WO 2007/049262 Al

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization International Bureau

(43) International Publication Date (10) International Publication Number 3 May 2007 (03.05.2007) PCT WO 2007/049262 Al

(51) International Patent Classification: (74) Agents: GATES,Marie Christina, Esther etal.; Tomkins A61K 31/167 (2006.01) A61K 31/4406 (2006.01) & CO., 5 Dartmouth Road, Dublin 6 (IE). A61K 31/19 (2006.01) A61P 25/00 (2006.01) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (21) International Application Number: AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, PCT/IE2006/000121 CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, (22) International Filing Date: 27 October 2006 (27.10.2006) JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LV,LY, MA, MD, MG, MK, MN, MW, MX, MY, (25) Filing Language: English MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 2005/0720 27 October 2005 (27. 10.2005) IE GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), (71) Applicant (for all designated States except US): BERAND European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, LIMITED [IE/IE]; UCD Conway Institute of Biomolecu- FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT, lar and Biomedica, 1Research, University College Dublin, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, Belfield, Dublin 4 (IE). GN, GQ, GW, ML, MR, NE, SN, TD, TG).

(72) Inventors; and Published: (75) Inventors/Applicants (for US only): REGAN, Ciaran, — with international search report M. [IE/IE]; c/o Berand Limited, UCD Conway Institute of — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of Biomolecular and Biomedica, 1, Research, University Col lege Dublin, Belfield, Dublin 4 (IE). FOLEY, Andrew, amendments G. [IE/IE]; c/o Berand Limited, UCD Conway Institute of For two-letter codes and other abbreviations, refer to the "G uid Biomolecular and Biomedica, 1, Research, University Col ance Notes on Codes and Abbreviations" appearing at the beg in lege Dublin, Belfield, Dublin 4 (IE). ning of each regular issue of the PCT Gazette.

(54) Title: Methods and compositions for the promotion of neuronal growth and the treatment of asociality and affective disorders

(57) Abstract: The invention relates to the use of at least one HDAC inhibitor in the manufacture of a medicament for promoting neuronal growth, to pharmaceutical compositions for promoting neuronal growth comprising at least one histone deacetylase inhibitor (HDAC) and to methods for promoting neuronal growth comprising administering to a patient in need of such therapy a pharmaceutically effective amount of at least one histone deacetylase inhibitor. Title Methods and compositions for the promotion of neuronal growth and the treatment of asociality and affective disorders.

Field of the Invention The present invention relates to methods of and compositions for promoting neuronal growth within the central nervous system of a mammal, and methods of and compositions for the treatment of asociality and affective disorders. The methods and compositions are based on histone deacetylase (HDAC) inhibitors.

Background to the Invention Asociality is a reluctance or inability to engage with other people and is a serious disability that plays a central role in a large range of disorders, and syndromes and results in impaired social and occupational interaction. Asocial tendancies manifest in a wide variety of symptoms such as avoidant behaviour, social withdrawal, social inhibition, impaired ability/ desire to communicate, alogia, abnormal social interaction, impairment of nonverbal behaviours, inability to develop peer relationships, inattention in social situations, hypersensitivity in social situations, restrictions in range and intensity of emotional expression, decreased responsiveness or receptiveness to the external world, and avolition.

A broad definition of affective disorders are mental disorders characterized by a consistent, pervasive alteration in mood, affecting the stability of thought, emotion, and behaviour and include a range of anxiety disorders, bipolar disorder, and depression. There is an obvious overlap between the two areas defined here, namely when asocial behaviour stems from various anxieties, or depressive states. Anxiety disorder refers to several different forms of abnormal anxiety, fear, phobia and nervous condition, in which the state of fear and mental tension is inappropriate and interferes with normal social and occupational functioning. The feeling of fear, apprehension and worry is often accompanied by physical sensations that may take the form of a panic attack.

Diseases which can be treated by the method of the present invention in regards to social dysfunction and affective instability include, but are not limited to, selective mutism, schizophrenia, avoidant personality disorder, social phobia, Attention Deficit- Hyperactivity Disorder (ADHD), major depressive disorder, post traumatic stress disorder, reactive attachment disorder, Fragile X, specific phobias, separation anxiety disorder, generalised anxiety disorder, panic disorder, agoraphobia, substance-related anxiety disorders, anorexia nervosa/bulimia, obsessive compulsive disorder, sexual function disorders, body dysmorphic disorder, adjustment disorder, and the Pervasive Developmental Disorders (PDDs; Rett's disorder, Asperger's disorder, autism, disintegrative disorder, and PDD Not Otherwise Specified including atypical autism disorder [Muhle et al, (2004) Pediatrics 113, 472-486; DSM-IV-TR, 4th edition, American Psychiatric Association]). The present invention may also be implicated in the treatment of affective spectrum disorders. The affective spectrum is a grouping of related psychiatric and medical disorders, which may accompany bipolar, unipolar, and schizoaffective disorders at statistically higher rates than would normally be expected. These disorders are identified by a common positive response to the same types of pharmacologic treatments. They also aggregate strongly in families and may therefore share common heritable underlying physiologic anomalies. Affective spectrum disorders that may benefit from treatment by the proposed method include attention-deficit hyperactivity disorder, bipolar disorder, body dysmorphic disorder, bulimia nervosa, cataplexy, dysthymia, general anxiety disorder, hypersexuality, impulse-control disorders, obsessive-compulsive disorder, panic disorder, posttraumatic stress disorder, premenstrual dysphoric disorder, social phobia, and Tourette's syndrome.

The medial temporal lobe, comprising the olfactory cortex, the amygdala and the hippocampus, is an area long associated with mediating emotional responses, fear, and in maintaining euthymia. Predictably, abnormalities in these areas are linked to bipolar disorder, depression and anxiety disorders. Functionally, increased activation in medial temporal lobe regions has been observed in patients suffering from panic attacks, specific and social phobias, anorexia nervosa, bipolar disorder, and depression (Veltman et al, (2004) Psychiatry Res. 132, 149-158; Furmark et al, (2002) Arch. Gen. Psychiatry 59, 425-433; Gordon et al (2001) J. Pediatr. 139, 51-7; Strakowski et al, (2005) MoI. Psychiatry 10, 105-1 16). Concordantly, positron emission tomography (PET) scans of phobic individuals has shown increased cerebral blood flow in the medial temporal lobe during exposure to the specific stressor (Veltman et al, (2004) Psychiatry Res. 132, 149-158; Furmark et al, (2002) Arch. Gen. Psychiatry 59, 425-433), and, similarly, patients with anorexia nervosa experienced increased blood flow to the medial temporal lobe, along with increased anxiety and heart rate, on exposure to high calorie foods (Gordon et al, (2001) J. Pediatr. 139, 51-7). These neuroimaging data are consistent with the wealth of evidence indicating that the medial temporal lobe especially the amygdala and hippocampus is crucially involved in the regulation of anxiety related behaviours, including social anxiety (Davidson et al, (2000) Psychol. Bull. 126, 890-909). Additionally, there is evidence of functional disconnectivity of the medial temporal lobe in patients with Asperger's syndrome during fearful face processing (Welchew et al, (2005) Biol. Psychiatry 57, 991-8)

Moreover, evidence of structural abnormalities also exists in these brain structures. The amygdala and cerebellum have been reported as abnormal in bipolar disorders, while for anxiety disorders, the temporal lobe was found to be abnormally reduced in panic disorder, and abnormal hippocampus shrinkage was shown in posttraumatic stress disorder (Bambilla et al, (2002) Epidemiol. Psichiatr. Soc. 11, 88-99). Recent findings suggest that the size of amygdala, hippocampus, and corpus callosum may also be abnormal in autistic patients (Brambilla et al, (2003) Brain Res. Bull. 61, 557- 69). There are reports of decreased hippocampal volume in depression and bipolar disorder, as well as decreased neurogenesis (Harrison (2002) Brain 125, 1428-49). It is possible that impaired neurogenerative capability in these regions underlie the deficits in emotional regulation, for example Laeng et al, ((2004) J. Neurochem. 91, 238-251) suggest that the therapeutic effects of the mood stabiliser VPA in bipolar disorder is due to the promotion of neurogenesis, via increasing cell number, neurite outgrowth, and neuronal differentiation. Therefore structural improvement by promotion of neurogenesis may be beneficial in these types of disorder.

Polysialylated neural cell adhesion molecule (NCAM PSA) has been demonstrated to be a neuroplastic marker associated with neurogenesis, neurite outgrowth and de novo synapse formation. In fact, its presence is required for learning, and synaptic plasticity. In particular it is associated with transient changes in the medial temporal lobe during memory consolidation following various forms of learning and experience. Interestingly, mice deficient in NCAM, as well as exhibiting deficits in spatial learning, show morphological and behavioural abnormalities, including increased social aggressiveness. This was attributed to an enhanced neuronal activity in medial temporal lobe regions and a resultant increase in social stress in these mutant animals as evidenced by an increase in corticosterone levels (Stork et al, (1997) Eur. J. Neurosci. 9, 1117-25).

NCAM PSA expression can be induced under a wide range of conditions and is, like any other inducible component, subject to effects on gene regulation. Epignetics is the study of factors that change the phenotype without changing the genotype, for example, chromatin remodelling leading to altered gene expression patterns. One factor mediating epigenetic control concerns post-translational modification of histones. Histone acetylation is the best studied of these modifications, with a balance being maintained through addition of acetyl groups via histone acetyl tranferases and removal of these groups by histone deacetylases (HDACs) (Thiagalingam et al. [2003] Ann. N .Y. Acad. Sci. 983, 84-100).

Administration of the HDAC inhibitors VPA or trichostatin A (TSA) in vitro increases mRNA and protein expression of both NCAM and the polysialtransferase enzymes responsible for the addition of PSA moieties to NCAM (Lamped et al. (2005) MoI. Pharmacol. 68, 193-203). Moreover, in animals, VPA analogue administration increases NCAM polysialylation in the hippocampus and improves performance in models of learning and memory (Murphy et al. (2001) J. Neurochem. 78, 704-714). Additionally, there is evidence of a HDAC inhibitor acting as a euthymic mediator; VPA, a HDAC inhibitor, is used as a mood stabiliser in the treatment of bipolar disorder. Furthermore, Phiel et al., ((2001) J. Biol. Chem. 276, 36734-41) suggest that it is the HDAC inhibitory activity of VPA that may account for its efficacy in the treatment of bipolar disorder. In concordance, another common antiepileptic and mood stabilising drug, carbamazepine, has recently been shown to have the ability to inhibit HDACs.

Conventional methods of treating asociality and affective disorders are wide ranging, including behavioural and psychological therapies as well as drug therapies. In the case of affective disorders, conventional treatments for depression include both psychological and pharmacological therapy. The first antidepressants, serendipitously discovered, were the monoamine oxidase inhibitors which increased synaptic availability of serotonin, dopamine and noradrenaline, closely follwed by the tricyclic antidepressants which effected only noradrenaline and serotonin. As a result it was proposed that depression results from a central deficiency of monoaminergic function (Wong and Licinio (2004) Nature Rev. Drug Disc. 3, 136-151), and antidepressants typically target these systems. Development of novel antidepressants has, until recently, been limited to increasing tolerability, safety and selectivity of monoaminergic modulators, resulting in the various Selective Reuptake Inhibitors (SRIs; selective serotonin reuptake inhibitors (SSRIs), Serotonin noradrenergic reuptake inhibitors (SNaRIs), Noradrenaline reuptake inhibitors (NaRIs), Noradrenaline and dopamine reuptake inhibitors (NDRIs) and Noradrenergic and specific serotinergic antidepressants (NaSSA)). Not only is there a long timecourse before onset of benefit, all of these drugs induce many side-effects such as dry mouth, constipation, weight gain/ loss, impaired thinking, tiredness, diminished sexual function and nervousness. Most recent developments in antidepressants have centred around the idea that chronic stress and resultant activation of the hypothalamus- pituitary-adrenal axis may increase susceptibility to depression. Consequentially, substance P antagonists, and corticotrophin releasing hormone (CRH) antagonists, have been successfully used in Phase I, clinical trials, but are still being developed for better pharmacological profiles. Treatments for bipolar disorder include mood stabilisers such as lithium and the anticonvulsants Valproic acid and carbamazepine, as well as various combinations of antidepressants. Lithium. can induce weight gain, tremors, thyroid abnormalities, and gastrointestinal problems, while Valproic acid and carbamazepine can also induce weight gain, tremors, and gastrointestinal adverse events, as well as alopecia, sedation, and thrombocytopenia. Atypical antipsychotics are also prescribed for manic symptoms. However, there is no generally accepted successful treatment for such disorders.

As regards anxiety disorders, the most common treatment is benzodiazepines which modulate the GABAergic system, that influences the activity of other neurotransmitters such as that of the serotonergic system. While these drugs may be effective in certain conditions, they do have certain inherent problems, such as dependence at therapeutic doses after a brief period of use, toxicity when taken in conjunction with other medications, psychomotor retardation/slowing, memory impairment, tolerance, and suicidal ideation. SSRIs are also approved for the treatment of anxiety disorders.

Treatment of asociality from a pharmacological perspective has to date been inadequate; current treatments are palliative rather than remedial. In the case of Pervasive Development Disorders (PDDs), drugs target the secondary symptoms (aggression and anxiety) or aberrant behaviours (hyperactivity, temper, stereotypies, self-injury), not the primary symptoms such as abnormal communication and social behaviour or impaired cognition. All current drugs were developed for other purposes and only later tried in autistic patients, these include SSRIs, atypical antipsychotics, and antidepressants.

Conventional methods of treating nerve damage include administration of nerve growth factors (NGF), Insulin and insulin-like growth factors, fibroblast growth factor (FGF) among others, and piperazine and piperidine derivatives have been suggested as a potential agent for preventing neuronal damage. However, hothere is no generally accepted agent for use in promoting neuronal growth.

HDACs are generally known in the treatment of cancers, haematological disorders, autosomal dominant disorders, genetic related metabolic disorders and neurodegenerative diseases. WO 2005/009349 relates to methods of treating or preventing neurological disorders by administering DNA methylation inhibitors, either alone or in conjunction with a histone deacetylase inhibitor. Thus while HDACs are known, it has not previously been shown that HDAC inhibitors are capable of enhancing social interactions and in promoting neuronal growth.

Object of the Invention

It is thus an object of the present invention to provide new methods and compositions for promoting neuronal growth and preventing and/or treating neuronal damage associated with neuropathologic conditions. In particular, it is an object to provide methods and compositions for improving or enhancing robustness of the CNS. By 'robustness treatments' is meant the enhancement of the ability of the brain to strengthen synaptic contacts between nerve cells in a manner that resists degenerative processes associated with aging or the course of psychiatric or neurological illness. Therapeutic enhancements in expression of NCAM PSA parallel concomitant cognitive improvements in animal models (Murphy et ah, Neuropsychopharmacology, 2005 Epub ahead of print; Foley et ah, J Neurosci Res 82 2005 245). A further object of the invention is to provide new methods and compositions for the treatment of asociality and affective disorders. In particular, it is an object to provide methods and compositions for mood enhancement.

Summary of the Invention

According to the present invention there is provided a composition for promoting neuronal growth comprising a histone deacetylase inhibitor (HDAC). The invention also provides methods for promoting neuronal growth comprising administering to a patient a histone deacetylase inhibitor

In a further aspect the invention provides a composition for the treatment of asociality and affective disorders comprising a histone deacetylase inhibitor (HDAC). The invention also provides methods for the treatment of asociality and affective disorders comprising administering to a patient a histone deacetylase inhibitor

In another aspect the invention provides use of HDAC inhibitors in the manufacture of a medicament for promoting neuronal growth and in the manufacture of medicaments for the treatment of asociality and affective disorders.

Histone deacetylase inhibitors or HDAC inhibitors, as that term is used herein are compounds that are capable of inhibiting the deacetylation of histones in vivo, in vitro or both. As such, HDAC inhibitors inhibit the activity of at least one histone deacetylase. HDAC inhibitors suitable for use in the present invention can be categorised into six general classes: 1) hydroxamic acid derivatives; 2) short-chain fatty acids; 3) epoxy and non-epoxy ketone-containing cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and 6) miscellaneous HDAC inhibitors. However, any other class of compound capable of inhibiting histone deacetylases is suitable for use in the invention. Examples of such HDAC inhibitors of each class include but are not limited to:

1) Hydroxamic acid derivatives such as:

3C1-UCHA: 6-(3-Chlorophenylureido) carpoic hydroxamic acid; A-161906: CAS Registry No. 191228-04-3 (7-[(4'-cyano[l,l'-biphenyl]-4-yl)oxy]-N- hydroxy- heptanamide); ABHA: CAS Registry No. 18992-11-5 (N,N'-dihydroxy-nonanediamide); AAHA: (Azelaic-l-hydroxamate-9-anilide); CRA-A: CAS Registry No. 756486-62-1 (3'-[(dimethylamino)carbonyl]-N~hydroxy-

5'-[(4-methylbenzoyl)amino]- [ 1, 1'-Biphenyl]-4-acetamide); CBHA: CAS Registry No. 174664-65-4 (N-hydroxy-3-[3-(hydroxyamino)-3-oxo-l- propenyl]- benzamide); JNJ16241 199; 2-[4-(naphthalen-2-ylsulfonyl)piperazin-l-yl]pyrimidine-5- carbohydroxamic acid LAQ-824: CAS Registry No. 591207-53-3 (N-hydroxy-3-[4-[[(2-hydroxyethyl)[2- (1H-indol-3 -yl)ethyl]amino]methyl]phenyl] -2-propenamide) ; MS344: CAS Registry No. 251456-60-7(4-(dimethylamino)-N-[7-(hydroxyamino)-7- oxoheptyl] -benzamide); Oxamflatin: CAS Registry No. 151720-43-3 (N-hydroxy-5-[3- [(phenylsulfonyl)amino]phenyl]-2-penten-4-ynamide); Pyroxamide: CAS Registry No. 382180-17-8 (N-hydroxy-N'-3-pyridinyl- octanediamide); PXDlOl: CAS Registry No. 414864-00-9 (N-hydroxy-3-[3- [(phenylamino)sulfonyl]phenyl]- 2-propenamide); SAHA: CAS Registry No. 149647-78-9 (N-hydroxy-N'-phenyl-octanediamide); SBHA: CAS Registry No. 38937-66-5 (N,N'-dihydroxy-octanediamide); Scriptaid: CAS Registry No. 287383-59-9 (N-hydroxy-l,3-dioxo-lH- Benz[de]isoquinoline-2(3H)-hexanamide); TSA: CAS Registry No. 58880-19-6 (7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6- dimethyl-7-oxo-2,4-heptadienamide); and Tubacin: CAS Registry No. 537049-40-4 (N-[4-[(2R,4R,6S)-4-[[(4,5-diphenyl-2- oxazolyl)thio]methyl]-6-[4-(hydroxymethyl)phenyl]-l,3-dioxan-2-yl]phenyl]-N'- hydroxy-octanediamide).

2) Short-chain fatty acids derivatives such as: 4-Phenyl butyrate: CAS Registry No. 4346-18-3 (phenyl ester butanoic acid); AN-9: CAS Registry No. 1221 10-53-6 (Pivanex; (2,2-dimethyl-l-oxopropoxy) methyl ester butanoic acid); Sodium butyrate: CAS Registry No. 156-54-7 (Butanoic acid, sodium salt); Sodium phenyl butyrate ; Valproic Acid: CAS Registry No. 99-66-1 (2-propyl-pentanoic acid); Isovalerate: CAS Registry No. 5711-68-2 (3-methyl-butanoic acid); Valerate: CAS Registry No. 10023-74-2 (Pentanoic acid); Propionate: CAS Registry No. 72-03-7 (Propanoic acid); Butyramide: CAS Registry No. 541-35-5 (Butanamide); Isobutyramide: CAS Registry No. 563-83-7 (2-methyl-propanamide); Phenylacetate: CAS Registry No. 7631-42-7 (Benzeneacetic acid); 3-bromopropionate: CAS Registry No. 16336-88-2 (3-bromo-propanoic acid); and Tributyrin: CAS Registry No. 60-01-5 (1,2,3-propanetriyl ester-butanoic acid).

3) CyclicTetrapeptides such as: a) epoxy-ketone containing: HC-toxin cyclic tetrapeptide: CAS Registry No. 83209-65-8 (Cyclo[L-alanyl-D- alanyl-( αS,2S)-α-amino-η-oxooxiraneoctanoyl-D-prolyl]); Trapoxin A: CAS Registry No. 133155-89-2 (cyclic tetrapeptide (Cyclo[(αS,2S)-α- amino-η-oxooxiraneoctanoyl-L-phenylalanyl-L-phenylalanyl-(2R)-2- piperidinecarbonyl] ). b) non epoxy-ketone containing: Apicidin: CAS Registry No. 183506-66-3 (Cyclo[(2S)-2-amino-8-oxodecanoyl-l- methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidinecarbonyl]); Chlamydocin CAS Registry No. 53342-16-8 (Cyclo[2-methylalanyl-L-phenylalanyl- D-prolyl-( αS,2S)-α-amino-η-oxooxiraneoctanoyl]); CHAPs CAS Registry No. 618056-29-4 (general; Cyclo[(2S)-2-amino-8- (hydroxyamino)-8-oxooctanoyl-L-phenylalanyl-L-phenylalanylprolylj); and Depsipeptide: CAS Registry No. 128517-07-7 (FK228; FK901228; Cyclo[(2Z)-2- amino-2-butenoyl-L-valyl-(3S,4E)-3-hydroxy-7-mercapto-4-heptenoyl-D-valyl-D- cysteinyl], cyclic (3-»5)-disulfide).

4) Benzamide derivatives such as: CI-994 CAS Registry No. 112522-64-2 (4-(acetylamino)-N-(2-aminophenyl)- benzamide); and MS-275 CAS Registry No. 209783-80-2 ([[4-[[(2-aminophenyl)amino]carbonyl]- phenyl]methyl]-3-pyridinylmethyl ester carbamic acid).

5) Electrophilic ketone derivatives such as: trifh αoromethyl ketones; alpha-keto amides; alpha-keto oxazoles; and alpha-keto heterocycles.

6) Miscellaneous HDAC Inhibitors such as: AOE (2-Amino-8-oxo-9,10-epoxydecanoic acid); Bromoacetamides;

Depudecin: CAS Registry No. 139508-73-9 (4,5 :8,9-dianhydro-l, 2,6,7, 11- pentadeoxy-D-threo-D-ido-undeco- 1,6-dienitol); Methyl sulfoxides; Mercaptoacetamides; N-formyl hydroxylamino; Psammaplins; Semi Carbazides; Sulfur containing cyclic peptides (SCOPs); Thiol derivatives; and others.

It is understood that the present invention includes any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers, polymorphs and prodrugs of the HDAC inhibitors described herein. This invention, in addition to the above listed compounds, is intended to encompass the use of homologues and analogues of such compounds. In this context, homologues are molecules having substantial structural similarities to the above-described compounds and analogues are molecules having substantial biological similarities regardless of structural similarities.

The invention also encompasses pharmaceutical compositions for promoting neuronal growth and for the treatment of asociality and affective disorders comprising pharmaceutically acceptable salts of the HDAC inhibitors with organic and inorganic acids, for example, acid addition salts which may, for example, be hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid and the like. Pharmaceutically acceptable salts can also be prepared from the above by treatment with inorganic bases, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The invention also encompasses pharmaceutical compositions comprising hydrates of the HDAC inhibitors. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

The HDAC inhibitors can be in a crystalline form, in amorphous form, and have any particle size. The HDAC inhibitor particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

Wherein said HDAC inhibitor is administered in the form of a pharmaceutical composition it may be prepared in admixture with one or more pharmaceutically acceptable excipients.

A pharmaceutical composition of the invention, which may be prepared suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusable solutions or suspensions or suppositories. Orally administrable compositions are generally preferred.

Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants and acceptable wetting agents. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colourants.

For parenteral administration, fluid unit dosage forms are prepared utilising a compound of the invention or a pharmaceutically acceptable salt thereof and a sterile vehicle. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the compound can be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspension in a sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound. The composition may contain from 0.1% to 99% by weight, preferably from 10 to 60% by weight, of the active material, depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors.

The present invention is illustrated by reference to the following Examples:

EXAMPLES

EXPERIMENTAL PROTOCOL

Animal maintenance Experimentally naϊve male Wistar rats were employed in all studies. The animals were purpose bred at the Biomedical Facility, University College Dublin, and maintained in standard laboratory conditions until the time of experimental use at postnatal day 64. Animals were introduced to the experimental holding rooms 5 days prior to the commencement of the study, housed in groups of six, and maintained at 22-24°C on a standard 12 hour light/dark cycle, with food and water available ad libitum. All animals were examined and weighed daily. All experimental procedures were approved by the Animal Research Ethics Committee of University College Dublin, and were carried out by Dr Andrew Foley and Dr Claire Barry, who retain the appropriate licences issued by the Irish Department of Health.

Drug administration

All studies were conducted using suberoylanilide hydroxamic acid (SAHA; Axxora Ltd. UK, batch number L 15566), the benzamide derivative 4-[[(2- aminophenyl)amino]carbonyl]-phenyl]methyl]-3-pyridinylmethyl ester carbamic acid (MS-275; Axxora Ltd. UK, batch number L14358/a) and the short-chain fatty acid valproic acid (VPA; Sigma Ltd. UK, batch number 064K1585). SAHA was administered via the intraperitoneal route (i.p.) at 5, 10 and 50 mg/kg in a 2 ml/kg dose volume of a 2-hydroxypropyl-beta-cyclodextrin vehicle (9g/L). In a second experimental cohort MS-275 was administered at 1 mg/kg in a 2 ml/kg dose volume of dH20 vehicle (solubility was assisted by first dissolving the MS-275 with a drop of 100% glacial acetic acid and adjusting the pH to 6.6 with the addition of NaOH).

VPA was administered at 150 and 250 mg/kg in a 2 ml/kg dose volume of dH2O vehicle. Test compounds were administered for 8 days prior to training in the water maze and 1.5 hours prior to each training and recall session. Treatment continued after the final recall test on day 15 of the experiment until day 2 1 when social approach-avoidance behaviour was assessed. The drug was administered 1.5 hours prior to its evaluation in the latter paradigm. Vehicle-treated controls were employed in each experimental cohort for comparison.

Quantitative analysis of NCAM PSA expression

(i) Ciyosection technique

Freshly dissected whole rat brain was carefully coated in optimum cutting temperature (OCT) compound and lowered into a Cryoprep freezing apparatus containing dry-ice- cooled n-hexane. The function of the OCT compound and n-hexane was to provide an even freezing of the tissue, thus avoiding freezing artefacts. Horizontal sections for all studies were cut semi-automatically or automatically on a Microm Series 500 ciyostat. Fresh, frozen brain sections (12µm) were cut at -15°C, while cryoprotected. AU sections were prepared on the day of the experiment and were not pre-cut and stored frozen. For the analysis of the NCAM PSA-positive hippocampal dentate granule cell layer/hilus border cells, 10 alternate sections were taken at a level equivalent to -5.6mm below Bregma (Paxinos and Watson, 1986), at which level this cell population was found to be maximal.

(ii) Immunohistochemicalprotocol

Horizontal cryosections were cut from the frozen tissue at various levels with reference to Bregma (see above), these were thaw-mounted onto glass slides, which were coated with poly-1-lysine diluted 1:1 in dH2O, and immersion fixed for 30 minutes with 70% ethanol. The sections were then washed twice for 10 minutes each in 0.1M phosphate buffered saline (PBS) and incubated for 20 hours in a humidified chamber at room temperature with the primary antibody diluted 1:500 in PBS containing 1% bovine serum albumin (w/v) and 1% normal goat serum (v/v) to reduce non-specific staining. The humidified chamber prevented the sections from evaporating. The primary antibody was a monoclonal raised against PSA, which was provided by Professor G, Rougon (CNRS UMR 6545, 13288 Marseille, France). On completion of the primary antibody incubation, the sections were washed twice for ten minutes each in PBS and incubated at room temperature for 3 hours in the humidified chamber with the secondary antibody, at a dilution factor of 1:100, again in PBS containing 1% BSA and 1% NGS. The secondary antibody was a goat anti-mouse IgM conjugated to fluorescein (FITC). Following the second incubation, the sections ware again washed twice for ten minutes each in PBS, mounted in the fluorescence enhancing medium Citifluor® and observed for fluorescence with a Leitz DM RB fluorescent microscope.

(iii) Quantitative evaluation of NCAM PSA expression

Quantitative image analysis was performed using the Leica Quantimet 500®, a P.C - based software package, which was connected to the fluorescence microscope with a high sensitivity CCD video camera. Each microscope lens was calibrated for length and area measurements using a lmm graticule. The total number of NCAM PSA- immunoreactive neurons on the right dentate granule cell layer/hilar border were counted in 7 alternate 12µm sections commencing -5.6mm from Bregma (Paxinos and Watson, 1986), to preclude double counting of the 5-10µm perikarya. Cell identification was aided by the use of the nuclear counter-stain propidium iodide (40ng/ml PBS; 60 sec). The number of cells was then divided by the total area of the dentate granule cell layer and multiplied by the average granule cell layer area for a p80 rat, which is 0.15 ± 0.01mm2 at this level. This was done for each section and a mean±SEM was calculated for each brain with the results expressed as PSA-positive cells per unit area. These results were then used to generate the mean±SEM for each animal group.

NCAM PSA-positive cell numbers were obtained from each animal group. Results were expressed as mean±SEM with at least 3-6 values per group and analysed by ANOVA or unpaired non-parametric, Student's t-test, as indicated.

Social approach-avoidance paradigm (i) Apparatus

The procedure employed was based on a previously published paradigm (Brodkin et al. [2004] Brain Research 1002, 151-157). In each trial, a test animal was evaluated for its social approach versus social avoidance towards a novel stimulus animal of equal size and age. Both stimulus and test animals were housed in groups (n=6) until time of assessment. Each test and stimulus animal was employed in only one trial. Behavioural testing was carried out in a specially constructed black Perspex apparatus. This rectangular box (65 cm long x 30 cm wide x 35 cm high) had two identical chambers (10 cm long x 30 cm wide x 35cm high) at either end that were separated from a central area by clear Perspex sheets with multiple, small, evenly placed holes over their surface. These chambers housed the stimulus animal, randomly assigned to left or right side chamber for each test, and the holes in the clear Perspex allowed for auditory, visual and olfactory interaction between the stimulus and test animals. The central portion of the apparatus was further divided by two black Perspex baffles with a 10 cm space to the outer wall. This created three equally sized interconnected areas (15 cm long x 30 cm wide x 35 cm high).

(ii) Social behaviour

At the beginning of each trial the entire apparatus was cleaned with 70% ethanol, dried and fresh sawdust bedding material added. The test animal was placed between the black Perspex baffles of the central area and allowed to freely explore all three areas during a 5-minute acclimatisation period. Exploratory behaviour was monitored using an overhead video camera linked to a computer. Time spent in each area was recorded when both head and forelimbs were within the particular area. At the end of the acclimatisation period the test rat was removed to a holding cage. The bedding material in the test apparatus was redistributed and the stimulus animal placed in one of the side chambers, now designated as the social area. The test animal was returned to the centremost area and the time spent in each of the three areas was again recorded for a 5-minute period.

(iii) Data analysis The times spent in each chamber of the apparatus were calculated and expressed as the mean ± SEM for the acclimatisation and social trials of the experiment and the presence of significant difference between treatments was determined by two-way ANOVA and post-hoc Bonferroni analysis. An additional parameter, approach- avoidance score was calculated by counting + 1 for each second spent in the social area, 0 for each second spent in the centre area (representing ambivalence between approach-avoidance), and - 1 for each second spent in the non-social area. Statistical difference was assessed by two-way ANOVA and post-hoc Bonferroni analysis. In all cases, values of p<0.05 were deemed to be significant.

Example 1: Effect of the histone deacteylase inhibitors suberoylanilide hydroxamic acid (SAHA), 4-[[(2-amino ρhenyl)amino]carbonyl]-phenyl]methyl]- 3-pyridinylmethyl ester carbamic acid (MS-275) and valproic acid (VPA) upon anxiety levels Postnatal day 64 animals (maintained in accordance with the general procedure outlined in section (a) above) were administered 5, 10 or 50 mg/kg suberoylanilide hydroxamic acid (SAHA), 1 mg/kg 4-[[(2-aminophenyl)amino]carbonyl]- phenyl]methyl]-3-pyridinylmethyl ester carbamic acid (MS-275), or 150 and 250 mg/kg valproic acid (VPA) via the intraperitoneal route (i.p.) for 8 days prior to study. Animals treated with either 5 or 10 mg/kg SAHA showed no change in spontaneous behaviour in an open-field apparatus (Table 1). Animals treated with 50 mg/kg SAHA, however, exhibited reduced exploration and activity in the open-field apparatus over the two days of evaluation. While VPA (150 and 250 mg/kg ) was without effect on openfield exploratory behaviour MS-275 ( 1 mg/kg) reduced exploration in the open-field on day one (Table 2).

TABLE 1

Treatment Locomotion

Day 1 Day 2

Vehicle 177.7±16.8 157±11.58 SAHA (5 mg/kg) 199.3±15.9 187±18.1 SAHA (10 mg/kg) 197.7±13.5 143±17.7 SAHA (50 mg/kg) 106.7±10.8 * 74±20.2

Treatment Rearing

Day l Day 2

Vehicle 36.3±3.2 25.7±3.5 SAHA (5 mg/kg) 35.3±3.3 29±3 SAHA (10 mg/kg) 37.3±4.6 27.7±4.7 SAHA (50 mg/kg) 19.3±2.9 16±4.5

Treatment Grooming

Day l Day 2

Vehicle l±0.7 1.7±0.6 SAHA (5 mg/kg) 0.8±0.4 0.8±0.4 SAHA (10 mg/kg) 1.3±0.7 l±0.7 SAHA (50 mg/kg) 0 * 0

Data represents mean ± SEM for each parameter over a 5-minute period in the open- field apparatus. Locomotion was quantified as the number of lines crossed, rearing as number of upright stances, and the frequency of a grooming behaviour. * P<0.05 versus vehicle, Student's t-test; n=6 in all cases.

TABLE 2

Treatment Locomotion

Day 1 Day 2

Vehicle 163.0±4.9 105.0±3.3 MS-275 ( 1 mg/kg) 147.7±5.9 116.3±17.1 VPA (150 mg/kg) 159.3±4.7 105.3±7.6 VPA (250 mg/kg) 156.7±6.9 101.3±4.6

Treatment Rearing

Day 1 Day 2

Vehicle 28.6±1.8 18.7±1.4 MS275 ( 1 mg/kg) 27.3±2.4 17±2.2 VPA ( 150 mg/kg) 36.0±2.2 19.3±1.4 VPA (250 mg/kg) 30.7±1.6 19.7±1.6

Treatment Grooming Day l Day 2

Vehicle 0.8±0.4 0.8±0.5 MS275 ( 1 mg/kg) 0.7±0.3 1.2±0.4 VPA (150 mg/kg) 0.8±0.4 1.0±0.3 VPA (250 mg/kg) 0.5±0.2 1.2±0.3

EXAMPLE 2: Effect of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA), 4-f[(2-aminophenyl)amino1carbonyI1-phenyl1methvIl- 3-pyridinylmethyl ester carbamic acid (MS-275) and valproic acid (VPA) upon social interaction in a rodent model of social approach-avoidance In order to determine the influence of the histone deacetylase inhibitors, SAHA, MS- 275 and VPA on social interaction, the social approach-avoidance paradigm, as described in sections (d) above, was employed. During the acclimatisation period, see table 3 below, all animal treatment groups explored the three areas of the apparatus. Analysis by one-way ANOVA indicated animals treated with SAHA at either 10 or 50 mg/kg spent significantly greater time exploring the left and right side areas as compared to the central area (F[2,17]=4.5 and 4.1; P=0.03, 10 and 50 mg/kg, respectively). Likewise, treatment with MS-275 ( 1 mg/kg) increased exploration of the left and right side areas (Table 4; F[2,17]=8.9; P=0.003, whereas vehicle- and VPA-treated animals explored all areas equally. No bias between left and right areas was observed in any treatment group. TABLE 3

Time spent in area (seconds)

Treatment left centre right

Vehicle 82±7.3 104.8+-1 1.5 113.2±9.4 SAHA (5 mg/kg) 88.5±15.5 114.7+3 95.8+15.7 SAHA (10 mg/kg) 104.5±10.9 73.2+12.5 122.3±1 1.8 * SAHA (50 mg/kg) 88.8±30.5 52.3+7.5 158.8+34.3 * Data represents mean ± SEM average time spent by the test animal in each area of the apparatus (seconds) during a 5-minute acclimatisation trial. Increased exploration of left or right areas, as compared to centre, is indicated by an asterisk, Bonferroni posi- hoc test, P<0.05, n=6 in all cases.

TABLE 4

Time spent in area (seconds)

Treatment left centre right

Vehicle 107.2±14.5 95.5±15. 1 97.3±23.5 MS-275 ( 1 mg/kg) 105.2±10.4 * 71.3+8.8 123.5+7.1 * VPA (150 mg/kg) 116.5±8.1 94.5+7.8 89.0+11.5 VPA (250 mg/kg) 95.8+.21.9 83.0+10.5 121.2+24.7

Data represents mean ± SEM average time spent by the test animal in each area of the apparatus (seconds) during a 5-minute acclimatisation trial. Increased exploration of left or right areas, as compared to centre, is indicated by an asterisk, Bonferroni post- hoc test, P<0.05, n=6 in all cases.

In the social-interaction trial, see tables 5 and 6 below, vehicle-treated animals tended to spend more time in the social side of the apparatus, but this was not significant (Student's t-test, p=0.08 and 0.18 versus non-social). However, SAHA treatment, at 5, 10 and 50 mg/kg, resulted in a significant increase in time spent in the social chamber compared to vehicle-treated controls. Analysis by one-way ANOVA indicated animals treated with SAHA at all doses spent significantly greater time exploring the social side of the apparatus (F[2,17]=60, 10.5 and 29.1; PO.0001, P=0.03, and PO.0001, 5, 10 and 50 mg/kg, respectively). Moreover, two-way ANOVA analysis indicated increased social interaction as compared to vehicle controls (F[2,30]=23.2, 10.2, 21.6; pO.OOOl, P=0.0004, PO.0001; 5, 10 and 50 mg/kg, respectively). Animals treated with MS-275 ( 1 mg/kg) significantly increased the time exploring the social area of the apparatus (Table 6; F[2,17]=52.7; PO.OOOl) and two-way ANOVA analysis indicated increased social interaction as compared to vehicle control (F[2,30]=14.7; P<0.0001). VPA treatment did not influence social interaction at either dose evaluated.

TABLE 5

Time spent in area (seconds)

Treatment social centre non-social JO Vehicle 130±13.3 97.5±18.5 72.5±26.5 SAHA (5 mg/kg) 208±16.2 53.7+1 1.8 38.3+6.1 SAHA (10 mg/kg) 173.2±20.4 61.0+22.7 65.8+14.6 SAHA (50 mg/kg) 207.7±23 .2 * 74.5+20.5 17.8+4.7 15

Data represents mean ± SEM average time spent in each area of the apparatus (seconds) during a 5-minute social interaction trial. Individual values significantly different from the corresponding vehicle-treated control, analysed by Bonferroni post- 2 hoc test, are indicated by an asterisk, P<0.05; n=6 in all cases.

TABLE 6

Time spent in area (seconds) 5 Treatment social centre non-social

Vehicle 118.0±14.0 100.2±24.9 81.8±20.9 MS-275 ( 1 mg/kg) 180.8±12.5 * 63.0+9.2 56.2+6.2 30 VPA (150 mg/kg) 82.8±15.63 123.3+48.8 93.8±41.6 VPA (250 mg/kg) 92.5±17.6 139.8±34.8 67.7±23.9

Data represents mean ± SEM average time spent in each area of the apparatus 35 (seconds) during a 5-minute social interaction trial. Individual values significantly different from the corresponding vehicle-treated control, analysed by Bonferroni post- hoc test, are indicated by an asterisk, P<0.05; n=6 in all cases.

0 An approach-avoidance score was also calculated for each treatment group as described in section (d)(iii) above, whereby SAHA and MS-275 treatment were found to significantly improve social interaction, with higher positive scores representing a preference for social approach. One-way ANOVA analysis indicated a significant effect of SAHA-treatment on social approach-avoidance score at all dose groups (F[3,23]=4.37; p=0.016 versus vehicle control). VPA was without effect on approach-avoidance score.

TABLE 7

Treatment approach-avoidance score

Vehicle 57.5±37.7 SAHA (5 mg/kg) 169.7±2 1.5 * SAHA (10 mg/kg) 107.3±27.3 SAHA (50 mg/kg) 189.8±26.6

Data represents mean ± SEM approach-avoidance score during a 5-minute social interaction trial. Individual values significantly different from the vehicle-treated control, analysed by Bonferroni post-hoc test, are indicated by an asterisk, P<0.05; n=6 in all cases.

TABLE 8

Treatment approach-avoidance score

Vehicle 36.2±25.4 MS-275 ( 1 mg/kg) 124.7±17.5 VPA (150 mg/kg) - 11.0±39.6 VPA (250 mg/kg) 24.8±23.6

Data represents mean SEM approach-avoidance score during a 5-minute social interaction trial. Individual values significantly different from the vehicle-treated control, analysed by Bonferroni post-hoc test, are indicated by an asterisk, P<0.05; n in all cases.

EXAMPLE 3: Effect of the historic deacetylase inhibitors suberoylanilide hydroxamic acid (SAHA) , 4-ff(2-aminophenyl)amino1carbonyI]-phenyl]m ethyIl- 3-pyridinyimethyl ester carbamic acid (MS-275) and valproic acid (VPA) upon neuronal cell growth within the hippocampus

Postnatal day 64 animals (maintained in accordance with the general procedure outlined in section (a) above) were administered 5 mg/kg suberoylanilide hydroxamic acid (SAHA) ), 1 mg/kg 4-[[(2-aminophenyl)amino]carbonyl]-phenyl]methyl]-3- pyridinylmethyl ester carbamic acid (MS-275), or 150 mg/kg valproic acid (VPA) via the intraperitoneal route (i.p.) for 2 1 days. The final drug treatment was administered 8 hours prior to animal sacrifice. Vehicle-treated controls (2-hydroxypropyl-beta- cyclodextrin) or dH2O were employed for comparison. NCAM PSA expression was then quantified for each of the treatment groups of animals in accordance with the general procedure detailed in sections (d)(i)-(iii) above.

The resultant data obtained was analysed as described in section (d)(iv) above and SAHA treatment was found to significantly increase the frequency of polysialylated neurons in the subventricular zone of the rat hippocampal dentate gyrus, as detailed in Table 1 below. These polysialylated neurons are represented by fluorescent cells located at the granule cell layer/hilar border and their dendrites extend into the molecular layer of the hippocampal dentate gyrus. Both MS-275 ( 1 mg/kg) and VPA (150 mg/kg) treated animals exhibited a trend towards increased basal frequency of polysialylated neurons in the subventricular zone of the rat hippocampal dentate gyrus (Table 10; Student's t-test, p=0.07 versus vehicle control).

TABLE 9

Treatment PSA immunopositive cell frequency

Vehicle 120.1±7.6 SAHA (5 mg/kg) 157.3±12.8

Data represents mean ± SEM NCAM PSA immunopositive cells in the polymorphic layer of the hippocampal dentate gyrus. Significant difference from vehicle control is indicated by an asterisk, Student's t-test, P<0.05; n=6 in all cases.

TABLE 10 Treatment PSA immunopositive cell frequency

Vehicle 64.4±3.1 MS-275 ( 1 mg/kg) 94.1±7.2 * VPA (150 mg/kg) 82.0±3.2 *

The inventors of the described invention present evidence that the HDAC inhibitor SAHA increases NCAM PSA in the dentate gyrus of normal animals, and furthermore, exerts a disinhibitory effect; in a measure of tendency for social investigation, SAHA pre-treatment resulted in increased attentiveness towards a novel animal. Likewise, MS-275 improves social interaction and significantly increased hippocampal NCAM PSA expression. VPA-treated animals did not differ from vehicle-treated controls in social approach behaviour but significantly increased the expression of NCAM polysialylated neurons.

That VPA exhibits a different profile to SAHA and MS-275 may be due to the fact that it is a weaker inhibitor of histone deacetlyase (HDAC). The ICs0 of VPA for the inhibition of HDACl in vitro is reported in the millimolar range, whereas both SAHA and MS-275 exhibit the same inhibition at nanomolar concentrations (Phiel et al, (2001) J Biol Chem 276 36734; Thiagalingham et al, (2003) Ann NY Acad Sci 983 84; Vannini et al, (2004) Proc Natl Acad Sci USA 101 15064).

Moreover, as VPA has an acute LD50 of 500-1000 mg/kg in rats, this limited the upper dose safely employed to achieve a significant level of HDAC inhibition and thus evaluate the effect of VPA-mediated HDAC inhibition on asociality without incurring side effects. However, a series of VPA analogues with more potent HDAC inhibitory properties has recently been reported (Eikel et al, (2006) Chem Res Toxicol 19 272). Moreover, one such analogue, pentyl-4-yn-VPA has also been shown to increase the frequency of NCAM PSA immunopositive cells in the hippocampus of adult rats at a lower dose range (Murphy et al, (2001) J Neurochem 78 704). Therefore short-chain fatty acid molecules with potent HDAC inhibition may yet be efficacious in the treatment of asociality.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. CLAIMS

1. Use of at least one HDAC inhibitor in the manufacture of a medicament for promoting neuronal growth.

2. A pharmaceutical composition for promoting neuronal growth comprising at least one histone deacetylase inhibitor (HDAC).

3. A method for promoting neuronal growth comprising administering to a patient in need of such therapy a pharmaceutically effective amount of at least one histone deacetylase inhibitor

4. Use of at least one HDAC inhibitor in the manufacture of a medicament for the treatment of asociality and affective disorders.

5. A pharmaceutical composition for the treatment of asociality and affective disorders comprising at least one histone deacetylase inhibitor (HDAC).

6. A method for the treatment of asociality and affective disorders comprising administering to a patient in need of such therapy a pharmaceutically effective amount of at least one histone deacetylase inhibitor.

7. The use, a pharmaceutical composition or a method of treatment as claimed in any preceding claim wherein the HDAC inhibitor is selected from the group consisting of a hydroxamic acid derivative, a short chain fatty acid, a cyclic tetrapeptide, a benzamide derivative, an electrophilic ketone derivative or any other class of compounds capable of inhibiting histone deacetylases.

8. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein the HDAC inhibitor is a hydroxamic acid derivative selected from the group consisting of :- 6-(3~Chlorophenylureido) carpoic hydroxamic acid; 7-[(4'-cyano[ 1,1'-biphenyl]-4-yl)oxy]-N-hydroxy-heptanamide; N,N'-dihydroxy-nonanediamide; Azelaic-l-hydroxamate-9-anilide; 3'-[(dimethylamino)carbonyl]-N-hydroxy-5'-[(4-methylbenzoyl)amino]-[l,r- Biphenyl]-4-acetamide; N-hydroxy-3-[3-(hydroxyamino)-3-oxo-1-propenylj-benzamide; N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(lH-indol-3-yl)ethyl]amino]methyl]phenyl]-2- propenamide; 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]-benzamide; N-hydroxy-5-[3-[(phenylsulfonyl)amino]phenyl]-2-penten-4-ynamide; N-hydroxy-N'-3-pyridinyl-octanediamide; N-hydroxy-3-[3-[(phenylamino)sulfonyl]phenyl]-2-propenamide; N-hydroxy-N'-phenyl-octanediamide; N,N'-dihydroxy-octanediamide ; N-hydroxy-l,3-dioxo-lH-Benz[de]isoquinoline-2(3H)-hexanamide; 7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; and (N-[4-[(2R,4R,6S)-4-[[(4,5-diphenyl-2-oxazolyl)thio]methyl]-6-[4-(hydroxy - methyl)phenyl]-l,3-dioxan-2-yl]phenyl]-N'-hydroxy-octanediamide

9. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein said HDAC inhibitor is a short Chain Fatty Acid selected from the group consisting of 4-Phenyl butyrate, 2,2-dimethyl-l-oxopropoxy) methyl ester butanoic acid, Sodium butyrate, Sodium phenyl butyrate, Valproic Acid, isovalerate, valerate, propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, and tributyrin.

10. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein the HDAC inhibitor is a Cyclic Tetrapeptide selected from the group consisting of HC-toxin cyclic tetrapeptide, Trapoxin A, Apicidin, Chlamydocin, CHAPs, Depsipeptide, and FR225497.

11. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein the HDAC inhibitor is a Benzamide derivative selected from the group consisting of CI-994, MS-275 and a 3'-amino derivative of MS-27-275. 12. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein the HDAC inhibitor is an electrophilic ketone derivative selected from the group consisting of trifluoromethyl ketones, alpha-keto oxazoles, alpha-keto heterocycles and alpha-keto amides. 5 13. The use, a pharmaceutical composition or a method of treatment as claimed in claim 7 wherein the HDAC inhibitor is selected from the group consisting of 2- Amino-8-oxo-9,10-epoxydecanoic acid); Bromoacetamides; Depudecin: (4,5:8,9- dianliydro-1,2,6,7, 11-pentadeoxy-D-threo-D-ido-undeco-l ,6-dienitol); Methyl 10 sulfoxides; Mercaptoacetamides; N-formyl hydroxylamino; Psammaplins; Semi Carbazides; Sulfur containing cyclic peptides (SCOPs); and Thiol derivatives.

14. The method of any of claims 3, 6 or 8 to 13, wherein the HDAC inhibitor is administered orally, parenterally, intraperitoneally, intracerebroventricularly, ]5 intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, via inhalation, vaginally, intraoccularly, locally, subcutaneously, intraadiposally, intraarticularly, intrathecally.

15. The composition of any of claims 2, 5 or 8 to 13, wherein the HDAC inhibitor is 0 in a slow release dosage form. A. CLASSIFICATION OF SUBJECT MATTER , , INV. A61K31/167 A61K31/19 A61K31/4406 A61P25/00

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K A61P

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used) EPO-Internal , BIOSIS, EMBASE, CHEM ABS Data, WPI Data, PAJ

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

LAENG PASCAL ET AL: "The mood stabilizer 1-7,9, valproic acid stimulates GABA neurogenesis 14,15 from rat forebrain stem cells" JOURNAL OF NEUROCHEMISTRY, vol. 91, no. 1 , October 2004 (2004-10), pages 238-251, XP002421163 ISSN: 0022-3042 cited in the application abstract

-/--

Further documents are listed in the continuation of Box C. See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date 1 or priority date and not in conflict with the application but "A document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention • 1 E" earlier document but published on or after the international 1X1 document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to "L1 document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another 1Y1 document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the 1O" document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu¬ other means ments, such combination being obvious to a person skilled "P" document published prior to the international filing date but in the art. later than the priority date claimed '&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

21 February 2007 15/03/2007

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk TeI. (+31-70) 340-2040, Tx. 3 1 651 epo nl, Fax: (+31-70) 340-3016 RODRIGUEZ-PALMERO, M C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

MURPHY KEITH J ET AL: 1-7,9, "Pentyl-4-yn-valproic acid enhances both 14,15 spatial and avoidance learning, and attenuates age-related NCAM-medi ated neuroplastic decline within the rat medial temporal lobe" JOURNAL OF NEUROCHEMISTRY, vol. 78, no. 4 , August 2001 (2001-08), pages 704-714, XP002421164 ISSN: 0022-3042 cited in the application abstract

JEONG M R ET AL: "Valproic acid, a mood 1-15 stabilizer and anticonvulsant, protects rat cerebral cortical neurons from spontaneous cell death: a role of h istone deacetylase inhibition" FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 542, no. 1-3, 8 May 2003 (2003-05-08), pages 74-78, XP004422726 ISSN: 0014-5793 abstract page 78, column 1 , paragraph 1

PHIEL C J ET AL: "Hi stone deacetylase is 1-15 a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 276, no. 39, 25 July 2001 (2001-07-25), pages 36734-36741, XP002386585 ISSN: 0021-9258 cited in the application abstract

BEUTLER A S ET AL: "Carbamazepine is an 1-15 inhibitor of histone deacetylases" LIFE SCIENCES, PERGAMON PRESS, OXFORD, GB, vol. 76, no. 26, 13 May 2005 (2005-05-13), pages 3107-3115, XP004858562 ISSN: 0024-3205 abstract

WO 03/083067 A2 (BRIGHAM & WOMENS HOSPITAL 1-3,5, [US]; DANGOND FERNANDO [US]) 7-15 9 October 2003 (2003-10-09) page 6 , lines 17-25 examples 1,6 claims 1,2,16-19

_ / C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

WO 02/090534 A (UNIV CALIFORNIA [US]; 1-15 STEFFAN JOAN S [US]; THOMPSON LESLIE M [US]; MAR) 14 November 2002 (2002-11-14) page 7 , paragraph 4 examples 1-5 claims

WO 2005/009349 A2 (SUPERGEN INC [US]; 1-15 LYONS JOHN [GB]; CHANG LUCY [US]) 3 February 2005 (2005-02-03) cited in the application claims 1,2,14-19; examples 1-4

TREMOLIZZO L ET AL: "Valproate and HDAC 1-3,5, inhibition: A new epi genetic strategy to 7-15 mitigate phenotypic severity in ALS?" AMYOTROPHIC LATERAL SCLEROSIS AND OTHER MOTOR NEURON DISORDERS 2005 NORWAY, vol. 6 , no. 3 , September 2005 (2005-09), pages 185-186, XP009078979 ISSN: 1466-0822 page 185, column 1 , paragraph 1

HOCKLY EMMA ET AL: "Suberoylanilide 1-3,5, hydroxamic acid, a h istone deacetylase 7-15 inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 100, no. 4 , 18 February 2003 (2003-02-18), pages 2041-2046, XP002389453 ISSN: 0027-8424 abstract

LANGLEY B ET AL: "REMODELING CHROMATIN 1-3,5, AND STRESS RESISTANCE IN THE CENTRAL 7-15 NERVOUS SYSTEM: HISTONE DEACETYLASE INHIBITORS AS NOVEL AND BROADLY EFFECTIVE NEUROPROTECTIVE AGENTS" CURRENT DRUG TARGETS. CNS & NEUROLOGICAL DISORDERS, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 4 , no. 1 , February 2005 (2005-02), pages 41-50, XP009048265 ISSN: 1568-007X abstract

P X WO 2006/010749 A2 (JANSSEN PHARMACEUTICA 1-15 NV [BE]; VAN BRANDT SVEN FRANCISCUS ANN [BE]; VA) 2 February 2006 (2006-02-02) page 18, line 23 - page 19, line 15

-/-- C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

MONNERET C : "Hi stone deacetylase 1-15 inhibitors" EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, EDITIONS SCIENTIFIQUE ELSEVIER, PARIS, FR, vol. 40, no. 1 , January 2005 (2005-01), pages 1-13, XP004708959 ISSN: 0223-5234 the whole document Box Il Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This International Search Report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

1. [_χj Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

Although claims 3 , 6 , 7-15 are directed to a method of treatment of the human/animal body, the search has been carried out and based on the alleged effects of the compound/composition.

2. Claims Nos.: because they relate to parts of the International Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically:

3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box III Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

1. As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims.

2. I As all searchable claims could be searched without effort Justifying an additional fee, this Authority did not invite payment of any additional fee.

3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.:

4. I I No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest I I The additional search fees were accompanied by the applicant's protest.

No protest accompanied the payment of additional search fees. Patent document Publication Patent family Publication cited in search report date member(s) date

WO 03083067 A2 09-10-2003 AU 2003226014 A l 13-10-2003

WO 02090534 A 14-11-2002 EP 1390491 A l 25-02-2004

WO 2005009349 A2 03-02-2005 CA 2532922 A l 03-02-2005 EP 1651164 A2 03-05-2006

WO 2006010749 A2 02-02-2006 AR 050189 A l 04-10-2006