EVALUATION OF THE POTENTIAL RISK OF DRUG INDUCED MOOD DISTURBANCE AND SUICIDE: USE OF A DEDICATED PLATFORM

Abstract

The present invention relates to in vitro methods for the determination of the potential toxicity of test compounds. The invention also comprises in vitro methods for the selection of therapeutical compounds useful for the treatment of pathology related to an alteration of the mechanism of the mRNA editing of ADAR dependent A to I mRNA editing of the serotonin 2C receptor (5HTR2C). Finally, the present invention is directed to the kits and tools for the implementation of these methods. The invention is of special utility in the pharmaceutical industry for analysis of the toxicity profile or the screening of compounds involved in drug development and/or in pharmaceutical compositions.

Claims

1-21. (canceled)

22. An in vitro method, comprising: a) obtaining a biological sample containing mammal cells wherein said mammal cells are cell lines of human origin which exhibit a regular and constitutive expression of editing enzymes ADAR1a, ADAR1b and ADAR2 and of serotonin 2C receptor (5HTR2C); b) contacting said mammals cells with a compound to be tested; c) determining in a cellular RNA extract of said biological sample: the editing profile giving the mean proportion of each identified isoform of the 5-HT2CR mRNA measured in the cellular RNA extract, and the quantitative expression of said editing enzymes ADAR1a, ADAR1b and ADAR2; d) comparing the results obtained in step c) between said treated cells with the compound to be tested and non-treated control cells; and e) determining potential toxicity or side-effects of a test compound after administration thereof in a patient.

23. The method according to claim 22, wherein in step c), the editing profile of each identified isoform of the 5HTR2C mRNA and the quantitative expression of said editing enzymes ADAR1a, ADAR1b and ADAR2 are determined in the same cellular extract.

24. The method according to claim 23, wherein in step c), when the editing profile of each identified isoform of the 5HT2CR mRNA and the quantitative mRNA expression of said editing enzymes ADAR1a, ADAR1b and ADAR2 are determined in the same cellular extract, they are determined in the same total RNA cell extract.

25. The method according to claim 22, wherein in step c), the analysis of the results of the determination of the editing profile comprises the determination of the activity indexes of the editing enzymes ADAR1a, ADAR1b and ADAR2.

26. The method according to claim 22, wherein in step a), said mammal cells are cells lines capable of expressing at least one 5HT2CR isoform exhibiting at least the editing site A edited, one 5HT2CR isoform exhibiting at least the editing site B edited, one 5HT2CR isoform exhibiting at least the editing site C edited, one 5HT2CR isoform exhibiting at least the editing site D edited and one 5HT2CR isoform exhibiting at least the editing site E edited, when said mammal cell is treated by a drug capable to alter the edition of the 5HT2CR.

27. The method according to claim 22, wherein in step a), said mammal cells are cell lines from human, mouse or rat.

28. The method according to claim 22, wherein in step a), said mammal cells exhibit a regular and constitutive expression of the 5HT2CR, ADAR1a, ADAR1b and ADAR2 enzymes.

29. The method according to claim 22, wherein in step a), said mammal cells are from a neuroblastoma cell line.

30. The method according to claim 22, wherein the potential toxicity or side-effects of said test compound after its administration in a patient related to the alteration of the mRNA editing the 5HT2CR is selected from the group consisting of mental disorders, schizophrenia, depression, depressed suicide and abnormal feeding behaviour.

31. The method according to claim 22, wherein in step b) said mammals cells are cultivated in presence of the compound to be tested in a medium suitable for the culture of said mammal cells.

32. The method according to claim 22, wherein in step b) said mammals cells are cultivated in presence of the compound to be tested for at least 1 hour before the step c) of determining in the same cellular extract the editing profile of each identified isoform of the 5HT2CR mRNA and the quantitative expression of said editing enzymes ADAR1a, ADAR1b and ADAR2.

33. The method according to claim 22, wherein the compound to be tested is further administered in vivo to an animal model, preferably a mouse or a rat, suitable to test the same compound and wherein the potential toxicity or side-effects of said test compound after its administration in said animal model can be evaluated by determining the alteration of the mRNA editing of the 5HT2CR and/or the ADAR isoforms expressed in total blood and/or skin sample, or in brain.

34. An in vitro method of predicting the potential toxicity of test compounds or for the selection of therapeutic compounds useful for the treatment of pathology related to an alteration of the mechanism of the mRNA editing of ADAR dependent A to I mRNA editing of the serotonin 2C receptor (5HT2CR), said method comprising: (a) screening compounds on a mammal cell line which exhibit a regular and constitutive expression of the 5HT2CR, ADAR1a, ADAR1b and ADAR2 enzymes for their ability to alter the 5HT2CR edition, these compounds being known to have or not toxicity or side-effects; (b) based on said screening, selecting a panel of reference members, said panel comprising members which differ with respect to their ability to alter the 5HT2CR edition; (c) screening a test compound of unknown activity relative to said 5HT2CR edition to determine its effect on the alteration on the 5HT2CR edition, thereby obtaining the edition profile of the 5HT2CR; (d) comparing the edition profile of the 5HT2CR; (e) predicting the potential toxicity of test compounds or selecting the test compound as potential therapeutic compounds useful for the treatment of pathology related to an alteration of the mechanism of the mRNA editing 5HTR2C, based on the assumption that the alteration of the 5HTR2C edition resulting from the test compound will be similar to that of reference compound, wherein screening steps on said mammal cell line for their ability to alter the 5HT2CR profile edition, corresponds to step e) of claim 22 and wherein the editing profile of each identified isoform of the 5HT2CR mRNA and the quantitative expression of said editing enzymes ADAR1a, ADAR1 b and ADAR2 are determined.

35. An in vitro method according to claim 34, wherein in step a) said mammal cell line is a neuroblastoma cell line.

36. An in vitro method according to claim 34, wherein in step b) said panel comprises members which differ with respect to their toxicity or side-effects.

37. An in vitro method according to claim 34, wherein step c) comprises the determination of the ADARs expression for said test compound.

38. An in vitro method according to claim 34, wherein step d) comprises the determination of the ADARs expression for said test compound and for said panel of references.

39. An in vitro method according to claim 34, comprising the determination of the ADARs expression.

40. A method according to claim 34, wherein the compound to be tested is administered in vivo to a mouse or a rat.

41. A kit for determining potential toxicity or side-effects of a test compound after its administration in a patient or for the selection of a therapeutic compounds useful for the treatment of pathology related to an alteration of the mechanism of the mRNA editing of ADAR dependent A to I mRNA editing of the 5HTR2C, said kit comprising: a) mammal cells from a cell line wherein said cells express the editing enzymes ADAR1a, ADAR1b and ADAR2 and the serotonin 2C receptor (5HTR2C) and b) two sets of primers for measuring by a quantitative (Q) PCR involving a nested type PCR comprising two rounds of PCR each isoform of the 5-HT2CR mRNA which can be present in a RNA extract of said mammal cells; and c) a set of primers for measuring by a quantitative Q-PCR the quantitative expression of the editing enzymes ADAM a, ADAR1b and ADAR2.

Description

LEGEND TO THE FIGURES

[0130] FIGS. 1A and 1B: Effect of human interferon 24 hours application on ADAR1a mRNA concentration of SH-SY5Y cells: determination of EC50%. ADAR1a RNA Expression in SH-SY5Y cells (Q-PCR, Applied TaqMan probes ref: Hs 01020780_m1).

Reference gene: GAPDH.

[0131] FIGS. 2A-2C: The modifications of editing profile reflect the alteration of the expression of ADAR1a in SH-SY5Y cultured cell treated by INF alpha.

[0132] FIG. 2A. The ΔΔCTs of the ADAR1a mRNA of treated dishes (n=8) versus controls (n=8) are plotted versus LOG10 of the applied concentrations of INF α. A best fit of the hyperbolic relationship adjusted by least square method allowed to calculate the maximum effect and the IC 50% estimated as their Mean ±SD (n=40).

[0133] FIG. 2B. The same experiment was done by amplification of the 5-HT2cR mRNA and the increase in the editing alteration was followed by the significant decrease of the NE isoform after identification of each individual editing profile. The results are calculated as for A from individual values (n=40).

[0134] FIG. 2C. The results concern the relationship of the positive variation of the percentage represented in the profile distribution by the sum of the 3 edited isoforms AB, ABC and AC versus the INF α concentration. The best fit estimated the maximal effect and IC 50% from the individual values of measurements (n=40) as in A and B. The IC 50% are expressed as IU/ml of culture medium. The maximal effects determined for ADAR1a mRNA as ΔΔCT normalized to controls gives a mean calculated value of QR of 10,56 when the mean controls is normalized to 1. In FIGS. 2B and 2C the y plot correspond to the mean values of the absolute variation versus controls ±SEM. The allowable error for the best fitting was 0.01%.

[0135] FIG. 3: A cell model as a basis to evaluate a possible psychiatric risk. The receptor (5-HT2cR) mRNA editing>>signatures<< has been evaluated in 3 limbic cerebral structures (dorsal prefrontal (DPFCx), anterior cingular (ACCx) and entorhinal (Entorhinal Cx) cortices of depressed/suicide patients. This signature corresponded to the variations of the distribution of the respective proportions of each identified edited isoforms (see table 7) when their mean values were compared to those found in the control group of patients. They were first classified by their algebraic mean of individual delta, then treated by component statistical analysis. Each component was defined as the proportion of edited isoforms in which for example A and B sites, or A and C or A, B and C, etc. were found edited. The Black and grey rectangles respectively represent a positive and negative mean variation of the defined component when it was identified as significant (p<0.05).

[0136] FIG. 4: Typical example of in vitro profiling of drugs by following the alteration of the activity of editing as detected from given statistical components analysis of the editing profiles of 5-HT2cR mRNA in SH-SY5Y cultured cells. For each tested molecules the components of the signature which were found significant are represented by black (positive variations) and grey (negative variations). For each component the alteration can affect the positive and/or negative parts of the signature and can be also found altering their total sum. The choice of the components is directly derived from the comparison of the analysis performed in suicide patients and INF α treated SH-SY5Y cultured cells presented in FIG. 3. The * indicates that the corresponding molecule has been the object of a FDA Psychiatric Alert for mood disturbance and suicide risk during treatment.

[0137] FIG. 5: ADAR1 mRNA and protein expression in IFNα-treated SH-SY5Y cells. Protein extracts SDS transferred nitrocellulose membrane. Bands corresponding to both constitutive (p110) and inducible (p150) ADAR1 isoforms are shown with arrows. Protein standards are also depicted (MW 250, 150 and 100 kD.

EXAMPLES

Example 1

Cell Culture and Pharmacological Treatment

[0138] Among ten different cell lines screening the SH-SY5Y human neuroblastoma cell line was selected as the most interesting when used in the following conditions.

[0139] The SH-SY5Y Human neuroblastoma cell line was purchased from ECACC (EC94030304 from the European Collection of Cell Cultures (ECACC)). The cell line SH-SY5Y is a thrice-cloned neuroblastoma, originally from SK-N-SH and first reported in 1978. A neuroblast-like subclone of SK-N-SH, named SH-SY, was subcloned as SH-SY5, which was subcloned again as SH-SY5Y (Biedler J L et al. Cancer Res. 1978;38:3751-7). Cells were cultured in high glucose D-MEM medium (Sigma, ref D6546) supplemented with 10% dialysed FCS (PAA, ref. A15-507, lot A50708-0050), 2 mM Glutamine (Sigma, G7513) and a 1× mix of Antibiotic-Antimycotic Stabilized (Sigma, ref. A5955) at 37° C. under a humidified atmosphere of 5% CO2. The day preceeding drug or hIFNα treatment, SH-SY5Y cells were plated in 6-well plates at a density of 10.sup.6 cells/well. One 6-well plate was used per experimental condition (concentration or treatment duration). The day after plating, culture medium was removed and cells were incubated for 24 hours with a 10 μM solution of the compound to be tested molecules or a 1000 IU/ml solution of hIFNα (PBL biomedical laboratories). For hIFNα dose-response experiment cells were incubated with 1, 10, 100, and 1000 IU/ml of hIFNα solutions. For hIFNα time-course experiment cells were treated for 24, 48, or 72 hours with a 1000 IU/ml solution of hIFNα. After the different treatments, cells were directly lysed in RLT lysis buffer and total RNA purified according to manufacturer's protocol (Qiagen, RNeasy Plus mini kit, ref 74134). Total RNA was then reverse transcribed with Thermoscript RT-PCR system Plus Taq (Invitrogen, 11146-032) and the resulting cDNA used for CE-SSCP and quantitative real-time PCR.

Example 2

[0140] In vivo Protocol for mIFNα and Drug Treatment

[0141] For the mIFNα experiment, 8 males (Balb/cJ mice, Charles Rivers) were injected i.p route once with 10000 IU of mIFNα (PBL biomedical laboratories). Control mice were injected same route with sterile Phosphate Buffer Saline. 8 hours after injection animals were sacrified by decapitation and total blood, ventral skin and brain collected. Total RNAs were purified with Mouse RiboPure Blood RNA Isolation kit (Ambion, ref 1951) for blood, TRIzol reagent (Invitrogen, ref 15596-026) -after tissue disruption- for skin, and RNeasy lipid mini kit (Qiagen, ref 74804) for brain. Total RNA was then reverse transcribed with Thermoscript RT-PCR system Plus Taq (Invitrogen, 11146-032) and the resulting cDNA used for CE-SSCP and quantitative real-time PCR. The method used for SSCP determinations was already described for mouse and human samples (see patent PCT/EP 2008/057519 filed on Jun. 13, 2008).

[0142] For antipsychotic, antidepressant, and suicide warning drugs, the different compounds were first dissolved in the vehicle (DMSO/Ethanol/Water: 50%/15%/35%) and administered to male Balb/cJ mice through Alzet pumps (Alzet, ref 2002, ordered from Charles River, France) to have a continuous and homogenous drug delivery. The Control group was composed of 8 mice treated with the vehicle alone. The test groups were composed of 8 mice treated with compounds at a dose of 3.5 or 7.0 mg/kg/day dissolved extemporaneously in the vehicule. After 15 days of drugs delivery through Alzet pumps animals were sacrified by decapitation. As previously described samples of total blood and skin, and brain were collected. Total RNA were purified and reverse transcribed as mentioned above.

Example 3

[0143] Total Profile of Distribution of all the Expressed Edited and Non Edited Isoforms of the 5-HT2cR mRNA; Quantification of 5-HT2cR (total) and ADARs mRNA Expression by Real-Time PCR Analysis
3A) Total Profile of Distribution of all the Expressed Edited and Non Edited Iso Forms of the 5-HT2Cr mRNA by Non Denaturing Capillary Electrophoresis by Single Strand Conformational Polymorphism

[0144] (CE-SSCP) (see also International PCT patent application WO 2008/152146, Example 2 and FIG. 1)

a): Obtention of the Complete Editing Profile from One Sample of Brain Tissue

[0145] Total RNA was extracted and purified from tissue or cell extracts, according to manufacturer's specifications (Qiagen RNeasy, Mini Kit). The quantity and purity of the extracted RNA were assessed by measuring both the absorbance at 260 nm and the 260/280 nm ratio with a GeneQuant spectrophotometer (PharmaciaBiotech). In order to eliminate possible contamination by genomic DNA, 8 μl of each RNA (between 88 ng and 1.3 μg) were then treated with 1 unit of DNase I (Invitrogen) for 15 min at room temperature in a final volume of 10 μl. The reaction was stopped by adding 1 μl of 25 mM EDTA and then heated for 10 min at 65° C. The reverse transcription of DNAse I-treated RNAs (10 μl) was performed using 15 units of ThermoScript reverse transcriptase (ThermoScript RT-PCR System, Invitrogen) and Oligo(dT) primers at a final concentration of 2.5 μM.

[0146] A first PCR reaction (final volume 25 μl) resulting in a 250 bp fragment, was then carried out on 1 μl of the reverse transcription products with 0.2 unit of Platinum Taq DNA polymerase (ThermoScript RT-PCR system, Invitrogen) and specific primers (forward primer: 5′-TGTCCCTAGCCATTGCTGATATGC-3′ (SEQ ID No. 1) and reverse primer: 5′-GCAATCTTCATGATGGCCTTAGTC-3′ (SEQ ID No. 2); final concentration of each 0.2 μM) located on exon IV and exon V of the Human 5-HT2cR cDNA, respectively. After a denaturing step at 95° C. for 3 min, the PCR was brought to its final point after 35 cycles (15 s at 95° C.; 30 s at 60° C.; 20s at 72° C.), and a final elongation step of 2 min at 72° C. Aliquots of the amplification products were used to check the product on a 2% agarosc analytic gel.

b) Second PCR and Separation of Single-Strand cDNA Fragments by Capillary Electrophoresis (CE)

[0147] 1 μl of a 1/50 dilution of the RT-1.sup.st PCR products, or the 250 bp cDNA amplified from plasmids harboring the thirty-two standard of human 5-HT2cR (or 5HT2CR) isoforms, were used as templates for an additive nested-PCR. These 32 standards, corresponding to the non-edited (NE) and edited isoforms of human 5-HT2cR. Amplifications were performed in a final volume of 20 μl with HPLC-purified fluorescent primers (forward primer: FAM-ATGTGCTATTTTCAACAGCGTCCATC-3′ (SEQ ID No. 3); reverse primer: VIC-GCAATCTTCATGATGGCCTTA-3′ (SEQ ID No. 4); final concentration of each 0.2 μM), and 0.2 unit of Platinum Pfx DNA polymerase (Invitrogen).

[0148] The VIC-labelled reverse primer hybridizes to a complementary sequence of the 5-HT2c receptor identical in human, mouse and rat. On the other hand, although used with human samples, the sequence of the FAM-labelled foward primer was designed to be as close as possible to that of the mouse. More precisely, T residues in positions 5 and 6 of the human oligonucleotide sequence (positions 1133 and 1134 of human reference U49516) were changed into G and C, respectively.

[0149] Simulations of stochastic folding pathways of both strands of the PCR product obtained with the two primers described above were carried out with the Kinefold server (kincfold.curic.fr). They showed that the lowest free-energy structures obtained for forward and reverse strands—the edited region embedded in the loop of a stem-loop structure, and able to hybridize with a complementary sequence located elsewhere in the whole structure after folding of the stem—were very close to that calculated for a mouse nested-PCR product successfully used for Mouse samples. This set of primers was shown to be optimal for conformational analysis of human 5HTR2C mRNA editing by non denaturing capillary electrophoresis by single strand conformational polymorphism (CE-SSCP).

[0150] The amplified fragment is 127 bp-long. As for RT-PCR, after an initial denaturing step of 5 min at 94° C., the amplification reaction was brought to an end with 35 cycles (15 s at 94° C.; 30 s at 55° C.; 20 s at 68° C.) and a final elongation step of 2 min at 68° C. Again, quality of the 127 bp-long amplified fragments were assessed on a 2% agarose gel before subsequent analysis in a 3100 Avant Genetic Analyser (Applied Biosystem).

[0151] Fluorescent PCR products corresponding to standard isoforms (1 μl of a 1/100 dilution in DEPC treated water) and samples (1 μl of a 1/30 dilution) diluted in 11 μ1 of deionized formamide were added to a mixture of ROX labelled migration standards (MWG-BIOTECH, AG) (0.5 μl each) covering the whole range of the electrophoregram retention times. These ROX standards were used for CE calibration and subsequently to obtain correct superimposition of standards and samples peaks. After denaturing for 2 min at 95° C., samples were immediately chilled on ice. Non-denaturing CE was carried out in an ABI PRISM 3100-Avant Genetic Analyser (Applied Biosystems) through 80 cm-long capillaries filled with 7% “POP Conformational Analysis Polymer” (Applied Biosystems), 1× TBE and without glycerol. After a pre-run performed at 15 kV for 3 min, samples were injected for 15 s at 2 kV, and electrophoresis was run for 105 min at 15 kV at a controlled temperature of 20° C. Under these conditions, each of the thirty-two possible iso forms were clearly resolved as a result of the single ssDNA conformation obtained with either the FAM-labelled or the VIC-labelled strand. The different retention times were used for unambiguous identification of the iso forms.

c) Identification and Relative Quantification of Each Isoform in Each Brain Sample

[0152] The Electrophoretic Signal was then processed using an in-house software. First, the time basis of electrophoretic profiles of each sample was adjusted using the ROX-labelled strands of the migration standards. This allowed FAM- and VIC-labeled strands to precisely deconvolute the standards and samples signals in a unique time basis. Background was then adjusted and subtracted and then total area under each signal normalized.

[0153] The relative proportion of each iso form was processed by a best fitting of each deconvoluted and normalized analytical signal of the brain samples. It was performed by the iterative adjustment of the integrated signal represented by the 32 similarly deconvoluted and normalized standard analytical signals. The calculation was based on the hypothesis that the SSCP signal

[00001]$S\left(t\right)=\underset{i=1}{\overset{N}{.Math.}}.Math.g_i.Math.R_i\left(t\right)$

in which R.sub.i (t), with i ∈ {1, . . . , N}, are the standard signals and g.sub.i the % of each of them in the signal. The best fit minimized the sum of squares due to error (SSE)

[00002]$\mathrm{SSE}=\int {\left[S\left(t\right)-\underset{i=1}{\overset{N}{.Math.}}.Math.g_i.Math.R_i\left(t\right)\right]}^2$

and was controlled by the least square statistical analysis.

[0154] The result of this best fitting was statistically evaluated after calculation of the r.sup.2 value such as

[00003]$r^2=1-\frac{\mathrm{SSE}}{\mathrm{SSM}}$

in which SSM is the Sum of Square about Mean such as

[00004]$\mathrm{SSM}=\underset{i=1}{\overset{t}{.Math.}}.Math.{\left(S\left(t\right)-\stackrel{\_}{S}\right)}^2.$

The maximum theoretical best fit would give an r.sup.2=1.

[0155] All experiments were carried out under blind conditions and all samples were assayed in the same batch for RT-PCR and second PCR reactions. The best fitting results yielded a specific editing profile for each individual sample, which was determined by the percentage of each edited and non edited form of the total analytical signal. These initial values were used for statistical analyses.

[0156] This method gives the proportion of each expressed mRNA iso form expressed as the percentage of the total of 5-HT2c receptor present in the extract.

3B) Quantification of 5-HT2CR and ADARs mRNA Expression by Real-Time PCR Analysis

[0157] In order to quantify levels of 5-HT2CR, ADAR1 and ADAR2 mRNA expression in SH-SY5Y cells or in prefrontal cortex, total blood and skin of Balb/cJ mice, first-strand cDNA was synthesized by reverse transcription and subjected to TaqMan quantitative real-time PCR analysis (Applied Biosystems). All probes and primers used for the quantitative PCRs were from Applied Biosystems (Gene Expression Assays, Assay-On-Demand) (see Table 1, Applied Biosystems primers and probes references):

[0158] These probes and primers could be easily designed, whether it is necessary, by the skilled person in view of the well known and disclosed nucleic sequences of the human and mouse gene encoding the 5-HT2cR, ADAR1, constitutive and inducible isoforms, and ADAR protein.

TABLE-US-00011 TABLE 1 ADAR1 constitutive ADAR1 5-HT2cR isoform or total inducible isoform ADAR2 Balb/cJ mice Mm 00434127_ml Mm 00508001_ml Mm 00507997_ml Mm 00504621_ml Mm 00507998_ml Human tissues Hs 00968672_ml Hs 01017596_ml Hs 01020780_ml Hs 00210562_ml And Hs 00968671_ml SH-SY5Y cells

[0159] Human GAPDH (product no. 4326317E; Applied Biosystems) or mouse GAPDH (product no. 4352339E; Applied Biosystems) were included in each multiplex PCR as an internal control. Real-time PCR and subsequent analysis were performed with a 48-well block StepOne RT PCR system (Applied Biosystems). Quantification of target gene expression in all samples was normalized to GAPDH expression by the equation Ct (target)−Ct (GAPDH)=ΔCt, where Ct is the threshold cycle number. The mean ΔCt value of samples from untreated mice or cells was determined and used as a reference point for the samples corresponding to treated animals or cells. Differences between untreated and treated animals or cells, including individual variation were calculated by the equation (ΔCt (individual treated samples)—ΔCt (mean of untreated samples)=ΔΔCt). Changes in target gene expression (n-fold) in each sample were calculated by 2.sup.−ΔΔCt, from which the means and standard deviations (SD) were derived.

[0160] In order to improve the sensitivity of the selected cell line to detect a significant alteration of the editing process the process was applied to the evaluation of human interferon and of 17 molecules on which FDA had concentrated the warning Box.

Example 4

[0161] Relation Concentration-Effect on the Expression of ADAR1a of the Human Interferon Applied on SH-SY5Y cells during 24 hours

(See FIGS. 1A and 1B)

[0162] The concentration of 1000 IU/ml was chosen for additional evaluation of the distribution of edited iso forms in controls and interferon treated cells the result is presented on Table 2.

TABLE-US-00012 TABLE 2 Analysis of the editing profile obtained after 24 hours of treatment by 1000 IU of human interferon alpha. The editing profile gives the mean proportion (%) ± SEM (n = 6) of each identified isoform of the 5-HT2cR mRNA (=100%) measured in the cellular RNA. These isoforms are classified in function of the algebraic delta when compared are the control and the INF treated groups. The obtained signature is tested for significance and a multiple evaluation of the activity of the editing enzymes is then proposed by classifications of the products of the enzymes activities. The first classification (edited sites) calculates the percentage of edition of each of the editing sites: A, B, C, D and E, found in each part of the signature. When the total distribution is used, the result given in “total” corresponds in fact to a result which could be obtained by the primer extension method. The enzyme indexes were calculated from the editing iso forms due to the action of ADAR1 alone (ADAR1). to the combined action of ADAR 1 and of ADAR 2 (ADAR1+2). from the exclusive action of the ADAR2 (ADAR2). A final classification expressed the activity by measuring the % represented by all the iso forms for which have been implicated ADAR 1 action (ADAR I) or ADAR2 (ADAR II). Other classifications are also possible (not shown here) for the evaluation of some specific editing action (for example to identified the proportion of products of the enzyme activities in which the sites C and E have been found edited. or A and C, A and B, A and B and C etc.). It is interesting to note that the non edited isoform (NE) is also significantly reduced. Editing Profle Signature IFN IFN Isoforms Ctls SEM 1000UI SEM Isoforms Δ % Student Ctls SEM 1000UI SEM Δ % Student NE (INI) 54.6 1.9 37.5 3.5 ADE (VDV) −89.2 0.00002 Global signature A (VNI) 29.5 1.3 32.3 2.5 ABCD (VSV) −82.4 0.21792 NE (INI) 54.6 1.9 37.5 3.5 −31.3 0.0008 B (MNI) 3.4 0.3 3.5 0.8 BD (MNV) −75.9 0.00002 Sum of 37.3 1.9 57.5 3.5 54.2 0.0002 C (ISI) 2.5 0.5 2.2 0.4 BCE (MGI) −69.2 0.12449 Delta > 0 AB (VNI) 1.8 0.8 2.9 0.9 ABD (VNV) −60.5 0.13164 Sum of 8.1 0.4 4.5 0.5 −44.3 0.0001 ACE (VGI) 1.4 0.3 0.8 0.2 D (INV) −59.8 0.00221 Delta < 0 D (INV) 1.3 0.2 0.5 0.1 CE (IGI) −43.9 0.34397 Edited sites BD (MNV) 1.1 0.1 0.3 0.1 ABDE (VDV) −41.7 0.36296 Delta > 0 AC (VSI) 0.9 0.6 13.5 3.4 ACE (VGI) −40.2 0.07315 A 33.6 2.1 53.6 3.9 59.3 0.0006 AE (VDI) 0.6 0.4 1.1 0.3 NE (INI) −31.3 0.00082 B 5.8 0.7 9.1 1.6 56.6 0.0447 ABD (VNV) 0.4 0.1 0.2 0.2 DE (IDV) −28.3 0.06225 C 1.2 0.6 15.8 3.6 1191.6 0.0013 ADE (VDV) 0.4 0.0 0.04 0.0 C (ISI) −14.2 0.28715 D 0.4 0.2 2.0 0.6 409.8 0.0125 DE (IDV) 0.4 0.0 0.27 0.0 B (MNI) 2.6 0.46177 E 1.1 0.6 2.2 0.6 99.4 0.1233 AD (VNV) 0.3 0.2 1.40 0.5 A (VNI) 9.5 0.17352 Delta < 0 ABE (VDI) 0.2 0.2 0.29 0.1 ABE (VDI) 25.8 0.40553 A 2.4 0.4 1.1 0.4 −54.1 0.0224 BE (MDI) 0.2 0.1 0.76 0.7 AB (VNI) 63.7 0.18693 B 1.8 0.3 0.5 0.2 −71.3 0.0014 CE (IGI) 0.2 0.2 0.13 0.1 AE (VDI) 69.2 0.17984 C 4.3 0.6 3.2 0.5 −27.2 0.0793 ABC (VSI) 0.2 0.1 1.62 0.3 CD (ISV) 123.2 0.24804 D 3.9 0.2 1.4 0.3 −64.9 0.0000 ABCD (VSV) 0.1 0.1 0.03 0.0 BE (MDI) 228.5 0.24235 E 2.5 0.3 1.3 0.3 −46.9 0.0083 CD (ISV) 0.1 0.0 0.19 0.1 AD (VNV) 342.7 0.03539 Total BCE (MGI) 0.1 0.0 0.02 0.0 ABC (VSI) 758.7 0.00104 A 36.1 2.3 54.7 3.9 51.7 0.0011 ABDE (VDV) 0.1 0.1 0.04 0.0 AC (VSI) 1324.2 0.00235 B 7.7 0.8 9.6 1.7 25.8 0.1596 100 100 C 5.6 0.8 18.9 3.6 240.1 0.0022 D 4.3 0.4 3.4 0.6 −20.6 0.1098 E 3.6 0.7 3.5 0.6 −2.6 0.4621 Enzyme activity index A Delta > 0 ADAR1+ 36.9 2.0 56.0 3.1 51.6 0.0002 ADAR1+2+ 0.3 0.2 1.8 0.6 485.6 0.0181 ADAR2+ 0.1 0.0 0.2 0.1 123.2 0.2480 NE Delta < 0 ADAR1− 1.5 0.2 0.9 0.2 −41.5 0.0552 ADAR1+2− 4.9 0.5 2.8 0.3 −42.1 0.0021 ADAR2− 1.7 0.2 0.8 0.1 −52.9 0.0005 NE 54.6 1.9 37.5 3.5 −31.3 0.0008 Sum ADAR1 38.4 2.1 56.8 3.0 48.1 0.0003 ADAR1+2 5.2 0.3 4.7 0.8 −10.2 0.2604 ADAR2 1.8 0.2 1.0 0.2 −44.7 0.0052 NE 54.6 1.9 37.5 3.5 −31.3 0.0008 Enzyme activity index B Delta > 0 ADAR I+ 37.2 1.9 57.8 3.5 55.3 0.0002 ADAR II+ 0.4 0.2 2.0 0.6 409.8 0.0125 NE+ Delta < 0 ADAR I− 6.4 0.5 3.7 0.5 −42.0 0.0016 ADAR II− 6.6 0.4 3.6 0.3 −44.9 0.0001 NE− 54.6 1.9 37.5 3.5 −31.3 0.0008 Sum ADAR I 43.6 2.0 61.5 3.6 41.1 0.0007 ADAR II 7.0 0.3 5.7 0.7 −19.1 0.0487 NE 54.6 1.9 37.5 3.5 −31.3 0.0008

[0163] It is clear that the INF treatment induces a strong and significant alteration of the editing profile which indicates that as expected demonstrates an important increase in the ADAR 1 activity and a decrease in the ADAR2 activity.

[0164] In the same samples the level of expression of the ADARs were measured by QPCR and the results are summarized on the following Table 3 and compared to those obtained after evaluation of the editing profile.

TABLE-US-00013 TABLE 3 Comparison of expression and activity indexes of ADAR1 and ADAR2 enzymes after INF treatment of the selected cell line. The results are given in % of variation versus controls (n = 6). ENZYME ACTIVITY INDEX (% Δ) Q−PCR (% Δ) ADAR ADAR ADAR ADAR ADAR ADAR ADAR ADAR 1 1+2 2 I II NE 1a 1b 2 IFN 24 H 48.05* −10.25 −44.75* 41.07* −19.08 −31.30* 970* 0 68 *The asterisk marked area and bold numbers indicate a significant variation (p < .05).

[0165] The next table summarizes the result after similar experiences performed with the same protocol to determine the eventual alteration of editing after application for 24 hours of 10 micromolar concentrations of 17 molecules which has been indicated by FDA alertness as presenting a risk of suicide induction when chronically used. These molecules belong to several chemical and different families. However they present a significant alteration of editing of 5-HT2cR. 11 of these molecules present a significant alteration of the expression of editing enzymes. The others induce significant changes in the activity of these enzymes which can be easily detected by using the same cellular samples (see Table 4).

TABLE-US-00014 TABLE 4 The molecules indicated as presenting a suicide risk by the FDA significantly alter the editing enzymes expression and/or their editing action on 5-HT2cR mRNA. This can be easily detected by their application on a dedicated cell line (SH-SY5Y). ENZYME ACTIVIY INDEX QPCR: RNA expresion (% of variation) (% of variation) ADAR ADAR ADAR ADAR ADAR ADAR ADAR ADAR 1 1+2 2 NE I II EC 1a 1b 2 Fenfluramine 4.2 −43.3* 0.7 4.5 −4.8 −34.8*    7   23   27 Rimonabant 13.3 2.4 −3.7 −10.4 11.3 1.2  −43*  −32*  −7 Carbamazepine −3 38.8* 26 −3.8 3 36.4*    52*  −62*  −42* Felbamate 6.3 −13.9 −24.6 −1.2 2.4 −16 −13  −1    1 Gabapentin 19.2 −28.5 −26.7 −8.3 10.1 −28.1*  −42*  −34*  −21* Lamotrigine 5.9 −24.6 −23 0.9 0.1 −24.3 −26  −26* −32 Levetiracetam 12 −36.6* 10.9 −3.1 2.8 −27.4  −9 −11 −17 Oxcarbazepine 1.1 −25.6 −25.9 4.8 −3.9 −25.6 −65.2* −28 −18 −15 Pregabalin 2.5 −8.7 −6.2 −0.1 0.4 −8.2 −33  −43* −36 Topiramate 14.4 −19.5 11.4 −8 8 −13.5  −44*  −46*  −60* Zonisamide 11.9 9.7 −7.8 −10.5 11.5 6.3  −24*  −19* −23 Bupropion −16.3* 42.6 68.9 5.3 −8.9* 34.7  −66*  −51*  −40* Citalopram −5.7 70.2* 70.1* −7.4* 5.3 70.2* −18 −2    64* Desipramine −8.6 5.7 15.5 5.9 −6.6 7.5   29 −38 −12 Imipramine −11.6* 146.4* 62.5 −13* 11.3 130.4*   34    0  111* Trazodone 0 89.3* 28.4 −13.4 12.9 77.7 −60 −40    5 Olanzapine −23.2 84.9* 9.2 7 −7.6 70.5*  −54*   20    92*

[0166] It becomes obvious that the dedicated cell line, when observed with the set of techniques allowing a rapid and complete measurement of these parameters represent a new model for the pre-clinical evaluation of the eventual risk of these molecules to produce altered mood by a chronic alteration of the 5-HT transmission.

Example 5

[0167] Choice of a Cell Line for in vitro Predictive Effect of Molecules
a-Criteria for Selection.

[0168] To be eligible for the in vitro screening of molecules the cell line must validate the main following points: [0169] to be from Human origin; [0170] to express the 5-HT2cR receptor at a range allowing a reproducible evaluation of the editing profile in control conditions; [0171] to express the editing enzymes in relative steady states similar to those observed in normal cortical structures in the Human brain.

b-A Proposal for a Best Choice.

[0172] Among ten different cell lines, the SH-SY5Y Human neuroblastoma cell line was selected as the most interesting when used in the following conditions.

[0173] The SH-SY5Y Human neuroblastoma cell line was purchased from ECACC. Cells were cultured in high glucose D-MEM medium (Sigma, ref D6546) supplemented with 10% dialysed FCS (PAA, ref A15-507, lot A50708-0050), 2 mM Glutamine (Sigma, ref G7513) and a 1× mix of Antibiotc-Antimycotic Stabilized (Sigma, ref A5955) at 37° C. under a humidified atmosphere of 5% CO2. The day preceeding hIFNα or drug treatment, SH-SY5Y cells were plated in 6-well plates (Corning, Multiwell Plate, 6 well, Corning CellBIND Surface, ref 3335) at a density of 5; 7 or 9.10.sup.6 cells/well for a 72-, 48- or 24-hour treatment respectively. Six or eight wells of 6-well plates were used per experimental condition (control, concentration or treatment duration). The day after plating, culture medium was removed and cells were incubated for 24 or 48 hours with a 10 μM solution of the to be tested molecules or a 1000 IU/ml solution of hIFNα (PBL biomedical laboratories). For hIFNα dose-response experiment, cells were incubated with 1, 10, 100, 1000, or 10000 IU/ml of hIFNα solutions. [For hIFNα time-course experiment cells were treated for 24, 48 or 72 hours with a 1000 IU/ml solution of hIFNα. In the case of the 72-hour treatment points, medium was changed for both controls and hIFNα-treated cells after 48 hours of culture]. Cells were then directly lysed in RLT lysis buffer and total RNA purified according to manufacturer's instructions (Qiagen, RNeasy Plus mini kit, ref 74134). Total RNA was then reverse-transcribed with Thermoscript RT-PCR system Plus Taq (Invitrogen, ref 11146-032) and the resulting cDNA used for CE-SSCP and quantitative real-time PCR.

[0174] When possible, total protein extracts were also prepared for western-blotting. Briefly, cells from eight wells corresponding to control or treatment procedures, were lysed in 600 μl of RIPA buffer (150 mM NaCl, 10 mM Tris-HCl pH8, 5 mM EDTA, 1% Triton X100, 0,1% sodium deoxycholate) supplemented with 1 mM PMSF and Complete Mini protease inhibitor cocktail (Roche, ref. 11836153001). Cell lysates were sonicated 3×15 seconds on ice, rocked for at least 2 hours at 4° C. and then spun at 100 g for 10 min at 4° C. Insoluble pellets were resuspended in 40 μl of 2× Laemmli loading buffer and protein concentrations were quantified with a Quant-IT Protein Assay kit (Invitrogen, ref. Q33211). After sonication and denaturation for 5 min at 70° C., 75 μg of insoluble protein extracts in Laemmli buffer were loaded on a 12% denaturing polyacrylamide gel. Migration and electro-transfer of protein extracts were further performed according to standard procedures. For ADAR1 proteins detection (both constitutive and inducible forms), nitrocellulose membranes were blotted with a L-15 affinity purified goat polyclonal antibody (Santa Cruz, ref. sc-19077).

c-Steady State of the Expression Editing Enzymes and 4-HT2cR in SH-SY5Y in Control Conditions.

[0175] It is illustrated on the table 5.

TABLE-US-00015 TABLE 5 Relative expression of ADAR1a, ADAR1b, ADAR2 and 5-HT2cR mRNA in the SH-SY5Y neuroblastoma, and HTB-14 astrocytoma cell lines and in a pool of Human brain. cerebral cortex total RNA. Cells lines were purchased from ECACC (SH- SY5Y, ref 94030304) and ATCC (HTB-14, ref HTB-14). Cerebral cortex total RNA was purchased from Clontech (ref 636561). Quantification of mRNA levels of expression was performed by TaqMan quantitative real-time PCR analysis (Q-PCR) on a Applied Biosystems StepOnePlus ™ 96-well apparatus (Applied Biosystems, ref 4376592). All probes and primers used for Q-PCR were from Applied Biosystems (Gene Expression Assays, Assay-On-Demand): 5-HT2cR (Hs 00968672_m1). ADAR1 p110 constitutive isoform (Hs 01017596_m1), ADAR1 p150 inducible isoform (Hs 01020780m1), ADAR2 (Hs 00210562_m1). Human GAPDH (Applied Biosystems. ref 4326317E) was included in each multiplex Q-PCR as an internal control. RQ (Relative Quantitation) were calculated as described by the furnisher. In each tissue or cell line. expression of the ADARla gene was taken as a reference and its RQ equal to 1. RQ RQ RQ RQ RNA Origin ADAR1a ADAR1b P ADAR2 P 5-HT2cR P SH-SY5Y 1 48.10 <0.0001 1.78 0.02 0.02 <0.0001 cells Pool of cortex 1 40.19 <0.0001 0.24 0.000889 1.31 0.044 RNAs HTB-14 1 20.27 <0.0001 4.23 0.000018 cells

[0176] It is important to note that when compared with the ADAR1a iso form (taken here as reference) which is the inducible isoform of the ADAR1, the constitutive isoform mRNA of ADAR1 (ADARib) is 48 fold more expressed and that the same ratio was found in the Human cortex. In SH-SY5Y cells as in Human cerebral cortex total RNA, a reproducible quantity of specific mRNA coding for the 5-HT2cR can be identified. That was not the case for HTB14 cells in which the mRNA coding for the receptor was not expressed in a range allowing constant expression.

[0177] Another point was to verify the capacities of these enzymes to respond to classical models of selective induction and to reveal the complex cooperative activity to generate the products of the editing profiles. The validation of these criteria are illustrated on the following two tables.

TABLE-US-00016 TABLE 6 ADAR1 mRNA and protein expression in IFNα-treated SH-SY5Y cells. SH-SY5Y cells were cultured for 48 hours in presence of IFNα at a concentration of 1000 IU/ml. After treatment, total RNA and protein extracts were prepared as described in Materials and Methods. Quantification of mRNA levels of expression was performed by TaqMan quantitative real-time PCR analysis (Q-PCR) on a Applied Biosystems StepOnePlus ™ 96-well apparatus (Applied Biosystems, ref 4376592). Probes used for Q-PCR were from Applied Biosystems (Gene Expression Assays, Assay-On-Demand): ADAR1 p110 constitutive isoform (Hs 01017596_ml), ADAR1 p150 inducible isoform (Hs 01020780_ml). Human GAPDH (Applied Biosystems, ref 4326317E) was included in each multiplex Q-PCR as an internal control. RQ were calculated as described by the furnisher. Expression of the ADAR1a gene was taken as a reference and its RQ equal to 1. Protein extracts were resolved by SDS-PAGE on a 12% acrylamide denaturing gel and transferred to a PROTRAN BA 85 nitrocellulose membrane (Whatman, ref 10 401 197). For ADAR1 proteins detection, nitrocellulose membranes were blotted with a L-15 affinity purified goat polyclonal antibody (Santa Cruz, ref. sc-19077). Bands corresponding to both constitutive (p110) and inducible (p150) ADAR1 isoforms are shown with arrows. Protein standards are also depicted (Precision Plus Protein Standards, Bio-Rad, ref. 161-0363). Bands corresponding to proteins of interest were scanned with the Li-Cor Odissey apparatus and further quantified with the MCID software (see FIG. 5). The optical density (OD) obtained for each scan and each experimental condition is shown between brackets (see Table 6). By convention, the OD of ADAR1a isoform in untreated cells was taken as a reference and equal to 1. OD protein RQ mRNA OD protein (western-blot) RQ mRNA IFN-treated cells (western blot) IFN-treated cells Untreated cells (1000 IU/ml-48 h) Untreated cells (1000 IU/ml-48 h) ADAR1a 1 10,3 1 12,1 (22,12 OD) (268 OD) ADAR1b 21,7 116,2 117,1 (2570 OD) (2590 OD)

[0178] This experiment clearly demonstrates that in the culture conditions expressed above the chosen cell line respond to the induction produced by INFα can be observed at the enzymatic protein level with the same specificity and amplitude ratio than those predicted by the mRNA quantification.

[0179] The analysis of the distribution of the products of these editing enzymes (index of their activities) was performed by using the SSCP-CE technology previously described in Example 3A and which allows to quantify in one single assay from a sample of total RNA, the total profile of distribution of all the expressed edited and non edited isoforms of the 5-HT2cR mRNA. The table 8 gives an example of editing profiles obtained from 3 limbic cortical structures and SHSY5Y cell line in control conditions.

[0180] The capacity to rapidly measure these distributions is a pre-requisite to correctly investigate the modifications of the activities of editing enzymes which could be produced by pathological states or application of molecules.

Example 6

The Interferon Model: Its Interest to Orientate a Specific Strategy to Compare Tested Molecules

[0181] As previously indicated the interferon a treatment, mainly to treat Hepatitis C, can trigger serious alteration of mood in a large proportion of patients (30 to 50%). It was thus interesting to analyse the effect of INF a upon our selected cell line since (see table 7) this molecule is known to be a strong inducer of ADAR1a inducible isoform of ADAR1.

[0182] In a first step the effect of a range of concentrations was evaluated by measuring the degree of expression of this enzyme by QPCR of its specific mRNA. An additional set of experiments was performed to analyse the effect of the product on the editing profile which was considered as a powerful index of editing enzymes activities. The results are summarized on the FIG. 1 and table 4 (see also table 7).

[0183] These results have clearly indicated that the induction of ADAR1a was selective and led to a significant alteration of the editing profile mainly concentrated to the AB, ABC and C isoforms. In control conditions these isoforms represented 7,5% of the total specific 5-HT2cR (see table 7). When 1000 IU/ml were applied to the medium for 48 hours, this proportion was found to be 26%. It was thus clear that the induction of ADAR1a affected mainly the production of these isoforms.

[0184] In order to statistically analyse these alteration of the profiles and in order to refer to the fact that the comparison was in fact limited to an alteration of a normalized distribution we first classified the observed variations of the mean proportion of the expressed isoforms in function of their algebraic mean delta. This classification was defined as the “signature” of the global modification observed. The variations of the two parts of this signature were then tested by variance analysis. Then the analysis was completed by a component analysis considering groups of iso forms for which a significant alteration of proportions could be detected.

[0185] As an example of this process we have decided to use as a reference, a group of significant components deduced from the analysis of the comparison of the signatures obtained from editing profiles of control subjects and depressed suicide patients.

[0186] The result of such an analysis is presented on FIG. 3.

[0187] Note that, from this analysis, 4 components significantly and positively varied in the 3 human brain structures and 9 components shared a positive significant variation in the 2 prefrontal cortices. The last column of the table illustrates the result of the same analysis performed in SH-SY5Y cultured cells (human origin) after application during 48 hours of human INF alpha at the concentration of 1000 IU/ml. It is interesting to remark the similarity of the signature when compared to components found altered in the 3 cortical structures in depressed-suicide patients. It was thus possible to reasonably propose the use of the same criteria after editing profiling of reference molecules to see if some of them have the capacity to induce similar alteration of the 5-HT2c editing mRNA as observed after INFα application.

Example 7

[0188] Use of the In Vitro Platform to the Detection of Reference Molecules Inducing Similar Alteration of the Editing Profile to that Observed After INFα Treatment

[0189] We decided to use this kind of component analysis to different classes of typical molecules having or not been subjected to FDA alerts concerning mood alteration and suicide risk. An in vitro screening of the editing profile was performed after application of each of them on cultured SH-SY5Y cells for 48 hours at a concentration of 10 μM. The analysis of the editing profiles of 5-HT2c mRNA was performed and analysed with the same set of components defined above (see FIG. 3).

[0190] An example of such a classification is given on FIG. 4 and allowed to identify several molecules, belonging to different therapeutic classes, with an activity of 5-HT2cR mRNA editing similarly altered, and potentially presenting, by the fact, a potential risk to induce similar secondary psychiatric effects. Note that Rimonabant and Taranabant belong to this family together with some anti-depressant, anti-psychotic and anti-convulsant molecules.

[0191] This kind of analysis of the editing profiles gives the most sensitive criteria allowing to class by “in vitro” screening, molecules sharing common alterations of m RNA editing of a given target. The choice of the components is not limited and can be oriented by several criteria. When this target, here the 5-HT2cR, is directly implicated in the control of mood, circadian rhythms, pain, eating behavior etc. . . . , this evaluation can be considered as a valid tool for testing predictive risks of eventual adverse secondary effects for new molecules under pre-clinical investigations.