WO2016026978A1

WO2016026978A1 - Methods and pharmaceutical compositions for treating drug addiction

Info

Publication number: WO2016026978A1
Application number: PCT/EP2015/069304
Authority: WO
Inventors: Guillaume Sandoz; Yannick COMOGLIO
Original assignee: Universite Nice Sophia Antipolis; Centre National De La Recherche Scientifique; INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2014-08-22
Filing date: 2015-08-24
Publication date: 2016-02-25

Abstract

The present invention relates to methods and pharmaceutical compositions for treating drug addiction, preferably alcoholism, ecstasy addiction or cocaine addiction, in a subject in need thereof. In particular, the present invention relates to a method for treating drug addiction in a subject in need thereof comprising administering the subject with a TREK agonist, particularly a TREK1 and/or TREK2 agonist.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATING DRUG ADDICTION

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositions for treating drug addiction in a subject in need thereof.

BACKGROUND OF THE INVENTION

Addiction is a primary, chronic disease of brain reward, motivation, memory and related circuitry. Dysfunction in these circuits leads to characteristic biological, psychological, social and spiritual manifestations. This is reflected in an individual pathologically pursuing reward and/or relief by substance use and other behaviors.

Addiction is characterized by inability to consistently abstain, impairment in behavioral control, craving, diminished recognition of significant problems with one's behaviors and interpersonal relationships, and a dysfunctional emotional response. Like other chronic diseases, addiction often involves cycles of relapse and remission.

Without treatment or engagement in recovery activities, addiction is progressive and can result in disability or premature death (definition of the American Society of

Addiction Medicine).

Drug addiction is a chronic and relapsing condition with a multifactorial etiology that includes genetic, neurobiological, psychological and environmental components. For a long time, psychological and behavior therapies have been considered the most effective long-term treatments. However, understanding of neurobiological mechanisms now permits to envisage promising pharmacotherapies for drug addiction. Since some decades, the research of medications for treating addictive disorders has increased. Although there are now promising therapeutic approaches for drug addiction, only a few of medications are approved for use in human; these treatments show benefit effects only for a few population of patients and important side effects. Thus, there is a substantial need for innovative therapeutic strategies for drug addictions.

Alcoholism is an example of drug addiction and constitutes a real health problem. The management of alcoholism is today still a stake for scientists and physicians, as the available treatments are not effective enough or involve important side effects.

Baclofen is a GABAB agonist drug approved for muscle spasms and cerebral spasticity. The use of baclofen is proposed for alcohol withdrawal symptoms and as an abstinence-promoting agent in alcohol-dependent subjects although its benefit effects are controverted; this molecule is also used for the treatment of cocaine addiction. However, baclofen shows several types of side effects such as sedation, nauseas, muscular weakness and pain and depression. Thus, new therapeutic approaches that permit to increase the ratio benefit/side effect are still needed.

In 2012, Sandoz et al. showed that TREKl contributes to the hippocampal

GABAB response using baclofen (Sandoz et al, Neuron, 2012 June 21; 74(6): 1005- 1014). This document shows that TREKl is an indirect target of baclofen. However, it does neither show nor suggest that TREKl and/or TREK2 is a target of alcohol. SUMMARY OF THE INVENTION

The present invention relates to a TREK agonist for use for treating drug addiction.

In a preferred embodiment, the present invention relates to a TREK agonist for use for treating alcoholism, more preferably chronic alcoholism.

In another preferred embodiment, the present invention relates to a TREK agonist for use for treating ecstasy or cocaine addiction.

In a particular embodiment, said TREK agonist is a direct TREK agonist.

Preferably, the TREK agonist is a TREKl and/or a TREK2 agonist, preferably a direct

TREKl and/or TREK2 agonist.

In another particular embodiment, the TREK agonist is not a GABAB agonist.

In a further preferred embodiment, the TREK agonist does not bind a GABAB receptor.

The invention further relates to a pharmaceutical composition comprising at least one TREK agonist of the invention for treating drug addiction, preferably alcoholism, ecstasy addiction or cocaine addiction.

The invention also provides a method for screening candidate compounds useful for treating drug addiction, preferably alcoholism, ecstasy addiction or cocaine addiction comprising a step (a) of testing each of the candidate compounds for its ability to activate at least one TREK channel and a step (b) of positively selecting the candidate compound(s) capable of activatiing said at least one TREK channel.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. TREKl is inhibited by protracted but not acute primary alcohol application. (A, B) Effect of acute primary alcohol application on TREKl current. (A) Representative example of TREKl current stability following brief (~1 minute) primary alcohol application in HEK 293T cells. (B) Summary of effect of acute primary alcohol application on TREKl current. Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). Inset, normalized TREKl current density after acute primary alcohol application. (C) Effect of protracted primary alcohol application on TREKl current in HEK 293T cells. Current was elicited by voltage-ramps (from - 100 to 50 mV, Is in duration). Inset, normalized TREKl current densities after acute primary alcohol application. (D) Effect of protracted butan-2-ol application. Insets, TREKl current densities before and after protracted butan-2-ol application are shown. (E) Summary of normalized TREKl current densities after protracted alcohol application. Student's t tests (*P < 0.05) shows the difference between TREKl and TREKl after ethanol, butan-l-ol or butan-2-ol application. The numbers of cells tested are indicated in parentheses. (F) Protracted ethanol application decreases native TREKl photocurrent in hippocampal neurons. Left, representative examples of TREKl photocurrent with (black trace) and without (gray trace) protracted (>1 hour) ethanol application. Right, average normalized TREKl photocurrent amplitudes with and without protracted ethanol application (>lhours).

Figure 2. TREKl is potentiated by PLD2 in a PA-dependent manner (A, B) TREKl is potentiated by PLD2 co-expression. (A) Representative traces showing that PLD2 co-expression increases TREKl current. (B) Bar graph showing the TREKl current densities in the presence or absence of co-expression of PLD2. Student's i test *** < 0.001) shows the difference between TREKl and TREKl co-expressed with PLD2. The numbers of cells tested are indicated in parentheses. (C-F) Primary alcohols and FIPI abolish the potentation of TREKl current by PLD2. Representative traces showing effects of protracted ethanol (C), butan-l-ol (D), or FIPI (E) application on TREKl current in the presence of PLD2. Insets, TREKl current densities before and after treatments. (F) Summary of normalized amplitude of TREKl current in the presence of PLD2 after protracted treatment with alcohol, FIPI or both. Student's t tests (*P < 0.05, **P < 0.01) show the difference between TREKl co-expressed with PLD2 and after ethanol, butan-l-ol or butan-2-ol application. Student's t tests (#P < 0.05) show the difference between TREKl and TREKl co-expressed with PLD2 after either ethanol or butan-l-ol or butan-2-ol application. The numbers of cells tested are indicated in parentheses. (G, H) A catalytically inactive mutant of PLD2 (PLD2- K758R) decreases TREKl current. (G) Representative traces showing that coexpression of PLD2-K758R decreases TREKl current. (H) Summary of TREKl current densities in the presence or absence of co-expressed PLD2-K758R and before and after protracted primary alcohol application are shown. Student's t tests (*P < 0.05) show the difference between TREKl and TREKl co-expressed with PLD2-K578R with or without protracted primary alcohol exposure. The numbers of cells tested are indicated in parentheses.

Figure 3. Model of channel regulation by PLD2. PLD2 is associated with TREKl and creates a microdomain rich in PA (gradient of PA is represented by a green arrow) that activates the channel at rest. Alternatively, primary alcohols (ROH) compete with water in the catalytic site of PLD2 which also leads to a reduction of the local PA concentration near the channel and causes a decrease in TREK channel activity.

Figure 4. TREKl is inhibited by MDMA application. TREKl activity is measured in HEK293T cells expressing TREKl by the patch clamp technique. (A) Representative TREKl current obtained by a ramp from -100 to +50 mV before (light grey in the figure, called "- MDMA"), after (dark grey in the figure, called "+ MDMA ") and after washout (curve in black in the figure, called "WASH") of 2.5mM of MDMA. (B) Bar graph showing the effect of an application of 2.5 mM of MDMA: A One Way Repeated mesure ANOVA test followed by a post-hoc test (Holm-Sidak) show a significant inhibition of TREKl activity with MDMA application.

Figure 5. TREK agonists reverse the inhibition induced by ethanol. (A) (left) representative TREKl current before and after ethanol incubation. (Middle) representative TREKl current after ethanol incubation and then after application of ML67-13. (Right) same as in the middle for ML67-18. (B) Bar graph representing the TREKl current for the different condition showed in (A). DETAILED DESCRIPTION OF THE INVENTION

The inventors highlighted the possible use of the TREK channels TREK1 and TREK2 as a direct therapeutic target for treating alcoholism.

They show that the phospholipase D2 (PLD2) specifically potentiates the effect of these channels by producing phosphatidic acid (PA) through hydrolysis of phosphatidylcholine that is present in the environment of said channels. They further show that the phospholipase D2 specifically activates TREK channels by a direct association and that the activity of TREK channels depends on phosphatidic acid production.

The primary alcohols compete with the binding of water to the catalytic site of

PLD2 leading to a decrease of the local concentration of phosphatidic acid in the environment of TREK channel. That consequently involves a decrease of TREK activity.

Thus, the inventors surprisingly showed a direct action of alcohol on TREK channels activity and thus propose to directly target TREK channels for treating alcoholism.

This inhibition of TREK channels can be prevented by baclofen but its action is indirect and via GABAB receptors. An action on GABAB receptor implies highly undesirable side effects that could be prevented by a direct action on TREK channels.

The inventors showed that TREKl is also inhibited by MDMA (also called ecstasy).

Thus, a first object of the invention relates to a TREK agonist for use for treating drug addiction.

As used herein, the term "drug addiction" refers to the compulsive use of chemicals (drugs, alcohol, etc.) and the inability to stop using them despite all the problems caused by their use. Drug addiction is considered as a disease. The term "substance dependence" may also be used.

As used herein, the term "drug addiction" encompasses addiction or dependence to any addictive substance; it includes, but is not limited to, alcohol addiction (also called alcoholism), opioid addiction, sedative, hypnotic or anxiolytic addiction (including benzodiazepine and barbiturate addictions), cocaine addiction, cannabis addiction, amphetamine addiction, ecstasy addiction; hallucinogen addiction, nicotine addiction. Preferably, said drug addiction is chosen among alcoholism, ecstasy addiction and cocaine addiction.

In a preferred embodiment, the invention relates to a TREK agonist for use for treating alcoholism.

As used herein, the term "alcoholism" or "alcohol addiction" has its general meaning in the art and refers to addiction to ethanol particularly contained in alcoholic drinks, which is considered as a disease, specifically an addictive illness, by the World Health Organization (WHO). The WHO differentiates two types of alcoholism: acute alcoholism and chronic alcoholism.

In a more preferred embodiment, the invention relates to the treatment of chronic alcoholism.

The term "chronic alcoholism" has its general meaning in the art and corresponds to an excessive regular drink associated with a state of dependency. The WHO defines the state of dependency when some symptoms of the trouble have persisted during at least one month or have repeatedly occurred over a prolonged period of time.

In another preferred embodiment, the invention relates to a TREK agonist for use for treating ecstasy or cocaine addiction.

The terms "ecstasy addiction" and "cocaine addiction" refer to addiction to said drugs.

The term "ecstasy" or "MDMA" (3,4-methylenedioxy-N-methylamphetamine) has its general meaning in the art and refers to a well-known empathogenic drug of the phenethylamine and amphetamine classes of drugs.

The term "cocaine" (benzoylmethylecgonine) has its general meaning in the art and refers to a well-known drug which is a tropane alkaloid that is obtained from the leaves of the coca plant. This addictive drug is considered as a hard drug.

As used herein, the term "TREK" or "TREK channels" has its general meaning in the art and refers to a subfamily of K₂p channels, more particularly to TREKl and TREK2.

As used herein, the term "TREKl" has its general meaning in the art and refers to the potassium channel subfamily K member 2 and that in humans is encoded by the KCNK2 gene. It is also called TREK-1, KCNK2, K_2p2.1, TPKC1. The term may include naturally occurring TREKl and variants and modified forms thereof. TREKl may be from any source, but typically is a mammalian (e.g., human and non-human primate) TREKl, particularly a human TREKl . An exemplary human native TREKl amino acid sequence is provided in NP 001017424 (GenPept database) and an exemplary human native TREKl mRNA sequence is provided in NM 001017424 (GenBank database).

As used herein, the term "TREK2" has its general meaning in the art and refers to the potassium channel subfamily K member 10 encoded by the KCNK10 gene. The protein encoded by this gene, also called K₂pl0.1, is a potassium channel containing two pore-forming P domains. The term may include naturally occurring TREK2 and variants and modified forms thereof. TREK2 may be from any source, but typically is a mammalian (e.g., human and non-human primate) TREK2, particularly a human TREK2. An exemplary human native TREK2 amino acid sequence is provided in NP 066984 (GenPept database) and an exemplary human native TREKl mRNA sequence is provided in NM 021161 (GenBank database).

The term "TREK agonist" (particularly "TREKl agonist" and "TREK2 agonist") encompasses any compound, naturally-occurring or synthetic, that specifically activates or promotes the biological activation of TREK channels (particularly TREKl and TREK2), thereby increasing TREK channels biological activity (particularly TREKl and/or TREK2 biological activity).

As used herein, the term "TREK channels biological activity" refers to an activity exerted by TREK channels, particularly TREKl and/or TREK2 which can be assessed by measuring the TREK channel current (particularly TREKl and/or TREK2 current) by well-known techniques. Such biological activity can be determined in vivo, in situ or in vitro, according to standard techniques and procedures. Particularly, the biological activity of TREK channels (particularly TREKl and/or TREK2) may be determined by measure of the current of said TREK channels by electrophysiology, more particularly by the two-microelectrode voltage-clamp technique or patch clamp technique. Other techniques may be used, for example using a fluorescent reporter of membrane potential. One of this fluorescent reporter is the DiSBAC. The DiSBAC fluorescence intensity is sensitive to membrane potential. Therefore, if a drug is active on a TREK channel, the fluorescence changes. The cell-based assay using DiSBAC which has been applied to TRAAK (another K2P channel) is well described in Marion et al, 2014, Jun 19 ;157(7): 1565-76. In a preferred embodiment, a TREK agonist of the invention is a TREK1 and/or TREK2 agonist. According to the invention, a TREKl and/or TREK2 agonist has an agonist activity on TREKl, on TREK2 or on both TREKl and TREK2.

Such an activity can be a direct activity, such as an association with a TREK channel ("TREK"), or an enzymatic activity on a second protein, or an indirect activity, such as a cellular process mediated by interaction of TREK channels with a second protein or a series of interactions as in intracellular signaling.

Particularly, said TREK agonist is a direct TREK agonist, such as phosphatidic acid.

As used herein, a "direct TREK agonist" refers to a compound that has a direct activity on TREK channels, particularly TREKl and/or TREK2, and that does not have an activity on a second protein upstream or downstream. Typically the direct TREK agonist is a direct TREKl and/or TREK2 agonist, i.e. a direct TREKl agonist, a direct TREK2 agonist, or a direct TREKl and TREK2 agonist.

Preferably, according to the invention, said TREK agonist does not have an activity on, typically does not bind, a GABAB receptor, and is not a GABAB agonist.

Preferably, the TREK agonist is not baclofen.

This direct activity on TREK channels and particularly without activity on GABAB receptor advantageously allows the decrease or inhibition of any side effects associated with GABAB activation. Such kind of side effects is observed with baclofen, which is a GABAB agonist.

TREK agonists are well known in the art and novel TREK agonists may be found within numerous classes of compounds, including small molecules, antibodies, peptides, nucleic acid molecules, aptamers, and the like. A TREK agonist may be a synthetic or natural compound; it may be a single molecule or mixture or complex of different molecules.

In one embodiment, the TREK agonist (particularly a TREKl and/or TREK2 agonist) is a small organic molecule.

The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In one embodiment, the TREK agonist of the invention is a caffeate ester which activates TREK1 channel described in Rodrigues N. et al, European Journal of

Medicinal Chemistry, 2014.

In a particular embodiment, said TREK agonist is a compound of formula (I):

• Rl is CN or CH₂NH₃ ⁺C1^"

• X is O, NH, CH₂.

In another particular embodiment, said TREK agonist is a compound of formula (I) wherein:

•

• X is O, NH or CH2.

In further particular embodiment, said TREK agonist is a compound of formula (I) wherein:

• Rl is CN

• X is O.

In a preferred embodiment, said TREK agonist is cinnamyl 3,4-dihydroxyl-a- cyanocinnamate, which has the following formula (II):

In another preferred embodiment, said TREK agonist is a compound of formula (I) wherein:

• Rl is CH₂NH₃ ⁺C1^"

• R2 is H, and

• X is NH,

and thus is a compound of formula (III):

In another preferred embodiment, said TREK agonist is the compound of formula (IV):

In another preferred embodiment, said TREK agonist is the compound of formula (V):

In another preferred embodiment, said TREK agonist is the compound of formula (VI):

In another particular embodiment, the TREK agonist is a compound illustrated herein below of formula:

I),

o

r (VIII),

wherein :

• A, when present, is CR3R4, where R3 and R₄, identical or different, are selected from H and an alkyl group from CI to C5, for example C2, C3 or C4, preferably C(Me)_2;

• Ri and R₂, identical or different, are selected from H, halogen atoms, a (C1-C5) alkoxy group, preferably OCH3 (OMe), and a cyano group (CN). The term "halogen" by himself or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (CI), bromine (Br), or iodine atom (I). A preferred halogen atom is selected from CI and Br. Each of Ri and R2 are preferably identical and preferentially located on position 3, 6 (see herein below ML67-17 and ML67-18) ;

• L is a substituted or unsubstituted aliphatic molecule (linear chain) including 2 to 5 carbon elements, for example 3 or 4 carbon elements, which is saturated or not, typically a substituted or unsubstituted alkane, alkyne or alkene, for example selected from methane, ethyne, ethene, ethane, propyne, propene, propane, 1, 2-butadiene, 1- butine, butene, butane and n-pentane; or a substituted or unsubstituted aliphatic cyclic molecule (cyclo-alkane) including 3 to 7 carbon elements, for example 4, 5 or 6 carbon elements, for example selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane. The substituted or unsubstituted aliphatic cyclic molecule can be satured or not. When saturated, it is typically a substituted or unsubstituted cyclo-alkyne or cyclo-alkene. L can also be a substituted or unsubstituted heteroalkyl including 2 to 4 carbon elements, or heterocycloalkyl including 3 to 6 carbon elements, further including at least one element selected from oxygen, nitrogen, sulfur and chlorine.

When substituted, L can be bound to one or several substituents consisting in a linear or cyclic aliphatic compound such as herein above defined, typically a linear aliphatic compound comprising 1 to 24 carbon elements or a cyclic aliphatic compound comprising one cycle comprising 3 to 7 carbon elements, for example 4, 5 or 6 carbon elements, or several such cycles, said linear or cyclic aliphatic compound optionally comprising at least one element selected from oxygen, nitrogen, sulfur and chlorine.

In a preferred embodiment, the TREK agonist of formula (VII) is the compound of formula (IX) also herein identified as ML67-17 [trans-3-(3,6-dichloro-9H-carbazol- 9-yl)cyclobutanecarboxylic acid] :

(IX)

In another preferred embodiment, the TREK agonist of formula (VII) is the compound of formula (X) also herein identified as ML67-18 [9-(2-(lH-tetrazol-5- yl)ethyl-3,6-dichloro-9H-carbazole]:

In a further preferred embodiment, the TREK agonist of formula (VII) is the compound of formula (XI) also herein identified as ML67-13 [(3-(3,6-dibromo-9H- carbazol-9-yl)propanoic acid] :

Methods for producing ML67-13, ML67-17 and ML67-18 are well known by the skilled person. Examples of methods for producing ML67-17 and ML67-18 are provided by inventors in the experimental part.

Particularly preferred direct TREK agonists according to the present invention, in particular TREK1 and/or TREK2 agonists, are compound of formula VII or VIII, in particular a compound of formula VII selected from ML67-13, ML67-17 and ML67-18, even more preferably from ML67-17 and ML67-18.

In another particular embodiment, the TREK agonist is 2-Aminoethoxydiphenyl borate, which is known to activate TREK channels (Beltran L. et al, Front Pharmacol., 2013).

Alternatively, the TREK agonist (particularly TREK1 or TREK2 agonist) may consist in an antibody (the term including "antibody portion").

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of TREK. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immuno stimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

In one embodiment, the antibody is selected from a monoclonal antibodya polyclonal antibody, a humanized antibody, a chimeric antibody and any functional portion thereof.

As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments and single chain antibodies.

In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Came lid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody(R)". According to the invention, sdAb can particularly be llama sdAb.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

According to the invention, such an antibody (or fragment thereof) has an agonistic effect towards TREK channels (particularly TREK1 and/or TREK2).

In another embodiment the TREK agonist (particularly TREKl and/or TREK2 agonist) is an aptamer.

Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against TREK as above described, the skilled man in the art can easily select those promoting TREK function.

According to the invention, such an aptamer has an agonistic effect towards TREK channels (particularly TREKl and/or TREK2).

In another embodiment the TREK agonist may consist in a polypeptide or a nucleic acid which has an agonistic effect towards at least one TREK channel (particularly TREKl and/or TREK2).

Agonistic activity or effect of a candidate compound towards TREK channels, particularly TREKl and/or TREK2 channels, may be determined by any well-known method in the art, particularly by screening methods of the invention described below.

A further object of the invention relates to a pharmaceutical composition comprising at least one TREK agonist of the invention for the treatment of drug addiction.

In a preferred embodiment, said pharmaceutical composition is used for the treatment of alcoholism.

In a more preferred embodiment, said pharmaceutical composition is used for the treatment of chronic alcoholism.

In another preferred embodiment, said pharmaceutical composition is used for the treatment of ecstasy or cocaine addiction.

In one embodiment, said at least one TREK agonist is a TREKl and/or TREK2 agonist.

In a particular embodiment, said at least one TREK agonist is a direct TREK agonist, typically a direct TREKl and/or TREK2 agonist, i.e. a direct TREKl agonist, a direct TREK2 agonist, or a direct TREKl and TREK2 agonist.

In a preferred embodiment, said at least one TREK agonist is not a GABAB receptor agonist.

In another preferred embodiment, said at least one TREK agonist does not bind a GABAB receptor.

Typically, the TREK agonist is not baclofen.

The TREK agonist of the present invention is typically combined with pharmaceutically acceptable excipient(s), and optionally at least one sustained-release matrix, such as a biodegradable polymer, to prepare a pharmaceutical composition. "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 being, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical composition contains at least one vehicle which is a pharmaceutically acceptable vehicle for a formulation capable of being injected. This may be in particular an isotonic, sterile, saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or a dry, especially a freeze-dried composition which upon addition, depending on the case, of sterilized water or physiological saline, permits the constitution of an injectable solution. 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. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention 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. The polypeptide (or nucleic acid encoding thereof) 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 are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several 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. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the subject being treated and on the subject's condition. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

A further object of the invention relates to a method for treating drug addiction in a subject in need thereof, said method comprising administering said subject with an effective amount of at least one TREK agonist of the invention.

In a preferred embodiment, said method is for treating alcoholism.

In a more preferred embodiment, said method is for treating chronic alcoholism. In another preferred embodiment, said method is for treating ecstasy or cocaine addiction.

In another embodiment, said at least one TREK agonist is a TREKl and/or TREK2 agonist.

In a particular embodiment, said at least one TREK agonist is a direct TREK agonist, typically a direct TREKl and/or TREK2 agonist, i.e. as a direct TREKl agonist, a direct TREK2 agonist, or a direct TREKl and TREK2 agonist.

In a preferred embodiment, said at least one TREK agonist does not bind a GABAB receptor.

Typically the TREK agonist is not baclofen.

According to the invention, the TREK agonist of the invention is administered to the patient in a therapeutically effective amount.

By a "therapeutically effective amount" is meant a sufficient amount of the compound of the present invention (e.g. TREK agonist) to treat the disease (i.e. alcoholism) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors/conditions including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; as well as upon any other factors well known in the medical art. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

A further object of the invention relates to a method for screening drugs for use for treating drug addiction, preferably alcoholism, ecstasy addiction or cocaine addiction, comprising the steps of testing compound(s) for its/their ability to be a TREK agonist [typically to bind and activate (open) a TREK channel], and selecting positively the compound(s) that is/are identified as TREK agonist(s).

In a preferred embodiment, said at least one TREK agonist is not a GABAB receptor agonist. Typically the TREK agonist is not baclofen.

In a preferred embodiment, said at least one TREK agonist does not bind a GABAB receptor. In some embodiments, the screening method of the invention comprises a first step of determining whether the candidate compound is able to bind at least one TREK channel, particularly TREKl and/or TREK2.

Methods for determining whether a candidate compound binds to a protein are well known in the art. Binding of the candidate compound to at least one TREK channel, particularly TREKl and/or TREK2, can be detected by any of a number of methods known to those of skill in the art. For example, in some embodiments, the candidate compounds are labelled with a detectable label (e.g., a fluorescent label, a colorimetric label, a radioactive label, a spin (spin resonance) label, a radio-opaque label, etc.). The membrane comprising the protein of interest (i.e. TREK channel, particularly TREKl and/or TREK2) is contacted with the candidate compound, typically washed, and then the membrane is screened for the detectable label indicating association (binding) of the test agent with the protein of interest. In some embodiments a secondary binding moiety (e.g. bearing a label) is used to bind and thereby label the bound test agent, or to bind the protein in which case association of the label on the secondary agent with the label on the test agent indicates binding of the test agent to the protein. In the latter case, in some embodiments, the selected label on the test agent and the selected label on the secondary agent can be labels that undergo fluorescent resonance energy transfer (FRET) so that excitation of one label results in emission from the second label thereby providing an efficient means of detecting association of the labels. In some embodiments, a competitive binding assay is used. In such assays, a "competitive" agent known to bind to the protein of interest is also utilized. The competitive agent can be labelled and the amount of such agent displaced when the bilayer containing the protein of interest is contacted with a test agent provides a measure of the biding of the test agent. Methods of detecting specific binding are well known and commonly used, e.g. in various immunoassays. Any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168) are well suited to detect test agent binding to proteins in a lipid bilayer. For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

In some embodiments, the screening method of the present invention is performed in screening cells. Typically, said screening cells are neurons, such as neurons derived from PC 12 cells or are macrophages, such as Raw267.4; COS-7 and HEK293 cells may also be used. In some embodiments, the screening cells retain a first DNA that encodes at least one TREK channel (particularly TREKl and/or TREK2), for example two TREK channels Method for introducing a nucleic acid sequence encoding for a protein of interest are well known in the art.

In some embodiments, the screening method consists in determining whether the candidate compound is able to induce hyperpolarisation of the screening cells. The step of contacting the screening cells with a candidate compound is accomplished for example by adding the candidate compound to the saline solution used for the patch clamp assay. The step of contacting a screening cell with a candidate compound for example includes contacting the cell simultaneously with a plurality of candidate compounds simultaneously, such as two or more candidate compounds, or three or more candidate compounds, etc.

Methods for analysing hyperpolarization of a screening cell are well known in the art and typically involve patch-clamp technique. Indeed, single channel ion currents are studied using the patch-clamp technique (see, e.g., Neher and Sakmann (1976) Nature, 260: 799-802; Sakmann and Neher (1983) Single Channel Recording, Plenum New York), in which a glass pipette filled with electrolyte (i.e. saline solution) is used to contact the membrane surface and measure ionic current. Various chip-based patch clamping methods are also known (see, e.g., Fertig et al. (2002) Appl. Phys. Letts., 81 : 4865-4867). In some embodiments the use of an ex vivo system comprising two chambers separated by a lipid bilayer, that contains channel of the present invention (i.e. TREK channel, particularly TREKl and/or TREK2). The conductance across the lipid bilayer is monitored continuously. Typically, the TREK channel (particularly TREKl and/or TREK2) is activated so that the resting membrane potential becomes deeper in the negative direction, or in other words, so that the negative potential increases. The resting membrane potential is preferably deepened in the negative direction to a degree that does not affect cell viability. The induced hyperpolarization is -5; -6; -7; -8; -9; -10; -11; -12; -13; -14; -15; -16; -17; -18; -19; -20; -21; -22; -23; -24; - 25; -26; -27; -28; -29; -30; -31; -32; -33; -34; -35; -36; -37; -38; -39; -40; -41; -42; -43; -44; -45; -46; -47; -48; -49; or -50 mV. In some embodiments, the induced hyperpolarization is compared to a predetermined reference value and when the hyperpolarization induced by the candidate compound is higher than the predetermined reference value then the candidate compound is selected. Typically the predetermined reference value represents the hyperpolarization determined in the absence of the candidate compound or the hyperpolarization induced by a reference compound (for example cinnamyl 3,4-dihydroxyl-a-cyanocinnamate).

In some embodiments, a phenotypic assay could be used for analysing hyperpolarization of a screening cell. Such a phenotypic assay may consist in the cell- based fluorescence assay coupled with image acquisition by automated confocal microscopy. Briefly this assay consists in measuring changes in the fluorescent intensity of a potential-sensitive fluorochrome when the screening cells are contacted with the candidate compound. The potential-sensitive fluorochrome as used herein may be any of the types that are generally available in the art concerned and a suitable one may be selected from among the following: styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs; cyanine- or oxonol-based potential-sensitive fluorochromes comprising DiSC's, DiOC's, DiIC's, DiBAC's, and DiSBAC's; and rhodamine-derived potential-sensitive fluorochrome such as Rh 123, TMRM, and TMRE. In the present invention, it is more preferred to use styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs, which are specifically exemplified by di-8-ANEPPS, di-4-ANEPPS, RH-237, RH-1691, di-5- ASP, RH-160, RH-421, RH-795, di-4-ANEPPDHQ, ANNINE-5, and ANNINE-6, and a preferred potential-sensitive fluorochrome may be selected from among these. For fluorescence assay, any type of fluorescent microscope that can be used in the art concerned may be applied in the present invention and a typical example is 1X71 (OLYMPUS Corporation). In the Examples that follow, 1X71 (OLYMPUS Corporation) was used as a fluorescent microscope and combined with a suitable light source unit such as a mercury lamp (OLYMPUS Corporation) or an LED assembly (OLYMPUS Corporation). For the purposes of capturing fluorescent images, imaging and numerical calculations, any models of analysis software for imaging and numerical calculations that can be used in the art concerned may be applied in the present invention. In some embodiments, confocal images are recorded on an automated fluorescent confocal microscope Opera™ (Evotec). Each image is then processed using dedicated image analysis software. In some embodiments, the screening cells (e.g. Raw267.4 cells) are stained with the potential-sensitive fluorochrome for a sufficient time. Then the extracellular dye is washed away and image acquisition is performed (Tl). The candidate compound is then added to the assay plate, which is incubated for an additional sufficient time. A second image acquisition is then performed (T2). Automated image analysis determined the average intensities for each well and the intensity ratio is calculated ([T2int] / [Tlint]). Accordingly, in some embodiments, the screening method of the present invention comprises the steps of i) bringing the candidate compound into contact with screening cells of the present invention previously labeled with potential-sensitive fluorochrome, ii) measuring a test value representative of the fluorescent intensity of the potential-sensitive fluorochrome, iii) comparing the test value measured at step ii) with a predetermined reference value and iv) positively selecting the candidate compound as a TREK agonist when the test value measured at step ii) is at least equal to the predetermined reference value or higher than the predetermined reference value. Typically, the predetermined reference value is the value measured in the absence of the candidate compound or the value measured while using a compound of reference (for example cinnamyl 3,4-dihydroxyl-a- cyanocinnamate).

Typically, the candidate compound may be selected from the group consisting of peptides or polypeptides, peptidomimetics, small organic molecules, antibodies, aptamers and nucleic acids. For example the candidate compound according to the invention may be selected from a library of compounds previously synthesized, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesized de novo. In some embodiments, the candidate compound may be selected from small organic molecules.

The screening methods of the invention are very simple. It can be performed with a large number of candidate compounds, serially or in parallel. The method can be readily adapted to robotics. For example, the above assays may be performed using high throughput screening techniques for identifying candidate compounds for developing drugs that may be useful to the treatment or prevention of pain. High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 384-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system. Thus, large libraries of candidate compounds may be assayed in a highly efficient manner. More particularly, stably-transfected cells growing in wells of micro-titer plates (96 well or 384 well) can be adapted to high through-put screening of libraries of compounds. Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes containing the transgenic cells described hereinabove.

In some embodiments, the candidate compound(s) that has/have been positively selected may be subjected to further selection steps in view of further assaying its/their properties in vitro assays or in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans. Any well- known animal model may be used for exploring the in vivo therapeutic effects of the screened candidate compound(s). For example, the therapeutic activity of the screened candidate compounds can be determined by using an animal model of drug addiction (such as animal model of alcohol, ecstasy or cocaine addiction). Once again the screened candidate compound may be compared to a reference compound such as baclofen. If the screened candidate compound provides the same effects or even better effects than the reference compound, said candidate compound could be typically selected for further clinical investigation.

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

EXAMPLES

• Example 1

Material and methods

Molecular Biology and Gene Expression. HEK293T cells were transiently co- transfected using Lipofectamine 2000 (INVITROGEN) with a total of 1.6 μg of DNA total per 18 mm diameter cover slip. For co-expression of TREKl, TREK2, or TRAAK with PLD2, PLD1, or PLD2-K758R cells were transfected with a ratio of 1 :4. For co- expression of TREKl and TREKl -PCS a ratio of 1 :3 or 1 :5 was used. All channel DNA was used in the pIRES2eGFP vector. TREKl -PCS was previously made by PCR and introduced into the pIRES2eGFP expression vector. Phospholipase D (PLD) variants were used in the pCGN vector. Chimeras between TREKl (mouse, DDBJ/EMBL/GenBank accession No. U73488) and TRAAK (mouse, DDBJ/EMBL/GenBank accession No.NM_008431) were generated by PCR. The carboxyl-terminal domain (Ctd) of TRAAK was replaced by the corresponding Ctd of TREKl (starting at Gly 293) to form TRAAK/Ct-TREKl and the cytoplasm Ctd of TREKl was replaced by the corresponding Ctd of TRAAK (starting at Gly 255) to form TREK 1 / Ct-TRAAK. The tandem between PLD2 and TRAAK was made by PCR. The PLD2 was fused to the N-terminus of TRAAK. Hippocampal neurons were transfected at 9 DIV using the calcium phosphate method. Each 12 mm covers lip received 1 μg of TREKl -PCS DNA and 1 μg of PLD2-K758R DNA or empty vector. Cell Culture. HEK293 cells were maintained in DMEM with 5% FBS on poly-L- lysine-coated glass coverslips. Dissociated hippocampal neurons were obtained from postnatal rats (PO-1) and plated at 75,000 cells/coverslip on poly-L-lysine-coated glass coverslips (12 mM). Neurons were maintained in media containing MEM supplemented with 5% fetal bovine serum, B27 (INVITROGEN), and GlutaMAX (INVITROGEN).

Electrophysiology. HEK293 cell electrophysiology was performed 24-72 h after transfection in solution containing (in mM): 145 NaCl, 4 KC1, 1 MgCk, 2 CaCk and 10 HEPES. Glass pipettes of resistance between 3 and 6 ΜΩ were filled with intracellular solution containing (in mM): 140 KC1, 10 Hepes, 5 EGTA, 3 MgCk, pH 7.4. Cells were voltage clamped using an Axopatch 200A (Molecular Devices) amplifier in the whole cell mode. Currents were elicited by voltage-ramps (from -100 to 50 mV, Is in duration) and the current density was calculated at 0 mV. For alcohol experiment, cells were incubated at least one hour before recording. For inside out patch experiments, the internal solution contained (in mM): 155 KC1, 5 EGTA and 10 HEPES and the external solution contained (in mM) 140 KC1, 10 Hepes, 5 EGTA, 3 MgCk, pH 7.4. To confirm the PA-sensitivity of the TREK channel subfamily, 5μΜ PA was applied directly to the patches.

Hippocampal neuron whole cell patch clamp electrophysiology was performed 3-6 days after transfection (DIV 12-15 for cultured neurons). For voltage and current clamp experiments in cultured neurons, extracellular solution contained (in mM): 138 NaCl, 1.5 KC1, 1.2 MgCk, 2.5 CaCk, 10 glucose, 5 Hepes, 0.002 TTX, pH 7.4. Intracellular solution contained (in mM): 140 K-Gluconate, 10 NaCl, 5 EGTA, 2 MgCk, 1 CaC12, 10 Hepes, 2 MgATP, 0.3 Na₂GTP, pH 7.2. Only cells with a resting potential <-45 mV were analyzed.

Optogenetic and electrophysiology. The inventors expressed an engineered TREKl subunit that renders native TREKl subject to block by a light-controlled switch. This engineered "photoswitchable conditional subunit", or TREK1-PCS, contains two modifications. The first modification is the introduction of a cysteine attachment site (S121C) near the external mouth of the pore for the covalent attachment of a photoswitchable tethered blocker (PTB). This PTB is called MAQ (Maleimide Azobenzene Quaternary ammonium). In response to 380 nm light, the tethered MAQ isomerizes from a stable trans configuration to a metastable cis configuration and allows the quaternary ammonium moiety to block the channel pore. Block is removed by illumination at 500 nm, which isomerizes MAQ back to the trans configuration. The second modification is a deletion in the TREK1 C terminal domain that retains TREK1- PCS homomultimeric channels inside the cell. When the TREK1-PCS is expressed in a cell that natively expresses wild-type (WT) TREK1, heteromeric TREK1-PCS/WT channels co-assemble, reach the cell surface and are subject to photo-block. For photoswitching experiments, illumination was controlled using a Polychrome V monochromator (TILL Photonics) through a 20x objective. pClamp software was used for both data acquisition and control of illumination. To conjugate MAQ, cells were incubated in 20-40 μΜ MAQ for 40 minutes in the dark at room temperature in standard extracellular cell buffer for either HEK293 cells or hippocampal neurons. Cells were washed thoroughly with extracellular buffer before beginning experiments. Immunocytochemistry. Transfected cells on coverslips were fixed with PBS containing 4% paraformaldehyde (15 min at 21-22°C), then permeabilized with PBS and 0.1% Triton X-100 (PBST) and blocked for 1 h with 5% horse serum (HS) in PBST. Primary and secondary antibodies were diluted in PBST and 5% HS and incubated for 1 h at 21-22°C. Three 5-min washes with PBST were carried out between each incubation step and at the end of the procedure. Coverslips were mounted in Dako^® Fluorescent Mounting medium (Dako Corporation, Carpinteria, CA, USA). The following antibodies were used: rabbit anti-TREKl polyclonal antibodies rat monoclonal antibody 3F10 against hemagglutinin (HA) epitope (Roche Diagnostics), goat anti-rabbit IgGs conjugated to Alexa Fluor^® 488 (Molecular Probes Europe BV, Leiden, The Netherlands), donkey anti-rat IgGs conjugated to Alexa Fluor^® 594 (MOLECULAR PROBES EUROPE BV, Leiden, The Netherlands). Microscopy analysis and data acquisition were carried out with an Axioplan 2 Imaging Microscope (CARL ZEISS, Le Pecq, France). Immunoprecipitation and Western blot analysis. HEK293T cells were homogenized in PBS containing saponin (0.5% w/v), Triton X-100 (0.5% w/v) and protease inhibitors (Roche Diagnostics, Basel, Switzerland). Lysates were clarified by centrifugation at 20,000g for 30 min. Anti-TREKl antibodies were immobilized on protein A-Sepharose 4B fast flow columns (Sigma, Saint Louis, MO, USA). Immunoprecipitated proteins were separated on 10%> SDS polyacrylamide gel and blotted onto nitrocellulose membranes (Hybond-C extra, AMERSHAM BIOSCIENCES, Freiburg, Germany). Detection was carried out using rat monoclonal antibody 3F10 against hemagglutinin (HA) (ROCHE DIAGNOSTICS).

Results

TREKl is inhibited by protracted but not acute primary alcohol application.

TREK channels can be stimulated by phospholipids, including directly applied (phosphatidic acid) PA but so far there has been no determination of whether such PA- mediated activation is regulated. Since alcohols target Phospholipase D (PLD) and PLD catalyzes the production of PA, the inventors wondered if alcohol might modulate TREK channels through PLD. A diverse population of potassium channels is directly regulated by ethanol, including BK, SK, Kv and GIRK. The inventors initially investigated the possible regulation of TREKl by alcohols in a heterologous system. They first tested primary alcohols and found that acute application of either 0.25 % butan-l-ol (27 mM) or 0.6 % ethanol (104 mM) for ~1 minute did not modify TREKl current in HEK 293T cells (Figure 1A, B). However, protracted (>1 hour) application of either of these primary alcohols reduced TREKl current by around 50% (Figure 1C, F; current densities were 39 ± 5 pA/pF for TREKl, 18 ± 5 pA/pF for TREKl + ethanol; P<0.05 and 18 ± 4 pA/pF for TREKl + butan-l-ol; P<0.05). They then tested secondary alcohols and found that, unlike ethanol or butan-l-ol, protracted application of 0.25 %> butan-2-ol did not modify TREKl current (Figure ID, E; current density was 43 ± 6 pA/pF, P>0.4).

The inventors then investigated the potential regulation of native TREKl current by alcohol in primary cultures of hippocampal neurons. They expressed an engineered TREKl -Photoconditionnal subunit (TREKl -PCS) to endow light sensitivity to the native TREKl channels. As in HEK293T cells, protracted (>1 hour) application of 0.6% of ethanol reduced TREKl current by around 70% compared to untreated cells (Figure IF). These results suggest that primary alcohols modulate native and heterogeneously expressed TREKl channels via an indirect mechanism, such as a metabolic effect on a second messenger that regulates TREKl .

PLD2-mediated potentiation of TREKl current is reversed by protracted primary alcohol treatment and the PLD inhibitor FIPI. The inventors next asked whether the observed effects of alcohol on TREKl could be mediated by PLD. To address this question, they first set out to determine if PLD can regulate TREK activity. They tested this by co-expressing TREKl and PLD2. They found that PLD2 co-expression increased TREKl current by more than 4-fold (Figure. 2A, B; current densities for TREKl and TREKl +PLD2 were 19 ± 2 pA/pF and 86 ± 9 pA/pF, respectively; PO.001).

Since the production of PA by PLD2 is inhibited by primary alcohols, they wondered if protracted treatment with primary alcohols would affect the potentiation of TREKl by PLD2. In the presence of co-expressed PLD2, protracted application of either ethanol or butan-l-ol reduced TREKl current by 71% (Figure 2C, D; current densities were 39 ± 5 pA/pF for TREKl alone, 139 ± 21 pA/pF for TREKl + PLD2, 24 ± 5 pA/pF for TREKl + PLD2 + ethanol, and 22 ± 5 pA/pF for TREKl + PLD2 + butan-l-ol; P<0.01 for TREKl + PLD2 vs. TREKl + PLD2 + ethanol and P<0.01 for TREKl + PLD2 vs. PLD2 + butan-l-ol). This inhibition is reversed after washout within ~ 30 min. Notably, the current densities observed for TREKl co-expressed with PLD2 and treated with ethanol or butan-l-ol were not significantly different than the current amplitude for TREKl expressed alone after primary alcohol incubation (Figure 2F; P>0.4 for TREKl + ethanol vs. TREKl + PLD2 + ethanol and P>0.5 for TREKl + butan-l-ol vs. TREKl + PLD2 + butan-l-ol). Consistent with the previous section, the secondary alcohol butan-2-ol failed to modify TREKl current when co-expressed with PLD2 (Figure 2F; current density was 127 ± 44 pA/pF, P>0.7). In summary, primary but not secondary alcohols suppressed the ability of PLD2 to enhance current by TREKl (Figure 2F). Since primary alcohols, but not secondary alcohols, can serve as alternative substrates in PLD-catalyzed transphosphatidylation to produce phosphatidylalcohols instead of PA, these results suggest that the inhibition of TREKl by primary alcohols is mediated by inhibition of the production of PA by PLD2. To confirm that the effect of alcohol on TREKl is directly linked to inhibition of PA production, the inventors used the recently developed, specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). Incubation for one hour with FIPI reduced TREKl + PLD2 current by 76% to a level similar to primary alcohol incubation (Figure 2E, F; current density was 19 ± 4 pA/pF for TREKl + PLD2 + FIPI, P<0.01 for TREKl + PLD2 vs. TREKl + PLD2 + FIPI). Furthermore, coapplication of primary alcohol and FIPI did not show an additional inhibitory effect indicating that both treatments may work through the same mechanism (Figure 2F; current density was 24 ± 6 pA/pF, P>0.4 for TREKl + PLD2+ FIPI vs. TREKl + PLD2 + FIPI + EtOH and P>0.6 for TREKl + PLD2+ EtOH vs. TREKl + PLD2 + FIPI + EtOH). In addition, FIPI reduced TREKl current densities to similar levels with or without PLD2 coexpression as was also observed for primary alcohol treatment.

These results strongly support the idea that PLD2 potentiates TREKl channel activity through production of PA.

PLD2-mediated potentiation of TREKl requires basic residues in the TREKl C terminus. The results so far indicate that the regulation of TREKl channels by PLD2 depends on the production of PA by PLD2. To further test this idea, the inventors turned our attention to the portion of the TREKl channel that is known to be essential for PA regulation, and where PA has been conjectured to bind. Stimulation of TREKl by PA depends on five positively charged residues in the TREKl carboxyl-terminal domain (Ctd) and the negative charge of the phosphate group of PA. This modulation can be eliminated by mutation of the positively charged residues to produce "TREKl - pentaA".

To test if the ability to sense phospholipids is required for TREKl to be potentiated by PLD2, they examined the effect of PLD2 co-expression on TREKl -pentaA. Unlike in wild-type TREKl, TREKl -pentaA was not potentiated by PLD2 co-expression.

Together with the suppression of PLD2 modulation of TREKl by primary alcohols and FIPI, this result argues that enzymatic production of PA by PLD2 is required for stimulation of TREKl .

A catalytically inactive mutant of PLD2 decreases TREKl current. These findings that protracted exposure to primary alcohols and FIPI reduces TREKl current in cells transfected with only TREKl, that the magnitude of this suppression is far greater when PLD2 is co-expressed and that the current that remains after primary alcohol or FIPI treatment is the same whether or not PLD2 is co-expressed, suggest that endogenous PLD2 tonically stimulates TREKl and that this stimulation is suppressed by primary alcohols or FIPI. In order to test if TREKl is regulated by endogenous PLD2, the inventors co-expressed a catalytically inactive mutant of PLD2 (PLD2-K758R). Co- expression of PLD2-K758R significantly decreased the TREKl current (Figure 2G, H; current densities were 51 ± 7 pA/pF and 28 ± 3 pA/pF for TREKl and TREKl + PLD2-K758R, respectively; P<0.05). This suppression was similar to that elicited by protracted application of primary alcohols and FIPI. Moreover, protracted primary alcohol application did not further inhibit TREKl current when PLD2-K758R was co- expressed (Figure 2H; current densities were 30 ± 4 pA/pF for TREKl + PLD2-K758R + ethanol and P>0.6 and 29 ± 4 pA/pF for TREKl + PLD2-K758R + butan-l-ol; P>0.7) which is consistent with a dependency of primary alcohol regulation of TREK on PLD2.

The ability of the over-expressed catalytically-inactive form of PLD2 to prevent endogenous wild-type PLD2 from stimulating TREKl could be explained by competition for localization to the vicinity of TREKl . The inventors therefore examined this possibility by asking if the channel and enzyme directly associate.

PLD2, but not PLD1, specifically regulates TREKl through direct interaction. Having found that PLD2 modulates TREKl, the inventors asked whether a related phospho lipase D, PLD1, has the same effect. Whereas co-expression of PLD2 significantly increased TREKl current (current densities were 168 ± 28 pA/pF for TREKl + PLD2 versus 41 ± 8 pA/pF for TREKl alone; P<0.001), co-expression of PLD1 had no effect on TREKl current (current density was 58 ± 9 pA/pF for TREKl + PLD1; P>0.6).

They asked whether the ability of PLD2 but not PLD1 to stimulate TREKl could be accounted for by direct association of only PLD2 with the TREKl channel. PLD2 was co-immunoprecipitated with TREKl, but PLD1 was not. As a control, in the absence of TREKl expression, anti-TREKl antibodies did not precipitate PLD2. Furthermore, using immunocytochemistry, they found that TREKl and PLD2 co-localize in HEK293T cells, but TREKl and PLD1 do not. In addition, PLD2-K758R is also coIP with TREKl, confirming the competition hypothesis with an endogenous PLD2 to induce the TREKl current decrease (Figure 2G).

Taken together, these results indicate that PLD2 interacts with TREKl but that PLD1 does not and that this explains the exclusive modulation of the channel by PLD2. PLD2 is able to potentiate TREK2, but not TRAAK. Having observed that PLD2 interacts with and regulates TREK1 but that PLD1 does not, the inventors asked whether the interaction and modulation extend to other members of this K₂p subfamily of channels. To test this, they co-expressed PLD2 with TREK2 and the more distantly related TRAAK channel. TREK2 and TRAAK, like TREK1, are lipid and mechano- gated and display the same PA-sensitivity as TREKl . PLD2 co-expression significantly increased TREK2 current (current densities were 24 ± 3 pA/pF for TREK2 alone and 144 ± 29 pA/pF for TREK2 + PLD2; P>0.01) but did not significantly affect TRAAK current (current densities were 8 ± 3 pA/pF for TRAAK alone and 8 ± 3 pA/pF for TRAAK + PLD2; P>0.8). Consistent with these results, PLD2 was co- immunoprecipitated with TREK2, but not with TRAAK. As a control, in the absence of TREK2 expression anti-TREK2 antibodies did not precipitate PLD2, confirming the specificity of the assay. In addition, immunocytochemistry of PLD2 and TREK2 showed co-localization, while TRAAK and PLD2 showed no overlap. Futhermore, they found that PLD1 co-expression does not affect TREK2 current ( current densities were 31 ± 11 pA/pF for TREK2 alone and 39 ± 21 pA/pF for TREK2 + PLD1; P>0.9 for TREK2 alone vs. TREK2 + PLD1). These results suggest that PLD2 interacts with, and hence regulates, TREK2, which is closely related to TREKl, but not the more distantly related TRAAK.

Having found that TREK2 resembles TREKl, the inventors asked if it is also similar in the ability to be inhibited by protracted exposure to primary alcohols. They found that, like TREKl, TREK2 current is reduced by protracted exposure to primary alcohols; and, moreover, that this effect is potentiated when PLD2 is over expressed along with TREK2. Unlike TREKl and TREK2, TRAAK, which is not potentiated by PLD2 over- expression, is not sensitive to protracted exposure to primary alcohols. In summary, TREKl and TREK2 are similar to one another and differ from the other TREK channel family member TRAAK in the ability to be specifically regulated by primary alcohols via PLD2 through specific interaction between channel and enzyme.

To further test the idea that direct interaction between enzyme and channel is necessary for the channel to be potentiated, the inventors forced an interaction between TRAAK and PLD2 by fusing the proteins to one another to produce a PLD2-TRAAK tandem. PLD2-TRAAK showed significantly increased current compared to TRAAK alone (current densities were 5.5 ± 2 pA/pF for TRAAK alone and 51 ± 13 pA/pF for PLD2- TRAAK; P<0.01). Similarly to TREK1 and TREK2, protracted application of ethanol reduced the current density of PLD2-TRAAK to the amplitude observed for TRAAK alone indicating that PLD2-mediated production of PA is required for the potentiation observed with PLD2-TRAAK. These results are consistent with the notion that anchoring of PLD2 to the channel enables it to regulate the channel's activity via its local enzymatic activity.

The inventors next investigated to which part of the channel PLD2 binds. They hypothesized that this interaction may take place in the Carboxy terminal domain (Ctd) because this is the major part of TREK channels that is accessible to the cytosol and this domain is highly conserved between TREK1 and 2. To test this hypothesis, they designed chimeras between TRAAK and TREKl to see if they could transfer PLD2 sensitivity to TRAAK. The Ctd of TRAAK was replaced by the corresponding Ctd of TREKl to form TRAAK/Ct-TREKl and the Ctd of TREKl was replaced by the corresponding Ctd of TRAAK to form TREKl/Ct-TRAAK. Unlike wild type TRAAK, TRAAK/Ct-TREKl was sensitive to PLD2 (current densities were 12 ± 3 pA/pF and 36 ± 7 pA/pF for TRAAK/Ct-TREKl and TRAAK/CtTREKl + PLD2, respectively; P<0.05). However, TREKl/Ct-TRAAK was not sensitive to PLD2 co-expression (current densities were 26 ± 4 pA/pF and 29 ± 2 pA/pF for TREKl/Ct-TRAAK and TREKl/Ct-TRAAK + PLD2 respectively, P>0.8).

These results indicate that the specificity of PLD2 depends on the TREKl Ctd, because the Ctd specifically binds to PLD2.

PA targets native TREKl channels though physical coupling with endogenous PLD2 in hippocampal neurons. TREK channels are natively expressed in the hippocampus where they contribute to the response to GABAB receptor activation and are inhibited by protracted primary alcohol application (Figure IB). Accordingly, the inventors wondered if hippocampal TREKl channels are regulated by endogenous PLD2 and if this can explain their alcohol sensitivity. To investigate this regulation in the native hippocampal TREKl channels, they co-expressed the catalytically inactive mutant of PLD2, PLD2-K758R, along with the TREKl -PCS.

In cultured hippocampal neurons transfected with the TREKl -PCS, alternating illumination between 380 nm and 500 nm modulated the resting membrane potential by 4.3 ± 0.9 mV. Co-expression of PLD2-K758R decreased this voltage change significantly (E; 1.3 ± 0.2 mV; P<0.01). Consistent with this, in voltage clamp at a holding potential of -20 mV, TREK1-PCS transfected neurons had photocurrents of 20 ± 4 pA and co-expression of PLD2-K758R reduced the photocurrents to 4.8 ± 1.7 pA (F; P<0.01) as was observed for protracted EtOH application.

These results show that in hippocampal neurons native TREK1 and PLD2 co-assemble and that this co-assembly leads to a tonic increase in TREK1 activity.

Conclusion. The inventors demonstrated that protracted application of primary alcohols inhibits TREK1 and TREK2 and provided evidence that this effect is mediated by PLD2 rather than direct interaction between alcohols and the channel. When exposed to primary alcohols such as ethanol or butan-l-ol, PLD2 catalyzes the production of the biologically inactive phospholipids PEtOH or P-l-BtOH rather than PA (Figure 3). Consistent with the hypothesis that regulation of TREK by alcohols is mediated by removal of tonic stimulation by PLD2, PLD2 is insensitive to secondary alcohols, which were also unable to regulate TREK1 or TREK2. They confirmed this mechanism by showing that primary alcohols also prevent the potentiation effect of PLD2 and that a catalytically inactive mutant of PLD2 is unable to potentiate TREKl and renders TREKl insensitive to protracted alcohol treatment. This may be a general mechanism by which ethanol can induce long term physiological changes by changing the phospholipid composition of the membrane. · Example 2

Having highlighted the possible use of the TREK channels TREKl and TREK2 as a direct therapeutic target for treating alcoholism, the inventors explored the effect of MDMA (ecstasy) on TREK channel.

They showed that TREKl is inhibited by MDMA by the technique of patch clamp described in the example 1 (Figure 4). The inhibition of TREKl by MDMA is rapid and reversible, and thus seems to be direct.

Cocaine having generally the same mechanisms of action and working, the inventors reasonably think that cocaine also inhibits TREK channels, particularly TREKl . The inventors thus explore the effect of cocaine on TREK channel by the technique of patch clamp described in the example 1.

In conclusion, the inventors showed that TREK channels could be used as a direct therapeutic target in the treatment of alcoholism and further showed that this new therapeutic approach could be used in other drug addictions such as ecstasy and cocaine addiction.

• Example 3

Material and methods

Molecular Biology and Gene Expression

HEK293T cells were transiently transfected using Lipofectamine 2000 (Invitrogen) with a total of 1.6 μg of DNA total per 18 mm diameter cover slip. TREKl channel DNA was used in the pIRES2eGFP vector. They were used for electrophysiological studies 1 day after injection.

For xenopus oocytes, dcfol!iculated Xetwpus oocytes were injected with cRNA encoding mouse TREKl (5 ng). They were used for electrophysiological studies 2-4 days after injection.

Cell Culture

HEK293 cells were maintained in DMEM with 5% FBS on poly-L-lysine-coated glass coverslips. Dissociated hippocampal neurons were obtained from postnatal rats (PO-1) and plated at 75,000 cells/coverslip on poly-L-lysine-coated glass coverslips (12 mM). Neurons were maintained in media containing MEM supplemented with 5% fetal bovine serum, B27 (Invitrogen), and GlutaMAX (Invitrogen).

Electrophysiology

HEK293 cell electrophysiology was performed 24-72 h after transfection in solution containing (in mM): 145 mM NaCl, 4 mM KC1, 1 mM MgCk, 2 mM CaCk and 10 mM HEPES. Glass pipettes of resistance between 3 and 6 ΜΩ were filled with intracellular solution containing (in mM): 140 KC1, 10 Hepes, 5 EGTA, 3 MgCk, pH 7.4. Cells were voltage clamped using an Axopatch 200A (Molecular Devices) amplifier in the whole cell mode. Currents were elicited by voltage-ramps (from -100 to 50 mV, Is in duration) and the current density was calculated at 0 mV.

For two electrode voltage clamp, in a 0. -ml. perfusion chamber, a single oocyte was impaled with two standard microelectrod.es (1-2.5 ΜΩ resistance) filled with 3 M KG and maintained under voltage clamp by using a Dagan TEV 200 amplifier, in standard ND96 solution. (96 mM NaCl, 2 mM KC1, 1.8 mM CaCk, 2 m : MgCk, and 5 mM Hepes, I I 7.4, with NaOH). Stimulation of the preparation, data acquisition, and analysis were performed using pClamp software (Molecular Devices). To test the effect on the inhibition induced by 100 ml ethanol, inventors first have incubated the cells for an hour prior to test the agonists. They called "reversion" when the agonist bring back the channel activity to the same level as the activity observed in the absence of ethanol. for the same agonist.

Results

Different agonists were tested either in Xenopus oocytes (TEVC) or in HEK cells (patch-clamp). Using 5 μΜ of each molecules, ML67-17 allows a 4.2 fold increase of the channel activity in the HEK expression system, ML67-18 allows a 3 fold increase of the channel activity in the Xenopus oocytes and a 9 fold increase of the channel activity in the HEK expression system and ML67-13 allows a 3 fold increase of the channel activity in the Xenopus oocytes and a 17 fold increase of the channel activity in the HEK expression system. Inventors further observed a 4.3 fold increase of the channel activity in the HEK expression system while using 5μΜ of ML42 (amantadine derivative). ML67-18 and ML67-13 have further been identified as capable of reversing the ethanol TREK1 inhibition.

These results confirm that TREK agonists can be efficiently used for treating drug addiction. Exam le 4: Synthesis of ML67-17

MLW-17

(85% trans)

i) NaBH₄, THF, MeOH; ii) TsCl, pyridine, CH₂C1₂; iii) 3,6-dichlorocarbazole, NaH, DMF; iv) LiOH, MeOH, THF. 3,6-dichlorocarbazole. A round bottom flask was charged with 9H-carbazole (20.0 g, 119.6 mmol) and dichloromethane (200 mL) and the mixture was stirred at 0 °C. Sulfuryl chloride (9.69 mL, 119.6 mmol) was slowly added at that temperature. The dark reaction mixture was stirred at 0 °C for 2h and then diluted with CH₂Cl₂and aq. NaHC0₃. The organic layer was separated and washed with aq. NaHS0₃, brine, and dried (Na₂S0₄). The solution was then filtered and concentrated to afford the crude product as thick oil. This was recrystallized from hexanes/ethyl acetate to afford 3,6- dichlorocarbazole (12) as a white solid (15.2 g, 54%). 1H NMR (300 MHz, DMSO- 6): 5 11.56 (s, 1H), 8.27 (d, J=l .8 HZ, 2H), 7.50 (d, J=8.7 Hz„ 2H), 7.39 (d, J=8.7 Hz, 2H). tert-butyl 3-(tosyloxy)cyclobutanecarboxylate (3). A mixture of tert-butyl 3- oxocyclobutanecarboxylate (1, 1.50 g, 8.8 mmol) in THF:MeOH (3 : 1 , 16 mL) was added dropwise to a stirring slurry of sodium borohydride (0.167 g, 4.4 mmol) in THF (8 mL) in round bottom flask cooled in an ice bath. The mixture was stirred at 0-5 °C for two hours. Water was added dropwise (10 mL) followed by aq. Na₂C0₃, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with brine and dried over sodium sulfate.

After filtration, the organic layer was concentrated to give the crude tert-butyl 3- hydroxycyclobutanecarboxylate (2) as a white semi-solid (2.3 g, 100%), which was used in the next step without purification. p-Toluenesulphonyl chloride (4.201 g, 0.022 moles) was added to a stirring solution of crude tert-butyl 3 hydroxycyclobutanecarboxylate (2, 2.30 g, 8.8 mmol) in dry pyridine (10 mL) and CH₂C1₂(20 mL) at 0 °C. The mixture was allowed to warm to room temperature and stirred under nitrogen overnight. The solvent was then removed under reduced pressure and the residue was partitioned between ethyl ether (100 mL) and 0.5 N aq. HC1 (20 mL). The organic layer was separated and washed with saturated NaHC03and brine, and dried (Na₂S0₄). After filtration, the solvent was removed under reduced pressure and the residue purified by silica gel flash chromatography (0-50% EtOAc-hexane) to afford tert-butyl 3- (tosyloxy)cyclobutanecarboxylate (3) as a colorless oil that slowly solidified at room temperature (2.6 g, 90% yield over 2 steps). 1H NMR (300 MHz, CDC13): δ 7.79 (d, 2H, J = 8.4 Hz), 7.35 (d, 2H, J = 8.1 Hz), 4.72 (m, 1H), 2.60-2.30 (m, 8H), 1.44 (s, 9H). cis and trans-tert-butyl 3-(3,6-dichloro-9H-carbazol-9-yl)cyclobutanecarboxylate

(4). To a stirred solution of 3,6-dichlorocarbazole (5, 694 mg, 2.94 mmol) in dry DMF (15 mL) under nitrogen was added 60% sodium hydride in mineral oil (127 mg, 3.19 mmol). The reaction mixture was stirred at room temperature for 20 min and then at 60°C for 30 minutes, and then cooled to rt. Solid tert-butyl 3- (tosyloxy)cyclobutanecarboxylate (3, 800 mg, 2.45 mmol) was added and the reaction mixture was stirred at 60 °C overnight. The reaction mixture was then cooled to room temperature and quenched with water and extracted with ethyl acetate. The organic layer was separated and washed with brine, dried (Na₂S0₄), filtered, and concentrated. The crude residue was purified over silica gel (0-20%> ethyl acetate/hexane) to afford cis-4 as a light yellow syrup which solidified on standing (220 mg), and then trans-4as colorless syrup which solidified on standing (580 mg). cis-4: 1H NMR (300 MHz, CDC13): δ 7.99 (d, 2H, J = 1.8 Hz), 7.47 (AB, 2H, J = 8.7 Hz), 7.41 (AB d, 2H, J = 8.7, 1.8 Hz), 5.45 (m, 1H), 3.35-3.15 (m, 3H), 3.00-2.80 (m, 2H), 1.57 (s, 9H). trans-4: 1H NMR (300 MHz, CDC13): δ 7.99 (d, 2H, J = 2.1 Hz), 7.66 (d, 2H, J = 9.0 Hz), 7.43 (dd, 2H, J = 8.7, 2.1 Hz), 5.08 (m, 1H), 3.40-3.20 (m, 2H), 3.02 (5 peaks, 1H, J = 8.7 Hz), 2.90-2.75 (m, 2H), 1.56 (s, 9H).

trans-3-(3,6-dichloro-9H-carbazol-9-yl)cyclobutanecarboxylic acid (ML67-17). A mixture of trans-tert-butyl 3-(3,6-dichloro-9H-carbazol-9-yl)cyclobutanecarboxylate (trans-4, 320 mg, 0.82 mmol) and lithium hydroxide monohydrate (336 mg, 8.2 mmol) in THF-MeOH (1 : 1, 10 mL) was stirred at room temperature overnight and then concentrated. The residue was treated with water and adjusted to pH ~ 3 with 2N aq.

HC1. The solids that precipitated out of this solution were collected by filtration, washed with water, and dried in air to afford the title compound (275 mg, >95% yield; 85% trans). 1H NMR (300 MHz, DMSO- 6): δ 8.33 (d, 2H, J = 2.1 Hz), 7.87 (d, 2H, J

= 9.0 Hz), 7.47 (dd, 2H, J = 9.0, 2.1 Hz), 5.32 (m, 1H), 3.15-2.90 (m, 3H), 2.85-2.65

(m, 2H); LCMS m/z 331.8 (M-l). • Example 5: Synthesis of ML67-18

i) B1-CH₂CH₂CN, NaH, DMF; ii) NaN₃, NH₄CI, DMF.

3-(3,6-dichloro-9H-carbazol-9-yl)propanenitrile (6). A round bottom flask was charged with 3,6-dichlorocarbazole (5, 175 mg, 0.74 mmol), sodium hydride (27 mg, 1.11 mmol) and DMF (5 mL) under nitrogen. The reaction mixture was stirred at 60° C for 30 minutes and 3-bromopropionitrile (62 μΐ_^, 0.74 mmol) was added and the reaction mixture stirred at 60° C overnight. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, and washed with water and brine. The organic solvents were removed under reduced pressure and the residue purified by flash chromatography over silica gel (0-30% ethyl acetate- hexanes) to give 3-(3,6-dichloro- 9H-carbazol-9-yl)propanenitrile (6) as white solid (140 mg, 65%). 1H NMR (300 MHz, CDC 13) δ 8.00 (dd, J=2.1 &0.6 MHz, 2H), 7.49 (d, J=2.1 Hz, 1H), 7.46 (d, J=1.8 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 4.63 (t, J=6.9 Hz, 2H), 2.86 (t, J=6.9 Hz, 2H).

9-(2-(lH-tetrazol-5-yl)ethyl-3,6-dichloro-9H-carbazole (ML67-18). A mixture of 3- (3,6-dichloro-9H-carbazol-9-yl)propanenitrile (6, 140 mg, 0.48 mmol), sodium azide (94 mg, 1.45 mmol) and ammonium chloride (104 mg, 1.94 mmol) in DMF (5 mL) was stirred at 120 °C for 6h, after which time LCMS analysis indicated complete reaction. The reaction mixture was diluted with EtOAc (50 mL), washed with brine, dried over Na₂S0₄, filtered, and concentrated. The crude residue was purified by flash chromatography over silica gel (0-10% MeOH/CH₂Cl₂) to afford 9-(2-(lH-tetrazol-5- yl)ethyl-3,6-dichloro-9H-carbazole (ML67-18) as a beige solid (165 mg, 93%). 1H NMR (300 MHz, CDC 13) δ 8.23 (d, 1H, J = 1.5 Hz), 8.13 (d, 1H, J = 1.8 Hz), 7.25 (t, 2H, J = 8.1 Hz), 7.05 (t, 1H, J = 7.5 Hz), 6.83 (d, 2H, J = 7.8 Hz), 3.35 (s, 3H), 3.13 (t, 4H, J = 7.2 Hz), 1.31-1.20 (m, 4H), 1.13-0.99 (m, 4H), 0.80 (t, 6H, J = 7.2 Hz); LCMS m/z 444.2 (MH+).

Claims

1. A TREK agonist for use for treating drug addiction wherein said TREK agonist is not baclofen.

2. The TREK agonist according to claim 1 for use for treating alcoholism.

3. The TREK agonist according to claim 2 for use for treating chronic alcoholism.

4. The TREK agonist according to claim 1 for use for treating ecstasy or cocaine addiction.

5. The TREK agonist according to any one of claims 1-4, wherein said agonist is a TREK1 and/or TREK2 agonist.

6. The TREK agonist according to any one of claims 1-5, wherein said agonist is a direct TREK agonist, in particular a direct TREKl and/or TREK2 agonist.

7. The TREK agonist according to any one of claims 1-6, wherein said agonist is a compound of formula:

wherein:

• A, when present, is CR3R4, where R3 and R₄, identical or different, are selected from H and an alkyl group from CI to C5, preferably C(Me)₂;

H · B is COOH or ^M ; • Ri and R2, identical or different, are selected from H, halogen atoms, preferably CI or Br, a (C1-C5) alkoxy group, preferably OCH3 (OMe), and a cyano group (CN); and

• L is a substituted or unsubstituted aliphatic compound including 2 to 5 carbon elements, said aliphatic compound being saturated or not, or a substituted or unsubstituted aliphatic cyclic compound including 3 to 7 carbon elements.

8. The TREK agonist according to any one of claims 1-7, wherein said agonist is not a GABAB agonist.

9. A pharmaceutical composition comprising a TREK agonist according to any one of claims 1-8 for treating drug addiction.

10. The pharmaceutical composition according to claim 9 for treating alcoholism.

11. The pharmaceutical composition according to claim 10 for treating chronic alcoholism.

12. The pharmaceutical composition according to claim 9 for treating ecstasy or cocaine addiction.

13. A method for screening candidate compounds useful for treating drug addiction comprising a step (a) of testing each of the candidate compounds for its ability to activate at least one TREK channel and a step (b) of positively selecting the candidate compound(s) capable of activating said at least one TREK channel.

14. The method according to claim 13, wherein the step (a) consists in testing each of the candidate compounds for its ability to activate TREKl and/or TREK2 channels.

15. The method according to claim 13 or 14, wherein said method is used for screening candidate compounds useful for treating alcoholism or for treating ecstasy or cocaine addiction.