ANAESTHETIC INDUCED NEUROEXCITATION

FIELD OF THE INVENTION

The present invention relates generally to anaesthesia, and in particular to the side effects of neuroexcitation associated with general anaesthetic agents. The invention also relates to methods and compositions for reducing such side effects and to methods, compositions and combined products for inducing and maintaining general anaesthesia with reduced side effects associated with neuroexcitation.

BACKGROUND

One significant side effect of general anaesthetic agents that is yet to be addressed is neuroexcitation induced by the administration of such agents. Neuromotor excitation resulting in involuntary myoclonic movement such as muscle twitching, limb movement and/or vocalisation during anaesthesia or recovery from anaesthesia is unpleasant and inconvenient in patients and, when used for veterinary purposes with large animals, poses a significant risk for injury to attending personnel. Neuromotor excitation may be provoked by sensory stimuli during recovery from anaesthesia, such as by light or sound.

In a number of cases, the side effect of neuroexcitation has prevented general anaesthetic agents with otherwise large therapeutic windows from being used as an anaesthetic agent for humans. Other compounds have been restricted to specific veterinary use only. One such class of general anaesthetic agent is the neurosteroidal general anaesthetics. Neurosteroids used as general anaesthetic agents for medicinal and veterinary applications possess a number of desirable properties. Neurosteroidal general anaesthetics have modest hemodynamic effects such that they generally demonstrate little or no depression of the cardiovascular or respiratory system. Neurosteroids are removed rapidly from the body by hepatic metabolism and elimination. Neurosteroids have a wide margin for safety and can reliably produce a good level of surgical anaesthesia lasting 30 to 60 minutes. Several neurosteroids provide the option of administration via a number of routes including intravenous and intramuscular and can be safely readministered in top-up doses. However, a common side effect of several known neurosteroid general anaesthetic agents is the occurrence of excitation with extraneous muscle movements affecting all parts of the body.

Accordingly, there exists a need to develop methods, compositions and combinations that address the unwanted side effect of neuron excitation to enable medical and veterinary practitioners to benefit from the desirable properties associated with general anaesthetic agents such as neuro steroidal general anaesthetic agents.

SUMMARY OF THE INVENTION

It has now been found that neuromotor excitation associated with neuro steroidal anaesthetics can be reduced by co-administration of the general anaesthetic with a compound that inhibits cannabinoid type 1 (CB 1) receptor mediated responses leading to neuroexcitation.

Accordingly, in one aspect the present invention provides a method of reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent, said method comprising the administration of an effective amount of a compound that inhibits CB 1 receptor mediated responses leading to said neuroexcitation.

In another aspect the present invention provides a method of reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent, said method comprising the administration of an effective amount of a CB 1 receptor inhibitor.

In a further aspect the present invention provides the use of a compound that inhibits CB 1 receptor mediated responses in the manufacture of a medicament for reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent. In another aspect the present invention provides the use of a CB 1 receptor inhibitor in the manufacture of a medicament for reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent. In another aspect the present invention provides a pharmaceutical composition comprising a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent.

In yet another aspect the present invention provides a combination comprising an effective amount of a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent.

In a further aspect the present invention provides a compound that inhibits CB 1 receptor mediated responses for use in reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent.

The present invention also provides a method for producing anaesthesia in a subject comprising administering a general anaesthetic agent to the subject and controlling, reducing or eliminating neuroexcitation by administering before, during or after administration of the general anaesthetic agent a compound that inhibits CB 1 receptor mediated responses.

These and other aspects of the present invention will become more apparent to the skilled addressee upon reading the following detailed description in connection with the accompanying Examples and claims.

BRIEF DISCRIPTION OF THE DRAWINGS

Figure 1. Graphical representation of the response of evoked IPSCs recorded from single motor neurons to alfaxalone alone and in combination with CB 1 receptor inhibitor AM251. Figure 2. Graphical representation of the effect of alfaxalone on spontaneous IPSC frequency of hypoglossal motor neurons, when administered alone or in combination with the CB 1 receptor inhibitor AM251. Figure 3. Graphical representation of the effect of alfaxalone on evoked or spontaneous IPSC frequency of hypoglossal motor neurons, when administered alone or in combination with a CB 1 receptor inhibitor NESS0327.

Figure 4. Graphical representation of the effects of pre-treatment of Wistar rats with CB 1 receptor inhibitor NESS0327 prior to alfaxalone anaesthesia.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification that neuroexcitation caused by the administration of general anaesthetic agents occurs through the suppression of the inhibitory pathway targeting motor neurons.

Without intending to limit the invention by theory, it is hypothesised that general anaesthetics decrease synaptic inhibition of motor neurons mediated by glycine receptors, which leads to enhanced motor neuron excitability resulting in psychomotor excitation. A second hypothesis is that this reduction of synaptic inhibition is due to generation of endogenous endocannabinoids by motor neurons activated by general anaesthetics. Endocannabinoids then activate CB 1 receptors on synaptic terminals releasing glycine, with a consequent reduction in glycine release. It has now been found that the unwanted side effect of neuroexcitation associated with general anaesthetics can be greatly reduced by co-administration of a compound that inhibits CB 1 receptor mediated responses leading to the said neuroexcitation.

The present invention thereby advantageously provides safe methods for administering general anaesthetic agents without the unwanted neuroexcitation side effect associated with neuro steroidal general anaesthetic agents. It will appreciated that the compound that inhibits CB l receptor mediated responses leading to the said neuroexcitation may act at any site or at multiple sites in the pathway between the up-regulation of native agonists, for example, endogenous endocannabinoids, and downstream signal transduction pathways and cellular responses resulting from the activation of the CB l receptor. It is envisaged that in one embodiment, the inhibition of CB l receptor mediated responses may be achieved by administering to a subject a compound that prevents the up-regulation of native CB 1 agonist, for example, a compound that inhibits the motor neurons activated by general anaesthetic agents. It is also envisaged that the compound that inhibits CB l receptor mediated responses may act by removing an excess of native CB l agonist, for example, by binding to the native ligand preventing it from acting on the CB 1 receptor. In other embodiments it is envisaged that the compound that inhibits CB l receptor mediated responses will act by binding directly to the CB l receptor and inhibiting the native agonist from activating the receptor. In further embodiments it is also envisaged that CB l receptor mediated responses may be inhibited by administering to a subject a compound that selectively inhibits the downstream signal transduction pathway that results in neuroexcitation, for example, the signal transduction pathway that down-regulates glycine release. In a preferred embodiment, the compound that inhibits CB l receptor mediated responses leading to neuroexcitation will be a CB l receptor inhibitor.

"Cannabinoid type 1 receptor inhibitors" or "CB l receptor inhibitors" of the present invention are compounds that bind to and inhibit the activation of the CB l receptor by native agonists such as endogenous endocannabinoids and include compounds that act as antagonists of the CB l receptor as well as reverse agonists of the receptor. Certain compounds (e.g. SR 141716) that were originally classified as selective antagonists are now considered to act as inverse agonists rather than pure antagonists. Whereas antagonists act by blocking the activation induced by agonist binding at the receptor, inverse agonists also occupy the receptor and function by decreasing the constitutive level of receptor activation in the absence of an agonist. CB 1 receptor inhibitors include, but are not limited to Rimonabant (SR 141716 or SR 141716A), AM251, AM281, SR144528, NESS0327, AM4113, AM6527, 0-2654, LY320135, taranabant, CP272871. Examples of publications disclosing such compounds include WO/2009/053553; Reggio, P. H., Curr. Pharm. Des., 2003, 9, 1607-33; Muccioli, G. G. and Lambert, D. M., Curr. Med. Chem., 2005, 12, 1361- 94.

In accordance with the present invention, a compound that inhibits CB 1 receptor mediated responses may be administered in combination with any general anaesthetic agent known in the art that has as a side effect the induction of neuroexcitation in a subject being anaesthetised. Examples of such agents include but not limited to, barbiturate general anaesthetics, benzodiazepine general anaesthetics, hypnotic agents and neurosteroidal general anaesthetics. In a preferred embodiment, the general anaesthetic agent is a neurosteroidal general anaesthetic agent.

Neurosteroidal general anaesthetic agents are neuroactive steroid compounds that are believed to elicit their effects by interacting with neurotransmitter-gated ion channels such as the GABAA receptor. Specific neurosteroidal general anaesthetics include, but are not limited to alfaxalone, alphadolone, eltanolone, hydroxydione, minaxolone, Org 20599 or Org 21465. Examples of publications disclosing such agents include Ramsay, A.E., et al. BMJ 1974, 2, 656-9; Sear, J. W., J. Clin. Anesth., 1996, 8, 91S-8S; McNeill, G. M., et al. The Lancet, 1979, 73-4; Robertson, J. D. and Williams, A. W., Anaesthesia, 1961, 16, 389- 409; Sneyd, J. R., et al. Br. J. Anaesth., 1997, 79, 427-32;

Another embodiment of the invention provides a method of co-administering a compound that inhibits CB 1 receptor mediated responses with general anaesthetic agents whereby neuroexcitation side effects associated with the general anaesthetic are reduced.

The effective amount of the compound that inhibits CB 1 receptor mediated responses and the general anaesthetic agent may, as appropriate, be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination. As an example, the method of reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent may comprise (i) administration of a CB l receptor inhibitor in free or pharmaceutically acceptable salt form and (ii) administration of the general anaesthetic agent in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order. The individual components of the combination of the invention can be administered separately at different times during the course of anaesthesia or concurrently in divided or single combination forms.

It will appreciated that the method and timing of administration of the compound that inhibits CB 1 receptor mediated responses and the general anaesthetic agent will depend on a number of factors including the length of the procedure, the nature of the subject and the properties of the compound that inhibits CB l receptor mediated responses and the anaesthetic agent, including the durations of action of the compound and the anaesthetic, and the time between administration and onset of action for each. In some cases it is envisaged that the inhibitor of CB 1 receptor mediated responses will be administered prior to the induction of anaesthesia as a premedicant. It is also envisaged that the inhibitor of CB l receptor mediated responses may be administered towards the end of the procedure or period of anaesthesia. In other embodiments it is envisaged that the inhibitor of CB l receptor mediated responses and the anaesthetic agent will be administered at the same time, as separate components or as a combination, when anaesthesia is initially induced. After induction of anaesthesia with the anaesthetic agent it is also possible for anaesthesia to be maintained with a different anaesthetic agent, and the compound that inhibits CB l receptor mediated responses could be administered with, or combined with, either or both of those agents.

Those skilled in the art will be familiar with premedicant regimes prior to the induction of anaesthesia. In one embodiment the compound that inhibits CB l receptor mediated responses will be administered between about 120 minutes and about 1 minute prior to the administration of the general anaesthetic. In a further embodiment the compound that inhibits CB 1 receptor mediated responses will be administered between about 60 minutes and about 2 minute prior to the administration of the general anaesthetic agent. In another embodiment the compound that inhibits CB l receptor mediated responses will be administered between about 30 minutes and about 5 minutes prior to the administration of the general anaesthetic agent.

The compound that inhibits CB l receptor mediated responses is administered to the subject in a treatment effective amount. As used herein, a treatment effective amount is intended to include at least partially attaining the desired effect, or reducing to an acceptable level the degree of observable myoclonus, or halting altogether the neuroexcitation induced by the administration to a subject of a general anaesthetic agent. The compound is administered before, during or after initial administration of the anaesthetic agent such that it is capable of reducing the neuroexcitation induced by the anaesthetic.

As used herein, the term "effective amount" refers to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 μg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage. The compound that inhibits CB l receptor mediated responses may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition or pharmaceutical composition. Accordingly, in one embodiment the present invention provides a pharmaceutical composition comprising a compound that inhibits CB 1 receptor mediated responses with at - Si - least one pharmaceutically acceptable adjuvant, carrier or diluent. In a preferred embodiment, the compound that inhibits CB 1 receptor mediated responses will be a CB 1 receptor inhibitor. The CB 1 receptor inhibitor may be in any suitable form, including the form of a pharmaceutically acceptable salt.

As used herein, the phrase "pharmaceutically acceptable carrier" includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals, birds, reptiles and amphibians. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colouring agents, release agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

A pharmaceutical composition is generally formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, intraperitoneal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, or liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion or by the use of surfactants. Prevention of the action of microorganisms can be achieved by incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally comprise an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurised container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished with nasal sprays or suppositories. The compounds can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It may be advantageous in some circumstances to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein preferably refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

In one embodiment the present invention provides a pharmaceutical composition comprising a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent together with at least one pharmaceutically acceptable adjuvant, carrier or diluent. The pharmaceutical compositions comprising a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent together with at least one pharmaceutically acceptable adjuvant, carrier or diluent may be prepared as described above.

Pharmaceutical compositions of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds. In another embodiment, the present invention provides that use of a compound that inhibits CB 1 receptor mediated responses in the manufacture of a medicament for reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent. In preferred embodiments, the compound that inhibits CB 1 receptor mediated responses is a CB 1 receptor inhibitor.

Other methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995).

It will be appreciated that pharmaceutical compositions comprising a compound that inhibits CB 1 receptor mediated responses in admixture with an anaesthetic agent and pharmaceutical compositions comprising the anaesthetic agent alone, will be formulated in manner that is suitable for the production of anaesthesia. It is envisaged that such compositions may be liquid compositions suitable for parenteral administration such as intravenous, intramuscular, intraperitoneal or subcutaneous administration. It is also envisaged that compositions comprising an anaesthetic agent may be in the form of a powder suitable for reconstitution into an injectable liquid solution. In other embodiments the pharmaceutical compositions will be formulated in manner that is suitable for inhalation, for example, as gaseous compositions or as volatile liquid compositions. In a preferred embodiment the composition will be formulated for parenteral administration. In a particular preferred embodiment it is envisaged that the composition will comprise the anaesthetic agent alfaxalone and the pharmaceutically acceptable carrier hydroxypropyl-β- cyclodextrin in an aqueous liquid solution. Such anaesthetic formulations are known in the art and are available to clinicians, for example, under the tradename Alfaxan ^® .

The present invention also provides the use of an effective amount of a compound that inhibits CB 1 receptor mediated responses for reducing neuroexcitation induced by the administration to a subject of a general anaesthetic agent. It would be understood by those skilled in the art that the present invention is applicable to any mammalian species, including, but not limited to, human, canine, feline, equine, bovine, porcine, ovine, murine. The present invention is also applicable to non- mammalian species including, but not limited to birds, reptiles, and amphibians.

In one embodiment, the compound that inhibits CB 1 receptor mediated responses and general anaesthetic agent will be administered separately. In other embodiments the compound that inhibits CB 1 receptor mediated responses will be administered prior to the neurosteroidal general anaesthetic, for example, as a premedicant.

In a further embodiment the present invention provides a combination comprising an effective amount of a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent. Each of the components of the combination may be formulated separately, and may independently be in the form of a suitable compound in free or pharmaceutically acceptable salt form, or may be in the form of a pharmaceutical composition, or may be formulated for administration to a subject by any route including parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal; oral (e.g., inhalation); transdermal (topical), transmucosal, vaginal and rectal administration. The term "combination", as used herein refers to a composition or kit of parts where the combination partners as defined above can be dosed dependency or independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The combination partners can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combination can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patients. The effective dosage of the general anaesthetic agent employed in compositions or combinations of the present invention will vary depending on the particular agent or pharmaceutical composition employed and the mode of administration, the species being anaesthetised, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion and any drug combination. Thus, the dosage regimen for the general anaesthetic is selected in accordance with a variety of factors including the route of administration and the renal and hepatic function of the patient. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of the single active ingredients required to induce or maintain anaesthesia. Optimal precision in achieving concentration of the general anaesthetic within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the active ingredients' availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of the active ingredients. In some embodiments the effective amount of the general anaesthetic agent will be an amount effective to induce anaesthesia in a subject in need thereof. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively anaesthetise a subject, including the general anaesthetic agent employed, the species of the subject being anaesthetised, the age, sex, weight and general physical condition of the subject, the mode of administration. By balancing these features it is well within the general skill of a medical or veterinary practitioner to determine appropriate dosages. By way of example, however, suitable dosages for inducing anaesthesia may be in the range of about 0.1 μg per kg of body weight to about 1 g per kg of body weight. A typical dosage is in the range of 0.1 mg to 1 g per kg of body weight, such as is in the range of 0.2 mg to 1 g per kg of body weight. In one embodiment, the dosage may be in the range of 0.2 mg to 500 mg per kg of body weight. In another embodiment, the dosage may be in the range of 0.5 mg to 250 mg per kg of body weight. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight, such as up to 50 mg per body weight. In other embodiments the general anaesthetic agents will be in an amount effective to maintain anaesthesia in a subject in need thereof. The appropriate dosage required to maintain anaesthesia in a subject will vary depending on, e.g. including the general anaesthetic agent employed, the species of the subject being anaesthetised, the age, sex, weight and general physical condition of the subject, the mode of administration. By balancing these features it is well within the general skill of a medical or veterinary practitioner to determine appropriate dosages.

In certain embodiments anaesthesia will be maintained in a subject by supplemental administration of bolus doses of the one or more neurosteroidal general anaesthetic agents at dose rates, for example, in the range of about 0.1 μg per kg of body weight to about 1 g per kg of body weight. A typical dosage is in the range of 0.1 mg to 1 g per kg of body weight per dosage, such as is in the range of 0.2 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 0.2 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 0.5 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

In further embodiments, anaesthesia will be maintained in a subject by as constant rate infusion of the general anaesthetic agent at a dose rate, for example, in the range of 0.1 mg to 1 g per kg of body weight per hour, such as is in the range of 0.2 mg to 1 g per kg of body weight per hour. In one embodiment, the dosage may be in the range of 0.2 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 0.5 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

In a further embodiment the present invention provides a kit comprising an effective amount of a compound that inhibits CB 1 receptor mediated responses and a general anaesthetic agent. A "kit" or "kit of parts" refers to a combination of components which may include a container for containing the pharmaceutical compositions and may also include divided containers such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art that is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a resealable bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle that is in turn contained within a box.

An example of such a kit in relation to solid dosage forms is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil that is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening. It maybe desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, veterinarian, pharmacist or other health care. When the kit contains separate compositions, a dose of one or more compositions of the kit can consist of one tablet, capsule, bottle, vial, or ampoule while a dose of another one or more compositions of the kit can consist of several tablets, capsules, bottles, vials or ampoules.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Examples of the procedures used in the present invention will now be more fully described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES Example 1. In vitro electrophysiological analysis of the effects of alfaxalone on hypoglossal motor neurons.

The responses of single hypoglossal motor neurons to alfaxalone alone and in the presence of the CB 1 receptor inverse agonist AM251 were measured using whole cell patch clamp electrophysiological recordings from in vitro slices of juvenile (10-18 day old) rat brain tissue with techniques known in the art over a range of relevant alfaxalone doses (100 nM to 10 μΜ).

Brain slice preparation and electrophysiological recording methods.

Electrophysiological recordings were made from transverse brainstem slices containing the hypoglossal motor nucleus, prepared from 10 to 16 day-old Wistar rats of either sex obtained from the University of Queensland institutional breeding colony. Animal care and handling was approved by the University of Queensland animal ethics committee, and was in accordance with university and national guidelines. In brief, animals were deeply anaesthetized with sodium pentobarbitone (100 mg/kg by intra-peritoneal injection) and then rapidly decapitated. The brainstem was rapidly removed, glued onto a metal tray and support block of solid agar with cyanoacrylate glue (Supafix, Selleys) and 300 μιη thick transverse slices made on a vibratome (D.S.K Microslicer DTK- 1000, Ted Pella, USA) in sucrose-substituted, ice-cold Ringer solution containing (in mM): 260 Sucrose, 10 Glucose, 3 KC1, 1.25 NaH ₂ P0 ₄ , 5 MgCl ₂ , 1 CaCl ₂ gassed with 95% 0 ₂ -5% C0 ₂ . Slices were incubated in the same solution at 35°C for 1 hour, then transferred to Ringer solution containing (in mM): 130 NaCl, 26 NaHC0 ₃ , 10 Glucose, 3 KC1, 1.25 NaH ₂ P0 ₄ , 1 MgCl ₂ , 2 CaCl ₂ , gassed with 95% 0 ₂ -5% C0 ₂ , pH 7.4, and maintained at room temperature (20-22° C) for up to 7 hours while recordings were made. Individual slices were transferred to a small recording chamber, and submerged in Ringer solution flowing continuously into the chamber via gravity from a reservoir and being removed by suction.

Whole cell patch clamp recordings were made from identified hypoglossal motor neurons using infrared video microscopy (Nikon Eclipse E600FN microscope, Australia and Hamamatsu C2400-07ER Newvicon video camera, Hamamatsu, Japan). Motor neurons had a large soma (>25 microns in diameter) within the motor nucleus boundary, more than two main dendrites, and a whole cell capacitance value >20 pF. Patch electrodes (tip resistance of 3-5 ΜΩ when filled with the intracellular solution below) were pulled from borosilicate glass capillaries (Vitrex, Modulohm AS, Denmark) and filled with an intracellular solution containing (in mM): 120 CsCl, 4 NaCl, 10 HEPES, 4 MgCl ₂ , 0.001 CaCl ₂ , 10 EGTA, 2 Mg ₂ ATP, 0.3 Tris-GTP (pH 7.3). Patch electrodes were not Sylgard- coated or fire-polished; a high resistance seal (>1GQ) with the motor neuron membrane was always obtained before entering whole cell patch clamp mode. Motor neuron membrane potential was voltage clamped at -60mV or more negative. Signals were recorded and low pass filtered at 5 kHz with an Axopatch ID patch clamp amplifier (Axon Instruments, Foster City, CA), sampled at 20 kHz (Digidata 1200A, Axon Instruments), then stored on a PC computer. Series resistance was always <20 ΜΩ and was compensated by 60-80% using amplifier circuitry. Recording methods were very similar to those previously described (Bellingham, M. C. and Berger, A. J., J Neurophysiol, 1996, 76, 3758-70; Ireland, M. F., et al., Neurosci, 2004, 128, 269-80). Paired inhibitory synaptic currents with an inter- stimulus interval of 50 ms were evoked by electrical stimulation (single electrical pulse duration of 0.1 ms, intensity 5-100 V) of the reticular formation lateral to the border of the hypoglossal motor nucleus with a bipolar concentric wire electrode (FHC, Bowdoin, Maine USA) at a frequency of 0.1 Hz. All evoked responses were inhibitory, as they were recorded in the presence of the non- NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μΜ) and the NMDA glutamate receptor antagonist DL-amino-5-phosphonovaleric acid (DL-APV, 50 μΜ) which together block all glutamatergic ionotropic synaptic receptors on hypoglossal motor neurons (Bellingham, M. C. and Berger, A. J., Neurophysiol, 1996, 76, 3758-70; O'Brien, J. A. et al, Neuron, 1997, 9, 919-27), and the evoked responses could be completely blocked by co-application of the glycine receptor antagonist strychnine HC1 (20 μΜ) and the GABA A receptor antagonist bicuculline methiodide (5 μΜ) (Singer, J. H., et al, J Neurophysiol, 1998, 80, 2608-20; O'Brien, J. A. and Berger, A. J., J Neurophysiol, 1999, 82, 1638-41). In some experiments, either strychnine or bicuculline were applied alone, to record isolated inhibitory synaptic events mediated by GABA A or glycine receptors respectively.

Electrophysiological Data Analysis

Inhibitory synaptic currents were recorded and measured using pCLAMP8.2 software (Axon Instruments, Foster City, CA, USA). Changes in evoked current amplitude were assessed by comparison of the mean amplitude of 8-12 consecutive synaptic currents immediately prior to drug application (control data block) with the same number of consecutive synaptic currents showing the largest mean amplitude change after drug application (test data block). When multiple drugs were applied, control measurements for each of the successively added drugs were taken from the period immediately before application of each individual drug, except where indicated in the results. Paired pulse ratio (PPR) was determined by averaging the evoked synaptic currents in the control block, and in the test block, then dividing the second averaged synaptic current amplitude by the first averaged synaptic current amplitude in each block; this method avoids spurious paired pulse facilitation which can be produced by calculating PPR as the average of individual synaptic current amplitude ratios (Kim, J. and Alger, B. E., 2001, 21, 1638-41). All data are given as mean + SEM and were compared statistically with a Students two-tailed paired t test except where noted, with statistical significance accepted at P<0.05. All statistical tests were done with Prism 5.0 (Graphpad, CA, USA). These methods are as previously described for analysis of excitatory synaptic currents (Bellingham, M. C. and Berger, A. J., Neurophysiol, 1996, 76, 3758-70; Ireland, M. F., et al, Neurosci, 2004, 128, 269-80).

Miniature synaptic currents were recorded in continuous epochs of 2 minutes, with cadmium chloride (100 μΜ) added to block calcium-dependent neurotransmitter release (Scanziani, M. et al., Neuron, 1992, 9, 919-27). Miniature synaptic currents were detected and measured off-line with Clampfit 10 software (Axon Instruments) using a sliding template algorithm (Clements, J. D. and Bekkers, J. M., 1997, 73, 220-29). Measured miniature synaptic current parameters were peak amplitude, 10-90% rise time (the time between 10% and 90% of peak amplitude during the synaptic current rising phase), half width (the time between 50% of peak amplitude during the synaptic currentrise and decay phases) and the time interval between successive synaptic currents. Cumulative frequency distributions of miniature synaptic current parameters recorded under different conditions in the same neuron were statistically compared with the Kolmogorov-Smirnov (K-S) test, using Clampfit 9 (Axon Instruments). The mean values of each parameter for each neuron recorded in different conditions were compared statistically with a Students two-tailed paired t test except where noted, with statistical significance accepted at <0.05. Spontaneous synaptic currents were recorded without cadmium chloride present and analyzed in the same way.

Chemicals

Most chemicals used in electrophysiological experiments were obtained from Sigma (NSW, Australia) with the exception of alfaxalone and of cyclodextrin, which were obtained from Jurox Australia (Rutherford, NSW). All drugs were dissolved in stock solutions; the vehicle of these stock solutions was double distilled water, dimethyl sulfoxide or ethanol. An appropriate volume of the stock solution was then added to the Ringer solution to achieve the desired final concentration after equilibration. The drug stock solution concentration was always at least 1000 times higher than the final concentration of the drug in Ringer solution; thus, the maximal vehicle concentration in the Ringer solution was never >0.1% by volume. Application of the vehicles at 0.1% volume did not alter inhibitory synaptic currents. The solution flow rate was 1-2 mL/minute; with a bath volume of <1 mL, drug concentration reached equilibrium in the bath within 5 minutes and all drugs were applied for a minimum of 8 minutes before measurements were made, to ensure drug concentration had reached equilibrium levels.

Results

Alfaxalone significantly decreased amplitude of inhibitory synaptic currents (IPSC, raw current value normalized to baseline values before alfaxalone) at concentrations ranging between 100 nM to 10 μΜ (Figure 1A); the CB l receptor inverse agonist AM251 blocked effects of 1 μΜ alfaxalone (Figure 1C). Alfaxalone significantly decreased IPSC PPR at concentrations between 1 μΜ and 10 μΜ (Figure IB; AM251 blocked effects of 1 μΜ alfaxalone (Figure ID).

As shown in Figure 1, alfaxalone caused a dose-dependent decrease in the size of inhibitory synaptic currents (IPSCs) evoked by local electrical stimulation in the brain slice (Figure 1A), consistent with increased motor activity and muscle twitching as alfaxalone dose increases. Indirect measurement of glycine synaptic release probability using the technique of paired pulse ratio (PPR) (Kim, J. and Alger, B. E., 2001, 21, 1638-41), shows that PPR is significantly reduced by alfaxalone (Figure IB). This change in PPR indicates that alfaxalone decreases glycine release by synapses, rather than the response of the postsynaptic glycine receptor to synaptically released glycine. Figure 1 shows that co-application of AM251, a CB l receptor inverse agonist, completely blocked the effect of alfaxalone on inhibitory synaptic input size and PP ratio (Figure 1C and D). This result indicates that alfaxalone causes activation of CB 1 receptors located on synaptic terminals releasing glycine, and the effect of CB 1 receptor activation is to decrease glycine release probability loss. Example 2. In vitro electrophysiological analysis of the affects of alfaxalone on evoked or spontaneous IPSC frequency of hypoglossal motor neurons.

The effects of alfaxalone on evoked or spontaneous IPSC frequency of single hypoglossal motor neurons (HM), were measured using whole cell patch clamp electrophysiological recordings from in vitro slices of juvenile (10-12 day old) rat brain tissue. Alfaxalone was applied to the HM alone and in combination with CB 1 receptor inverse agonist AM251, or CBI receptor competitive antagonist NESS0327.

In vitro motor neuron slice preparation

Whole cell recordings were performed using in vitro brain stem slices from juvenile Wistar rats of either sex (10-12 days old). Rats were anaesthetised using sodium pentobarbitone (100 mg/kg IP). When deep anaesthesia was established, the rat was swiftly decapitated. The skull, cerebrum, cerebellum and the neck muscles were removed to expose the brainstem. For cutting, the brainstem was placed into an icy-cold bath of artificial cerebrospinal fluid (aCSF), which was bubbled with carbogen (95% 0 ₂ , 5% C0 ₂ ). Transverse slices at a thickness of 300 μΜ were cut with a DSK Microslicer DTK- 1000 (TED Pella Inc) and incubated for 35-50 min in the same aCSF at 35°C. The slices were then maintained at room temperature (19-21°C) in a maintenance aCSF (see solutions), bubbled with carbogen.

Solutions

The artificial cerebrospinal fluid (aCSF) solution used for cutting and initial incubation of slices contained (in mM) 130 NaCl, 26 NaHC0 ₃ , 3 KC1, 5 MgCl ₂ , 1 CaCl ₂ , 1.25 NaP0 ₄ , 10 Glucose. The maintenance aCSF solution, was a similar solution with the exception of 2 CaCl ₂ and 1 MgCl ₂ . The patch pipette internal solution contained (in niM) 120 CsCl, 4 NaCl, 4 MgCl ₂ , 0.001 CaCl ₂ , 10 N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), 10 Caesium ethylene glycol-bis( -aminoethyl ether)-N,N,N,N-tetra-acetic acid (EGTA), pH adjusted to 7.2 with CsOH and osmolality was adjusted to 290-300 mOsM with sucrose. 3-adenosine 5 '-triphosphate (ATP-Mg) and 0.3 guanosine 5-triphosphate-tris (hydroxymethyl) aminomethane (GTP-Tris) was added to the internal solution before use.

DL-2-amino-5-phosphonopentanoic acid (APV, Sigma, 50μΜ) and l,2,3,4-Tetrahydro-6- nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt hydrate (NBQX) disodium salt hydrate (Sigma, 10 μΜ) were added into the external bath solutions to block N-methyl-D-aspartate (NMD A) and non-NMDA glutamate receptor activity (both DL-a- amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainate receptors). l(S),9(R)-(-)-Bicuculline methchloride (Sigma, 5 μΜ) was added to the external bath solution to block GABAA receptor activity.

Drugs

3-a-hydroxy-5-a-pregna-l l,20-dione (alfaxalone, Jurox Pty Ltd) was dissolved in hydroxypropyl substituted β-cyclodextrin (HPCD, Jurox Pty Ltd) to a ratio of 1:8 to make a stock concentration of 10 mM alfaxalone, then diluted to the required bath concentration of 100 nM, 1 μΜ, and 10 μΜ. The stock solution solvent (DMSO, ethanol or HPCD) was always diluted by a factor of 1000 or greater in the external bathing solution, and had no effect when applied alone at these concentrations. Application of drugs via the bathing fluid was always for >2 min; the time taken to completely exchange the recording chamber solution was typically <40 s. The alfaxalone dose response study was only applied to one HM per slice.

Electrophysiological recordings

Brainstem slices were submerged in a mounted microscope chamber with a volume of ~ 0.5 mL and were continuously superfused with maintenance aCSF at a rate of 1.5-2 mL/min. Patch electrodes were pulled from thin-walled borosilicate glass capillary tubes without a filament (Vitrex Medical) on a two-stage electrode puller (PP-83, Narishige); patch electrodes had a final DC resistance of 2-3 ΜΩ when filled with the internal solution and a tip diameter of 1-2 μιη by visual inspection. Recordings were performed at room temperature (19-21°C) with the patch electrode connected to the headstage of an Axopatch ID patch-clamp amplifier (Axon instruments). HMs were visually identified by their size, shape, location in the nXII, and whole cell capacitance (>20pF). Whole cell recordings were obtained by the "blow and seal" method, where positive pressure (10-15kPa) was maintained in the pipette to allow surrounding neuropil to be cleaned away as the pipette tip is guided onto the surface of the target HM. The program pCLAMP 8 (Axon Instruments) was used to apply voltage commands and record whole cell currents and measure responses. Spontaneous and evoked inhibitory postsynaptic current (IPSC) activity was recorded with the motor neuron voltage clamped at a membrane potential of -60mV. For evoked IPSC recordings, a bipolar concentric stimulation electrode (Frederick Haer Company) was placed in the reticular formation ventrolateral to the border of the hypoglossal motor nucleus, and a stimulus current of 0.5- 1.1mA and 0.1 ms duration was applied to reliably evoked an IPSC with consistent IPSC amplitudes. The recorded signal was amplified (2-20x) and low pass filtered with a cut-off frequency of 2 kHz by the Axopatch ID amplifier before digitization with a 16-bit digitizer (Digidata 1320A, Axon Instruments) and recording on a PC hard disk (Dell Optiplex, running Windows XP Professional). Data was acquired as episodic sweeps of 1.04 sec duration for evoked IPSCs, or as continuous blocks of data of 2 min duration for spontaneous IPSCs. For evoked IPSCs, the first stimulus pulse was preceded by a short (20 ms) voltage step of -10 mV, to monitor input resistance (Rn) and series resistance. Results

Treatment of HM with a cannabinoid receptor inverse agonist or with a cannabinoid receptor competitive antagonist attenuates the reduction in amplitude of evoked or spontaneous inhibitory synaptic inputs observed after treating HM with alfaxalone. Treatment of HM with alfaxalone was shown to produce a dose dependent reduction in amplitude of electrically evoked inhibitory synaptic inputs to the HM (Figure 1A). Alfaxalone was also demonstrated to produce a dose dependent decrease in the frequency of spontaneous inhibitory synaptic inputs to HM (Figure 2A).

Treatment of HM with AM251, an inverse agonist at cannabinoid receptors (Figure 1C), or with NESS0327, a competitive antagonist at cannabinoid receptors (Figure 3A), has no effect on evoked IPSC amplitude alone, but in both instances treatment of HM with AM251 or NESS0327 was demonstrated to attenuate the effect of alfaxalone on evoked IPSC amplitude. Similarly, AM251 (Figure 2C) or NESS0327 (Figure 3D) have no effect on spontaneous IPSC frequency alone, but both compounds are able to block the effect of alfaxalone on spontaneous IPSC frequency.

Example 3. Analysis of the effects of premedication of Wistar rats prior to alfaxalone anaesthesia.

In vivo anaesthesia

Adult Wistar rats of either sex (6 males and 6 females) were weighed and then injected subcutaneously with Alfaxan CT (Jurox Australia) (20 mg/kg) for all groups. The vehicle and NESS0327 premedication groups also received an intraperitoneal injection of either NESS0327 (0.05 mg/kg) dissolved in DMSO or an equivalent volume of DMSO alone, given 15 minutes prior to the Alfaxane injection. All rats received each of these treatments over a period of 1 week, with a minimum of 24 hours between treatments. The experimenter was blind to the premedication composition.

Measured anaesthesia parameters

The index to measure muscle twitching was scored as 0, 1, 2, or 3, with the criteria below:

Twitch Score Description

Relaxed body position

None

No spasmodic movements

(- or O)

Mild Short-lived minor muscle contractions (+ οΠ) Relaxed body position

Localised to the facial region or extremities

Short-lived minor muscle contractions

Moderate

Multiple regions affected at one time

(++ or 2)

Increased muscle tone

Full body pronounced muscle contractions

Severe

Increased muscle tone

(+++ or 3)

Marked startle response to auditory or physical stimulus

The time taken from alfaxalone injection to being able to place the rat on its back (dorsal recumbency) was noted as the induction time, while the time taken from injection to the rat spontaneously righting itself to resting on its sternum (sternal recumbency) was taken as the recovery time. Immobilization time was the difference between induction time and recovery time. Arterial blood 0 ₂ saturation and respiration rate were measured with a NellcorN-20E pulse oximeter and a clip- style probe attached to a hind paw. This oximeter can only measure heart rate up to 250 bpm, and thus could not measure heart rates >250 bpm in these rats. Normal resting heart rates in Wistar rates range between 250-600 bpm; heart rates below 250 bpm were never seen during the experiments.

Results

The administration of NESS0327 as a premedication prior to alfaxalone anaesthesia significantly reduced muscle twitching during the induction and recovery phases of anaesthesia (Figures 4A and 4B). Pre-treatment with NESS0327 was also demonstrated to reduce the time taken from alfaxalone injection to dorsal recumbency (Figure 4C). Premedication with NESS0327 had no effect on the time from alfaxalone injection restoration of sternal recumbency or righting (Figure 4D), or the duration of immobilisation (Figure 4E). Premedication with NESS0327 had no detrimental effect on arterial 0 ₂ saturation (Figure 4F) or respiration rate (Figure 4G) during alfaxalone anaesthesia.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.