Department of Anaesthesia, Monash University, Clayton, Victoria, Australia 3168*Corresponding author
Accepted for publication: December 18, 2000
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Abstract |
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Br J Anaesth 2001; 86: 7048
Keywords: anaesthetics i.v., alphadolone
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Introduction |
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Experiments reported previously, showed that subanaesthetic doses of Saffan® produce powerful antinociception in rats.4 This is because of an interaction of one of the components of the mixture with spinal cord GABAA receptors even though the drug was given i.p.4 Investigations showed that all the sedative and anaesthetic properties of the mixture are a result of the alphaxalone content and all of the antinociceptive properties are a result of the alphadolone content. It is possible that the difference in the activity profile of alphadolone described in those experiments compared with what was reported in the literature during the development of Althesin® may be because of the route of administration. In the early reports on the development of the i.v. anaesthetic combination of alphaxalone and alphadolone the individual agents were probably tested by i.v. administration. By contrast, the experiments reporting antinociceptive effects with no sedation utilized i.p. administration of alphadolone.4 Thus, in the latter experiments the parent drug would have to pass through the liver and be prone to metabolic transformation before gaining access to the general circulation and the rest of the body. The experiments reported here were performed, first of all to ascertain if i.v. alphadolone did cause anaesthesia as no documentation exists for the effects of this drug given alone. The second aim was to produce a dose response curve for the antinociceptive effect of i.p. alphadolone and to determine if much higher doses than those used previously could cause sedation when given i.p. Finally, intragastric administration was investigated as this also delivers the drug first to the liver prior to the drug gaining access to the rest of the body. These experiments might indicate a potential use of the drug as an orally-administered analgesic. The highest dose of alphadolone that could be administered intragastrically was investigated for sedative and antinociceptive effects.
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Materials and methods |
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I.v. administration
Four male Wistar rats (weight 100 g) were given 0.25 ml i.v. injections of alphadolone acetate solution (10 mg ml1; dissolved in 10% Cremophor EL®), into the lateral tail vein using a Hamilton microsyringe and 27-gauge needle. A stopwatch was used to time the following scoring of sedation every 15 s:
0no righting reflex;
1righting reflex present but no spontaneous locomotion;
2spontaneous locomotion observed but uncoordinated;
3normal locomotion and exploratory behaviour.
These scores were plotted against the time of the observation for each rat.
I.p. administration
Dose response relationship for antinociception
Thirty male Wistar rats (weight 150200 g) were given a range of doses of alphadolone (0.06, 0.6, 6.0, and 60 mg per kg1 i.p.; n=4, 10, 15 and 4 experiments at each dose, respectively). The drug was suspended in saline solution and injected in a volume of 2 ml kg1. The rats were observed continuously for signs of sedation such as normal startle reactions to tapping on the Plexiglass restrainer in which they were placed and attempts at exploratory behaviour in the confined space. Antinociceptive thresholds were measured in the tail using noxious electrical current (ECT) as described previously.5 ECT measurements were made every 5 min until three stable (pre-drug) readings had been obtained. The i.p. injection of alphadolone was then given and the ECT measurements were continued every 5 min for 30 min thereafter. The antinociceptive effect for each dose was calculated as a ratio of control (pre-drug) values as previously described.5 These values were combined for each alphadolone dose and plotted as means (SEM) to produce a dose response curve.
Intragastric administration
An observer blinded randomized crossover study in five rats
Alphadolone was suspended in 1.0 ml normal saline at a concentration of 375 mg ml1. This was the highest concentration of drug that could be given to a rat in an intragastric injection. Five male Wistar rats (weight 150200 g) with lumbar intrathecal catheters implanted as described previously,6 were randomized to receive on two different occasions on different days either saline or alphadolone given intragastrically in a volume of 2 ml kg1 (750 mg kg1). This high dose of alphadolone was chosen to assess whether administration of the drug by the gastrointestinal tract could cause sedation and antinociceptive effects. This was a crossover study such that on the following day if a rat had received saline on the first occasion then it received alphadolone on the second occasion and vice versa. This dosing was randomized and the observer making measurements of nociceptive thresholds was blinded to the nature of the treatment. In each experiment, the electrical current thresholds for nociception (ECT) were measured in the neck and the tail every 5 min until three stable readings were obtained. At that stage either saline or alphadolone 750 mg kg1 suspended in saline was instilled into the stomach via an oesophageal needle. Five minutes later and every 5 min thereafter, ECT measurements were performed in the skin of the tail and neck. At 60 min, 10 pmol of bicuculline (a GABAA receptor antagonist) dissolved in 5 µl 6% dextrose solution was injected intrathecally down a Portex catheter that had been implanted previously in the lumbar subarachnoid space to lie adjacent to the most caudal segments of spinal cord responsible for tail innervation.
ECT measurements were continued for tail and neck electrodes every 5 min for a further 30 min. ECT results for tail and neck electrodes for each treatment (vehicle or alphadolone) at each time point were combined as means (SEM) to produce time response curves.
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Results |
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I.p. administration
It can be seen from Fig. 1 that there was a dose-related increase in antinociceptive effect with a maximum response of approximately 2.2x control (pre-drug) values. There were no overt signs of sedation even at the highest dose of i.p. alphadolone 60 mg kg1. Rats exhibited normal startle reflexes and exploratory behaviour, even after release from the restrainer 30 min after the i.p. injection when the full antinociceptive effects assessed by tail ECT were still present.
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Discussion |
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Previous experiments have also shown that the antinociceptive effects of Saffan® which were because of the alphadolone content of the anaesthetic mixture were caused by an interaction of the neurosteroid with spinal cord GABAA receptors as intrathecal injection of the GABAA receptor antagonist bicuculline reversed the antinociceptive effects.4 GABAA antagonists also reversed the spinally-mediated antinociceptive effects of intrathecal injections of water-soluble aminosteroids.7 Results presented in this paper also show that injection of bicuculline onto the most caudal segments of the spinal cord totally reversed the antinociceptive effects in the tail, the region of the body innervated by the most caudal segments of the spinal cord. As bicuculline is a GABAA antagonist and as the neck thresholds remained unchanged, it may be conclude that non-sedating doses of intragastric alphadolone produced long-lasting antinociceptive effects mediated by spinal cord GABAA receptors even though the drug was given intragastrically.
The effect of sex and adrenal steroids on the modulation of neuronal activity, dendritic structure, and behaviour has been well documented.810 These effects result from the regulation of intracellular receptors which direct changes in protein synthesis.1012 Therefore, the physiological action of these compounds occurs slowly over hours or days.12 13 Pioneering work by Seyle 50 yr ago demonstrated that certain pregnanes and androstanes have potent anaesthetic and anticonvulsant effects occurring within a few minutes of administration.14 Since these early findings, it has become apparent that some steroids can interact directly with a surface membrane receptorcomplex to cause a change in central nervous system (CNS) activity.15 16 Investigators studying the steroid anaesthetic alphaxalone (3-hydroxy-5
-pregnan-11,20-dione) found that this drug can allosterically positively modulate the
-aminobutyric acid (GABA) receptor. In subsequent studies alphaxalone and other neuroactive steroids were shown to enhance the inhibitory effect on neuronal excitability of GABA acting at the GABAA receptor complex.1720
It is now well established that GABA generally and receptors of the GABAA subclass are involved in spinal cord control of nociception.2123 Several different subtypes of GABAA receptor involved with spinal cord antinociceptive systems have been described.6 Thus, there is the possibility for these GABAA receptors to be targeted selectively by analgesic compounds. This effect might be achieved without sedation if they are significantly structurally different from the receptors in the brain that are responsible for the sedative, anxiolytic, anticonvulsant, and anaesthetic effects of a wide variety of compounds.9 2428 This idea is supported by the observation of antinociception after recovery from the anaesthetic and sedative effects of propofol, an i.v. anaesthetic drug that causes its sedative and anaesthetic effects by positive modulation of GABAA receptors.29 It might, therefore, be expected that two structurally different neurosteroids could interact with different receptors. There is also some suggestion from in vitro studies that neurosteroid modulation of native GABAA receptors obtained from the brain and spinal cord may be different.30 However, the molecular structures of alphaxalone and alphadolone are very similar and there are no suggestions in published reports of any differences between these two compounds in activity profiles.
Reports in the literature concerned with the development of Althesin® suggest that the alphadolone acetate component of Saffan® is present in the mixture merely to improve the solubility of alphaxalone (the active ingredient) in Cremophor EL.2 It is also reported that alphadolone is an i.v. anaesthetic like alphaxalone but with one-third the potency.1 The results of this study, in which alphadolone was given i.v. and all four rats were anaesthetized support those statements. This seems to conflict with the results of the other experiments reported in this paper which show that doses of alphadolone up to 100 mg kg1 given i.p. and 750 mg kg1 intragastrically caused no sedative effects.
It is clear, however, that the drug is absorbed in rats after i.p. and intragastric administration because antinociceptive effects follow such treatment. One possible explanation is that metabolic transformation of the molecule deactivates anaesthetic properties but preserves or imparts antinociceptive properties. In rat brain membranes a well-defined structure-activity relationship has been shown for pregnanes. Studies have shown that the 3-hydroxyl group on the steroid is required for anaesthetic activity whilst a carbonyl group at position C17 confers greater potency.3133 Glucuronide and sulfates have been described to be major metabolites of these neurosteroids.34 These metabolites may be produced by reaction at the 3
-hydroxyl group in both alphaxalone and alphadolone, and also at the hydroxyl group in the 21 position on the alphadolone molecule after de-acetylation. A drug injected i.p. or given via the gastrointestinal tract is first presented to the liver and neurosteroids are known to have significant first pass metabolism at the liver. As the only difference in the molecular structure between alphadolone and alphaxalone is the hydroxyl group at the 21 position it is suggested that metabolism at this group in the liver is responsible for the differences observed in their biological activity after i.p. and intragastric administration. This metabolite is analgesic and not anaesthetic, a subject worthy of further investigation.
We conclude that the antinociceptive effect of alphadolone after i.p. and gastrointestinal administration may be because of production of a metabolite of the parent compound. This causes spinally-mediated antinociceptive effects by interaction with spinal cord GABAA receptors. As this effect occurs in the absence of sedation, this suggests a possible clinical use of alphadolone or its metabolite in the management of pain syndromes.
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Acknowledgements |
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