WO2007029063A2

WO2007029063A2 - Treatment and/or prevention of pervasive developmental disorders

Info

Publication number: WO2007029063A2
Application number: PCT/IB2006/002068
Authority: WO
Inventors: Henry Markram; Tania Rinaldi; Tamila Markram; Maria Rodriguez Bravo; Brandi Mattson; Karina Kulangara
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2005-07-29
Filing date: 2006-07-28
Publication date: 2007-03-15
Also published as: WO2007029063A3

Abstract

The present invention concerns a method for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention.

Description

Methods for treating and/or preventing Pervasive Developmental Disorders in a subject.

FIELD OF THE INVENTION

The present invention concerns a method for treating and/or preventing Pervasive Developmental Disorders in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention performance.

BACKGROUND OF THE INVENTION

Autism spectrum disorders is now recognized as a major neurological developmental disorder affecting children starting during the first 5 years of life. Leo Kanner, in 1943, first described children that do not make eye contact, are impaired in language, apparently lack the need or skill for social interaction, indulge in repetitive behavior, display sensory perceptual abnormalities, are extremely sensitive to novel situations or environments displaying a resistance to change, and they do not seek comfort during stress. Most autistics also suffer from severe sensory distortions, gastrointestinal and eating disorders as well as sleep, movement, metabolic and immune disorders, and about half experience epileptic episodes. Autism is a broad-spectrum disorder with a wide range of symptoms.

Classically autism is considered to be on one end of the spectrum of an even broader category of Pervasive Developmental Disorders (PDD). Some types of autism that have been described include Typical Autism, Atypical Autism/PDD, Autistic Savants, Asperger's Syndrome, and autism associated with Kanner's Syndrome, fragile X, Rett and Down's syndromes. The overall prevalence of the broadest category of abnormal developmental progression is thought to occur in 1 in 300 children, 1 in 500 children will be born on the autistic spectrum and 1 in 1000 with severe autism. Autism has a clear genetic predisposition which is based on the fact that the probability of an identical twin having autism is around 50% as apposed to 0.5% in the general population. The probability of autism in Down's Syndrome is also 10%. It seems that the genes involved in these developmental disorders do not directly cause these autistic spectrum disorders, but impart a vulnerability to external triggers, especially teratogens during pregnancy. The wide range of possible symptoms have led researches to speculate multiple triggers including viruses, vaccinations, bacterial, immune deficiency, gastrointestinal disorders, toxic insults at neurologically sensitive periods of brain development, and many more.

Typically, developmental disorders are thought to be caused by impaired cognitive functions. While brain imaging studies have most often implicated the cerebellum in autism, abnormalities in many other brain areas have been reported, including limbic areas, thalamus, hypothalamus, the brain stem, the neocortex, and even the pineal gland. In accordance with the spectrum of symptoms and potential causes of autism, a variety of intervention treatments have emerged, including behavioral, sensory, sensory-motor integration, cognitive enhancing therapies, and various nutritional and pharmacological treatments. Aside from helping by acting on selective symptoms, the efficacy of pharmacological treatments is, in general, very poor. While many children with autism do improve to some extent with behavioral intervention programs, most autistics will be unable to live independently and more than half will never learn to speak correctly. While the autistic phenotype is highly heterogenous, common features are high level cognitive abnormalities in memory, perception and attention. Surprisingly, Applicants have shown that the autistic brain is not impaired in many key cognitive abilities, but rather that the handicap arises from excessive brain functions and capabilities. In particular, behavioral experiments have revealed that memory processes are enhanced making it difficult for the autistic to uncouple prior associations leading to rigidity in social adaptation and communication. Perceptual processes are enhanced making the autistic overly sensitive to sensory stimulation and attention is enhanced, making autistics inflexible to shifting attention easily and fluidly in a rapidly changing sensory world. Electrophysiological experiments have also revealed a massive increase in the probability of synaptic connections between pyramidal neurons in different neocortical layers indicating that the local microcircuitry in the autistic neocortex is hyper-connected. The electrophysiological experiments also reveal an altered and greatly enhanced form of synaptic plasticity, which is thought to underlie memory formation as well as a large increase in the amount of NMDA-receptor triggered currents, which causes plasticity. Protein assays revealed an increase in the NMDA receptor levels in particular subunits NR2A and NR2B, indicating that there is a massive increase in currents that can be triggered by NMDA receptors in autism. Electrophysiological data also show a compensatory decrease in excitability of neurons. There has been some progress in the treatment of Pervasive Developmental Disorders (PDD), in particular autism, by using N-rnethyl-D-aspartate receptor (NMDA) antagonists such as ketamine or dextrometorphan. For example, U.S. Patent 6,362,226 discloses a method of treating autism in a patient, said method comprising administering to the patient an effective amount of a glutamine level reducing agent, a glycine level reducing agent or combinations thereof. Optionally, an NMDA receptor antagonist can also be administered to the patient in combination of a glutamine level reducing agent and/or glycine level reducing agent.

In the same way, U.S. Patent 4,994,467 relates to a method that is provided for treating autism and other pervasive developmental disorders in children by the administration of a therapeutically effective amount of an NMDA receptor antagonist.

However, the use of these NMDA receptor antagonists is not ideal. NMDA antagonism by, for example, Ketamine is a commonly used aneasthetic and low doses of NMDA antagonism can cause dissociative disorders and psychoses. U.S . Patent Application N° 2004/0067978 Al discloses the use of mGluR antagonists, preferably selective for mGluR5 receptors, in the hippocampus, for the treatment and prevention of disorders, including Fragile X, autism, mental retardation, schizophrenia and Down's Syndrome. However, this disclosure is based on the finding that mGluR-5 receptor activation causes depression of synaptic connections in the neocortex and they have the goal of trying to increase synaptic plasticity and memory processes. The applicants have found that the opposite is required in the treatment of autism. A possible treatment for the autistic hippocampus would be to activate rather than block the mGlur-5 receptor in the hippocampus. The applicants also found that mGlur-5 receptor results in very different effects in different brain regions. Activation of the group I metabotropic receptors, including mGluR-5, in the neocortex, underlies the formation and disconnections of synaptic connections and they found that antagonizing the mGluR-5 receptor can retard this circuit remodeling in the neocortex. Antagonizing mGlur-5 receptors, specifically in the neocortex, is therefore a good potential treatment for autistic neocortex, and for a very different reason than proposed by U.S. Patent Application N° 2004/0067978 Al. In addition, the treatment would be required chronically from as early after birth as possible to retard the formation of circuits and prevent the hyperconnectivity and resulting symptoms. Despite the disclosure of the foregoing patents and patent applications, there remains therefore significant room for improvement in the treatment of Pervasive Developmental Disorders and in particular autism.

Therefore, it is an object of the present invention to provide new treatment modalities for the treatment of Pervasive Developmental Disorders which have a good safety profile, a good specificity, only low or no side effects and the possibility to retreat, whenever necessary.

This object has been achieved by providing a method for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by either blocking synaptic strengthening, enhancing synaptic weakening, reducing synapse formation, enhancing synaptic breakdown, decreasing network excitability, increasing the level of network inhibition, reducing the supply of dopamine to the neocortex, blocking calcium channels, reducing intracellular calcium levels, and/or blocking the somatostatin receptor 2.

SUMMARY OF THE INVENTION

The present invention concerns a method for treating and/or preventing a Pervasive

Developmental Disorder in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by either blocking synaptic strengthening, enhancing synaptic weakening, reducing synapse formation, enhancing synaptic breakdown, decreasing network excitability, increasing the level of network inhibition, reducing the supply of dopamine to the neocortex, blocking NMDA receptors, blocking calcium channels, reducing intracellular calcium levels, and/or blocking the somatostatin receptor 2. A further object of the present invention is to provide a kit for the treatment for a

Pervasive Developmental Disorder comprising a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention and instructions to use. Another object of the present invention is to provide a diagnostic kit. The kit includes antibody markers to perform immunohistochemical stainings to test for the alteration in specific proteins levels and gene expression changes found in the autistic brain.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 depict the Hyperconnectivity. Fig. 1 a shows the increased probability of direct connection between layer 5 pyramidal cells (PCs) in a cluster (P = 0.008, Chi-square). Fig. 1 b, No hyperconnectivity at longer distances (P = 0.52, Chi-square). Fig. 1 c represents the parameters of the synaptic connection with a model of dynamic synaptic transmission: the absolute synaptic strength A (P = 0.023, Student's t-test), the probability of release Pr (P = 0.17, Student's t-test), the time constant of synaptic depression!) (P = 0.61, Student's t-test). The weakened synapses were consistent with the decreased synaptic conductances evoked by minimal stimulation (n = 36 cells (control), 45 cells (treated); P < 0.002 for all stimulation amplitudes, Student's t-test). Fig. 1 d shows the increased number of di-synaptic connections between thick tufted layer 5 PCs (via interneuron (M) between the two PCs) (P < 0.0001, Chi-square). Data show mean ± s.e.m.

Figures 2 depict the Hyper-reactive network. Fig. 2 a represents the MEA stimulation with 16 electrodes (50Hz Poisson train, 300 ms) in layer 5 thick tufted pyramidal neurons. Fig. 2 b, shows the integral of responses to MEA stimulation in current-clamped PCs as a function of stimulation amplitude. Fig. 2 c represents the number of spikes elicited by the MEA stimulation as a function of the stimulation amplitude. Fig. 2 d represents the number of network events elicited by the stimulation as a function of stimulation amplitude. The upper traces represent the response of a whole-cell patched layer 5 pyramidal neuron, with an emphasis on what is measured for each graph. Fig. 2 e shows the MEA stimulation with 16 electrodes (50Hz Poisson train, 300 ms) in layer 2/3 pyramidal neurons. Fig. 2 f, g, h show the charge responses to MEA stimulation: global (V_hoi_d = -80 mV) (b), excitatory (V_hoi_d = -57 mV) (c) and inhibitory (V_hoid = +10 mV) (d) response curves. The upper traces represent the responses of the whole-cell patched layer 2/3 pyramidal cells. The grey area is the charge measured for the dose response curves. Data show mean ± s.e.m. (*, P < 0.05; **, P < 0.01).

Figures 3 depict the Hypoexcitability of pyramidal cells. Fig. 3 a shows an example of currents injected and corresponding voltage measurements in layer 2/3 pyramidal cells from control (blue) and VPA-treated (red) rats. Mean of injected current to threshold for layer 2/3 (P = 0.00001) and layer 5 cells (P = 0.05). Fig. 3 b is an Example of the current-frequency (IF) curve for layer 2/3 pyramidal cells. Mean of IF slope (linear fit) for layer 2/3 (P = 0.011), and layer 5 cells (P = 0.019). Fig. 3 c is an example of wider second action potential of layer 2/3 cells. Mean of second action potential half width for layer 2/3 (P = 0.049) and layer 5 cells (P = 0.17). All statistics were made with the Student's Mest; data show mean ± s.e.m.

Figures 4 represent the Anatomy of layer 5 pyramidal cells. Fig. 4 a shows an example of Sholl analysis of a layer 5 thick tufted pyramidal cell. Fig. 4 b represents the Sholl analysis of the axonal (ASI; P = 0.62, Kolmogorov-Smirnoff) and dendritic (DSI; P = 0.23, Kolmogorov- Smirnoff)) tree intersections. Fig. 4 c shows the surface of the soma (406 ± 40 μm² for control, 340 ± 29 μm² for treated; P = 0.12, Student's f-test). Bouton density (0.15 ± 0.02 μm^" ¹ for control, 0.12 ± 0.01 μm^"1 for treated; P = 0.20, Student's t-test). Spine density (0.13 ± 0.01 μm^'1 for control, 0.15 ± 0.02 μm^"1 for treated; P = 0.48, Student's r-test). Fig. 4 d is a picture of layer 5 somatosensory cortex, as used for pyramidal cell density counting. Thick tufted pyramidal cells were recognized by their characteristic apical dendrite, soma shape and size. The density was calculated in a volume of 180 μm x 180 μm x 30 μm. Fig. 4 e represents layer 5 pyramidal cell density (52 ± 1 xlO³ mm^"3 for control, 54 ± 2 xlO³ mm^"3 for treated; P = 0.28, Student's t-test). Fig. 4 f is an Example of the reconstruction of a connected pair of layer 5 pyramidal cells in the VPA-treated rats. The green stars represent the putative synapses. Data show mean ± s.e.m.

Figures 5 depict the enhanced CarnKII, NR2A and NR2B protein expression levels. Examples of Western blot gels and mean values reported as percentage of control. Fig. 5 a shows Western blots of CREB (n = 6, 7), CamKH (n = 11, 11) and ERK (n = 12, 12). Fig. 5 b, shows Western blots of AMPA receptor subunits GIuRl (n = 8, 9), GluR2 (n = 8, 8) and GluR3 (n = 8, 8). Fig. 5 c shows Western blots of NMDA receptor subunits NRl (n = 8, 8), NR2A (n = 8, 8), NR2B (n = 7, 8). Data show mean ± s.e.m. (*, P < 0.05; **, P < 0.01). Figures 6 represent the Enhanced NMDA mediated synaptic currents. Fig. 6 a shows the Experimental scheme and example of response in the postsynaptic cell before (total) and after blocking of AMPA receptors with NBQX (NMDA). The difference between the control and NMDA traces give the AMPA trace. Fig. 6 b shows the Peak currents of AMPA and NMDA responses. Fig. 6 c represents the Charge of total, NMDA and AMPA traces; percentage of NMDA ( = NMD A/total) and AMPA/NMDA ratio. Data show mean ± s.e.m. (*, P < 0.05; **, P < 0.01).

Figures 7 depict the Enhanced postsynaptic plasticity. Fig. 7 a,b,c show the LTP with extracellular stimulation in layer 2/3 pyramidal neurons, a, Experimental scheme, pairing protocol and example of mean response in a patched cell, before and after pairing . b, Example of response amplitude as a function of time. The grey line represents the timing of the pairing protocol, c, Mean of percentage increase in the amplitude of the response to extracellular stimulation after pairing. Fig. 7 d, e show the LTP in direct connections between layer 5 pyramidal neurons, d, Experimental scheme, pairing protocol and example of mean responses in the postsynaptic cell before and after pairing, e, Mean of percentage change after pairing of the absolute synaptic efficacy A, the probability of release Pr and the time constant for depression D. The absolute values of A, Pr and D before and after pairing are displayed in Supplementary Fig 3. Data show mean ± s.e.m. (*, P < 0.05).

Figure 8 shows enhanced fear memories after 1, 34 and 69 days and shows reduced ability to extinguish fear memories after 3 days of de-conditioning .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by either i) blocking synaptic strengthening, ii) enhancing synaptic weakening, iii) reducing synapse formation, iv) enhancing synaptic breakdown, v) decreasing network excitability, vi) increasing the level of network inhibition, vii) reducing supply of dopamine to the neocortex, viii) blocking the somatostatin receptor 2, wherein compositions i) to viii) are administered separately or concurrently in different combinations.

As used herein, "a" or "an" means "at least one" or "one or more."

The term "comprising" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

"Pervasive Developmental Disorder", "Pervasive Developmental Disorders" or

"PDD" refer to a group of disorders characterized by delays in the development of socialization and communication skills. Parents may note symptoms as early as infancy, although the typical age of diagnoses is before 3 years of age. Symptoms may include problems with using and understanding language; difficulty relating to people, objects, and events; unusual play with toys and other objects; difficulty with changes in routine or familiar surroundings, and repetitive body movements or behavior patterns.

Usually, the PDD to be treated are selected from the group comprising Fragile X, autism, mental retardation, schizophrenia and Down's Syndrome.

Particularly, the PDD is autism. In such case, autism comprises Typical Autism, Atypical Autism/PDD, Autistic Savants, Asperger's Syndrome, and autism associated with Kanner's Syndrome, fragile X, Rett and Down's syndromes.

"Concurrently" as used herein means that the compositions of the invention are coadministered either by administering individual compositions or by administering a cocktail of said compositions.

As used herein "neocortex" refers to the evolutionary newest brain region that comprises almost 80% of the human brain. It is the upper mantel covering the paleo and archi cortices. It is the part of the brain the processes sensations, motor actions and high level cognitive functions such as perception, attention and memory.

Usually, the term "subject" refers to any animal classified as a mammal including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, horses, cats, cows, monkeys, etc. Preferably, the mammal is a human.

As used herein, the various forms of the term "modulating" include stimulation (e.g. increasing or upregulating a particular response or activity) and inhibition (e.g. decreasing or downregulating a particular response or activity).

"Synaptic connectivity" refers to the connections formed between neurons to transmit information. A single synaptic connection is comprised of multiple synapses, typically 5-10.

"Synaptic plasticity" refers to changes in the strength of synaptic connections. Long term synaptic plastic changes are also called either Long Term Potenatiation (LTP) or Long Term Depression (LTD) depending on the direction of the change.

"Administering", as it applies in the present invention, refers to contact of a pharmaceutical, therapeutic, diagnostic agent or composition, to the subject, preferably a human.

A "therapeutically effective amount" is an amount effective to ameliorate or prevent the symptoms, or prolong the survival of, the subject being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

An "agonist" is a molecule which activates a certain type of receptor. For example, glutamate molecules act as agonists when they excite excitatory amino acid (EAA) receptors.

By contrast, an "antagonist" is a molecule which prevents or reduces the effects exerted by an agonist on a receptor.

Many naturally occurring neurotransmitters are agonists, since they activate the receptors they interact with. By contrast, artificial and/or exogenous drugs may be agonists or antagonists. For example, NMDA antagonists are drugs that can suppress excitatory activity of glutamate or glycine at NMDA receptors.

As used herein a "blocker" refers to an antagonist which is a chemical compound that binds to a receptor or ion channel to prevent its function.

Although agonist and antagonist drugs are generally thought to interact directly with receptors to achieve their effects, such effectivity may not result from direct interaction but may involve intermediate steps or compounds.

Hence, the present invention is not limited to mechanisms acting directly on affected receptors involved or thought to be involved in autistic disorders. Rather, any effect of the drugs on receptors or more generally on metabolism or symptoms of the disorder is contemplated as part of the present invention.

The term "drug" refers to chemical, peptidic or natural compounds which, when incorporated in the composition of the invention, or even alone, are capable of reducing memory, perception and/or attention by either blocking synaptic strengthening, enhancing synaptic weakening, reducing synapse formation, enhancing synaptic breakdown, increasing network excitability, decreasing the level of network inhibition, reducing supply of dopamine to the neocortex, blocking alpha 1 noradrenergic receptors, or blocking the somatostatin receptor 2. There are several classes of drugs of different origin and with different modes of action e.g. they can be agonists, antagonists, inhibitors, enhancers, blockers or down regulators.

As supported in the examples, Applicants have completed a number of studies which allow a better understanding of the hyper connectivity associated with PDD. Applicants have shown that hyperconnectivity is not due to excessive growth of axons or dendrites or boutons or synapses. Instead a novel form of target dominance seems to be activated.

The Target Dominance (TD) mechanism determines the number of target neurons contacted by a neuron. The higher the target dominance the more strongly the network of neurons are coupled causing the network to become autonomously active and difficult to control by regulatory mechanisms, such as those from the prefrontal cortex that control executive functions. The predicted consequence is that the local microcircuits of the neocortex become overly sensitive, hyper-reactive, autonomously active and difficult to control. At the cognitive level this is predicted to translate into severe abnormalities in perception, attention and memory. The perceptual abnormality is proposed to be due to oversensitivity and over- reactivity of the local circuits. The attention abnormality is proposed to be due to the autonomous local circuits which can not be controlled by attention mechanisms. The memory abnormality is proposed to be due excessive storage of memories in the hyper-connected networks causing behavioral rigidity and inflexibility to new environments.

There is no generalized hypertrophy of synapses to cause an increase in synaptic connections on existing neurons and add synapses onto new targets, instead many more neurons are contacted with less synapses on each neuron. The mechanism of Target Dominance is therefore activated to contact as many neurons as possible. Accordingly, reversing this mechanism is a man novel strategy for treating PDD and in particular autism.

Applicants have also shown which key molecules are involved in the TD mechanism. Additionally, they have shown that it is possible to change the target profile of neurons, i.e. the set of neurons that each neuron targets. Experience and intense excitation of the circuit (equivalent to an intense experience) can enhance TD resulting in hyperconnectivity. Accordingly, Applicant's can therefore induce hyper-connectivity in the normal brain with intense activation.

In normal life, this is likely to be a transient process during which memories can be stored more effectively, but in the case of Autism, the circuit seems to always be in this primed state. Since the excitation that causes the hyper-connectivity is mediated by glutamate the receptors that mediate TD are novel therapeutic targets to treat PDD.

Usually, the therapeutically effective amount of a composition of the present invention capable of reducing memory, perception and/or attention by blocking synaptic strengthening comprises a drug selected from the group comprising a muscarinic receptor antagonist, acetylcholin esterase enhancer, N-methyl-D-aspartate (NMDA) receptor antagonist, second messenger blocker, calcium channel blocker, group IE metabotropic glutamatergic receptor agonist (group IH mGluR), intracellular calcium buffer molecule and/or a combination thereof. Exemplary muscarinic receptor antagonists, aimed at reducing the supply of acetylcholine to the neocortex, comprise, but are not limited to:

Muscarinic antagonists having a selectivity for muscarinic receptors Mi > ML₂; e.g. Nitrocaramiphen hydrochloride (2-Diethylaminoethyl l-(4- nitrophenyl)cyclopentanecarboxylate hydrochloride),

Muscarinic M^ antagonists: e.g. 4-DAMP (l_l>l-Dimethyl-4-diphenylacetoxypiperidinium iodide),

Antagonist-receptor complex stabilisers: e.g. W-84 dibromide (Hexamethylene-bis-[dimethyl-(3- phthalimidoρropyl)ammonium]dibromide),

Muscarinic antagonist: e.g. Ipratropium bromide (e«Jo,j>'«X±)-3-(3-Hydroxy-l-oxo-2-phenylpropoxy)-8-methyl-8- (l-methylethyl)-8-azoniabicyclo[3.2.1]octane bromide),

M₄ selective muscarinic receptor antagonist: e.g. Tropicamide (jV-Ethyl-3-hydroxy-2-phenyl-N-(pyridinylmethyl)propanamide)₅

Selective Mj_ muscarinic antagonist: e.g. Pirenzepine dihydrochloride (5,ll-Dihydro-l l-[(4-methyl-l-piperazinyl)acetyl]-6/i- pyrido[2,3-b][l,4]benzodiazepin-6-one dihydrochloride),

Selective M? antagonists: e.g. AF-DX 116 (l l-[[2-[(Diethylamino)methyl]-l-piρeridinyl]acetyl]-5,ll-dihydro-6H^"- pyrido[2,3-b][l,4]benzodiazepin-6-one) or Otenzepad,

Potent selective M^ antagonists: e.g. Telenzepine dihydrochloride (4,9-Dihydro-3-methyl-4-[(4-methyl-l-piperazinyl)acetyl]- 10/7-thieno[3 ,4-b] [ 1 , 5]benzodiazepin- 10-one dihydrochloride), Selective high affinity muscarinic Mi receptor antagonist (K_t = 0.94 nM).

Potent MTZM₄ antagonists: e.g. AF-DX 384 (ll-ttl-tCDiethylamino^nethy^-l-piperidinyllacetylJ-SJl-dihydro-όH- pyrido[2,3-b] [ 1 ,4]benzodiazepin-6-one) or Otenzepad,

Non-selective muscarinic antagonists: e.g. Scopolamine hydrobromide ((α,S)-α-(Ηydroxymethyl)benzeneacetic acid

(1 a,2b,4b, 5α,7&)-9-metliyl-3-oxa-9-azatricyclo[3.3.1.02,4]non-7-yl ester hydrobromide),

M?-selective antagonists: e.g. (5)-(+)-Dimetbindene maleate (iV,iV-Dimethyl-3-[(15)-l-(2-pyridinyl)ethyl]-lH-indene-2- ethanamine maleate),

Enantiomer that is a subtype-selective M₂ muscarinic receptor antagonist (pKt values are 7.08, 7.78, 6.70 and 7.00 for Mi, M₂, M₃ andM₄ receptors respectively). Also Hi histamine receptor antagonist (pK_t = 7.48).

Selective M₄ antagonists: e.g. PD 102807 (3₅6a,ll,14-Tetrahydro-9-methoxy-2-methyl-(12H)-isoquino[l,2- 6]pyrrolo[3,2_:/][l,3]benzoxazine-l-carboxylic acid, ethyl ester),

Selective M₄ muscarinic receptor antagonist. ICso values are 91, 6559, 3441, 950 and 7412 nMfor human M₄, Mj, M₂, M₃, and Ms receptors respectively

Other Muscarinic receptor inhibitors such as Type 1 and Type 2 inhibitors.

Modulators of cholinergic function, usually acetylcholine esterase enhancers: such as Cholinesterase inhibitor (e.g. Ambenonium dichloride, Galanthamine hydrobromide, Tacrine hydrochloride), ACh release Stimulators (e.g. MR 16728 hydrochloride, Cisapride), Cholinesterase inhibitor (e.g. Physostigmine hemisulfate), ACh transport inhibitor (e.g. (±)- Vesamicol hydrochloride), Presynaptic cholinergic modulator (e.g. PG-9 maleate), Presynaptic cholinergic modulator (e.g. SM-21 maleate). Exemplary mGluR Group HI (mGluR 4, 6, 7 and 8) agonists comprise, but are not limited to:

L(+)-2-amino-4-phosρhonobutyric acid (L- AP4), (S)-3,4-dicarboxyphenylglycine (DCPG), selective mGluR8 subunit (a) agonist,

(R,S)-4-Phosρhonophenylglycine ((R₅S)-PPG),

(lS,3R,4S)-l-Aminocyclopentane-l,2,4-tricarboxylic acid (ACPT-I), a selective agonist at the group III mGluR4 subunit (a),

(3RS,4RS)-1- Aminocyclopentane-l,3,4-tricarboxylic acid ((±)-ACPT IH).

Applicant have shown that the NMDA receptor does not determine TD, but determines the strength of existing synapses. It is well known that NMDA receptors control the strength of synapses, but it was not known that target selection can be made without NMDA. The involvement of NMDA receptors in plasticity of existing synapses is therefore likely to be important in the memory processes after neurons are contacted and in the case of Autism after the TD mechanism has been activated. Indeed, Applicant's shown that the plasticity was much stronger at autistic synapses and that 1) the NMDA receptor-mediated current is massively increased (functional index of NMDA receptor activity), that 2) the phosphorylation of the NRl NMDA receptor subunit (the obligatory protein subunit) is greatly enhanced in the animal model of autism, 3) and that the 2A and 2B subunits are overexpressed.

Exemplary NMDA receptor antagonists comprise, but are not limited to:

NMDA Site antagonists such as:

DL- AP7 (DL-2-Amino-7-phosphonoheptanoic acid),

DL-AP5 (DL-2-Amino-5-ρhosphonopentanoic acid), D-AP5 (D-(-)-2-Amino-5-phosphonopentanoic acid), L-AP5 (L-(+)-2-Amino-5-phosphonopentanoic acid), D-AP7 (D-(-)-2-Amino-7-phosphonoheptanoic acid),

(RS)-CP? ((i?5)-3-(2-Carboxypiperazin-4-yl)-proρyl- 1 -phosphonic acid), (R)-CP? (3-((/?)-2-Carboxypiperazin-4-yl)-propyl- 1 -phosphonic acid), Shows some selectivity for NR2A-containing receptors

(i?)-4CPG ((i?)-4-Carboxyρhenylglycine), .

LY 235959 ([SiS-CSα^α^ό^αα^-Decahydro-β-CphosphonomethyOSisoquinolinecarboxylic acid))

Competitive NMDA antagonists such as:

CGS 19755 (cw-4-[Phosphomethyl]-piperidine-2-carboxylic acid),

SDZ 220-581 ((^-α-Amino^'-chloro-S-CphosphonomethyOt^r-biphenyy-S-propanoic acid),

SDZ 220-040 ((5)-α-Amino-2',4'-dichloro-4-hydroxy-5-(phosρhonomethyl)-[l,r-biphenyl]- 3 -propanoic acid),

CGP 37849 ((E)-(±)-2-Amino-4-methyl-5-phosphono-3-pentenoic acid),

CGP 39551 ((E)-(±)-2-Amino-4-methyl-5-pliosphono-3-pentenoic acid ethyl ester)

Glycine Site Antagonists such as: CNQX (6-Cyano-7-nitroquinoxalme-2,3-dione),

7-Chlorokynurenic acid (7-Chloro-4-hydroxyquinoline-2-carboxylic acid),

ACBC (l-Aminocyclobutane-l-carboxylic acid),

(S)-(-)-HA-966 ((S)-(-)-3-Ammo-l-hydroxypyrrolidin-2-one),

5,7-Dichlorokynurenic acid (5,7-Dichloro-4-hydroxyquinoline-2-carboxylic acid), L-701 ,252 (7-Chloro-3-(cycloρropylcarbonyl)-4-hydroxy-2(lH)-quinolinone),

L-689,560 (trans-2-Carboxy-5,7-dichloro-4-phenylaminocarbonylamino- 1 ,2,3 ,4- tetrahydroquinoline),

Felbamate (2-Phenyl-l,3-propanedioldicarbamate),

L-701,324 (7-Chloro-4-hydroxy-3-(3-ρhenoxy)ρhenyl-2(lH)-quinolinone), CGP 78608 hydrochloride ([(lS)-l-[[(7-Bromo-l,2,3,4-tetrahydro-2,3-dioxo-5- quinoxalinyl)methyl]amino]ethyl]phosphonic acid hydrochloride).

Ion Channel Antagonists such as: (±)-l-(l,2-Diphenylethyl)piperidme maleate,

Memantine (hydrochloride 3,5-Dimethyl-tricyclo[33 J J3,7]decan-l-amine hydrochloride), Dizocilpine ((+)-MK 801 maleate), (5S, 10R)-(+)-5-Methyl- 10, 1 l-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine maleate,

(-)-MK 801 maleate ((5R,10S)-(-)-5-MethyM0,ll-dihydro-5H-dibenzo[a,d]cylcoheρten- 5,10-imine maleate),

Loperamide hydrochloride (4-(4-Chloroρhenyl)-4-hydroxy-N,N-dimethyl-a,a-diphenyl-l- piperidinebutanamide hydrochloride),

Also a Ca²⁺ channel blocker; at low micromolar concentrations it blocks broad spectrum neuronal HVA Ca²⁺ channels and at higher concentrations it reduces Ca²⁺ flux through NMDA receptor operated channels Remacemide hydrochloride (2- Amino-N-(1 -methyl- l,2-diphenylethyl)acetamide hydrochloride),

Uncompetitive NMDA receptor antagonist; blocks ion channel and allosteric modulatory site (IC₅₀ = 8-68 mM).

IEM 1460 (N,N,N,-Trimethyl-5-[(tricyclo[3.3.1.13,7]dec-l-ylmethyl)amino]-l- pentanaminiumbromide hydrobromide), shows selectivity between subtypes, blocking GluR2 subunit-lacking (Ca²⁺ -permeable) cerebellar receptors more potently than GluR2-containing hippocampal receptors (IC so values are 2.6 and 1102 mM respectively.

Norketamine hydrochloride (2-Amino-2-(2-chlorophenyl)cyclohexanone hydrochloride), (K₁ = 3.6 JtM for displacement Of[³H]-MK 801 in rat brain).

N20C hydrochloride (2-[(3,3-Diphenylpropyl)amino]acetamide hydrochloride)

Selective, non-competitive NMDA receptor antagonist (IC₅₀ — 5 mM); binds to the receptor- associated ion channel and prevents glutamate-induced Ca²⁺ influx.

Polyamine Site Antagonists such as:

N-(4-Hydroxyphenylacetyl)spermine N-(N-(4-Hydroxyphenylacetyl)-3-ammoρropyl)-(N'-3- aminopropyl)- 1 ,4-butanediamine,

N-(4-Hydroxyphenylproρanoyl) spermine trihydrochloride (N-(N- (4Hydroxyphenylpropanoyl)-3-aminopropyl)-(N'-3-ammopropyl)-l,4-butanediamine trihydrochloride ),

Arcaine sulfate (N,N'-1,4-Butanediylbisguanidine sulfate),

Ifenprodil hemitartrate (2-(4-Benzylpiperidino)-l-(4-hydroxyphenyl)-l-propanol hemitartrate),

Synthalin sulfate (N₅N'- 1 , 10-Decanediylbisguanidine sulfate), Eliprodil (a-(4-Chlorophenyl)-4-[(4-fluorophenyl)methyl]-l-piperidineethanol)

Non-competitive NR2B-selective NMDA antagonist. Also s ligand and Ca²⁺ channel blocker. Other NMDA selective Antagonists such as:

Ro 25-6981 maleate ((αR,βS)-α-(4-Hydroxyphenyl)-β-methyl-4-(ρhenylniethyl)-l- piperidinepropanol maleate) Potent and selective activity-dependent blocker of NMDA receptors that contain the NR2B subunit. IC₅₀ values are 0.009 and 52 aMfor cloned receptor subunit combinations NR1C/NR2B andNRlC/NR2A respectively.

Bilobalide, a constituent of Ginkgo Biloba, Lipocortin-1, Cerebral Fluid Zinc level elevators

Spermidines.

Preferably, the NMDA antagonists are selective for NMDA receptor subunits NR2A and/or NR2B.

Applicants have also shown that blocking group 3 metabotropic glutamate receptors

(mGluRs) while intensely activating the circuit, causes synapses to get stronger indicating that these receptors normally serve to keep synapses from becoming to too strong. Activating these receptors therefore prevent the hyper-plasticity associated with the hyper-connectivity. Group 3 mGluRs act by inhibiting the cyclic AMP pathway, which normally activates the Protein Kinase A (PKA) enzyme. PKA phosphorylation of the 2B NMDA receptor subunit is excessive and thus activating the group 3 receptor prevents this phosphorylation and reduces the NMDA mediated current.

As used herein, "second messengers" are molecules that relay signals received at receptors on the cell surface such as the arrival of protein hormones, growth factors, etc. to target molecules in the cytosol and/or nucleus. There are 3 major classes of second messengers: cyclic nucleotides {e.g., cAMP and cGMP), inositol trisphosphate (IP₃) and diacylglycerol (DAG) and calcium ions (Ca²⁺). Preferably, the second messenger of the present invention is the Ca²⁺ ion. Calcium ions are important intracellular messengers since they are probably the most widely used intracellular messengers. The applicants have evidence for an increase in current triggered by NMDA receptors. Some of this current may be a high voltage activated channel that does not inactivate. Such channels are typically from the L and N type calcium channel classes. In response to many different signals, a rise in the concentration of Ca²⁺ in the cytosol triggers many types of events such as e.g. release of neurotransmitters at synapses (and essential for the long-term synaptic changes that produce Long-Term Potentiation (LTP) and Long-Term Depression (LTD). Therefore, encompassed by the present invention is also an intracellular Ca²⁺ levels blocker or reducer such as Acetoxymethyl (AM) or Acetate Esther versions of buffers, intracellular calcium release blockers, gene therapeutic agents that induce enhanced expression of calcium buffers, Signal Transduction Agents (e.g. CaM kinase II inhibitors, CaM kinase IV inhibitors, Myosin light chain kinase inhibitors), Calcium Signalling Agents, Calcium Binding Protein Modulators (e.g. Calmodulin antagonists), agents blocking the phosphorylation of subunit NR2B, CREB, GIuRl on residue Ser 831 (a protein required for trafficking of AMPA receptors) , or ERK 1 or 2.

Also encompassed by the present invention is a composition capable of reducing memory, perception and/or attention by reducing intracellular calcium changes comprising a drug selected from the groups comprising the NMDA receptor antagonists, the non-inactivating Calcium channels antagonists, the blockers of intracellular calcium release, a blocker of the metabotropic receptor class of neurotransmitters that are linked to the intracellular release of calcium such as, for example, the metabotropic glutamate receptors from group I, and/or a combination thereof. The present invention also envisioned a composition capable of reducing memory, perception and/or attention by preventing release of calcium from intracellular stores by blocking the activation of alpha 1 adrenergic receptors, said composition comprises a drug selected from the group comprising a noradrenergic receptor antagonist, a noradrenergic uptake enhancer, and/or a combination thereof.

Exemplary calcium channel blockers comprise, but are not limited to: Ca²⁺ channel blocker fL-type)

Nimodipine (l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2- methyloxyethyl 1-methylethyl ester),

Nitrendipine (l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-ρyridine dicarboxylic acid ethyl methyl ester), Verapamil hydrochloride (α-[3-[[2-(3,4-Dimethoxyphenyl)ethyl]methylamino]propyl]-3,4- dimethoxy- α -(l-methylethyl)benzeneacetonitrile hydrochloride),

Diltiazem hydrochloride (2S-cis)-3-(Acetyloxy)-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2- (4-methoxyphenyl)- 1 ,5-benzothiazepin-4(5H)-one hydrochloride), Gabapentin (l-(Aminomethyl)cyclohexaneacetic acid),

Nifedipine (l,4-Dihydro-2,6-dimethyl-4-(2-nitroρhenyl)-3,5-ρyridinedicarboxylic acid dimethyl ester),

(S)-(+)-Niguldipine hydrochloride ((S)-l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5- pyridinedicarboxylic acid, 3-(4,4-diphenyl-l-piρeridinyl)propyl methyl ester hydrochloride),

(R)-(+)-Bay K 8644 ((4R)- 1 ^-Dihydro^^-dimethyl-S-nitro^-tl-trifluoromethyOphenylJ-S- pyridinecarboxylic acid methyl ester),

SR 33805 (oxalate 3,4-Dimethoxy-N-methyl-N-[3-[4-[[l-methyl-3-(l-methylethyl)-lH- indol-2-yl]sulfonyl]ρhenoxy]propyl]benzeneethanamine oxalate), Potent Ca²⁺ channel antagonist; binds allosterically to the ai-subunit ofL-type Ca²⁺ channels (K_d = 2OpM), at a site distinct from other types of blocker.

Isradipine (4-(2, 1 ,3 -Benzoxadiazol-4-yl)- 1 ,4-dihydro-2,6-dimethyl-3 ,5-pyridinecarboxylic acid methyl 1-methylethyl ester),

Ca²⁺ channel blocker (TST, P and O-type) ω-Conotoxin MVIIC, ω-Conotoxin GVIA, Ziconotide

(R)-(-)-Niguldipine hydrochloride ((R)- 1 ,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5- pyridinedicarboxylic acid), 3-(4,4-diphenyl-l-piρeridinyl)propyl methyl ester hydrochloride,

SKF 96365 (hydrochloride l-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl- lH-imidazole hydrochloride),

Ruthenium Red (Ammoniated ruthenium oxychloride).

L-T ype calcium channel blockers, such as those from the dihydropyridine, phenylalkylamines and benzothiazepine chemical classes.

Amlodipine (Norvasc™, Lotrel™),

Felodipine (Plendil™, Lexxel™),

Isradipine (Dynacirc™),

Nicardipine (Cardene™), Nifedipine (Adalat™, Procardia™),

Nimodipine (Nimotop ™),

Nisoldipine (Sular™), Verapamil (Isoptin™), Diltiazem (Angizem, Altiazem), Bepridil (Vascor™).

Blockers of Intracellular Calcium Release

Blockers of ryanodine receptors (RyRs) and inositol l,4,5-(-trisphosphate receptors (IP₃Rs),

Alphal -adrenergic receptor blockers,

Ryanodine which blocks the Ryanodine receptor and blocks intracellular calcium release.

Usually, the therapeutically effective amount of a composition of the present invention capable of reducing memory, perception and/or attention by enhancing synaptic weakening includes also a drug selected from the group comprising a metabotropic glutamatergic receptor (mGluR) antagonist, a CREB gene down regulator, a CREB phosphorylation blocker and/or a combination thereof.

Applicants have shown that when the group 1 metabotropic receptors, and more specifically the mGluR5 receptor is blocked, TD is prevented. It is known that mGluR5 triggers a signaling pathway called the Inositol Phosphate Pathway. This pathway is a controlled by Ca2+ influx into neurons which occurs during electrical activity and attenuating this pathway is therefore also a potential target for treating autism.

In case the drug is a metabotropic glutamatergic receptor (mGluR) antagonist, then preferably the metabotropic glutamatergic receptor (mGluR) antagonists are mGluR Group I antagonists. Preferably, mGluR Group I antagonists will selectively target the neocortex, most preferably by differential affinities of the antagonists. For example, mGluR5, a member of the niGluR Group I, triggers a signaling pathway called the Inositol Phosphate Pathway. This pathway is a controlled by Ca2+ influx into neurons which occurs during electrical activity and attenuating this pathway is therefore also useful for reducing memory, perception and/or attention by enhancing synaptic weakening. Exemplary mGluR Group I antagonists comprise, but are not limited to: DL-AP3 (DL-2-Amino-3-phosphonopropionic acid),

(S)-4-Carboxy-3-hydroxyphenylglycine, (S)-4-Carboxyphenylglycine (S)-3-Carboxy-4-hydroxyphenylglycine (RS)-MCPG ((RS)- α -Methyl-4-carboxyphenylglycine),

(S)-MCPG ((S)-a-Methyl-4-carboxyphenylglycine),

ADDA ((RS)-l-Aminoindan-l,5-dicarboxylic acid),

E4CPG ((RS)-α-Ethyl-4-carboxyphenylglycine),

PHCCC (N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-la-carboxamide ), (IC₅₀ - 3 mM)

CPCCOEt (7-(Hydroxyiniino)cyclopropa[b]chromen-la-carboxylate ethyl ester), (IC₅₀ = 6.5 mM)

SIB 1893 (2-Methyl-6-(2-ρhenylethenyl)pyridine), IQo = 0.3 mM at HmGIu₅,

SIB 1757 (6-Methyl-2-(ρhenylazo)-3-ρyridinol), 0.4 mM at HmGIu₅

LY 367385 ((S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid ),

IC₅O = 8.8 mMfor blockade of quisqualate-induced phosphoinositide hydrolysis vs. > 100 mMfor mGlu_5a

MPEP hydrochloride (2-Methyl-6-(phenylethynyl)pyridine hydrochloride), IC₅O ⁼ 36 nM at the mGlu₅ receptor

HexylHIBO (α-Amino-4-hexyl-2,3-dihydro-3-oxo-5-isoxazolepropanoic acid),

Kb values are 140 and 110 mM at mGlu]_a and mGlu_5a receptors respectively.

(S)-HexylHIBO ((αS)-α-Amino-4-hexyl-2,3-dihydro-3-oxo-5-isoxazolepropanoic acid), K_b values are 30 and 61 mMat mGluj_a and mGlu_5a receptors respectively MATIDA (α-Amino-5-carboxy-3-methyl-2-thioplieneacetic acid 3-),

IC₅₀ = 6.3 mMat rat mGlui_a). Displays ≥ 40-fold selectivity over other receptors: mGlus, mGlu₂, mGlu_4a (IC₅₀ > 300 niM), NMDA and AMPA (IC₅₀ = 250 niM).

ACDPP hydrochloride (3-Amino-6-chloro-5-dimethylamino-N-2- pyridinylpyrazinecarboxamide hydrochloride),

Antagonist at the mGlu₅ receptor (K_t = 295 nM). Inhibits human mGlu₅ receptor-mediated calcium flux with an IC₅0 of 134 nM

Since it has been shown that CREB (Cyclic-AMP Response Element Binding) proteins in neurons are involved in the formation of long-term memories and that they are necessary for the late stage of long term potentiation (LTP) the present invention also envisioned either to down regulate the CREB gene or to block the phosphorylation CREB protein. Down regulating the CREB gene could, for example, occur by using gene regulating mechanisms that limits the transcript level by either suppressing transcription of CREB gene (transcriptional gene silencing), by activating a sequence-specific RNA degradation process (posttranscriptional gene silencing i.e. PTGS/RNA interference), by activating the CREM gene or by the phosphorylation of CREB such as for example on residue 142 or 123.

Usually, the therapeutically effective amount of a composition of the present invention capable of reducing memory, perception and/or attention by reducing synapse formation comprises a drug selected from the group comprising a NCAM expression inhibitor, a synaptophysin expression inhibitor, a cell-to-cell protein expression inhibitor, a receptor recycling inhibitor, and/or a combination thereof.

Cell-to-cell proteins can be selected from the group comprising ephrins, cadherins and cathenins.

Also encompassed in the present invention is a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by enhancing synaptic breakdown that comprises a drug selected from the group comprising a NOGO expression enhancer, a blocker that prevents the binding of polysialic acid (PSA) to NCAMs such as endoneurarainidase-N (endo-N), and/or a combination thereof.

Also encompassed in the present invention is a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by reducing network excitability that comprises a drug that inhibits potassium or calcium channels.

The neurons in autistic animals display a damped down excitability. This is due to the over expression of a variety of potassium channels.

Preferably, said drag will act by blocking the SK2 calcium sensitive potassium channels, or by blocking the BK Ca sensitive potassium channel, or by blocking voltage-gated postassium channels such as the ion channel encoded by the Kv 3.2 and/or the Kv4 beta gene gene, or by blocking L, N, P and/or Q -type calcium channels.

Also encompassed in the present invention is a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by increasing the level of network inhibition comprises a drag selected from the group comprising a cannabinoid receptor 1 inhibitor, a gamrna-amino butyric acid A (GABA-A) agonist or attenuator, a gamma-amino butyric acid B (GABA-B) agonist or attenuator and/or a combination thereof. In case the gamma-amino butyric acid A (GABA-A) agonist is a GABA-A agonist or attenuator then it is selected from the benzodiazepine groups.

In case the GABA-B inhibitor is a GABA-B agonist, then it is selected from the group comprising, but not limited to:

4-Amino-3-(4-chlorophenyl)butanoic acid ((RS)-Baclofen),

(R)-4-Amino-3-(4-chlorophenyl)butanoic acid ((R)-Baclofen),

CGP 35024 ,

CGP44532,

3-Aminopropyl(methyl)phosphinic acid (SKF 97541). "Cannabinoid receptor 1" or CBl are found primarily in the brain, specifically in the basal ganglia and in the limbic system, including the hippocampus. They are also found in the cerebellum and in both male and female reproductive systems. CBl receptors appear to be responsible for the euphoric and anticonvulsive effects of cannabis. This receptor has also been implicated in anxiety.

Gene expression analysis indicates that there is an increased expression of the cannabinoid receptor 1, suggesting that autistics are able to "self-administer" anxiety reducing behavior. Inhibition of the cannabinoid receptor 1 will therefore encourage external reward seeking behavior.

Also encompassed in the present invention is a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by reducing the supply of dopamine to the neocortex that comprises a drug selected from the group comprising a dopamine receptor inhibitor, a dopamine uptake enhancer, and/or a combination thereof. The hyper-connectivity found by the Applicants and the resistance to unlearning new associations suggest that the attentional mechanisms are too strong in Autism. Dopamine is a neurotransmitter that enhances attention in attention deficit disorders and it is therefore desirable to provide the inverse therapy in autism, namely to block dopamine effects.

Applicants have also shown an increase in the somatostatin receptor 2 by Gene expression analysis. This receptor is activated by the release of the neuropeptide somatostatin from a special type of inhibitory neuron called a Martinotti cell. The Martinotti cell is crucial for maintaining electrical balance in the neocortex and imbalance could also account for the high tendency towards seizures and epilepsy in autism. An increase in the expression of the receptor indicates that these interneurons can exert a stronger inhibitory effect, which appears to be a compensatory effect to prevent hyper-excitability. Blocking this receptor in combination could force the hyper-connectivity in reverse. Therefore, also encompassed in the present invention is a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by blocking the somatostatin receptor 2 that comprises a drug selected from the group comprising neuropeptide receptor blockers, somatostatin synthesis blockers, and/or a combination thereof. Preferably, in addition to at least one drug as described herein, the composition may contain one or more pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitates processing of the active compounds into preparation which can be used pharmaceutically. The composition may contain, for example, 0.1 to 99.5%, more preferably 0.5 to 90%) of the drug.

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

The form of administration of the composition maybe systemic and/or topical and/or via the nasal cavity. For example, administration of such a composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.

The composition comprising a drug, as described herein, as an active agent may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support. In addition the matrix may be comprised of a biopolymer.

Sustained-release preparations maybe prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.

It is understood that the suitable dosage of the composition of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.

The appropriate dosage form will depend on the disease, the drug, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, nasal inhalers, solutions, ointments and parenteral depots. The selected dosage level will depend upon a variety of factors including the activity of the particular compound, or combination of compound, of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular reuptake inhibitors employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a composition of the invention will be that amount of the composition which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the composition of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

In case the composition of the invention are associated with an antidepressant drug such as selective serotonin-reuptake inhibitors (SSRIs), an anticonvulsive drug, or an antipsychotic drug, or and combinations thereof, the dosage level may vary accordingly.

It is also understood that the described method not only applies to autistic children or autistic adults but also to pregnant non-autistic women in order to prevent the appearance of autistic conditions in the fetus, hi such a case, the therapeutically effective amount of a composition capable of reducing memory, perception and/or attention according to the present invention will be administered as soon as a diagnosis is possible. It is not currently possible to diagnose an autistic fetus, but a gene expression screen of amitotic fluid may reveal the changes found in autism. Treatment must start as early as possible to prevent the formation and consolidation of this disorder during development.

Preferably, treatment will start within the first three years of the children, but because plasticity of the circuitry continues well beyond adulthood, the prescribed treatment regime can be administered effectively at any age.

Also encompassed by the present invention is the use of a composition of the invention in the preparation of a medicament for treating and/or preventing a Pervasive Developmental Disorder.

It is another embodiment of the invention to provide a kit for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof, said kit comprising a therapeutically effective amount of a composition of the present invention capable of reducing memory, perception and/or, optionally with reagents and/or instructions for use.

The kit of the present invention may further comprise a separate pharmaceutical dosage form comprising an antidepressant drug such as selective serotoriin-reuptake inhibitors (SSRIs), an anticonvulsive drug, or an antipsychotic drug, and combinations thereof. Generally, the Kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent is a composition of the present invention. The label or package insert indicates that the composition is used treating and/or preventing a Pervasive Developmental Disorder, such as autism.

Alternatively, or additionally, the Kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Electrophysiology

The valproic acid rat model of autism

Wistar Han rats (Charles River Laboratories, L'Arbresle, France) were mated, with pregnancy determined by the presence of a vaginal plug on embryonic day 1 (El). The sodium salt of valproic acid (NaVPA, Sigma) was dissolved in 0.9% saline for a concentration of 150 mg/ml, pH 7.3. The dosing volume was 3.3 ml/kg, the dosage was adjusted according to the body weight of the animal on the day of injection. Treated rats received a single ip injection of 500 mg/kg NaVPA on gestation day GD 12.5, control rats were untreated [I]. Delivery of this dose to rats during embryogeneis has been shown to result in maximum levels of total valproic acid in maternal plasma in less than 1 hour, with a mean plasma elimination half life of 2.3 h [2].

Dams were housed individually and were allowed to raise their own litters. The offsprings were used for experiments on postnatal day 12 to 16 (PN12-PN16).

Acute slice preparation

All experimental procedures were carried out according to the Swiss federation rules for animal experiments. Offspring (PN12 to PN16) were rapidly decapitated and sagittal neocortical slices (300 μm) were sectioned on a vibratome (HR2, Sigmann Elektronik) filled with iced extracellular solution. Optimal slices, running parallel to the apical dendrites of pyramidal cells, were selected for recording. Slices were incubated for 30 minutes at 35⁰C and then at room temperature until transferred to the recording chamber (room temperature or 34⁰C). The extracellular solution contained (niM): 125 NaCl, 2.5 KCl₅ 25 glucose, 25 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂ and 1 MgCl₂. Neurones in somatosensory cortex were identified using IR-DIC microscopy, with an upright microscope (Olympus Bx51Wl, fitted with a 60x/0.90 W objective, Olympus, Switzerland). Recorded neurons were selected up to 70 μm below the surface of the slice.

Electrophysiological recording

Simultaneous whole-cell recordings from clusters of up to four neurons (pipette resistance 4 to

10 MΩ) were made and signals were amplified using Axopatch 200B amplifiers (Axon Instruments). Neurones were submitted to different type of protocols depending on what properties were being looked at. Voltages (in I-clamp mode) or current (in V-clamp mode) were recorded with pipettes containing (mM): 100 potassium gluconate, 20 KCl, 4 ATP-Mg, 10 phosphocreatine, 0.3 GTP, 10 Hepes (pH 7.3, 270-300 mOsm) and 0.5% biocytin (Sigma).Membrane potentials were not corrected for the junction potentials between pipette and bath solution ( ~10 mV).

Intrinsic properties of the cells:

During whole-cell patch-clamping, neurones were submitted to a series of somatic current injection designed to capture their key active and passive electrical properties. Recordings were sampled at intervals of 10-400 μs using Igor (Wavemetrics, Lake Oswego, OR, USA), digitized by an ITC- 18 interface (Instrutech, Great neck, NY, USA) and stored for off-line analysis. The results were calculated as a mean of 89 cells from control rats and 100 cells from NaVPA treated rats. Connectivity: Synaptic direct connections were examined by eliciting short trains (8 pulses) of precisely timed action potentials (APs) at different frequencies (20 to 70 Hz) followed by a recovery test response 500 ms later. The average synaptic response to this stimulation protocol allows the extraction of the basic parameters of the synaptic connection with a model of dynamic synaptic transmission (Ase, the absolute strength of the connection; U, equivalent to average P; d, the time constant to recover from depression) [3] The number of analyzed connections was 41 for control slices, 33 for treated slices.

For the observation of disynaptic connectivity, neighboring thick tufted layer V pyramidal cells (PCs) were recorded and stimulated with high frequency action potential (AP) trains (15 regular pulses at 70Hz). When the cells were connected via an intermediate interneuron, inhibitory responses were observed on neighboring PCs. The amplitude, latency and half- width of these responses were approximated with a Gaussian fit (control cells: n = 275, treated cells: n = 208 polysynaptic connections).

Long term potentiation :

To check long term potentiation, a connection was, five minutes after a testing period (same protocol as described above ), stimulated with a pairing protocol consisting of Poisson train stimulation of high frequency (50Hz) to the postsynaptic cell and of low frequency (5Hz) to the presynaptic cell, for 30 seconds. The connection was tested after two Poisson pairing sessions, each 15 minute for 30 minutes (control connections: n = 15, treated connections: n = 13).

MEA stimulation: Multi-site extracellular stimulations were performed using a multi-electrode array (MEA) made of 60 3D Pt electrodes (Ayanda Biosystems, EPFL, Switzerland), on top of which acute brain slices were glued with a solution of nitrocellulose (0.14 mg/ml in ethanol). The responses of these stimulations were recorded in whole-cell patched layer 2/3 pyramidal cells. For the study of cell response with minimal network stimulation, 8 different MEA electrodes were stimulated non- simultaneously (single pulse of 0.5 to 1. IV) and current were recorded in V-clamped cells (holding voltage between -80 and -4OmV). Total synaptic, excitatory and inhibitory conductances could then be calculated according to the method described in [4]. 38 control cells (n = 420 inputs) and 49 treated cells (n = 480 inputs) were used for the analysis. For the study of strong network stimulation, 16 MEA electrodes were stimulated simultaneously with a Poisson train (50Hz, 300 ms) at increasing stimulation amplitude

(normalized to the response amplitude). Whole-cell V-clamped cells filled with Qx-314 were then recorded at -80, -57 and +10 mV to get the contribution of respectively all synaptic components, of inhibitory components and excitatory components. 12 control and 12 treated cells were kept for the analysis.

Pharmacological compounds; N-Methyl-D-aspartate receptors (NMDARs) were blocked using 20 μM D-2-amino-5- phosphonopentanoic acid (D-APV, Sigma). L-amino-3-hydroxy-5-methyl-4- isoxazolepropionate receptors (AMPARs) were blocked using 10 μM 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX disodium salt, Sigma), μ-aminobutyric acid A receptors (GABA_ARS) were blocked using 20 μM bicuculline (Sigma).γ-aminobutyric acid B receptors (GABA_BRS) were blocked using 100 μM CPG-35348 (Tocris). Lidocaine n-ethylbromide quaternary salt (QX-314, 5 μM, Sigma) was used to block voltage-gated channels.

Histological procedures and 3-D reconstruction After recording, slices were fixed for 24h in cold 0.1M phosphate buffer (PB, pH 7.4) containing 2% paraformaldehyde and 0.3% saturated picric acid. Thereafter, slices were rinced several times in PB, and transferred in phosphate buffer 3% H₂O₂ for 30 min to block endogeneous peroxidases. They were then incubated overnight at 4°C in biotinylated horseradish peroxidase conjugated to avidin according to the manufacturer's protocol (ABC- Elite, Vector Laboratories, Peterboroug, UK). Following incubation, sections were washed several times in PB and developed with diaminobenzidine under visual control. The reaction was stopped by transferring the slices into PB. After washing in the same buffer, slices were mounted in aqueous mounting medium (IMMCO Diagnostics, hie).

3-D neurone models were reconstructed from five control and five treated stained cells using Neurolucida system (MicroBrightField Inc., USA) and a bright-field microscope (Olympus).

1. Ingram, J.L., et al., Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism. Neurotoxicol Teratol, 2000. 22(3): p.

319-24.

2. Binkerd, P.E., et al., Evaluation of valproic acid (VPA) developmental toxicity and pharmacokinetics in Sprague-Dawley rats, Fundam Appl Toxicol, 1988. 11(3): p. 485- 93. 3. Markram, H., Y. Wang, and M. Tsodyks, Differential signaling via the same axon of neocortical pyramidal neurons. Proc Natl Acad Sci U S A, 1998. 95(9): p. 5323-8. 4. Borg-Graham, LJ., C. Monier, and Y. Fregnac, Visual input evokes transient and strong shunting inhibition in visual cortical neurons. Nature, 1998. 393(6683): p. 369- 73.

Behavioral Testing

Overview

Several lines of behavioural tests were conducted on a total of 120 adolescent (approximately 6 weeks old at the beginning of experiments) and adult (approximately 12 weeks old at the beginning of experiments) on male and female, treated and untreated rats. The testing focused on perception, learning and memory capabilities and involved the following tests: Startle response and pre-pulse inhibition

- Fear conditioning and extinction - Water maze.

Animals

Male and female Wistar rats (own breeding), 6 to 12 weeks old at the beginning of the experiments, were housed in groups of three to five per cage, under light (12 hr light/dark cycle; light on at 7 A.M.)-and temperature (22± 2° C)-controlled conditions. Food and water were available ad libitum. All animals were handled for 5 minutes every day for 3 days preceding the first day of the behavioural tests. Animal care procedures were conducted in accordance with the guidelines set by the European Community Council Directives (86/609/EEC) and Ecole Polytechnique Federate de Lausanne (EPFL), Switzerland.

Startle response and pre-pulse inhibition

The startle apparatus (Columbus Instruments, Ohio) consisted of aplastic, transparent cage, equipped with a movable platform floor attached to a sensor, which recorded vertical movements of the floor. A loudspeaker was suspended above the cage, and it was placed in a soundproof box. A transient force resulting from up-and-down movements of the floor, evoked by a startle reaction to acoustic stimuli, was recorded by PC using a recording window of 200 ms measured from the onset of the acoustic stimulus. The amplitude of a startle response was defined as a difference between the average force detected within a recording window and the force measured immediately before the stimulus onset. The threshold was set at 20 g and allowed for correct evaluation of the maximum response in all the animals tested.

The experiment started with an adaptation period during which the animals were placed in experimental cages for 5 min and exposed to a 70 dB background white noise. Following habituation, baseline startle responses were recorded by confronting the rats with 5 tone pulses (15 dB, with a random intertrial interval of 10-30 sec). Immediately afterwards a series of prepulse-pulse stimulation was initiated consisting of 12 pulse alone trials, and 4 prepulse- pulse combinations, each made up by 6 trials. The combinations consisted of either a 78 or 86 dB prepulse tone preceding the main pulse by either 30 or 120 ms. Each trial was separated by a random intertrial interval of 10 to 30 sec. The trial order was random. The experiment concluded with a series of 5 pulses alone, the same as during baseline recordings.

Prepulse inhibition was calculated as the percentage of inhibition of the startle amplitude evoked by pulse alone: ((pulse-prepulse)/pulse) x 100.

Fear conditioning

Training and testing took place in a rodent observation cage (30 x 37x 25 cm) that was placed in a sound-attenuating chamber. The side walls of the observation cage were constructed of stainless steel, and the door of Plexiglas. The floor consisted of 20 steel rods through which a scrambled shock from a shock generator (model LI 100-26 Shocker, LETICA LC, Madrid, Spain) could be delivered. Each observation cage was cleaned with a 0.1% acetic acid solution before and after each session. Ventilation fans provided a background noise of 68 dB, and a 2OW white light-bulb illuminated the chamber.

On the training day for auditory fear conditioning rats were exposed to a conditioning chamber during 160 sec, followed by three presentations of a tone-shock pairing in which the tone (20 sec, 85dB sound at 1000Hz) co-terminated with a footshock (1 mA, 1 sec). The inter-tone interval was 40 sec and the conditioning session lasted 5.5 min. On the next day, the animals were re-exposed to the context in which they originally received the tone-footshock pairings but in the absence of tones and shocks. One day after the context test, the rats were put in a novel context for 8 min in which they were re-exposed to the tone, but not the shocks, continuously during the last 5 minutes of the test (tone test). Remote memories for both, context and tone, were re-assessed one and again two months after fear-conditioning.

Subsequently, rats were re-conditioned to the tone (0.5 mA, 1 sec) in order to re-establish the fear memory and underwent a two-day extinction procedure consisting of a 30 min protocol per day, in which 20 sec tone intervals alternated with 40 sec no-tone intervals. Over the two days, rats were exposed to 60 tone stimulations in total. One day and one month after extinction training animals were re-tested for their tone memory using the same protocol as in the previous tone tests.

Using a time-sampling procedure every 2 sec, each rat was scored blindly as either freezing or active. Freezing was defined as behavioural immobility except for movement needed for respiration.

Water maze

Training and testing took place in a black, circular water tank (170 cm in diameter, 50 cm high, custom-made in the house) surrounded by grey curtains containing several spatial cues (e.g. plastic flowers, geometrical patterns, poster or clothes attached to the curtains and walls). The water temperature was 25° C +/- 1 ° C.

For the spatial training task, a 12 cm in diameter escape platform was placed with the top surface 1.5 - 2.5 cm below the water level at one of four positions in the pool. Each trial was initiated by placing the animal in one of four randomly chosen locations near the wall of the tank. Animals were allowed to search for the hidden platform for a maximum of 90 sec. If an animal reached the platform before the maximum time, the trial was stopped. In case the animal did not find the platform during this time, it was guided by the experimenter towards it and in both cases it was left on the platform for further 30 sec. Each learning day consisted of four consecutive learning trials, with 30 sec intertrial intervals. In total a session would last maximally 8 min. At the end of a learning session the animal was dried and taken to a recovery cage placed under a red light and subsequently returned to its home cage. Training consisted of a total of four learning sessions. One day and approximately three weeks later, spatial memory was tested in probe trials by removing the platform from the pool and placing the animal for 90 sec into the pool. After the second probe trial animals received two more learning sessions on consecutive days in order to re-establish a strong spatial memory. The following day, the platform was changed to the opposite position and rats underwent a final reversal learning session, the protocol being the same as above.

Learning was assessed as the latency to reach the platform. Memory performance was estimated by means of variables such as time spent, distance moved in the target quadrant as opposed to the opposite quadrant and the frequency of platform position crossing. All behaviour and learning and memory variables were registered by means of a video tracking system (Etho vision, Noldus).

Statistical analysis All results were expressed as mean ± SEM and analyzed using repeated measure ANOVA or one-way ANOVA where appropriate. Differences between different conditions were further evaluated for significance with Tukey's post hoc test. In all cases significance of results was accepted at p <0.05.

Genomics

Single cell cytoplasm harvesting using Patch-Clamp electrodes, RNA isolation/amplification and gene expression quantification (Q-PCR and/or oligonucleotide microarrays)

All experimental procedures were carried out according to the Swiss federation guidelines for animal experiments. Wistar rats (13-16 days old) were rapidly decapitated and neocortical slices (sagittal; 300 mm thick) were sectioned on a vibratome (DSK, Microslicer, Japan) filled with iced extracellular (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, and 1 MgCl₂. Neurones were identified using IR-DIC microscopy as previously described. Somatic whole-cell recordings of layer 5 pyramidal cells (pipette resistance-3 MΩ) were employed for harvesting their cytoplasmic contents. Pipettes were filled with RNase free intracellular solution, containing (mM) 100 potassium gluconate, 20 KCl, 4 ATP-Mg, 10 phosphocreatine, 0.3 GTP and 10 Hepes (pH 7.3, 310 mOsmol, adjusted with sucrose). The intracellular solution was prepared under RNAse free conditions: water was autoclaved; glassware and pH meter were cleaned with NaOH (10N) and chemicals were opened from the first time using gloves and RNAse free tools. After preparation, the intracellular solution was tested for RNAse contamination. Right after whole cell configuration, cell cytoplasm was aspirated into the recording pipette under visual control by applying gentle negative pressure. Only cells in which the seal was intact throughout the recording, and whose nucleus was not harvested, were further processed. The electrode was then withdrawn from the cell to form an outside-out patch that prevented contamination as the pipette was removed. The tip of the pipette was broken and the contents of the pipette expelled into a test tube by applying positive pressure. The contents of cells harvested from the same animal where pooled at the end of the day and the RNA was cleaned and isolated using silica-gel-membrane columns (RNeasy Micro Kit, Qiagen) and stored at -80⁰C before further processing.

RNA messenger was reverse transcribed using an oligo-dT and linearly amplified using a two round amplification kit (Epicenter) The quality of the amplified RNA was controlled on a Nano LabCbip (Agilent Technologies) and samples with high quality aRNA were either a) labeled and hybridize on oligonucleotide microarrays (Agilent and/or Affymetrix); or b) tested for the quantitative expression of selected genes using real time PCR (Applied Biosystems).

Laser capture microscopy (LCM), RNA isolation/amplification and gene expression quantification (Q-PCR and/or oligonucleotide microarrays)

AU experimental procedures were carried out according to the Swiss federation guidelines for animal experiments. Wistar rats (14 days old) were rapidly decapitated and their brains (entire or one hemisphere) were immediately frozen in 2-methylbutane-dry ice mix, and stored at - 80⁰C.

Coronal 8-12 um sections were cut on a cryostat and thaw mounted on PEN-membrane coated or non-coated glass slides. Sections were stained with brief Nissl stain for neuronal identification: 70% ethanol 1 min, water 5 dips, 1% cresyl violet 20 s, water 5 dips, processing through graded ethanols 70/95/100% (30 s each) and xylene 5 min. Neurons were captured using two LCM systems: AutoPix LCM system (Arcturus) and PALM MicroLaser System (P.A.L.M. Microlaser Technologies): Laser capture microdisection using the AutoPix LCM system (Arcturus). After Nissl staining, layer 5 pyramidal cells (somatosensory cortex and prefrontal cortex) were dissected using the AutoPix LCM system (Arcturus), adjusting the voltage and duration of the laser beam to allow capture of neuronal cytoplasm without adjacent tissue. The captured cells were collected onto CapSureTM HS caps covered with a thermoplastic film. The harvested cells were solubilized from the film in extraction buffer provided in the Arcturus Pico PureTM RNA isolation kit for 30 min at 40⁰C and stored at -80⁰C.

Laser capture microdisection using the PALM® MicroLaser System (P.A.L.M. Microlaser Technologies).

Right after Nissl staining, layer 5 pyramidal cells (somatosensory cortex and prefrontal cortex) on polyethylene naphthalate membrane coated slices were dissected using the PALM MicroLaser System (P.A.L.M. Microlaser Technologies). The captured cells were collected onto PALM AdhesiveCaps. The harvested cells were solubilized in extraction buffer provided in the RNeasy Micro Kit, Qiagen and stored at -8O⁰C.

At the end of each microdisection section part of the remaining brain slices was scraped from the glass slides and the RNA was isolated. All procedures were done under RNAsefree conditions.

Before amplification the quality of the scraped brain controls was controlled by detecting 18S and 28S bands on RNA 600 Nano LabChip (Agilent Technologies). The captured material (pooled of single neurons) was DNAse I treated and the RNA was purified using silica-gel- membrane columns (Pico PureTM RNA isolation kit, Arcturus or RNeasy Micro Kit, Qiagen) We performed double round amplification of the isolated mRNA with the RiboAmp RNA amplification kit, according to manufacturer's instructions (Arcturus). The quality of the amplified RNA was controlled on a Nano LabChip (Agilent Technologies) and samples with high quality aRNA were either a) labeled and hybridize on oligonucleotide microarrays (Agilent and/or Affyrnetrix); or b) tested for the quantitative expression of selected genes using real time PCR (Applied Biosystems).

Data analysis Oligonucleotide microarrays (Agilent and Asymetrix) were analyzed (probe level data preprocessing, quality checks, normalization, and visualization) using the RACE (Remote Analysis Computation for gene Expression data suite), a collection of web tools designed to assist with the analysis of DNA microarray data and results.

Immunoblotting

Preparation of brain membrane extracts

Rat brains were homogenized in 8 volumes of cold homogenization buffer A (0.32M sucrose, 1OmM HEPES/KOH, and the following protease inhibitors 0.3mM PMSF, 0.7μg/ml Pepstatin, 2μg/ml Aprotinin, 2μg/ml Leupeptin) using a motor driven glass-teflon homogenizer. The homogenate was spun at 450Og in a Beckman centrifuge to remove the pelleted nuclear fraction, the supernatant was spun at lOOOOOg for 40min. in a Beckman ultracentrifuge. The pellet was resuspended in buffer B (20 niM HEPES/KOH pH7.4, 2 mM EDTA, 2 mM EGTA, 0.1 mM DTT) containing 0.1 M KCl, 0.3mM PMSF, 0.7μg/ml

Pepstatin, 2μg/ml Aprotinin, 2μg/ml Leupeptin. The suspension was rehomogenized using a glass-teflon homogenizer and spun at lOOOOOg for 40min. Membrane pellets were lysed in buffer B (20 mM HEPES/KOH pH7.4, 2 mM EDTA, 2 mM EGTA, 0.1 mM DTT) containing 0.1 M KCl, 1% Triton XlOO, 0.3mM PMSF, OJμg/ml Pepstatin, 2μg/ml Aprotinin, 2μg/ml Leupeptin for 30min. at 4⁰C and spun at 100¹OOOg for 40min. 2 g of rat brain was lysed in a volume of 5 ml, yielding a concentration of 8 mg/ml.

Preparation of brain slices

Brain slices of the somatosensory cortex from NaVPA treated and control animals were homogenized in sucrose buffer (0.32M sucrose, 1OmM Hepes protease inhibitors 0.3 mM PMSF, 0.7 μg/ml Pepstatin, 2 μg/ml Aprotinin, 2 μg/ml Leupeptin) at 800rpm with an eppendorf pellet pistel. Triton was added to a final concentration of 1% and the homogenate rolled for 5min. at 4°C. The homogenate was spun 5min. at full speed and 4⁰C. The supernatant was assayed for the protein concentration using a Bradford dosage kit (Biorad) and Lammli buffer 2x (4% SDS, 6OmM Tris pH6.8, 10% glycerol, 5% D-mercaptoethanol) was added. Samples were kept at -20°C. Preparation of region specific brain extracts

Rats (n=5 males, VPA treated; n=5 males, saline treated) were sacrificed by rapid decapitation. Their brains were extracted and frozen in isopentane (-50⁰C) within 45 sec of decapitation and stored at -80°C until dissection. One-millimeter coronal slices of brain were cut in a cryostat (Leica ) kept at -20⁰C. Tissue punches were obtained from primary somatosensory cortex, secondary somatosensory cortex, and prefrontal cortex, and then sonicated in 1% sodium dodecyl sulfate (SDS). Protein concentrations of the samples were determined using the bichinconinic acid assay (Pierce Chemical Company; Rockford, IL). Sample concentrations were equalized by diluting with 1% SDS and loading buffer 4X (16% glycerol, 8% SDS, 3% Tris, 20% beta-mercaptoethanol, and 0.5% bromophenol blue) to yield a final protein concentration of 1.33 mg/mL.

SDS-Page Electrophoresis

Samples were subjected to SDS-polyacrylamide gel electrophoresis (10% acrylamide/0.27% N, N'-methylenebisacryalamide resolving gel) for 50 min at 185 volts. For each electrophoresis run, increasing amounts of protein pooled from the brain region being tested were run alongside the individual samples and used to produce a standard curve. Proteins were transferred electrophoretically to rmmobilon-P membranes (Millipore Corp; Bedford, MA) at 75 volts for 70 min. Blots were stained with Ponca Red and then rinsed with 0.1 M PBS for 10 min. Membranes were incubated in blocking buffer (Odyssey blocking buffer diluted in 0.1 M PBS) for 1 hour at room temperature. Membranes were then incubated overnight at 4 ⁰C with primary antibody diluted in blocking buffer with 0.1% TweerώO.

After incubation with primary antibodies, blots were rinsed in 0. IM PBS with 0.1 % Tween20 and were processed with fluorescent secondary antibodies (Molecular Probes, Rockland

Chemicals) diluted blocking buffer with 0.1% Tween20. The blots were rinsed in 0.1 M PBS for 1 hour. Fluorescence from the blots was detected by digital scanning in transparency mode using the Odyssey system. Band intensities were quantified using Odyssey software. Band intensities from test samples were compared to the band intensities from the standard curve.

Immunohistochemistry Rats were deeply anesthetized with pentobarbital (65 mg/200-350 gm). Rats were then perfused transcardially with 50 ml of 0.1 M sodium phosphate with 1.0 niL of heparin (1000 units/mL), pH 7.4 followed by 100 ml of 4% paraformaldehyde in 0.1 M sodium phosphate, pH 7.4. Brains were removed from the skull and post-fixed for 24 hr at 4°C before being transferred to 20% sucrose in 0.1 M sodium phosphate, pH 7.4, for 48 hr at 4⁰C. Brains were frozen in powdered dry ice and stored at -20° C.

Coronal sections (40 μM) were cut in a cryostat, collected in ethylene-glycol cryoprotectant, and stored at -80° C until further processing. To control for variability that might be introduced during immunocytochemical processing, sections for each treatment group were processed at the same time, such that each batch contained tissue from saline treated rats and valproic acid treated rats. The tissue was allowed to come to room temperature for one hour. Sections were rinsed 3 X 3 minutes in tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and 0.1 % Triton followed by a 20 minute rinse in 0.7% H2O2 diluted in TBST. Sections were rinsed 3 X 3 minutes in TBST and placed in blocking buffer for 1 hr at 22⁰C. The blocking buffer consisted of 10% normal serum in TBST. Sections were incubated overnight at 4°C in primary antibody diluted in 10% normal rabbit serum in TBST. After sections were washed in TBST, they were processed using a standard secondary detection system with ABC and nickel DAB.

Neurons were counted bilaterally at no less than two different anatomic levels for each anatomic structure (medial PFC, primary somatosensory cortex, and secondary somatosensory cortex). Bright-field images of immunoreactive-postive soma were captured using a digital camera and automatically counted using a computerized system.

Results

The Applicants for the first time ever performed experiments to examine the cellular, synaptic and local circuit changes in an animal model of autism as well as novel behavioral experiments. The applicants also carried out genomic and proteomic studies which reveal the molecules and genes involved in the synaptic, cellular, circuit abnormalities as well as those underlying the behavioral alterations in memory, perception and attention.

Weaker synapses in the Autistic Neocortex

The weak single fiber responses and large response to strong stimulation predicts that the synaptic connections between the excitatory neurons are also weaker in Autism. Applicants therefore recorded from pairs of excitatory pyramidal neurons to study directly the synaptic properties. Indeed, they found that synaptic connections between layer 5 pyramidal neurons were weaker (Figure Ic).

Applicants have shown that when the neocortical microcircuit is stimulated with weak stimulation to activate only one or a few fibers, that lower excitation was produced in the neocortex of autistic animals (Figure Ic).

Increased Total Synaptic Currents in Autistic Neocortex

When Applicants increased the stimulation strengths used to maximally excite the neocortical tissue so that they can study the delivery of synaptic current by the total synaptic pool, Applicants found that much greater excitatory and inhibitory synaptic current is produced by the autistic neocortex (Figure 2). The figure shows a dose-response curve in which the neocortical microcircuit was stimulated in multiple sites at progressively greater stimulus strengths. The response increases to produce a sigmoid response curve. The autistic microcircuit was hyper-excitable in terms of both excitatory and inhibitory voltage as can be seen by the generation of more voltage at lower stimulus strengths and reaching peak responses considerably higher than in control animals (Figure 2). When the traces were normalized to the maximum, the slope was also found to be slightly steeper for the excitation (not shown), indicating that the autistic neocortex is also hyper-reactive to stimuli. When Applicants compared the ratio of excitation to inhibition during the stimulations, they showed that the autistic microcircuit becomes more unbalanced at higher stimulation strengths with excess excitation. These results may explain the hyper-sensitivity to sensory stimulation that is found in the autistic spectrum disorder and the tendency to epilepsy that is found in 38% of autistics. Hyperconnectivity in the Autistic Neocortex

The weaker individual synapses and higher total current that they can deliver indicates that there are many more synapses formed in the autistic neocortex. Applicants therefore examined this directly by recording from pairs of neurons and determined the probabilities that neurons are connected. Indeed, they found that the probability of forming direct connections between pyramidal neurons nearly doubles (Figure Ia). This is a massive increase since these P to P connections constitute already most of the synapses in the neocortical column (NCC). Applicants also found that the probability to find double synaptic junctions from a pyramidal neuron to an inhibitory neuron and then to a second excitatory neuron was also greatly increased to over 55% (Figure Id). These results provide direct proof that the autistic neocortex is locally hyper-connected. Such local hyperconnectivity also implies that excess synapses are taken by the local connection which would cause a disconnection of the individual neocortical columns from neighbouring columns and distant brain regions. This would lead to self-autonomous modules of neurons in the brain which will be difficult to control by higher brain areas such as attentional mechanisms leaving the autistic "frozen" in its own world. Reducing the local hyper-connectivity will allow long range fibers to enter the columns and restore flexible command of perception, attention and memory.

Increased NMDA-triggered Currents in the Autistic Neocortex.

NMDA receptors are special forms of excitatory transmitter receptors activated by glutamate and are essential for many memory processes. During intense stimulation, synapses are fully activated and it is possible to record the amount of NMDA triggered current that the NCC can produce. The NMDA receptor channel allows the flow OfNa⁺, K⁺ and Ca²⁺ ions. When the cell is held at high potentials then only Ca²⁺ influx can be detected. Additionally NMDA receptors open neighbouring Calcium channels to allow additional Calcium inflow. Applicants found that the amount of NMDA receptor-mediated current at high potentials is greatly increased in Autism indicating a massive enhancement of Ca²⁺ ion influx triggered by NMDA receptors (Figure 6). Such Ca²⁺ influx is known to trigger synaptic learning processes and is important for memory formation. These results therefore suggest that the Autistic neocortex is primed to exaggerated synaptic learning which could lead to hyper-memories. Protein analysis indicate that the NR2 subunits, NR2A and NR2B, are upregulated (Figure 5), which explains the increased NMDA receptor-induced currents.

Altered synaptic plasticity in the Autstic Neocortex. When two cells are forced to become active together at the same time the synaptic transmission changes. This is known as synaptic plasticity. The applicants found that there is a massive enhancement of a postsynaptic form of plasticity in the autistic (Figure 7). This is most likely due to the increased levels of Calcium into the postsynaptic neuron as found triggered by the NMDA receptors.

Enhanced Fear Memories in Autism

Applicants examined behavioral changes in the autistic model. In addition to the usual behavioral tests for anxiety and social interactions, they performed memory tests which were never performed before on autistics. In particular they wanted to explore fear memories because autistics have enhanced reactions to novel situations that may bee fearful. A link between fear and autism has not previously been reported. Applicants also wanted to determine the degree to which the autistic model retains a memory and how easy it is to reverse a memory. They therefore decided to test behaviorally the memory and memory extinction capabilities in the autistic model. The test they used was classical fear conditioning where a tone is presented followed by an electric shock to the plate on which the rats stand. They found that the Autistic animals remembered the fear memory for much better over a period of 2 months (data not shown). The results are consistent with an increased level of NMDA triggered calcium currents in the Autistic neocortex which suggest that there is more powerful learning and hence they remember events better and for longer. This finding is also consistent with the hyper-connectivity since this would make learning an association much easier because there are more synapses that can be involved.

Impaired Extinction Learning in Autism Applicants then retrained the animals to fully re-instate the memory and began a series of extinction protocols where the tone is given but no shock follows. The results indicate that the autistics are significantly impaired in their ability to reverse the prior associative conditioning (data not shown).

This suggests that there is a deficit in synaptic depression in autism, which is required to reverse the learning. In addition, the hyper-connectivity may make it easier to remember but more difficult to unlearn prior associations because too many synapses are involved. These experiments indicated that autistics have hyper-memories and that their memories become so fixed that they are unable to re-learn a new association. This could also explain their inflexibility to un-learn previous associations and re-learn new ones that - become relevant and hence their need and demand for extremely structured daily routines. Since many events may require only transient associations to be dropped the next day, this tendency could form the basis of a severe social handicap.

Decreased Pyramidal Neuron Excitability

When Applicants examined the electrical properties of the excitatory neurons they found that the excitability was significantly decreased. In particular, they found that the increase in spiking activity as progressively more current is injected into the neuron, referred to as the current-discharge relationship, was decreased . The maximal discharge when the neurons reach threshold is also greatly depressed. Finally the amount of current required to reach the discharge threshold is significantly increased making it more difficult for the neurons to fire spikes (Figure 3).

These results suggest that the neurons may be trying to compensate for the hyper-excitability caused by the hyper-connectivity by reducing their excitation. These effects could be due to the decreased expression of sodium channels, increased expression of various potassium channels and also result from the increased calcium currents. This is a significant finding since this depressed activity would oppose the hyper-excitable effects of the enhanced connectivity in the microcircuit.

Protein changes in Autism

Applicants carried out a course-grained gene expression analysis in which proteins from somatosensory cortex from controls and autistics were assayed using SDS-page electrophoresis and immunoblotting. They found a number of protein deficits, but some are particular highly relevant to the hyperconnectictivity disorder. In particular, the subunits 2A and B that are required to build special subtypes of NMDA receptors were up regulated. Another protein alteration is relevant to the hyper-learning disorder: Applicants have shown increased levels of CamKII, a key kinase involved in learning and memory processes, in the treated rats. These results support the excessive learning due to increased intracellular calcium levels (Fig 5).

These protein data further support the results that a major deficit in autism is a hyperconnectivity, excessive learning and memory processes, hand hypo-neuronal excitability in the neurons of the neocortex.

This hyperconnectivity of local microcircuits of neurons may be throughout the brain, but the effects in the neocortex will be devastating for the cardinal cognitive deficits in autism that result from hyper-perception, attention and memory.

Claims

1. A method for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof comprising the step of modulating the synaptic connectivity in the neocortex by administering a therapeutically effective amount of a composition capable of reducing memory, perception and/or attention by either i) blocking synaptic strengthening, ii) enhancing synaptic weakening, iii) reducing synapse formation, iv) enhancing synaptic breakdown, v) decreasing network excitability, vi) increasing the level of network inhibition, vii) reducing the supply of dopamine to the neocortex, viii) blocking the somatostatin receptor 2, wherein compositions i) to viii) are administered separately of concurrently.

2. The method of claim 1, wherein the composition capable of reducing memory, perception and/or attention by blocking synaptic strengthening comprises a drug selected from the group comprising a muscarinic receptor antagonist, acetylcholin esterase enhancer, N- methyl-D-aspartate (NMDA) receptor antagonist, second messenger blocker, calcium channel blocker, intracellular calcium blocker, group IH metabotropic glutamatergic receptor agonist (group IH mGluR) and/or a combination thereof.

3. The method of claim 1 , wherein the composition capable of reducing memory, perception and/or attention by enhancing synaptic weakening comprises a drug selected from the group comprising a group I metabotropic glutamatergic receptor (group I mGluIO antagonist, a CamKII gene down regulator, and/or a combination thereof.

4. The method of claim 1, wherein the composition capable of reducing memory, perception and/or attention by reducing synapse formation comprises a drug selected from the group comprising an NCAM expression inhibitor, a synaptophysin expression inhibitor, a cell- to-cell protein expression inhibitor, a receptor recycling inhibitor, and/or a combination thereof.

5. The method of claim 1 , wherein the composition capable of reducing memory, perception and/or attention by enhancing synaptic breakdown comprises a drug selected from the group comprising a NOGO expression enhancer, a PSA-NCAM binding blocker, and/or a combination thereof.

6. The method of claim 1, wherein the composition capable of reducing memory, perception and/or attention by network excitability comprises a drug that inhibits potassium and/or calcium channels.

7. The method of claim 1, wherein the composition capable of reducing memory, perception and/or attention by decreasing the level of network inhibition comprises a drug selected from the group comprising a cannabinoid receptor 1 inhibitor, a gamma-amino butyric acid A (GABA-A) inhibitor, a gamma-amino butyric acid B (GABA-B) inhibitor and/or a combination thereof.

8. The method of claim 1 , wherein the composition capable of reducing memory, perception and/or attention by reducing the supply of dopamine to the neocortex comprises a drug selected from the group comprising a dopamine receptor inhibitor, a dopamine uptake enhancer, and/or a combination thereof.

9. The method of claim 1, wherein the composition capable of reducing memory, perception and/or attention by blocking the somatostatin receptor 2 comprises a drug selected from the group comprising neuropeptide receptor blockers, somatostatin synthesis blockers, and/or a combination thereof.

10. The method of any of the preceding claims, wherein the Pervasive Developmental Disorder is selected from the group comprising Fragile X, autism, mental retardation, schizophrenia and Down's Syndrome.

11. The method of claim 9, wherein the Pervasive Developmental Disorder is autism selected from the group comprising Typical Autism, Atypical Autism/PDD, Autistic Savants, Asperger's Syndrome, and autism associated with Kanner's Syndrome, fragile X, Rett and Down's syndromes.

12. A pharmaceutical composition for treating and/or preventing a Pervasive Developmental Disorder in a subject in need thereof, said pharmaceutical composition being capable of reducing memory, perception and/or attention by either i) blocking synaptic strengthening, ii) enhancing synaptic weakening, iii) reducing synapse formation, iv) enhancing synaptic breakdown, v) decreasing network excitability, vi) increasing the level of network inhibition, vii) reducing the supply of dopamine to the neocortex, viii) blocking the somatostatin receptor 2.

13. The pharmaceutical composition of claim 12 characterized in that it is administered separately or concurrently.

14. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by blocking synaptic strengthening, said pharmaceutical composition comprises a drug selected from the group comprising a muscarinic receptor antagonist, acetylcholin esterase enhancer, N-methyl-D-aspartate (NMDA) receptor antagonist, second messenger blocker, calcium channel blocker, intracellular calcium blocker, group III metabotropic glutamatergic receptor agonist (group IE mGluR) and/or a combination thereof.

15. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by enhancing synaptic weakening, said pharmaceutical composition comprises a drug selected from the group comprising a group I metabotropic glutamatergic receptor (group I mGluR) antaRonist, a CamKII gene down regulator, and/or a combination thereof.

16. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by reducing synapse formation, said pharmaceutical composition comprises a drug selected from the group comprising an NCAM expression inhibitor, a synaptophysin expression inhibitor, a cell-to-cell protein expression inhibitor, a receptor recycling inhibitor, and/or a combination thereof.

17. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by enhancing synaptic breakdown, said pharmaceutical composition comprises a drug selected from the group comprising a NOGO expression enhancer, a PSA-NCAM binding blocker, and/or a combination thereof.

18. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by network excitability, said pharmaceutical composition comprises a drug that inhibits potassium and/or calcium channels.

19. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by decreasing the level of network inhibition, said pharmaceutical composition comprises a drug selected from the group comprising a cannabinoid receptor 1 inhibitor, a gamma-amino butyric acid A (GABA-A) inhibitor, a gamma-amino butyric acid B (GABA-B) inhibitor and/or a combination thereof.

20. The pharmaceutical composition of claims 12 or 13 composition capable of reducing memory, perception and/or attention by reducing the supply of dopamine to the neocortex , said pharmaceutical composition comprises a drug selected from the group comprising a dopamine receptor inhibitor, a dopamine uptake enhancer, and/or a combination thereof.

21. The pharmaceutical composition of claims 12 or 13 capable of reducing memory, perception and/or attention by blocking the somatostatin receptor 2 neocortex, said pharmaceutical comprises a drag selected from the group comprising neuropeptide receptor blockers, somatostatin synthesis blockers, and/or a combination thereof.

22. Use of a composition of claims 12 to 21 in the preparation of a medicament for treating and/or preventing a Pervasive Developmental Disorder.