WO2010034960A1

WO2010034960A1 - Non-human animals having psychiatric disorders, method for obtaining same, and use thereof for drug screening

Info

Publication number: WO2010034960A1
Application number: PCT/FR2009/051853
Authority: WO
Inventors: Marc Merten
Original assignee: Universite Henri Poincare Nancy 1; Centre Hospitalier Et Universitaire De Nancy (Chu)
Priority date: 2008-09-29
Filing date: 2009-09-29
Publication date: 2010-04-01
Also published as: EP2353016A1; FR2936527A1

Abstract

The invention relates to non-human animals having invasive developmental disorders and/or psychiatric disorders, to the method for obtaining same, and to the use thereof for drug screening capable of substantially preventing, reducing, or eliminating the invasive developmental disorders and/or psychiatric disorders.

Description

Non-human animals with psychiatric disorders, their method of obtaining and their use for screening drugs.

The present invention relates to non-human animals with pervasive developmental disorders and / or psychiatric disorders, their method of obtaining and their use for the screening of drugs capable of substantially preventing, reducing or canceling invasive development and / or psychiatric disorders.

Autism is a pathology characterized by withdrawal and loss of social relationships. In this disease, we speak rather of a syndrome that includes a very heterogeneous population of patients with behavioral disorders. Autism is a disease that occurs in early childhood (usually between 2 and 6 years old) and is diagnosed by specialists and based on the existence of behavioral disturbances. Although this disease has a great diversity in its manifestations, the main symptoms are at the sociocultural level. It is mainly a delay of language and disorders in social interactions. There is also neophobic behavior, motor stereotypy and anxiety.

The incidence of this disease is important. In France, autism affects 100,000 people and almost 150,000 with pervasive development disorders according to the Ministry of Health and Solidarities (April 07). Statistically, about 1 in 300 children (and 1/165 in the US) have autism-like disorder and the estimated growth is 4000 new cases per year in France. The number of patients is 1.5 million in the USA, as much in Europe with according to the CDC a growth rate of 20% per year. The generally accepted global rate is 10%.

It is clearly recognized that the etiology of this disease is not psychological but organic, which explains the low effectiveness of current therapies. However, the biological cause is not yet known. Genetic studies show that chromosomes 6.7 and 15 could be involved in some cases of autism. However, when a genetic cause is mentioned, the frequency of appearance of a pathology remains relatively constant from one generation to another. However, if certain genes or chromosomal regions are possibly associated with this disease, the implication of these genes in the disease is not demonstrated and they are currently rather considered as susceptibility genes. On the other hand, the impressive increase in the number of cases in recent years strongly suggests the involvement of environmental causes. Many hypotheses have been proposed to identify the genes or environmental factors involved in the disease, but no experimental study has yet demonstrated this. The etiology of the pathology remains as yet unknown and many research teams continue their efforts to identify it. In fact, there is no biological marker of autism that is currently validated.

Psychiatric therapies undertaken to date have been disappointing. Educational, pedagogical and psychotherapeutic interventions sometimes have very variable results from one child to another and only concern a small percentage of cases. In the same way, the current drug therapies of this disease are palliative and / or symptomato logical and do not solve any psychiatric attack of these patients. The possible therapeutic interventions to fight against this disease are therefore very limited. There are no drugs that cure autism. Since some psychiatric symptoms are similar to some in other conditions, active drugs used in schizophrenia, hyperactivity and / or anxiety are sometimes prescribed with modest results and poorly reproducible.

Among the reasons for this weakness of therapeutic possibilities, one of the most important is the ignorance of etiology. The scientific community is still searching for the causative agents of the disease, and reliable models of studies would make significant and beneficial progress in therapy.

To study this disease and to find preventive or curative means supposes to have models of study. Autism is a disease for which an animal model is particularly difficult to obtain. Indeed, animals can not be studied to evaluate language delays, and do not have in their brain regions comparable to those that manage verbal communication in humans. However, the mouse Mus musculus, which is the most commonly used experimental animal model, is a social species with a high degree of social interaction and communication (Poole TB, Morgan HDR, 1976). territorial behavior of laboratory mice musculus L.) in small complex areas. Anim. Bahavior, 24: 476-80). Thus, nonverbal forms of communication and social relations are not only well known and well documented in this species but may also be subject to quantitative analysis.

At present, there is no precise animal model that faithfully reproduces man's autism (Tordjman S, Drapier D, Bonnot O, Graignic R, Fortes S, Cohen D, Millet B, Laurent C, Roubertoux PL (2007), Animal models falling to schizophrenia and autism: validity and limitations, Behav Genet., 37: 61-78), even if there are animal models of the deficit of social games (Bolivar VJ, Walters SR , JL Phoenix. (2006). Assessing autism-like behavior in mice: Behav Brain Res., 176: 21-26). In the few available models, the neurobehavioral disorders described are of genetic origin (Moy SS, Nadler JJ (2008), Advances in behavioral genetics: mouse models of autism, Mol. Psychiatry., 13: 4-26). Some other models describe links between an autoimmune deficiency and a predisposition to neuro-behavioral disorders close to those of autism (Wills S, Cabanlit M, Bennett J, Ashwood P, Amaral D, Van de Water J. ( 2007), Autoantibodies in Autism Spectrum Disorders (ASD), Ann NY, Acad Sd., 1107: 79-91), although this remains conjectural.

An animal model of autism will be a model in which behavioral characteristics typical of autism can be observed with sufficient conceptual analogy. A model may be considered relevant if several symptoms of this disease can be clearly identified. On the other hand, certain specific aspects can not be included in evaluation criteria to be measured in an animal model because of the great differences of species.

One of the aims of the invention relates to the use of compounds for preparing animals with pervasive developmental disorders and / or psychiatric disorders. One aspect of the invention is to provide a method for causing pervasive developmental disorders and / or psychiatric disorders and for providing a corresponding animal model. One of the other aspects of the invention is to provide a method for the screening of compounds that may be of interest in the prevention and / or treatment of pervasive developmental disorders and / or psychiatric disorders.

One aspect of the invention is the use of compounds for preparing a composition for creating pervasive developmental disorders and / or psychiatric disorders in a non-human mammal.

The present invention relates to the use of at least one compound of formula I below:

in which :

* n = 0 or 1,

Ra represents -CH ₂ OH, -COOH, or -NR 1 R 2, with R 1 and R 2 representing, independently of one another, a hydrogen atom or an alkyl group of 1 to 3 carbon atoms,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising a group -NH- and / or

CO-,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, for the preparation of a nonhuman mammal presenting disorders invasive development and / or psychiatric disorders. "Pervasive developmental disorders" refers to mental disorders as defined by DSM IV (Diagnostic and Statistical Manual - Revision 4, published by the American Psychiatric Association in 1994). These disorders include autistic disorders (Kanner autism), Rett's syndrome, childhood disintegrative syndrome, Asperger's syndrome, and non-specified pervasive developmental disorders. Some of these disorders are considered as early psychoses by the French classification of mental disorders in children and adolescents.

"Psychiatric disorders" refers to conditions that disrupt a person's thinking, feelings or behavior sufficiently strongly to make their social integration problematic or cause them suffering.

The present invention also relates to the use of at least one compound of formula (I) described above, for the preparation of a composition intended to create pervasive developmental disorders and / or psychiatric disorders in a non-mammalian mammal. human, to which said composition has been administered.

The present invention relates to non-human mammals having pervasive developmental disorders and / or psychiatric disorders in which at least one compound having the following formula (I) has been administered:

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group, Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders.

It has surprisingly been found that the administration of at least one of the compounds of formula I makes it possible to obtain animals with behavioral problems in the following tests: the open field test, the emergence test , the test of the new object and a test revealing the social interactions.

These tests are usually used in models of studying the effects of toxic or pharmacological substances on behavioral modifications on animal models. These tests reveal the effects of a substance on general exploratory activities, reactions to a new context, and social interactions. They also make it possible to appreciate the anxiogenic effects of the evaluated substances.

These tests are therefore well suited for a first approach of a possible "autistic" effect of the compounds of formula I, since autism is revealed in part by a maladjustment of the reactions to the physical or social environment.

The GABAergic system is disrupted in certain cases of autistic pathology. The administration of at least one of the compounds of formula (I) makes it possible to obtain animals having in their brains calpain concentrations ranging from 1.2 to 8 μg per gram of tissue, in particular from 1.5 to 6 μg per gram of tissue, particularly from 2 to 4 μg per gram of tissue, and / or concentrations of GABA (gamma-amino-butyric acid) ranging from 2 to 20 μmol / g of tissue, in particular from 3 to 10 μmol gram of tissue, especially from 3 to 6 μmol / g of tissue, particularly from 3 to 4 μmol / g of tissue.

The concentration of calpain observed in the brain of a control animal is generally about 1 μg per gram of tissue. The concentration of calpain observed in the brain of an animal model to which a compound according to the present invention has been administered is greater than the concentration observed in a control animal.

The concentration of GABA observed in the brain of a control animal is generally about 1 to 2 μmol / gram of tissue.

The concentration of GABA observed in the brain of an animal model to which a compound according to the present invention has been administered is greater than the concentration observed in a control animal.

By "GABAergic System" is meant the neurological system of which GABA is the mediator Gamma-amino-butyric acid (GABA) is the main inhibitory neurotransmitter of the central nervous system (brain and spinal cord). Its function is to reduce the nervous activity of the neurons on which it is fixed. It would be involved in many physiological processes such as memory, sleep or drug addiction. A real obstacle to the transmission of nerve impulses, GABA serves among other things to control the fear or anxiety manifested by neuronal excitement. Thus, it appears that dysfunction of the GABAergic system is at the origin of many disorders of the nervous system (Bettler et al, 2004). While hyperactivity of this system is associated with schizophrenia, hypoactivity can lead to epileptic seizures, anxiety, depressive states and sleep disorders.

It has been found that said disorders and / or disorders extend beyond the elimination of the administered compounds. It has also been found that the use of compounds according to the present invention causes irreversible effects on the animal model for a time sufficient to perform meaningful behavioral experiments. The effects being irreversible for at least about 1 month, at least about 2 months, including about 3 months, including about 4 months, particularly about 5 months, particularly at least 6 months.

By "animal model" is meant a non-human animal having a condition similar to a human condition and serving as a model for the study of this condition. An animal model is a model for the study of baseline data on the biology or behavior, or in which one can study a pathological process spontaneous or induced, the latter having one or more aspects in common with an equivalent phenomenon in humans or other animal species.

By "responsible for the above disorders and / or disorders" it is indicated that only the compound administered causes the disorders and / or disorders observed. Without administration of this compound, the animal would be healthy and would not have the disorders and / or disorders mentioned above.

It is important to note that pervasive developmental disorders and / or psychiatric disorders are induced by the chemical compound administered and not by genetic modification.

According to an advantageous embodiment of the invention, the non-human mammal is chosen from canids, felids, mustelids, suids, primates, rodents, preferably from the rat, hamster, gerbil, chinchilla, guinea pig, rabbit, monkey, ferret, pig, dog, or cat, and is especially a mouse.

According to another advantageous embodiment of the invention, pervasive developmental disorders and / or psychiatric disorders belong to the group comprising autism, developmental delays, or neurodegenerative diseases such as Alzheimer's disease or the disease. from Parkinson.

By "autism" is meant a set of clinical signs that may be of several etiologies and / or several pathological mechanisms. Autism is characterized by severe developmental alterations in the following three areas: verbal and nonverbal communication, social interactions, behaviors, interests and activities that are restricted and stereotyped.

"Developmental delay" refers to a deficiency, to varying degrees, of intellect, mental faculties, perception and behavior. It can be associated with other physical or mental disorders or occur alone. A delay in the development of mental faculties can be linked to a chromosomal anomaly, a dysmorphic syndrome, a genetic disease, a morphological abnormality of the nervous system. central, to complications related to prematurity, cultural-familial mental retardation, environmental and drug factors, or endocrine causes.

By "neurodegenerative diseases" is meant a disease that affects the functioning of the brain or more generally the nervous system gradually during its evolution. This can be more or less long (from a few weeks to several years). The consequence for the patient is a gradual, often irreversible, alteration of nerve function that can lead to death. Examples include leukoaraiosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, multiple sclerosis, lysosomal diseases and progressive supranuclear palsy.

According to an advantageous embodiment of the invention, in the non-human mammal, at least one compound having the following formula (I) has been administered:

in which :

* n = 0 or 1,

Ra represents -COOH,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH 3 group; Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted with a OH group, said chain optionally comprising an -NH- and / or -CO- group,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders. According to an advantageous embodiment, the present invention relates to the use of at least one compound of formula (I) below:

in which :

* n = 0 or 1, * Ra represents -CH ₂ OH, -COOH, or -NR1R2, with R1 and R2 representing, independently of one another, a hydrogen atom or an alkyl group of 1 to 3 atoms of carbon,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, for the preparation of a composition intended to create invasive disorders development and / or psychiatric disorders in a non-human mammal, to which said composition has been administered, said compound being responsible for the aforesaid disorders and / or disorders.

According to another advantageous embodiment of the invention, in the non-human mammal, at least one compound having the following formula (II) has been administered:

where n, Ra and Z are such that

* n = 0 or 1,

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compound being responsible for the aforesaid disorders and / or disorders.

According to another embodiment, the present invention relates to the use of at least one compound of formula (II) below

where n, Ra and Z are such that

* n = 0 or 1,

Ra represents -CH ₂ OH, -COOH, or -NR 1 R 2, with R 1 and R 2 representing, independently of one another, a hydrogen atom or an alkyl group of 1 to 3 carbon atoms, Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compound being responsible for the aforesaid disorders and / or disorders.

According to another advantageous embodiment of the invention, in the non-human mammal, at least one compound having the formula (I) or (II) has been administered, in which: n = 0 or 1,

2. Ra represents -COOH,

3. Z is - CH ₂ -, -CHOH-, -CHOH-CH ₂ -, -CH ₂ -CHOH-, -CH ₂ -CH ₂ -, - CH ₂ - CO-, or -CO-NH-CH ₂ -, said compound being responsible for the aforesaid disorders and / or disorders.

According to another embodiment, the present invention relates to the use of at least one compound of formula (I) or (II), in which

* n = 0 or 1,

Ra represents -COOH, * Z represents -CH ₂ -, -CHOH-, -CHOH-CH ₂ -, -CH ₂ -CHOH-, -CH ₂ -CH ₂ -, - CH ₂ -

CO-, or -CO-NH-CH ₂ -,

for the preparation of a composition for creating pervasive developmental disorders and / or psychiatric disorders in a non-human mammal, to which said composition has been administered, said compound being responsible for the aforesaid disorders and / or disorders.

According to another advantageous embodiment of the invention, in the non-human mammal, at least one compound having the formula (I) or (II) has been administered, in which:

* n = 1,

Ra represents -COOH,

* Rb represents -CH ₃ , Z represents -CH ₂ -CHOH- said compound being responsible for the aforesaid disorders and / or disorders.

According to another embodiment, the present invention relates to the use of at least one compound corresponding to formula (I) or (II), in which:

* n = 1,

Ra represents -COOH,

* Rb represents -CH3,

Z represents -CH ₂ -CHOH- to prepare a composition for creating pervasive developmental disorders and / or psychiatric disorders in a non-human mammal.

According to another advantageous embodiment of the invention, in the non-human mammal has been administered at least one compound of formula (II) chosen from one of the following compounds:

Vanillylglycine Vanillic acid Vanillylpyruvic acid

Vanillylpropionic acid Vanillyllactic acid 3-methoxy-4-hydroxyphenyl-3-hydroxypropionic acid, said compound being responsible for the aforesaid disorders and / or disorders.

Vanillylglycine is a hepatic detoxification product of vanillic acid. The liver transforms vanillic acid by glycination to facilitate its elimination from the body.

The presence of vanillylglycine is an indicator of the presence of vanillic acid.

According to another advantageous embodiment of the invention, in the non-human mammal, a compound of the following formula has been administered:

(3-methoxy-4-hydroxyphenyl-3-hydroxypropionic acid, or vanillylhydracrylic acid)

said compound being responsible for the aforesaid disorders and / or disorders. According to another embodiment, the present invention relates to the use of a compound of the following formula:

COOH

CH ₀

(3-methoxy-4-hydroxyphenyl-3-hydroxypropionic acid, or vanillylhydracrylic acid) for preparing a composition for creating pervasive developmental disorders and / or psychiatric disorders in a non-human mammal.

(vanillic acid)

said compound being responsible for the aforesaid disorders and / or disorders

According to another embodiment, the present invention relates to the use of a compound of the following formula:

(Vanillic acid) for preparing a composition for creating pervasive developmental disorders and / or psychiatric disorders in a non-human mammal.

The invention also relates to a method for obtaining a non-human mammal having pervasive developmental disorders and / or psychiatric disorders, comprising a step of administering to a non-human mammal at least one compound in formula (I) below:

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders,

and optionally a comparison step between: the non-human mammal obtained at the end of the preceding step, and the aforementioned non-human mammal before administration of the compound, or another healthy non-human mammal of the same species as the one to which the above compound has been administered, in which no compound is administered.

By "comparison step" is meant the linking of at least two sets of data and the evaluation of observed discrepancies between these sets of data.

A compound for inducing pervasive developmental disorders and / or psychiatric disorders is administered to a non-human mammal. In order to verify that said compound does induce the aforesaid disorders and / or disorders in the above-mentioned non-human mammal, the mammal to which the compound has been administered is compared with a reference. Said reference may be the aforesaid non-human mammal before the above-mentioned compound is administered to it, or another mammal of the same species to which the aforesaid compound has not been administered.

The comparison is made by means of tests or analyzes making it possible to obtain values characterizing the behavior of the non-human mammal and / or the level of said compound or of one of its metabolites in the biological fluids of the non-human mammal .

The compound may be the aforesaid compound inducing disorders and / or disorders, a metabolite resulting from the degradation or the biotransformation of the aforesaid compound inducing disorders and / or disorders, or any other endogenous compound whose amount varies in response to administration of the aforesaid compound inducing disorders and / or disorders.

Said biological fluids that can be especially blood, urine, cerebrospinal fluid, saliva. Said tests or analyzes may be performed before administration of the compound inducing the pervasive developmental disorders and / or psychiatric disorders; thus the values obtained in the non-human mammal before administration serve as internal references. The reference may also be the values obtained in the above tests and analyzes on another healthy mammal of the same species, these are then external references.

By comparing the differences between the values obtained during the tests and analyzes carried out on the aforesaid non-human mammals before and after administration of the aforesaid compound, it is possible to determine whether the above-mentioned compound makes it possible to induce the aforesaid disorders and / or disorders. .

According to an advantageous embodiment, in the method of the invention, the non-human mammal is chosen from canids, felids, mustelids, suids, primates, rodents, preferably from the rat, hamster, gerbil, chinchilla, the guinea pig, the rabbit, the monkey, the ferret, the pig, the dog, or the cat, and is in particular a mouse.

According to another advantageous embodiment, in the method of the invention the compound is administered to the non-human mammal by digestive, cutaneous, rectal, respiratory, for example by inhalation, instillation, or nebulization of a spray on the mucous membranes preferably parenterally, for example by intravenous, intraarterial, intrathecal, intraperitoneal, epidural, subcutaneous, intradermal or intramuscular injection,

According to an advantageous embodiment of the invention, the amount of compound administered to the non-human mammal is from 0.01 mg / kg to 100 mg / kg, in particular from 1 mg / kg to 50 mg / kg, the amount of compound administered preferentially being 10 mg / kg.

Depending on the case, the quantity of compound administered may be from 1 mg / kg to 50 mg / kg or from 20 mg / kg to 100 mg / kg. In the case of prolonged exposure the doses may be 0.01 mg / kg at 10 mg / kg, in particular 0.1 mg / kg at 5 mg / kg, the quantity of compound administered preferentially being 1 mg / kg.

The method of the invention relates in particular to the production of a mouse presenting pervasive developmental disorders and / or psychiatric disorders, which method optionally comprises a step of comparison between: the mouse obtained at the end of the administration of vanillic acid and the mouse before administration of vanillic acid, or a healthy mouse in which no compound, especially vanillic acid, is administered.

According to another embodiment, the method of the invention relates in particular to the production of a mouse presenting pervasive developmental disorders and / or psychiatric disorders, which method comprises a step of administration to the mouse of 0 , 01 mg / kg to 100 mg / kg, in particular 5 mg / kg to 50 mg / kg, preferably 10 mg / kg of vanillylhydracrylic acid, and optionally a comparison step between: the mouse obtained at the end of the administration of vanillylhydracrylic acid, and the mouse before administration of vanillylhydracrylic acid, or a healthy mouse in which no compound, particularly vanillylhydracrylic acid, is administered.

According to another advantageous embodiment, the method of the invention relates in particular to the production of a mouse presenting pervasive developmental disorders and / or psychiatric disorders, which method comprises a step of administering the mouse to 0.01 mg / kg to 100 mg / kg, in particular 10 mg / kg to 50 mg / kg, preferably 20 mg / kg of a mixture in proportions ranging preferably from 1/10 to 10/1 of vanillic acid and vanillylhydracrylic acid, in particular at a rate of 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid. and optionally a comparison step between: the mouse obtained after the administration of the mixture of vanillic acid and vanillylhydracrylic acid, and - the mouse before administration of the mixture of vanillic acid and vanillylhydracrylic acid, or with a healthy mouse in which no compound, in particular a mixture of vanillic and vanillylhydracrylic acids, is administered.

The invention also relates to the use of a non-human mammal with pervasive developmental disorders and / or psychiatric disorders, for the screening of therapeutic agents capable of substantially preventing, reducing or canceling invasive development and / or psychiatric disorders, in which at least one compound having the following formula (I) has been administered:

in which :

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH ₃ group; Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted with an OH group, said chain optionally comprising an -NH- and / or -CO- group,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders, and a therapeutic agent capable of substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders, and

optionally comprising a step of comparison between: the non-human mammal obtained after the administration of the aforesaid compound and therapeutic agent.

the aforesaid non-human mammal to which the above-mentioned compound has been administered, but the above-mentioned therapeutic agent has not been administered, or another healthy non-human mammal of the same species as that administered with the aforesaid compound, to which the above-mentioned compound has been administered, but the aforesaid therapeutic agent has not been administered.

A non-human mammal, to which at least one compound inducing pervasive developmental disorders and / or psychiatric disorders has been administered, is treated with a therapeutic agent whose effect on the aforesaid pervasive developmental disorders is to be evaluated and / or or psychiatric disorders.

In order to judge the efficacy of this therapeutic agent, behavioral tests, and / or tests on biological samples, and / or samples of biological material are performed on the aforesaid non-human mammal. The results obtained during the above tests and / or samples are compared with the results obtained for the same tests and / or samples obtained on a reference non-human mammal.

The aforementioned non-human reference mammal may be in particular the same non-human mammal that has been administered the aforementioned compound inducing the aforesaid disorders and / or disorders prior to administration of the aforesaid therapeutic agent, it is an internal reference . It may be another non-human mammal of the same species, having undergone the administration of the compound inducing the aforesaid disorders and / or disorders; it is then an external reference. The differences observed during the above tests and / or samples, between the non-human mammal treated with the above-mentioned compound and the above-mentioned therapeutic agent, and the non-human mammal serving as a reference, make it possible to determine whether the therapeutic agent has an effect behavioral disorders induced by the compound.

By "screening" is meant the identification of molecules with new, logically active biological properties. Screening refers to a set of techniques that make it possible to check the biological properties of a large number of molecules according to standardized tests applicable to a large quantity of samples.

By "therapeutic agents" is meant all the compounds suitable for combating diseases, restoring and preserving health; such as for example drugs, vaccines. According to an advantageous embodiment, the invention relates to the use of a non-human mammal, in particular a mouse, presenting pervasive developmental disorders and / or psychiatric disorders, for the screening of therapeutic agents capable of preventing substantially reducing or canceling Pervasive Developmental Disorders and / or Psychiatric Disorders, wherein a non-human mammal, including a mouse, has been administered from 0.01 mg / kg to 100 mg / kg, including 5 mg / kg at 50 mg / kg, preferably 10 mg / kg at 20 mg / kg of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging preferably from 1/10 to 10/1, said compound being responsible for the aforesaid disorders and / or disorders, and a therapeutic agent capable of preventing, reducing or substantially canceling the pervasive disorders of development and / or psychiatric disorders, and

optionally comprising a step of comparison between: the non-human mammal, in particular a mouse, obtained after the administration of vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, and the aforesaid agent therapeutic, and

the aforesaid non-human mammal, in particular a mouse, to which vanillic acid or vanillylhydracrylic acid has been administered or a mixture of these two acids, but the aforesaid therapeutic agent has not been administered, or

another non-human mammal, in particular a healthy mouse of the same species as that administered with vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, to which vanillic acid or vanillylhydracrylic acid; or a mixture of these two acids was administered, but the above therapeutic agent was not administered.

The present invention relates to a method of screening for therapeutic agents capable of substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders from a non-human mammal administered:

at least one compound of formula (I) below:

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

CO-,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders, and

at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive disorders of development and / or psychiatric disorders,

comprising: at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from the aforesaid non-human mammal in which said compound and said therapeutic agent are administered, a step of behavioral study of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered,

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered,

* a whole animal study by imaging technique.

and optionally a comparison step between: the non-human mammal to which said compound and therapeutic agent have been administered, and

the aforesaid non-human mammal to which the compound has been administered, but the therapeutic agent has not been administered, or

another healthy non-human mammal, of the same species as that administered with the aforesaid compound, to which the above-mentioned compound has been administered, but the above-mentioned therapeutic agent has not been administered.

By "an assay step" is meant any series of measurements made on fluid samples from the aforesaid non-human mammal.

By "sample from the aforesaid non-human mammal" is meant a quantity of liquid, for example blood, urine, cerebrospinal fluid, saliva, collected or collected on the aforesaid non-human mammal.

"Behavioral study stage" means any series of measurements carried out on the aforesaid non-human mammal, comprising a phase of evaluation and scoring of a set of observable reactions in the aforesaid non-human mammal placed in its living environment, or in any other environment, and under given circumstances.

By "tissue study" is meant observation and analysis of a set of tissues to characterize a change or difference between the observed tissue and tissue of the same type from a reference healthy sample. By "cell study" is meant observation and analysis of a set of cells to characterize a change or difference between the observed cells and cells of the same type from a reference healthy sample.

"Whole animal imaging study" means an observation or analysis of an entire animal by imaging techniques to characterize a change or difference between an entire organism, or an organ thereof, and another reference organism, or organ of that other organism serving as a reference.

In the above-mentioned tissue studies, cell studies and whole animal studies, it is possible to carry out proliferation, differentiation, migration, synaptogenesis, remodeling, receptor, enzymology, immunocytochemistry, immunohistochemistry, imaging (electroencephalography, computed tomography, nuclear magnetic resonance, single photon emission computed tomography, positron emission tomography, radiography, FRET, scanner, ...), stereotaxis, proteomics, transcriptomics, interactomics, metabolomics, neurotransmitters, cellular and molecular pharmacology, intracellular messengers, transcription factors, molecular biology, electrophysiology, pro or anti-apoptotic effects, plasticity synaptic, efficiency and strength of synaptic transmission or even physical studies (microscopic, ultrasonographic, thermographic, magnetoencephalographic, scintigraphic, ...) or biochemical (electrophoresis, Western-blotting, chromatography, ...). All these techniques are conventional and known to those skilled in the art.

By "organs" is meant a set of tissues contributing to the achievement of a physiological function.

By "tissues" is meant a set of cells that are not necessarily identical but of the same origin, grouped into a functional unit, that is, contributing to the same function.

According to an advantageous embodiment for the screening method of the invention, a non-human mammal is used which has been administered: at least one compound of formula (I) below:

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH ₃ group,

comprising: administering to said non-human mammal at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive development disorders and / or psychiatric disorders, and

at least one of the steps selected from:

* a whole animal study by imaging technique.

and optionally a comparison step between: the non-human mammal to which said compound has been administered and said therapeutic agent, and

The aforesaid compound and the aforesaid therapeutic agent can advantageously be administered at the same time or at different times.

The time interval between the two administrations may vary from 1 second to several weeks, in particular from 1 minute to 1 week, preferably 24 hours.

The time interval between the last of the two administrations and the dosage step, and / or tissue study, and / or cell, and / or may vary from 1 second to several weeks, including 1 minute to 1 week preferably 24 hours.

According to an advantageous embodiment for the screening method of the invention, use is made of:

administering to a non-human mammal at least one compound of formula (I) below:

in which :

* n = 0 or 1,

administering to said non-human mammal at least one therapeutic agent capable of preventing, reducing or substantially canceling the pervasive disorders of the development and / or psychiatric disorders, and

at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal in which said compound and therapeutic agent are administered; a behavioral study step of the aforesaid non-human mammal in which the above-mentioned compound and therapeutic agent are administered, a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal in which the above-mentioned compound and therapeutic agent are administered,

* a whole animal study by imaging technique.

According to an advantageous embodiment in the screening method of the invention, it has been administered to a non-human mammal, in particular to a mouse:

from 0.01 mg / kg to 100 mg / kg, in particular from 5 mg / kg to 50 mg / kg, preferably from 10 mg / kg to 20 mg / kg of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging from 1/10 to 10/1, in particular at a rate of 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the above disorders and / or disorders, and

which process comprises - at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal, in particular a mouse, in which said compound is administered and therapeutic agent, a step of behavioral study of the aforesaid non-human mammal, in particular a mouse, in which the above-mentioned compound and therapeutic agent are administered,

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal, in particular a mouse, in which the above-mentioned compound and therapeutic agent are administered,

* a whole animal study by imaging technique.

and optionally a comparison step between: the non-human mammal, in particular a mouse, obtained after the administration of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids, and of the aforesaid agent therapeutic, and

According to another advantageous embodiment in the screening method of the invention, it has been administered to a non-human mammal, in particular to a mouse:

from 0.01 mg / kg to 100 mg / kg, in particular from 5 mg / kg to 50 mg / kg, preferably from 10 mg / kg to 20 mg / kg of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging from 1/10 to 10/1, especially at 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the above disorders and / or disorders, and

which method comprises: injecting said non-human mammal, in particular a mouse, with at least one therapeutic agent capable of preventing, reducing or canceling Substantially pervasive developmental disorders and / or psychiatric disorders, and

at least one of the steps chosen from: * a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal, in particular a mouse, in which the above-mentioned compound and therapeutic agent are administered,

a step of behavioral study of the aforesaid non-human mammal, in particular a mouse, in which the above-mentioned compound and therapeutic agent are administered,

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal, in particular a mouse, in which the above-mentioned compound and therapeutic agent are administered, a study on whole animals by imaging technique.

According to another advantageous embodiment in the screening method of the invention, the method comprises:

administration of 0.01 mg / kg to 100 mg / kg, in particular 5 mg / kg to 50 mg / kg, preferably 10 mg / kg to 20 mg / kg of vanillic acid or of acid vanillylhydracrylic acid or a mixture of these two acids in proportions ranging from 1/10 to 10/1, especially at 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the aforesaid disorders and / or disorders, said non-human mammal, including a mouse, and

injecting said non-human mammal, in particular a mouse, with at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive disorders of the development and / or psychiatric disorders, and

at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal, in particular a mouse, in which said compound is administered and therapeutic agent,

a step of behavioral study of the aforesaid non-human mammal in which the above-mentioned compound and therapeutic agent are administered,

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal in which the above-mentioned compound and therapeutic agent are administered,

* a whole animal study by imaging technique.

another non-human mammal, in particular a healthy mouse of the same species as that administered with vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, to which the acid vanillic or vanillylhydracrylic acid or a mixture of these two acids was administered, but the above therapeutic agent was not administered.

said method comprises: a step of behavioral study of said non-human mammal, in particular a mouse, in which said compound and therapeutic agent are administered,

another non-human mammal, in particular a healthy mouse of the same species as that administered with vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, to which vanillic acid or vanillylhydracrylic acid; or a mixture of these two acids has been administered, but the above therapeutic agent has not been administered, According to another embodiment, the screening method of the invention, relates to the evaluation of a method capable of substantially preventing, reducing or canceling the pervasive disorders of the development and / or psychiatric disorders, the method comprises :

administering to a non-human mammal, in particular to a mouse, at least one compound corresponding to the following formula (I):

in which :

the application of a method capable of preventing, reducing or substantially canceling the pervasive disorders of development and / or psychiatric disorders,

said method optionally comprises a step of comparison between: the non-human mammal obtained after the administration of the compound and the application of the method, and

the aforesaid non-human mammal to which the compound has been administered, but no method has been applied, or another healthy non-human mammal of the same species as that to which the compound has been administered and applied the method, to which the compound was administered, but no method was applied.

"Evaluation" refers to any action aimed at assessing, estimating the value or technical efficiency of a method.

"Method likely to prevent, reduce or substantially cancel the developmental disorders and / or psychiatric disorders" means any method, administration of compounds, physical or mental stimulation method, particular diet, vaccination, allowing prevent, reduce or eliminate the discomfort, suffering or physical or psychological disorders caused by pervasive developmental disorders and / or psychiatric disorders.

Description of figures

Figure 1. Open field test

This Tuckey diagram describes six pairs paired boxes in three vertical series along the x-axis. The y-axis represents the number of moves recorded per mouse during an open field test. Each series of boxes comprises a clear box representing the so-called control mice, and a dark box representing the mice injected with the mixture AV (vanillic acid) + VH (vanillyl acid dracrylic acid).

The first set of 2 boxes denoted J1 represents the results on day 1, the second series of boxes denoted J2 represents the results on day 2, the third series of boxes denoted J3 represents the results on day 3. The three days are consecutive. Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box.

The fine extensions at the ends of each box represent the 1st and 9th deciles. The points above and below each box represent the maximum values.

The differences are significant for p <0.05 (*).

Figure 2. Movements in the clear compartment during the seat preference test

This Tuckey diagram describes four vertical boxes along the abscissa axis. The ordinate axis represents the number of recorded movements of the mice in the clear compartment during a preference test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for so-called VH mice (administration of vanilly lhy dracrylic acid). The fourth box denoted AV + VH represents the results for mice called AV + VH (administration of vanillic acid and of acid vanilly lhy dracry lique).

Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box. The fine extensions at the ends of each box represent the 1st and 9th deciles.

The points that can appear above the boxes represent the maximum values. When this point is not represented, it means that the point of the decile is superimposed on the point of the maximum value. It is noted that the mice to which the AV compounds have been administered and the mixture

AV + VH move less in the clear compartment than control mice.

Figure 3. Total mouse movements in the place preference test

This Tuckey diagram describes four vertical boxes along the abscissa axis.

The y-axis represents the number of displacements, per mouse, recorded in the two light and dark compartments during a preference-of-place test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice

(administration of vanillic acid). The third box labeled VH represents the results for mice known as VH (administration of vanillyl dracrylic acid). The fourth box rated

AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillyl dracrylic acid). Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box.

The fine extensions at the ends of each box represent the 1st and 9th deciles. The points that can appear above and below the boxes represent the maximum values. When this point is not represented, it means that the point of the decile is superimposed on the point of the maximum value.

It is noted that the mice to which the compounds AV, VH and the AV + VH mixture were administered have a reduced exploratory activity compared with the so-called control mice. Figure 4. Time of presence of the mice in the clear compartment during the preference test of place.

This Tuckey diagram describes four vertical boxes along the abscissa axis.

The y-axis represents the times (in seconds) of presence recorded in the clear compartment during a preference test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box.

The fine extensions at the ends of each box represent the 1st and 9th deciles.

The points that can appear above and below the boxes represent the maximum values. When this point is not represented, it means that the point of the decile is superimposed on the point of the maximum value.

It is noted that the mice to which the AV compounds and the AV + VH mixture have been administered spend less time in the clear compartment than the control mice.

Figure 5. Exit time of the mouse heads during the emergence test.

This Tuckey diagram describes four vertical boxes along the abscissa axis. The y-axis represents the times (in seconds) of the exit of the heads of the mice out of the dark enclosure, recorded during the emergence test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

The fine extensions at the ends of each box represent the 1st and 9th deciles.

Figure 6. Exit time of the two front legs of the mice during the emergence test

This Tuckey diagram describes four vertical boxes along the abscissa axis.

The y-axis represents the times (in seconds) of output of the two front legs of the mice out of the dark enclosure, recorded during the emergence test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice

(administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box rated

AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid). Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box.

It should be noted that the mice to which the AV + VH mixture has been administered take longer to partially leave the enclosure than the so-called control mice. Figure 7. Complete exit time of the mice out of the dark enclosure during the emergence test

This Tuckey diagram describes four vertical boxes along the abscissa axis.

The y-axis represents the times (in seconds) of complete output of the mice out of the dark enclosure recorded during the emergence test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box (horizontal line at the top of the graph) denoted AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

The fine extensions at the ends of each box represent the 1st and 9th deciles.

It is noted that the mice to which the compounds AV and VH have been administered come out very late from the dark chamber. The mice administered the AV + VH mixture will not leave the dark enclosure.

Figure 8. Time of contact of the mice vis-à-vis the new object during the test of the new object

This Tuckey diagram describes four vertical boxes along the abscissa axis. The ordinate axis represents the delay (in seconds) of contact of the mice vis-à-vis the new object, recorded during the test of the new object. The first box labeled control represents the results for so-called control mice (no administered product), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

The fine extensions at the ends of each box represent the 1st and 9th deciles.

It is noted that the results are not significantly different whatever the treatment.

Figure 9. Number of mouse contacts with the new object when testing the new object

This Tuckey diagram describes four vertical boxes along the abscissa axis. The y-axis represents the number of mouse contacts with the new object, recorded during the test of the new object. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid). Each box represents an interquartile range (+ or - a quartile to the median value) for the measure represented, the median value is indicated by a horizontal line inside the box.

It should be noted that the number of contacts is very low in the mice to which the compounds AV, VH or the mixture AV + VH have been administered.

Figure 10. Number of contacts of mice with a new congener during the social interaction test.

This Tuckey diagram describes four vertical boxes along the abscissa axis. The y-axis represents the number of contacts of mice with a new congener, recorded during the social interaction test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

The fine extensions at the ends of each box represent the 1st and 9th deciles.

Figure 11. Contact time of mice to a new congener during the social interaction test

This Tuckey diagram describes four vertical boxes along the abscissa axis. The y-axis represents the contact time of the mice vis-à-vis a new congener, recorded during the social interaction test. The first box labeled control represents the results for so-called control mice (no product administered), the second box denoted AV represents the results for so-called AV mice (administration of vanillic acid). The third box denoted VH represents the results for mice called VH (administration of vanillylhydracrylic acid). The fourth box labeled AV + VH represents the results for mice called AV + VH (administration of vanillic acid and vanillylhydracrylic acid).

The fine extensions at the ends of each box represent the 1st and 9th deciles.

Note that the number of contacts is significantly increased in VH mice.

Experimental part

On the animal (in whole or on the head alone), on the brain (in whole or in isolated parts, either freshly obtained or in organotypic culture), on tissue sections, on isolated neurons or on neurons in culture cell, it is possible to perform proliferation, differentiation, migration, synaptogenesis, remodeling, receptor, enzymology, immunocytochemistry, immunohistochemistry, imaging studies (electroencephalography, computed tomography, nuclear magnetic resonance, by single photon emission computed tomography, positron emission tomography, radiography, FRET, scanner, ...), stereotaxis, proteomics, transcriptomics, interactomics, metabolomics, neurotransmitters cell and molecular pharmacology, intracellular messengers, transcription factors, molecular biology, electrophysiology, pro or anti-apoptotic effects, plasti synaptic city, efficiency and strength of synaptic transmission or even physical studies (microscopic, ultrasonographic, thermographic, magnetoencephalographic, scintigraphic, ...), biochemical (electrophoresis, Western blotting, chromatography, ...) or behavioral.

Several examples illustrate these studies. They will be carried out mainly on isolated cells in culture, on tissue sections or on the whole animal.

It is understood that the techniques and examples described below are described to facilitate the implementation of the invention by those skilled in the art.

In any case, the stated techniques should not be considered as a limitation of the present invention, since all variants of said techniques are equally applicable to the present invention. I Biological tests

1. Biological material

1.1. Primary culture of neurons.

The hippocampus is a key element of the limbic system and memory processes. It has been suggested that this region of the brain might be involved in autism. All experimental procedures and protocols performed on animals follow the recommendations of the Animal Ethics Committee. The dissected hippocampi are dissociated with a solution of papain (Worthington) for 45 min at 37 ° C with gentle shaking. An alternative may be dissociation using a 0.025% (weight / volume) trypsin solution with gentle shaking. The resulting cell suspension is centrifuged and the pellet resuspended in culture medium (for example Neurobasal-A plus B27) and placed in culture flasks coated with a matrix composed of poly-D-lysine / laminin. The substrate may optionally be poly-L-Lysine and the medium may contain different concentrations of K ⁺ . Optionally, a 0.05% (w / v) solution of DNase can be used beforehand to catabolize DNA from dead cells. The neurons are cultured in a 5% CO ₂ incubator and 36 ° C. Eventually, the next day, cytosine {alpha} -D-arabinofuranoside (AraC) is added (at 5 μM) to prevent proliferation of glial cells. The culture medium is changed every 4 days.

1.2. Organotypic tissue culture.

The hippocampi (or other region of the brain) are dissected from anesthetized mice that have been or have not been treated with a compound described by the patent FR2899973. They are then cut transversely in thick slices of about 400 microns using a microtome. The sections are then deposited on a Millicell-CM sterile membrane (Millipore) and cultured in a CO ₂ incubator (5%) and at a temperature of 36 ° C. A culture medium (Neurobasal-A plus B27, Invitrogen, Paris) containing 20% horse serum is used for the first 4 days. Serum concentration is then gradually decreased to 5% in 48 hours. Then after a 24 hour incubation period in serum-free medium, the sections are used either for electrophysiological measurements, or for immunocytochemistry, or for isotopic markings. 2. Techniques

2.1. Immunocytochemical measurements. The sections or cells in culture which have (or have not) been incubated with the compounds mentioned in the patent FR2899973, are rinsed with PBS buffer (Phosphate Buffered Saline) and then fixed with a solution of 4% paraformaldehyde containing 4% of sucrose. After several rinses with PBS and then with PBS containing 10 mM NH ₄ Cl, the sections or cells are incubated for 1 hour with a blocking / permeabilization solution (PBS containing 0.5% beef serum and 0.1 % Triton X-100). Then the sections or cells are exposed to a solution containing antibodies diluted in the blocking / permeabilization solution. These antibodies are directed against different proteins or membrane or intracellular receptors. These receptors are neurotransmitter receptors or receptors of the TRCPs family (Transducin Repeat Containing Protein). The sections or cells are then incubated with a solution containing a second labeled antibody. This labeling is either fluorescent (Rhodamine, Texas Red, FITC, ...) or enzymatic (alkaline phosphatase, peroxidase, ...) or it may be a biotin labeling which will be revealed by an avidin conjugate or streptavidin.

2.2. RT-PCR.

Tissue sections or aliquots of cells in culture are analyzed by RT-PCR (Reverse Transcription Polymerization Chain Reaction) using commercial kits and conventional techniques of molecular biology known to those skilled in the art (Molecular cloning: a laboratory manual by T. Maniatis, EF Fritsch, J. Sambrook (1982) CoId Spring Harbor, NY: CoId Spring Harbor Laboratory Press). The expression of neurotransmitter receptors or receptors of the TRCPs family may for example be sought.

2.3. Measurements of cell viability. Cells in culture are incubated with variable concentrations and / or variable times with one or more compounds cited in patent FR2899973 in combination or not with antagonists or other pharmacological effectors and compared with untreated control cells. At the end of the treatment, the cell viability is evaluated using standard cell viability tests known to those skilled in the art (Basic cell culture protocols, (Methods in Molecular Biology), (3rd Ed.); JW Pollard and JM Walker, Eds, Humana Press).

2.4. Determination of changes in intracellular calcium concentrations. This measurement involves fluorescent markers which are for example of the type

Fluo, Indo or Fura (Handbook of Fluorescent Probes and Research Products, Molecular Probes, RP Haugland). The principle is an incubation of cells with a permeant fluorescent marker in a balanced buffer optionally containing Pluronic which facilitates the incorporation of the fluorescent probes. After the incubation period (usually 30 min), the cells are rinsed with PBS and exposed to effectors in culture medium or in saline at 37 ° C. The variations in fluorescence intensity are visualized by means of a camera and a fluorescence microscope at the excitation and emission wavelengths characteristic of the fluorescent probes used. The effectors used may be either neurotransmitters, related molecules, the compounds described in FR2899973 or potential antagonists. This variation in fluorescence intensity is converted into intracellular calcium variations by the formula of Tsien et al. (T his RY, Pozzan T, Rink TJ Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator, J. Cell Biol 1982, 94 (2): 325-334.) added a calcium ionophore (A23187 or ionomycin) and then EGTA (Ethylene Glycol Tetraacetic Acid). These intracellular calcium variations demonstrate receptor coupling with an intracellular calcium system serving as a secondary messenger and showing 1) the presence of a primary receptor of the tested effector and 2) that calcium is a secondary messenger to this receptor.

2.5. Measurements of enzymatic activity of calpain.

Calpains are cysteine proteases, non-lysosomal, whose activity, papain type, is dependent on calcium. These enzymes are responsible for calcium-dependent neutral proteolysis in a reducing environment. It is a family of proteins often associated with cellular inhibitors that play a key role in several cellular functions and in particular in synaptic plasticity and apoptosis. These proteins appear to be associated with certain neurodegenerative pathologies such as Alzheimer's and Parkinson's disease (Vosler PS, Brennan CS, Chen J. Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration, Mol Neurobiol 2008 38 (1): 78- 100) and probably also in autism. In fact, in these diseases, the activation of these calpains could be due to high levels of calcium and leads to hydrolysis of some proteins or even their total degradation. The activity of calpain is determined using a commercial kit (Axxora, Calpain Activity Assay Kit, ref: BV-K240-100). The principle of the assay is the fluorometric detection of the hydrolysis of a fluorogenic substrate measured with a fluorimeter.

2.6. Electrotransfer.

10 μg of protein solution (from cell or tissue lysate) are electrophoresed in a polyacrylamide gel in the presence of SDS (Sodium Dodecyl Sulfate) and then electrotransfered (Western-Blott) on a nitrocellulose membrane in a transfer buffer ( for example: 39 mM glycine, 48 mM Tris, and 20% methanol).

After saturation of the membrane with a solution enriched in proteins (milk, serum, ...) it is exposed to a solution of primary antibodies which are then revealed with a solution of labeled secondary antibodies. This technique makes it possible to visualize the proteins of interest, in particular membrane receptors or proteins involved in intracellular signaling.

2.7. Production and / or secretion of glutamate. Neurotransmitters (including GABA (Gamma Amino Butyric Acid) and glutamate) are both messengers of communication between neurons and key players in brain building. They may be involved in some cases of autism (Collins AL, MaD, WH Whitehead, Martin ER, HH Wright, Abramson RK, J Hussman, Haines JL, ML Cuccaro, Gilbert JR, Pericak-Vance MA. GABA receptor subunit genes in multiple ethnic groups Neurogenetics, 2006; 7 (3): 167-174). The amounts of glutamate released into the culture medium or the tissue survival medium are determined using a commercial kit. (For example the Molecular Probes Amape Red Glutamic Acid / Glutamate Oxidase Assay kit). The principle is a fluorometric measurement involving fluorochrome Amplex Red, glutamate oxidase, glutamate pyruvate transaminase, alanine and peroxidase. After incubation at 37 ° C., the fluorescence of the reaction medium is measured (emission wavelength: 590 nm and excitation wavelength: 555 nm). 2.8. Electrophysiology techniques.

These techniques make it possible to measure the electrical behaviors of tissues or nerve cells during their stimulation or during the addition of different molecules.

These conventional techniques, known to those skilled in the art, are widely used for research in Neurobiology (The Neuronal Microenvironment Editor (s): Alan A. Boulton, Glen B.

Baker, Wolfgang WaIz, University of Saskatchewan, Saskatchewan Canada, Springer).

2.9. Cytology and histology.

The organs studied (in particular the brain) are taken from the animal after anesthesia and fixed with a fixing solution (for example 4% paraformaldehyde in PBS) and then included in paraffin. They are cut into thin slices of a few microns. These sections are then stained (for example with a mixture of hematoxylin and eosin) and studied morphologically with an image analysis system. According to another alternative, these sections are prepared and observed by electron microscopy techniques, using conventional cytology and histology techniques known to those skilled in the art.

(Neurons: Methods and Applications for the Biologist) Posted by Peter J. Hollenbeck and

James R. Bamburg. Academy Press, 2003, Amsterdam).

2.10. 3D imaging of the brain. Noninvasive imaging techniques applied to small animals (microRMN,

Positron emission tomography (PET), CT scan, ...) are currently technologies of choice in the exploration of various human pathologies, in particular in the cerebral area. Due to the non-invasive nature of the exploration and growing development of new labeled radio molecules, these techniques are of particular interest for neurobiology studies involving the use of appropriate ligands. In particular, these various imaging techniques are used to determine the target brain areas of the compounds described in FR2899973 and to study the effect of potential pharmacological antagonists. II Behavioral tests

1. Definitions

1.1. Mouse says control

Mice injected intraperitoneally with a solution composed of a physiological buffer. This buffer (PBS) has the following composition: 135 mM NaCl, 10 mM NaHPO ₄ ZNa ₂ HPO ₄ , pH 7.4. This solution serves both injection control, and solution to dissolve the compounds of interest tested.

1.2. VH mice

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved vanillylhydracrylic acid (VH) at a concentration of 10 mg / kg.

1.3. AV mouse

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved vanillic acid (VA) at a concentration of 10 mg / kg.

1.4. Mouse AV + VH

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved both vanillylhydracrylic acid (VH) and vanillic acid (VA), both at the concentration of 10 mg / kg.

1.5. VPA mouse

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved vanillylpropionic acid (VPA) at a concentration of 10 mg / kg.

1.6. VG Mouse

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing in a dissolved manner vanillylglycine (VG) at a concentration of 10 mg / kg.

2. Description of the tests

Four tests were performed: the open field test, the emergence test, the new object test and a test revealing the social interactions. These tests are usually used in study models of the effects of toxic or pharmacological substances on behavioral modifications on animal models. These tests reveal the effects of a substance on general exploratory activities, reactions to a new context, and social interactions. They also make it possible to appreciate the anxiogenic effects of the evaluated substances. These tests are thus quite well adapted for a first approach of a possible "autistic" effect of compounds since autism reveals itself partly by a maladjustment of the reactions to the physical or social environment.

These tests are designed to determine the possible behavioral effects of vanillylhydracrylic acid (VH), vanillic acid (VA), vanillylpropionic acid (VPA), and vanillylglycine (VG) using mouse SWISS white males Rjθrl (cd-l) aged 4-5 weeks.

The products are diluted in phosphate buffered saline and administered intraperitoneally (100 μL) in the mice at concentrations of 10 mg / kg for each product. The mice are observed 30 minutes after the injection. In order to establish a possible cumulative effect, the mice are subjected to a daily injection for three days and analyzed consecutively over this period of 3 days.

For example, in the studies of vanillylhydracrylic acid and vanillic acid, four cohorts of mice are treated with either placebo (phosphate buffered saline), VH, or vanillic acid. alone or with both compounds, VH and vanillic acid. Groups of 10 mice per trial (AV, VH or VH + V A) are analyzed and compared to the groups of 10 placebo-treated mice.

In practice, the ^1st day and ^2nd day it performs the test of the open field and the emergence test. Then the ^3rd, it performs the test of the open field test the new object and that of social interaction. The apparatus for the open-field, new object and social interaction test consists of a square (plastic) box, 37 cm wide and 17 cm high that contains 16 squares of equal dimension drawn on the bottom of the box. This is illuminated by the ambient light.

2.1. The open field test

The mice are placed individually in the center of the box and their movements and movements are monitored for 5 min and this for 3 days in a row. The number of total (exploratory activity measurement) and central (crossed) squares (the least anxiety zone) is quantified. We also note the number of straightening on the two hind legs, the number of grooming and scraping (scratching), the duration of the moments of fixity, as well as the number of poop and urine (criteria also expressing a possible feeling of anxiety).

2.2. The test of the new object This test is carried out only the 3 ^rd day immediately after the test of the open field leaving the mouse in the box. This makes it possible to condition the mouse to the paradigm of the open box (open field) before placing the new object there. The object is a metal cylinder 6.5 cm in diameter and 7 cm high, placed at a distance from the mouse and outside its field of vision. Care must be taken to place the object on a square adjacent to a square bordering the box. Thus the object is placed on a central square but close to the edge of the box. The latency before ¹ contact with the object and the number of contact therewith are quantified for a period of 5 minutes under ambient light.

2.3. The social interaction test This test is performed immediately after the previous test and also only the ^3rd day. A

^2nd mice of the same sex is placed in the center of the remote box and out of the visual field of the mice tested; the interactions are then followed for 5 min. One contact is considered positive if the nose of the mouse studied is at a distance less than 2 mm from the other mouse. All types of contacts are taken into consideration.

2.4. The emergence test

This test is performed both day ^1: a small dark box (8 x 8 cm) having only a small opening is placed at the center of a larger box, different from that used for the open field test ( to avoid any recognition by the mouse of its environment). The mouse is placed in the small box and we note the time it takes to get out. The passage times of the head, of the 2 front legs and then of the four legs are quantified for a maximum duration of 5 minutes.

2.5. The place preference test

In this test, the mice are placed in the presence of 2 compartments, one clear, the other dark, separated by a guillotine door. Each compartment (30x30 cm and 17 cm high) is divided into 9 squares drawn on the floor. The number of squares crossed per compartment is counted, as are the number of passages between the two compartments and the time spent in each compartment for a total duration of 5 minutes. The whole is lit by the ambient light.

III Biological analyzes

The GABAergic system is disrupted in certain cases of autistic pathology. In these cases, the concentrations of GABA are modified by the administration of the compounds according to the present invention and in particular AV, VH, VPA, and VG, or mixtures of these compounds, and these modifications participate in the differences in behavior observed.

1. Definitions

1.1. Mouse says control

1.2. VH mice

1.3. AV mouse

1.4. Mouse AV + VH

1.5. VPA mouse

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved acid vanillylpropionic acid (VPA) at a concentration of 10 mg / kg.

1.6. VG Mouse

Mice injected intraperitoneally with a solution (100 microL) composed of a physiological buffer (PBS) containing dissolved vanillylglycine (VG) at a concentration of 10 mg / kg.

2. Principle

2.1. GABA

The GABA assay is based on the use of two enzymes GABGT (for gamma-aminobutyrate glutamate transferase) and SSDH (semialdehyde succinic dehydrogenase) according to the following scheme:

GABA + 2-oxo-glutarate> - succalic semialdehyde + succinic semialdehyde glutamate + beta-NADP → succinate + NADPH

The use of both enzymes in the presence of 2-oxo-glutarate and beta-NADP at pH

8.6 and at room temperature can determine the amount of GABA in a sample that is proportional to the amount of NADPH produced. This NADPH is easily measurable by its absorbance at 340 nm, (its molar absorption coefficient being 6220 at this wavelength). The GABAse used is a commercial preparation (Sigma-Aldrich Chimie SARL) obtained from Pseudomonas fluor escens containing the two enzymes.

2.2. calpain

Calpain is a neutral intracellular cysteinyl protease whose activity depends on calcium. It is very present in the brain where it has many cellular substrates. These include cytoskeleton and membrane proteins, membrane receptors, and myelin sheath proteins. The activity of calpain leads to limited hydrolysis of these proteins and is therefore involved in the control of many important intracellular systems related to calcium-dependent processes such as synaptic transmission, neurotransmitter secretion, growth and differentiation. neuronal. It would appear that this protease plays an important role in the mediation of the calcium signal in a biochemical and physiological cell response.

The dosage of calpain is followed by the degradation of the substrate into a fluorescent product according to the following reaction: Succinyl-Leu-Tyr-7-amido-4-methylcoumarin> Succinyl-Leu-Tyr + 7-amido-4-methyl coumarin

Calpain is able to cleave the N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin substrate after the tyrosine residue to release the fluorescent methylcoumarin. The fluorescence is measured with an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The use of the inhibitor of calpain I (N-acetyl-Leu-Leu-Norleucinal) and the inhibitor of calpain II (N-acetyl-Leu-Leu-Methioninal) makes it possible to determine the respective shares of these two calpains in the results obtained.

3. Description of dosages

The brains of mice are removed at the latest 2 min after the sacrifice of the animals (obtained by overdose with pentobarbital 200 mg / kg ip) and immersed in liquid nitrogen. The brains are then stored at -80 ^° C. until they are used.

The brains of mice are sonicated in 100 μl of 100 mM phosphate buffer solution, pH 8.6 in Eppendorf tubes and the tissue suspension is centrifuged at 10000 g for 15 min at 4 ° C. The supernatant is recovered.

3.1 GABA

The dosage of GABA is carried out on the supernatant. The supernatant is added to the wells (20 .mu.l of supernatant per well) of a 96-well microplate containing 130 .mu.l of a 100 mM phosphate buffer solution pH 8.6. The phosphate buffer solution contains 2-mercaptoethanol (3 mM), 2-oxo-glutarate (5 mM), NADP (1.5 mM) and 0.02 U (Units) GABAse. A GABAse Unit is defined as being able to convert 1 micromole of GABA to succal semialdehyde and then succinate per minute with a stochiometric reduction of NADP + at pH 8.6 and 25 ° C.

The reaction is monitored with a microplate reader at 340 nm as a function of time (over 60 min). The kinetics makes it possible to determine the precise concentration of GABA of the samples.

3.2 Calpain

The same supernatant is used for the determination of calpain activity of tissue lysates.

The protein concentration of the tissue lysate is determined by the method of

Bradford. A volume of 50 μL supernatant is deposited in each well of a 96-well plate and the volume of each well is supplemented to obtain a total volume of 100 μL with a solution of 100 mM HEPES, pH 7.4. HEPES solution contains 1 mmM DTT

(Dithiotheritol) and 2 mM CaCl ₂ . 10 μL of the substrate solution (from a stock solution in dimethylformamide) are then added to each well to obtain a final concentration of 150 μM. Alternatively, specific inhibitors of calpains I and II (respectively N-acetyl-Leu-Leu-Norleucinal and N-acetyl-Leu-Leu-Methioninal) are added (5 μL, 100 μM final). In this case, the initial protein volume of each well is supplemented to a volume of

95 μL.

The OD is measured every 5 minutes for 1 hour on a fluorescence plate reader equipped with a 350 nm excitation filter and a 450 nm emission filter. The DO = f (time) curves are plotted and compared with those obtained with commercial pure calpain (0.1 U / ml).

The GABA and calpain values are normalized to the amount of protein contained in the Bradford-determined tissue lysate. IV Examples

The behavioral studies and assays described in the examples were performed under standardized laboratory conditions. Thus, the main symptoms characterizing subjects with autism that can be studied in animals have been selected: space exploration capabilities, neophobia, anxiety and social interactions.

Autism is defined only by behavioral variations of the subjects.

1. Examples of biological effects in point administration

These measurements are intended to determine the possible biological repercussions of the compounds involved in the present invention, and in particular of vanillylhydracrylic acid (VH) and vanillic acid (VA), of vanillylpropionic acid (VPA), and of vanillylglycine

(VG) using white male SWISS Rjθrl (cd-1) mice aged 4-5 weeks after single administration in mice as a model.

The products are diluted in phosphate buffered saline and administered intraperitoneally (100 μL) in the mice at concentrations of 10 mg / kg for each product. The mice are studied 60 minutes after the injection.

60 minutes after the injection, the mice are euthanized, and the rapidly removed brain is immediately ground in the presence of a phosphate buffered saline (PBS) containing protease inhibitors (Sigma). The ground material is then centrifuged (10,000 rpm, 10 minutes) and the supernatant is recovered to determine:

1) the concentration of proteins by the Bradford technique since all the results are expressed per mg of protein, 2) the activity of calpain.

The calpain concentrations observed ranged from 2 to 4 μmol / g of tissue.

3) the concentration of glutamate,

4) the concentration of the compounds which have been injected into the mice,

5) the concentration of GABA, The concentrations of GABA observed ranged from 3 to 4 μmol / g of tissue.

2. Examples of biological effects in chronic administration

These measurements are intended to determine the possible biological repercussions of the compounds involved in the present invention, and in particular of vanillylhydracrylic acid (VH) and vanillic acid (VA), of vanillylpropionic acid (VPA), and of vanillylglycine (VG), using as a model male white SWISS Rjθrl (cd-1) mice aged 4-5 weeks after chronic administration in mice. The products are diluted in phosphate buffered saline and administered intraperitoneally (100 μL) in the mice at concentrations of 10 mg / kg for each product.

The mice are subjected to three successive injections over three consecutive days and then to three other successive injections over three consecutive weeks. The mice are studied 60 minutes after the last injection. The mice are then euthanized, and the rapidly removed brain is immediately ground in the presence of a phosphate buffered saline (PBS) containing protease inhibitors (Sigma). The ground material is then centrifuged (10,000 rpm, 10 minutes) and the supernatant is recovered to determine:

1) the concentration of proteins by the Bradford technique since all the results are expressed per mg of protein, 2) the activity of calpain,

The calpain concentrations observed ranged from 2 to 4 μmol / g of tissue.

3) the concentration of glutamate,

4) the concentration of the compounds which have been injected into the mice,

5) the concentration of GABA, the observed GABA concentrations ranging from 3 to 4 μmo / g of tissue. 3. Examples of behavioral effects in point administration

Example 1: Open field test

In this example the effect of three injections on three consecutive days of the compounds VH, AV, AVP, VG or mixture AV + VH was determined. The different behavioral observations were made 30 minutes after the injection.

The open field test shows a significant difference in the number of total squares crossed by AV + VH compared to control mice (Figure 1). On the other hand, no difference is found for VH mice, AVP mice, or AV mice. In addition, it has been found that the control mice explore their environment whereas the VH, AV, AVP or AV + VH mice seek instead to flee it. Thus, the exploratory abilities of the mice are modified by administering a mixture of the two compounds AV and VH.

Example 2: Emergence test

The emergence test makes it possible to determine a potential anxiogenic effect of a compound.

1) After a first administration, the control mice take the head out of the dark chamber after 6 +/- 5 sec, the bust after 11 +/- 7 sec and exit completely after

14 +/- 9 sec. As a result, they continue their exploratory behavior of their new environment. AV mice left the head after 2.2 +/- 1.2 sec, the bust after 5.4 +/- 4.4 sec and the whole body after 11.2 +/- 5.4 sec. The VH mice emerge after 4.6 +/- 3.2 sec, the bust after 8.6 +/- 4.2 sec and the whole body after 18 +/- 15 sec. The AV + VH mice left the head after 3.0 +/- 1.8 sec, the bust after 6.4 +/- 4.8 sec and the whole body after 9.2 +/- 3.2 sec. No significant difference was found between control mice and treated mice (VH, AV, and AVP).

2) After the second administration (the next day), the so-called control mice pull the head of the dark chamber after 1.8 +/- 0.2 sec, the bust after 3.0 +/- 0.4 sec and exit completely after 5 +/- 1.2 sec. This time shorter than the day before these three parameters is explained by the fact that the mice already know this environment, which thus shows their rapid capacity for habituation, their ability to memorize their environment and the decrease of the fear generated by a known situation. The mice AV leave the head after 2.6 +/- 0.8 sec, the bust after 12.2 +/- 7.0 sec and the complete body after 14.0 +/- 5.2 sec. No significant behavioral variation was observed with mice after a single administration. The VH mice left the head after 7.4 +/- 4.2 sec, the bust after 10.4 +/- 4.8 sec; on the other hand, they never left their dark walls completely. The AV + VH mice leave the head after 7.2 +/- 4.2 sec, the bust after 93 +/- 66 sec and do not come out of the dark speaker either.

This test shows a clear effect of the two compounds AV and VH, this effect seeming additive. These effects are different: the AV compound prevents habituation whereas the compound VH is clearly anxiogenic. The compound VPA shows a similar effect to the compound VH.

Example 3: Test of the new object

This test is based on the spontaneous tendency of rodents to preferentially explore a new object. The so-called control mice react quickly to the introduction of the object into their environment since the first contact is after 5.4 +/- 2.4 sec and during the 5 min of the experiment they have 24 +/- 5 , 2 contacts with this one. The time of first contact of the AV mice with the new object is longer (14.8 +/- 7.6sec) and the number of contacts a little less (20.6 +/- 5.2). VH mice also had longer initial contact time (15.0 +/- 8.2 sec) but unchanged contact counts (25.2 +/- 5.8). In contrast, the time of first contact is longer in AV + VH mice (25 +/- 12sec) and the number of contacts significantly reduced (7.8 +/- 3.4).

In this test, the increases in the delay before the first contact with the new object as well as a decrease in the number of contacts is indicative of an anxiogenic effect of the compounds administered. This effect is additive when the AV and VH compounds are administered together.

The effect of the VPA compound was tested according to the same protocol. VPA mice show similar behavior to VH mice.

4. Examples of behavioral effects in chronic administration

In these examples, the compounds (AV, VH, VPA, and AV + VH mixture) were administered to the mice for three consecutive days and then three more times three weeks consecutively. Two weeks later, preference tests of place, emergence, the new object and social interaction, have been carried out.

Example 1. Place preference test

The mice to which the compounds AV, VH, VPA or the mixture AV + VH were administered explore much less a new environment unlike so-called control mice. This decrease in the exploration of injected mice (AV, VH, VPA or AV + VH mixture) is no longer manifested when these are put back into their original cage, that is to say in their usual environment. . In these latter conditions, their motor and / or exploratory behavior is identical to that of the so-called control mice. By cons, when they are out of their cage and deposited on the ground, unlike so-called control mice that move spontaneously, injected mice (AV, VH, VPA or AV + VH mixture) have either a behavior flight, or they remain motionless. This behavioral change was quantified by the place preference test. This test is based on the spontaneous tendency of rodents to explore their environment and to avoid light (photophobia). The device consists of two compartments, one clear, the other dark, separated by a guillotine door. Placed in this two-color space preference test chamber, a device designed to quantify both the exploratory behavior of animals and the anxiety that this exploration generates, control mice and VG mice explore their new environment. In this test, the movements in each compartment, the number of passes from one compartment to another and the respective times spent in each compartment are measured. The data are reported in FIGS. 2 to 4 (AV mice, VH and AV + VH mixture). The measurement of the total number of displacements shows that the compounds reduce the exploratory activity of the mice and that the combination of the two products has an additive effect. The calculation of the number of trips made in the light compartment and the time spent in this compartment is a sign of fear compared to an environment considered more hostile (mice are nocturnal animals naturally fearing lighted places). It is found that the mice to which the compounds were administered (AV, VH, VPA or AV + VH mixture) are more afraid of an environment considered hostile than the control mice.

Example 2. Emergence test

The emergence test is another test revealing the possible anxiogenic effect of a product. The mice are introduced into a small dark chamber placed in a larger clear enclosure. Exit times of the head, both front legs and finally the body whole are noted. Under these experimental conditions, the mice are subjected to a choice, that of the security of the dark enclosure and that of the curiosity to discover a new but uncertain environment. Figures 5 to 7 show that the control mice do not stay very long in the dark chamber, they come out completely after 10 sec. The mice to which the compounds were administered (AV, VH, VPA or the mixture AV + VH) leave the head and the two front legs at times close to those of the so-called control mice. This means that they have similarly become aware of the presence of an external environment, but unlike so-called control mice, they will not explore it, or very late. This result shows an anxiogenic effect of the administered compounds.

Example 3. Test of the new object

In this test, the latency time before the first contact and the number of contacts are noted. Figures 8 and 9 show the results obtained. The mice at which the compounds were administered (AV, VH, VPA or the AV + VH mixture) come into contact with the new object half as much as the control mice. These results rise either a significant decrease of the natural curiosity of the mice, or an anxiety effect of the products.

Example 4. Social interaction test

In this test, the mice are placed in the open field enclosure of another non-congenerous mouse from another cage, and to whom Calmivet (0.25 mg / kg, Vétoquinol Laboratory) was administered. It is a neuroplegic tranquilizer used sometimes as anesthetic. The mice treated with Calmivet at this dose lose only very little of their exploratory activity, but show a total disinterest with respect to a new congener. Thus, the contact time and the number of contacts measured in this test come solely from the initiative of the mice tested. The number of contacts decreases significantly in the mice to which the compounds were administered (Figure 10). The time of first contact is also increased in VH mice (FIG. 11) and AVP mice. It is not significantly in AV mice and in AV + VH mice. The nature of the contacts, on the other hand, is very different in the mice to which the compounds have been administered. In no case did the control mice show signs of aggressiveness vis-à-vis the new mouse. This is not the case for AV, VH, AVP and AV + VH mice. These all had aggressive behavior that is to say they beat their tail (sign aggressiveness in mice), and systematically attack and bite the other mouse on approach. Signs of prostration often accompany this aggressive behavior, they are placed in an angle of the open field and do not move any more.

The behaviors of VH mice and AVP mice, in the experimental situations described above, resemble those of autistic subjects: neophobia and impoverished exploration, avoidance of social interactions. The results of this study show that the mice to which the test compounds were administered can be used as an animal model of autism.

Claims

claims

1) Use of at least one compound of formula I below:

in which: n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, for the preparation of a nonhuman mammal presenting disorders invasive development and / or psychiatric disorders.

2) non-human mammal with pervasive developmental disorders and / or psychiatric disorders, wherein has been administered at least one compound of formula (I) below:

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

CO-,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders.

3) non-human mammal according to claim 2, selected from the rat, hamster, gerbil, chinchilla, guinea pig, rabbit, monkey, ferret, pig, dog, or cat, and is in particular a mouse.

4) non-human mammal according to claim 2 or 3, wherein the pervasive developmental disorders and / or psychiatric disorders belong to the group comprising autism, developmental delays, or neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.

5) Non-human mammal according to one of claims 2 to 4, wherein has been administered at least one compound of formula (I) below:

in which :

* n = 0 or 1,

Ra represents -COOH, * Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH ₃ group,

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders.

6) non-human mammal according to one of claims 2 to 5, wherein has been administered at least one compound of formula (II) below:

wherein n, Ra, and Z are as defined in claim 2 or 5, said compound being responsible for the aforesaid disorders and / or disorders. 7) non-human mammal according to one of claims 2 to 6, wherein has been administered at least one compound of formula (I) or (II), wherein:

* n = 0 or 1,

* Ra represents -COOH, * Z represents - CH ₂ -, -CHOH-, -CHOH-CH ₂ -, -CH ₂ -CHOH-, -CH ₂ -CH ₂ -CH ₂ -

CO-, or -CO-NH-CH ₂ -, said compound being responsible for the aforesaid disorders and / or disorders.

8) non-human mammal with pervasive developmental disorders and / or psychiatric disorders according to one of claims 2 to 6, wherein has been administered at least one compound of formula (I) or (II), in which :

* n = 1,

Ra represents -COOH,

* Rb represents -CH ₃ , * Z represents -CH ₂ -CHOH- said compound being responsible for the aforesaid disorders and / or disorders.

9) non-human mammal according to claim 6, wherein has been administered at least one compound of formula (II) selected from one of the following compounds:

Vanillylglycine Vanillic acid Vanillylpyruvic acid

10) non-human mammal according to claim 6, in which a compound of the following formula has been administered:

(3-methoxy-4-hydroxyphenyl-3-hydroxypropionic acid, or vanillylhydracrylic acid) said compound being responsible for the aforesaid disorders and / or disorders.

11) non-human mammal according to claim 6, in which a compound of the following formula has been administered:

(vanillic acid) said compound being responsible for the aforesaid disorders and / or disorders

12) Process for obtaining a non-human mammal according to one of claims 2 to 11, comprising a step of administering to a non-human mammal, at least one compound corresponding to the following formula (I) :

in which :

* n = 0 or 1,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group,

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders, and optionally a comparison step between: the non-human mammal obtained at the end of the preceding step, and the aforesaid non-human mammal before administration of the compound, or another healthy non-human mammal of the same species as that to which the above compound has been administered, in which no compound is administered.

13) The method of claim 12, wherein said non-human mammal is selected from the rat, hamster, gerbil, chinchilla, guinea pig, rabbit, monkey, ferret, pig, dog, or cat, and is especially a mouse.

14) The method of claim 12 or 13, wherein the compound is administered parenterally, cutaneously, digestive, rectal or respiratory, preferably parenterally.

15) Method according to one of claims 12 to 14, wherein the amount of compound administered is from 0.01 mg / kg to 100 mg / kg, especially 1 mg / kg to 50 mg / kg, preferably 10 mg / kg .

The method for obtaining a non-human mammal according to claim 12, wherein the mammal is a mouse and comprising a step of administering to the mouse of

0.01 mg / kg to 100 mg / kg, in particular 1 mg / kg to 50 mg / kg, preferably 10 mg / kg of vanillic acid, and possibly a step of comparison between: the mouse obtained at the end of the administration of vanillic acid and - the mouse before administration of vanillic acid, or a healthy mouse in which no compound, in particular vanillic acid, is administered.

17) A method for obtaining a non-human mammal according to claim 12, wherein the mammal is a mouse and comprising a step of administering to the mouse of

0.01 mg / kg to 100 mg / kg, in particular 1 mg / kg to 50 mg / kg, preferentially 10 mg / kg of vanillylhydracrylic acid, and possibly a step of comparison between: the mouse obtained at the end of the administration of vanillylhydracrylic acid, and the mouse before administration of vanillylhydracrylic acid, or a healthy mouse in which no compound, in particular vanillylhydracrylic acid, is administered.

18) A method for obtaining a non-human mammal according to claim 12, wherein the mammal is a mouse and comprising a step of administering to the mouse from 0.01 mg / kg to 100 mg / kg, in particular 1 mg / kg at 50 mg / kg, preferably 20 mg / kg of a mixture in proportions ranging from 1/10 to 10/1 of vanillic acid and of vanillylhydracrylic acid, in particular at the rate of 10 mg / kg of vanillic acid and 10 mg / kg vanillylhydracrylic acid. and optionally a comparison step between: the mouse obtained after the administration of the mixture of vanillic acid and vanillylhydracrylic acid, and the mouse before administration of the mixture of vanillic acid and vanillylhydracrylic acid, or with a healthy mouse in which no compound, in particular a mixture of vanillic and vanillylhydracrylic acids, is administered.

19) Use of a non-human mammal with pervasive developmental disorders and / or psychiatric disorders for the screening of therapeutic agents capable of substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders, in which at least one compound having the following formula (I) has been administered:

in which :

* n = 0 or 1, Ra represents -CH ₂ OH, -COOH, or -NR 1 R 2, with R 1 and R 2 representing, independently of one another, a hydrogen atom or an alkyl group of 1 to 3 carbon atoms,

optionally comprising a step of comparison between: the non-human mammal obtained at the end of the administration of the aforesaid compound and the aforesaid therapeutic agent; the aforesaid non-human mammal to which the aforesaid compound has been administered, but the aforesaid therapeutic agent; was not administered, or

another healthy non-human mammal of the same species as that administered with the aforesaid compound, to which the above-mentioned compound has been administered, but the aforesaid therapeutic agent has not been administered.

20) The use according to claim 19 of a non-human mammal, in particular a mouse, presenting with pervasive developmental disorders and / or psychiatric disorders, for the screening of therapeutic agents capable of preventing, reducing or reducing substantially canceling the pervasive developmental disorders and / or psychiatric disorders, wherein a non-human mammal, including a mouse, has been administered from 0.01 mg / kg to 100 mg / kg, especially 5 mg / kg to 50 mg / kg kg, preferably 10 mg / kg to 20 mg / kg of vanillic acid or vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging from 1/10 to 10/1, said compound being responsible for the aforesaid disorders and / or disorders, and a therapeutic agent capable of substantially preventing, reducing or canceling the pervasive developmental disorders and / or psychiatric disorders, and

21) A method of screening for therapeutic agents capable of substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders from a non-human mammal that has been administered:

at least one compound of formula (I) below:

in which :

Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group, said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders, and

comprising: - at least one of the steps selected from:

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered, a whole animal study by imaging technique,

and optionally a comparison step between: the non-human mammal to which the aforesaid compound and the aforesaid therapeutic agent have been administered, and

the aforesaid non-human mammal to which the compound has been administered, but the therapeutic agent has not been administered, or another healthy non-human mammal of the same species as that administered with the aforesaid compound, to which the above compound was administered, but the above therapeutic agent was not administered.

A method of screening for therapeutic agents that can substantially prevent, reduce or cancel Pervasive Developmental Disorders and / or Psychiatric Disorders from a non-human mammal that has been administered: at least one following formula (I):

in which :

said compounds being optionally in the form of salts or when there is at least one asymmetric carbon, in the form of isomers alone or in a racemic mixture, said compound being responsible for the aforesaid disorders and / or disorders, and comprising: administering to said non-human mammal at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive development disorders and / or psychiatric disorders, and

at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from the aforesaid non-human mammal in which said compound and said therapeutic agent are administered,

a step of behavioral study of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered,

* a whole animal study by imaging technique,

23) A method of screening therapeutic agents capable of substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders comprising:

administering to a non-human mammal at least one compound of formula (I) below:

in which :

* n = 0 or 1,

at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from the aforesaid non-human mammal in which said compound and said therapeutic agent are administered, a step of behavioral study of the aforesaid non-human mammal in which the aforementioned compound and the aforesaid therapeutic agent are administered, a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered,

* a whole animal study by imaging technique,

24) A method of screening according to claim 21, a therapeutic agent capable of preventing, reducing or substantially canceling the pervasive developmental disorders and / or psychiatric disorders, from a non-human mammal, including a mouse, which was administered:

comprising at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said mammal non-human, in particular a mouse, in which the aforementioned compound and the aforesaid therapeutic agent are administered,

a step of behavioral study of the aforesaid non-human mammal, in particular a mouse, in which the aforementioned compound and the aforesaid therapeutic agent are administered,

a tissue and / or cell study carried out on organs or tissues of the aforesaid non-human mammal, in particular a mouse, in which the aforementioned compound and the aforesaid therapeutic agent are administered,

* a whole animal study by imaging technique,

and optionally a comparison step between: the non-human mammal, in particular a mouse, obtained after the administration of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids, and of the aforesaid agent therapeutic, and * the aforesaid non-human mammal, including a mouse, which has been administered vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, but the aforesaid therapeutic agent has not been administered, or

25) A method of screening according to claim 22, a therapeutic agent capable of preventing, reducing or substantially canceling the pervasive developmental disorders and / or psychiatric disorders, from a non-human mammal, including a mouse, which was administered:

from 0.01 mg / kg to 100 mg / kg, in particular 5 mg / kg to 50 mg / kg, preferably 10 nig / kg to 20 mg / kg of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging from 1/10 to 10/1, in particular at a rate of 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the above disorders and / or disorders, and comprising: injecting said non-human mammal, especially a mouse, with at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive disorders of development and / or psychiatric disorders, and

at least one of the steps selected from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal, in particular a mouse, in which said compound is administered and the aforesaid therapeutic agent,

* a whole animal study by imaging technique.

another non-human mammal, in particular a healthy mouse of the same species as that administered with vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, to which vanillic acid or vanillylhydracrylic acid; or a mixture of these two acids was administered, but the above therapeutic agent was not administered. 26) A method of screening according to claim 23, a therapeutic agent capable of preventing, reducing or substantially canceling the pervasive developmental disorders and / or psychiatric disorders, from a non-human mammal, including a mouse, comprising:

the administration of 0.01 mg / kg to 100 mg / kg, in particular 5 mg / kg to 50 mg / kg, preferably 10 mg / kg to 20 mg / kg of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids in proportions ranging from 1/10 to 10/1, in particular at the rate of 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the aforesaid disorders and / or disorders, to the aforesaid non-human mammal, in particular a mouse, and

injecting said non-human mammal, in particular a mouse, with at least one therapeutic agent capable of substantially preventing, reducing or canceling the pervasive disorders of the development and / or the psychiatric disorders, and

at least one of the steps chosen from:

a step of blood and / or urine measurement, in vitro, of said compound or of one of its metabolites from a sample from said non-human mammal, in particular a mouse, in which said compound is administered and the aforesaid therapeutic agent, a step of behavioral study of the aforesaid non-human mammal in which the aforesaid compound and the aforesaid therapeutic agent are administered,

and optionally a comparison step between: the non-human mammal, in particular a mouse, obtained after the administration of vanillic acid or of vanillylhydracrylic acid or of a mixture of these two acids, and of the aforesaid therapeutic agent, and

27) A method of screening according to claim 21, a therapeutic agent capable of preventing, reducing or substantially canceling the pervasive developmental disorders and / or psychiatric disorders, from a non-human mammal, including a mouse, which has been administered: from 0.01 mg / kg to 100 mg / kg, especially 5 mg / kg to 50 mg / kg, preferably 10 mg / kg to 20 mg / kg of vanillic acid or vanillylhydracrylic acid or a mixture of these two acids in proportions ranging from 1/10 to 10/1, in particular at the rate of 10 mg / kg of vanillic acid and 10 mg / kg of vanillylhydracrylic acid, said compound or mixture of compounds being responsible for the aforesaid disorders and / or disorders, and

comprising: a step of behavioral study of the aforesaid non-human mammal, in particular a mouse, in which the aforesaid compound and the aforesaid therapeutic agent are administered,

another non-human mammal, in particular a healthy mouse of the same species as that administered with vanillic acid or vanillylhydracrylic acid or a mixture of these two acids, to which vanillic acid or vanillylhydracrylic acid; or a mixture of these two acids has been administered, but the above therapeutic agent has not been administered,

28) Use of a non-human mammal with pervasive developmental disorders and / or psychiatric disorders, for the evaluation of a method for substantially preventing, reducing or canceling pervasive developmental disorders and / or psychiatric disorders, to which non-human mammal has been administered at least one compound corresponding to the following formula (I):

in which: n = 0 or l,

Rb represents an alkyl group of 1 to 3 carbon atoms, preferably a -CH3 group, Z represents a hydrocarbon chain of 1 to 3 carbon atoms, in particular of 1 or 2 carbon atoms, optionally substituted by an OH group, said chain optionally comprising an -NH- and / or -CO- group,

applied a method that is likely to prevent, reduce or substantially reverse pervasive developmental disorders and / or psychiatric disorders, and

optionally comprising a step of comparison between: the non-human mammal obtained after the administration of the compound and the application of the method, and

the aforesaid non-human mammal to which the compound has been administered, but no method has been applied, or

another healthy nonhuman mammal, of the same species as that administered the compound and applied the method, to which the compound was administered, but no method was applied.