An analgesiometry system for use in rabbits with some preliminary data on the effects of buprenorphine and lofentanil

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Laboratory Animals (19) 22, 217-222 217 An analgesiometry system for use in rabbits with some preliminary data on the effects of buprenorphine and lofentanil R. WOOTTON*, G. CROSSt, S. WOOD* and C. D.

1 Laboratory Animals (19) 22, An analgesiometry system for use in rabbits with some preliminary data on the effects of buprenorphine and lofentanil R. WOOTTON*, G. CROSSt, S. WOOD* and C. D. WESTt Divisions of Comparative Medicine and Bioengineering, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ, UK Summary A low cost infrared skin heating system has been designed to measure the efficacy of analgesics in rabbits. Following construction of a prototype, it was used to assess the effect of buprenorphine given subcutaneously and per rectum. Buprenorphine administered subcutaneously has a rapid onset of action, but its duration (-10 h) appears slightly shorter than has been suggested previously; rectal administration appears to prolong its effect. Preliminary data show that lofentanil has a longer duration of action than buprenorphine and it may prove, therefore, to be a valuable long-acting analgesic in the rabbit. Keywords: Analgesiometry; Rabbits; Buprenorphine; Lofentani/ Assessment of the potential analgesic activity of drugs is normally undertaken in animals and a large number of different tests have been described (for reviews see Evans, 1964; Taber, 1974), which involve a range of noxious stimuli including electrical stimulation, pressure, heat and chemical irritants. While virtually all analgesic drugs have been developed for clinical use in man, their analgesic activity is usually assessed Present address: Department of Medical Physics, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London WI2 OHS, UK tpresent address: Bellhouse Medical Products, Abingdon OXI4 IDY, UK IPresent address: Imperial Chemical Industries, Macclesfield, SKIO 4TG, UK Received July 197; accepted 23 September 197 first only in small rodents; final testing is then carried out in human subjects. For various reasons, there has been a growing awareness of the need to alleviate pain in laboratory animals and in animals presenting as patients in veterinary clinical practice (Yoxall, 197; Baumberger, 193;Flecknell, 194; Morton and Griffiths, 195; Taylor, 195; Wright et al., 195; Association of Veterinary Teachers and Research Workers, 196; British Veterinary Association Animal Welfare Foundation, 196). However, the use of analgesics in these circumstances is often hampered by lack of information about their potency in non-rodent species. We have therefore designed a simple analgesiometer for the assessment of the analgesic activity of compounds in larger animals. The analgesiometer utilizes a thermal stimulus to the skin. Following the construction of a prototype, it was used in the rabbit for a preliminary evaluation of the analgesic activity of buprenorphine (Temgesic, Reckitt and Colman, Hull) given both subcutaneously and per rectum, and lofentanil, an extremely long-acting opioid analgesic (Janssen, 192). Materials and methods A nalgesiometer The analgesiometer consists of two parts, a power unit and a hand-held beam projector unit connected by a cable (Fig. 1). The power unit also contains the infrared (Lr.) power calibration components and carries a mounting socket for the hand-held unit. The hand-held unit contains the optical system, a motor-driven shutter assembly and an electronic timer.

Wootton, Cross, Wood & West 21 Fig. t. Photograph of the skin heating system. The power unit is on the left and the hand-held infrared projector unit on the right.

2 Wootton, Cross, Wood & West 21 Fig. t. Photograph of the skin heating system. The power unit is on the left and the hand-held infrared projector unit on the right. The white light aiming beams can be seen on the skin of the subject../'i-r ~ The light source is a 12 V quartz halogen slide projector lamp of 100 W power (Type Al/215 Philips, Croydon). Two dichroic mirrors are positioned, equally spaced about the lamp (Fig. 2). Light from the lamp falls on the mirrors and is reflected through 90, passing through pin hole collimators to produce two cold light beams which intersect O 14 m in front of the lens assembly. The hot i.r. light from the lamp passes through the mirror and is absorbed by a cover. The cover screens the electronics box, and shields both the operator and subject from the intense light and heat produced by the lamp. The i.r. heat beam is produced by a lens and filter assembly. A plano-convex condenser lens of 50 mm focal length is focused on the lamp filament so that light passing through the lens forms a parallel beam. This beam passes through energy,. Dichroic mirror,'...1pin hole.. Fig. 2. Diagram of the beam projector unit.

A rabbit analgesiometer 219 another plano-convex lens, focused to form the filament image at 0 14 m. A glass i.r. pass filter cuts out most of the white light, thus producing an i.r. heat beam.

3 A rabbit analgesiometer 219 another plano-convex lens, focused to form the filament image at 0 14 m. A glass i.r. pass filter cuts out most of the white light, thus producing an i.r. heat beam. The motor-driven shutter normally blocks off the heat beam between the lamp and the lens assembly. The shutter is driven by a slipping clutch and controlled by a trigger switch mounted on the handle of the beam projector unit. When the trigger is depressed the shutter is lowered so that heat reaches the subject. When the switch is released, the shutter rises automatically and occludes the heat beam. The trigger switch is connected to an electronic timer which displays elapsed skin heating time to the operator. The power in the heat beam can be checked by a meter mounted on the power unit. The beam projector fits into a socket on the power unit and when the trigger is depressed, the heat beam illuminates a photodetector mounted in the meter box behind an integral i.r. pass filter. The meter reading is proportional to the i.r. power falling on the photodetector. Experimental design New Zealand White rabbits of both sexes weighing between 2 and 4, 7 kg were used. A double-blind crossover design was employed in which each animal received in random order either placebo (for a control measurement) or the test substance; neither the animal nor the observer knew which substance was being administered. Analgesic test substances were administered at the following dose rates: (i) buprenorphine injection (Temgesic, Reckitt and Colman; 300/-tg/ml) subcutaneous 0 1 ml/kg; (ii) buprenorphine tablets (Temgesic; Reckitt and Colman; 200/-tg) per rectum; (iii) lofentanil injection (5 /-tg/ml) subcutaneous 0'1 ml/kg. Saline was used as the placebo for the injections and a commercially-produced tablet placebo (supplied by Hammersmith Hospital Pharmacy), which contained an inert lactose material, as the placebo for the tablets. Measurement procedure Approximately 24 h before the experiment, the back of the rabbit was shaved and depilated, and a grid of 1x 1mm squares drawn on the skin with a permanent marker. The rabbit was placed in a restraining box, adapted so that the animal was firmly held at the neck with its back exposed. The beam projector was aimed at a single target.square. By moving the projector unit so that it was perpendicular to the skin and adjusting the distance, the two cold light beams could be made to coincide on the target at a reproducible source-to-skin distance. The beam projector was then clamped in position using a retort stand and the trigger depressed. As soon as the rabbit's skin twitched the trigger was released, which automatically stopped the i.r. beam and displayed the time to response. (An alarm device was incorporated into the beam projector which sounded a buzzer after 20 s. Whether or not the rabbit had responded with a skin twitch, heating was discontinued if the alarm sounded in order to prevent skin damage.) Readings were taken prior to administration of the test substance and then at approximately hourly intervals, subsequently, throughout an -12 h period. Statistical methods Exponential curves were fitted using the Maximum Likelihood Program of Ross (190). Results Subcutaneous buprenorphine Ten New Zealand White rabbits weighing 3' 1to 4, 7 kg were studied. Figure 3 shows the mean (± SEM) time before the twitch response was observed, after subtracting the mean time in the placebo experiment, i.e. the response variable represents the additional effect due to buprenorphine alone. The peak response occurred at approximately 3 h postinjection and the subsequent fall in the twitch time was approximately exponential, with a halflife of 4,7 h (95070 confidence limits 3, 4-7' 4 h).

4 220 Wootton, Cross, Wood & West 2 Fig. 4. Effect of buprenorphine per rectum (approximately 60 "g/kg). Points are the mean (± SEM) in 6 animals. 10 ~ 6.r= B 'il 4 I- 10 ~ 6.r= B 'il 4 I Time (h) Fig. 3. Effect of subcutaneous buprenorphine (30 "g/kg). Points are the mean time (± SEM) before the twitch response was observed in 10 animals, after subtracting the placebo response, Time Fig. 5. Effect of subcutaneous lofentanil (0' 5 "g/kg). Points are mean (± SEM) in 6 animals. (h) Time (h) 12 Buprenorphine per rectum Three New Zealand White rabbits weighing 3, 1 to 3, 3 kg were studied. Figure 4 shows the mean time before the skin twitch response was observed. Subcutaneous lofentanil Six New Zealand White rabbits weighing 2 to 4, 7 kg were studied. Figure 5 shows the mean time before the twitch response was observed. The peak response occurred at approximately 2 h postinjection and the subsequent fall in response time was approximately exponential, with a halflife of,9 h (95070 confidence limits 6, h). Measurements made on the day after injection confirmed our clinical impressions that there was still a significant effect of lofentanil: the mean time to skin twitch was 2 7 s (SEM 1 2) at approximately 25 h postinjection. Discussion This paper describes the design and construction of a simple, low-cost analgesiometer and its use in the rabbit. Initial trials with the analgesiometer confirmed that it could be used to provide a rapid method of measuring the efficacy and duration of action of opioid analgesics in rabbits. Radiant heat stimulation is, of course, only one possible method by which analgesic action can be investigated and does not necessarily mimic the more complex nociceptive stimuli which follow surgery. However, its advantage in contrast with other techniques which have been applied in this species, for example electrical stimulation of the tooth pulp (Piercey & Schroeder, 190), is that only minimal preparation of the animal is required prior to study. Previous use of radiant heat in the rabbit appears to have involved considerable physical restraint of the animals, and utilized head withdrawal following heating of the nose and mouth as the 'pain threshold' (Zhongfu et al., 191) or ear withdrawal following heating (McCallister et al., 196). The present technique requires minimal restraint of the animal and uses a skin twitch response as the end point, the response being obtained well before any damage occurs. Since the instrument

A rabbit analgesiometer 221 has its own focusing device, it is likely to be suitable for use in a number of larger animals, such as the cat, dog, sheep and pig.

5 A rabbit analgesiometer 221 has its own focusing device, it is likely to be suitable for use in a number of larger animals, such as the cat, dog, sheep and pig. The results of the preliminary study of the analgesic action of buprenorphine and lofentanil in the rabbit suggest that both of these drugs may be suitable for clinical use in this species. Buprenorphine has been advocated as a suitable opioid analgesic for the control of postoperative pain in a number of animal species, including the rabbit (Flecknell, 194). Clinical experience of its use by the authors suggested that it was an effective drug, but no objective assessment as to the suitability of the dose rate, or the duration of action, could be made. Administration of buprenorphine in the present study produced analgesia which persisted for -10 h. This period is slightly shorter than that suggested previously (Flecknell, 194), but nevertheless confirms reports in other species of the relatively long duration of action of this agent in comparison with other opioids (Cowan et al., 1977;Livingston et al., 196). Buprenorphine administered per rectum as a suppository had a less marked analgesic action, but the analgesic effect appeared to persist for a longer period. The rectal route may therefore be useful for postoperative pain relief, since a tablet could be inserted at operation. Lofentanil has been shown to have a prolonged duration of action in rodents (Janssen, 192) and our preliminary results obtained in the rabbit indicate that it has a similar prolonged action in this species. If this opioid became available commercially, it could be the agent of choice for use as a postoperative analgesic in animals, since its duration of action would enable the provision of effective pain relief based on daily drug administration alone. Conclusion The analgesiometer described in this paper is relatively simple to construct and easy to use. The response of other species to thermal stimulation of the skin awaits evaluation, but results of our preliminary study in the rabbit suggest that the instrument may be useful in a range of larger, non-rodent species for the evaluation of the duration of action of opioid analgesics. Acknowledgments The authors thank Dr M. J. Halsey (Division of Anaesthesia, CRC) for supplying the lofentanil and Dr P. A. Flecknell (University of Newcastle upon Tyne) for much helpful advice and encouragement. References Association of Veterinary Teachers and Research Workers (196) Guidelines for the recognition and assessment of pain in animals. Veterinary Record 11, Baumberger A (193) The relief of pain in animals: a report on the practical aspects of administering analgesia to experimental animals. Schweizer Archiv fur Tierheilkunde 125,17-29 (Ger.) British Veterinary Association Animal Welfare Foundation (196). 'The detection and relief of pain in animals.' Second British Veterinary Association Animal Welfare Foundation Symposium, British Veterinary Association Animal Welfare Foundation, London Cowan A, Lewis JW & McFarlane IR (1977) Agonist and antagonist properties of buprenorphine, a new anti nociceptive agent. British Journal of Pharmacology 60, Evans WO (1964) A critical review of some new methods in animal analgesiometry. Journal of New Drugs Flecknell PA (194) The relief of pain in laboratory animals. Laboratory Animals 1, Janssen PAJ (192) Potent, new analgesics, tailor-made for different purposes. Acta Anaesthesiologica Scandinavica 26, Livingston A, Nolan A & Waterman A (196) The relationship between analgesia induced by, and plasma concentration of, buprenorphine in the sheep (Abstract). British Journal of Pharmacology, 363P McCallister LW, Lipton JM, Giesecke AH & Clark WG (196) The rabbit ear-withdrawal test: a new analgesiometric procedure. Pharmacology, Biochemistry and Behaviour 25, Morton DB & Griffiths PHM (195) Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record 116, Piercey MF & Schroeder LA (190) A quantitative analgesic assay in the rabbit based on the response to tooth pulp stimulation. Archives Internationales de Pharmacodynamie et de Therapie 24,

222 Wootton, Cross, Wood & West Ross GJS (190) MLP, Maximum Likelihood Program. Harpenden, Herts: Rothamsted Experimental Station Taber RI (1974) Predictive value of analgesic assays in mice and rats.

6 222 Wootton, Cross, Wood & West Ross GJS (190) MLP, Maximum Likelihood Program. Harpenden, Herts: Rothamsted Experimental Station Taber RI (1974) Predictive value of analgesic assays in mice and rats. Advances in Biochemical Psychopharmacology, Taylor PM (195) Analgesia in the dog and cat. In Practice Wright EM, Marcella KL & Woodson JF (195) Animal pain: evaluation and control. Laboratory Animals 14, Yoxall AT (197) Pain in small animals-its recognition and control. Journal of Small Animal Practice 19, Zhongfu Z, Minyi D, Wenying W, Ying J & Jisheng H (191) Effect of intracerebral microinjection of naloxone on acupunture- and morphine-analgesia in the rabbit. Scientia Sinica 24,

A METHOD FOR TESTING ANALGESICS IN

Brit. J. Pharmacol. (1952), 7, 433. A METHOD FOR TESTING ANALGESICS IN BY From the Department of Pharmacology, Oxford University MICE (Received March 18, 1952) The methods available for testing the analgesic