WO2009071725A2

WO2009071725A2 - Use of valproate for the treatment of fear and phobia in subjects with alzheimer's disease

Info

Publication number: WO2009071725A2
Application number: PCT/ES2008/000765
Authority: WO
Inventors: Carlos Alberto Saura Antolin; Catalina Casas Louzao; Lydia GIMÉNEZ LLORT; Judit ESPAÑA AGUSTÍ
Original assignee: Universitat Autónoma De Barcelona
Priority date: 2007-12-07
Filing date: 2008-12-05
Publication date: 2009-06-11
Also published as: WO2009071725A3; ES2325824A1; ES2325824B1

Abstract

The invention relates to the use of valproate for the production of a drug for the treatment of fear and phobia in a subject with Alzheimer's disease. The invention also relates to a method for the treatment of fear and phobia in a subject with Alzheimer's disease, whereby the subject is administered an effective quantity of valproate.

Description

USE OF VALPROATE FOR THE TREATMENT OF FEAR AND FOBIA IN SUBJECTS WITH ALZHEIMER'S DISEASE

Field of the Invention

The present invention relates to the use of valproate for the manufacture of a medicament for the treatment of fear and phobia in subjects suffering from Alzheimer's disease.

Background of the invention

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and neuropsychiatric symptoms, such as delusions, agitation, apathy, aggressiveness, anxiety and phobia. The AD represents today one of the main global public concerns since it has become the most common form of insane disease that appears at a late stage of life, which requires a huge amount of human, medical and economic resources. The true burden of this disease for societies is only emerging now, considering that life expectancy is growing steadily and the increasing number of people reaching an old age (Alzheimer's Disease: A Physician's Guide to Practical Management. Brigitte Zoeller ed. Lit. Richter, Ralph W. Richter).

Fear and anxiety are normal experiences that healthy subjects can experience, but when these feelings become unbearable and disproportionate to the situation, they can represent an anxiety disorder. The feeling of apprehension and fear appear, in anxiety, along with physical symptoms, such as palpitations, sweating and feeling of stress Unlike the brief and benign anxiety caused by a stressful event, such as the presentation of a business or a first date, anxiety disorders are chronic, unforgiving, and can progressively get worse if they are not treated. Anxiety disorders affect 35-50% of people with mild cognitive deficits and AD (Ferretti et al., 2001; Moran et al., 2004).

Anxiety disorders include phobias, such as social phobia (or social anxiety disorder) and specific phobias, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, and generalized anxiety disorder.

Phobias are the most common type of anxiety disorder. Social phobias involve fears of performing certain behaviors before other people, such as public speaking. Specific phobias are characterized by fears and the evasion of specific objects or situations, as well as the recognition that fears are not justified. The phobias can develop in different ways: they can be learned by directly having a negative experience with an object or situation; or indirectly when observing someone showing fear or hearing repeated warnings. Biological factors, such as having a sympathetic nervous system that is too reactive and slow to get used to false signs of fear, can increase people's likelihood of developing an anxiety disorder. Once people have developed a phobia, their fear is maintained by a number of factors, including the relief they feel from avoiding the feared object or situation, and the cognitive prejudices that cause them to focus on threats and interpret situations in the way more threatening Valproic acid is an antiepileptic compound described in the Physician Desk Reference, 52nd ed. , page 421 (1998). After oral ingestion in the gastrointestinal tract, the acid group dissociates to form a carboxylate group, that is, a valproate ion. It is also used as sodium valproate, which is described in detail in The Merck Index, 12th ed. , page 1691 (1996). You can find more detailed descriptions in the Physicians' Desk Reference, 52 Ed. , page 417 (1998). Documents have also been published that study the effect of valproate in the treatment of anxiety disorder with variable results.

On the one hand, it has been observed that this compound reduces the symptoms of anxiety and psychosis that are induced after re-exposure to life-threatening experiences in panic disorders and post-traumatic stress (PTSD). (Davis et al., 2000. Comprehensive review of the psychiatric uses of valproate. J Clin Psychopharmacol 20, 1S-17S). Primeau F. et al. (Can J Psychiatry. 1990 Apr; 35 (3): 248-50) also support the hypothesis that valproate is useful in the treatment of panic disorders. The same application of valproate in the treatment of panic disorder has also been described by Keck PE et al. (Biol Psychiatry. 1993 Apr 1; 33 (7): 542-6).

In addition, the effectiveness of valproate in social phobia was described in Kinrys G et al. (Int Clin Psychopharmacol. 2003 May; 18 (3): 169-72); and in Townsend MH et al. (Compr Psychiatry. 2005 Sep-Oct; 46 (5): 368-70), where patients with psychotic disorders treated with valproate had anxiety, tension and excitation results of the BPRS (Brief Psychiatric Rating Scale) scale, as well as the total results of agitation of BPRS. On the other hand, the effectiveness of valproate in the treatment of different psychiatric symptoms in different types of patients has been qualified in the following studies. Woodman et al. (J Clin Psychiatry. 1994 Apr; 55 (4): 134-6) suggest that valproic acid may be effective for the treatment of panic disorder in patients diagnosed with primary DSM-II-R (Diagnostic and Statistical Manual of Mental Disorders , 3rd ed. By the American Psychiatric Association) of panic disorder; while the effect on phobic evasion is not significant.

The comprehensive review of the psychiatric uses of valproate by Davis et al. (J Clin Psychopharmacol. 2000 Feb; 20 (1 Suppl 1): 1S-17S) concludes that valproate shows the most promising efficacy in the treatment of mood and anxiety disorders, with a possible efficacy in the treatment of agitation and impulsive aggression, and a less convincing therapeutic response in the treatment of psychosis and alcohol withdrawal or dependence.

Bowden CL. (Expert Rev Neurother. 2007 Jan; 7 (1): 9-16) indicates a more defined profile of the usefulness of sodium valproate in bipolar disorders, mainly for basic characteristics d4 manic symptomatology (for example, impulsivity, hyperactivity and irritability) , with little evidence of benefits for anxiety or psychosis. Valproate seems effective in other behavioral disorders included in the domains of bipolar disorders, such as schizophrenia.

However, and to our knowledge, none of these previous studies investigated the use of valproate in reducing fear and phobia in patients with AD. Only preliminary pilot studies suggest that valproate improves the rate of agitation and reduces aggressive behavior in demented patients (Porsteinsson et al., 2003; Sival et al., 2004). Needless to say, with Alzheimer's disease being the most common cause of dementia, many other diseases can also cause dementia.

It is pertinent to note that the selection of subjects with AD as a specific group of patients to study for the treatment of fear and phobia is supported by the fact that the brain regions involved in the irruption of anxiety in patients with AD compared to Other types of patients are different (see, for example, J Neuropsychiatry Clin Neurosci. 2006 FaIl, 18 (4): 521-8; Neurobiology of mental illness Charney and Nestler, Oxford University Press, Table 42: 1, page 655; and J Neuropsychiatry Clin Neurosci. 2006 FaIl, 18 (4): 521-8).

In an effort to elucidate the neural and pathological mechanisms that lead to anxiety and fear in AD, it has been surprisingly observed that valproate effectively reduces conditioned and unconditioned fear responses in transgenic mouse models of the disease of Alzheimer, demonstrating that a short-term treatment with valproate has anxiolytic effects in subjects with AD. To a large extent, it has been observed that while valproate significantly reduced fear responses in subjects with AD, it had no effect on control subjects subjected to the same experimental and stimulation conditions, thus finding and unexpectedly a new therapeutic utility for valproate based on the subgroup of patients. Summary Description of the Invention

The present invention relates to the use of valproate for the manufacture of a medicament for the treatment of fear or phobia in a subject with Alzheimer's disease. The present invention also relates to a method for the treatment of fear or phobia in a subject with Alzheimer's disease, which comprises administering to said subject with Alzheimer's disease an effective amount of valproate.

In one embodiment, the present invention relates to the use of valproate for the manufacture of a medicament for the treatment of fear associated with anxiety disorder in a subject with Alzheimer's disease.

In another embodiment, the present invention relates to the use of valproate for the manufacture of a medicament for the treatment of phobia associated with an anxiety disorder in a subject with Alzheimer's disease, wherein said phobia is a specific phobia or A social phobia.

In another embodiment, the present invention relates to the previous uses of valproate, in which the subject with Alzheimer's disease is in an early stage of said disease of

Alzheimer's

In another embodiment, the present invention relates to the previous uses of valproate in which the treatment of fear or phobia comprises the prophylaxis of said medium or phobia.

In another embodiment, the present invention relates to a dosage schedule in its use for the manufacture of a medicament for the treatment of fear or phobia in a subject with Alzheimer's disease, in particular for the treatment of fear. conditioned or unconditioned, in which said treatment comprises the daily administration of effective or therapeutically optimal doses of 1 to 40 mg / kg of total body weight of valproate, approximately 20 to 30 mg / kg, 10 to 20 mg / kg , or about 16, 17, 18 or 19 mg / kg.

Description of the figures

Figure 1. Increased neophobia in APP and 3xTg-AD transgenic mice

The experimental design of the behavior consisted of the manipulation for three days before a training session of the neophobia followed by the context-based fear test (CFC) and the aquatic labyrinth of

Morris (MWM) as described in Materials and

Procedures (A). APPi _n d (B) transgenic mice,

APP _Sw , i _nd (C), 3xTg-AD (D) and wild-type mice

(WT) of the same bait (n = 8-9) of 6 months of age were tested in a new medium of bright light. Freezing behavior was assessed as a measure of neophobia for 3 minutes. All groups show similar levels of freezing during the first minute (Tl). However, APPi _nd , APP _Sw , m _d , and 3xTg-AD mice show a significant increase in freezing during the second (T2) and third (T3) minute compared to control mice (APP _Ind ; F ( 1.42) = 10.2, p <0.003; APP _Sw , i _nd : F (1.48) = 13.7, p <0.0005; 3xTg-AD: F (1, 42) = 10.1; p <0.003). * p <0.05, ** p <0.01.

Figure 2. Altered fear responses in the context-conditioned fear test in APP and 3xTg-AD mice. Freezing responses of APPi _nd (A), APP _Sw , md (B), 3xTg-AD (C) and their control (WT) transgenic mice of the same 6-month bait were evaluated. life in the fear test conditioned to the context. The three transgenic groups show increasing levels of immobilization immediately after receiving an electric shock (Immediate). APPi _nd (n = 9) and control (n = 9) mice show similar levels of immobilization (approximately 40%) at 24 hours, while APP _Sw , m _d (n = 9) and 3xTg- mice AD (n = 8) show significantly higher levels of immobilization (-60-80%). * p <0.05, ** p <0.005. Figure 3. Spatial memory deficits in APP and 3xTg-AD mice of 6 months of age.

Morris's water maze consisted of a 5-day training in the hidden platform version of the task (4 tests per day) followed by a test (1 minute) on the last day. (A, D, G) The analysis of the distances traveled (feed length) reveals statistically significant differences between the APP mice _Ind (n = 7) (A), APP _Sw , _Ind (n = 9) (D) and 3xTg -AD (n = 8) (G) and the corresponding 6-month-old non-transgenic control mice (p <0.001). These data indicate that APP _Ind , APPa ,, ^ and 3xTg-AD transgenic mice showed learning deficits during MWM training. (B, E, H). Percentage of time spent in each quadrant during the test performed 2 hours after the last day in the Morris water maze in transgenic mice

APPm _d (B), APP _Sw , _Ind (E) and 3xTg-AD (H). In the tests all control groups showed a significantly higher level of occupancy of the target quadrant (T) compared to other quadrants (p <0.001), while the transgenic mice APPi _nd , APP _Sw , in _d and

3xTg-AD spent less time searching for the virtual target platform (p> 0.05). (C, F, I). Number of crosses at each point of the virtual platform during the test in the aquatic labyrinth of Morís in transgenic mice APPm _d (C), APP _Sw , i _nd (F) and 3xTg-AD (I). The APPmd / APP _Sw , ind and 3xTg-AD transgenic mice cross the target platform less frequently than control mice. The number of crossings of the target platform with respect to the crossings on the other platforms was significantly higher in control mice (p <0.0001) compared to APPi _{n <} a, APP _Sw , ind Y 3xTg-AD mice. These data indicate that the 6-month APP and 3xTg-AD mice show a spatial memory deficit. OP, opposite quadrant; AR, adjacent right; T, target quadrant; AL, adjacent left. * p <0.005, ** p <0.001

Figure 4. Accumulation of intraneuronal Aβ in the tonsil of transgenic APP and 3xTg-AD mice. (A, B, C) Nissl staining of coronal sections shows cortical and tonsil structures (boxes) in APPind / APP _Sw , ind and 3xTg-AD mice, 6 months old. Scale bar: 200 μm

(D, E, F) Enlarged images of adjacent brain sections of the left panels (A, B and C) were stained with an anti-Aβ antibody (6ElO) to reveal the presence of intraneuronal Aβ in the tonsil of mice

APPmd / APP _Sw , ind and 3xTg-AD. Scale bar: 50 μm

(G, H, I) Enlarged images of the basolateral tonsil shown on the left panels (D, E, F). The images show the accumulation of Aβ as small points in the soma and projections of some neurons (arrow), but it was absent in large pyramidal neurons (arrowhead) in APP _Ind and APP _{SW / Ind} mice. However, in 3xTg-AD mice, intraneuronal Aβ was mainly detected in the soma of pyramidal neurons

(arrow), while it was absent in small interneurons of the basolateral tonsil (arrowhead). Image captured at 40 x. Scale bar: 10 μm (J, K, L) Coronal brain sections of non-transgenic control mice of the same bait were stained with anti-Aβ antibody (6E10). Microscope analysis revealed the absence of intraneuronal Aβ that stains tonsil neurons in non-transgenic control mice. Scale bar: 10 μm (M, N) The tonsil sections of transgenic APP _Sw , i _nd and 3xTg-AD mice were stained with anti-Aβ40 antibody (2G3). Microscope analysis revealed the presence of intraneuronal Aβ40 in these transgenic mice. Scale bar: 10 μm Figure 5. Effects of valproate on immobilization behavior in APP _Ind mice after exposure to mild and aversive stimuli.

(A) The percentage of immobilization time in mice 6 months of age treated with vehicle or valproate was determined in the bright light neophobia test. Vehicle or valproate (200 mg / kg) was administered for three consecutive days and 30 minutes before training. APPm _d mice (n = 7) treated with vehicle showed an increase in immobilization levels compared to control mice (n = 8) treated with vehicle. Daily administrations of valproate significantly reduced immobilization of APP _Ind mice (n = 10) compared to APPi _nd mice or vehicle treated control mice. The data represent the average percentage of immobilization ± SEM * p <0.02, ** p <0.005.

(B) Control mice (WT) and APPm _d treated with vehicle and valproate were tested in a fear task conditioned to an acoustic stimulus. Vehicle-treated _{Ind Ind} mice showed an increase in immobilization levels immediately after electric shock compared to control mice. Valproate treatment significantly reduced the immediate immobilization of APP _Ind mice. No significant differences were observed between vehicle-treated control mice and control mice. APPm _d treated with valproate. The data represent the average percentage of immobilization ± SEM * p <0.02.

(C) In the task of fear conditioned to an acoustic stimulus, APPma mice treated with vehicle were more immobilized for 24 hours after training during the pre-conditioned stimulus (pre-

CS) compared to control mice. Valproate significantly reduced the immobilization response of control and APPi _nd mice during the pre CS stimulus. When exposed to CS, the immobilization time of valproate-treated APPm _d mice decreased significantly with respect to vehicle-treated APPmd and control mice. * p <0.05, ** p <0.03.

Description of the invention

The present invention relates to the use of valproate for the manufacture of a medicament for the treatment of fear or phobia in a subject with Alzheimer's disease. The present invention also relates to valproate for use in the treatment of fear or phobia in a subject with Alzheimer's disease. These uses of valproate include the administration to a subject with Alzheimer's disease of an amount effective to combat fear or phobia. The present invention also relates to a method of treating fear or phobia in a subject with Alzheimer's disease, which comprises administering to said subject with Alzheimer's disease an effective amount of valproate. Valproate refers to the ionic form of valproic acid (2-propylpentanoic acid) in water. Valproic acid has the following structure:

The valproate ion absorbed is absorbed and produces the therapeutic effect. As such, any reference to "valproate" or "valproate compound" should be construed to include a compound that dissociates in the gastrointestinal tract, or in the in vitro dissolution medium, to produce a valproate ion. As such, valproate includes, but is not limited to, valproic acid, any of the various salts of valproic acid described below, any of the prodrugs of valproic acid described below, any hydrated form of the compounds mentioned above, as well as Any combination thereof. Valproic acid is commercially available from Abbott Laboratories of abbot Park, Hl, USA. The procedures for their synthesis are described in Oberreit, Ber. 29, 1998 (1896) and Keil, Z. Physiol. Chem. 282, 137 (1947), the content of which is incorporated into the present invention by reference.

Suitable pharmaceutically acceptable salts of basic valproic acid include, but are not limited to, cations based on alkaline or alkaline earth metals, such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and cations of quaternary amine and ammonium, which include ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, and the like. Other representative organic amines useful for salt formation by base addition include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

Sodium valproate is a popular salt of valproic acid. The procedures for the preparation of sodium valproate can be found in US 4,988,731 and US 5,212,326, the contents of which are incorporated herein by reference. Like valproic acid, sodium valproate also dissociates in the gastrointestinal tract to form a valproate ion.

Compounds comprising combinations of sodium valproate and valproic acid (divalproex sodium, for example) are bioavailable and therapeutically active sources of valproate. The non-stoichiometric compound known as divalproex sodium is described in US Patent 4,988,731.

In addition to the compounds mentioned above, one skilled in the art would readily understand that the carboxylic group of the valproate compound can be functionalized in different ways. This includes the formation of compounds that are readily metabolized in vivo to produce valproate, such as valproate amide (valproimide), as well as other pharmaceutically acceptable amides and esters of the acid (i.e., prodrugs).

"Pharmaceutically acceptable ester" refers to esters that maintain, after hydrolysis of the ester bond, the biological efficacy and properties of the carboxylic acid and are not biologically or in any case undesirable. For a description of pharmaceutically acceptable esters as prodrugs, see Bundgaard, E., ed. , (1985) Design of Prodrugs, Elsevier Science Publishers, Amsterdam. These esters are usually formed from the corresponding carboxylic acid and an alcohol. In general, the formation of esters can be performed by conventional synthetic techniques (see, for example, March Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York p. 1157 (1985) and references cited therein, and Mark et al. Encyclopedia of Chemical Technology, John Wiley & Sons, New York (1980)). The alcoholic component of the ester will generally comprise (i) a C2-1 ₂ aliphatic linear or branched alcohol with one, two or three double bonds; or (ii) a C ₇ - I ₂ aromatic or heteroaromatic alcohol. The present invention also contemplates the use of those compositions that are esters, as described in the present invention, and at the same time are pharmaceutically acceptable salts thereof.

"Pharmaceutically acceptable amide" refers to those amides that maintain, after hydrolysis of the amide bond, the biological efficacy and properties of the carboxylic acid and are not biologically or in any case undesirable. For a description of pharmaceutically acceptable amides as prodrugs, see Bundgaard, E., ed. , (1985) Design of Prodrugs, Elsevier Science Publishers, Amsterdam.

These amides are usually formed from the corresponding carboxylic acid and an amine. In general, amide formation can be performed by conventional synthetic techniques (see, for example,

March Advanced Organic Chemistry, 3rd Ed., John Wiley &

Sons, New York p. 1157 (1985) and Mark et al. Encyclopedia of Chemical Technology, John Wiley & Sons, New York

(1980)). The present invention also contemplates the use of those compositions that are amides, as described in the present invention, and at the same time are pharmaceutically acceptable salts thereof.

In one embodiment, the present invention relates to the use of valproate for manufacturing. of a medication useful for the treatment of fear associated with an anxiety disorder in a subject with Alzheimer's disease. In one embodiment, fear is selected from conditioned and unconditioned fear. In another embodiment, the present invention relates to the use of valproate for the manufacture of a medicament useful for the treatment of phobia associated with an anxiety disorder in a subject with Alzheimer's disease. In one embodiment, the phobia is selected between specific phobia and social phobia.

The term "fear" in the present invention refers to a disturbing emotion caused by a sign of real danger - be it damage, pain and the like - being said danger caused by an imaginary or real situation. A fear response appears by exposure to an innate or unconditional danger signal - or stimulus. This innate / unconditional danger signal or stimulus, which is a reflection of the fear of dying, is strongly connected in the brain and includes: the unknown (which reflects the fear of dying in new circumstances), heights (reflecting the fear of die from a fall), enclosed spaces (reflecting the fear of dying when trapped), open spaces (reflecting the fear of dying from not having a place to hide), repellent bugs (reflecting the fear of dying from predators of the earth) and something that comes out of our visual field (which reflects the fear of dying from air predators)

In anxiety, mental and physical tension is very similar to the symptoms experienced with fear, but with an important difference. With anxiety, there is usually nothing that is really happening right here or there to trigger tension. The tension comes from anticipation of future danger or something bad that could happen - there is no danger that is happening now.

The term "phobia" in the present invention refers to a persistent, irrational and excessive fear of objects or situations. It is an inappropriate fear response that does not provide an evolutionary advantage and causes physiological changes that cause restlessness and dysfunction. The phobias are learned and triggered by various objects or situations, that is, conditioning stimuli (CS), such as bugs, colors, numbers, light, darkness, bridges, tunnels, elevators, airplanes, to name a few, which are not associated I get an imminent danger.

As such, phobias are fundamentally different from fear, in the fact that the latter is triggered by a stimulus of innate or unconditioned fear (US), and instead, they carry an imminent real danger associated with them.

An innate or unconditioned stimulus that leads to a fear response in the presence of another object or situation - that is, the conditioning stimulus, sets the stage for the generation of the phobia. For example, when traveling on a bridge (which is the CS); Someone could look down and see the height (which is the US). It is the height that causes a person to become afraid of dying from the fall. This appears at the subconscious level; one is not fully aware of why he is afraid, however, he consciously realizes that he is on a bridge, if the neural landscape is "primed", the bridge is then associated with the fear response. Thus, when an image of a bridge is brought to consciousness, a fear response occurs.

The fear system has been systematically explored using the fear test Pavlov conditioning (Fanselow and LeDOux, Neuron, VoI. 23, 229-232, June 1999). In this laboratory model for the study of phobias, fear responses, and their treatments, animals learn to fear a previously neutral stimulus (i.e. the conditioned stimulus: CS) due to its association with an aversive stimulus (i.e., the unconditioned stimulus: US), such as an electric shock in the foot. Conditional fear requires learning and produces stereotypical immobilization behavior, a period of immobility in surveillance that can be measured and used for research purposes. In this paradigm, immobilization responses can be triggered by two different types of CS, acoustic tone and context, each of which requires different neuroanatomic substrates (Kim, et al., 1992; Phillips, et al., 1992) . The conditioning to an acoustic stimulus, in which the CS is a tone, depends on the amygdala, and the contextual conditioning, in which the CS is a new environment, depends on the hippocampus and the amygdala. After . of several CS pairings with the discharge, the animal reacts with fear to the CS, just as the bridge (CS) was capable of producing fear. It is the anticipation of the discharge (the CS) that produces fear, not the discharge itself. . (The Neurobiological Basis of Peripheral Sensory Stimulation for Modulation of Emotional Response by Ronald A. Ruden, MD, Ph.D. March, 2005).

Another embodiment of the present invention relates to the previous uses of valproate, in which the subject with Alzheimer's disease is at an early stage of uncomplicated onset of AD, at an early onset with illusions, at an early onset with a depressive mood, in a late start not complicated, in a late start with illusions, in a late start with a humor depressant. Particularly, the present invention relates to the aforementioned uses of valproate, in which the subject with Alzheimer's disease is at an early stage of said Alzheimer's disease.

A further embodiment of the present invention relates to the previous uses of valproate in which the treatment of fear or phobia comprises the prophylaxis of said fear or phobia. The term "treatment" of fear or phobia refers to, among others, the reduction or relief of one or more symptoms in a subject, the prevention of one or more symptoms of worsening or progression, induction for recovery or improvement of prognosis. , and / or prevention of the disease in an individual who is free of it, as well as the slowdown or reduction of the progression of the existing disease. For a given subject, the improvement of a symptom, its worsening, regression, or progression can be determined by an objective or subjective measurement. The efficacy of the treatment can be measured as an improvement in morbidity or mortality (for example, the lengthening of the survival curve for a selected population). Prophylactic procedures (for example, prevention or reduction of the incidence of relapse) are also considered as treatment and are a particular embodiment of the present invention.

In another embodiment, the present invention relates to the aforementioned uses of valproate, in which the subject to be treated can be any animal or human. In particular, disease models in mammals, especially humans, and rodent or primate models can be treated. Thus, both veterinary and medical procedures are contemplated. In a further embodiment, the present invention relates to the aforementioned uses of valproate for the manufacture of a medicament for the treatment of fear or phobia in a subject with Alzheimer's disease, where valproate is formulated in a pharmaceutical composition comprising a therapeutically effective amount of valproate, and a pharmaceutically acceptable carrier. A therapeutically acceptable amount in this context is an amount sufficient to act prophylactically against, to stabilize or reduce the symptoms of fear and phobia in subjects with AD.

Therefore, valproate can be formulated in various pharmaceutical forms for administration purposes. As appropriate compositions, all compositions commonly used for the systematic administration of drugs can be cited. To prepare the pharmaceutical compositions of the present invention, an effective amount of valproate as an active ingredient is combined in intimate mixing with a pharmaceutically acceptable carrier, which can take a wide variety of forms depending on the manner of preparation desired for administration. These pharmaceutical compositions are desirable in the form of unit doses suitable, particularly, for administration by oral, rectal, transdermal, inhalation, or parenteral route (ie, subcutaneous, intravenous, intramucuslar or intraperitoneal). Valproate can be administered by the oral route in solid dosage forms, such as tablets, capsules and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. The pharmaceutical compositions of the present invention can also be determined parenterally, in sterile liquid dosage forms. In the preparation of the compositions in oral dosage form, any of the usual pharmaceutical means such as, for example, water, glycols, oils, alcohols, and the like can be used in the case of oral liquid preparations, such as suspensions, syrups , elixirs, emulsions and solutions; or solid carriers, such as starch, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, tablets, capsules and tablets. Due to their ease in administration, tablets and capsules represent the most advantageous oral unit dose forms, in which case solid pharmaceutical carriers are obviously used.

Valproate can also be administered in controlled, sustained or slow-release oral dosage forms, such as those described in US2005276850, EP1219295, EP1216704 or US5589191.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous solutions or suspensions for sterile isotonic injection. Injectable solutions, for example, can be prepared so that the carrier comprises saline solution, glucose solution or a mixture of saline solution and glucose solution. Injectable suspensions can also be prepared so that appropriate liquid carriers, suspending agents and the like can be used. Also included are preparations in solid form that are intended to be converted, shortly before use, into liquid form preparations. In compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, whose additives do not introduce a significant detrimental effect. on the skin. These suitable additives may be antioxidants, preservatives, stabilizing agents, emulsifiers, salts to influence osmotic pressure and / or buffer substances. It is especially advantageous to formulate the pharmaceutical compositions mentioned above in unit dosage form to facilitate administration and dosage uniformity. The unit dosage form as used in the present invention refers to physically discrete units suitable as unit doses, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including shaved or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and multiple segregates thereof. In a further embodiment, the present invention relates to a dosage regimen of valproate in its use for the manufacture of a medicament for the treatment of fear or phobia in a subject with Alzheimer's disease. In one embodiment, the valproate dosage schedule provided in the present invention is particularly useful for the treatment of conditioned or unconditioned fear. A daily oral dosage for the treatment of fear or phobia, in particular conditioned or unconditioned fear, comprises the daily administration of effective or therapeutically optimal doses of 1 to 40 mg / kg of total body weight of valproate, from about 20 to 30 mg / kg, 10 to 20 mg / kg, or approximately 16, 17, 18 or 19 mg / kg. The duration of administration of sodium valproate in the treatment of conditioned fear and Unconditioned in a subject with Alzheimer's disease can vary from 1 to 10 days, preferably from 2 to 7 days, more preferably for 4 days. As such, the present invention provides an acute treatment of fear or phobia with valproate in a subject with Alzheimer's disease.

Valproate can be used as a single therapeutic agent or in combination with other therapeutic agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are administered simultaneously or sequentially at different times, or the therapeutic agents can be administered as a single composition. The combination therapy or therapy in the definition of the use of valproate and another pharmaceutical agent, is intended to include the administration of each drug in a sequential manner in a pattern that will provide beneficial effects of the combination of drugs, and it is also intended that comprise co-administration of these drugs in a substantially simultaneous manner, such as in a single dosage form having a fixed proportion of these active drugs or in separate multiple dosage forms for each drug. Specifically, administration of valproate may occur in conjunction with additional therapies known to those skilled in the art in the treatment of AD, such as, for example, galantamine, donepezil, rivastigmine, memantine and the like.

Accordingly, the present invention relates to the use of valproate for the manufacture of a medicament useful for the treatment of fear or phobia in a subject with AD, wherein said medicament is used in a combination therapy, said said comprising. Combination therapy preferably valproate and an anti-AD drug, such as galantamine, donepezil, rivastigmine, memantine and the like.

The present invention is illustrated by the following examples, which should not be construed as limiting.

Examples

Materials and procedures Transgenic mice

APP _Ind transgenic mice (H6 line) and APP _Sw , ind (J9 line) (C57BL / 6) expressing the mutant human APP695 isoform comprising the FAD (V717F) or Swedish (K670N / M671L) / Indiana Indiana mutations (V717F) under the expression of the PDGFβ neuronal promoter have been previously described (Mucke et al., 2000). Mice were obtained by crossing APP _Ind or APP _Sw , _Ind heterozygous with non-transgenic mice (WT). OxTg-Ad triple-transgenic mice; 129 / C57BL / 6) comprising mutations in PSl (M146V) and expressing mutant human APP (KM670 / 671NL) and Tau (P301L) under the control of the Thyl promoter .2 were previously described (Oddo et al., 2003) . Homozygous and non-transgenic control (WT) 3xTg-AD mice were used in this study. Unless otherwise indicated, the APP _{In <} a, APP _w , m _d and 3xTg-AD transgenic mice used in this study were male and of the same age. Mice were stored and maintained in a 12-hour light / dark cycle and were given ad limitum access to food and water.

Fear conditioned on context and acoustic tone

The mice used for all behavioral tests were of the same bait and were 6-7 months old. The conditioned fear test was performed as described above with some modifications (Saura et al., 2005). The mice were handled individually for 3 minutes daily for 3 days before the behavioral tests. For contextual fear conditioning, the mice were placed in a new training chamber (15.9 x 14 x 12.7 cm) equipped with a white homemade light and a stainless steel grid floor (Med Associates Inc, St. Albans, VT). White light illumination had been previously described to have angiogenic effects in rodents (Walter and Davis, 1997a). The mice were placed in the chamber for 3 minutes and the spontaneous immobilization responses of the mice were videotaped and used as a measure of neophobia behavior. Immediately afterwards, the mice received an electric shock on the foot (unconditioned stimulus: US; 1 s / 1 mA). After discharge, the mice were left in the chamber for 2 minutes (immediate immobilization) and returned to their cages. Fear conditioning was tested 24 hours after training for 4 minutes in the same conditioning chamber. The immobilization, which was defined as a complete cessation of all movement except for breathing, was assessed and analyzed automatically using the Video Freeze Software system (Med Associates, Inc.). In the fear conditioned to an acoustic stimulus, the mice were allowed to explore the conditioning training chamber for 3 minutes before the onset of the acoustic tone (conditioned stimulus: CS) (2,800 Hz and 80 dB; 30 s). A discharge was administered to the foot (0.8 mA, 2 s) at the end of the acoustic stimulus presentation and immobilization was measured for 2 minutes immediately after discharge (immediate immobilization). The immobilization behavior was examined 24 hours after training in a new environment before (pre-CS; 3 minutes) and during the presentation of the tone (Cs; 4 min). The new medium consisted of the modified conditioning chamber with a white Plexiglas floor and two black sheets on the walls. In addition, the lights in the room were changed to red lighting and a new smell was added. The immobilization responses were measured by the Video Freeze Software (Med Associates, Inc.).

Morris Water Maze The water maze test was performed in a circular pool (90 cm in diameter) that contained a hidden platform (6.5 cm in diameter) (Saura et al., 2004). For each test, the mice were placed in the pool at one of the four starting points in a pseudorandom order. Each mouse was given four daily tests (5 days) with a maximum test duration of 60 s. and an interval between tests of 15 minutes. The mice were allowed to find the submerged platform, otherwise they were manually guided to the platform and remained there for 10 seconds. After this, the mice were placed in a cage until the start of the next test. A 1-minute test was conducted 2 hours after training on day 5 to assess memory retention. The visible platform test was carried out in the same pool but without the visible guides and the platform was raised over the water and marked with a flag. Each mouse was subjected to four tests for each platform location, which moved to the four positions of the quadrant. The result of the hidden and visible versions of the aquatic labyrinth test was recorded on video and automatically analyzed using SMART software (PanLab S. L., Barcelona, Spain).

The behavioral test experimenters were blind to the mouse genotypes. Valproate treatments

APP _Ind transgenic mice and the same 6-month-old WT bait were treated (ip) with a vehicle (0.9% NaCl) or valproate (200 mg / kg; Sigma, St. Louis, MO) dissolved in Saline solution. Valproate is a saturated branched chain fatty acid that penetrates the blood-brain barrier. The drug was administered daily for 3 days and 30 minutes before the behavioral tests. On day 4 all groups trained in the fear test conditioned to an acoustic stimulus as described above. The dosage regimen of valproate of 200 mg / kg administered to rodents corresponding to 16 mg / kg of human body weight following the conversion methods described in "Science and Technology in animal protection and experimentation", McGraw-Hill, Madrid (2001 ), Zuñiga, J. M, Tur, JA, Milocco, S. N, and Piñeiro, R .; and in "Experimental procedures in pharmacology and toxicology", pp 489-512, Chapter 18, by Giráldez-Dávila, A and Romero-Vidal, A.

Immunohisto <iuymic and histology

The dissected brains were quickly fixed in 10% buffered formalin at 4 ° C for 2 hours before being paraffin embedded. Coronal or sagittal brain sections (5-10 μm) were deparaffinized in xylene, rehydrated and incubated with 3% hydrogen peroxide. Sections were incubated in 60% formic acid for 6 minutes to allow recovery of antigens, washed in Tris HCl (0.1 M) and incubated at 4 ° C overnight with anti-Aβ 6ElO monoclonal antibody ( 1: 1,000; Signet, Dedham, MA) or anti-Aβ40 C-terminal 2G3 (1: 1,000). Sections were incubated with secondary anti-mouse antibodies biotinylated (1: 200; Vector Laboratories, Burlingame, CA) and were developed using the avidin-biotin peroxidase reagent and the Vectastain Elite ABC kit (Vector Laboratories) (Saura et al., 2005). Sections 5 were incubated with a Harri formulation hematoxylin solution, rinsed in a Scott water solution and dehydrated in ethanol solutions. For double marking stains, dewaxed sections were pretreated with citrate solution

10 for the recovery of antigens (Biogenex, San Ramón, CA) and incubated overnight (4 ° C) with anti-Aβ 6E10 or CaMKIIa monoclonal antibodies (a-1, 1: 750; Santa Cruz Biotechnology) and antibodies polyclonal mouse VGAT (1: 200; Sigma), p-CaMKI (Thr

15 286) (1: 200; Santa Cruz Biotechnology), NMDARl (1: 200; Chemicon, Temecula, CA) or GABAAbeta 3 (1: 250; Abcam, Cambridge, UK). Sections were incubated at room temperature for 1 hour with secondary goat antibodies Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes,

20 Eugene, OR) and Hoescht (Saura et al., 2005). For Nissl staining, the dewaxed sections were incubated in cresyl violet solution for 12 minutes, stained in dilute acetic acid and dehydrated in ethanol solutions. The sections are

25 mounted on slides and analyzed with a Nikon Eclipse 9Oi microscope.

Statistic analysis

Statistical analysis was performed using a 30 or 1-tailed variance analysis (ANOVA). Comparisons between the groups were made using an ANOVA followed by a post hoc Scheffe test using the SuperANOVA program. Data were presented as mean ± SEM. For all tests, the differences with p <0.05 were considered significant.

Results Neophobia food in transgenic APP and 3x Tg-AD mice

Anxiety symptoms, such as fear and phobia, are characteristic neuropsychiatric features of AD that have been described in several lines of APP transgenic mice (Janus and Westaway, 2001). In order to study neophobic behavior and determine its association with neuropathological changes in AD, the innate (unlearned) immobility behavior associated with fear obtained through a new environment or context consisting of a light test chamber was analyzed first bright (see, figure IA for experimental design). This essay has previously been used as a paradigm to measure innate or unconditional fear in rodents (Walker and Davis,

1997a). At 6 months of age, transgenic mice

APPm _d and APP _{SW / Ind} and the corresponding control mice

Non-transgenic (WT) showed a time-dependent increase in immobilization responses in the new medium (Figures IB, C). The two-tailed ANOVA analysis revealed a significant main effect of the genotype (APP _Ind , F (1, 42) = 10.2, p <0.003; APP _Sw , _Ind , F (1, 48) = 13.7, p < 0.0005) and time (APPi _nd , F (2, 42) = 17, p <0.0001; APP _Sw , ind, F (2, 48) = 7.1, p <0.002), while the interaction Genotype x time was not significant (p = 0.007). Compared to control mice, total immobilization responses increased significantly in APP _Ind (~ 2-fold; p <0.0005) and APP _Sw , md (~ 4-fold; p <0.003) mice. The fear responses appear to be specific and reflect the neophobic behavior since locomotor activity

(speed) and exploratory motivation among mice

APPind / APP _Sw , md and control were similar in the Morris water maze (see below). To rule out the possibility that immobilization behavior in APP transgenic mice could be due to a specific strain (i.e., C57BL / 6), mice were examined

3xTg-AD (129 / C57BL / 6) at a similar age using our unconditional fear paradigm. Statistical analysis of the immobilization responses revealed significant differences between the groups (F (1, 42) = 10.1; p <0.003) and a time interval effect (F (2, 42) = 4.27; p <0.02) (Fig. ID). 3xTg-AD mice also showed a significant (~ 4-fold) increase in total freezing responses compared to control mice (p <0.003). These results are consistent with previous results that showed a temporary cessation of exploratory activity and an increase in neophobia in APP transgenic mice (Hsiao et al., 1995; Moechars et al., 1999).

APP and 3xTg-AD transgenic mice show altered immobilization responses in contextual conditioned fear Our previous results suggested an increase in unconditioned or innate fear in APPmd, APP _Sw , md and 3xTg-AD transgenic mice. To determine if similar fear responses were observed when mice were exposed to an aversive stimulus, the immobilization behavior related to fear was measured in the contextual conditioned fear test, a task that depends on the tonsil and the hippocampus (Phillips and LeDoux, 1992). In this task, the mice were allowed to explore a test chamber before releasing a single electric shock in the foot. The mice associate the neutral context (conditioned stimulus: CS) with the aversive event (unconditioned stimulus: US), so that when exposed to the same environment later, the animals show anticipated fear responses (immobilization). The immobilization behavior obtained immediately after the presentation of the foot discharge is a measure of the unconditional response, while the freezing obtained by the CS represents the conditioned response (Rosen, 2004). In comparison with the control mice of the same bait, the APP _Ind and APP _Sw mice, in _d showed an increase in immobilization levels immediately after discharge in the foot (Figures 2A, 2B). The two-tailed ANOVA analysis revealed a significant main effect of the genotype (APP _Ind , F (1, 28) = 5.6, p <

0.02; APP _Sw , _Ind , F (1, 32) = 12.6, p <0.001) and time

(APP _Ind , F (1, 28) = 32.7, p <0.0001; APP _Sw , _Ind , F (1, 32) =

13.5, p <0.001). Planned statistical comparisons revealed an increase in immediate immobilization responses in APP _Sw mice, ind (P <0.01) and an increase, but not significant, in APPi _nd mice (p = 0.07). Similarly, APP _Sw , i _nd mice showed a significant increase in immobilization compared to control mice at 24 hours (p <0.02), while APPm _d mice showed no statistical differences (p = 0, 14). Notably, 3xTg-AD mice also showed an increase in freezing levels both immediately and 24 hours after foot discharge (Figure 2C). The two-tailed ANOVA revealed a significant main effect of the genotype (F (1.28) = 102; p <0.0001), time (F (1.28) = 30.9; p <0.0001) and of the genotype x time interaction (F (1.28) = 4.3; p <0.05). Post hoc analysis revealed significant differences in immobilization responses between control mice and 3xTg-AD immediately and 24 hours. after training (p <0.0001). Together, these results indicate an increase in the responses of conditioned and unconditioned fear in APP and 3xTg-AD transgenic mice. 5

Spatial memory deficits in APP and 3xTg-AD transgenic mice

The absence of long-term memory deficits in the contextual conditioned fear test caused the

10 investigation of the behavior of our transgenic mice in a task dependent on the hippocampus. APP transgenic mice have been shown to show learning deficits and age-related spatial memory in the Morris water maze (Hsiao et al.,

15 1996; Saura et al., 2005; Westerman et al., 2002). Therefore, after contextual fear conditioning, APP and 3xTg-AD mice and respective bait control mice were tested in the Morris water maze. In the platform version

20 hidden from the task, the performance of APPmd, APP _Sw , ind and its non-transgenic controls improved significantly during training days (day 1 versus day 5, p <0.001) (figure 3A, D), suggesting that all The groups were able to learn the task. However the

Two-tailed ANOVA revealed a significant main effect of the APPi _nd genotype, F (1, 65) = 40, p <0.0001; APP ₃ W, md, F (1, 80) = 12, p <0.001) and training day (APP _Ind , F (4, 65) = 16.6, p <0.0001; APP _Sw , i _nd , F (4.80) = 22.2, p <0.0001). APP _Ind mice

30 and APP _Sw , ind showed significantly longer distances (p <0.0001) and latencies (p <0.0001; not shown) compared to controls, although their performance improved with extensive training (Figures 3A, D). APP _Ind and APP _Sw , m _d mice showed swimming speed

35 similar to that of the control groups (H6, p>0.05; J9, p > 0.05) excluding the presence of motor deficits in these mice. In the post-training test, the control mice showed a significantly higher occupation of the target quadrant in relation to other quadrants (p <0.001), while the APPmd and APP _Sw , ind mice showed no preference (p> 0.05 ) (Figures 3B, E). Notably, the number of crossings of the target platform by APPmd (1.9 ± 0.5) and APP _Sw , ind (2.9 ± 0.8) mice was significantly lower than the controls (4.6 ± 0.4 for APPi _nd and 4.8 ± 0.9 for APP _{SW /} i _nd ) (p <0.001; Figures 3C, F). Control mice showed a significantly higher number of crosses on the target platform in relation to other platforms (p <0.0001), while APPi _nd and APP _{SW; Ind} mice did not show a preference for the platform

(p> 0.05). The deficits in the spatial memory of APP mice seem to reflect a learning or acquisition deficit instead of being caused by motor or motivational alterations since no significant differences were found in latencies and swimming speed during the visible platform task (p> 0.05; not shown).

The learning and spatial memory of 3xTg-AD mice in the water labyrinth task were analyzed below. During training, 3xTg-AD mice showed significantly longer distances compared to control mice (F

(1.65) = 12.2, p <0.001) (Fig. 3G). The performance of the control mice improved significantly during training (day 1 versus day 5, p <0.02), although, 3xTg-AD mice acted very poorly with significantly longer distances (day 1 versus day 5, p <0 , 87). In post-training tests, control mice showed significantly higher occupancy of the target quadrant compared to others. quadrants (p> 0.0005), while 3xTg-AD mice showed no such preference (p> 0.05) (Figure 3H). The number of crosses on the mice's target platform

3x-Tg-AD (3.4 ± 0.7) was significantly lower than those of non-transgenic mice (5.1 ± 0.9; p <0.04).

In addition, the number of crossings by the target platform in relation to the other supposed platforms was significantly higher in control mice

(p <0.005), while 3xTg-AD mice did not show a platform preference (p <0.05) (Figure 31). The performance of the control and 3x-Tg-AD mice in the visible platform task was similar (data not shown), suggesting that the spatial memory deficits of 3xTg-AD mice were not due to sensory or motor alterations. Together, these results indicate a hippocampus-dependent spatial memory damaged in APP and 3xTg-AD transgenic mice that show an anxiety phenotype.

Accumulation of intracellular Aβ in the tonsil of transgenic APP and 3x-Tg-AD mice

The accumulation of soluble and oligomeric forms of intracellular Aβ has been observed in AD brains and transgenic mice (Gouras et al., 2000; Oakley et al., 2006; Oddo et al., 2003). The intracellular accumulation of Aβ precedes the deposition of amyloids and other neuropathological features typical of AD, such as inflammatory responses and dystrophic neurites. Long-term spatial and contextual memory deficits in 3xTg-AD mice were associated with the accumulation of intraneuronal Aβ in the hippocampus and tonsil (Billings et al., 2005), two regions severely affected by amyloid pathology and neurofibrillar structure pathology in AD (Hyman et al., 1990). Since the hippocampus and the amygdala are critical regions that receive and store spatial and behavioral learning information Emotional related fear and anxiety (Fanselow and LeDoux, 1999; Rosen and Donley, 2006), respectively, investigated whether the accumulation of Aβ in those regions was associated with emotional responses of fear and memory damage in APP mice and 3xTg-AD. Immunohistochemical analysis of the brain sections of the mice tested in the behavioral tests demonstrated the presence of intracellular Aβ, but not of amyloid plaques, in the hippocampus and tonsil of APP and 3xTg-AD transgenic mice (Figure 4 and non-data shown). Intracellular Aβ was detected in the regions

CAl and CA2 / CA3 of hippocampal formation in transgenic mice APPm _d , APP _Sw , i _nd and 3xTg-AD of 6 months of age

(data not revealed) . Interestingly, intraneuronal Aβ and Ab40 were detected mainly, but not all, in the tonsil basallateral complex neurons in APP _Ind , APP _Sw _ _Ind and 3xTg-AD mice, while appearing to be absent in other tonsil regions ( Figs. 4D, E, F). Thus, while Aβ and Ab40 accumulated primarily in small neurons in APPm _d and APPsw.ind mice / only large neurons were apparently labeled with Aβ or Ab40 in 3xTg-AD mice (Figs. 4G, H, I, M , N). Aβ immunoreactivity was absent in the cortex, hippocampus and tonsil of control mice, indicating a lack of Aβ accumulation in these mice (Figs. 4J, K, L). Similar to the cortex, the projection neurons of the basolateral tonsil are glutamatergic neurons of the pyramidal type, while the spine-sparse neurons are interneurons (GABA) non-pyramidal γ-aminobutyric acid (McDonald, 2003). To identify specific neurons affected by Aβ, a double immunolabel was performed in brain sections with an anti-Aβ antibody (6E10) and markers for pyramidal glutamatergic neurons (CaMKIIa and p-CaMKIlα) and interneurons (GABA). ) used previously in morphological studies in the amygdala

(McDonald and Mascagni, 1996; McDonald et al., 2002). Notably, the double immunolabeling of brain sections of APP _Ind and APP _Sw , ind revealed a strong colocalization of Aβ with V-GAT positive neurons, while Aβ was apparently absent in CaMKIIa positive neurons. In contrast, intraneuronal Aβ was primarily co-located with pyramidal neurons containing p-CaMKIlα but not with V-GAT positive neurons in the basolateral tonsil of 3xTg-AD mice. Human Aβ was not detected in tonsil neurons in non-transgenic control mice. The quantification analysis confirmed that Aβ accumulated mainly in the erotic interneurons (GABA) in the basolateral tonsil in APP _Ind / APP _Sw , i _n d transgenic mice (APP _Ind : 93.4 ± 2.1%; APP _{Sw Ind} : 94.1 ± 2.2%) while modestly accumulating in pyramidal excitatory neurons (APP _Ind : 4.5 ± 1.1%; APP _{Sw Ind} : 5.4 ± 1.7%). On the other hand, Aβ accumulated in pyramidal glutamatergic neurons (91.7 ± 2.0%) but not in interneurons (1.9 ± 0.5%) in the basolateral tonsil in 3xTg-AD mice. Together, these results indicate the accumulation of Aβ in cell-specific neurons in the tonsil of APPind / APP _Sw , ind and 3xTg-AD mice. In addition, these data suggest a possible mechanistic union between the accumulation of Aβ in tonsil neurons and the fear and anxiety responses altered in transgenic AD mice.

Valproate reduces conditioned and unconditioned fear in APPi _nd transgenic mice.

The inhibitory erratic innervation (GABA) of pyramidal excitatory neurons in the basolateral tonsil is critical to control the excitability of pyramidal cells during emotional behavior, and in pathological conditions, such as depression and anxiety (McDonald et al., 2002; Sah et al., 2003). The role of GABA dysfunction in anxiety has emerged widely due to the benefits of pharmacological enhancers of GABAA receptor function or the synthesis of GABA or inhibitors of GABA degradation or reuptake (Nemeroff, 2003). The accumulation of Aβ in GABAergic interneurons of the basolateral tonsil of APPm _d and APP _SW transgenic mice _{; Ind} suggested that Aβ could reduce GABAergic inhibitory synaptic entry

10 in pyramidal cells. To test this hypothesis, it was tested whether short-term treatment with valproate, an anxiolytic agent that acts by increasing GABA levels (Nemeroff, 2003), would reverse the neophobic behavior observed in APPm _d transgenic mice. He

Analysis of the immobilization responses of the groups treated with vehicle - and valproate - in the bright light test chamber revealed significant differences between the groups (F (3.26) = 3.7, p <0.02). In accordance with the previous results

20 (Figure 1), vehicle-treated _{Ind Ind} mice showed an increase in immobilization levels compared to vehicle-treated control mice (p <0.02) (Fig. 5A). Interestingly, the administration of valproate significantly reduced the

25 levels of immobilization in APP _Ind mice (p <0.05), which acted similarly to control mice treated with both vehicle and valproate (p> 0.05) (Figure 5A). This result strongly suggests that valproate reduces unconditioned fear in APP _Ind mice. For

30 examining whether valproate was also able to reverse the tonsil-dependent fear responses in APP _Ind mice, the effect of valproate on the fear test conditioned to an acoustic stimulus was evaluated, a task that depends on the amygdala (Phillips and LeDoux,

35 1992). During the fear test conditioned to a stimulus acoustically, a neutral conditioned stimulus (CS; tone) is paired with an aversive unconditional stimulus (US; electric shock in the foot), so that the CS will only produce a response of the learned or conditioned emotional fear (immobilization). In accordance with the results of contextual conditioned fear (Figure 2A), vehicle-treated APP _Ind mice showed an increase in immobilization responses immediately after electric shock compared to vehicle-treated control mice (Figure 5B)

(p <0.02). Valproate did not affect immobilization levels in control mice (p> 0.05 versus vehicle), but significantly decreased immobilization levels in APP _Ind mice (p <0.02 versus vehicle), which were similar to those in mice Control treated with vehicle and valproate (p> 0.05) (Figure 5B). During pre-CS stimuli at 24 hours, immobilization responses of APP _Ind mice (32.4 ± 2.9%) increased slightly but not significantly compared to vehicle-treated control mice.

(21.4 ± 4.1%) (Figure 5C). Notably, valproate significantly reduced the immobilization responses of APP _Ind mice during pre-CS (p <0.02), while having no effect on control mice (p = 0.24). Notably, immobilization responses of APP _Ind mice treated with valproate were indistinguishable from control mice treated with vehicle and valproate (p> 0.05). In the presence of CS, vehicle-treated control and APP _Ind mice showed similar levels of immobilization (p = 0.27). However, while valproate did not affect immobilization in control mice (p = 0.36 versus vehicle), it did significantly reduce immobilization levels of APPm _d mice (p <0.03 versus vehicle) (Figure 5C). These results show that the Valproate treatment effectively reduces the responses of conditioned and unconditioned fear in APP transgenic mice, thus demonstrating that short-term treatment with valproate has anxiolytic effects on AD.

Claims

1.- Use of valproate for the manufacture of a medication for the treatment of fear or phobia 5 in a subject with Alzheimer's disease.

2. Use according to claim 1, wherein the fear or phobia are associated with an anxiety disorder.

3. Use according to claim 1, wherein the fear is selected between conditioned or unconditioned fear.

4. Use according to claim 1, wherein the phobia is selected from specific phobia and social phobia.

5. Use according to any of claims 15 1-4, wherein the subject with Alzheimer's disease is at an early stage of said disease of

Alzheimer's

6. Use according to any of the claims

1-5, in which the treatment of fear or phobia 20 comprises the prophylaxis of said fear or phobia.

7. Use according to any of the claims

1-6, in which the subject is a mammal.

8. Use according to claim 7, wherein the mammal is a human being. 25

9. Use according to claims 1 or 3, wherein the valproate is administered in a dosage schedule of 1 to 40 mg / kg / day for 2 to 7 days.

10.- Valproate for use in the treatment of fear or phobia in a subject with Alzheimer's disease. 30 11.- Process of treatment of fear or phobia, in a subject with Alzheimer's disease, which comprises administering to said subject with Alzheimer's disease an effective amount of valproate.