Short duration anaesthesia with medetomidine and ketamine in cynomolgus monkeys

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1 Short duration anaesthesia with medetomidine a ketamine in cynomolgus monkeys S. S. Young, A. M. Schilling, S. Skeans & G. Ritacco Department of Allergy, Schering-Plough Research Institute, Kenilworth, New Jersey, USA Summary Cynomolgus monkeys were anaesthetized with either intramuscular ketamine (10 mg/kg or intramuscular ketamine 2 mg/kg a medetomidine 50 Ilg/kg. Various physiological measurements were made once the animals were safe to hale a again 10 min later. Cardiovascular a respiratory function were well maintained with both regimens but the heart rate was lower a arterial-alveolar carbon dioxide gradient was higher in the animals that received medetomidine. In those animals that received medetomidine, atipamezole was given to reverse the medetomidine but there was no difference in recovery times between the two regimens. Anaesthesia was not entirely reliable with medetomidine/ketamine a we recomme caution when using this mixture. Keywords Reversible anaesthesia; non-human primates; cardiovascular function; respiratory function The cynomolgus monkey (Macaca fascicularis) is used quite widely in research for respiratory a toxicological work. Nonhuman primates present special hazards to halers, particularly the danger of bites a zoonotic infections. There are several techniques that allow relatively safe haling of conscious non-human primates (e.g. pole a collar system a primate chairs) but for many procedures it is safer for the haler a less stressful for the animal if it is anaesthetized. Ketamine is by far the most widely used drug for the iuction of anaesthesia in nonhuman primates. It has a combination of properties that make it the anaesthetic of choice in most circumstances: rapid iuction of anaesthesia after intramuscular injection, a wide safety margin, little or no cardiovascular a respiratory depression Correspoence to: Dr Simon Young, Schering Plough Research Institute, m(s K , 2015 Galloping Hill Road, Kenilworth, N! 07033, USA. Tel: , Fax: , simon.young@spcorp.com (Haskins et al. 1985)a reliable action. One problem with intramuscular ketamine is its relatively long duration of action. For very short procedures such as blood sampling or tuberculosis testing it would be desirable to have an agent with a more rapid recovery. It is difficult to do this with any agent given by the intramuscular route, because the slow absorption means that a large dose (relative to the intravenous route) has to be given in order to achieve anaesthetic blood levels. Sedative a anaesthetic drugs that are reversible with specific antagonists offer a way arou this pharmacokinetic problem. Antagonists are available for several classes of drugs including the benzodiazepines, opiates a alpha2-adrenergic agonists. Successful regimens that have been reported in laboratory animals include midazolam-fluanisone-fentanyl anaesthesia partially reversed with opioid antagonists, a medetomidine-fentanyl anaesthesia that is completely reversed with antagonists to both agents (Flecknell1996). Medetomidine is a relatively new alpharadrenergic agonist that Accepted 29 July 1998 Laboratory Animals Ltd.,

2 Medetomidinejketamine in primates 163 is reversible with the specific antagonist atipamezole. The aim of this study was to investigate the use of a combination of ketamine a medetomidine as a partially reversible intramuscular anaesthetic agent in cynomolgus monkeys. Materials a methods Animals Fifteen male cynomolgus monkeys were used in this study. They were captive bred at the Schering-Plough facility in Lafayette, New Jersey, USA a housed at the Schering- Plough Research Institute, Kenilworth, New Jersey, USA. Their mean weight was 6.2±2.0 kg (mean±sd) a their ages were approximately 3-10 years. The animals were housed in stainless steel primate cages in groups of two or three animals per cage, with the cage size appropriate for the number of animals, in a closed system with limited access. Temperature a relative humidity were kept at 22 C a 40%. Lighting was 12 h light (750 lumens) a 12 h dark, 07:00 h to 19:00 h. Air was HEPA filtered 100% fresh air with changes per hour. They were fed a staard monkey diet (Monkey Diet 5038, PMI Feeds Inc., St Louis, Missouri, USAl with reverse osmosis water ad libitum. Animals were quarantined for 6 weeks before entry to the colony a tested negative for tuberculosis, enteric pathogens, herpes viruses, haematological a blood chemistry disturbances a parasites. Animals uerwent a routine physical a haematological analysis every 6 months. An environmental enrichment programme ensured that the monkeys were provided with cage toys, television, foraging boards, fresh fruit a vegetables a other stimuli. The experimental protocol was approved by Schering-Plough's Animal Care a Use Committee. Procedure Experiments were performed in the morning after an overnight fast. Twelve of the animals were raomly assigned to receive either intramuscular ketamine (Ketaject, Phoenix Scientific Inc., St Joseph, Missouri, USA) at a dose of 10 mg/kg (KET treatment group) or intramuscular ketamine 2 mg/kg a medetomidine 50 /lg/kg (Domitor, Pfizer Animal Health, Exton, Pennsylvania, USA) mixed in the same syringe just before use (KET/MED treatment group). Three animals received intramuscular ketamine 4 mg/kg a medetomidine 50 /lg/kg (HIGH KET/MED treatment group). When the animal became recumbent a did not show purposeful movements in response to touch it was removed from the cage a placed in dorsal recumbency on a heated water blanket. The time from injection to removal from the cage (the iuction time) was recorded. Several physiological parameters were measured as soon as the monkey was removed from its cage a again 10 min later. Systolic, mean a diastolic pressure were measured iirectly (Dinamap Veterinary model 8300, Critikon, Tampa, Florida, USA) with a cuff arou the leg just above the tarsus or arou the humerus. Core temperature was measured with an oesophageal probe (Datex Cardiocap II, Datex Engstrom, Tewkesbury, Massachussetts, USA). E tidal carbon dioxide was measured with a sidestream analyser (Datex Cardiocap II), the probe being held just inside one nostril a the other nostril a mouth occluded. Arterial oxygen saturation was measured with a pulse oximeter (model 8500V, Nonin Medical Inc., Plymouth, Minnesota, USAI with the probe on the upper lip. An arterial blood sample was taken from the femoral artery uer anaerobic coitions, stored on ice a analysed within 2 h (ABL50,Radiometer America Inc., Paramus, New Jersey, USAI. The arterial-alveolar carbon dioxide gradient l[a-a]pco,) was calculated as the difference between the arterial a the e tidal carbon dioxide tensions. After the seco set of physiological measurements the animals that had received KET were kept on the heated blanket uer observation until they had to be returned to their cages (grasping reflexes a/or gross limb movements present). Animals that had received KET/MED or HIGH KET/MED were returned straight away to their cages a the medetomidine reversed with 250 /lg/kg atipamezole (Antisedan, Pfizer

3 164 Young et al. Animal Health, Exton, Pennsylvania, USA) i.m. Recovery time was defined as the time from the e of the physiological measurements (ketamine only) or the time from the injection of atipamezole lketamine/ medetomidine group) to the point at which the monkey sat up in its cage. Total anaesthesia time was defined as the time from the first injection of anaesthetic to the point at which the monkey sat up. Data from the six animals that received KET were compared statistically to animals that received KET/MED. Single data points (iuction/recovery times a blood gas data) were compared with unpaired t tests a physiological measurements were compared with repeated measures analysis of variance using staard statistical software (Statview a SuperANOVA, Abacus Concepts Inc., Berkeley, California, USA). Results The results are summarized in Tables 1-4. There were no significant differences in iuction, recovery a total anaesthesia times between the KET a KET/MED groups. The mean time to complete the study, from injection of the anaesthetic to moving the animal into recovery, was 16.5 min. Blood gas variables were not different between the KET a KET/MED groups but (a-a)peo. was significantly higher in the KET/MED group. The heart rate was significantly higher in the KET group compared to the KET/MED group with no change over time. E tidal CO 2 rose significantly in the KET/MED group with time. Mean a diastolic blood pressures a body temperature fell significantly with time with no difference between treatment groups. One animal in the KET/MED group (data not included) was so light that measurements were impossible a it was returned to its cage. Another animal in the same group required manual restraint to ensure that measurements could be made safely. Two animals in the KET group had spontaneous limb movements during measurements, but we were able to collect reliable data from these animals a the results were included in the analyses_ None of the animals vomited during the experiments. Discussion This study demonstrates that ketamine 2 mg/kg a medetomidine 50 Ilg/kg is an effective intramuscular anaesthetic mixture in cynomolgus monkeys with cardiovascular a respiratory effects similar to that of ketamine 10 mg/kg. We have some reservations about its safety a reliability. Table 1 Physiological variables in the ketamine 10 mg/kg treatment group (mean±sd) Variable Heart rate (bpm) Respiratory rate (bpm) Systolic blood pressure (mmhg) Mean blood pressure (mmhg) Diastolic blood pressure (mmhg) Temperature (0C) Arterial oxygen saturation (%) E tidal CO 2 (kpa) Arterial ph PaC0 2 (kpa) Pa02 (kpa) HCo 3 - (mm) Actual base excess(mm) (a-a)pco, (kpa) Immediate 142.2±20.3* 30.7± ± ± ± ± ± ± ± ± ± ± ±2.6 O.19±O.48* +10 min ± 22.7* 34.3± ± ±22.1 t 53.2 ± 14.4 t 37.S ± 1.0 t 92.S±7.8 S.58±O.36 : not determined; *Significantly different to ketamine 2 mgjkg + medetomidine 50 I'gjkg; tsignificant change with time

4 Medetomidine/ketamine in primates 165 Table 2 Physiological variables in the ketamine 2 mg/kg + medetomidine 50 l19/k9 treatment group (mean ± SO) Variable Heart rate (bpm) Respiratory rate (bpm) Systolic blood pressure (mmhg) Mean blood pressure (mmhg) Diastolic blood pressure (mmhg) Temperature (DC) Arterial oxygen saturation (%) E tidal CO 2 (kpa) Arterial ph P a C0 2 (kpa) P a 0 2 (kpa) HC0 3 - (mm) Actual base excess(mm) (a-a)pco, (kpa) Immediate 99.3± 10.9* 32.0± ± ± ± ± ± ± ± ± ± ± ± ± 0.40* + 10 min 90.5±4.8* 26.0± ± ± 12.0 t 49.0±8.0 t 37.6±0.4 t 93.5± ±0.38 t : not determined; *Significantly different to ketamine 10 mg/kg; tsignificant change with time Table 3 Physiological variables in the ketamine 4 mg/kg + medetomidine 50I19/kg treatment group (mean ± SO) Variable Heart rate (bpm) Respiratory rate (bpm) Systolic blood pressure (mmhg) Mean blood pressure (mmhg) Diastolic blood pressure (mmhg) Temperature (0C) Arterial oxygen saturation (%) E tidal CO 2 (kpa) Arterial ph P a C0 2 (kpa) P a 0 2 (kpa) HC0 3 - (mm) Actual base excess(mm) (a-a)pco, (kpa) Immediate 95.7± ± ± ± ± ± ± ± ± ± ± ± ± ± min 85.7± ± ± ± ± ± ± ±0.15 Note: values are descriptive only, no statistical comparison was performed between this a other treatment groups Table 4 Iuction. recovery a total anaesthesia times. Medetomidine. if given. was reversed with atipamezole (mean ± SO) Treatment Iuction time Recovery time Total anaesthesia (min) (min) time (min) Ketamine 10 mg/kg 4.0± ± ± 10.5 Ketamine 2 mg/kg + 4.3± ± ±3.8 medetomidine 50 J.lg/kg Ketamine 4 mg/kg + 3.2± ± ± 1.0 medetomidine 50 J.lg/kg

5 166 Young et al. Cardiovascular a respiratory effects The heart rate was lower in the KETjMED group. Bradycardia is a consistent feature of alpha2-adrenergic agonists (Cullen 1996) a is due to a combination of central reduction in sympathetic drive to the heart a reflex bradycardia following peripheral vasoconstriction (Keegan et al. 1995). In this study there was no difference in blood pressure between the KET a KETjMED groups a thus the bradycardia is likely to be central in origin, although an initial transient hypertension would not have been detected. Blood gas variables a arterial oxygen saturation were good for anaesthetized animals breathing room air. However, the mucous membranes in the KETjMED group appeared pale a cyanotic. This is commonly seen in other animals given medetomidine (Ra et al. 1996) a in the dog a cat it is due not to arterial hypoxaemia but to very low venous oxygen content secoary to a reduced cardiac output (FlecknellI9971. Serteyn et al. (1993) reported a fall in cardiac output from 194 to 84 mljkgjmin after giving ketamine 5 mgjkg a medetomidine 40 J.lgjkg intramuscularly to dogs. The significantly elevated la-alpco2 in the KET jmed group suggests that there was ventilation-perfusion mismatch which may be related to circulatory abnormalities. Arterial blood pressure was reported to be 121 mmhg systolic, 60 mmhg diastolic a 84 mmhg mean in conscious cynomolgus monkeys (Chester et al. 1992). The values in this study were lower but still acceptable for anaesthetized animals. The mean a diastolic pressures fell with time, possibly due to deepening of the plane of anaesthesia as the drugs were absorbed from the injection site. Iuction a recovery times The aim of this study was to develop an anaesthetic regimen with recovery times shorter than that of ketamine alone. The iuction a recovery times were not different between the two treatment groups a thus it appears that KETjMED has little advantage over KET. However, the mean duration of the study was 16.5 min a one would expect KET to be wearing off after this period. KETjMED will be most useful for very short (1-2 min) procedures where the recovery time (after reversal) is expected to be shorter than KET. It should be pointed out that the perceived advantages of KETjMED become more apparent when one considers the total time from iuction until the monkey is sitting up in its cage. One problem with the study design was that the KET group spent most of the recovery time on a heated blanket whereas the KETjMED a HIGH KETjMED groups recovered in their cages. Hypothermia will delay recovery from anaesthesia a the difference in recovery coitions may have influenced the recovery times. It was necessary for safety reasons to return the animals in the KETjMED a HIGH KETjMED groups to their cages immediately after injection of atipamezole, a thus the alternatives were to recover the KET group in their cages as well or place heated blankets in the cages for the KETjMED a HIGH KETjMED groups. There are problems with both these approaches. Recovering animals given KET in their cages invite further problems with hypothermia, a heating blankets placed in the cages would have been destroyed by the monkeys. In a different study KETjMED was used to immobilize 20 cynomolgus monkeys for oral dosing by stomach tube, a very short procedure suitable for this regimen. After oral dosing the monkeys were returned to their cages a atipamezole given i.m. The total time from injection of the anaesthetic agent to the animal sitting up in its cage (a thus safe to leave) was typically min. Although specific times were not recorded we had the impression that this was shorter than when KET was used. The time from injection of the atipamezole to the animal sitting up was usually 5 to 7 min. Side effects Vomiting is a common side effect of alpha2- adrenergic agonists in dogs a cats (Vainio 1989, Cullen 1996, Flecknell 1997). None of the monkeys in this study retched or vomited. In addition, retching a vomiting were

6 Medetomidinejketamine in primates 167 not seen in the 20 animals given KET/MED for oral dosing even though the procedure involved placing 2.5 ml/kg fluid in the stomach via an orogastric tube. Safety a reliability When working with non-human primate the safety of the halers is paramount a an anaesthetic that produces a reliably immobilized animal is needed, even at the expense of some cardiorespiratory depression. In pilot studies we fou that doses of medetomidine alone up to 100 J.lg/kg did not produce reliable sedation. More worrying was the fact that some animals appeared to be heavily sedated but could suddenly become aroused if stimulated. This effect is well known in horses sedated with alpha2-adrenergic agonists such as xylazine a detomidine (Hall & Clarke 1991). The addition of ketamine to medetomidine improved the reliability of the drug in cynomolgus monkeys at the expense of some loss of reversibility. However, one animal suddenly awakened with little warning after being given KET/MED. This lack of 'warning signs' such as limb movement is the biggest drawback to using KET/MED in cynomolgus monkeys a the doses used in this study should be regarded as minima. Additionally, the lack of warning of awakening limits the use of the mixture to short procedures. Morris a Hoare (1994) also advised caution in primates sedated with medetomidine. We gave three monkeys HIGH KET/MED a cardiorespiratory variables were acceptable although the Pa02 was lower a PaC02 higher, suggesting some respiratory depression (Table 3). This mixture deserves further investigation. There is little literature on the use of medetomidine in non-human primates. King (19941 reported that he had used ketamine 5 mg/kg a medetomidine 100 J.lg/kg in several species of monkey but gave no details. He also reported that a capuchin monkey required IS mg/kg ketamine a 266 J.lg/kg medetomidine for adequate sedation. Morris a Hoare (1994) reported that 10 J.lg/kg medetomidine caused sedation in cynomolgus monkeys a noted a considerable species difference in response to mede- tomidine. They suggested that atipamezole should not be given to animals soon after anaesthesia with ketamine/medetomidine because of the risk of excitement due to high levels of ketamine. Excitement after reversal was not seen in this study a we question whether it is a problem in primates, because ketamine is widely used as a sole anaesthetic in non-human primates. In contrast to medetomidine, xylazine (an older alpha2- adrenergic agonist) a ketamine has been used fairly extensively for anaesthesia in non-human primates. Turner et a1. (19961 used ketamine 10 mg/kg a xylazine 1 mg/kg intramuscularly in cynomolgus monkeys a Guel et al. (1991) used ketamine 4 mg/kg a xylazine 1 mg/kg intramuscularly in the same species. 1 mg/kg xylazine is equipotent to 14 ~g/kg medetomidine in the dog (Vainio et a ) but the relative potency of the two drugs in cynomolgus monkeys is not known. Our selection of a dose of SO ~g/kg medetomidine was based on the literature for dogs a pigs because reports of non-human primate use of medetomidine did not offer much guidance. Pilot studies using SO a then 100 J.lg/kg medetomidine showed that both doses produced heavy but unreliable sedation. Ketamine was therefore added at the lowest dose that produced reliable sedation. The relatively low doses of ketamine used in this a other studies of ketamine + alpha2-adrenergic agonist are insufficient on their own to immobilize a monkey. Thus when using atipamezole to reverse the medetomidine the medetomidine must be given to the animal when it is back in its cage, a after this the animal should be treated as if it were unanaesthetized. Typically, monkeys given KET/MED a reversed with atipamezole slept for about 7 min after administration of the atipamezole a then sat up. Some ataxia a nystagmus were present, due to the residual ketamine, but the animals were safe to be left unatteed. References Chester AE, Dorr AE, Lu K, Wood LD (1992) Noninvasive measurement of blood pressure in conscious cynomolgus monkeys. Fuamental a Applied Toxicology 19,64-8

7 168 Young et al. Cullen LK (1996) Medetomidine sedation in dogs a cats: a review of its pharmacology, antagonism a dose. British Veterinary Journal 152, Flecknell P (1996) Laboratory Animal Anaesthesia, 2 edn. Loon: Academic Press Limited, p. 163 Flecknell P (19971Medetomidine a atipamezole: potential uses in laboratory animals. Lab Animal February, 21-5 Guel RH, Letts LG, Gleich GJ (19911Human eosinophil major basic protein iuces airway constriction a airway hyperresponsiveness in primates. Journal of Clinical Investigation 87, Hall LW, Clarke KW (1991) Veterinary Anaesthesia, 9th edn. Loon: Balliere Tiall, pp Haskins SC, Farver TB, Patz JD (19851Ketamine in dogs. American Journal of Veterinary Research 46, Keegan RD, Greene SA, Bagley RS, Moore MP, Wei! AB, Short CE (1995) Effects of medetomidine administration on intracranial pressure a cardiovascular variables of isoflurane-anaesthetized dogs. American Journal of Veterinary Research 56, King A (1994) Anaesthesia of capuchin monkeys (letter). Veterinary Record 134, 559 Morris TH, Hoare C (1994) Sedation of exotic species (letter). Veterinary Record 134, Ra JS, Reynolds WT, Priest J (1996) Echocardiographic evaluation of the effects of medetomidine a xylazine in dogs. Australian Veterinary Journal 73,41-4 Serteyn D, Coppens P, Jones R, Verstegen J, Philipparts C, Lamy M (1993) Circulatory a respiratory effects of the combination medetomidineketamine in beagles. Journal of Veterinary Pharmacology a Therapeutics 16, Turner CR, Aresen CJ, Smith WB, Watson JW (1996) Characterization of a primate model of asthma using anti-allergyjanti-asthma agents. Inflammation Research 45, Vainio 0, Viihii-Vahe T, Palmu L (19891Sedative a analgesic effects of medetomidine in dogs. Journal of Veterinary Pharmacology a Therapeutics 12, Vainio 0 (1989) Introduction to the clinical pharmacology of medetomidine. Acta Veterinaria Scainavica 85, 85-8

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