PLA‐695 or carprofen groups at any measurement time points. Between group comparisons found significantly higher PVF and VI values in the carprofen group vs. the no treatment and perzinfotel groups at 2 and 4 hours. The PLA‐695 group had higher impulse values vs. no treatment at 2 hours.

Conclusions and Clinical Relevance: At the dose tested, perzinfotel did not significantly alter the SU induced lameness, while PLA‐695 did attenuate the lameness but not as completely as carprofen.

26 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

Multimodal pain control has been used in the management of both surgical and chronic pain syndromes.1‐3 Several approaches to multimodal pain control have been proposed including attempts to determine and suppress the inflammatory cascade of the pain profile, suppression of afferent pain pathways, and neuronal transmission inhibition.4 Neuronal transmission of nociceptive stimuli has been a focus of much recent research. Classes of drugs studied for potential benefit as centrally acting analgesics include , norepinephrine and serotonin reuptake inhibitors,5 neurokinin‐1 receptor antagonists,6,7 vanilloid receptor antagonists,8,9 cyclooxygenase (COX) inhibitors acting on constitutive spinal COX,1,10 N‐ 2,11‐17 methyl‐D‐aspartate (NMDA) receptor antagonists, and Phospholipase A2 (PLA2) inhibitors.18,19 Several NMDA receptor antagonists have been studied in pain control.3,11,12 The NMDA receptor itself is made up of three known subtypes: NR1, NR2 (A,B,C, and D), and NR3 (A and B).3,20 The NR2A and NR2B containing receptors have been shown to be associated with high conductance post‐synaptic depolarization, and the NR2B more specifically with less psychomimetic effects.20 Although many NMDA receptor antagonists selective for the NR2B site have been developed, perzinfotel, with a high affinity and selectivity to the glutamate site on the NR2A subunit receptors, has shown promise in early studies with rats, showing decreased hypersensitivity and decreased allodynia along with no extrapyramidal signs.11 Both NR2A and NR2B mRNA have shown up‐regulation of gene expression after inflammation supporting their role in central sensitization.14 To date, published studies in dogs have demonstrated that perzinfotel has a dose dependent lowering of the minimum alveolar concentration of isoflurane,2 and that a combination of perzinfotel with fentanyl produces a lower isoflurane MAC then either drug alone.15 21 PLA2 is an enzyme superfamily containing 15 known groups, and has long been known to act on phospholipid membranes, releasing fatty acid chains leading to arachidonic acid (AA)22 and the subsequent eicosanoids involved in corporeal homeostasis and the 23 inflammatory state. It is also known that production of AA substrate by PLA2 is the rate limiting step of eicosanoid formation24 and so has been a target for pharmaceutical development looking to inhibit this step with the eventual goal of providing analgesia through modulation of the inflammatory process. In addition to inflammation modulation, a cytosolic 25 presynaptic role of PLA2 in the transmission of nociceptive stimuli has been demonstrated. The purpose of the study reported here was to examine the ability of perzinfotel (a a NMDA receptor antagonist), and a proprietary PLA2 inhibitor (PLA‐695) to attenuate lameness in dogs created by an induced stifle urate synovitis model. The effectiveness of perzinfotel and PLA‐695 were evaluated using clinical and ground reaction force assessments of lameness in comparison with baseline, negative control and a positive control treatment.

27 CHAPTER 3

Materials and Methods

Animals – Eight adult purpose‐bred mixed‐breed dogs (4 males and 4 females) were used in the study. All dogs underwent physical exam and orthopedic exam as well as screening radiographs of both hips, stifles and elbows. Dogs were excluded from the study if there was evidence of any lameness or health concerns. All dogs were housed in the institution’s climate‐controlled animal housing facility and received routine immunizations and anthelmintics, were fed a commercially available maintenance diet and offered water ad libitum. The study was approved by the University of Georgia Institutional Animal Care and Use Committee (AUP # A2009‐10085).

Study Design – A blinded four way crossover study was performed. The different treatment groups for each dog that were randomly assigned included perzinfotel (10 mg/kg intramuscularly), PLA‐695 (10 mg/kg orally), Carprofen (4.4 mg/kg SQ; positive control), and no treatment (negative control). Treatments were given once daily for four days. Any adverse event observed such as ocular discharge, nasal discharge, coughing, emesis, diarrhea, sedation, depression, recumbency, reluctance to move, ataxia, salivation, rapid or labored breathing, muscle tremors, or convulsions was recorded. Synovitis was induced on the fourth day of treatment by intra‐articular injection of 1.0 ml of a 5.0 mg/ml solution of sodium urate (SU) prepared as previously described.12 Injections were administered in the designated stifle one hour before the last treatment dose. A single blinded observer (BT) evaluated ground reaction force measurements and performed a clinical lameness evaluation at each time point. Measurements were taken prior to SU injection (baseline) and at 2, 4, 6, 8, 12, and 25 hours after SU injection. A minimum of 21 days was observed between treatment periods (21 days between SU injections, with treatments starting three days prior).

Data Collection – Clinical lameness scoring was performed and recorded for each dog at each time point using a subjective lameness score as previously described.26 Ground reaction force (GRF) data was collected with two force platesb mounted in series, a dedicated computer, and software.c From the GRF data, peak vertical force (PVF) and vertical impulse (VI), peak craniocaudal braking force (PCBF) and craniocaudal braking impulse (CBI), and the peak craniocaudal propulsion force (PCPF), and craniocaudal propulsion impulse (CPI) were determined. All trials were performed at a trotting speed of 1.70 to 2.10 m/s and an acceleration of +/‐ 0.50 m/s/s by one of two experienced handlers (LR and SA). Each dog was trotted by the same handler within each period of the experiment. Trials were only accepted if there was a single rear limb footfall on each force platform while maintaining a standard trotting gait with no extraneous movements. At each time point for each dog, six observations were recorded for both rear legs (healthy and SU injected stifles). Subjective clinical lameness evaluation (Appendix 1)6 and acceptance of GRF data for each dog was performed by a single individual blinded to treatment assignment.

28 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

Statistical Analysis – Repeated‐measures ANOVAs were used to evaluate the difference of treatment (perzinfotel, PLA‐695, carprofen, and no treatment) on PVF, VI, PCBF, CBI, PCPF, CPI, and subjective lameness scores between groups and over time. The full model included factors for treatment, time, and a treatment by time interaction term. Multiple comparisons were adjusted for using Tukey’s test. An unstructured covariance model was used in all repeated measures models. All hypothesis tests were 2‐sided and the significance level was α = 0.05. The repeated measures analysis was performed using PROC MIXED in SAS.d

Results

Lameness Scores – The no treatment group had significantly higher scores at 2, 4, 6, and 8 hours when compared to baseline values (Figure 1).

Figure 1: Cumulative Lameness Scores for all groups.

N = 8, Mean ± SE Key for Figure 1 ± No treatment significantly different from baseline **PLA‐695 significantly different from baseline + Perzinfotel significantly different from baseline # Carprofen significantly different from no treatment ## Carprofen significantly different from perzinfotel Ф PLA‐695 significantly different from no treatment

The perzinfotel group had significantly higher scores at 2, 4, and 6 hours compared to baseline, while the PLA‐ 695 group had significantly higher scores at 2 and 4 hours compared

29 CHAPTER 3

to baseline. The carprofen group showed no difference in lameness score compared to baseline at any time point. Between group comparisons found the PLA‐ 695 group having significantly lower scores than the no treatment group at 2 hours. The carprofen group had significantly lower lameness scores than the no treatment group at 2, 4 and 6 hours as well as significantly lower scores at 2, 4, and 6 hours when compared to the perzinfotel group. Peak Vertical Force (PVF) – Compared to baseline values, both the no treatment and perzinfotel groups had significantly lower forces at 2 and 4 hours (Figure 2).

Figure 2: Peak vertical force data (as a percent body weight) for all groups.

N = 8, Mean ± SE See Figure 1 Key

No differences from baseline values were seen in the PLA‐ 695 or carprofen groups at any measurement time points. Between group comparisons found significantly higher forces in the carprofen group vs. the no treatment and perzinfotel groups at 2 and 4 hours. The PLA‐ 695 group had higher forces vs. no treatment group at 2 hours. Vertical Impulse (VI) ‐ Compared to baseline values, both the no treatment and perzinfotel groups had significantly lower impulse values at 2 and 4 hours (Figure 3). The PLA ‐ 695 group had significantly lower impulse values compared to baseline at 2 hours. No differences from baseline values were seen in the carprofen group at any measurement time points. Between group comparisons found significantly higher impulse values in the carprofen group vs. the no treatment and perzinfotel groups at 2 and 4 hours. The PLA ‐695 group had higher impulse values vs. no treatment at 2 hours. Peak Craniocaudal Braking Force (PCBF) – Compared to baseline, both the no treatment and perzinfotel groups had significantly lower forces at 2 and 4 hours (Figure 4). No differences from baseline forces were seen in either the PLA‐ 695 or carprofen group at any

30 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

measurement time points. Between group comparisons found significantly higher forces in the carprofen group at 2 and 4 hours compared to no treatment.

Figure 3: Vertical impulse force data (as a percent body weight/time) for all groups.

N = 8, Mean ± SE See Figure 1 Key

Figure 4: Craniocaudal peak braking force (as a percent body weight) for all groups.

N = 8, Mean ± SE See Figure 1 Key

Craniocaudal Braking Impulse (CBI) – The only change from baseline was a significant decrease in impulse value in the no treatment group at 2 hours (Figure 5). The only between group difference was a significant increase in the impulse values for the carprofen group compared to the no treatment group at 4 hours.

31 CHAPTER 3

Figure 5: Craniocaudal peak braking impulse (as a percent body weight/time) for all groups.

N = 8, Mean ± SE See Figure 1 Key

Peak Craniocaudal Propulsion Force (PCPF) – Compared to baseline, the perzinfotel group had significantly lower forces at 2 and 4 hours (Figure 6). Between group comparisons showed significantly higher forces for the PLA‐ 695 group vs. both no treatment and perzinfotel at 4 hours. The carprofen group had significantly higher forces at 2 hours compared to no treatment.

Figure 6: Craniocaudal peak propulsion force (as a percent body weight) for all groups.

N = 8, Mean ± SE See Figure 1 Key ◊ PLA695 significantly different from perzinfotel

32 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

Craniocaudal Propulsion Impulse (CPI) – Compared to baseline, the no treatment group had significantly lower impulse values at 2 hours, while perzinfotel had significantly lower impulse values at 4 hours (Figure 7). Between group comparisons showed significantly increased impulse values for both PLA ‐695 and carprofen vs. no treatment at 2 hours.

Figure 7: Craniocaudal peak propulsion impulse (as a percent body weight/time) for all groups.

N = 8, Mean ± SE See Figure 1 Key

Discussion

The current SU model induced a consistent mild to moderate lameness when evaluated by the subjective lameness system. With this subjective instrument, the lameness was statistically evident for 8 hours when compared to baseline scores. Interestingly, when examining the same dogs with objective vertical ground reaction force measurements, a consistent lameness was only documentable for the first 4 hours. Thus the model proved reliable for detecting changes in lameness for a relatively short period of time depending on the measurement used. Both evaluation methodologies found that dogs were beginning to return to baseline levels within 6 hours and were completely back to baseline levels by 12 hours. The urate dose used in the current study produced a much less severe lameness than higher urate doses previously described.6,26,27 These data show that by titrating the amount of urate given, one can vary the severity and duration of the lameness created. Perzinfotel displayed no significant attenuation of limb dysfunction caused by the sodium urate induced stifle synovitis. The differences noted by both subjective and objective evaluation methodologies between perzinfotel and the negative control (no treatment) were subtle at best. In view of previous clinical evidence indicating analgesic activity of perzinfotel,2,11,15 the lack of effect in this study is a bit surprising. However the lack of efficacy

33 CHAPTER 3

of perzinfotel in this model may be explained by several mechanisms. First, this failure may simply be a matter of a dose dependent response and the dose given in this study was insufficient to attenuate lameness in this model. Secondly, in the present study, perzinfotel was administered to the dogs once daily with three treatments prior, and the final dose one hour after urate induction. Thus it is not known whether the once daily dosing had any pre‐ emptive effect on central sensitization in these dogs. It is possible that the one hour lag in giving perzinfotel after urate induction allowed sufficient time for central sensitization to occur, thereby decreasing potential effect of the perzinfotel. In a previous study, epidural ketamine, a non‐selective NMDA receptor inhibitor, had no effect when given 12 hours after urate induced synovitis, but had improvement of ground reaction forces at two hours, if given at the same time.12 Timing of perzinfotel relative to noxious stimuli may also be important to achieve its desired effect of decreased hypersensitivity. Lack of effect of the perzinfotel in this model may also be due to its selectivity for the glutamate site of the NR2A subunit.11 It has been demonstrated that NR2A containing NMDA receptors are not present on the presynaptic primary afferent fibers in the dorsal root ganglion of the rat.16,20 Substance P, prostaglandins, adenosine, and glycine, as well as glutamate (which can enhance its own release) can be released from the presynaptic afferent fibers, and act on presynaptic and postsynaptic receptors.3,13,16 Stimulation of presynaptic NR2B containing receptors can facilitate and prolong transmission of nociceptive messages.16 It has also been demonstrated that NR2A knockout mice fail to show changes in acute and chronic pain related behaviors.16 Substance P and glutamate release cause influx of Ca2+ and Na+ with resultant activation of Protein Kinase C,3 driving tyrosine phosphorylation of the NMDA receptors.20 This causes release of the Mg2+, acting as a block from the NMDA receptors, decreases resting membrane potential, and allows for prolonged channel opening time.20 The end result is increased postsynaptic activity from the multiplicity of neurotransmitters in the dorsal root ganglion synapses. These processes may have allowed the perzinfotel treated dogs in this study to continue to exhibit pain and lameness. Finally, another possible reason is that like all NMDA antagonists, perzinfotel has not displayed antinociceptive effects, but rather has prevented hypersensitivity as in previous studies.11 Specifically, NR2A seems to play a larger role relative to NR2B in inflammatory states with up‐regulation of the NR2A subunits in areas of the brain and spinal cord as a result of peripheral inflammatory stimulus.11 Spinal NR2A subunits have been decreased in models of nerve pain.11 Thus, the present study may emphasize the importance of pre‐emptive treatment with NMDA antagonists for successful attenuation of inflammatory based pain syndromes. Perzinfotel may play an important role in analgesia when used synergistically with opioids or nonsteroidal anti‐inflammatory agents.3,15 PLA‐695 attenuated the lameness at both time points where the negative control values were different than baseline. Interestingly at no time point in the study did either the subjective or objective measurements note a difference between the PLA‐695 and the positive control group receiving carprofen. Almost all the different measurements placed the

34 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

PLA‐695 results between perzinfotel and carprofen. However PLA‐695’s lameness attenuation was not as complete as carprofen. The effectiveness of PLA‐695 may be related to its inhibition of AA metabolism to its subsequent eicosanoids, and resultant anti‐inflammatory effects. PLA2, as a superfamily, have been found to be involved in many roles including antimicrobial activity, bone formation, apoptosis, insulin secretion, sperm development, Wallerian degeneration, axon regeneration, as a marker of coronary disease in people, and as 21,28 an anti‐inflammatory. Of the four main types of PLA2 (secreted, sPLA2; cytosolic, cPLA2; calcium independent iPLA2; and platelet activating factor acetyl hydrolase / oxidized lipid protein associated, LpPLA2) it has been shown that cPLA2, and in particular group IVA is a central enzyme mediating generation of eicosanoids and subsequent inflammatory 21 processes. In addition cPLA2 has shown a role in neurotransmitter release by coupling with a multitude of receptors in the brain such as glutamate, dopamine, and serotonin amongst 18 25 others. A constitutive spinal PLA2 has been demonstrated in rats and monkeys. Although many cPLA2 inhibitors demonstrating enzymatic activity have been studied, many have no in vivo effect.19 It appears that PLA‐695 performs comparably to the positive control carprofen in the SU induced synovitis model of inflammatory pain.

PLA2 antagonists have shown dose dependent association with the outcome measures of 19 antihypersensitivity effects and PGE2 production. As clinical trials explore the analgesic effects of these compounds and effective PLA2 antagonists are found, appropriate dosing regimens and safety margins will need to be established along with any drug interactions. To date there have been no listed adverse affects of these compounds, although there is a report of a human deficient in cPLA (2‐alpha) that developed small intestinal ulceration and platelet 29 dysfunction. One interesting note is that opioids stimulate PLA2 in presynaptic afferent fibers leading to metabolites of 12‐lipoxygenase.1 These metabolites enhance the activity of voltage dependent K+ channels inhibiting GABAergic neurotransmission.1 Use of COX‐1, COX‐ 2, and specific 5‐LOX inhibitors shunt more metabolite through to the 12‐LOX pathway 30 synergistically enhancing the µ‐opioid based GABAergic release. PLA2 antagonists have been 30 shown to block this µ‐opioid effect. Concurrent use of PLA2 antagonists with µ‐opioids in a clinical setting may be contraindicated but as an analgesic on its own merit, it shows promise.

Footnotes a PLA‐695, Fort Dodge Animal Health, Princeton, NJ. b Model OR‐6‐6, Advanced Mechanical Technology Inc, Newton, Mass. c Acquire, version 7.3, Sharon Software, East Lansing, Mich. d SAS V 9.2, Cary, NC.

35 CHAPTER 3

References

1. Christie MJ, Connor M, Vaughan CW, et al. Cellular actions of opioids and other analgesics: implications for synergism in pain relief. Clin Exp Pharmacol Physiol 2000;27:520‐523. 2. Kushiro T, Wiese AJ, Eppler MC, et al. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2007;68:1294‐1299. 3. Pozzi A, Muir WW, Traverso F. Prevention of central sensitization and pain by N‐methyl‐D‐ aspartate receptor antagonists. J Am Vet Med Assoc 2006;228:53‐60. 4. Lamont LA. Multimodal pain management in veterinary medicine: the physiologic basis of pharmacologic therapies. Vet Clin North Am Small Anim Pract 2008;38:1173‐1186, v. 5. Jones CK, Peters SC, Shannon HE. Efficacy of duloxetine, a potent and balanced serotonergic and noradrenergic reuptake inhibitor, in inflammatory and acute pain models in rodents. J Pharmacol Exp Ther 2005;312:726‐732. 6. Punke JP, Speas AL, Reynolds LR, et al. Kinetic gait and subjective analysis of the effects of a tachykinin receptor antagonist in dogs with sodium urate‐induced synovitis. Am J Vet Res 2007;68:704‐708. 7. Hong SK, Han JS, Min SS, et al. Local neurokinin‐1 receptor in the knee joint contributes to the induction, but not maintenance, of arthritic pain in the rat. Neurosci Lett 2002;322:21‐24. 8. Cui M, Honore P, Zhong C, et al. TRPV1 receptors in the CNS play a key role in broad‐spectrum analgesia of TRPV1 antagonists. J Neurosci 2006;26:9385‐9393. 9. Liu DL, Wang WT, Xing JL, et al. Research progress in transient receptor potential vanilloid 1 of sensory nervous system. Neurosci Bull 2009;25:221‐227. 10. McCrory CR, Lindahl SG. Cyclooxygenase inhibition for postoperative analgesia. Anesth Analg 2002;95:169‐176. 11. Brandt MR, Cummons TA, Potestio L, et al. Effects of the N‐methyl‐D‐aspartate receptor antagonist perzinfotel [EAA‐090; [2‐(8,9‐dioxo‐2,6‐diazabicyclo[5.2.0]non‐1(7)‐en‐2‐yl)‐ ethyl]phosphonic acid] on chemically induced thermal hypersensitivity. J Pharmacol Exp Ther 2005;313:1379‐1386. 12. Hamilton SM, Johnston SA, Broadstone RV. Evaluation of analgesia provided by the administration of epidural ketamine in dogs with a chemically induced synovitis. Vet Anaesth Analg 2005;32:30‐39. 13. Liu H, Mantyh PW, Basbaum AI. NMDA‐receptor regulation of substance P release from primary afferent nociceptors. Nature 1997;386:721‐724. 14. Miki K, Zhou QQ, Guo W, et al. Changes in gene expression and neuronal phenotype in brain stem pain modulatory circuitry after inflammation. J Neurophysiol 2002;87:750‐760. 15. Ueyama Y, Lerche P, Eppler CM, et al. Effects of perzinfotel, fentanyl, and a perzinfotel:fentanyl combination on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2009;70:1459‐1464. 16. Marvizon JC, McRoberts JA, Ennes HS, et al. Two N‐methyl‐D‐aspartate receptors in rat dorsal root ganglia with different subunit composition and localization. J Comp Neurol 2002;446:325‐ 341. 17. Kudoh A, Takahira Y, Katagai H, et al. Small‐dose ketamine improves the postoperative state of depressed patients. Anesth Analg 2002;95:114‐118, table of contents. 18. Farooqui AA, Ong WY, Horrocks LA. Inhibitors of brain phospholipase A2 activity: their neuropharmacological effects and therapeutic importance for the treatment of neurologic disorders. Pharmacol Rev 2006;58:591‐620.

36 EFFECTS OF PERZINFOTEL AND PLA‐ 695 ON KINETIC GAIT AND SUBJECTIVE LAMENESS SCORES

19. Yaksh TL, Kokotos G, Svensson CI, et al. Systemic and intrathecal effects of a novel series of phospholipase A2 inhibitors on hyperalgesia and spinal prostaglandin E2 release. J Pharmacol Exp Ther 2006;316:466‐475. 20. Petrenko AB, Yamakura T, Baba H, et al. The role of N‐methyl‐D‐aspartate (NMDA) receptors in pain: a review. Anesth Analg 2003;97:1108‐1116. 21. Burke JE, Dennis EA. Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res 2009;50 Suppl:S237‐242. 22. Balsinde J, Winstead MV, Dennis EA. Phospholipase A(2) regulation of arachidonic acid mobilization. FEBS Lett 2002;531:2‐6. 23. Bergh MS, Budsberg SC. The coxib NSAIDs: potential clinical and pharmacologic importance in veterinary medicine. J Vet Intern Med 2005;19:633‐643. 24. Fitzpatrick FA SR. Regulated formation of eicosanoids. The Journal of Clinical Investigation 2001;107:1347‐1351. 25. Svensson CI, Yaksh TL. The spinal phospholipase‐cyclooxygenase‐prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol 2002;42:553‐583. 26. Cross AR, Budsberg SC, Keefe TJ. Kinetic gait analysis assessment of meloxicam efficacy in a sodium urate‐induced synovitis model in dogs. Am J Vet Res 1997;58:626‐631. 27. Millis DL, Weigel JP, Moyers T, et al. Effect of deracoxib, a new COX‐2 inhibitor, on the prevention of lameness induced by chemical synovitis in dogs. Vet Ther 2002;3:453‐464. 28. Farooqui AA, Horrocks LA. Phospholipase A2‐generated lipid mediators in the brain: the good, the bad, and the ugly. Neuroscientist 2006;12:245‐260. 29. Adler DH, Cogan JD, Phillips JA, 3rd, et al. Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction. J Clin Invest 2008;118:2121‐2131. 30. Vaughan CW, Ingram SL, Connor MA, et al. How opioids inhibit GABA‐mediated neurotransmission. Nature 1997;390:611‐614.

37 CHAPTER 3

Appendix 1 : Description of the scales used for the subjective clinical lameness evaluation of dogs with urate–induced synovitis of a stifle joint.

Stance 0 Normal stance 1 Slightly abnormal stance (favors limb but foot remains on floor) 2 Severely abnormal stance (holds limb off of floor) 3 Not able to stand

Lameness at walk 0 No lameness and full weight bearing observed on all strides; normal gait 1 Mild subtle lameness with partial weight bearing; dog may bear full weight on some strides and not others 2 Obvious lameness with partial weight bearing; dog is clearly lame on all strides 3 Obvious lameness with intermittent weight bearing; dog non‐weight bearing on some strides, partial weight bearing on others. Includes dogs that “toe touch” on some strides 4 Full non‐weight bearing lameness; bears no weight on any strides

Lameness at a trot 0 No lameness and full weight bearing observed on all strides; normal gait 1 Mild subtle lameness with partial weight bearing; dog may bear full weight on some strides and not others 2 Obvious lameness with partial weight bearing; dog is clearly lame on all strides 3 Obvious lameness with intermittent weight bearing; dog non‐weight bearing on some strides, partial weight bearing on others. Includes dogs that “toe touch” on some strides 4 Full non‐weight bearing lameness; bears no weight on any strides

Pain upon manipulation of affected joint through its normal range of motion 0 No pain elicited on palpation or movement of affected joint 1 Mild pain elicited (turns head in recognition) on palpation or movement of affected joint 2 Moderate pain elicited (pulls limb away) on palpation or movement of affected joint 3 Severe pain elicited on palpation or movement of affected joint (vocalizes or becomes aggressive, or will not allow palpation or movement of affected joint)

Total Score: ______(Adapted from Punke JP, Speas AL, Reynolds LR, et al. Kinetic gait and subjective analysis of the effects of a tachykinin receptor antagonist in dogs with sodium urate‐induced synovitis. Am J Vet Res 2007;68:704‐708.)

38

CHAPTER 4

Effects of perzinfotel, butorphanol and a butorphanol‐ perzinfotel combination on the minimum alveolar concentration of isoflurane in cats

Raphael J Zwijnenberg DVM, MVPHMgt* Carlos L del Rio DVM, PhD** Robert A Pollet, PhD* William W Muir DVM, PhD**

*Fort Dodge Animal Health, PO Box 5366, Princeton, NJ 08543 **QTest Labs, PO Box 12381, Columbus, OH 43212

Am J Vet Research 2010; 71 (11): 1270‐1276 CHAPTER 4

Objective – To determine the effects of perzinfotel, butorphanol and their combination on the minimal alveolar concentration (MAC) of isoflurane in cats.

Animals ‐ Seven healthy sexually intact cats, 4 males and 3 females, 12 ‐ 17 months of age and weighing 2.8 – 4.6 kg.

Procedures – Perzinfotel, saline, butorphanol and a combination of perzinfotel and butorphanol were administered to 6 cats before anesthesia. Cats, previously implanted with a telemetry device, were anesthetized with isoflurane in oxygen in an induction chamber and then orotracheally intubated. Anesthesia was maintained with isoflurane in oxygen. Heart rate (HR), arterial blood pressure, bispectral index (BIS) and inspiration and expiration concentrations of isoflurane were continuously monitored. The isoflurane MAC was determined twice during anesthesia.

Results – IV, IM and SQ administration of perzinfotel (2.5 – 15 mg/kg) significantly decreased mean isoflurane MAC by 43.3 – 68.0%. BIS was significantly increased at doses of 2.5 to 15 mg/kg perzinfotel and after the combination of 0.2 mg/kg butorphanol and 5 mg/kg perzinfotel IM. HR tended to be lower (not significant) and blood pressure was higher (significant for 5 mg/kg IM, 10 mg/kg IV and 10 mg/kg SQ) compared to placebo.

Conclusions and Clinical Relevance ‐ Perzinfotel decreased isoflurane MAC and increased BIS (Bispectral Index) and blood pressure values in anesthetized cats. The administration of perzinfotel as pre‐anesthetic medication prior to isoflurane anesthesia should improve anesthetic safety by reducing inhalant anesthetic requirements and improving cardiovascular function during anesthesia.

40 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

N‐methyl‐D‐aspartate receptors (NMDARs) are a class of glutamate‐gated ion channels that regulate Na and Ca transmembrane flux. NMDARs have received considerable attention experimentally and clinically because of their crucial roles in excitatory synaptic transmission, plasticity, and prevention of neurodegeneration in the central nervous system (CNS)1,2. There is considerable evidence that pain caused by peripheral tissue or nerve injury involves NMDAR activation3. NMDARs have been identified on myelinated and unmyelinated axons in peripheral somatic tissues4,5,6. NMDARs are comprised of NR1 (eight splice variants) and NR2 (A, B, C, and D subtypes) subunits with further variation possibly provided by the recently discovered NR3 (A and B) subunits. These subunits represent a class of structurally different binding sites with different affinities for agonists and antagonists. Recent evidence suggests that activation of NR2B and NR3A subtypes play important roles in perception of pain and neuronal injury, respectively1. NMDARs contain a variety of sites at which endogenous ligands and subunit selective drugs modulate receptor activity. Local injections of glutamate or NMDA result in nociceptive behaviors in rats that can be attenuated by the peripheral administration of NMDAR antagonists7,8,9,10. NMDAR antagonists have demonstrated effective alleviation of pain in both animal models and clinical situations11,12, however, their use as analgesics may be limited by side effects such as memory impairment13, psychotomimetic effects14, ataxia and motor incoordination15. Antinociceptive selective antagonists of NR2B‐containing NMDARs (e.g. ifenprofil) have a lower side‐effect profile compared with some other NMDA receptor antagonists12. Perzinfotel (EAA‐090) is a potent NMDAR antagonist16. In vivo characterization showed that perzinfotel was 10 times more potent at blocking NR2A‐ versus NR2B‐ or NR2C‐ containing NMDARs, and protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate‐ and NMDA‐induced neurotoxicity17. A single bolus dose of perzinfotel administered IV following permanent occlusion of the middle cerebral artery in the rat, reduced infarct size by 57%18. When compared with uncompetitive channel blockers (e.g., memantin, dizocilpine, and ketamine), a NR2B selective antagonist (e.g., ifenprodil) and other glutamate antagonists (e.g., selfotel, CCP, CGP‐39653), perzinfotel had superior therapeutic ratios for effectiveness in treating pain versus adverse behavioral effects18. The minimum alveolar concentration (MAC) of an inhalant anesthetic is defined as the amount of inhaled anesthetic required to prevent gross purposeful movement to a noxious stimulus in 50% of the subjects19. The MAC is a measure of anesthetic potency and provides a guide to the concentration of inhaled anesthetic needed to induce unconsciousness and immobility. NMDAR antagonists may reduce the amount of inhaled anesthetic needed to maintain anesthesia (anesthetic sparing) in addition to producing analgesic effects. Currently available NMDARS like ketamine reduce the MAC of isoflurane needed to maintain dogs under anesthesia by up to 25%20,21. Recently, perzinfotel has been shown to have similar or greater anesthetic‐sparing effects in dogs22,23. There are no prior reports of the effects of perzinfotel in cats.

41 CHAPTER 4

Butorphanol, a synthetic morphinan derivative and mixed agonist‐antagonist opioid analgesic, is frequently administered alone or in conjunction with other sedatives as a preanesthetic medication prior to general anesthesia in cats24,25. Current opinion suggests that butorphanol acts as a partial mu (u) agonist and antagonist, pure kappa (κ) agonist and delta (δ) antagonist although species differences have been noted26. One study demonstrated a 19% reduction in isoflurane requirements in cats administered butorphanol27. Bispectral Index (BIS) processing is a proprietary method for analyzing the degree of sedation and hypnosis28,29. Bispectral analysis examines the harmonic and phase relation of EEG signals and quantifies the amount of synchronization in the EEG. The BIS is a numeric value derived from the EEG and provides a reasonably accurate index of anesthetic depth and the presence or absence of consciousness30. Changes in BIS values are used to indicate a return to consciousness during inhalant anesthesia and to help identify differences between drug induced analgesic and hypnotic effects. The purpose of these studies was to determine the anesthetic sparing effects of perzinfotel in cats administered by IM, IV or SQ routes as pre‐anesthetic medication. We also evaluated the effects of IM perzinfotel after the administration of butorphanol to isoflurane anesthetized cats. MAC and BIS values were recorded in addition to measurements of heart rate (HR), arterial blood pressure, and the time to sternal recumbency after anesthesia.

Materials and Methods

Animal care and instrumentation: The studies were approved by the Institutional Animal Care and Use Committee of the study facility. Seven healthy sexually intact Domestic Shorthair cats, 4 males and 3 females, 12 – 17 months of age and weighing 2.8 – 4.6 kg, were the subjects of these studies. Two weeks before beginning study 1 each cat had been surgically implanted with a telemetry device that permitted the simultaneous and continuous monitoring of respiration, ECG, arterial (femoral artery) blood pressure, and body temperature.

Experimental design: Study 1: Six cats were submitted to seven different treatments (Table 1). All treatments were separated by a minimum washout period of 7 days. The first treatment (saline) was performed in order to determine baseline MAC (MACo) and other parameters during isoflurane anesthesia. Subsequent treatments were done in a Latin square cross‐over design (Table 2). Perzinfotel or saline (control) were administered as a treatment 30 minutes before induction to isoflurane anesthesia. Isoflurane MAC was determined twice for each treatment at approximately 30 minutes after anesthesia onset and 2 hrs later (MAC1 and MAC2, respectively).

42 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

Table 1: Description of treatments study 1 Treatment Dosing Rate

MACo Determination of baseline MAC1 & MAC2 (saline), no perzinfotel A 5 mg/kg IM B 10 mg/kg IM C 15 mg/kg IM D 10 mg/kg IV E 10 mg/kg SQ F Saline control

Table 2: Experimental design study 1 Cat number 07FPK1 08AAH3 08QBO5 07AQI1 07KQA2 08QDO5

MACo MACo MACo MACo MACo MACo

A E F D C B B A E F D C C B A E F D D C B A E F

Treatment* E D C B A F F E D C B A * All treatments were administered at an interval of ≥ 7 days

Study 2: Six cats were studied to evaluate a lower dose of perzinfotel and the combination of perzinfotel with butorphanol. The first treatment (saline) allowed the determination of the baseline MAC (MAC0) and other parameters in all cats to evaluate any possible confounding effects (i.e., lessening of anesthetic requirements) resulting from habituation to the laboratory environment, the noxious stimulation protocol, and/or the repeated anesthesia (i.e., temporal factors). Each cat was subsequently administered two different treatments (Table 3). Treatments were separated by a minimum washout period of 7 days. All treatments were evaluated in the same sequence for each cat (Table 4). During subsequent treatments, perzinfotel or butorphanol were administered 30 minutes before induction to isoflurane anesthesia. Isoflurane MAC was determined twice during each treatment at approximately 30 minutes after anesthesia onset and 2 hrs later (MAC1 and MAC2, respectively). For the first treatment, perzinfotel was administered IM at 2.5 mg/kg, 30 minutes before induction of anesthesia. For the second treatment, butorphanol (0.2 mg/kg IM) was administered 30 minutes before induction of anesthesia. After determination of isoflurane MAC1, perzinfotel (5 mg/kg IM) was administered 30 minutes before determination of MAC2. Due to dysfunction of one of the telemeters, one of the original 6 cats was replaced for the second study.

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Table 3: Description of treatments study 2

Treatment Dosing Rate

MACo Determination of baseline MAC1 & MAC2 (saline), no perzinfotel A 2.5 mg/kg IM perzinfotel B(1+2) 0.2 mg/kg IM butorphanol for MAC1, + 5 mg/kg perzinfotel IM for MAC2

Table 4: Experimental design study 2

Cat number 07FPK1 08QEB6 08QBO5 07AQI1 07KQA2 08QDO5 MAC MAC MAC MAC MAC MAC

o o o o o o A A A A A A ent*

Treatm B(1+2) B(1+2) B(1+2) B(1+2) B(1+2) B(1+2) * All treatments were administered at an interval of ≥ 7 days

Experimental procedures: Cats were fasted for 12 hours and water was withheld for 2 hours prior to the administration of treatment (pre‐medication) with either saline, perzinfotel, or butorphanol. The cats were induced to anesthesia with isoflurane in oxygen in an induction box and were orotracheally intubated and positioned in right lateral recumbency. Isofluranea in oxygen was used to maintain anesthesia through an out‐of‐circle, agent‐specific vaporizerb in a semi‐closed anesthetic circle rebreathing systemc. The end‐tidal carbon dioxide d concentration (ETco2; mm Hg) was maintained between 35 and 45 mm Hg by means of controlled breathing. The ECG, heart rate (beats/min), systolic (SAP), diastolic (DAP) and mean

(MAP) arterial pressure (mm Hg), ETISO (%), ETco2 (mm Hg), PO2 (mm Hg) by pulse oximetry (%), body temperature (ºC), inspiration and expiration concentrations of isoflurane were continuously monitorede,f. Heating padsg and hot water blankets were used during anesthesia to maintain body temperature between 37.5 and 38.5ºC.

Determination of isoflurane MAC: Isoflurane MAC was determined by delivering a noxious supramaximal electrical stimulus to the buccal mucosa18. Two 24‐gauge, 10‐mm insulated h stimulating electrodes were inserted 1 cm apart into the buccal mucosa at a location dorsal and caudal to the incisors. The opposite ends of the electrodes were connected to an electrical stimulatori that delivered a predetermined stimulus of 50 V, 5 Hz, and 10 ms duration. Stimulation continued for 1 min unless the cat showed gross purposeful movement before completion of the 1‐min stimulation. Lifting of the head and repeated movement of the limbs were considered gross purposeful movement. The ETISO was initially set at 1.5% for each cat’s first control MAC determination and at 1.2 times each cat’s control MAC value during subsequent days when experimental treatments were administered. If there was a negative response to the stimulus, the ETISO was decreased by 20% and allowed to equilibrate

44 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

for at least 15 min before applying the stimulus. This process was continued until the cat responded with gross purposeful movement. The ETISO was then increased by increments of 10% until the cat failed to demonstrate gross purposeful movement. The MAC was considered to be the average of the lowest ETISO value that did not produce gross purposeful 18 movement and the highest ETISO value that produced gross purposeful movement .

Determination of BIS: The BIS value was derived by continuously monitoring EEG activity. The EEG was obtained from platinum subdermal needle electrodes using a 3‐lead referential montage, arranged in a bifrontal configuration with the reference electrode positioned on the midline of the head rostral to the medial canthus of the eyes. The ground electrode was positioned on the midline in the atlantooccipital region30. EEG and BIS values were continuously acquired and displayed by use of a proprietary BIS monitorj with the high‐ frequency filter set at 70 Hz and the low‐frequency filter set at 2 Hz. The BIS number was automatically calculated and digitally displayed every 5 seconds and represented the EEG activity during the previous 60 seconds. Eight BIS values were recorded during a 2 min period before and after buccal mucosal stimulation.

Time to sternal recumbency: Time to sternal recumbency (min:sec) was determined to be the time between extubation (laryngeal cough reflex) and attainment of sternal recumbency. Time was measured with a digital clock.

Statistical analysis: Average values for MAC, BIS values and all cardio‐pulmonary parameters (at MAC level) for each animal and treatment were calculated and reported as the mean ± SD. For study 1, control values for all parameters were first compared with baseline. Since no significant differences were found (P>0.05), all drug treatments included in the Latin square were compared to each other and to the saline control. For study 2, the drug treatments were compared to each other and to the baseline. Control MAC’s for the two studies were compared to confirm equivalence and a combined analysis was conducted for the two studies. Comparisons were made using ANOVA, with LSmeans of groups compared to each other using a two‐sided Student’s t‐test at the 5% level of significance k.

Results

During study 1, control MAC values remained stable throughout the course of this study. The mean baseline MAC value for isoflurane was 1.54 ± 0.15 while the mean control MAC during the trial was 1.50 ± 0.17 (Table 5). The IV, SQ and IM administration of perzinfotel 30 min before induction to anesthesia significantly decreased the mean isoflurane MAC values by 58.0 – 68.0% for all doses of perzinfotel (Table 5). The decrease after administration of all different doses/routes of administration of perzinfotel was equivalent (P>0.05) and dose

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dependency was not demonstrated. For study 2, the IM administration of 2.5 mg/kg perzinfotel 30 minutes before induction to anesthesia significantly reduced (P<0.05) the isoflurane MAC value by 43.3% compared to control (Table 6). The IM administration of 0.2 mg/kg butorphanol 30 min before induction to anesthesia provided a significant reduction of isoflurane MAC by 15.3%, while IM administration of 5 mg/kg perzinfotel 30 min before measuring the second MAC, gave a significant reduction of MAC of 58.0% compared to baseline. Combining and analyzing the results from study 1 and study 2, a dose dependency was demonstrated. The dose of 2.5 mg/kg perzinfotel resulted in a significantly higher isoflurane MAC (P<0.05) compared to the higher (5, 10, 15 mg/kg) dose groups.

Table 5: MAC, HR and BIS; changes in these parameters relative to saline controls for six cats anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel. HR and BIS were measured at MAC level (Study 1).

MAC Heart rate Bispectral Index 1 Treatment N Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline baseline3 12 1.54±0.15 ‐ 173.7±27.6 ‐ 54.8±14. 5 ‐ Saline control 12 1.50±0.17a ‐ 177.6±35.4a ‐ 67.0±12.1b 5 mg/kg 12 0.63±0.08b ‐58.0% 161.6±15.2a ‐10.0% 75.7±12.6a 13.0% perzinfotel IM 10 mg/kg 12 0.51±0.13b ‐66.0% 163.8±13.0a ‐7.8% 76.8±6.1a 14.6% perzinfotel IM 15 mg/kg 12 0.52±0.20b ‐65.3% 163.4±9.3a ‐8.0% 77.9±3.8a 16.3% perzinfotel IM 10 mg/kg 12 0.48±0.04b ‐68.0% 166.3±11.8a ‐6.4% 77.3±3.0a 15.4% perzinfotel IV 10 mg/kg 12 0.62±0.19b ‐58.7% 163.2±15.6a ‐8.1% 75.3±4.0a 12.4% perzinfotel SQ 1N = Number of measurements. 2Mean ± Standard Deviation. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05). 3Since there was no statistical difference between the saline control and the saline baseline, the latter was not included in the analyses.

In study 1, the BIS values significantly increased with all perzinfotel treatments compared to saline control (Table 5). However, all doses and routes resulted in a similar increase of about 15% (P>0.05%). In study 2, the BIS values for doses of 2.5 mg/kg perzinfotel IM and the combination of 0.2 mg/kg butorphanol with 5 mg/kg perzinfotel IM were similar, and were

46 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

significantly higher (P<0.05) than saline control (Table 6). Butorphanol alone resulted in a non‐significant rise of BIS (P>0.05). These BIS values were measured at MAC level. During study 1, heart rate was 6.4 – 10.0% (non‐significant, P>0.05%) lower compared to saline treatment (control) heart rate in anesthetized cats after the various perzinfotel treatments (Table 5). During study 2, heart rate was slightly lower (4.5 – 6.6%) compared to control after treatments with perzinfotel, while heart rate was somewhat higher (4.3%) after the administration of butorphanol (Table 6). However, these changes were not significant (P>0.05). These HR values were measured at MAC level.

Table 6: MAC, HR and BIS; changes in these parameters relative to saline controls for six cats anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel and butorphanol. HR and BIS were measured at MAC level (Study 2).

MAC Heart rate Bispectral Index 1 Treatment N Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline control 12 1.50±0.17a ‐ 172.6±28.2a ‐ 66.7±11.7b ‐ 2.5 mg/kg 12 0.85±0.17c ‐43.3% 164.9±13.9a ‐4.5% 77.7±3.3a 16.5% perzinfotel IM 0.2 mg/kg 6 1.27±0.10b ‐15.3% 180.0±47.9a 4.3% 74.7±7.8a,b 12.0% butorphanol IM 0.2 mg/kg 6 0.63±0.08d ‐58.0% 161.2±24.5a ‐6.6% 80.8±5.3a 21.1% butorphanol + 5 mg perzinfotel IM 1N = Number of measurements. 2Mean ± Standard Deviation. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05).

During study 1, treatment with perzinfotel resulted in a consistent rise in DAP (11.8 – 16.4%), SAP (7.6 – 13.2%) and MAP (9.3 – 14.2%) relative to control during isoflurane anesthesia (Table 7). Rises in DAP, SAP and MAP were significant (P<0.05) for doses of 5 mg/kg IM, 10 mg/kg IV and 10 mg/kg SQ, while a dose of 10 mg/kg IM also gave a significant rise in DAP. In study 2, DAP, SAP and MAP rose 9.7%, 8.2% and 7.8%, respectively, after the administration of 2.5 mg/kg perzinfotel IM. After the administration of 0.2 mg/kg butorphanol IM, blood pressure values for DAP, SAP and MAP were 11.3%, 7.9 % and 10.1% lower then control, respectively, while administration of 5 mg/kg of perzinfotel IM following 0.2 mg/kg butorphanol caused a rise of blood pressures to just above control levels (1.4%, 4.4% and

47 CHAPTER 4

2.4%, respectively). All changes in blood pressure were not significant (P>0.05) during study 2. These blood pressure values were measured at MAC level.

Table 7: DAP, SAP and MAP; changes in these parameters relative to saline controls for six cats anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel. All these parameters were measured at MAC level (Study 1).

Diastolic arterial blood Systolic arterial blood Mean arterial blood Treatment N1 pressure pressure pressure Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline baseline3 12 75.3±11.9 ‐ 105.1±15.2 ‐ 89.2±14.2 ‐ Saline control 12 84.1±22.5b ‐ 113.8±25.9b ‐ 97.6±24.3b ‐ 5 mg/kg 12 95.9±19.6a 14.0% 127.3±20.5a 11.9% 109.9±19.9a 12.6% perzinfotel IM 10 mg/kg 12 95.7±18.3a 13.8% 124.8±22.2a,b 9.7% 108.5±19.5a,b 10.0% perzinfotel IM 15 mg/kg 12 94.0±11.9a,b 11.8% 122.4±17.4a,b 7.6% 106.7±13.6a,b 9.3% perzinfotel IM 10 mg/kg 12 97.7±12.5a 16.2% 128.8±10.3a 13.2% 111.5±11.8a 14.2% perzinfotel IV 10 mg/kg 12 97.9±15.3a 16.4% 126.7±21.7a 11.3% 109.6±15.8a 12.3% perzinfotel SQ 1N= Number of measurements. 2Mean ± Standard Deviation. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05). 3 Since there was no statistical difference between the saline control and the saline baseline, the latter was not included in the analyses.

The administration of perzinfotel alone produced some sedative effects during the 30 min prior to induction especially at the higher (10‐15 mg/kg IM) doses. The administration of perzinfotel indicated dose dependent increases in the time required to sternal recovery following extubation (Tables 9, 10). Control cats required 1:08 ± 0:35 min to reach a sternal position after anesthesia with isoflurane during study 1; perzinfotel pre‐medication with 5 mg/kg IM, 10 mg/kg IM, 10 mg/kg SQ and 10 mg/kg IV increased this time to 2:07 ± 1:57, 4:19 ± 3:29, 3:52 ± 2:19 and 6:40 ± 9:08 min, respectively (not different to control, P>0.05). Only the 15 mg/kg IM dose gave a significant (P<0.05) increase of sternal recovery time of 12:04 ± 12:17 min. During study 2, control cats required 1:23 ± 0.44 minutes to reach a sternal position. This time did not significantly change (P>0.05) after IM treatment with 2.5 mg/kg perzinfotel (1:28 ± 0:32 minutes) or the combination of 0.2 mg/kg butorphanol and 5 mg/kg

48 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

perzinfotel (4:56 ± 6:14 minutes). No adverse reactions were observed during anesthesia or during recovery.

Table 8: DAP, SAP and MAP; changes in these parameters relative to saline controls for six cats anesthetized with isoflurane following pretreatment with various doses and routes of perzinfotel and butorphanol. All these parameters were measured at MAC level (Study 2).

Diastolic arterial blood Systolic arterial blood Mean arterial blood Treatment N1 pressure pressure pressure Mean2 Change Mean2 Change Mean2 Change (%) (%) (%) Saline control 12 78.7±17.6a ‐ 106.1±23.4a ‐ 91.0±20.3a ‐ 2.5 mg/kg 12 86.3±16.9a 9.7% 114.8±18.1a 8.2% 98.1±17.7a 7.8% perzinfotel IM 0.2 mg/kg 6 69.8±15.7a ‐11.3% 97.7±16.5a ‐7.9% 81.8±16.3a ‐10.1% butorphanol IM 0.2 mg/kg 6 79.8±18.1a 1.4% 110.8±25.2a 4.4% 93.2±21.2a 2.4% butorphanol + 5 mg perzinfotel IM 1N= Number of measurements. 2Mean ± Standard Deviation. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05).

Table 9: Time from extubation to sternal recumbency (Study 1)

Treatment N1 Time (min:sec)2 Saline baseline3 6 2:04 ± 2:28 Saline control 6 1:08 ± 0:35b 5 mg/kg perzinfotel IM 6 2:07 ± 1:57b 10 mg/kg perzinfotel IM 6 4:19 ± 3:29b 15 mg/kg perzinfotel IM 6 12:04 ± 12:17a 10 mg/kg perzinfotel IV 6 6:40 ± 9:08a,b 10 mg/kg perzinfotel SQ 6 3:52 ± 2:19b N1= number of measurements. 2Significantly (P<0.05) different compared to control. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05). 3Since there was no statistical difference between the saline control and the saline baseline, the latter was not included in the analyses.

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Table 10: Time from extubation to sternal recumbency (Study 2)

Treatment N1 Time (min:sec)2 Saline control 6 1:23 ± 0:44a 2.5 mg/kg perzinfotel IM 6 1:28 ± 0:32a 0.2 mg/kg butorphanol + 5 mg/kg 6 4:56 ± 6:14a perzinfotel IM N1= number of measurements. 2Significantly (P<0.05) different compared to control. Means and standard deviations without the same letter grouping in each column are significantly different (P<0.05).

Discussion

These results demonstrate the anesthetic sparing effects of perzinfotel in cats. All doses of perzinfotel decreased isoflurane MAC values relative to saline controls, regardless of route of administration. IM doses of 5, 10 and 15 mg/kg of perzinfotel did not demonstrate dose dependent reductions in isoflurane MAC values (58.0‐68.0% reduction of control, P>0.05), indicating that a dose‐plateau may have been reached. A dose of 2.5 mg/kg IM perzinfotel, however, lowered the isoflurane MAC significantly less (43.3% reduction of control), providing a dose dependent effect when compared to the higher doses (P<0.05). The isoflurane MAC reduction of 15.3% after administration of butorphanol is similar to that reported in earlier studies24. The addition of 5 mg/kg of perzinfotel IM to butorphanol treated cats resulted in an isoflurane MAC reduction of 58.0%, which was equivalent to 5 mg/kg perzinfotel IM alone. The MAC of an inhaled anesthetic required to prevent gross purposeful movement in 50% of the subjects in response to a supramaximal noxious stimulus is used as a clinical index of drug potency and a guide to selection of the inhalant anesthetic concentration required for general anesthesia19. The repeatability and stability over time of the control MAC values reported in these studies indicate that the measured decrease in isoflurane MAC values is scientifically appropriate. The decrease in isoflurane MAC values were associated with statistically significant increases in BIS values (P<0.05) for all doses and routes of perzinfotel and suggest a reduction in CNS and anesthetic associated depression but adequate analgesia to prevent gross purposeful movement to a noxious stimulus. A lack of change in BIS and hemodynamic values, among all treatment groups, when isoflurane concentration was held constant at 1.5% suggests that the decrease in the isoflurane concentration was responsible for the changes observed. Perzinfotel decreased HR in anesthetized cats compared to control (4.5 ‐ 10%), although not significantly. In dogs, perzinfotel tended to increase HR (although not significantly)22,23. The reason for this apparent discrepancy is unclear and might be species related.

50 EFFECTS OF PERZINFOTEL, BUTORPHANOL AND A BUTORPHANOL‐PERZINFOTEL COMBINATION

The DAP, MAP and SAP in these anesthetized cats were increased compared to saline controls for all perzinfotel treatments as well as for the butorphanol and perzinfotel combination. These increases were consistent for all doses of perzinfotel and were statistically significant during study 1 for doses of 5 mg/kg IM and 10 mg/ kg SQ and IV; the dose of 10 mg/kg IM provided a significant rise in DAP. Hypotension as a function of cardio‐ vascular management during anesthesia is found to be a significant factor in anesthetic mortality31. The ability to affect blood pressure could therefore potentially improve anesthetic safety. These hemodynamic effects seem to be secondary (dependent) effects and most likely due to a decrease in inhaled isoflurane concentrations. Recovery was significantly longer when cats were pretreated with perzinfotel only at the highest dose (15 mg/kg IM). These data suggest that perzinfotel may produce some immobilizing activity when combined with inhalant anesthetics and supports studies suggesting that NMDA receptor inhibition contributes part of the immobilizing activity of aromatic volatile anesthetics32. As recoveries were smooth and uneventful, these longer recovery times would be of little or no practical significance at a therapeutic dose in a clinical setting. Some of the results of these current studies are limited due to the number of animals. Increases in DAP, MAP and SAP, although a consistent finding throughout study 2, were not significant but may have been if larger numbers of animals had been used (increase of power). This first evaluation of perzinfotel in cats confirms the compound has the potential to reduce the amount of gaseous anesthetic required in cats with subsequent benefits on cardiovascular function. These studies provide guidance on the potentially useful dose range and warrants further evaluation in cats undergoing surgical procedures. Ketamine is an NMDA antagonist approved for use in feline medicine for restraint (11 mg/kg IM) and anesthesia (22‐33 mg/kg IM)33. In this study, perzinfotel was evaluated for a different use, as a pre‐medication, which resulted in significant anesthetic (isoflurane) sparing and an increase in blood pressure, thereby enhancing the cardiopulmonary safety during anesthesia. At a therapeutic dose of 5 mg/kg, there was negligible sedation. As compared to ketamine, perzinfotel can be administered IV, IM or SQ, and, at the dose range used in this study, is expected to provide much greater anesthetic sparing21 and produce less side effects such as dysphoria and difficult recoveries from anesthesia. In conclusion, pretreatment with perzinfotel (IM, IV, SQ) at doses ranging from 2.5 to 15 mg/kg or a combination of butorphanol and perzinfotel produced significant decreases in isoflurane MAC and was associated with significant increases in BIS and blood pressure in sexually intact male and female cats.

51 CHAPTER 4

Acknowledgements

The authors would like to thank Deborah Amodie BS for statistical analysis. This study was funded by Fort Dodge Animal Health.

Footnotes: a IsoFlo. Abbott Laboratories, North Chicago, ILL b Isotec 3, Ohmeda, Madison, Wis c LEI Medical, Boring, Ore d Veterinary Anesthesia Ventilateor Modle 2KIE, Hallowell Engineering and Manufacturing Corp, Pittsfield, Mass e DSI Physio Tel D70‐PCT transmitter, Data Sciences International, Saint Paul, Minn f Passport 2, Datascope, Montvale, NJ g T/Pump, Gaymar Industries Inc., Orchard Park, NY h Genuine grass platinum subdermal needle electrodes, Astro‐Med, Inc., West

Warwick, RI i Grass SD9 Stimulator, Grass Medical Instruments, Quincy, MA j A‐1000 EEG Monitor, Aspect Medical Systems, Inc., Newton, MA k SAS version 8.2, SAS Institute, Inc. (Cary, NC)

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9. Lawand NB, Willis WD, Westlund KN. Excitatory amino acid receptor involvement in peripheral nociceptive transmission in rats. Euro J Pharmacol 1997; 324: 169 – 177. 10. Chizh BA, Headley PM, Tzschentke TM. NMDA receptor antagonists as analgesics: focus on the NR2B subtype. TRENDS in Pharmacol Sci 2001; 22: 12: 636 – 642. 11. Fisher K, Coderre TJ, Hagen NA. Targeting N‐methyl‐D‐aspartate receptor for chronic pain management: preclinical animal studies, recent clinical experience and future research directions. J. Pain Symptom Manage 2000; 20:358 –373. 12. Hewitt DJ. The use of NMDA receptor antagonists in the treatment of chronic pain. Clin J Pain 2000; 16: S73 –S76. 13. Kim AH, Kerchner GA, Choi DW. (2002). Blocking Excitotoxicity. In Marcoux FW and Choi DW, eds. CNS . New York: Springer, 3‐36. 14. Pomarol‐Clotet E, Honey GD, Murray GK et al. Psychological effects of ketamine in healthy volunteers. Phenomenological study. Br J Psychiatry 2006; 189: 173–179. 15. Rockstroh, S; Emre M, Tarral A, and Pokorny R. Effects of the Novel NMDA‐Receptor Antagonist SDZ EAA 494 on Memory and Attention in Humans. Psychopharmacology 1996; 124: 261‐266. 16. Kinney WA, Abou‐Gharbia M, Garrison DT, Schmid J. Design and synthesis of (2‐[8,9‐Dioxo‐2,6‐ diazabicyclo[5.2.0]non‐1(7)‐en‐2‐yl]ethylphosphonic Acid) (EAA‐090), a potent N‐methyl‐D‐ aspartate antagonist, via the use of 3‐Cyclobutene‐1,2‐dione as an achiral ά‐amino acid bioisostere. J Med Chem1998; 41: 236 – 246. 17. Sun L, Chiu D, Kowal D et al. Characterization of two novel N‐methyl‐D‐aspartate antagonists: EAA‐ 090 (2‐[8,9‐Dioxo‐2,6‐diazabicyclo[5.2.0]non‐1(7)‐en2‐yl]ethylphosphonic Acid) and EAB‐318 (R‐ά‐ Amino‐5‐chloro‐1‐(phospohonomethyl)‐1H‐benzimidazole‐2‐propanoic Acid Hydrochloride). J Pharmacol and Experim Therap 2004; 310: 563 – 570. 18. Brandt MR, Cummons TA, Potestio L et al. Effects of the N‐methyl‐D‐aspartate Receptor Antagonist Perzinfotel [EAA‐090; (2‐[8,9‐Dioxo‐2,6‐diazabicyclo[5.2.0]non‐1(7)‐en2‐yl]ethyl]phosphonic Acid] on Chemically Induced Thermal Hypersensitivity. J Pharmacol and Experim Therap 2005; 313: 1379 – 1386. 19. Antognini JF, Cartens E. Measuring minimum alveolar concentration: more than meets the tail. Anesthesiology 2005; 103: 751‐758. 20. Solano AM, Pyendop BH, Boscan PL et al. Effect of intravenous administration of ketamine on the minimum alveolar concentration of isoflurane in anesthetized dogs. Am J Vet Res 2006; 67:21‐25. 21. Muir WW III, Wiese AJ, March PA. Effects of morphine, lidocaine, ketamine, and morphine‐ lidocaine‐ketamine drug combination on minimum alveolar concentration in dogs anesthetized with isoflurane. Am J Vet Res 2003; 64: 1155 – 1160. 22. Kushiro T, Wiese AJ, Eppler MC et al. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2007; 68: 12: 1294 – 1299. 23. Zwijnenberg RJG, Del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs when given as a pre‐anesthetic IV, IM or SQ and in combination with butorphanol. Am J Vet Research, accepted for publication 11 June 2009. 24. Lamont LA, Mathews KA. Opiods, Nonsteroidal Anti‐inflammatories, and Analgesic Adjuvants. In: Veterinary Anesthesia and Analgesia. 4th Ed. Oxford: Blackwell Science Ltd, 2007; 250‐251. 25. Hewson CJ, Dohoo IR, Lemke KA. Perioperative use of analgesics in dogs and cats by Canadian veterinarians in 2001. Can Vet J 2006; 47: 352 – 359. 26. Commiskey S, Fan LW, Ho IK, Rockhold RW. Butorphanol: effects of a prototypical agonist‐ antagonist analgesic on kappa‐opioid receptors. J Pharmacol Sci. 2005; 98(2):109‐116. 27. Ilkiw JE, Pascoe PJ Tripp LD. Effects of morphine, butorphanol, buprenorphine and U50488H on the minimum alveolar concentration of isoflurane in cats. AJVR 2002; 63: 8: 1198 – 120 28. Kissin I. Depth of anesthesia and bispectral index monitoring. Anesth Analg 2000; 90: 1114 – 1117.

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29. Johansen JW. Update on Bispectral Index monitoring. Best Practice & Research Clinical Anaesthesiology 2006; 20: 81‐99. 30. March PA, Muir WW III. Bispectral analysis of the electroencephalogram: a review of its development and use in anesthesia. Vet Anesth Analg 2005; 32: 241 – 255. 31. Arbous MS, Grobbee DE, van Kleef JW et al. Mortality associated with anaesthesia: a qualitative analysis to identify risk factors. Anaesth 2001; 56 (12):1141 – 53. 32. Sewell JC, Raines DE, Eger EI 2nd et al. A comparison of the molecular bases for N‐methyl‐D‐ aspartate‐receptor inhibition versus immobilizing activities of volatile aromatic anesthetics. Anesth Analg 2009; 108(1):168 – 75. 33. United States Food and Drugs Administration Website. Ketamine. Available at: http://www.fda.gov/AnimalVeterinary/Products/ApprovedAnimalDrugProducts/FOIADrugSummari es/ucm118102.htm. Accessed Oct 14, 2009.

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CHAPTER 5

Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure Measurement Techniques in Anesthetized Cats: Comparative Performance versus Implanted Telemetry.

Raphael J Zwijnenberg DVM, MVSc, MVPHMgt, MACVSc* Carlos L del Rio, PhD** Cobb R DVM, MACVSc Yukie Ueyama, DVM** William W Muir DVM, PhD**

*Pfizer Animal Health Australia, 38‐42 Wharf Road, West Ryde, NSW 2114 **QTest Labs, PO Box 12381, Columbus, OH 43212

Am J Vet Research, accepted for publication 25 May 2010 CHAPTER 5

Objective – The comparison of three methods of blood pressure measurements; invasive telemetry (DSI), semi‐invasive Vascular Access Port (VAP) technology and non‐invasive oscillometry (NIBP) in cats.

Animals ‐ Six healthy sexually intact cats, 3 males and 3 females, 12 ‐ 17 months of age and weighing 2.8 – 4.6 kg.

Procedures – Thirty days before the study all cats were implanted with telemeters and a VAP device. At 4 different time points and under normo‐ and hypertension, blood pressure was measured with the implanted devices and with NIBP. When comparing VAP and NIPB with DSI, VAP had a correlation coefficient (R2) between 0.8487 and 0.9972 while NIPB had a R2 between 0.7478 and 0.9689.

Conclusions and Clinical Relevance – Both VAP and NIBP showed a high degree of correlation with DSI as the gold standard. VAP showed a slightly higher degree of correlation compared to NIBP.

56 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

Multiple methods and techniques have been developed to measure blood pressure in animals. Both direct or invasive (arterial catheterization) and indirect or noninvasive methodologies are currently utilized in experimental and clinical trials. Radiotelemetry was developed as a reliable, method to monitor physiological functions including arterial blood pressure in awake and free moving laboratory animals including rats, dogs and cats1‐5. Implantable radiotelemetry transmitters are surgically connected to subcutaneously tunneled arterial catheters, are calibrated both before and after implantation, and have been demonstrated to accurately and reproducibly detect changes in arterial blood pressure for extended periods of time although bias of the zero reference value is always a concern. Alternatively subcutaneously tunneled arterial catheters can be connected to a vascular access port and a completely external and calibrated pressure sensing device utilized to continuously or intermittently record arterial blood pressure. Non‐invasive (Doppler, oscillometric, plethymographic) methodologies employ a cuff placed around a distal extremity such as a limb or a tail, a sphygmomanometer and methods for detection of blood flow (Doppler) or arterial pulsations. Most oscillometric methodologies have been validated against radiotelemetry as a gold standard in rats6. Validation of less invasive and non‐invasive techniques for measuring blood pressure is critical when performing clinical trials in client‐ owned animals such as cats and has obvious benefits in every day clinical veterinary practice.

Materials and Methods

Animal care and instrumentation: This study was approved by the Institutional Animal Care and Use Committee of the study facility. Six healthy sexually intact Domestic Shorthair cats, 3 males and 3 females, 12 – 17 months of age and weighing 2.8 – 4.6 kg, were the subjects of this study. Thirty days before beginning the study each cat had been surgically implanted with a radiotelemetry devicea as well as with a vascular access portb. For the telemetry device under general anesthesia with isoflurane at 1‐3% and strict aseptic conditions, the cats were implanted using a small‐animal radio transmittera with a weight of 11 g and a volume of 6 cc, providing telemetry signals for body temperature, arterial blood pressure through an anti‐ thrombotic gel‐filled catheter and a 2‐electrode lead ECG. The device was subcutaneously secured to the superficial abdominal musculature of the left flank via a ventral skin incision. The arterial pressure catheter was tunneled to the left hind limb, and introduced/placed in the abdominal aorta via the left femoral artery. The two ECG electrodes were tunneled subcutaneously and secured to the superficial muscles of the left and right sides of the chest wall using a lead II configuration. The surgical incisions were closed in two layers; the underlying musculature was closed with absorbable sutures, and the skin was closed with staples. During the same procedure VAP technology was performed by using a femoral artery indwelling catheter. This 20‐25 cm catheter, made out of soft compliant material, was implanted under strict aseptic conditions by performing a cut‐down over the right femoral artery, and introducing a 3F catheter, designed for chronic usec , into the abdominal aorta. This catheter was subcutaneously tunneled to a subcutaneous pocket on the animal’s right hip, where it was attached to the VAP and secured to the underlying fascia. The

57 CHAPTER 5

catheter/port was flushed with 2‐3ml of a locking‐solution of Taurolidine‐Citrate (TCS‐04c) in order to prevent both clotting and bacterial growth, and the incision was closed in layers with absorbable suture/staples. The VAP was used in conjunction with a wearable telemetry transmitterb to provide a less invasive telemetric arterial pressure signal. Throughout the duration of the study, both the VAP and the catheter was flushed with saline and locked periodically (~7 days) in order to ensure chronic patency. No major body cavity was opened/accessed during the VAP implantation procedures. After surgery the animals were allowed to recover for ≥ 30 days during which they were observed daily for signs distress while the wound sites were monitored for signs of infections. Prophylactic antibiotics (cephalexin 30 mg/kg) were administered at time of surgery (IV) and twice daily (PO for 3 days) after surgery. The skin incision staples were removed approximately 12 days after surgery. No signs of infection, pain or distress related to the surgery could be observed at any time. The DSI as well as the VAP technique permitted the simultaneous and continuous monitoring of ECG, arterial (femoral artery) blood pressure, and body temperature. During the study a volume‐pressure recording tail‐cuffd to measure Non‐Invasive Blood Pressure (NIBP) was also used. The cuff was placed around the base of the tail and the hair was not clipped over the measurement site. During this study, cuff sizes with a width between 2 – 2.5 cm and with a range between 3 – 8 cm were used.

Experimental design: Six cats were anesthetized with isoflurane at two time points (A and B), 4 weeks and 14 weeks. Recordings of arterial blood pressure were made utilizing all three techniques at normotension (MAP <85 mmHg) after stabilization of 15 minutes at two end‐tidal isoflurane (ETISO) concentrations (2.2% and 1.8%), and at hypertension (MAP>85 mmHg) 5 seconds immediately following an acute phenylephrine bolus (80 µg IV) while at 1.8% isoflurane. Additional measurements (C and D) were performed at 84.5 ± 0.7 days (12 weeks) and 91.5 ± 0.7 days (13 weeks) post implantation, during a minimum alveolar concentration (MAC) of isoflurane determination7. Recordings were made at 2.2% and at the MAC‐level of isoflurane (1.1 ± 0.5 % and 1.2 ± 0.5 %, respectively). At each time‐point, five concurrent determinations of arterial blood pressure were obtained and averaged for each method.

Hemodynamic monitoring: Arterial blood pressures were simultaneously recorded via DSI, VAP and NIBP techniques. DSI signals were captured by a telemetry receivera and relayed along with an atmospheric pressure referencea to a personal computer via a data exchange modela. Arterial blood pressure signals were obtained from the implanted VAP via non‐coring Huber needles, and a manometer through a calibrated fluid‐filled telemetered transducerb. Radio signals from these wearable transmitters were captured and converted back to an analog signal by a telemetry receiver. Meanwhile, NIBP measurements were derived via appropriately sized tail‐cuffs (15‐20mm) connected to a calibrated stand‐alone monitord. Telemetry measurements were collected immediately prior to the inflation of the tail cuff.

Data collection: Throughout the studies, the analog telemetered blood pressure signals were digitally sampled (1000Hz), recorded, visualized and analyzed using a common software

58 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

platforme. For all measurements recorded values included systolic (SAP), diastolic (DAP) and mean arterial (MAP) blood pressure.

Statistical analysis: Data are presented as means with standard deviations (mean ± SD). At each time‐point, the mean and standard deviation (SD) for the 5 repetitive measurements of DAP, MAP and SAP were calculated. In order to evaluate measurement repeatability at steady state, coefficients of variability (mean/SD) were also determined for data collected under anesthesia (2.2% and 1.8% isoflurane) at time‐points A and B; differences in repeatability (i.e., between mean coefficients of variation) were evaluated using a two‐way analysis of variance (ANOVA; methodology: DSI, NIBP, VAP; intervention: 2.2%, 1.8%) and Tukey post‐hoc tests. For all time‐points A, B, C and D inter‐methodology comparisons were made using both simple and multiple linear regression analyses over the aggregate of all the collected data points (i.e., all measurements and all time‐points); both slope and intersect differences were evaluated using multiple linear regression. In addition, the bias, precision, and accuracy of NIBP and VAP derived pressures (when compared against DSI readings) were evaluated via Bland‐Altman analysis8,9, as well as by Pearson’s and concordance correlations (respectively). In all cases, statistical significance was set (a priori) at p < 0.05, and DSI values were used as reference (i.e., as gold standard).

Results

A first analysis was made comparing the VAP and NIBP techniques with the often used and referenced DSI technique (industry gold standard): Recordings of time points A and B showed a high degree of similarity as the 95% confidence interval for the two measurements overlapped in 29 out of 32 time points (Table 1). The average coefficients of variability were lowest for DSI (Mean ± SD = 2.8 ± 2.5), intermediate for VAP (Mean ± SD = 4.0 ± 3.4) and highest for NIBP (Mean ± SD = 6.9 ± 3.6) (Table 2). Overall Bias, Precision (Pearson coefficient), Concordance coefficient and Accuracy (Bias correction factor) calculations show consistently higher values for the VAP vs. DSI comparison compared to the NIBP vs DSI comparison (Table 3). The correlation coefficient (R2) for time points A‐D between DSI and VAP was 0.9649 for MAP, 0.9372 for SAP and 0.9488 for DAP (Figure 1). The R2 for time points A‐D between DSI and NIBP was 0.8409 for MAP, 0.8029 for SAP and 0.8066 for DAP (Figure 1). When comparing MAP and DAP in multiple linear regression plots, the regression lines representing NIPB and VAP are clearly further apart at DAP (Figure 2). The 95% confidence intervals for NIBP are consistently wider (Figure 2). The Bland‐Altman plots also show consistently reduced bias and limits of agreement for VAP vs. DSI compared to NIBP vs. DSI (Figure 3).

59 CHAPTER 5

Table 1. Average systolic (SAP), diastolic (DAP) and mean (MAP) arterial pressures simultaneously derived via DSI, NIBP and VAP technologies readings both at baseline (awake), and under steady‐state isoflurane anesthesia over the course of the study. Recordings were made at 4 different time‐points: 4 (A) and 14 (B) weeks post‐implantation, as well as during MAC determinations performed 12 (C) and 13 (D) weeks post‐implant.

DSI NIBP VAP Time‐Point SAP DAP MAP SAP DAP MAP SAP DAP MAP 160 ± 116 ± 138 ± A n/a n/a n/a n/a n/a n/a 12 8 11 Baseline 159 ± 120 ± 138 ± B n/a n/a n/a n/a n/a n/a 20 16 18 47 ± A 76 ± 10 59 ± 12 86 ± 9 28 ± 2 46 ± 2 76 ± 6 45 ± 5 57 ± 4 12 B 65 ± 7 44 ± 9 53 ± 8 63 ± 4 42 ± 8 51 ± 6 83 ± 10 25 ± 4 45 ± 6 2.20% C 64 ± 7 42 ± 7 51 ± 7 77 ± 7 26 ± 5 46 ± 6 60 ± 13 39 ± 11 49 ± 11 48 ± D 71 ± 12 58 ± 13 77 ± 4 29 ± 4 48 ± 4 63 ± 8 42 ± 6 51 ± 7 14 48 ± 79 ± 10 60 ± 11 85 ± 9 29 ± 3 48 ± 4 80 ± 6 47 ± 5 59 ± 5 12 1.80%+ PE1 A 151 ± 101 ± 124 ± 162 ± 87 ± 117 ± 162 ± 107 ± 128 ± 18 25 23 23 24 24 22 26 21 47 ± 72 ± 13 57 ± 12 71 ± 4 45 ± 8 56 ± 5 85 ± 9 27 ± 4 46 ± 4 12 1.80%+ PE1 B 140 ± 95 ± 116 ± 146 ± 95 ± 118 ± 154 ± 107 ± 73 ± 10 17 16 16 16 20 18 18 21 112 ± 84 ± 109 ± 53 ± C 96 ± 23 76 ± 25 99 ± 33 80 ± 32 89 ± 33 25 22 25 20 MAC2 109 ± 80 ± 111 ± 51 ± 100 ± D 93 ± 33 75 ± 28 77 ± 33 88 ± 32 34 30 26 22 11 Blood pressures (in mm Hg) are mean ± standard deviation. 1: Acute phenylephrine (PE) IV bolus while stable (for 15min) at 1.8% end‐tidal isoflurane; data are reported before and during the bolus. 2: MAC levels were 1.1 ± 0.5 % and 1.2 ± 0.5 % for time‐points C and D (respectively).

60 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

Table 2. Average coefficients of variability (mean/SD) at two steady‐state isoflurane end‐tidal concentrations for 5‐consecutive mean arterial pressure (MAP) readings as derived via DSI, NIBP and VAP technologies. Aggregate data were taken from time‐points A and B.

DSI NIBP VAP 2.20% 2.7 ± 2.4 8.4 ± 2.2 4.0 ± 3.7 1.80% 2.9 ± 2.8 5.5 ± 4.3 3.9 ± 3.3 Mean ± SD 2.8 ± 2.5 6.9 ± 3.6 4.0 ± 3.4 (range) (0.5 – 10.5) (2.8 – 16.7) (0.5 – 11.6) P – value1 ‐ < 0.001 0.2 coefficients of variability (as %) are mean ± standard deviation. 1: NIBP, VAP vs. DSI.

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Table 3. Overall bias (i.e., absolute pressure difference), precision (Pearson’s correlation coefficient), concordance, and accuracy (bias correction factor) obtained when comparing NIBP (left, blue dots) and VAP (right, red squares) derived values against DSI readings for all systolic (SAP, top), diastolic (DAP, mid), and mean (MAP, bottom) arterial pressures recorded throughout the study (i.e., time‐points A‐D).

NIBP vs. DSI VAP vs. DSI P<0.051 SAP ‐10.1 ± 16.7 ‐2.5 ± 10.1 Y DAP 20.2 ± 12.7 ‐2.5 ± 7.2 Y Bias (mmHg) MAP 10.0 ± 13.3 ‐2.5 ± 6.5 Y p‐value2 < 0.001 NS

0.8960 0.9681 SAP Y (0.87 to 0.92) (0.96 to 0.98) 0.8981 0.9740 Pearson coefficient (precision) DAP Y (0.87 to 0.92) (0.97 to 0.98) 0.9170 0.9823 MAP Y (0.90 to 0.93) (0.98 to 0.99) 0.8632 0.9624 SAP Y (0.83 to 0.89) (0.95 to 0.97) 0.7060 0.9677 Concordance coefficient DAP Y (0.66 to 0.75) (0.96 to 0.98) 0.8756 0.9788 MAP Y (0.85 to 0.90) (0.97 to 0.98) SAP 0.9634 0.9941 ‐ Bias correction factor (accuracy) DAP 0.7862 0.9935 ‐ MAP 0.9548 0.9964 ‐ Bias data are mean ± standard deviation, while coefficients are presented with 95% confidence intervals (in parenthesis). 1: NIBP vs. VAP, i.e., non‐overlapping 95% confidence intervals; 2: SAP vs. DAP vs. MAP.

62 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

Figure 1. Linear regression plots comparing NIBP (left, blue dots) and VAP (right, red squares) derived pressures against DSI readings for all systolic (SAP, top), diastolic (DAP, mid), and mean (MAP, bottom) arterial pressures recorded throughout the study (i.e., time‐points A‐D). Both regression line (blue lines) and 95% confidence intervals (dashed lines) are presented.

NIBP vs. DSI VAP vs. DSI

SAP SAP 250 250

200 200

150 150

100 100 SAP Reference (DSI) Pressure (mmHg) Pressure (DSI) Reference (mmHg) Pressure (DSI) Reference 50 50

0 0 0 50 100 150 200 250 0 50 100 150 200 250 NIBP Pressure (mmHg) VAP Pressure (mmHg) SLOPE: 0.8844 (0.8329 to 0.9360), R2 = 0.8029 (P < SLOPE: 0.8880 (0.8587 to 0.9173), R2 = 0.9372 (P < 0.001) 0.001) INTERSECT: 2.3214 (‐3.5201 to 8.1629) INTERSECT: 8.4615 (5.3735 to 11.5495)

DAP DAP 250 250

200 200

150 150

100 100 DAP Reference (DSI) Pressure (mmHg) Pressure (DSI) Reference (mmHg) Pressure (DSI) Reference 50 50

0 0 0 50 100 150 200 250 0 50 100 150 200 250 NIBP Pressure (mmHg) VAP Pressure (mmHg) SLOPE: 0.9805 (0.9237 to 1.0373), R2 = 0.8066 (P < SLOPE: 0.9001 (0.8734 to 0.9268), R2 = 0.9488 (P < 0.001) 0.001) INTERSECT: 21.1268 (18.1489 to 24.1046) INTERSECT: 4.1290 (2.1844 to 6.0736)

MAP MAP 250 250

200 200

150 150

100 100 MAP Reference (DSI) Pressure (mmHg) Pressure (DSI) Reference (mmHg) Pressure (DSI) Reference 50 50

0 0 0 50 100 150 200 250 0 50 100 150 200 250 NIBP Pressure (mmHg) VAP Pressure (mmHg) SLOPE: 0.9302 (0.8826 to 0.9778), R2 = 0.8409 (P < SLOPE: 0.9408 (0.9179 to 0.9637), R2 = 0.9649 (P < 0.001) 0.001) INTERSECT: 14.8809 (11.2399 to 18.5218) INTERSECT: 2.2546 (0.2702 to 4.2390)

63 CHAPTER 5

Figure 2. Multiple linear regression plots comparing diastolic (DAP, left) and mean (MAP, right) arterial pressures as derived by NIBP (blue) and VAP (red) pressures against DSI readings for all values recorded throughout the study (i.e., time‐points A‐D). Both regression line (blue lines) and 95% confidence intervals (dashed lines) are presented. MAP DAP

250 250

200 200

150 150

100 100

50 50 Reference (DSI) Pressure (mmHg) Reference (DSI) Pressure(mmHg)

0 0 0 50 100 150 200 250 0 50 100 150 200 250 Measured (NIBP, VAP) Pressure (mmHg) Measured (NIBP, VAP) Pressure (mmHg)

NIBP NIBP VAP VAP

64 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

Figure 3. Bland‐Altman plots comparing NIBP (left, blue dots) and VAP (right, red squares) derived pressures against DSI readings for all systolic (SAP, top), diastolic (DAP, mid), and mean (MAP, bottom) arterial pressures recorded throughout the study (i.e., time‐points A‐D). Both bias (blue lines) and limits of agreement (±1.96SD, dashed lines) are presented.

NIBP vs. DSI VAP vs. DSI

SAP SAP 70 70

50 50

30 30 +1.96 SD 22.7 +1.96 SD 17.4 10 10 Mean Mean -2.5 SAP -10 -10 -10.1 -1.96 SD -22.4 -30 -30 -1.96 SD Pressure Difference VAP vs. DSI (mmHg) Pressure DifferencePressure NIBP DSI (mmHg) vs. -42.8 -50 -50

-70 -70 0 50 100 150 200 250 0 50 100 150 200 250 Reference Pressure (mmHg, Average for DSI and NIBP) Reference Pressure (mmHg, Average for DSI and VAP)

DAP DAP 70 70

50 +1.96 SD 50 45.2

30 30 Mean 20.2 +1.96 SD 10 10 11.7 -1.96 SD Mean -2.5 DAP -10 -4.7 -10 -1.96 SD -16.6 -30 -30 Pressure Difference VAP vs. DSI (mmHg) DSI VAP vs. Difference Pressure Pressure DifferencePressure DSI NIBP (mmHg) vs. -50 -50

-70 -70 0 50 100 150 200 250 0 50 100 150 200 250 Reference Pressure (mmHg, Average for DSI and NIBP) Reference Pressure (mmHg, Average for DSI and VAP)

MAP MAP 70 70

50 50

+1.96 SD 30 36.1 30

Mean +1.96 SD 10 10 10.0 10.1 Mean -2.5 MAP -10 -10 -1.96 SD -1.96 SD -16.0 -15.0

-30 -30 Pressure Difference VAP vs. DSI (mmHg) Pressure DifferencePressure NIBP DSI (mmHg) vs. -50 -50

-70 -70 0 50 100 150 200 250 0 50 100 150 200 250 Reference Pressure (mmHg, Average for DSI and NIBP) Reference Pressure (mmHg, Average for DSI and VAP)

65 CHAPTER 5

Discussion

The use of tail cuffs in dogs for the indirect measurement of blood pressure and the use of VAP blood pressure measurement has been shown to be clinically acceptable and precise10. There was good correlation and no statistical difference between the three methods examined in this study in felines. Certain differences between direct (both radiotelemetry and VAP technology) and indirect measurement are to be expected11. These differences can be explained because of the different artery used in the measurement12 (femoral artery versus coccygeal artery) and because the indirect measurement measure blood pressure as the movement of the arterial wall as a consequence of a pressure wave (NIBP) while the direct measurement methods measure this pressure wave directly (VAP and DSI). Also the attachment of the VAP catheter to a telemetry device could add some bias although because of constant calibration to atmospheric conditions the authors believe this source of bias to be minimal. Despite the expected differences, the overall association for both systems with DSI was high (Figures 1‐3). As expected the R2 of the tail cuff system (NIBP) was slightly lower compared to the VAP. In this design we chose not to clip the tail where the cuff was placed. When certain drug trials need to involve patient owned animals in order to achieve registration it is often undesirable to clip the tail at the site where a monitoring cuff is placed. Furthermore a past study failed to demonstrate significant differences between clipped and unclipped limbs13. Previous studies often evaluated oscillometric blood pressure monitoring in cats using a direct method as a comparison. One study didn’t find a good correlation between oscillometric and direct measurement13, while another study found that apart from a minor underestimation of SAP during normo‐ and hypertension, the oscillometric monitor yielded accurate measurements for DAP and MAP throughout the entire pressure range14. An evaluation of Doppler ultrasonic and oscillometric methods of indirect blood pressure measurement in cats found that in anesthetized cats oscillometric methods underestimated all BP parameters15. The American Heart Association has recommended tail‐cuffed blood pressure measurement for high‐throughput experimental designs. However, some tail‐cuff methods show a good agreement with radiotelemetry (the above used method) and others do not, indicating that each tail‐cuff method requires independent validation6. Both the Pearson and the concordance correlation coefficients in were lower in the NIPB/DSI comparison compared to the VAP/DSI comparison (Table 3). Especially for the measurement of DAP the bias correction factor (Table 3) seems to confirm less alignment. This could mean that the NIBP might be (slightly) less accurate with lower blood pressures. These findings seem to be at odds with a study in anesthetized dogs where a NIBP monitor lacked accuracy at high pressures16. Both NIBP and DSI systems are subject to a certain bias. It is expected that this bias is possibly lower in the VAP system of measuring blood pressure because of constant calibration to atmospheric conditions. The overlap of part of the regression lines in Figure 1 and 2 combined with high correlation coefficients however clearly indicates the validity of both

66 Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure

NIBP and DSI although the wider limits of agreement when using the NIBP device should warrant a certain degree of caution. Overall, these results demonstrate the high accuracy of both the VAP and the NIBP system and also confirmed the accuracy of the DSI system. VAP and NIBP may provide reliable alternatives for invasive telemetry in future animal studies. NIBP is currently widely used in clinical veterinary practice. This study demonstrates that NIBP is an accurate and reliable way to measure blood pressure in anesthetized cats.

Acknowledgements

This study was funded by Fort Dodge Animal Health.

Footnotes a DSI Physio Tel C50‐PXTtransmitter, Data Sciences International, Saint Paul, Minn b VAP, MTI/CP6titanium port, with a 3F IntiSilf Medical grade silicone rubber (Silastics) with atraumatic rounded tip (length : 20‐25cm), Access Technologies/emkaPACK radiotransmitter (EMKA Technologies) c Access Technologies, Skokie, IL d Cardell Model 9403 e IOX‐2‐Emka technologies f Microsoft Excel

References

1. Kramer K, Kinter LB. Evaluation and applications of radiotelemetry in small laboratory animals. Physiol Genomics 2003: 13; 197 – 295. 2. Zwijnenberg RJ, Muir III WW. Evaluation of the potential for interaction between a metaflumizone‐ amitraz combination and dexmedetomidine hydrochloride in dogs. Vet Therap 2009: 10 ;1‐2; 40 – 45. 3. Kushiro T, Wiese AJ, Eppler MC et al. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res 2007; 68: 12: 1294 – 1299. 4. Zwijnenberg RJG, Del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs when given as a pre‐anesthetic IV, IM or SQ and in combination with butorphanol. Am J Vet Research, accepted for publication 11 June 2009. 5. Wiese AJ, Muir WW. Anaesthetic and cardiopulmonary effects of intramuscular morphine, medetomidine and ketamine administered to telemetered cats. J Feline Med Surg. 2007; 9:150‐ 156. 6. Feng M, Whitesall S, Zhang Y et al. Validation of Volume‐Pressure Recording Tail‐Cuff Blood Pressure Measurements. Am J of Hypertension 2008; 21: 12: 1288 – 1291.

67 CHAPTER 5

7. Zwijnenberg RJ, Del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel, butorphanol and a butorphanol‐perzinfotel combination on the minimum alveolar concentration of isoflurane in cats. Am J Vet Research, accepted for publication 15 October 2009. 8. Myles PS, Cui J. Editorial I: Using the Bland‐Altman method to measure agreement between repeated measures. Br J of Anaesthesia 2007; 99: 3: 309 – 311. 9. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; i: 307 – 310. 10. Bodey AR. Systemic hypertension in the dog – Fact or fiction. Waltham/Ohio State University Symposium: Cardiology 1994; 44 ‐ 45. 11. Guyton AC. Vascular distensibility and functions of the arteries and venous system. In: Textbook of Medical Physiology (8th edn). Guyton AC (ed.). W.B Saunders, Philadelphia, PA, USA 1991; 162 – 163. 12. Haberman CE, Kang CW, Morgan JD, Brown SA. Evaluation of oscillometric and Doppler ultrasonic methods of indirect blood pressure estimation in conscious dogs. Can J Vet Res 2006; 70: 3: 211 – 217. 13. Branson KR, Wagner‐Mann CC, Mann FA. Evaluation of an Oscillometric Blood Pressure Monitor on Anesthetized Cats and the Effect of Cuff Placement and Fur on Accuracy. Veterinary Surgery 1997; 26: 347 – 353. 14. Pedersen KM et al. Evaluation of an oscillometric blood pressure monitor for use in anesthetized cats. JAVMA 2002; 221: 5: 646 – 650. 15. Haberman CE et al. Evaluation of Doppler Ultrasonic and Oscillometric Methods of Indirect Blood Pressure Measurement in Cats. Intern. J Appl Res Vet Med 2004; 2: 4: 279 – 289. 16. Deflandre CJA, Hellebrekers LJ. Clinical evaluation of the Surgivet V60046, a non invasive blood pressure monitor in anaesthetized dogs. Veterinary Anaesthesia and Analgesia 2008; 35: 13 – 21.

68

CHAPTER 6

Evaluation of the Potential for Interaction Between a Metaflumizone‐Amitraz Combination and Dexmedetomidine Hydrochloride in Dogs.

Raphael J Zwijnenberg DVM, MVPHMgt* William W Muir DVM, PhD**

*Fort Dodge Animal Health, PO Box 5366, Princeton, NJ 08543 **QTest Labs, PO Box 12381, Columbus, OH 43212

Vet Therap 2009; 10 (1‐2): 40 – 45. CHAPTER 6

Clinical Relevance

This study investigated the effects on cardiovascular parameters, if any, of a commercially available combination of metaflumizone and amitraz administered to healthy, telemetered beagles that were subsequently sedated with dexmedetomidine. Dogs were sedated first without any pre‐treatment and then after pre‐treatment with metaflumizone and amitraz. Baseline values of all parameters were within normal limits for all dogs before the first anesthetic event. At 10 and 20 minutes after onset of sedation, oxygen saturation as measured by pulse oxymetry was significantly higher for dogs that were pre‐treated with metaflumizone and amitraz. At all times after induction of sedation, blood pressure, heart rate and baseline body temperature for dogs pre‐treated with metaflumizone and amitraz were not statistically different from when they were not pre‐treated. In conclusion, prior treatment with metaflumizone and amitraz did not influence the hemodynamic response to dexmedetomidine in telemetered dogs.

70 EVALUATION OF THE POTENTIAL FOR INTERACTION

Introduction

A commercial combination of metaflumizone and amitraz for topical administration as an ectoparasiticide for dogs (ProMeris® for Dogs, Fort Dodge Animal Health) was recently approved for the US market. Some clinicians have theorized that amitraz could potentially interact with compounds that have α2‐adrenergic agonist activity if it were systemically absorbed following dermal application, even though no actual report of interaction of the metaflumizone‐amitraz combination and α2‐adrenergic agonists was found in the published literature. Although previously published pharmacokinetic data1 indicate transdermal absorption of amitraz is unlikely, the potential interaction remains a question in the minds of some clinicians. Metaflumizone is a chemical derived from the pyrazoline chemistry and is not believed to contribute to the aforementioned interaction because it acts by binding to voltage‐dependent sodium channels producing a relaxed paralysis in a broad range of important pest insects including lepidopterans, beetles, ants, and termites2. Metaflumizone demonstrates very low acute toxicity and relatively low toxicologic potential following subchronic oral exposure in 3 rats, mice and dogs and a battery of mammalian toxicology studies indicated no α2‐ adrenergic activity3. Repeated topical administration of metaflumizone‐amitraz causes no adverse health effects when used as recommended in dogs as young as 8 weeks of age4. Amitraz (N'‐(2,4‐dimethylphenyl)‐N‐[[(2,4‐dimethylphenyl)imino]methyl]‐N‐ methylmethanimidamide) is a formamidine acaricide first used in the 1970s to control mites and ticks that had developed resistance to conventional parasiticides5. Amitraz elicits a variety of behavioral changes in both argasid and ixodid ticks, often manifested as hyperactivity, leg waving and detaching behavior. These behavioral effects are thought to be secondary to the actions of amitraz on tick octopaminergic receptors6. In arthropods, formamidine‐acting compounds mimic the actions of the neurotransmitter octopamine, producing constant synaptic stimulation leading to tremor and/or convulsions followed by the death of the parasite7,8.

Dexmedetomidine, a potent α2‐adrenergic agonist, was recently approved as a sedative and analgesic in dogs and cats to facilitate clinical examinations, clinical procedures, minor surgical procedures, and minor dental procedures9,10,11. It is also indicated for use as a preanesthetic in dogs prior to general anesthesia11. The selection of dexmedetomidine for this 12 study was made based on its comparatively high selectivity for α2‐adrenergic receptors .

The similarity between octopaminergic receptors in invertebrates and α2‐adrenergic receptors in vertebrates has led some investigators to suggest that the formamidines may interfere with specific membrane receptors in vertebrates as well13. Binding studies have demonstrated, in fact, that these compounds can show a weak interaction with α2‐adrenergic receptors14,15.

α2‐Adrenergic sedatives such as xylazine, detomidine, medetomidine and dexmedetomidine are frequently administered in veterinary practice. It was theorized that

71 CHAPTER 6

amitraz, when given systemically to mammals, would bind with α2‐receptors producing 16 sedative effects that may exacerbate the effects of α2‐sedatives. This study was designed to investigate the cardiovascular effects, if any, of a topical metaflumizone‐amitraz combination administered cutaneously to telemetered dogs that were subsequently sedated with dexmedetomidine hydrochloride (Dexdormitor®, Pfizer Animal Health).

Materials and methods

Animal care and instrumentation: The study was approved by the Institutional Animal Care and Use Committee of the testing facility. Seven healthy, sexually intact beagles (four males and three females; age range, 18.5 to 31 months; weight range, 9.8 to 12.4 kg) were the subjects of this study. Each dog was equipped with a telemetry device (DSI PhysioTel D70‐PCT transmitter, Data Sciences International, Saint Paul, MN) that had been surgically implanted 2 months before the study began. The telemetry device permitted the simultaneous and continuous monitoring of respiratory rate, electrocardiogram (ECG), arterial (femoral artery) blood pressure, and body temperature.

Experimental design: The study was conducted in seven healthy beagles as a single cohort, with each dog serving as its own control. Each dog was sedated with dexmedetomidine hydrochloride twice (3 days apart), first without any pre‐treatment (control, day 0), and subsequently after being treated with metaflumizone‐amitraz (day 3). Topical metaflumizone‐ amitraz was administered 2 days after the initial anesthetic event (day 2), approximately 24 hours before the second anesthetic procedure. The timing for the second anesthetic event was selected to allow the ectoparasiticide product to distribute throughout the dogs’ hair coat. Temperature, heart rate (HR), respiratory rate, blood pressure and an estimation of oxygen saturation of hemoglobin as measured by pulse oxymetry(SpO2) were monitored prior to anesthesia induction (baseline), and every 10 minutes thereafter until reversal of anesthesia with atipamazole (Antisedan®, Pfizer Animal Health) 60 minutes after induction.

Experimental procedures: Dogs were fasted for 12 hours and water was withheld for 2 hours before dexmedetomidine was administered. A venous catheter was placed in a cephalic vein for administration of dexmedetomidine. Sedation was induced by bolus IV administration of the recommended dose (375 ug/m2) of dexmedetomidine given over approximately 60 seconds, in accordance with label directions. Once dexmedetomidine was administered, the dosing syringe was removed from the catheter, the catheter was flushed with saline, and a cuffed tracheal tube was placed. However, at no time during the experiments was there a need to mechanically ventilate the lungs because no sedation‐induced apnea was observed. During each of the two anesthesia events, the ECG; HR (beats/min), systolic (SAP), diastolic (DAP) and mean (MAP) arterial pressure (mm Hg); end tidal isoflurane and carbon

72 EVALUATION OF THE POTENTIAL FOR INTERACTION

dioxide concentrations (ETISO and ETco2, respectively); SpO2 (%); and body temperature (ºC) were monitored continuously (DSI PhysioTel D70‐PCT transmitter; and Passport 2, Datascope, Montvale, NJ). Heating pads (T/Pump, Gaymar Industries Inc., Orchard Park, NY) and hot water blankets were used during anesthesia to maintain body temperature between 37.5ºC and 38.5ºC.

Statistical analysis— Data are reported as the mean ± SD. Using the PROC MIXED Procedure (SAS 8.2, SAS Institute, Cary, NC), each parameter was analyzed by a repeated measures analyses of variance with a model that considered treatment as a fixed effect and time as the repeated factor. Treatment was tested with the residual error at the 5% level of significance. Least squares means of the data were calculated for each treatment group. Because the animals served as their own controls, the repeated measures analysis of variance F‐test was used to provide the two‐sided test to determine whether differences existed between treatments at the 5% level of significance.

Results

Baseline values for HR, SpO2, MAP, SAP and DAP were within normal limits for all dogs before the first anesthetic event. Baseline HR was slightly lower (not statistically significant) in dogs pre‐treated with the metaflumizone‐amitraz combination, but HRs became similar to those in untreated dogs within 10 minutes after dexmedetomidine administration (Figure 1).

All parameters were similar for all times (10‐60 minutes) after dexmedetomidine administration in dogs pre‐treated with metaflumizone‐amitraz except for two time points, 10 and 20 minutes after the onset of sedation, at which times SpO2 was significantly higher (Table 1).

73 CHAPTER 6

Figure 1: Heart Rate before (baseline) and after induction of anesthesia with dexmedetomidine either under control conditions (Pre‐Tx, closed circles) or 24 hours after treatment with metaflumizone‐amitraz (Post‐Tx, open circles)

Pre-Tx Post-Tx 110

100

90

80

HR (bpm) 70

60

50

40 ne in in in in in in li m m m m m m se 10 20 30 40 50 60 Ba

Time Point

74 EVALUATION OF THE POTENTIAL FOR INTERACTION

Table 1: Hemodynamic parameters before (BASELINE) and after anesthesia induction with dexmedetomidine either under control conditions (CTRL) or 24hr following topical treatment with a combination of metaflumizone and amitraz

Time after Induction1

BASELINE +10min +20min +30min +40min +50min +60min

CTRL 94 ± 6 72 ± 7 54 ± 3 57 ± 3 55 ± 4 55 ± 5 54 ± 6

HR 2 (bpm) M + A 76 ± 5 68 ± 6 61 ± 5 62 ± 5 64 ± 8 54 ± 3 63 ± 8

CTRL 93 ± 1 87 ± 2 91 ± 2 94 ± 1 93 ± 2 94 ± 2 95 ± 2 (%) SpO2 M + A 94 ± 1 92* ± 1 96* ± 1 94 ± 0 95 ± 1 95 ± 1 94 ± 1

CTRL 119 ± 5 148 ± 9 130 ± 8 117 ± 8 112 ± 6 108 ±6 106 ± 6 MAP

(mmHg) M + A 116 ± 7 147 ± 8 136 ± 7 128 ± 6 120 ± 8 117 ± 6 115 ± 6

CTRL 164 ± 5 189 ± 11 165 ± 9 155 ± 9 149 ± 9 143 ± 7 141 ± 8 SAP

(mmHg) M + A 160 ± 8 194 ± 11 173 ± 9 164 ± 7 159 ± 8 155 ± 7 153 ± 7

CTRL 92 ± 5 127 ± 8 110 ± 6 101 ± 5 94 ± 5 90 ± 5 87 ± 5 DAP

(mmHg) M + A 91 ± 6 131 ± 7 116 ± 6 108 ± 5 105 ± 4 98 ± 5 95 ± 4

CTRL 38 ± 0 38 ± 0 38 ± 0 38 ± 0 38 ± 0 38 ± 0 38 ± 0 Temp

(Celsius) M + A 38 ± 0 38 ± 1 38 ± 1 38 ± 1 38 ± 1 38 ± 1 38 ± 1 1 Time after anesthesia induction with dexmedetomidine. 2Metaflumizone and amitraz combination. * Statistically significant difference (P<0.05) from control

Discussion

Beagles have become a major model for pharmacologic safety studies, which are required by the International Conference on Harmonisation “Guidelines on Safety Pharmacology” (ICH S7A) finalized by the FDA in 2001.17 Cardiac electrophysiology in these dogs plays a decisive role in the selection of a potential “cardio‐safe” new chemical entity. Telemetry is a well‐ researched and validated method to study variations in hemodynamic parameters in beagles18 and the use of telemetry in the dogs in this study facilitated continuous, sensitive, precise and repeatable measurements of such physiological parameters like HR, MAP, SAP and DAP.

75 CHAPTER 6

The interaction between amitraz and dexmedetomidine in mammals was theorized based on the understanding that the binding pattern of these compounds may overlap due to the structural similarities of octopaminergic and adrenergic receptors.13 To date, there have been no reports of an actual clinical interaction between these compounds. This may be due to the lower affinity of amitraz to mammalian receptors and/or due to the fact that amitraz is not systemically absorbed after topical administration.1 19,20 Due to the similarity of octopaminergic and α2‐receptors, oral administration of amitraz can produce a sedative effect accompanied by lower HR and body temperature. Although baseline HR was lower (albeit not significantly different) when dogs were pre‐ treated with metaflumizone‐amitraz compared with when they were not pre‐treated, it is unlikely that this effect can be attributed to amitraz because body temperature was not decreased and there was no sign of sedation. As these measurements were taken 3 days apart, and HR may have been influenced by the behavioral state of the dogs. Also, the relatively small sample size of this study might be a contributing factor. It is noticeable that immediately after induction of anesthesia, HRs were similar for pretreated and not pretreated dogs.

The results demonstrate that the administration of the highly selective α2‐agonist dexmedetomidine to dogs that have been treated with this combination of metaflumizone‐ amitraz does not produce adverse side effects as investigated in this study. Values for HR, temperature and mean SAP and DAP after the administration of dexmedetomidine in dogs that had been pre‐treated with the metaflumizone‐amitraz combination were not different (P

> .05) than when the dogs were not pre‐treated. SpO2 was significantly higher at 10 and 20 min after onset of anesthesia in the dogs pretreated with metaflumizone‐amitraz; however, the difference was small and very likely to be clinically irrelevant. In conclusion, prior treatment (24 hours) with a combination of metaflumizone‐amitraz did not influence the hemodynamic response to dexmedetomidine in telemetered dogs. This confirms the safety of using an α2‐adrenergic agonist, such as dexmedetomidine, for anesthetizing dogs previously treated with this commercially available combination of metaflumizone and amitraz.

Acknowledgements

The authors would like to thank Dr. Carlos del Rio DVM, PhD for scientific support and Deborah Amodie BS for statistical analysis. This study was funded by Fort Dodge Animal Health.

76 EVALUATION OF THE POTENTIAL FOR INTERACTION

References

1. DeLay RL, Lacoste E, Mezzasalma T, Blond‐Riou F. Pharmcokinetics of metaflumizone and amitraz in the plasma and hair of dogs following topical application. Vet Parasitol. 2007;150:3:251‐257. 2. Takagi K, Hamaguchi H, Nishimatsu T, Konno T. Discovery of metaflumizone, a novel semicarbazone insecticide. Vet Parasitol. 2007;150:3:177‐181 3. Hempel K, Hess FG, Bögi C, Fabian E, Hellwig J, Fegert I. Toxicological properties of metaflumizone. Vet Parasitol. 2007;150:3:190‐5. 4. Heaney K, Lindahl RG. Safety of a topically applied spot‐on formulation of metaflumizone plus amitraz for flea and tick control in dogs. Vet Parasitol. 2007;150:3:225‐32. 5. Hollingworth RM. Chemistry, biological activity and uses of formamidine pesticides. Environ. Health Persp. 1976; 14: 57 – 69. 6. Page SW. Antiparasitic drugs. In: Maddison JE, Page SW, Church D, eds. Clinical Pharmacology. First ed. Amsterdam: Elsevier Health Sciences, 2002; 186. 7. Evans PD, Gee JD. Action of formamidine pesticides on octopamine receptors. Nature 1980; 287: 60‐62. 8. Nathanson JA. Charactarization of octopamine‐sensitive adenylate cyclase; Elucidation of a class of potent and selective octopamine‐2 receptor agonists with toxic effects in insects. Proc. Natl. Acad. Sci. USA 1985; 82: 599 – 603. 9. Bloor BC, Frankland M, Alper G, Raybould D, Weitz J, Shurtliff M. Hemodynamic and sedative effects of dexmedetomidine in dog. J Pharmacol Exp Ther. 1992; 263:2:690‐7 10. Granholm M, McKusick BC, Westerholm FC, Aspegrén JC. Evaluation of the clinical efficacy and safety of dexmedetomidine or medetomidine in cats and their reversal with atipamezole. Vet Anaesth Analg. 2006;33:4:214‐23. 11. Campagnol D, Teixeira Neto FJ, Giordano T, Ferreira TH, Monteiro ER. Effects of epidural administration of dexmedetomidine on the minimum alveolar concentration of isoflurane in dogs. Am J Vet Res. 2007;68:12:1308‐18. 12. Gerlach AT, Dasta JF. Dexmedetomidine: an updated review. Ann Pharmacother. 2007;41:2:245‐52. 13. Nathanson JA. Identification of octopaminergic agonists with selectivity for octopamine receptor subtypes. J Pharmacol Exp Ther. 1993;265:2:509‐15. 14. Costa LG, Murphy SM. Interaction of the pesticide chlordimeform with adrenergic receptors in mouse brain: An in vitro study. Arch. Toxicol. 1987; 59: 323 – 327. 15. Costa LG, Olibet G, Murphy SM. α2‐adrenoreceptors as a target for formamide pesticides; In vitro and in vivo studies in mice. Toxocol. Appl. Pharmacol. 1988; 93: 319 – 328. 16. Queiroz‐ Neto A, Zamur A, Gonçalves SC et al. Characterization of the antinociceptive and sedative effect of amitraz in horses. J. vet. Pharmacol. Therap. 1998; 21: 400 – 405. 17. Food and Drug Administration, HHS. International Conference on Harmonisation; guidance on S7A safety pharmacology studies for human pharmaceuticals; availability. Notice. Fed. Regist.2001; 66: 135: 36, 791 – 836. 18. Soloviev MV, Hamlin RL, Shellhammer LJ. Variations in Hemodynamic Parameters and ECG in Healthy, Conscious, Freely Moving Telemetrized Beagle Dogs. Cardiovasc. Toxicol. 2006; 6:1:51 – 62 19. Hugnet C, Buronrosse F, Pineau X, Cadoré JL, Lorgue G, Berny PJ. Toxicity and kinetics of amitraz in dogs. Am J Vet Res. 1996;57(10):1506‐10. 20. Hsu WH, Lu ZX, Hembrough FB. Effect of amitraz on heart rate and aortic blood pressure in conscious dogs: influence of , prazosin, tolazoline, and yohimbine. Toxicol Appl Pharmacol. 1986;84:2:418‐22.

77

CHAPTER 7

Discussion

CHAPTER 7

The results reported in Chapter 2 and Chapter 4 support the anaesthetic sparing effects of perzinfotel in both dogs and cats. In both species, all doses of perzinfotel investigated decreased isoflurane MAC values regardless of route of administration (IV, IM or SQ) or combination with the opioid agonist butorphanol. In contrast to the cat study, escalating IM doses of perzinfotel in the dog study demonstrated mild dose‐dependent reductions in isoflurane MAC values, which were augmented when perzinfotel was combined with butorphanol. These results indicate that with (lower) doses of 5, 10 and 15 mg/kg a dose‐ plateau may have been reached in cats. Only a dose of 2.5 mg/kg provided a dose‐dependent effect by lowering the isoflurance MAC significantly. The 0.2 mg/kg IM dose of butorphanol produced a similar smaller, but also statistically significant reduction in isoflurane MAC in both dogs and cats (Chapter 2,4). The MAC of an inhaled anaesthetic required to prevent gross purposeful movement in 50% of the subjects in response to a supramaximal noxious stimulus is used as a clinical index of drug potency and a guide to selection of the inhalant anaesthetic concentration required for general anaesthesia1. The repeatability and stability over time of the control MAC values reported in both the dog and cat studies indicate that the measured decrease in isoflurane MAC values is scientifically valid. The decrease in MAC values after pretreatment with perzinfotel was greater than the relatively small decrease in MAC after pretreatment with butorphanol. The decrease in isoflurane MAC values are associated with statistically significant increases in BIS values (increase in level of consciousness) and suggest a reduction in CNS and anaesthesia‐associated depression but adequate analgesia to prevent gross purposeful movement to a noxious stimulus. The lack of change in BIS and haemodynamic values, among all treatment groups, when isoflurane concentration was held constant at 1.5%, suggests that the change in the isoflurane concentration was the main factor responsible for the changes observed in both studies.

In both species, butorphanol produced comparatively minimal effects on isoflurane MAC and BIS values, although in both cats and dogs, arterial blood pressure tended to be decreased compared to control values. In dogs, the combination of perzinfotel and butorphanol produced the greatest decrease in isoflurane MAC (59%) while in cats higher doses of perzinfotel gave the greatest reduction of isoflurance MAC. In dogs, these results support previous studies suggesting that butorphanol produces minimal inhalant anaesthetic‐sparing effects in isoflurane‐anaesthetised dogs2. We did not perform the types of experiments required to determine if this drug interaction relative to isoflurane MAC reduction was additive or synergistic but did not observe an antagonistic effect3.

In dogs, perzinfotel increased HR for all doses of studied, although only at a dose of 30 mg/kg was the increase significant. The combination of perzinfotel and butorphanol decreased HR in anaesthetised dogs compared to control (‐9%), but increased HR compared to butorphanol alone (+12%) although not significantly. In cats, perzinfotel decreased the HR, although not significantly. The reason for this apparent discrepancy is unclear and might be species related.

80 DISCUSSION

The DAP, MAP and SAP in both anaesthetised dogs and cats were increased compared to untreated controls for all doses of perzinfotel that were administered. In dogs, this increase was only statistically significant for DAP for the highest dose of perzinfotel (30 mg/kg) compared to the untreated controls while for cats the increases in DAP, MAP and SAP were significant for doses of 5 mg/kg IM and 10 mg/kg SQ and IV; the dose of 10 mg/kg IM provided a significant rise in DAP. In dogs, the increases were statistically significant for all doses of perzinfotel administered alone compared to the administration of butorphanol alone. For both species, the combination of perzinfotel and butorphanol increased the DAP, MAP and SAP compared to butorphanol alone, an effect that was most likely due to the greater decrease in isoflurane concentration; this increase, however, was not statistically significant and the values were comparable to control values. Given its anaesthetic‐sparing effect, perzinfotel pre‐treatment tended to limit the decrease in arterial pressure normally associated with isoflurane anesthesia at MAC level. In contrast, butorphanol alone tended to result in a further lowering of blood pressure (MAP) by about 10% in cats to 18% in dogs relative to control (non‐significant). Butorphanol’s effects upon heart rate and blood pressure when administered in combination with perzinfotel or alone suggest that it possesses mild cardiovascular depressant activity in isoflurane anaesthetised dogs4. Further studies are required, however, to determine the dose‐dependent cardiovascular effects of perzinfotel alone and in combination with other opioid agonists in isoflurane anaesthetised dogs5 and cats. Overall, dogs and cats seem to have a different sensitivity to perzinfotel, which resulted in higher anaesthetic‐sparing effects at lower doses for cats compared to dogs. Hypotension as a function of cardiovascular management during anaesthesia is a significant factor in anaesthetic mortality6. The ability to affect blood pressure could therefore potentially improve anaesthetic safety. These haemodynamic effects seem to be secondary (dependent) effects and are most likely due to a decrease in inhaled isoflurane concentrations.

Recovery was longer when dogs or cats were pretreated with perzinfotel. These data suggest that perzinfotel may produce some immobilising activity when combined with inhalant anaesthetics and support studies suggesting that NMDA receptor inhibition contributes to part of the immobilising activity of aromatic volatile anaesthetics7. The longer recovery times observed in these studies are expected to be of little or no clinical significance in a clinical setting.

Some of the results previously discussed were not statistically significant compared to controls for either study. The authors are of the opinion that as increases in HR, DAP, MAP and SAP were a general and consistent finding throughout both studies, it was of clinical significance to mention and discuss these data. Results that are statistically significant are not necessarily biologically or clinically important (e.g. recovery times) and vice versa8,9.

81 CHAPTER 7

The analgesic properties of perzinfotel, PLA‐695 and carprofen (as a positive control) were tested in a sodium urate (SU) model that induced a consistent mild to moderate lameness when evaluated by the subjective lameness system (Chapter 3). With this subjective instrument, the lameness was statistically evident for 8 hours when compared to baseline scores. Interestingly, when examining the same dogs with objective vertical ground reaction force measurements, a consistent lameness was only documentable for the first 4 hours. Thus, the model proved reliable for detecting changes in lameness for a relatively short period of time depending on the measurement used. Both evaluation methodologies found that dogs were beginning to return to baseline levels within 6 hours and were completely back to baseline levels by 12 hours. The urate dose used in the current study produced a much less severe lameness than higher urate doses previously described15,16,17. These data show that by titrating the amount of urate given, one can vary the severity and duration of the lameness created. Perzinfotel displayed no significant attenuation of limb dysfunction caused by the sodium urate induced stifle synovitis. The differences noted by both subjective and objective evaluation methodologies between perzinfotel and the negative control (no treatment) were subtle at best. In view of previous clinical evidence indicating analgesic activity of perzinfotel18, the lack of effect in this study is surprising. The lack of efficacy of perzinfotel in this model, however, may be explained by several mechanisms. First, this failure may simply be a matter of a dose‐dependent response and the dose given in this study was insufficient to attenuate lameness in this model. Secondly, in the present study, perzinfotel was administered to the dogs once daily with three treatments prior, and the final dose one hour after urate induction. Thus, it is not known whether the once daily dosing had any pre‐ emptive effect on central sensitisation in these dogs. It is possible that the one hour lag in giving perzinfotel after urate induction allowed sufficient time for central sensitisation to occur, thereby decreasing potential effect of the perzinfotel. In a previous study, epidural ketamine, a non‐selective NMDA receptor inhibitor, had no effect when given 12 hours after urate induced synovitis, but had improvement of ground reaction forces at two hours, if given at the same time19. Timing of perzinfotel relative to noxious stimuli may also be important to achieve its desired effect of decreased hypersensitivity. Lack of effect of the perzinfotel in this model may also be due to its selectivity for the glutamate site of the NR2A subunit18. It has been demonstrated that NR2A containing NMDA receptors are not present on the presynaptic primary afferent fibers in the dorsal root ganglion of the rat20,21. Substance P, prostaglandins, adenosine, and glycine, as well as glutamate (which can enhance its own release) can be released from the presynaptic afferent fibres, and act on presynaptic and postsynaptic receptors20,22,23. Stimulation of presynaptic NR2B containing receptors can facilitate and prolong transmission of nociceptive messages20. It has also been demonstrated that NR2A knockout mice fail to show changes in acute and chronic pain‐related behaviours20. Substance P and glutamate release cause influx of Calcium (Ca2+) and Sodium (Na+) with resultant activation of Protein Kinase C23, driving tyrosine

82 DISCUSSION

phosphorylation of the NMDA receptors21. This causes release of (Mg2+), acting as a block from the NMDA receptors, decreases resting membrane potential, and allows for prolonged channel opening time21. The end result is increased postsynaptic activity from the multiplicity of neurotransmitters in the dorsal root ganglion synapses. These processes may have allowed the perzinfotel‐treated dogs in this study to continue to exhibit pain and lameness. Finally, another possible reason is that like all NMDA antagonists, perzinfotel has not displayed antinociceptive effects, but rather has prevented hypersensitivity as in previous studies18. Specifically, NR2A seems to play a larger role relative to NR2B in inflammatory states with up‐regulation of the NR2A subunits in areas of the brain and spinal cord as a result of peripheral inflammatory stimulus18. Spinal NR2A subunits have been decreased in models of nerve pain18. Thus, the present study may emphasise the importance of pre‐emptive treatment with NMDA antagonists for successful attenuation of inflammatory based pain syndromes. Perzinfotel may play an important role in analgesia when used synergistically with opioids or nonsteroidal anti‐inflammatory agents22,5. PLA‐695 attenuated the lameness at both time points where the negative control values were different than baseline. Interestingly at no time point in the study did either the subjective or objective measurements note a difference between the PLA‐695 and the positive control group receiving carprofen. Almost all the different measurements placed the PLA‐695 results between perzinfotel and carprofen. PLA‐695’s lameness attenuation, however, was not as complete as carprofen. The effectiveness of PLA‐695 may be related to its inhibition of AA metabolism to its subsequent eicosanoids, and resultant anti‐inflammatory effects. PLA2, as a superfamily, have been found to be involved in many roles including antimicrobial activity, bone formation, apoptosis, insulin secretion, sperm development, Wallerian degeneration, axon regeneration, as a marker of coronary disease in people, and as 24,25 an anti‐inflammatory . Of the four main types of PLA2 (secreted, sPLA2; cytosolic, cPLA2; calcium independent iPLA2; and platelet activating factor acetyl hydrolase / oxidised lipid protein associated, LpPLA2) it has been shown that cPLA2, and in particular group IVA is a central enzyme mediating generation of eicosanoids and subsequent inflammatory 24 processes . In addition cPLA2 has shown a role in neurotransmitter release by coupling with a multitude of receptors in the brain such as glutamate, dopamine, and serotonin amongst 19 26 others . A constitutive spinal PLA2 has been demonstrated in rats and monkeys . Although many cPLA2 inhibitors demonstrating enzymatic activity have been studied, many have no in vivo effect27. It appears that PLA‐695 performs comparably to the positive control carprofen in the SU induced synovitis model of inflammatory pain.

PLA2 antagonists have shown dose dependent association with the outcome measures of 27 antihypersensitivity effects and PGE2 production . As clinical trials explore the analgesic effects of these compounds and effective PLA2 antagonists are found, appropriate dosing regimens and safety margins will need to be established along with any drug interactions. To date there have been no listed adverse affects of these compounds, although there is a report

83 CHAPTER 7

of a human deficient in cPLA (2‐alpha) that developed small intestinal ulceration and platelet 28 dysfunction . One interesting note is that opioids stimulate PLA2 in presynaptic afferent fibres leading to metabolites of 12‐lipoxygenase29. These metabolites enhance the activity of voltage dependent potassium (K+) channels inhibiting GABAergic neurotransmission29. Use of COX‐1, COX‐2, and specific 5‐LOX inhibitors shunt more metabolite through to the 12‐LOX 30 pathway synergistically enhancing the µ‐opioid based GABAergic release . PLA2 antagonists 30 have been shown to block this µ‐opioid effect . Concurrent use of PLA2 antagonists with µ‐ opioids in a clinical setting may be contraindicated but as an analgesic on its own merit, it shows promise. The aim of this trial was to learn whether perzinfotel and/or PLA 695 have analgesic properties. PLA 695 showed a significant level of pain control in this model at a dose of 10 mg/kg. Dogs treated with PLA 695 vomited twice during this trial (both times a one‐time occurrence about 5 minutes post administration). The formulation of PLA 695 seemed to be dose limiting, however this was an experimental formulation. If other formulations can be identified, improved levels of pain control can be expected. As perzinfotel will be used in a pre‐anaesthetic protocol it would be useful to know whether this compound could be used for peri/post operative pain control. Although the SU synovitis model might not exactly mimic post‐surgical pain, pain control in this model should give a useful indication regarding the control of post‐surgical pain. Unfortunately the dose used in this trial was limited to 10 mg/kg as some dogs would start stumbling at higher doses and could not be tested on the force plate. At the dose of 10 mg/kg perzinfotel gave no side effects. However, if perzinfotel will be licensed for the use as a pre‐anaesthetic with anaesthetic sparing properties it will be useful to know if higher doses (e.g. 20 mg/kg) will have analgesic properties on their own or in combination with opiods or NSAIDS. The development of perzinfotel as a drug with an anaesthetic sparing claim will certainly require field trials in client‐owned animals. Because the use of telemeters (both internal and external) in client‐owned animals will be prohibited, it was necessary to validate other less invasive measurement methods, such as oscillometry through the use of a tail cuff (Chapter 6). The use of tail cuffs in dogs for the indirect measurement of blood pressure and the use of VAP blood pressure measurement has been shown to be clinically acceptable and precise31. There was also good correlation and no statistical difference between the three methods examined in our study in felines. Certain differences between direct (both radiotelemetry and VAP technology) and indirect measurement are to be expected32. These differences can be explained because of the different artery used in the measurement33 (femoral artery versus coccygeal artery) and because the indirect measurement measures blood pressure as the movement of the arterial wall as a consequence of a pressure wave (NIBP) while the direct measurement methods measure this pressure wave directly (VAP and DSI). Also the attachment of the VAP catheter to a telemetry device could add some bias although because of constant calibration to atmospheric conditions the authors believe this source of bias to be minimal.

84 DISCUSSION

Despite the expected differences, the overall association for both systems with DSI was high. As expected the R2 of the tail cuff system (NIBP) was slightly lower compared to the VAP. In this design we chose not to clip the tail where the cuff was placed. When certain drug trials need to involve client owned animals in order to achieve registration it is often undesirable to clip the tail at the site where a monitoring cuff is placed. Furthermore, a previous study failed to demonstrate significant differences between clipped and unclipped limbs34. Previous studies often evaluated oscillometric blood pressure monitoring in cats using a direct method as a comparison. One study didn’t find a good correlation between oscillometric and direct measurement34, while another study found that apart from a minor underestimation of SAP during normo‐ and hypertension, the oscillometric monitor yielded accurate measurements for DAP and MAP throughout the entire pressure range35. An evaluation of Doppler ultrasonic and oscillometric methods of indirect blood pressure measurement in cats found that in anaesthetised cats oscillometric methods underestimated all BP parameters36. The American Heart Association has recommended tail‐cuffed blood pressure measurement for high‐throughput experimental designs. However, some tail‐cuff methods show a good agreement with radiotelemetry (the above used method) and others do not, indicating that each tail‐cuff method requires independent validation37. Both the Pearson and the concordance correlation coefficients in were lower in the NIPB/DSI comparison compared to the VAP/DSI comparison. Especially for the measurement of DAP the bias correction factor seems to confirm less alignment. This could mean that the NIBP might be (slightly) less accurate with lower blood pressures. These findings seem to be at odds with a study in anaesthetised dogs where a NIBP monitor lacked accuracy at high pressures38. Both NIBP and DSI systems are subject to a certain bias. It is expected that this bias is possibly lower in the VAP system of measuring blood pressure because of constant calibration to atmospheric conditions. The overlap of part of the regression lines in Figure 1 and 2 in Chapter 5 combined with high correlation coefficients however clearly indicates the validity of both NIBP and DSI although the wider limits of agreement when using the NIBP device should warrant a certain degree of caution. Telemetry is a well‐researched and validated method to study variations in haemodynamic parameters in beagles10 and the use of telemetry in the dogs in this study facilitated continuous, sensitive, precise and repeatable measurements of such physiological parameters like HR, MAP, SAP and DAP. Cardiac electrophysiology in these dogs plays a decisive role in the selection of a potential “cardio‐safe” new chemical entity like perzinfotel, but also to study a possible interaction between compounds like amitraz and dexmedetomidine (Chapter 6). The interaction between amitraz and dexmedetomidine in mammals was theorised based on the understanding that the binding pattern of these compounds may overlap due to the structural similarities of octopaminergic and adrenergic receptors11. To date, there have been no reports of an actual clinical interaction between these compounds. This may be due to the

85 CHAPTER 7

lower affinity of amitraz to mammalian receptors and/or due to the fact that amitraz is not systemically absorbed after topical administration12. 13,14 Due to the similarity of octopaminergic and α2‐receptors , oral administration of amitraz can produce a sedative effect accompanied by lower HR and body temperature. Although baseline HR was lower (albeit not significantly different) when dogs were pre‐treated with metaflumizone‐amitraz compared with when they were not pre‐treated, it is unlikely that this effect can be attributed to amitraz because body temperature was not decreased and there was no sign of sedation. As these measurements were taken 3 days apart, and HR may have been influenced by the behavioural state of the dogs. Also, the relatively small sample size of this study might be a contributing factor. It is noticeable that immediately after induction of anaesthesia, HR’s were similar for pretreated and not pretreated dogs.

The results demonstrate that the administration of the highly selective α2‐agonist dexmedetomidine to dogs that have been treated with this combination of metaflumizone‐ amitraz does not produce adverse side effects as investigated in this study (Chapter 6). Values for HR, temperature and mean SAP and DAP after the administration of dexmedetomidine in dogs that had been pre‐treated with the metaflumizone‐amitraz combination were not different (P > .05) than when the dogs were not pre‐treated. SpO2 was significantly higher at 10 and 20 min after onset of anaesthesia in the dogs pretreated with metaflumizone‐amitraz; however, the difference was small and very likely to be clinically irrelevant.

References

1. Antognini JF, Cartens E. Measuring minimum alveolar concentration: more than meets the tail. Anesthesiology 2005; 103: 751‐758. 2. Ko JC, Lange DN, Mandsager RE, Payton ME et al. Effects of butorphanol and carprofen on the minimal alveolar concentration of isoflurane in dogs. J Am Vet Med Assoc. 2000; 217(7):1025‐8. 3. March PA, Muir WW III. Bispectral analysis of the electroencephalogram: a review of its development and use in anesthesia. Vet Anesth Analg 2005; 32: 241 – 255. 4. Tyner CL, Greene SA, Hartsfield SM. Cardiovascular effects of butorphanol administration in isoflurane‐O2 anesthetized healthy dogs. Am J Vet Res. 1989; 50(8):1340‐2. 5. Ueyama Y, Lerche P, Eppler M, Muir WWIII. Effects of Perzinfotel, Fentanyl, and a Combination of Perzinfotel with Fentanyl on the Minimum Alveolar Concentration of Isoflurane in Dogs. (accepted: AJVR‐08‐10‐0355.R2). 6. Arbous MS, Grobbee DE, van Kleef JW et al. Mortality associated with anaesthesia: a qualitative analysis to identify risk factors. Anaesth 2001; 56 (12):1141 – 53. 7. Sewell JC, Raines DE, Eger EI 2nd et al. A comparison of the molecular bases for N‐methyl‐D‐ aspartate‐receptor inhibition versus immobilizing activities of volatile aromatic anesthetics. Anesth Analg 2009; 108(1):168 – 75. 8. Petrie A, Watson P. The distinction between statistical and biological difference. In: Statistics for Veterinary and Animal Science. 1rst ed. Oxford: Blackwell Science Ltd, 2003; 74.

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9. Thrusfield M. Statistical versus clinical (biological) significance. In: Veterinary Epidemiology.2nd ed. Oxford: Blackwell Science Ltd, 2001; 202. 10. Food and Drug Administration, HHS. International Conference on Harmonisation; guidance on S7A safety pharmacology studies for human pharmaceuticals; availability. Notice. Fed. Regist.2001; 66: 135: 36, 791 – 836. 11. Nathanson JA. Identification of octopaminergic agonists with selectivity for octopamine receptor subtypes. J Pharmacol Exp Ther. 1993;265:2:509‐15. 12. DeLay RL, Lacoste E, Mezzasalma T, Blond‐Riou F. Pharmcokinetics of metaflumizone and amitraz in the plasma and hair of dogs following topical application. Vet Parasitol. 2007;150:3:251‐257. 13. Hugnet C, Buronrosse F, Pineau X, Cadoré JL, Lorgue G, Berny PJ. Toxicity and kinetics of amitraz in dogs. Am J Vet Res. 1996;57(10):1506‐10. 14. Hsu WH, Lu ZX, Hembrough FB. Effect of amitraz on heart rate and aortic blood pressure in conscious dogs: influence of atropine, prazosin, tolazoline, and yohimbine. Toxicol Appl Pharmacol. 1986;84:2:418‐22. 15. Punke JP, Speas AL, Reynolds LR, et al. Kinetic gait and subjective analysis of the effects of a tachykinin receptor antagonist in dogs with sodium urate‐induced synovitis. Am J Vet Res 2007;68:704‐708. 16. Cross AR, Budsberg SC, Keefe TJ. Kinetic gait analysis assessment of meloxicam efficacy in a sodium urate‐induced synovitis model in dogs. Am J Vet Res 1997;58:626‐631. 17. Millis DL, Weigel JP, Moyers T, et al. Effect of deracoxib, a new COX‐2 inhibitor, on the prevention of lameness induced by chemical synovitis in dogs. Vet Ther 2002;3:453‐4631. 18. Brandt MR, Cummons TA, Potestio L, et al. Effects of the N‐methyl‐D‐aspartate receptor antagonist perzinfotel [EAA‐090; [2‐(8,9‐dioxo‐2,6‐diazabicyclo[5.2.0]non‐1(7)‐en‐2‐yl)‐ethyl]phosphonic acid] on chemically induced thermal hypersensitivity. J Pharmacol Exp Ther 2005;313:1379‐1386. 19. Farooqui AA, Ong WY, Horrocks LA. Inhibitors of brain phospholipase A2 activity: their neuropharmacological effects and therapeutic importance for the treatment of neurologic disorders. Pharmacol Rev 2006;58:591‐620. 20. Marvizon JC, McRoberts JA, Ennes HS, et al. Two N‐methyl‐D‐aspartate receptors in rat dorsal root ganglia with different subunit composition and localization. J Comp Neurol 2002;446:325‐341. 21. Petrenko AB, Yamakura T, Baba H, et al. The role of N‐methyl‐D‐aspartate (NMDA) receptors in pain: a review. Anesth Analg 2003;97:1108‐1116. 22. Pozzi A, Muir WW, Traverso F. Prevention of central sensitization and pain by N‐methyl‐D‐aspartate receptor antagonists. J Am Vet Med Assoc 2006;228:53‐60. 23. Liu H, Mantyh PW, Basbaum AI. NMDA‐receptor regulation of substance P release from primary afferent nociceptors. Nature 1997;386:721‐724. 24. Burke JE, Dennis EA. Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res 2009;50 Suppl:S237‐242. 25. Farooqui AA, Horrocks LA. Phospholipase A2‐generated lipid mediators in the brain: the good, the bad, and the ugly. Neuroscientist 2006;12:245‐260. 26. Svensson CI, Yaksh TL. The spinal phospholipase‐cyclooxygenase‐prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol 2002;42:553‐583. 27. Yaksh TL, Kokotos G, Svensson CI, et al. Systemic and intrathecal effects of a novel series of phospholipase A2 inhibitors on hyperalgesia and spinal prostaglandin E2 release. J Pharmacol Exp Ther 2006;316:466‐475. 28. Adler DH, Cogan JD, Phillips JA, 3rd, et al. Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction. J Clin Invest 2008;118:2121‐2131.

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29. Christie MJ, Connor M, Vaughan CW, et al. Cellular actions of opioids and other analgesics: implications for synergism in pain relief. Clin Exp Pharmacol Physiol 2000;27:520‐523. 30. Vaughan CW, Ingram SL, Connor MA, et al. How opioids inhibit GABA‐mediated neurotransmission. Nature 1997;390:611‐614. 31. Bodey AR. Systemic hypertension in the dog – Fact or fiction. Waltham/Ohio State University Symposium: Cardiology 1994; 44 ‐45. 32. Guyton AC. Vascular distensibility and functions of the arteries and venous system. In: Textbook of Medical Physiology (8th edn). Guyton AC (ed.). W.B Saunders, Philadelphia, PA, USA 1991; 162 – 163. 33. Haberman CE, Kang CW, Morgan JD, Brown SA. Evaluation of oscillometric and Doppler ultrasonic methods of indirect blood pressure estimation in conscious dogs. Can J Vet Res 2006; 70: 3: 211 – 217. 34. Branson KR, Wagner‐Mann CC, Mann FA. Evaluation of an Oscillometric Blood Pressure Monitor on Anesthetized Cats and the Effect of Cuff Placement and Fur on Accuracy. Veterinary Surgery 1997; 26: 347 – 353. 35. Pedersen KM et al. Evaluation of an oscillometric blood pressure monitor for use in anesthetized cats. JAVMA 2002; 221: 5: 646 – 650. 36. Haberman CE et al. Evaluation of Doppler Ultrasonic and Oscillometric Methods of Indirect Blood Pressure Measurement in Cats. Intern. J Appl Res Vet Med 2004; 2: 4: 279 – 289. 37. Feng M, Whitesall S, Zhang Y et al. Validation of Volume‐Pressure Recording Tail‐Cuff Blood Pressure Measurements. Am J of Hypertension 2008; 21: 12: 1288‐1291. 38. Deflandre CJA, Hellebrekers LJ. Clinical evaluation of the Surgivet V60046, a non invasive blood pressure monitor in anaesthetized dogs. Veterinary Anaesthesia and Analgesia 2008; 35: 13 – 21.

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CHAPTER 8

Conclusion CHAPTER 8

Pretreatment with perzinfotel (IM, IV, SQ) at dosages ranging from 10 to 30 mg/kg produced significant, dose dependant decreases in isoflurane MAC values which were associated with improvement in BIS and haemodynamic values in sexually intact male and female telemetered and anaesthetised dogs. The isoflurane MAC reduction was augmented by the concomitant use of butorphanol and did not produce any adverse effects (Chapter 2). In sexually intact male and female telemetered and anaesthetised cats, pretreatment with perzinfotel (IM, IV, SQ) at doses ranging from 2.5 to 15 mg/kg or a combination of butorphanol and perzinfotel produced significant decreases in isoflurane MAC and was associated with significant increases in BIS and blood pressure (Chapter 4). The SQ, IM or IV administration of perzinfotel as a pre‐anaesthetic treatment prior to isoflurane anaesthesia improves anaesthetic safety in both dogs and cats by reducing inhalant anaesthetic requirements. In an evaluation of the analgesic properties of perzinfotel, PLA‐695 and carprofen in mixed breed dogs, both the negative control and perzinfotel groups had significantly lower peak vertical force (PVF) and vertical impulse (VI) at 2 and 4 hours compared to baseline values in an induced urate crystal synovitis model.. No differences from baseline values for PVF and VI were seen in the PLA‐695 or carprofen groups at any measurement time points. Between group comparisons found significantly higher PVF and VI values in the carprofen group versus the no treatment and perzinfotel groups at 2 and 4 hours. The PLA‐695 group had higher impulse values versus dogs without treatment at 2 hours (Chapter 3). A high accuracy of both the external telemetry (VAP) and oscillometry (NIBP) was demonstrated and also the accuracy of internal telemetry (DSI system) was confirmed in Chapter 5. VAP and NIBP may provide reliable alternatives for invasive telemetry in future animal studies. NIBP is currently widely used in clinical veterinary practice. This study demonstrates that NIBP is an accurate and reliable method to measure blood pressure in anaesthetised cats. In telemetered dogs, prior treatment (24 hours) with a combination of metaflumizone‐ amitraz did not influence the haemodynamic response to dexmedetomidine. This confirms the safety of using an α2‐adrenergic agonist, such as dexmedetomidine, for anaesthetising dogs previously treated with a commercially available combination of metaflumizone and amitraz (Chapter 6).

90

Samenvatting

Het ontdekken van moderne geneesmiddelen is een proces dat zich met name afspeelt in wetenschappelijke instituten en in farmaceutische bedrijven. Zodra een nieuw actief middel is ontdekt wordt er vaak zo snel mogelijk een octrooi aangevraagd. Ondanks dat men tegenwoordig meer inzicht heeft in ziekteprocessen op een moleculair en een physiologisch niveau, blijft het ontdekken van nieuwe geneesmiddelen een lang en kostbaar proces. In de humane geneeskunde duurt het gemiddeld 13 jaar en kost het ongeveer 1,5 miljard euro om een nieuw geneesmiddel op de markt te brengen. Nadat een nieuwe actieve stof is ontdekt volgt een ontwikkelings traject dat getypeerd wordt door een intensieve samenwerking van een farmaceutisch bedrijf en de overheid van het land waar men het product tracht te registreren. Doordat de eisen van de overheden alsmaar strenger worden, wordt onwikkelingsproces van nieuwe geneesmiddelen moeilijker en tijdrovender. In de Verenigde Staten nam het aantal nieuwe registraties met 50% af tussen 2003 en 2008 in vergelijking met de 5 jaar daarvoor. Na de ontdekking van perzinfotel (een sterke NMDA antagonist) in het wetenschappelijk onderzoeks laboratorium van Wyeth in Princeton, New Jersey, werd het middel in 2006 aangeboden aan Fort Dodge Animal Health toen perzinfotel niet voldeed aan de Wyeth verwachtingen met betrekking tot onder andere de controle van neuropathische pijn. Fort Dodge Animal Health heeft daaropvolgend perzinfotel getest op anaesthesie sparende eigenschappen: in sexueel intacte mannelijke en vrouwelijke honden werd perzinfotel (IM, IV of SC) in een pre‐anesthetisch protocol gebruikt met doses varierend van 10 tot 30 mg/kg (Hoofdstuk 2). Perzinfotel gaf een significante, dosis afhankelijke daling van de isofluraan MAC waarden. Deze daling was geassocieerd met een verbetering van de BIS en haemodynamische waarden. De isofluraan MAC reductie nam toe bij het gelijktijdig gebruik van butorphanol en leidde niet tot negatieve bijwerkingen. In sexueel intacte mannelijke en vrouwelijke katten leidde het gebruik van perzinfotel (IM, IV of SC) in doses varierend van 2,5 tot 15 mg/kg eveneens als bij het gebruik van de combinatie van perzinfotel en butorphanol tot significante dalingen van de isofluraan MAC (Hoofdstuk 4). Deze dalingen waren geassocieerd met signficante stijgingen in de BIS en de bloeddruk. Hieruit valt te concluderen dat het subcutane (SC), intramusculaire (IM) of intra‐ veneuze (IV) gebruik van perzinfotel in de hond en de kat in een pre‐anaesthetisch protocol kan resulteren in een toename van de veiligheid van de anaesthesie door een reductie in het gebruik van inhalatie gassen. Om de analgetische eigenschappen van perzinfotel te bestuderen werd perzinfotel tesamen met een ander potentieel analgetisch middel, PLA 695, uitgetest in een pijn model in honden (Hoofdstuk 3). Hierbij werd geconcludeerd dat perzinfotel geen significante

SAMENVATTING

pijnstillende werking had bij een dosis van 10 mg/kg, terwijl PLA 695 op 2 uur na toediening van de pijnstimulus door middel van injectie van uraat kristallen in het kniegewricht, wel een significante pijnstillende werking had bij een dosis van 10 mg/kg. Eveneens werden drie methoden van het meten van de bloeddruk bij de kat vergeleken (Hoofdstuk 5). Een externe invasieve methode, door middel van een vasculaire toegangspoort, en een externe non‐invasieve methode, door middel van een staart cuff, werden gevalideerd ten opzichte van de standaard methode (een interne invasieve methode). Hierbij werd geconcludeerd dat zowel de externe invasieve methode als de externe non‐ invasieve methode accuraat waren. Beide methodes kunnen in de toekomst als alternatief dienen voor de kostbare invasieve methode. In de diergeneeskunde praktijk wordt met name de externe non‐invasieve methode gebruikt. Deze studie bevestigt dat de bestudeerde staart cuff een accurate en betrouwbare methode is voor bloeddruk metingen van katten onder anaesthesie. Tenslotte werden honden die voorafgaand waren uitgerust met interne bloeddrukmeters behandeld met een combinatie van metaflumazone en amitraz (Hoofdstuk 6). Deze behandeling die vooraf ging aan de anaesthesie, had geen invloed op de haemodynamische waarden tijdens anaesthesie met dexmedetomidine. Dit bevestigt de veiligheid van het gebruik van een α2‐adrenerge agonist zoals dexmedetomidine als anaesthesie middel in honden die vooraf zijn behandeld met een commercieel verkrijgbare combinatie van metaflumazone en amitraz.

92

Acknowledgements

First and foremost I would like to thank Rami Cobb for offering me the opportunity and the support to pursue my long term dream of doing research. Rami, you personally made it possible for me to make the transition from a Technical Services Veterinarian in Sydney Australia to a Principal Lead Scientist II in the Fort Dodge Animal Health Facility in Princeton, NJ. I will never be able to thank you enough for your support. I also would like all my former Fort Dodge colleagues who supported me in my research projects; Mark Eppler, Doug Rugg, Bob Pollet, Albert Boeckh and Debbie Amodie; your support was invaluable. Also the support from my colleagues and friends at QTest Labs in Columbus, Ohio was invaluable for achieving all but one of the publications. Bill Muir, thank you so much for your guidance and Carlos DelRio, “muchissimas gracias” to you for all your hard work. I also would like to thank Ingrid van der Gaag for inspiring me at the right times to pursue my dreams of doing a PhD. Professor Jolle Kirpensteijn, the indignation on your face when I mentioned I might do this PhD through the University of Sydney, I will never forget. Isn’t Utrecht in the top 5 of veterinary science? You absolutely made it work for me. To do a PhD from the other side of the world, especially in combination with a new and challenging position is only possible with a supervisor who responds quickly to e‐mails and is very (pro‐)active; I thank you so very much for all your support over the last year. Last but not least I would like to thank my parents. Although you often had no idea why I was pursuing what I was pursuing, you were always there for me with a listening ear and loads of patience. Muchos gracias!

Curriculum Vitae

Raphael Zwijnenberg was born 22 December 1961 in Winterswijk, the Netherlands. After high school graduation in 1980, Raphael started studing Veterinary Medicine at Utrecht University. Raphael Zwijnenberg graduated as a DVM from this University in 1987. In 1988 Raphael completed a Postgraduate Masters of Veterinary Science degree (MVSc) in Tropical Animal Medicine and Animal Production in the Tropics, "IEMVT" in Paris, France. This was followed by a three‐year period working in the department of exotics of the University of Utrecht as a clinician and a pathologist. Raphael then lived in Israel for a year working for the Department of Agriculture as a research scientist on both bovine and ovine babesiosis. After returning to the Netherlands, Raphael worked in 2 separate self‐owned practices, combining practice work with a part‐time job in the Amsterdam Zoo for 4 years. In August 2002 Raphael immigrated to Australia where he accepted the position of a Technical Services Manager of the ethical (companion animals) division at Fort Dodge Animal Health in Sydney. In April 2005 Raphael graduated as a Master of Veterinary Public Health Management (MPHVMgt) at the University of Sydney and in July 2005 Raphael became a Member of the Epidemiology Chapter of the Australian College of Veterinary Scientists (MACVSc). On 1 September 2007 Raphael started working as a Principal Lead Scientist II in pharmaceutical discovery and development with Fort Dodge Animal Health in Princeton, New Jersey, USA. In this role Raphael quickly became accredited as a veterinarian in the USA, published several articles and was involved in the discovery of new drugs that are currently being considered by Pfizer Animal Health for further development. In December 2009, Raphael returned to Sydney to accept a position with Pfizer Animal Health as a New Product Marketing Manager. In this new position Raphael is responsible for evaluation of new products for the Pfizer Animal Health Australia Portfolio as well as the communication between research departments in Australia and internationally and the Australian marketing team.

List of publications

• Zwijnenberg RJG, Vulto AG, Miert ASJPAMv, Lumeij JT, 1992: Evaluation of anthelmintics, antiprotozoal drugs and ectoparasiticides for racing pigeons (Columba livia var. domestica) available in the Netherlands. J Vet Pharmacol Ther. 15 (4): 395 ‐ 408.

• Zwijnenberg RJG, Vulto AG, Miert ASJPAMv, Lumeij JT, 1992: Evaluation of antibiotics for racing pigeons (Columba livia var. domestica) available in the Netherlands. J Vet Pharmacol Ther. 15 (4): 364 ‐ 378.

• Zwijnenberg RJG, Zwart P, 1994: Squamous Metaplasia in the salivary glands of canaries (a case report). Vet Quarterly 16 (1): 60 ‐ 61.

• Lumeij JT, Zwijnenberg RJG, 1990: Failure of nitroimidazole drugs to control trichomoniasis in the racing pigeons (Columba livia domestica). Avian Pathology 19: 165 – 166.

• Zwijnenberg RJG, 2003: The recent availability in Australia of a vaccine to protect dogs against both coronavirus and Leptospira icterohaemorrhagiae. Aust Vet J 81 (12):731.

• Zwijnenberg RJG, 2005: Feline immunodeficiency virus vaccine issues. Aust Vet J 83 (4):215.

• Kann RK, Kyaw‐Tanner MT, Seddon JM, Lehrbach PR, Zwijnenberg RJG, Meers J, 2006: Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia. Aust Vet J 84 (4):112 – 116.

• Zwijnenberg RJG, Smythe LD, Symonds ML, Dohnt MF, Toribio J‐ALML, 2008: A cross‐ sectional study of canine leptospirosis in animal welfare populations in mainland Australia. Aust Vet J 86 (8): 317 ‐323.

• Kann R, Seddon J, Zwijnenberg R, Meers J, 2007. Feline immunodeficiency virus subtypes in domestic cats in New Zealand. New Zeal Vet J 55 (6):358 ‐ 360.

• Meers J, Kyaw‐Tanner M, Bensink Z, Zwijnenberg R, 2007. Genetic analysis of canine parvovirus from dogs in Australia. Aust Vet J 85 (10):392 ‐ 396.

• Zwijnenberg RJ, Del Rio CL, Pollet RA, Muir III WW, 2010. Effects of perzinfotel on the minimum alveolar concentration of isoflurane in dogs when given as a pre‐anesthetic IV, IM or SQ and in combination with butorphanol. Am J Vet Research 71 (6): 604‐ 609.

• Zwijnenberg RJ, Muir III WW, 2009: Evaluation of the potential for interaction between a metaflumizone‐amitraz combination and dexmedetomidine hydrochloride in dogs. Vet Therap 10 (1‐2): 40 – 45.

• Yamamoto KJ, Huang C, Zwijnenberg R, Motokawa K, Hohdatsu T. Prophylactic Efficacies and Mechanisms Of The Prototype And Commerial Dual‐Subtype Feline Immunodeficiency Virus (FIV) Vaccines. AIDS Vaccine 2009. Abstract.

• Pu M, Martin MM, Coleman JK, Brettas L, Zwijnenberg R, Abbott, JR, Yamamoto JK. Dual‐subtype (A+D) FIV vaccine efficacies and mechanisms of protection against global FIV subtypes and circulating recombinants. Vaccine: submitted for publication September 2009.

• Zwijnenberg RJ, Del Rio CL, Pollet RA, Muir III WW. Effects of perzinfotel, butorphanol and a butorphanol‐perzinfotel combination on the minimum alveolar concentration of isoflurane in cats. Am J Vet Research, accepted for publication 15 October 2009.

• Zwijnenberg RJ, Del Rio CL, Cobb R, Ueyama Y, Muir III WW. Evaluation of Oscillometric and Vascular‐Access‐Port Derived Arterial Blood Pressure Measurement Techniques in Anesthetized Cats: Comparative Performance versus Implanted Telemetry. Am J Vet Research, accepted for publication 25 May 2010.

• Budsberg S, Torres B, Zwijnenberg R, Clark J, Eppler M, Cathcart C, Reynolds L, Al‐ Nadaf S. Effects of perzinfotel and PLA‐ 695 on kinetic gait and subjective lameness scores in a sodium urate‐ induced synovitis model in dogs. Am J Vet Research, accepted for publication March 2010.

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