WO2004089351A2

WO2004089351A2 - Treatment of progressive neurological disorders comprising a mao inhibitor (miclobemid) and an acetylcholinesterase inhibitor (tacrin)

Info

Publication number: WO2004089351A2
Application number: PCT/GB2004/001546
Authority: WO
Inventors: Anne Jennifer Morton
Original assignee: Cambridge University Technical Services Ltd
Priority date: 2003-04-10
Filing date: 2004-04-07
Publication date: 2004-10-21
Also published as: GB0308382D0; WO2004089351A3

Abstract

The present invention relates to therapeutic compositions and methods of treating progressive neurological disorders, such as Huntington's disease (HD). Administration of a monoamine oxidase inhibitor such as moclobemide, in combination with an acetylcholine esterase inhibitor, such as tacrine, and, optionally, creatine, are shown herein to ameliorate symptoms in murine model systems.

Description

Methods and Means for the Treatment of Progressive Neurological Disorders

The present invention relates to therapeutic compositions and methods for the treatment of neurological disorders, in particular Huntington's disease (HD) .

Huntington's disease (HD) is a progressive adult-onset movement disorder (Harper PS, (1996) Huntington's Disease 2nd Edn London, Saunders) . The disorder also has serious psychiatric symptoms, including mood disorders, depression, and progressive cognitive decline into dementia .

HD is caused by an abnormally expanded CAG repeat in the HD gene. R6/2 transgenic mice carry the human HD mutation in the first exon of the gene (Mangiarini, L. et al (1996) Cell 87, 493-506) and display a progressive neurological phenotype similar to HD in which overt symptoms appear around 8-10 weeks of age. However, motor and cognitive deficits can be measured several weeks earlier than this (Carter, R. J. et al (1999) J Neurosci. 19, 3248-3257, Lione, L.A. et al . J. Neurosci .23 , 10428- 10437 (1999) ) suggesting that deleterious neuronal dysfunction occurs before symptoms are observed.

Using R6/2 mice as a model, the present inventors have unexpectedly discovered that a combination of two known therapeutic compounds produces a beneficial therapeutic effect in the treatment of in progressive neurological conditions such as HD and related conditions, which is not observed with either compound alone. A first aspect of the present invention provides a composition comprising a first agent which is a monoamine oxidase inhibitor and a second agent which is an acetylcholine esterase inhibitor.

Treatment with an inhibitor of either monoamine oxidase or acetylcholine esterase alone does not produce any significant beneficial effects. However, the combination of both of these two inhibitors, which reduces and/or prevents the breakdown of both acetylcholine (ACh) and norepinephrine (NE) (also called noradrenalin) within the brain of the subject, improves cognitive function in an HD model system (R6/2 mouse) .

Monoamine oxidase inhibitors prevent or reduce the breakdown of norepinephrine (NE) , serotonin (5- hydroxytryptamine) and dopamine in the brain of a subject. An inhibitor for use in accordance with the present invention may be an irreversible or, more preferably, a reversible inhibitor of monoamine oxidase, such as brofaromine .

A suitable monoamine oxidase inhibitor may be non- selective or may be specific for either the MAO-A or the MAO-B isoform of monoamine oxidase.

Examples of irreversible MAO-B inhibitors include deprenyl (selegiline) and rasagiline. Examples of irreversible MAO-A inhibitors include phenelzine and isocarbazid.

Most preferably, the monoamine oxidase inhibitor is a reversible and selective inhibitor of MAO-A isoenzyme, Suitable monoamine oxidase inhibitors include moclobemide (M) .

Suitable acetylcholine esterase inhibitors which prevent or reduce the breakdown of ACh in the brain of a subject, include tacrine (T) , donazepil, rivastigmine and galantamine .

In some preferred embodiments, the first agent is moclobemide (M) and the second agent is tacrine (T) .

These two agents have well described mechanisms of action and both are known to be well tolerated when used chronically.

Moclobemide (4-chloro-N- [2- (4-morpholinyl) -ethyl] benzamide) is an antidepressant (see No: 6309, Merck Index 12th edition (Merck __ Co, Whitehouse Station NJ 1996) and references therein, Fulton & Benfield (1996) Drugs 52 (6) 869) . The term ^Λmoclobemide' or M' as used herein encompasses variants and derivatives of moclobemide which possess the same pharmacological properties .

CH₂CH₂NHCO

Moclobemide

Tacrine (1, 2, 3, 4-Tetrahydro-9-acridinamine) is an irreversible ACh esterase inhibitor that prevents the breakdown of ACh (see No: 9199, Merck Index 12th edition (Merck & Co, Whitehouse Station, NJ 1996) and references therein) . The term ^λtacrine' or ^ΛT' as used herein also encompasses variants and derivatives of tacrine which possess the same pharmacological properties .

In addition to tacrine and moclobemide, creatine (C) , was also included in the experiments described herein. Creatine (N- (aminoiminomethyl) -N-methylglycine : see No: 2637, Merck Index 12th edition (Merck & Co, Whitehouse Station NJ 1996) and references therein) ) is a naturally occurring compound found in mammalian tissues such as the brain, muscles, retina and heart. It is phosphorylated in vivo by creatine kinase to generate phosphocreatine. The adminstration of creatine has been shown to increase brain phosphocreatine levels and improve the motor symptoms and survival of R6/2 mice (Ferrante et al (2000) J. Neuroscience 12 4389-4397) and creatine is currently being tested as a treatment in HD patients. Creatine was included in these experiments because it is likely to be self-administered by HD patients and so it is important to determine any effects associated with its interaction with the other drugs. The term ^x creatine' or ^λC as used herein also encompasses variants and derivatives of creatine which possess the same pharmacological properties . CH,

NH₂

Creatine

Compositions of the present invention may additionally comprise creatine (C) .

Another aspect of the invention provides a combination of a first agent which is a monoamine oxidase inhibitor and a second agent which is an acetylcholine esterase inhibitor, and optionally, creatine, for example for use in the treatment of a progressive neurological condition such as HD .

As described above, depression is a common symptom of HD, Compositions and combinations of the present invention may additionally comprise anti-depressant agent. Such agents may elevate global biogenic amine levels. Suitable antidepressants include selective serotonin re-uptake inhibitors (SSRIs) such as fluoxetine and fluvoxamine and inhibitors of NE and serotonin uptake such as clomiphamine (see Merck Index (1996) supra) .

Motor dysfunction is an important HD symptom. Compositions, combinations and methods of the present invention may further include agents which act on motor symptoms of HD. These may include novel classes of agents such as endocanabinoid uptake inhibitors (Lasrtres-Becker et al, (2002) Synapse 44, 23-35) such as AM404, agents with dual muscarinic receptor agonist/dopamine receptor antagonist properties (Veinbergs et al, (2001) Society for Neurosciences 31^st Annual Meeting, San Diego, Abstract 549) that improve abnormal spontaneous and drug-induced locomotor symptoms without impairing cognition, and cannabinoids, including endogenous cannabinoids such as anandamide .

Compositions and combinations, may include, in addition to tacrine, moclobemide and optionally other active ingredients such as creatine as described above, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient . The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous .

Compositions and combinations of the present invention may be useful in a method of treatment of the human or animal body, in particular a method for the treatment of a neurological disorder, in particular a progressive neurological disorder such as Huntington's Disease (HD) , AIDS dementia, Parkinson's disease and Alzheimer's disease.

The term "treatment," as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications) , in which a desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, and amelioration of the condition or its symptoms, in particular behavioural symptoms such as cognitive deterioration and/or degeneration. Treatment as a prophylactic measure (i.e. prophylaxis) is also included.

The present compositions may in particular be useful in the alleviation or amelioration of symptoms associated with progressive neurological disorders such as Huntington's disease, in particular cognitive deterioration or degeneration.

Other aspects of the present invention provide the use of a composition or combination as described above in the manufacture of a medicament for use in the treatment of a progressive neurological disorder or dementia, such as HD, and the use of tacrine, moclobemide and optionally creatine in the manufacture of a medicament for use in the treatment of a progressive neurological disorder or dementia, such as HD. Examples of methods of treating progressive dementias using such medicaments are described below.

Another aspect of the present invention provides a method for treatment of a progressive neurological disorder or dementia, such as Huntington's disease, comprising administering a therapeutically effective amount of tacrine and a therapeutically effective amount of moclobemide to an individual in need thereof .

Such a method may further comprise administering a therapeutically effective amount of creatine to said individual .

The term "therapeutically-effective amount," as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

Methods of the present invention may further comprise the administration of other agents, such as anti-depressants and agents which act on motor symptoms of dementia, as described above .

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.

The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations within the brain which achieve the desired effect . Further details of appropriate dosages are found in the British National Formulary (2000) Pub: British Medical Association & Royal Pharmacological Society of Great Britain. Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

The active compounds or composition comprising the active compounds may be administered to a subject by any convenient route of administration.

Routes of administration include, but are not limited to, oral, for example by ingestion, parenteral, for example, by cutaneous, subcutaneous or intravenous injection; or by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

While it is possible for tacrine and moclobemide to be administered separately (i.e. as discrete compositions administered separately to the same patient) , it is preferable to present them together as a single pharmaceutical composition (e.g., formulation) comprising both active ingredients together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials well known to those skilled in the art and optionally other therapeutic agents, which may include, for example, creatine.

In addition to pharmaceutical compositions and therapeutic methods, the present invention further provides methods of making a pharmaceutical composition comprising admixing tacrine and moclobemide, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein. Methods may further comprise admixing creatine together with the other ingredients .

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Compositions of the present invention may conveniently be formulated in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredients with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may, for example, be in the form of liquids, solutions, suspensions, emulsions, tablets, capsules, cachets, pills or ampoules. For parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , the active ingredients will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Typically, the concentration of the active ingredient in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound particular organs, such as the brain.

Pharmaceutical compositions for oral administration (i.e. by ingestion) may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose) , lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) , surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

A pharmaceutical composition produced by a method described herein may be used for treating a progressive neurological condition such as Huntington's disease (HD) .

Another aspect of the present invention pertains to a kit for use in the treatment of a progressive neurological condition such as Huntington's disease, comprising (a) tacrine and moclobemide, preferably provided as a single pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the composition and/or active compounds.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents referenced in this specification are incorporated herein by reference .

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

Figure 1 shows performance of R6/2 and WT mice in the Morris water maze (vehicle-treated WT (light circles) , WT triple treated (dark circles) , R6/2 vehicle treated (dark diamonds) R6/2 triple treated (light diamonds) ) . Mice were trained over 4 sessions using a visible platform

(black bar) and tested with an invisible platform (open bars) . Latency (a) , pathlength (b) , swimming speed (c) and the thigmotaxis (d) were examined.

Figure 2 shows the effects of triple drug therapy on the performance of R6/2 mice in a T-maze alternation test (WT mice: light circles, vehicle treated R6/2 mice: dark diamonds, triple treated R6/2 mice: light diamonds). The total number of trials taken to reach criterion (a) , the total number of errors made (b) and the percentage of these errors that were perseverative (c) are shown.

Figure 3 shows the effect of triple treatment (tacrine/moclobemide/creatine) on various measures of the R6/2 phenotype. (WT vehicle treated mice: light circles, triple treated WT mice: dark circles, vehicle treated R6/2 mice: dark diamonds, triple treated R6/2 mice: light diamonds) .

Experimental Materials and Methods

Animals A colony of R6/2 transgenic mice was established in the Department of Pharmacology, University of Cambridge and maintained by backcrossing onto CBAxC57/BL6 FI mice. Mice were housed in cages of mixed genotype with 12 hours light and dark cycles in a temperature controlled room. All cognitive testing was performed on female mice.

Weights of mice were recorded from 5 weeks either twice weekly (creatine treatment) or daily (all other treatments) .

Drugs Tacrine (Sigma) was used at 2 doses (2.7 mg/kg and 3.2 mg/kg) and prepared in 0.9% saline. During our pilot studies we found that tacrine at the 3.2 mg/kg dose was associated with acute side effects (reduced locomotor activity) that appeared for 30 min after the drug was given. Therefore for most of the studies described here, a lower dose of 2.7 mg/kg was used. Moclobemide (10 mg/kg: Roche (manerix) ) was prepared in a small volume of ethanol and saline. Tacrine and moclobemide were administered via the intraperitoneal route. Creatine, which is readily available from commercial sources, was added to the diet by dissolving it in the water used to prepare the wet mash. Drugs were administered as single, double (tacrine/creatine, moclobemide/creatine, tacrine/moclobemide) or triple (tacrine/moclobemide/creatine) treatments .

Behavioural testing T-maze al ternation

The protocol of T-maze alternation was used as previously described (Lione et al 1999 supra) . Briefly, a T-maze was formed by blocking 5 arms of an 8 arm radial maze to form a T-shape. Mice were habituated in the maze for 6 mins over 3 consecutive days, so they would run reliably and find banana flavoured food pellets (20 mg) in wells placed at the end of each arm. All mice were trained for 2 days to perform 15 forced alternations. During the testing phase mice were placed in the start arm with both side arms baited. When a mouse entered an arm (defined when all four feet were inside the arm) , it was confined to its chosen arm by lowering a guillotine door. Mice were then returned to the start arm and the mice had to perform 10 alternations each session. An error was noted when a mouse returned to the arm it had previously visited, a perseverative error was recorded when the same error was made more than once .

Morris water maze

Spatial and nonspatial learning were assessed in a Morris water maze as previously described (Lione et al 1999 supra) . A circular white pool (diameter, 120 cm; height 50 cm) was filled to a depth of 25 cm with water and rendered opaque with nontoxic white paint. Four positions were designated N, S, E, W around the pool. A circular platform (diameter, 10 cm) was submerged 0.5 cm below the surface of the water. Mice were given 4 trials each day over 17 sessions from 9.3- 12 weeks of age. On the first 4 sessions mice were trained to swim to the submerged platform made visible by a high contrast black marker placed on the platform. Both the start position (N, S ,E ,W) and the position of the platform (NE, SE, SW, NW) were psuedorandomised across trials. Mice were allowed 60 seconds to find the platform, and those that did not reach the platform were placed on the platform by hand. From sessions 5-13 mice the high contrast marker was removed, and the platform placed in the SE quadrant. From sessions 14-17 the high contrast marker was reintroduced to identify the platform location, and as before placed in different locations across trials. All trials were analysed using HVS software (HVSImage, Buckingham, UK) .

Rotarod

Training consisted of placing mice aged 4.5 weeks on the rotarod at 24 rpm for 4 trials over 3 consecutive days . Mice were tested at speeds of 5, 8, 15, 24, 33 and 44 rpm at weekly intervals from 5 weeks of age.

Glycosuria Mice were placed on a clean acetate sheet. Most mice urinated spontaneously, otherwise firm but gentle pressure was applied to the abdomen to induce micturition. Urine was tested twice weekly using Diastix reagent sticks (Bayer PLC, Newbury UK) which provided a semi-quantitative analysis of glycosuria. Detection of glycosuria was considered to be indicative of diabetic status .

Glucose Challenge

Mice were fasted for 12 hours and then given a glucose challenge 1.5 g/kg intraperitoneally. Blood samples were taken before glucose was administered, and then 1 hour after the challenge. A drop of blood collected from the anaesthetised tail tip and blood glucose was measured using an automated glucometer (Bayer Diagnostics, Newbury, UK) .

Statistics

All behavioural data was analysed using repeated measures ANOVA with genotype and treatment as between subject factors, treated groups were compared to control values. Onset of glycosuria and survival were analysed with the log rank test. A Student's t-test was used for comparing blood glucose measurements. The numbers of diabetic mice per group were compared by the Chi squared test . A critical value of P<0.05 was used throughout this study.

Results

Two cognitive tasks were used in this study, the Morris water maze and a food rewarded T-maze alternation task. Drug treatment was started at a presymptomatic time point (4 weeks of age) because it has been shown previously that R6/2 mice show impairments in spatial learning before they show overt symptoms. Drugs were administered either individually, or as double (tacrine/creatine, moclobemide/creatine, tacrine/moclobemide) or triple (tacrine/moclobemide/creatine) combination treatments.

Cognitive function was first assessed in the Morris water maze. Training started post-symptomatically (at 9.5 weeks) , with mice being taught to swim towards a visible platform. All groups of mice displayed evidence of having learned the task, as indicated by their reduced latency and pathlength (Fig. la & b) across test sessions (P<0.001 for both). As expected, R6/2 mice took longer to escape (Fig la) swam significantly more slowly than WT mice (Fig lc) , and took a slightly longer path to the visible platform used for training (Fig lb) . This deficit was exacerbated by a tendency to swim close to the pool edge (thigmotaxis : Fig id) However, both R6/2 and WT mice showed nonspatial learning, with both groups showing a reduction in both latency (P<0.001) and pathlength (P<0.001) during the training period. Note also that when the visible platform was reintroduced during the final 4 sessions (11.4-12 weeks of age), both groups successfully recalled this task without significant deterioration in performance. Between 10 and 11.5 weeks of age, spatial learning was tested using an invisible platform. This revealed a significant cognitive deficit in R6/2 mice, who took a significantly longer pathway to the platform and performed the task much more slowly than WT mice (P<0.001). After 6 weeks of treatment with tacrine/moclobemide/creatine, R6/2 mice performed significantly better on the spatial tasks such as escape latency (Gene, Fχ.₅₂=349.70; Drug F_ι.₅₂=5.02; Gene x Days x Drug

all P<0.05) and path length (Gene

Drug Fι.₅₂=5.77,-all P<0.05) than vehicle- treated mice (Figs, la-c) . There was also significant improvement following treatment with some double treatments. Treatment with tacrine/moclobemide reduced pathlength (P<0.05) and latency (P<0.001), and small but significant effects were also found with tacrine/creatine (pathlength and latency (P<0.05 for both) and moclobemide/creatine treatment (P<0.05 latency only).

None of the individual treatments improved performance in this task. In all experiments, WT mice swam significantly faster than R6/2 groups (P<0.001). However, improved swimming did not account for the improvements in performance in Morris water maze performance by tacrine/moclobemide/creatine-treated R6/2 mice, since none of the treatments improved R6/2 swim speed (Fig lc) .

The improvement in the performance of the tacrine/moclobemide/creatine-treated mice is likely to be due to a change in strategy used to find the hidden platform. When the platform was hidden, R6/2 mice exhibited significantly more thigmotaxis (swimming around the perimeter of the pool) than wild-type mice (P<0.001) (figure Id) . This activity reduced the probability of the R6/2 mice locating the platform and accounted for the increase in path length and latency.

Tacrine/moclobemide/creatine treatment significantly reduced thigmotaxis in R6/2 mice (Gene, F_ι.₅₂=77.12; Drug Fi.52=15.00; Gene x Days x Drug Fι.₅₂=25.14; all P<0.001), and small but significant effects on thigmotaxis were also found with double treatments of tacrine/moclobemide (P<0.05), tacrine/creatine (P<0.05) and moclobemide/creatine (P<0.01). Individual treatments did not alter thigmotaxic behaviour (figure Id) .

Notably, the combined treatment with tacrine and moclobemide produced beneficial effects equivalent to triple (tacrine/moclobemide/creatine) treatment. No additional benefit was observed when creatine was also administered.

The effect of drug combinations on performance in a second maze, the T-maze, was then examined. As previously, the mice were treated from 4 weeks of age, but this time, cognitive performance was assessed in a T- maze alternation task between 5.5 and 10 weeks of age. R6/2 mice showed significant impairments in this task (Fig 2a) . Whereas WT mice gradually improved their performance with time (P<0.001), R6/2 mice deteriorated across test sessions, with the mice needing progressively more trials to complete the task (P<0.001). Analysis of the first 10 trials showed that R6/2 mice displayed a gradual deterioration in the alternation task (Fig. 2b) , until by 8 weeks of age, mice were performing at just below chance level. However, by the end of the testing period when the mice were 10 weeks of age, R6/2 mice were performing significantly below chance. This worsening of performance coincided with the onset of overt phenotype and suggested that the mice might be making a particular kind of error, rather than just failing at the task. The nature of the errors made by the mice were therefore analysed. R6/2 mice displayed a steady increase in perseverative errors (Fig 2c; P<0.001). By contrast, although WT mice made errors, these were rarely of a perseverative nature.

Treatment with tacrine/moclobemide/creatine markedly improved the performance of R6/2 mice in the T-maze, as shown by a reduction in the number of trials to complete the task (Group x Day F₃₈.₇ss= 6.06 P<0.001), the percentage of correct alternations in the first 10 trials on each day (F₃₈.₇₆₆=5.00, P, 0.001) and the numbers of perserverative errors made (F₃₈.₇66= .16, P, 0.001). The triple treatment did not restore the performance to the level of the WT mice, but rather prevented the deterioration of performance by significantly reducing the number of perseverative errors . When the drugs were administered individually, there was no improvement seen in either number of trials taken to reach criterion or in the number of perseverative errors made.

It seems likely that the improvement in performance in both Morris water^" maze and the T-maze resulted from the tacrine/moclobemide/creatine-treated mice adopting a different strategy to solve the tasks. In particular, vehicle-treated R6/2 mice displayed an increase in perseverative errors that was prevented by tacrine/moclobemide/creatine treatment. In the T-maze, perseveration is unlikely to result from impaired working memory, since if this was the case, performance should have reverted to chance .

Improvements in T-maze performance were attributable to the combined action of tacrine and moclobemide.

Tacrine/moclobemide/creatine treatment also improved performance in the Morris water maze via a change in strategy used to find the hidden platform, since thigmotaxis was significantly reduced by tacrine/moclobemide/creatine treatment. While few studies have examined the neurochemistry or neuroanatomy of thigmotaxis in mice, the absence of thigmotaxis in fimbria fornix lesioned rats points towards a non- hippocampal origin of this type of behaviour (Devan & White (1999) J^". Neuroscience 7, 2789-2798) Indeed, lesions of the medial striatum but not the dorsal striatum were associated with increased thigmotaxis. This suggests (a) that an underlying dysfunction in this striatal subregion as well as the hippocampus of R6/2 mice may be responsible for the behaviour, and (b) this deficit can be improved by tacrine/moclobemide/creatine treatment . Notably that there is pathology in both the hippocampus and striatum in presymptomatic mice (Morton et al (2000) J. Neurocy tol .29 , 681-705) .

The perseverative behaviours that improved in the R6/2 mouse after tacrine/moclobemide/creatine treatment, have human correlates that are found in HD. Visuospatial deficits are one of the earliest cognitive changes in HD (Lawrence, A. D. et al (1996) Brain. 119, 1633-1645), and perseverative behaviour has been reported in HD patients in Wisconsin card sorting tests (Backman et al (1997) Brain 120, 2207-2217, Josiassen, R. C. et al (1983) Arch Neurol . 40, 791-796), attention set shifting tests (Lange, K. W. et al (1995) J Neurol Neurosurg Psychiatry. 58, 598-606, Lawrence 1996 supra) , and spatial working memory tasks (Lawrence, 1996 supra) . Obsessive compulsive disorder, arguably considered as a type of perseverative behaviour, is also found in HD patients (Harper, 1996 supra, Anderson, K.E. et al (2001 Am. J. Psychiatry 158, 799-801) .

A number of other parameters, including motor performance on the rotorod, onset of diabetes and glucose sensitivity, body weight and survival (Fig. 3), were assessed to determine whether or not the drug treatment improved other aspects of R6/2 mouse phenotype.

R6/2 mice showed normal growth until about 11 weeks of age when body weight dropped dramatically irrespective of drug treatment (transgene x days

P, 0.001; drug, transgene x drug and transgene x drug x days all n.s. )

Apart from the onset of diabetes, which was significantly delayed (Fig. 3c: Log rank=5.258, P<0.01), and a small improvement in survival (see below) , none of these factors were significantly affected by tacrine/moclobemide/creatine treatment. R6/2 mice develop diabetes (Hurlbert et al, (1999) Diabetes 48 649-651), and in our colony about 75% of mice are diabetic by death at 16-17 weeks of age. Tacrine/moclobemide/creatine treatment significantly delayed the onset of glycosuria (P<0.05), although it did not alter the percentage of mice eventually developing glycosuria (23/29 compared to 48/58 of vehicle-treated R6/2 mice) . Notably, tacrine/moclobemide/creatine did not simply delay glycosuria onset, rather there was a slowing of appearance of diabetes. After fasting, R6/2 mice treated with tacrine/moclobemide/creatine or creatine alone displayed higher blood glucose concentrations but no other differences were found between R6/2 groups (Fig. 3c) . The delayed in onset of glycosuria produced by tacrine/moclobemide/creatine is unlikely to underlie the improvement in cognitive function, since (i) double treatment with tacrine and moclobemide enhanced cognition without affecting glycosuria, (ii) the onset of glycosuria did not correlate with deterioration of cognitive performance or changes in synaptic plasticity (Murphy, K. P. et al (2000) J Neurosci. 20, 5115-5123) and (iii) there was no difference in the number of mice that developed diabetes between R6/2 groups.

Survival was improved by tacrine/moclobemide/creatine treatment (Fig 3b; Log rank=5.258, P<0.01) and also by tacrine (from 111+2 (N=23) to (119+3 (N=24) days) . The cause of death of R6/2 mice is at present unknown, and the mechanism underlying these improvements in survival are also unknown. However, although improvements in survival are commonly used to show beneficial effects of drugs, it should be noted that our untreated animals already survive longer than R6/2 mice from other studies where beneficial drug effects have been reported. For example, in a recent study showing that cystamine improved survival in R6/2 mice the number of days the mice survived increased from 92+12 to 103±9 days (Karpuj et al. Nature Medicine 8, 143-149 (2002)). However, the mean survival of vehicle-treated mice in this study was already 109+1 days (N=95) , nearly a week longer than the drug-improved group in their study. A number of factors affect R6/2 survival, in particular improved access to food and environmental enrichment (Carter, R. J. et al (2000) Mov Disord. 15, 925-937) . Increased survival following our drug treatments, particularly creatine, was not expected because, while creatine treatment improves survival of R6/2 mice housed in standard condition with food given as hard pellets, (Ferrante et al, 2000 supra) , the effect is abolished when hard food is supplemented with mash. This indicates that in animals that are sub- optimally nourished, creatine acts as an energy supplement. In accordance with this, a significant increase of body weight after creatine treatment was observed in the present experiments, particularly in mice aged 12-15 weeks of age. However, creatine treatment had no beneficial effect on any other parameter, as all animals in this study were given mash-supplemented diets.

While marked improvements in cognitive function were found after tacrine/moclobemide/creatine treatment, we did not find any beneficial effect of any of the drugs used on motor performance on the rotorod (Figure 3d: all comparisons between treated and untreated, n.s.) . R6/2 mice showed a progressive impairment at greater ages for any given rotation speed compared to WT mice. This was expected, since DA has been shown to be the primary neurotransmitter which modulates motor dysfunction in R6/2 mice (Hickey, M. A, et al . (2002). J^". Neurochem. , in press) , and DA remains relatively unchanged by the drugs used.

No histopathological changes were observed in brains of mice treated with the drugs. However, this was also expected, since all mice were used for survival studies and therefore died of their phenotype.

In contrast to tacrine and moclobemide, the role of creatine in the beneficial effects observed after tacrine/moclobemide/creatine treatment is not clear. The combination of tacrine and moclobemide is responsible for the cognitive enhancing effects, since tacrine/moclobemide produced equivalent improvements to the triple treatment in the Morris water maze, and creatine is not essential. However, no deleterious effects of creatine were observed either alone or in combination with any other drug and this is significant because it indicates that tacrine and moclobemide could be used safely in patients who were independently taking creatine supplements.

Claims

Claims :

1. A composition comprising a first agent which is a monoamine oxidase inhibitor and a second agent which is a acetylcholine esterase inhibitor.

2. A composition according to claim 1 wherein the first agent is a reversible and selective inhibitor of MAO-A isoenzyme

3. A composition according to claim 2 wherein the first agent is moclobemide and the second agent is tacrine.

4. A composition according to claim further comprising a pharmaceutically acceptable excipient .

5. A composition according to any one of the preceding claims comprising creatine.

6. A composition according to any one of the preceding claims for use in a method of treatment of the human or animal body

7. A composition according to any one of claims for use in the treatment of Huntington's Disease (HD) .

8. Use of a composition according to any one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of HD

9. Use of a monoamine oxidase inhibitor and an acetylcholine esterase inhibitor in the manufacture of a medicament for use in the treatment of HD.

10. Use according to claim 9 wherein the monoamine oxidase inhibitor is a reversible and selective inhibitor of MAO-A isoenzyme

11. Use according to claim 9 or claim 10 wherein monoamine oxidase inhibitor is moclobemide and the acetylcholine esterase inhibitor is tacrine.

12. Use according to any one of claims 9 to 11 wherein the medicament comprises creatine.

13. A method for the treatment of HD comprising administering a therapeutically effective amount of tacrine and a therapeutically effective amount of moclobemide to an individual in need thereof .

14. A method according to claim 13 further comprising administering creatine to said individual .

15. A method of making a pharmaceutical composition comprising admixing tacrine and moclobemide and a pharmaceutically acceptable excipient.

16. A method according to claim 15 wherein the composition is for the treatment of HD.

17. A method according to claim 15 or claim 16 further comprising admixing creatine to said composition.