WO2011067225A1

WO2011067225A1 - Compositions targeting cb1 receptor for controlling food intake

Info

Publication number: WO2011067225A1
Application number: PCT/EP2010/068466
Authority: WO
Inventors: Giovanni Marsicano; Luiggi Bellocchio
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2009-12-01
Filing date: 2010-11-30
Publication date: 2011-06-09
Anticipated expiration: 2012-06-01

Abstract

The present invention relates to compositions and methods for controlling food intake exploiting the synergistic effects of CBl receptor agonists with GABA receptor or glutamate receptor agonists, respectively. More particularly, the present invention relates to compositions and method for reducing food intake and/or body weight in a mammal comprising administering to said mammal a pharmaceutically low amount of at least one CBl receptor agonist and an amount of at least one glutamate receptor agonist. The present invention also relates to compositions and method for elevating food intake and/or body weight in a mammal comprising administering to said mammal a pharmaceutically high amount of at least one CBl receptor agonist and an amount of at least one GABA receptor agonist.

Description

COMPOSITIONS TARGETING CB1 RECEPTOR FOR CONTROLLING

FOOD INTAKE

FIELD OF THE INVENTION

The present invention relates to compositions and methods for controlling

(reducing or elevating) food intake using CB 1 receptor agonists

BACKGROUND OF THE INVENTION

Obesity is widely recognized as a serious health problem that is increasing in prevalence across the United States and the world. According to the 1998 National Institute of Health (NIH) Clinical Guidelines on the Identification, Evaluation and Treatment of Overweight and Obesity in Adults, an estimated 97 million people in the US are classified as either overweight or obese. The medical and other costs related to obesity have risen considerably in the last two decades. In addition, many pets or companion animals, such as dogs or cats, have become obese and their owners may seek veterinary treatment to cure their obesity and associated medical problems.

Agents that have been or are currently being used for the treatment of obesity include phenylpropanolamine, dexfenfluramine, phentermine/fenfluramine, sibutramine and orlistat. Unfortunately, all of these drugs have serious adverse effects and dexfenfluramine and fenfluramine have been withdrawn because of toxicity associated with valvular heart disease in a small subset of patients.

Thus, there is a therapeutic need for safer and more effective compounds to treat obesity.

Much attention has been focused in the last ten years on the endocannabinoid system for its potential for pharmacologic manipulation to treat obesity. The endocannabinoid system comprises endogenous ligands commonly referred to as endocannabinoids (anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glyceryl ether (noladin ether), virodhamine), and two cannabinoid receptor subtypes (CB1 and CB2). Marijuana and the major plant cannabinoid, delta(9)tetrahydrocannabinoid, have been implicated in the changes of feeding behavior in both man and animals. Recently, researchers have shown that in partially satiated animals, the administration of the naturally occurring cannabinoids anandamide and 2-arachidonyl glycerol increases food intake. It is believed that these endocannabinoids stimulate the CB1 and CB2 receptors, which alter glucose and lipid metabolism in both liver and adipose tissue and, most notably, help to regulate energy balance and body weight.

The cannabinoid CB1 receptor has received the greatest attention with respect to appetite and body weight regulation, leading to the development of a new class of appetite suppressants and/or weight regulating drugs that appear to work by selectively blocking CB1 receptors. The discovery of the first selective CB1 receptor antagonist was reported several years ago. This antagonist compound, [Lambda]/-(piperidin-l - yl)-5-(4- chlorophenyl)- 1 -(2,4-dichlorophenyl)-4-methyl- 1 /-/-pyrazole-3-carboxamide

(SR141716A or rimonabant), has been shown to have anorectic efficacy and produce a sustained reduction in body weight. SR141716A is the hydrochloride of SR 141716. See U.S. Patent No. 6,344,474.

It is theorized that SR141716A binds to CB1 receptors and competitively antagonizes many of the CB1 receptor-mediated effects of cannabinoids or stabilize an inactive form of the receptor. Thus, synthesis of an antagonist such a SR141716A that selectively binds to CB1 receptors without producing cannabimimetic activity in vivo suggests that recognition and activation of cannabinoid receptors are separable events.

Finally, there are potential side effects associated with treatment with CB1 antagonists. In human trials, the most common side effects of SR141716A (5 and 20 mg doses) were nausea, dizziness, arthralgia and diarrhea [13]. The use of this drug was withdrawn from European market in 2008 for psychiatric side effects, such as anxiety and depression.

CB1 receptors are expressed in brain regions controlling food intake, where they presynaptically regulate both excitatory and inhibitory neurotransmission [1-4]. Given the opposite roles of glutamatergic and GABAergic transmission in feeding behaviour [5,6], the expression of CB1 in both types of neurons suggests that the endocannabinoid system (ECS) might also induce opposite effects on this function of the brain. Consistent with this idea, pharmacological treatments with CB1 agonists lead to biphasic effects, with low-to- moderate doses exerting hyperphagia, and moderate-to-high doses causing hypophagia [7]. This hypophagic effect is generally considered as an unspecific consequence of the hypo locomotion caused by high doses of these drugs [7]. An alternative unexplored possibility is that endogenous and/or exogenous cannabinoids might activate different sets of CB1 receptors expressed in different neuronal populations to oppositely control food intake. Accordingly, it is an object of the present invention to provide alternative and improved compositions and methods for controlling food intake.

SUMMARY OF THE INVENTION

Agonists of the cannabinoid receptor CBl exert biphasic effects on food intake, with low dose having a hyperphagic effect and high doses a hypophagic one, respectively. The inventors found that these effects are due to a selective decrease of glutamatergic and GABAergic transmission, respectively. The inventors found also surprisingly that the effects of low doses (hyperphagic) are transformed into hypophagic ones by the combination of the drugs with allosteric activators of glutamatergic receptors. Conversely, the hypophagic effects of higher doses become hyperphagic upon simultaneous administration of an allosteric activator of GABA-A receptors. The side effects of CBl agonists are likely due to their action on glutamatergic transmission. Therefore, the use of low doses of CBl agonists combined with allosteric modulators of glutamatergic transmission (e.g. D-cyclo-serine, DCS, that is used in clinics for treating tubercolosis), could have a strong hypophagic effect, avoiding any side effect of CBl antagonists (e.g. rimonabant, Accomplia from Sanofi) and blunting psychotropic effects of CBl agonists, like Delta-9-tetrahydrocannabinol (THC). The invention is the combination of cannabinoid CBl agonists with either of these two classes of drugs: allosteric enhancers of glutamate receptors and allosteric enhancers of GABA-A receptors.

Furthermore, the doses used in the present invention for allosteric enhancers of glutamate receptors and allosteric enhancers of GABA-A receptors are 10-15 time less that the doses normally used in the clinics to exert their psychotropic effects (i. e. D-cycloserine is clinically used to at a dose of 50-250 mg to treat Obsessive Compulsive Disorder, schizophrenia, phobias, whereas diazepam is used at doses of 5-40 mg to treat anxiety and neuropsychiatric disorders).

Preclinical results presented here show that doses of D-cycloserine and diazepam that have no effect on food intake are able to alter this function if combined with THC. Therefore, there is a synergistic effect of CBl agonists with allosteric enhancers of GABA or glutamate receptors, allowing the use of low doses of drugs. For these reasons, the invention should strongly reduce possible side effects of any of the drugs used alone. Thus, the present invention relates to a pharmaceutical composition comprising a low amount of at least one CBl receptor agonist and an amount of at least one glutamate agonist and optionally a pharmaceutically acceptable carrier.

This pharmaceutical composition comprising a low amount of at least one CBl receptor agonist and an amount of at least one glutamate agonist is for use in the prevention or the treatment of obesity, obesity related diseases, overweight or overeating in a mammal in need thereof.

The present invention also relates to a pharmaceutical composition comprising a high amount of at least one CBl receptor agonist and an amount of at least one GABA agonist and, optionally a pharmaceutically acceptable carrier.

This pharmaceutical composition comprising a high amount of at least one CBl receptor agonist and an amount of at least one GABA agonist is for use in the prevention or the treatment of anorexia cachexia syndrome (ACS), underweight or under eating in a mammal in need thereof.

The present invention also relates to a kit containing:

a) a CBl receptor agonist, and

b) a Glutamate receptor agonist,

as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of obesity and obesity related disease.

The present invention also relates to a kit containing:

a) a CBl receptor agonist, and

b) a GABA agonist,

as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of anorexia cachexia syndrome (ACS).

The present invention also relates to a method for the prevention and/or the treatment of obesity, obesity-related disease, overweight or overeating in a mammal in need thereof, comprising administering the individual with a prophylactically or therapeutically effective quantity of a CBl receptor agonist at low amount and a glutamate receptor agonist, most preferably an allosteric enhancers of NMD A receptor.

The present invention also relates to a method for the prevention and/or the treatment of anorexia cachexia syndrome (ACS), underweight or under eating in a mammal in need thereof, comprising administering the individual with a prophylactically or therapeutically effective quantity of a CBl receptor agonist at high amount and a GAB A receptor agonist, most preferably an allosteric enhancers of GAB A A receptor.

DESCRIPTION OF THE INVENTION

"Definition"

Throughout the specification, several terms are employed and are defined in the following paragraphs.

The term "CBl receptor" has its general meaning in the art and may include naturally occurring CBl receptor and variants and modified forms thereof. The CBl receptor can be from any source, but typically is a mammalian (e.g., human and non- human primate) CBl, particularly a human CBl . CBl receptors include for example, two iso forms: a long isoform (Accession No NP-057167) and a shorter one truncated in the NH2 terminal part corresponding to a splice variant (Accession No NP- 149421).

The term "CBl receptor agonist" refers to any CBl receptor agonist (direct agonist or allosteric agonist) that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in activation or up-regulation of a biological activity associated with activation of the CBl receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to CBl receptor of its natural ligands (endocannabinoids). Such CBl receptor agonists include any agent that can help CBl receptor activation or any of the downstream biological effects of CBl receptor activation. For example, such a CBl receptor agonist can act by occupying the ligand binding site or a portion thereof of the CB 1 receptor, thereby making the receptor more accessible to its natural ligand so that its normal biological activity is enhanced. CBl receptor agonist also refers to "indirectly CBl receptor active agent" which means a compound able to inhibit the degradation or the uptake of endocannabinoids, thereby enhancing the activation of CBl receptors (for examples of such compounds, see the reviews [21] and [22], which are incorporated by reference).

A number of agonists with significant selectivity for CBl or CB2 receptors have been developed [14, 15]. Examples of most selective CBl receptor agonists include the eicosanoid cannabinoids, anandamide and 2-arachidonoyl-glycerol. Reference may be made also to DELTA-9-THC, WIN55212-2, HU-210 and CP55,940, which are mixed CB1/CB2 receptor agonists. Important CBl -selective agonists include the anandamide analogues, R- (+)- methanandamide, arachidonyl-20-chloroethylamide (ACEA), arachidonyl- cyclopropylamide (ACPA) and 0-1812. Another CBl -selective agonist of note is 2- arachidonyl glyceryl ether (noladin ether).

Examples of CBl agonist are also described in the several reviews [23, 24, and 25] and are incorporated here by reference.

The term "selective CBl receptor agonist" as used herein refers to a compound able to activate selectively CBl receptors and not any other receptor such as CB2 receptors.

The term "non selective CBl receptor agonist" as used herein refers to compound natural or not which has the capability to activate CB2 receptors but also CBl receptors.

Agonist activity toward CBl receptor must be determined by any well known method in the art. For example, the discovery and pharmacological validation of new cannabinoid receptor agonists relies on the availability of suitable bioassays [14, 15]. For CBl receptor agonists, the most commonly used in vivo bioassay is the mouse tetrad, in which their ability to produce hypokinesia, hypothermia, catalepsy in the Pertwee ring test and antinociception in the tail-flick or hot plate test is determined in the same animal. There are no standard in vivo bioassays for CB2 receptor agonists. As to established in vitro bioassays for CBl and CB2 receptor agonists, these all involve the use of membrane or tissue preparations that contain CBl and/or CB2 receptors, expressed either naturally or after transfection [14, 15].

Among the most commonly used of these bioassays are binding assays that measure the ability of test compounds to displace a radiolabeled cannabinoid receptor ligand such as [3H]CP55940 from membranes obtained from CBl and/or CB2 receptor- expressing cells or tissues. As to commonly used functional in vitro bioassays, some of these measure the effects of test compounds on CBl or CB2 receptor signalling, for example stimulation of binding to G proteins of the hydrolysis-resistant GTP analogue [35S]GTPgS, Gi/o-mediated inhibition of basal or drug-induced cyclic AMP production and elevation of intracellular free Ca²⁺, which is presumably a Gs-mediated effect. The bioassay of CBl receptor agonists can also be performed with isolated nerve-smooth muscle preparations such as the mouse vas deferens. These bioassays exploit the ability of cannabinoid agonists to act through neuronal CBl receptors to produce a concentration- related inhibition of electrically-evoked contractile transmitter release and hence of the contractions resulting from this release. Strategies commonly used to validate effects as CB1 or CB2 receptor-mediated rely on the availability of selective CB1 and CB2 receptor antagonists (Cannabinoid CB1 and CB2 receptor antagonists), of cells or tissues that express either CB1 or CB2 receptors (but not both these receptor types), or of animals or tissues from which CB1 and/or CB2 receptors have been genetically deleted.

Alternatively, other binding assays may be used. In particular binding assays with tritiated CB1/CB2 agonist may be carried out on membranes prepared from rat forebrain (for CB1) or from frozen mouse spleen (for CB2). Reference may be made for instance to the assay described in the US patent specification US2006030563.

The pharmaceutical compounds of the present invention are in preferred embodiment, for GABA receptor agonist and Glutamate receptor agonist, are allosteric enhancer.

An "allosteric modulation" of a receptor results from the binding of allosteric modulators at a different site (regulatory site) other than of the endogenous ligand (orthosteric ligand) and enhances or inhibits the effects of the endogenous ligand. It normally acts by causing a conformational change in a receptor molecule, which results in a change in the binding affinity of the ligand. By this way, an allosteric ligand "modulates" its activation by a primary "ligand" and can be thought to act like a dimmer switch in an electrical circuit, adjusting the intensity of the receptor's activation.

The different definitions of allosteric ligand are:

"Ago-allosteric modulator": proposed term for 'a ligand that functions both as an agonist on its own and as an allosteric modulator of the efficacy (co-agonist) and/or the potency of the orthosteric ligand'. The effect of the ago-allosteric modulator can be positive with regard to both efficacy and potency, but might also be negative or inhibitory in terms of, for example, potency while being positive in terms of efficacy.

"Allosteric agonist": 'a ligand that is able to mediate receptor activation in its own right by binding to a recognition domain on the receptor macro molecule that is distinct from the primary (orthosteric) site' - as defined and differentiated from allosteric enhancer by the IUPHAR committee on quantitative pharmacology [16].

"Allosteric enhancer": 'a modulator that enhances the affinity and/or efficacy of the orthosteric ligand while having no effect on its own' - as defined by the IUPHAR committee on quantitative pharmacology [16].

In the context of the invention, the term "treating" or "treatment" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in the treatment of obesity, overweight or overeating in a mammal in need thereof, or in the treatment of anorexia, underweight or under eating in a mammal in need thereof.

According to the invention, the term "subject" or "individual" to be treated is intended for a human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate). Preferably, the subject is a human.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- so lid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

"Composition and method of treatment of obesity and obesity related disorder"

The present invention provides methods and compositions (such as pharmaceutical compositions) for treating obesity and/or obesity-related disorders.

Thus, an object of the invention is pharmaceutical composition comprising a low amount of CB1 receptor agonist and an amount of glutamate receptor agonist for use in the prevention or the treatment of obesity and/or obesity-related disorders, overweight or overeating in a mammal in need thereof.

In preferred embodiment glutamate receptor agonist is an allosteric enhancer of glutamate receptor, more preferably an allosteric enhancer of NMD A receptor.

The term "a low amount of CB1 receptor agonist" means using dose with a limited or no psychotropic effect of CB1 agonists wherein the amount of at least one CB1 agonist is supplied at a dosage level inferior to 5 mg.

By way of example, the compositions and use at low dose of CB1 agonist according to the invention may be administered at a dose of about 0.05 to 5 mg for humans

(or about 0.5 to 1.75 mg/kg body weight for mice).

The term "glutamate receptor" refers to any receptor that binds and is activated by the neurotransmitter glutamate. Glutamate receptors can be divided into two groups according to the mechanism by which their activation gives rise to a postsynaptic current: lonotropic glutamate receptors and Metabotropic glutamate receptors. lonotropic glutamate receptors include NMDA, AMPA and Kainate receptors, form the ion channel pore that activates when glutamate binds to the receptor. Metabotropic glutamate receptors (mGluR) indirectly activate ion-channels on the plasma membrane through a signalling cascade that involves G proteins.

Preferably the glutamate receptor of the invention is selected more specifically from the NMDA receptor and AMPA receptor.

The term "glutamate receptor agonist "refers to any glutamate receptor, agonist (direct agonist or allosteric agonist) that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in activation or up-regulation of a biological activity associated with activation of the glutamate receptors in the patient, including any of the downstream biological effects otherwise resulting from the binding to glutamate receptor of its natural ligand (glutamic acid). Such glutamate receptor agonists include any agent that can help glutamate receptor activation or any of the downstream biological effects of glutamate receptor activation. Typically, the glutamate receptor agonist is an allosteric enhancer of glutamate receptor, and more preferably an allosteric enhancer of NMDA receptor.

The term "amount of glutamate receptor agonist" means using dose with a limited or no psychotropic effect of glutamate agonists wherein the amount of at least one glutamate agonist is supplied at a dosage level inferior to 25 mg.

By way of example, the compositions and use of an amount of glutamate receptor agonist according to the invention may be administered at a dose of about 5 to 25 mg for human.

The term "NMDA receptor" (NMDAR) means a specific type of ionotropic glutamate receptor. NMDA (N-methyl D-aspartate) is the name of a selective agonist that binds to NMDA receptors but not to other glutamate receptors. The NMDA receptor is distinct in two ways; first that it is both ligand-gated and voltage-dependent, and second that it requires co-activation by two ligands, glutamate and glycine.

"Activation of NMDA receptors" requires binding of glutamate or aspartate (aspartate does not stimulate the receptors as strongly). In addition, NMDARs also require the binding of the co-agonist glycine for the efficient opening of the ion channel, which is a part of this receptor. D-serine has also been found to co-agonize the NMDA receptor with even greater potency than glycine. In addition, a third requirement is membrane depolarization. The term "allosteric enhancers of glutamate" means an allosteric NMDA enhancer or an allosteric AMPA enhancer. A variety of accepted tests are used to determine whether a given agent is a positive modulator of an AMPA or an NMDA receptor. The primary in vitro assay is measurement of the enlargement of the excitatory postsynaptic potential (EPSP) in in vitro brain slices, such as rat hippocampus brain slices. Modulators useful in the present embodiments are agents that cause an increased ion flux through the AMPA or NMDA receptor complex channels. Increased ion flux is typically measured as at least a 10% increase in decay time, amplitude of the waveform and/or the area under the curve of the waveform and/or a decrease of at least 10% in rise time of the waveform, for example.

The term "Allosteric enhancers of NMDA receptor " may affect any of a number of interactions among the NMDA receptor, glycine and glutamate

The NMDA receptors exhibit a variety of modulatory sites and, in particular, exhibit the binding site for the amino acid glycine. Several compounds acting at the glycine site of the NMDA receptor have been proposed as cognitive enhancers such as D- serine and D-cycloserine, for example. Further, inhibitors of glycine uptake exert similar effects as glycine, and are proposed as cognitive enhancers. Drugs acting as positive modulators of NMDA receptors are also termed «nemdakines"

Exemple of "allosteric enhancers of NMDA receptor" include L-alanine, D-alanine, D- cycloserine, N-methylglycine, L-serine, D-serine, Ν,Ν,Ν-trimethylglycine, 3-amino-l- hydroxypyrrolid-2-one (HA966), (R)-(N-[3-(4^,-fluorophenyl>3-(4^*- phenylphenoxy)propyl])sarcosine (ALX5407), N-methyl-N-[3 -[(4- trifluoromethyl)phenoxy]-3 -phenyl-propyl] glycine (ORG 24598), a polyamine, neurosteroid, a salt thereof, an ester thereof, a precursor thereof, a metabolite thereof, a derivative thereof, a racemic mixture thereof, or a combination thereof, for example.

Such positive modulators may have a mechanism of action as follows.

"Agonist at the glycine site": Positive modulators at the glycine site are likely located on the NR1 subunit of the NMDA receptor. Glycine acts as a co-agonist with glutamate; neither glutamate nor glycine alone can activate the NMDA receptor. While glutamate increases the rate of dissociation of glycine from the NMDA receptor, the partial agonist at the glycine site HA966 reduces the affinity of glutamate for the NMDA receptor also by increasing its dissociation rate. Since binding of glutamate and glycine are necessary for channel opening and thus, for synaptic activation, the influence of one by the other has necessarily an impact on the transition states of the kinetic scheme. Positive modulators of the glycine site include D-serine, L-alanine, L-serine, 3-amino-l- hydroxypyrrolid-2-one (HA966), D-cycloserine, and derivatives thereof. Blockers of glycine uptake/transport: Positive modulators of the glycine transporter site that block the re-uptake/transport of glycine out of the synaptic cleft, thereby increasing concentrations of glycine include (R)-(N-[3-(4'-fluorophenyl)-3-(4'- phenylphenoxy)propyl])sarcosine (ALX5407) , N-methyl-N- [3 -[(4-trifluoromethyl)phenoxy] -3 -phenyl-propyl] glycine (ORG 24598), and derivatives thereof.

Compounds acting at other modulatory sites of the NMD A receptor complex: Positive modulators of channel sites include agents that reduce activity for Mg2+, for PCP, for MK801, and for ketamine, for example. Positive modulators of sites on the NR2 subunits include agents that reduce activity for Zn2+, and for protons. Positive modulators at the NR2 subunits include polyamines such as spermine, spermidine, neomycine, for example, that enhance synaptic activity by preventing the proton-induced inhibition of receptor activity; neurosteroids, in particular, pregnenolone sulphate acts on a segment of the extracellular domain next to a transmembrane portion called SMDl (steroid modulatory domain 1); ATP and derivatives thereof. Further positive modulators of the NR2 subunits include agents for preventing Ca2+ dependent calmodulin-sensitive and calmodulin- insensitive inactivation of NMD A receptor activity. Additional positive modulators of the NR2 subunits signal intracellular proteins that convey to the second messenger cascade via the proteins anchored to the postsynaptic density (PSD95) and other mechanisms downstream leading to receptor trafficking.

The term "AMPA receptor" means the a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptor (also known as AMPA receptor, AMPAR, or quisqualate receptor) is a non-NMDA-type ionotropic transmembrane receptor for glutamate that mediates fast synaptic transmission in the central nervous system (CNS). Its name is derived from its ability to be activated by the artificial glutamate analog, AMPA. AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system.

A category of drugs, termed "ampakines" acts as positive modulators of AMPA receptors (PARMs). Ampakines have been proposed as cognitive enhancers due to their ability to facilitate learning in a variety of tasks in mammals and humans. Ampakines are in clinical trials to treat various indications including mild cognitive impairment (MCI) associated with aging. A positive modulator of an AMPA receptor likely acts on a transition state of the AMPA receptor complex by reducing deactivation, slowing channel closing, accelerating channel opening, reducing/blocking desensitization or accelerating the recovery from desensitization, for example. Modulation may occur at or near the dimer interface, at levels downstream of the receptor channel involving proteins linked to the postsynaptic densities (PSD) and to proteins engaged in the cascade of second messengers and even further downstream to transcriptional and translational mechanisms involving, among others, CREB.

A positive modulator of an AMPA receptor may also be an agent having activity for reducing an effect of a negative modulator. For example, an agent having activity for soaking up protons is a positive modulator since protons promote receptor desensitization. An agent that deactivates thiocyanate is also a positive modulator of AMPA receptors. A positive modulator of an AMPA receptor may also be an agent that reduces the effect of a noncompetitive antagonist (also called negative allosteric modulators) and derivatives thereof such as 1-4-aminophenyl- methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466) (Vizi et al, 1996, CNS Drug Rev. 2:91-126), quinoxaline-7sulphonamide, NS102, 5-nitro-6,7,8,9-tetrahydrobenzo[g]-2,3-dione-3-oxime (SYM-2206), (7-acetyl-5- (4-aminophenyl)-8-methyl- 8,9-dihydro-7H-l,3-dioxolo[4,5-b][2,3]benzodiazepine (Talampanel), 3-(2-chloro-phenyl)-2- [2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6- fluoro-3H-quinazolin-4-one (CP-465022), or chemical analogs using the catalyst approach as cited by Barreca et al. (2003, J. Chem. Inf. Comput. ScL 43:651-655).

Positive Modulators of AMPA Receptors: For a general review of AMPA receptor modulators [17].

Positive modulators of AMPA receptors include, for example, an azepine, a benzamide, benzoylpiperidine, benzoylpyrrolidine, benzoxazine, benzothiadiazide, benzothiadiazine, biarylpropylsulfonamide, pyrrolidinone, pyrroline, tetrahydropyridine, phenoxyacetamide, sulfur-containing organic nitrate ester, lectin, a salt thereof, an ester thereof, a precursor thereof, a metabolite thereof, a derivative thereof, a racemic mixture thereof, or a combination thereof.

The term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg/m²). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m², or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m². An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m² or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m². A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg/m² to less than 30 kg/m² or a subject with at least one comorbidity with a BMI of 25 kg/m² to less than 27 kg/m². The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian- Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m². An "obese subject" in these countries refers to a subject with at least one obesity- induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m². In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg/m2 to less than 25 kg/m².

The term "obesity-related disorders" encompasses disorders that are associated with, caused by, or result from obesity. Examples of obesity-related disorders include overeating and bulimia, diabetes, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, breast, prostate, endometrial and colon cancer, heart disease, cardiovascular disorders, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack. Other examples include pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass. Further examples of obesity- related disorders include metabolic syndrome, also known as syndrome X, insulin resistance syndrome, type II diabetes, impaired fasting glucose, impaired glucose tolerance, inflammation, such as systemic inflammation of the vasculature, atherosclerosis, hypercholesterolemia, hyperuricaemia, as well as secondary outcomes of obesity such as left ventricular hypertrophy. Obesity-related disorders also include the liver abnormalities associated with obesity such as non-alcoholic fatty liver disease (NAFLD) a rising cause of cirrhosis associated to obesity and metabolic syndrome. Indeed, NAFLD can present as simple steatosis or evolve towards inflammation and steatohepatitis (NASH), with a 20 % risk of cirrhosis after 20 years. "Dyslipidemia" is a major risk factor for coronary heart disease (CHD). Low plasma levels of high density lipoprotein (HDL) cholesterol with either normal or elevated levels of low density (LDL) cholesterol is a significant risk factor for developing atherosclerosis and associated coronary artery disease in humans. Dyslipidemia is often associated with and caused by obesity.

Preferred obesity-related disorders may be in particular selected from the group consisting of dyslipidemia, non-insulin-dependent diabetes mellitus, insulin resistance, metabolic syndrome, coronary heart disease, atherosclerosis and non-alcoholic fatty liver disease.

Preferably, obesity and obesity-related diseases are not of genetic origin. In particular, obesity and obesity-related diseases induced by overeating, high fat diet, and/or hyperglycaemic diet are preferably contemplated.

Finally, the present invention also relates to a method for the prevention and/or the treatment of obesity, obesity related disease, overweight or overeating in a mammal in need thereof, comprising administering the individual with a prophylactically or therapeutically effective quantity of CB1 receptor agonist at low dose and glutamate receptor agonist, most preferably allosteric enhancers of NMD A receptor

In its broadest meaning, the term "treating" or "treatment" refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

In particular, "treatment" of obesity and obesity-related disorders may refer to the administration of the compounds or combinations of the present invention to reduce or maintain the body weight of an obese subject. One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the compounds of the present invention. Another outcome of treatment may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of treatment may be decreasing the occurrence of and/or the severity of obesity-related diseases. Another outcome of treatment may be to maintain weight loss.

In particular, "prevention" of obesity and obesity-related disorders may refer to the administration of the compounds of the present invention to reduce or maintain the body weight of a subject at risk of obesity. One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the compounds of the present invention. Another outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and/or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be to prolong resistance to weight gain. Another outcome of prevention may be to prevent weight regain. Moreover, if treatment is commenced in already obese subjects, such treatment may prevent the occurrence, progression or severity of obesity-related disorders.

Furthermore, the present invention related to a glutamate receptor agonist, preferably an allosteric enhancer of glutamate receptor, more preferably an allosteric enhancer of NMD A receptor in order to allow use of a CB1 receptor agonist in the prevention or the treatment of obesity, and obesity related disorder, overweight or overeating in a mammal in need thereof.

"Composition and method of treatment of anorexia cachexia syndrome"

The present invention provides methods and compositions (such as pharmaceutical compositions) for treating anorexia cachexia syndrome (ACS), underweight or under eating in a mammal in need thereof.

Thus, an object of the invention is a pharmaceutical composition comprising a high amount of at least one CB1 receptor agonist as a high dosage and an amount of at least one GABA receptor agonist for use in the prevention or the treatment of anorexia cachexia syndrome (ACS), underweight or under eating in a mammal in need thereof.

In a preferred embodiment, the amount of said CB1 agonist is supplied at a dosage level superior to 5 mg.

Typically said CB1 agonist according to the invention may be administered at a dose of about 5 to 20 mg for human (or about 1.75 to 5 mg/kg body weight for mice).

The term "GABA receptor" means class of receptors that respond to the neurotransmitter gamma-aminobutyric acid (GABA), the chief inhibitory neurotransmitter in the vertebrate central nervous system. There are two classes of GABA receptors: GAB A- A and GABA_B. GAB A- A receptors are ligand-gated ion channels (also known as ionotropic receptors), whereas GABA_B receptors are G protein-coupled receptors (also known as metabotropic receptors).

The active site of the GAB A- A receptor is the binding site for GAB A and several drugs such as muscimol, gaboxadol, and bicuculline. The protein also contains a number of different allosteric binding sites which modulate the activity of the receptor indirectly. These allosteric sites are the targets of various other drugs, including the benzodiazepines, nonbenzodiazepines, barbiturates, ethanol, neuroactive steroids, inhaled anaesthetics, and picrotoxin, among others.

The term "GABA receptor agonist "refers to any GABA receptor agonist that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in activation or up-regulation of a biological activity associated with activation of the GABA receptors in the patient, including any of the downstream biological effects otherwise resulting from the binding to GABA receptor of its natural ligand (GABA). Such GABA receptor agonists include any agent that can help glutamate receptor activation or any of the downstream biological effects of glutamate receptor activation. Most preferred gaba receptor agonist of the invention is an allosteric enhancer of GABA-A receptor.

Examples of GABA-A Agonists include but are not limited to : gaboxadol, isoguvacine, isonipecotic acid, muscimol.

Examples of GABA-A allosteric enhancers may be selected from the group consisting of: barbiturates, benzodiazepines (like diazepam : Valium®), carisoprodol, ethanol, etomidate, glutethimide, kavalactones, L-theanine, meprobamate, neuroactive steroids, nonbenzodiazepines, propofol, volatile/inhaled anaesthetics.

The term "amount of GABA receptor agonist" means using dose with a limited or no psychotropic effect of GABA agonist wherein the amount of at least one GABA agonist is supplied at a dosage level inferior to 1 mg.

By way of example, the compositions and use of an amount of GABA agonist according to the invention may be administered at a dose of about 0.1 to 1 mg for human.

The term "Cachexia" refers to a state of general ill health and malnutrition. It is often associated with and induced by malignant cancer, and it is characterized by loss of appetite, loss of body mass, especially lean body mass, and muscle wasting.

Cachexia is a syndrome characterized by an involuntary loss of weight and may include one or more of progressive loss of both fat and skeletal muscle, refractoriness of weight loss to increase nutritional input, elevated resting energy expenditure (REE), decreased protein synthesis, altered carbohydrate metabolism, hyper-catabolism/increased degradation of muscle via the ATP-ubiquitin-proteasome pathway of proteolysis and of adipose tissue via lipo lysis, asthenia, anemia, chronic fatigue, nausea, and loss of bone mass. Typically, at least 5% or 5 pounds of pre-illness body weight must have been lost before the patient is diagnosed with cachexia.

Of course, one or more of the above symptoms may or may not be present in a given subject depending on the underlying disease or condition associated with it and of the treatment already received by the subject for treating the underlying disease or condition. The above symptoms or physiological conditions may also be present at various degrees. Cachexia may or may not be associated with anorexia.

"Anorexia" refers simply to a loss of appetite, whether brought on by medical or psychological factors. Anorexia is often closely associated with, and generally contributes to, the cachexia seen in patients with advanced cancers

Anorexia is a medical term for appetite loss. Manifestations of anorexia include a decreased sense of taste and smell of food, early satiety, a decreased sense of hunger and even outright aversion of food.

The terminology Anorexia-Cachexia Syndrome (ACS) is a generic term used by physician as a diagnostic of patients having either anorexia or cachexia. As used herein therefore, ACS designates anorexia or cachexia. Diseases or conditions associated with or likely to be associated with ACS, include but are not limited to, cancer, immunodeficiency disorders such as AIDS, other infectious diseases including viral, bacterial and parasitic diseases, sepsis, rheumatoid arthritis and chronic diseases of the bowel, liver, kidneys, lungs and heart including congestive heart failure and chronic organ failure. It can also manifest itself as a condition in aging or as a result of physical traumas and burn injuries.

More specifically, diseases, conditions or disorders that are typically associated with cachexia include, but are not limited to, cancer, AIDS, liver cirrhosis, diabetes mellitus, chronic renal failure, chronic obstructive pulmonary disease, chronic cardiac failure, immune system diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), tuberculosis, cystic fibrosis, gastrointestinal disorders (e.g., irritable bowel syndrome and inflammatory bowel disease), Parkinson's disease, dementia, major depression, anorexia nervosa, an aged condition and sarcopenia. More typically, the diseases, conditions or disorders that are associated with cachexia include, but are not limited to, cancer, AIDS, liver cirrhosis, chronic renal failure, chronic obstructive pulmonary disease, chronic cardiac failure, immune system diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), tuberculosis, cystic fibrosis, gastrointestinal disorders (e.g., irritable bowel syndrome and inflammatory bowel disease), Parkinson's disease, dementia, major depression, anorexia nervosa, an aged condition and sarcopenia. Cachexia is a strong independent risk factor for morbidity and mortality. For example, cancer cachexia occurs in about half of all cancer patients and is more common in patients with lung and upper gastrointestinal cancers. Cancer patients with an involuntary 5% weight loss have a shorter median survival rate than patients with stable weight. Cancer patients with weight loss can respond poorly to chemotherapy and also can require increased chemotherapy treatments. The fact that a large proportion of cancer patients have cachexia, coupled with the demonstrated relationship between cachexia and mortality, has provided impetus for the search into underlying mechanisms and therapies that might prevent or reverse cachexia and provide a model for identifying additional therapies.

An "underlying disease or condition" is a disease or condition that is associated with ACS or that is likely to be associated with ACS.

As used herein Cancer Anorexia-Cachexia-Syndrome (CACS) is intended to include any form of cancer associated with ACS or likely to be associated with ACS. Non- limiting examples of cancers that are most often associated with ACS include gastric cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer lung cancer, prostate cancer, colon cancer, non-Hodgkin's lymphoma, sarcoma, acute non- lymphocytic leukaemia and breast cancer.

In an embodiment, a subject in need thereof is a subject diagnosed with ACS or having a disease or condition that is likely to be associated with ACS. Subjects having cancer or AIDS are examples of likely candidates.

In an embodiment, a subject in need thereof is a subject suffering from cancer. In another embodiment, the subject in need thereof is a subject suffering from cancer but which has not yet developed ACS.

In an embodiment, a subject in need thereof is a subject suffering from an immunodeficiency such as AIDS.

In a further embodiment, the subject in need thereof is a subject which has lost at least 5%, 8%, 10%, 12%, 15% or more of his/her initial weight prior to the onset of ACS. In another embodiment, the subject in need thereof is a subject which has lost at least 5%, 8%, 10%, 12%, 15%) or more of his/her weight within a six-month period.

In a further embodiment, the subject in need thereof is a subject that is desirous of increasing his/her appetite and/or weight.

In yet another embodiment, a subject in need thereof is a subject undergoing therapy for the underlying disease or condition which is associated with ACS or likely to be associated with ACS.

Thus, in one aspect of the present invention the pharmaceutical composition comprising CB1 agonist and GAB A- A agonist is administered prior to the onset of ACS as a preventive measure.

In another aspect of the present invention the pharmaceutical composition of the present invention is administered in combination with a drug or drugs used to treat the underlying disease or condition. In a further aspect, the composition of the present invention is administered once the subject has been diagnosed with ACS. In another embodiment, the composition of the present invention is administered in combination with one or more other drugs or food supplements used for the prevention and/or treatment of ACS.

A further object of the invention relates to a method for the prevention and/or the treatment of anorexia, cachexia, underweight or under eating in a mammal in need thereof in a mammal in need thereof, comprising administering the individual with a prophylactically or therapeutically effective quantity of CB1 receptor agonist at high dose and GABA-A receptor agonist, most preferably allosteric enhancers of GABA-A receptor.

Furthermore, the present invention related to an agonist GABA, most preferably an allosteric enhancer of GABA-A receptor, in order to allow the use of a CB1 receptor agonist in the prevention or the treatment of ACS, underweight or under eating in a mammal in need thereof.

Pharmaceutical composition and kits

The invention also relates to a pharmaceutical composition comprising at least one

CB1 receptor agonist as defined above and at least one glutamate receptor agonist as defined above, and optionally a pharmaceutically acceptable carrier. The invention also relates to a pharmaceutical composition comprising at least one CB 1 receptor agonist as defined above and at least one GAB A receptor agonist as defined above, and optionally a pharmaceutically acceptable carrier.

Preferably the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

The expression "pharmaceutically acceptable carrier" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- so lid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can notably be formulated for an intravenous, intramuscular, subcutaneous, mhaled/mirfmasal, oral and rectal administration, and the like.

The pharmaceutical compositions of the invention may contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

To prepare pharmaceutical compositions, an effective amount of the receptor agonist may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The form is preferably sterile and is fluid to the extent that easy syringability exists. It is preferably stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

A composition of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

By way of example, the compositions 1 of the invention may be administered for a low amount of CBl agonist at a dose of about 0.05 to 5 mg or about 0.1 to .5 mg or about 0.2 to 5 mg or about 0.5 to 5 mg for human.

By way of example, the compositions 2 of the invention may be administered for a high amount of CBl agonist at a dose of about 5 to 20 mg/kg body weight or about 5 to 15 mg or about 5 to 10 mg for human.

The invention also relates to a kit containing:

a CB 1 receptor agonist as defined above,

a Glutamate receptor agonist as defined above;

as combined preparation for simultaneous, separate or sequential use in the prevention or treatment of obesity and or obesity related disease.

According to preferred embodiments, said glutamate agonist is an allosteric enhancers of glutamate receptor, more preferably an allosteric enhancer of NMD A receptor.

The invention also relates to a kit containing:

a CB 1 receptor agonist as defined above,

a GABA receptor agonist as defined above;

According to preferred embodiments, said glutamate agonist is an allosteric enhancer of GABA-A receptor

A first receptor agonist can be administered prior to, concomitantly with, or subsequent to the administration of the second receptor agonist to a subject which had, has, or is susceptible to obesity related disease or ACS. The CBl agonist and the GABA agonist or glutamate agonist molecules are administered to a subject in a sequence and within a time interval such that the first receptor agonist can act together with the second receptor agonist to provide an increased benefit than if they were administered otherwise. Preferably, the binding molecules are administered simultaneously to the subject with an obesity related disease or ACS. Also preferably, the molecules are administered simultaneously and every day to said patient.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES

FIG. 1. Deletion of CB1 from cortical glutamatergic or GABAergic neurons results in opposite phenotypes in fasting-induced food intake, due to altered GABAergic or glutamatergic transmission, respectively, (a) Glu-CBl-KO mice (lacking CB1 receptors from cortical glutamatergic neurons) exhibit a decrease in food intake under vehicle treatment (left black and white bars). An ineffective dose of the NMD A receptor antagonist MK-801 (0.03 mg/kg, solid and dashed black bars) abolishes the phenotype of Glu-CBl- KO mice (white bars), (b) GABA-CB1-KO mice (lacking CB1 receptors from GABAergic neurons) display a hyperphagic phenotype (left black and white bars). An ineffective dose of the GABA-A receptor antagonist picrotoxin (0.3 mg/kg, solid and dashed black bars,) abolishes the phenotype of GABA-CB1-KO (white bars). N=6-15 per group.

FIG. 2. The doses of 1 mg/kg and 2.5 mg/kg THC induce a hyperphagic and a hypophagic effect in wild-type C57BL/6NCrl mice, respectively, which do not depend on altered locomotion. Dose response of THC effects in fasting-refeeding experiments in wild-type mice. Note the clear biphasic effect of the drug. The hyperphagic (1 mg/kg) and the hypophagic 2.5mg/kg) effects of THC are not accompanied by alterations in locomotor activity (figure not shown).*, p<0.05, **, p<0.01 as compared to vehicle. N=4-6 per group.

FIG 3 Schematic representation of the rationale for pharmacological experiments using THC in combination with allosteric modulators of NMD A or GABA-A receptors, (a) Under normal conditions, presynaptic action potentials and [Ca2+] levels regulate the synaptic release of GABA or glutamate. The presynaptic release of the neurotransmitter activates postsynaptic receptors and induces signalling in the postsynaptic neurons. The intensity of this signalling depends on the amount of neurotransmitter released presynaptically. (b) The administration of THC activates presynaptic CB1 receptors, decreases presynaptic [Ca2+] levels, and, thus, reduces the release of the neurotransmitter and postsynaptic signalling, (c) The co-administration of allosteric enhancers of NMD A and GABA-A receptors (D-cyclo-serine, DCS, and diazepam, DZP,) would "restore" the postsynaptic levels of activity of these receptors, thereby compensating the effects of the low and high doses of THC, respectively. FIG. 4. Dose response studies of the effects of the NMD A receptor allosteric enhancer D-cyclo-serine (DCS, a), and of the GABAA receptor allosteric enhancer diazepam (DZP, b) on fasting-induced food intake in wild-type C57BL/6CtrlN mice. *, p<0.05 as compared to respective vehicle controls. Dashed black bars, doses chosen for experiments in combination with THC, as described in the main text. N=4-8 per group.

FIG. 5. The hyperphagic and hypophagic effects of THC depend on glutamatergic and GABAergic transmission, respectively. (a,b) Allosteric enhancement of glutamatergic (NMDA) and GABAergic (GABA-A) neurotransmission reverts the hyperphagic and hypophagic effects of 1 and 2.5 mg/kg THC in C57BL/6-N mice, respectively, (a) An ineffective dose of D-cyclo-serine (DCS, 3 mg/kg,) reverts the hyperphagic effect of THC 1 mg/kg into a hypophagic effect, whereas the hypophagic effect of 2.5 mg/kg THC is unaltered by the simultaneous administration of DCS. (b) An ineffective dose of diazepam (DZP, 0.3 mg/kg.) does not interfere with the hyperphagic effect of 1 mg/kg THC, but it reverts the effect of 2.5 mg/kg THC from hypophagia to hyperphagia (dashed blue/violet bar), (c) The effects of DCS and DZP are evident also in combination with an intermediate dose of the CB1 agonist. THC 1.75 mg/kg has no effect of food intake per se (intermediate dose, compare with Fig. 2). However, in association with DZP or DCS, it results in a hyperphagic or a hypophagic effect, respectively.

*, p<0.05, p<0.001 as compared to vehicle treatments. N=6-10 per group.

EXAMPLE 1 MATERIALS

Animals

All experimental procedures were approved by the Committee on Animal Health and Care of INSERM and French Ministry of Agriculture and Forestry (authorization number, 3306369). Mice, aged 2-5 months, were maintained under standard conditions with food and water ad libitum. In most experiments male mice were used. Due to low availability of animals, male and female mice were used in the experiments with double Glu/GABA-CBl mutants. No significant difference was observed between genders in these mice (not shown). CB1 mutant mice (CB1-KO, Glu-CBl-KO and GABA-CB 1 -KO) were obtained, maintained and genotyped as described [18,19,8,20]. All lines were in a mixed genetic background, with a predominant C57BL/6NCrl contribution. All animals used in experiments involving mutant mice were littermates. For total CB1-KO mice, the parents of experimental animals were always heterozygous for the mutation. For conditional mutants, obtained using the Cre/loxP system, Cre-positive/CBlflox/flox males were bred with Cre-negative/CBlflox/flox females (i.e. phenotypically wild-type), in order to avoid potential influence of the mother's genotype on the adult phenotype of the experimental animals. To control for the potential influence of Cre expression on the observed phenotypes of conditional CB1 mutant mice, Cre-positive and Cre-negative littermate mice were derived from Cre-positive/CBl-WT males crossed with wild-type C57BL/6NCrl female mice. Dlx 5/6-Cre mice and NEX-Cre were the controls for the Cre effect of GABA-CB1-KO and Glu-CBl-KO, respectively [8,20]. Wild-type C57BL/6NCrl were purchased from JANVIER (France). Experimenters were always blind to genotypes and/or treatments.

Generation of double mutants Glu-GABA-CBl-KO

Glu-CBl-KO mice were crossed with GABA-CB1-KO mice in order to obtain a first generation with male mice bearing deletion in both glutamatergic and GABAergic neurons. These males were bred with CBlflox/flox females. The resulting litters contained either wild-type, Glu-CBl-KO, GABA-CB1-KO, or Glu/GABA-CBl-KO littermates, which were used for experiments.

The mice were genotyped by PCR, using the following primers: NEX-CRE forward primer for Glu-CBl-KO: TCTTTTTCATGTGCTCTTGG, Dlx5/6-CRE forward primer for GABA-CB1-KO: AGCAATCGCACTCACAACAGA, CRE reverse for both lines: CGCGCCTGAAGATATAGAAGA. CBlf/f alleles were detected as described [19].

Drugs

All drugs were purchased from SIGMA Aldrich (France). Stocks of all drugs were prepared in ethanol. The injectable solutions were prepared just before the experiments, by diluting the stock solutions into sterile distilled H20 (final ethanol concentration, 2%). The control vehicle solution was distilled H20 with 2% ethanol. Drugs or vehicle were injected 30 minutes before refeeding (see below). Behavioural tests

Fasting-induced food intake. Animals were single housed under a normal dark/light cycle (light on 7 A.M., light off 7 P.M.) for at least 7 days before the experiments. 2 hours after the light onset, animals were food deprived for 24 hours and then given free access to a pre-weighted amount of standard chow (Standard Rodent Diet A03, SAFE, France). Food intake was recorded 1 and 2 hours after refeeding[9]. In these conditions, spillage of food was minimal. It was however controlled by inspection of litter and calculated as not eaten food.

THC effect on locomotor activity. Locomotor activity in THC dose-response experiments was recorded during the first hour of refeeding by an automated system (Mice Actimetry System, IMETRONIC, France) and expressed as number of bins/h.

Palatable food intake. 2 hours after the light onset, ad libitum fed animals were presented with a pre-weighted pellet of palatable food (Happycookies for rodents, Vitakraft, Germany), together with a pre-weighted pellet of normal chow. Both pellets were placed onto the floor of the mouse home cage. After 30 minutes, both pellets were removed and weighed, after controlling for spillage (minimal in all experiments). All animals showed an approximate 100% preference for the palatable food (data not shown). Therefore, only data related to the consumption of palatable food are presented. Very similar results were obtained using the 5TUL diet (AIN-76A, Test Diet, Richmond, USA) (data not shown).

Immunohistochemical detection of CB1 receptors in the brain

Animals were deeply anaesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Brains were removed, post fixed 1 h in 4% PFA/ PBS and incubated over night in 20%> sucrose at 4°C. Brains were frozen and cut to 30 μιη slides in a cryostat (Microm HM 500M, Microm Microtech, Francheville, France). Slides were stored at -20°C in cryoprotection solution (25%o glycerol, 25% ethylene glycol, 0.5x PBS) until being processed for immunohistochemistry. Sections were washed five times for 10 min in PBS and incubated in blocking buffer (5% normal goat serum, 2.5% BSA, 0.3% Triton X-100) for 1 hour at room temperature. The primary antibody (LI 5 polyclonal rabbit anti CB1, a generous gift of Ken Mackie, Indiana University, Bloomington, IN, U.S.A.) was diluted 1 : 1000 in PBS containing 1% normal goat serum, 0.1 % BSA and 0.1 % Triton X-100. The slides were incubated 48 hours with the primary antibody at 4°C, washed five times for 10 min with PBS containing 0.1% Triton X-100. For signal detection we used the ABC kit of Vector Laboratories (Burlingame, CA, U.S.A.) followed by DAB staining according to manufacturer's protocols. After staining, sections were put onto slides and let dry over night. After ethanol dehydratation and xylene treatment, the slides were mounted with Eukitt and coverslipped (Kindler GmbH, Freiburg, Germany). The slides were analyzed with an Olympus SZX10 stereomicroscope (Olympus, France).

Statistical analysis

Data were analysed using one- or two-way ANOVA (using genotype and treatment as variables and post-hoc tests), or Student's t-test, where appropriate. Graphs and statistics were generated by GraphPad Prism 4.03 (U.S.A.) and GBstat vlO (U.S.A.).

EXAMPLE 2: Genetics and pharmacology studies

RESULTS

To dissect the roles of CB1 receptors on excitatory or inhibitory transmission, we recently generated conditional mutant mice, wherein the expression of the receptor lacks either in all the cells of the body (CB1-KO), in cortical glutamatergic (Glu-CBl- KO), or in GABAergic neurons (GABA-CB1-KO) [8]. In fasting-refeeding experiments, food intake was lower in CB1-KO mice as compared to wild-type littermates, confirming previous observations [9]. A similar phenotype was observed in Glu-CBl -KO (Fig. la), whereas GABA-CB1-KO displayed a hyperphagic phenotype (Fig. lb). These phenotypes were also observed when fed animals were exposed to a palatable food, confirming the effects of the mutations on stimulated feeding. No alterations in fasting-induced food intake were found in the appropriate genetic controls of CB 1 conditional mutant mice (not shown). These results suggest that the orexigenic functions of the ECS depend on the control of glutamatergic transmission, whereas the control of GABAergic activity leads to an ECS-dependent decrease in food intake. As CB1 activation is generally associated with a decrease in neurotransmitter release [1,2,10], the opposite phenotypes of Glu-CBl -KO and GABA-CB1-KO mice are likely due to increased activity of glutamatergic and GABAergic neurotransmission, respectively.

Consistently, the acute administration of an ineffective dose of the NMDA receptor antagonist MK-801 abolished the phenotype of Glu-CBl- KO mice (Fig. la). Similarly, an ineffective dose of the GAB A- A receptor antagonist picrotoxin abolished the phenotype of GABA-CB1-KO mice (Fig. lb). To check whether the two genetic mutations could phenotypically compensate each other, we generated double mutant mice bearing deletion both in cortical glutamatergic and in GABAergic neurons (Methods). Food intake was increased in single GABA-CB1-KO and decreased in Glu-CBl-KO mutant littermates. In contrast, double Glu/GABA-CBl-KO mice ate the same amount of food as wild-type littermates. Altogether, these results show that CB1 on cortical glutamatergic neurons exert an orexigenic function through the acute modulation of glutamatergic transmission, whereas CB1 receptors in GABAergic neurons mediate hypophagia through the acute reduction of GABAergic transmission.

As expected, the CB1 agonist A9-tetrahydrocannabinol (THC) exerted a biphasic effect on food intake in fasting-refeeding experiments (Fig. 2). To investigate the neuronal mechanisms of this biphasic effect, we administered a hyperphagic (1 mg/kg) or a hypophagic dose (2.5 mg/kg) of THC to CB1 mutant mice and their wild-type littermate controls. Whatever the dose, THC was inactive in CBl-KO mice. The hyperphagic dose of THC bore no effect in Glu-CBl-KO mice, whereas the higher dose decreased food intake in these mutants.

Conversely, the lower dose of THC significantly increased food intake in GABA- CB1-KO mice, whilst the higher dose was inactive in these mice.

Importantly, these doses did not alter locomotor activity, demonstrating that locomotion was not involved in the effects of the drug in our experimental conditions. Hence, these results indicate that the orexigenic effect of low doses of THC are mediated by CB1 receptors expressed in cortical glutamatergic neurons, whereas the hypophagic effect of higher doses specifically involves CB1 receptors on GABAergic neurons.

To rule out potential confounding mutant-related developmental alterations [11,12], acute pharmacological treatments were applied to wild-type C57BL/6NCrl mice. The activation of CB1 receptors by THC in either glutamatergic (low doses) or GABAergic neurons (high doses) should lead to a decreased release of the respective neurotransmitter, thereby reducing, but not abolishing, GABAergic and glutamatergic signalling, respectively [1,2] (Fig. 3a,b). Theoretically, these effects could be compensated by simultaneous treatments with allosteric enhancers of the respective receptors, which would strengthen the effects of the remaining synaptic neurotransmitters (Fig. 3c). Dose-response experiments allowed selecting ineffective doses of the allosteric enhancers of NMD A receptors, D-cyclo-serine, and of GABA-A receptors, diazepam. Fig. 4 shows that the two drugs bear a hypophagic and a hyperphagic effect, respectively. However, these effects are exerted at the minimal doses of 10 mg/kg for DCS and 1 mg/kg for DZP. Lower doses (3 mg/kg of DCS and 0.3 mg/kg of DZP) did not alter food intake. Therefore, these doses were chosen and used in combination with THC to identify possible synergistic effects. DCS fully reverted the hyperphagic effect of 1 mg/kg THC, significantly reducing the food intake of the mice receiving the two drugs, but it did not alter the hypophagic effect of the high dose of THC (Fig. 5a). Conversely, DZP failed to change the hyperphagic outcome of the low dose of THC, but it fully reverted the hypophagic effect of 2.5 mg/kg THC to a significant increase of food intake (Fig. 5b). Interestingly, DZP and DCS were also able to transform the effect of an ineffective intermediate dose of the CB1 agonist. THC at 1.75 mg/kg did not alter food intake in starved wild-type animals (Fig. 5c). However, the combination with DZP and DCS resulted into frankly hyperphagic and hypophagic effects, respectively (Fig. 4c).

These findings reveal two unexpected opposite brain functions of CB1 receptor activation in the regulation of food intake. First, the limited amount of CB1 receptors expressed on cortical glutamatergic neurons is responsible for the well-known orexigenic function of the ECS. Endogenous or exogenous cannabinoids very likely mediate this function by acting at CB1 receptors on terminals of cortical glutamatergic neurons, which project to sub-cortical areas involved in the regulation of feeding. Second, the majority of CB1 receptors, which are expressed on GABAergic neurons, mediate a previously unknown inhibitory role of the ECS on food intake. Inhibition of GABAergic transmission in cortical and/or sub-cortical regions is likely to be responsible for this function of the ECS. Importantly, ineffective doses of the NMDA allosteric enhancer DCS and of the GABA-A allosteric enhancer DZP were able to decrease and increase food intake if combined with different doses of THC, respectively. This clearly indicate a so-far unsuspected synergistic action of cannabinoid drugs with modulators of glutamatergic and GABAergic transmission in the control of stimulated food intake.

The overall promoting effects of the ECS on ingestive behaviour seem the end product of tightly regulated opposing functions. It is presently unknown how endogenous and/or exogenous CB1 agonists could "select" the neuronal type where they exert these opposite functions. The pharmacology of CB1 receptors might vary according to the neuronal populations where they are expressed. For instance, cortical glutamatergic neurons contain very limited amounts of the receptor [3], which could be maximally stimulated by low doses of agonists, whereas GABAergic terminals containing large amounts of CBl protein [3] might need higher concentrations of agonists to display a significant effect.

In conclusion, these findings open an unforeseen avenue for the development of pharmacological treatments of food-related disorders, able to target CBl on specific cell types and to exploit synergistic actions of cannabinoids with glutamatergic and GABAergic modulators.

REFERENCES

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

1. A pharmaceutical composition comprising a low amount of at least one CB1 receptor agonist and an amount of at least one Glutamate agonist and optionally a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1 for use in the prevention or the treatment of obesity, obesity related disorder, overweight or overeating in a mammal in need thereof.

3. The pharmaceutical composition according to claim 1 to 2 wherein Glutamate receptor agonist is an allosteric enhancer of glutamate receptor, more preferably an allosteric enhancer of NMD A receptor.

4. The pharmaceutical composition according to claim 1 to 3, wherein an amount of at least one Glutamate receptor agonist is comprise between 5 to 25 mg for human.

5. The pharmaceutical composition according to claim 1 to 4, wherein a low amount of at least one CB1 receptor agonist is comprise between 0.05 to 5 mg for human.

6. A pharmaceutical composition comprising a high amount of at least one CB1 receptor agonist and an amount of at least one GABA receptor agonist and optionally a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5 for use in the prevention or the treatment of anorexia cachexia syndrome, underweight or under eating in a mammal in need thereof.

8. The pharmaceutical composition according to claim 6 to 7 wherein said GABA receptor agonist is an allosteric enhancer of GABA-A receptor.

9. The pharmaceutical composition according to claim 6 to 8, wherein an amount of at least one GABA receptor agonist is comprise between 0,1 to 1 mg for human.

10. The pharmaceutical composition according to claim 6 to 9, wherein a low amount of at least one CB1 receptor agonist is comprise between 5 to 20 mg for human.

11. The pharmaceutical composition according to claim 7 to 10 wherein anorexia cachexia syndrome is associated with a disease selected from the group consisting of : cancer, AIDS, liver cirrhosis, diabetes mellitus, chronic renal failure, chronic obstructive pulmonary disease, chronic cardiac failure, immune system diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), tuberculosis, cystic fibrosis, gastrointestinal disorders (e.g., irritable bowel syndrome and inflammatory bowel disease), Parkinson's disease, dementia, major depression, anorexia nervosa, an aged condition and sarcopenia.

12. A kit containing:

a) a CB1 receptor agonist, and

b) a Glutamate receptor agonist,

13. A kit according to claim 12 wherein glutamate receptor agonist is an allosteric enhancer of glutamate receptor, more preferably an allosteric enhancer of NMD A receptor.

14. A kit containing:

a) a CB1 receptor agonist, and

b) a GABA receptor agonist,

as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of anorexia cachexia syndrome and anorexia cachexia syndrome related disorder.

15. A kit according to claim 14 wherein said GABA receptor agonist is an allosteric enhancer of GABA-A receptor.