U.S. patent application number 14/297933 was filed with the patent office on 2014-12-04 for evaluation of the potential risk of drug induced mood disturbance and suicide: use of a dedicated platform.
The applicant listed for this patent is Alcediag. Invention is credited to Laurent Cavarec, Jean-Francois Pujol, Laurent Vincent, Dinah Weissmann.
Application Number | 20140356875 14/297933 |
Document ID | / |
Family ID | 40435151 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140356875 |
Kind Code |
A1 |
Weissmann; Dinah ; et
al. |
December 4, 2014 |
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 1 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.
Inventors: |
Weissmann; Dinah; (Paris,
FR) ; Pujol; Jean-Francois; (Paris, FR) ;
Vincent; Laurent; (Bondoufle, FR) ; Cavarec;
Laurent; (Vincennes, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alcediag |
Peynier |
|
FR |
|
|
Family ID: |
40435151 |
Appl. No.: |
14/297933 |
Filed: |
June 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13140325 |
Jun 16, 2011 |
8771953 |
|
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PCT/EP09/67464 |
Dec 17, 2009 |
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14297933 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 2600/158 20130101;
G01N 2800/303 20130101; C12Q 1/6881 20130101; C12Q 2600/142
20130101; G01N 33/942 20130101; G01N 2800/304 20130101; G01N
33/5014 20130101; G01N 2800/302 20130101; C12Q 1/6883 20130101;
G01N 33/5058 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2008 |
EP |
08305963.4 |
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. 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 5HT2C
receptor; b) contacting said mammals cells with a compound to be
tested; c) determining in a cellular RNA extract: 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) selecting a therapeutic compound 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
24. 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.
25. The method according to claim 24, 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.
26. 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.
27. The method according to claim 23, wherein in step c), the
quantitative expression of said editing enzymes ADAR1a, ADAR1b and
ADAR2 is determined by the measure of the mRNA expression of said
editing enzymes or by the measure of said editing enzymes protein
expressed in the cellular extract.
28. 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.
29. The method according to claim 22, wherein in step a), said
mammal cells are cell lines from human, mouse or rat.
30. 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.
31. The method according to claim 22, wherein in step a), said
mammal cells are from a neuroblastoma cell line.
32. 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.
33. The method according to claim 23, wherein the treated pathology
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.
34. 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.
35. 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.
36. The method according to claim 22, wherein in step c), the
editing profile giving the mean proportion of each identified
isoform of the 5HT2CR mRNA measured in the cellular RNA extract is
measured by a CE-SSCP method comprising two rounds of PCR, and
wherein the first round of PCR is carried out by the following sets
of primers: for mouse or rat mammal cell lines: TABLE-US-00018
Forward: (SEQ ID N.sup.o 1) 5'-TGTCCCTAGCCATTGCTGATATGC-3',
Reverse: (SEQ ID N.sup.o 2) 5'-GCAATCTTCATGATGGCCTTAGTC-3',
for human cell lines: TABLE-US-00019 Forward: (SEQ ID N.sup.o 1)
5'-TGTCCCTAGCCATTGCTGATATGC-3', Reverse: (SEQ ID N.sup.o 2)
5'-GCAATCTTCATGATGGCCTTAGTC-3';
and wherein the second round of PCR is carried out by the following
set of primers: for mouse or rat mammal cell lines: TABLE-US-00020
Forward: (SEQ ID N.sup.o 5) 5'-TTTGTGCCCCGTCTGGAT-3', Reverse: (SEQ
ID N.sup.o 6) 5'-GCCTTAGTCCGCGAATTG-3',
for human cell lines: TABLE-US-00021 Forward: (SEQ ID N.sup.o 3)
5'-ATGTGCTATTTTCAACAGCGTCCATC-3', Reverse: (SEQ ID N.sup.o 4)
5'-GCAATCTTCATGATGGCCTTA-3'.
37. The method according to claim 22, wherein in step c), the pair
of primers specific for the ADAR mRNA PCR amplification are
selected from the group consisting of: for human ADAR1-150 isoform
mRNA amplification: TABLE-US-00022 Forward: (SEQ ID N.sup.o 7)
5'-GCCTCGCGGGCGCAATGAATCC-3', Reverse: (SEQ ID N.sup.o 8)
5'-CTTGCCCTTCTTTGCCAGGGAG-3',
for human ADAR1-110 isoform mRNA amplification: TABLE-US-00023
Forward: (SEQ ID N.sup.o 9) 5'-CGAGCCATCATGGAGATGCCCTCC-3',
Reverse: (SEQ ID N.sup.o 10) 5'-CATAGCTGCATCCTGCTTGGCCAC-3',
for human ADAR2 mRNA amplification: TABLE-US-00024 Forward: (SEQ ID
N.sup.o 11) 5'-GCTGCGCAGTCTGCCCTGGCCGC-3', Reverse: (SEQ ID N.sup.o
12) 5'-GTCATGACGACTCCAGCCAGCAC-3'.
38. 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 c) 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, ADAR1b and ADAR2 are
determined.
39. 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.
40. 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 ADAR1a, ADAR1b and ADAR2.
41. A kit according to claim 40, further comprising a panel of
references as selected in claim 17 b) and/or the edition profile of
the 5HT2CR and the ADARs expression for said panel of
references.
42. The method according to claim 35, wherein in step b) said
mammals cells are cultivated in presence of the compound to be
tested for at least 5 hours.
43. The method according to claim 35, wherein in step b) said
mammals cells are cultivated in presence of the compound to be
tested for at least 10 hours.
44. The method according to claim 35, wherein in step b) said
mammals cells are cultivated in presence of the compound to be
tested for at least 16 hours.
45. The method according to claim 35, wherein in step b) said
mammals cells are cultivated in presence of the compound to be
tested for at least 24 hours.
46. An in vitro method according to claim 38, wherein in step a)
said mammal cell line is a neuroblastoma cell line.
47. An in vitro method according to claim 38, wherein in step b)
said panel comprises members which differ with respect to their
toxicity or side-effects.
48. An in vitro method according to claim 38, wherein step c)
comprises the determination of the ADARs expression for said test
compound.
49. An in vitro method according to claim 38, wherein step d)
comprises the determination of the ADARs expression for said test
compound and for said panel of references.
50. An in vitro method according to claim 38, comprising the
determination of the ADARs expression.
51. A method according to claim 38, wherein the compound to be
tested is administered in vivo to a mouse or a rat.
Description
[0001] 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.
[0002] Toxicity is the major reason for abandoning candidate
therapeutic molecules during preclinical and clinical development.
To our knowledge, at the present time there is a need to provide
with tests by which to rapidly determine the toxicity profile of a
compound in man. In general, such tests are long and costly and are
only partially satisfactory. For example, animal toxicity is far
from being a reflection of human toxicity. Furthermore, the small
number of patients enrolled in clinical trials does not
systematically allow identification of toxicities associated with a
small, specific population. The development, perfection and use of
such tests should make it possible to identify and remove toxic
compounds from the development process as far upstream as possible.
In this manner new drugs could be marketed sooner and at a lesser
cost to drug companies and, in turn, to health care organizations
and consumers. In addition, such tests might also make it possible
to detect some toxicities which currently come to light only during
the post-marketing period.
[0003] Genetic association studies, knockout mice and postmortem
analysis have suggested the implication of the serotonin 2C
receptor (HTR2C) in neuropsychiatric disorders. Firstly, a
functional allelic polymorphism (Cys23Ser) of HTR2C is associated
with depression and bipolar disorder (Lerer et al., 2001, Mol.
Psychiatry, 6: 579-585). Secondly, mice lacking the 5-HT2C
serotonin receptor exhibit spontaneous convulsions, cognitive
impairment and abnormal control of feeding behavior (Tecott et al.,
1995, Nature, 374:542-546). These animals are also hyperresponsive
to repeated stress (Chou-green et al., 2003, Physiol. Behav.,
79:217-226). Thirdly, in postmortem brains of patients affected by
bipolar disorder or schizophrenia, the RNA expression of the 5-HT2C
serotonin receptor is down-regulated (Iwamoto et al., 2004, Mol.
Psychiatry, 9: 406-416; Castensson et al., 2003, Biol. Psychiatry,
54: 1212-1221) RNA editing of HTR2C is also thought to be involved
in the pathophysiology of mental disorders and the action of
antidepressants (Seeburg, 2002, Neuron, 35: 17-20). Five adenosine
residues (named A, B, C, D and E or C') are edited in a region
coding for the second intracellular loop of the 5-HT2C serotonin
receptor and can lead to amino-acid substitutions at three
different positions of the receptor sequence. The combinational
substitution of these amino residues generates up to 24 different
HTR2C protein isoforms with different G-coupling efficiencies
(Price et al., 2001, J. Biol. Chem., 276: 44663-44668). In mice,
when compared with C57BL/6 and 129sv inbred strains, the BALB/c
strain exhibits a different basal forebrain neocortical 5-HT2C
pre-mRNA editing pattern that may underlie their difference in
stress reactivity. Moreover, the BALB/c mice exhibit stress-induced
changes in 5-HT2C pre-mRNA editing ressembling those detected in
brains of depressed suicide victims (Englander et al., 2005, J.
Neurosci., 25: 648-651). Actually, in postmortem brains, altered
RNA editing of HTR2C has been reported in patients with
schizophrenia, depression and those who committed suicide
(Niswender et al., 2001, Neuropsychopharmacology, 24: 478-491;
Sodhi et al., 2001, Mol. Psychiatry, 6:373-379; Gurevich et al.,
2002, Neuron, 34: 349-356; Dracheva S. et al. 2008, Molecular
Psychiatry 13, 1011-10). Additionally interferon a is used in
hepatitis C treatment but symptoms of depression often appear` as a
side effect of this molecule in patients and Yang et al. have
demonstrated that this molecule strongly alters the editing of
5HT2C receptor (see ref. in Tohda et al., 2006, J. Pharmacol Sci,
100, 427-432).
[0004] Previous studies have shown that the 5-HT2C serotonin
receptor is mainly expressed in the brain, particularly in choroid
plexus, prefrontal cortex, limbic areas, basal ganglia and
hypothalamus (Tohda et al., 1996, Neurosci. Res., 24: 189-193;
Julius et al., 1988, 241: 558-564; Pasqualetti et al., 1999,
Neuroscience, 92: 601-611). This brain specific pattern of
expression restricts investigations of potential links existing
between HTR2C RNA editing and psychiatric condition to postmortem
studies.
[0005] New method were recently developed which allows to quantify
from total tissue RNA the complete editing profile of 5-HT2CR by
one single assay (Poyau et al., 2007, Electrophoresis, 28,
2843-52). It determines the percentage of each edited and non
edited isoforms in the fraction of specific mRNA contained in the
tissue sample. It is well adapted to the evaluation of editing
variations in specific brain regions in Mouse, Rat and Man. This
Kind of technology has been successful to analyse the editing
profile in primary cultured neural cells (Chanrion B., et al.,
Molecular Pharmacol, 2008, 73, 748-57).
[0006] The inventors main interest of this evaluation is to allows
to extract from the profile an index of the activity of the editing
isoenzymes the ADARs 1a, 1b, ADAR2 since ADAR1 isoenzymes edit the
sites A, B and also C and E and the ADAR2 can edit the sites D and
C and E. It is thus actually admitted that an isoform in which the
sites A and/or B are edited have been transformed by the ADARs I
isoenzymes, acting alone if the D site is not edited (the edited
isoforms: A, AB, ABC, ABCE, ABE, AC, ACE, AE, B, BC, BCE, BE), or
acting together with ADAR2 if the isoforms presents also the edited
D site (the edited isoforms: ABCD, ABCDE, ABD, ABDE, ACD, ACDE, AD,
ADE, BCD, BCDE, BD, BDE, C, CE, E).
[0007] The iso forms in which the sites A and/or B are not edited
and the D site is edited are considered as the result of the action
of ADAR2 alone.
[0008] Together to this analysis of the complete editing profile of
the 5-HT2C R, the inventors have demonstrated that an evaluation of
the expression of the ADARs isoenzymes as a complementary approach
is particularly well adapted to the evaluation of the general
editing context of dysregulation of the editing machinery. It
includes the quantitative and qualitative analysis of ADARs
isoenzymes expression (mRNA, i.e. by quantitative RT-PCR, and/or
the corresponding encoded protein, i.e. by the western
blotting).
[0009] Considering the special warning of the Food and Drug
Administration (FD) about suicide risk to epilepsy drug, to
antidepressants and antipsychotics. For example, recently the
weight-loss drug Acomplia.TM. (rimonabant) was found as triggering
suicidal behavior and other psychological side effects in some
patients, these known neuropsychiatric side effects rendering
difficult for the FDA and the European Medicines Agency to see a
positive risk-benefit ratio for this new drug and finally
recommended pulling it from the market because of its side
effects.
[0010] As mentioned above, there is a need to provide with tests
which can rapidly determine the toxicity or potential side-effects
profile of a drug in man, particularly before the post-marketing
period.
[0011] It is the reason why the inventors have decided to develop a
dedicated platform which could be proposed to evaluated, for
example at a pre-clinical stage, the potential effect of new
therapeutic molecules on the editing regulation since editing has
been already found altered in patients suffering from depression,
psychosis or having committed suicide.
[0012] The inventors have demonstrated that:
[0013] using particular mammal cells lines, particularly human
cells lines, which expressed the editing enzymes ADAR1a, ADAR1b and
ADAR2 and the 5-HT2C receptor; and
[0014] comparing between control cells and treated cells with the
compound to be tested [0015] a) the activity of these editing
enzymes (obtained by analysis of the editing profile giving the
mean proportion of each identified iso form of the 5-HT2CR mRNA
measured in the cellular RNA extract); and/or [0016] b) the
quantitative expression of these editing enzymes ADAR1a, ADAR1b and
ADAR2 (i.e. by Q-PCR or by western blotting), [0017] it is possible
to predict the eventual risk of these compounds to produce altered
mood by a chronic alteration of the 5-HT transmission.
[0018] The present invention now describes rapid, effective methods
by which to determine the potential toxicity of efficiency of test
compounds, as well as the tools and kits for the implementation of
such methods.
[0019] Thus, the present invention is directed to an in vitro
method for the determination or for the prediction of the potential
toxicity or side-effects of a test compound which can result after
its administration in a patient, comprising the following steps
of:
a) obtaining a biological sample containing mammal cells wherein
said mammal cells express the editing enzymes ADAR1a, ADAR1b and
ADAR2 and the serotonin 2C receptor (5HTR2C); b) contacting said
mammals cells with the compound to be tested; c) determining in the
same cellular extract:
[0020] the editing profile giving the mean proportion of each
identified isoform of the 5-HT2CR mRNA measured in the cellular RNA
extract, and/or
[0021] 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
or cells contacted with a control compound.
[0022] In the context of the invention, the term "toxicity" refers
to any adverse and/or side effect of a compound on the metabolism
of a cell or a tissue and more generally any alteration in
metabolism that can result in a harmful effect of the compound on
the patient, particularly in the context of the present invention
the potential risk of drug induced mood disturbance and
suicide.
[0023] The term "test compound" refers in general to a compound to
which a test subject is exposed. Typical test compounds will be
small organic molecules, typically drugs and/or prospective
pharmaceutical lead compounds, but can include proteins, peptides,
polynucleotides, heterologous genes (in expression systems),
plasmids, polynucleotide analogs, peptide analogs, lipids,
carbohydrates, viruses, phages, parasites, and the like.
[0024] The term "control compound" refers to a compound that is not
known to share any biological activity with a test compound, which
is used in the practice of the invention to contrast "active"
(test) and "inactive" (control) compounds during the derivation of
Group Signatures and Drug Signatures. Typical control compounds
include, without limitation, drugs used to treat disorders distinct
from the test compound indications, vehicles, inactivated versions
of the test agent, known inert compounds, and the like.
[0025] In a second aspect, the present invention is also directed
to an in vitro method for the selection of a 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 5-HTR2C comprising the following steps
of:
[0026] a) obtaining a biological sample containing mammal cells
wherein said mammal cells expressing the editing enzymes ADAR1a,
ADAR1b and ADAR2 and the 5-HT2C receptor;
b) contacting said mammals cells with the compound to be tested; c)
determining in the same cellular extract:
[0027] the editing profile giving the mean proportion of each
identified isoform of the 5-HT2CR mRNA measured in the cellular RNA
extract, and/or
[0028] 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
or cells contacted with a control compound; and e) selecting the
tested compound, whether this tested compound exhibits the
alteration or the non alteration of the HT2CR editing profile
and/or of the editing enzymes ADAR1a, ADAR1b and ADAR2 activities
which is desired to obtain.
[0029] In a preferred embodiment, the present invention is directed
to an in vitro method for the determination or for the prediction
of the potential toxicity or side-effects of a test compound or for
the selection of a therapeutical compounds, wherein step c)
comprises determining in the same cellular extract:
[0030] the editing profile giving the mean proportion of each
identified isoform of the 5-HT2CR mRNA measured in the cellular RNA
extract, and, optionally
[0031] the quantitative expression of said editing enzymes ADAR1a,
ADAR1b and ADAR2.
[0032] In an even more preferred embodiment, the present invention
is directed to an in vitro method for the determination or for the
prediction of the potential toxicity or side-effects of a test
compound or for the selection of a therapeutical compounds, wherein
step c) comprises determining in the same cellular extract:
[0033] the editing profile giving the mean proportion of each
identified isoform of the 5-HT2CR mRNA measured in the cellular RNA
extract, and
[0034] the quantitative expression of said editing enzymes ADAR1a,
ADAR1b and ADAR2.
[0035] In a preferred embodiment, in step a) of the methods of the
present invention, said mammal cells are capable of expressing of a
significant number of edited iso forms of 5HT2CR mRNA demonstrating
that these ADAR1a, ADAR1b and ADAR2 enzymes are active to control
the production of several edited iso forms of the receptor in these
cells.
[0036] More preferably, these mammal cells are able to present at
least the sites A, B and also C and E edited, also more preferred
are mammals cells which can present at least 1, preferably 2, 3, 4,
5, 6, 7, 6, 9, 10, 11 and 12, edited ADAR 1 isoforms selected from
the group consisting of A, AB, ABC, ABCE, ABE, AC, ACE, AE, B, BC,
BCE, BE edited isoforms, together with at least 1, preferably 2, 3,
4, 5, 6, 7, 6, 9, 10, 11, 12, 13 14 and 15 isoforms presenting also
the edited D site selecting from the group of the edited isoforms
ABCD, ABCDE, ABD, ABDE, ACD, ACDE, AD, ADE, BCD, BCDE, BD, BDE, C,
CE and E.
[0037] In a preferred embodiment of the methods of the present
invention in step a), said mammal cells are cells lines,
particularly from human, mouse or rat.
[0038] In a more preferred embodiment of the methods of the present
invention in step a), said mammal cells are mammal cells lines,
particularly from human, mouse or rat, which exhibit a regular and
constitutive expression of the 5HT2CR, ADAR1 and ADAR2 enzymes,
preferably ADAR1a, ADAR1b and ADAR2 enzymes.
[0039] The characteristic "exhibit a regular and constitutive
expression of the 5HT2CR, ADAR1 and ADAR2 enzymes, preferably
ADAR1a, ADAR1b and ADAR2 enzymes" is very important in the sense
where the expression of the content of the 5HT2CR, ADAR1 and ADAR2
enzymes are to be constant (regular) when the cells are cultured
and untraited (control cells) in order to have reliable comparative
data with traited cells (robustness and reliability of the
assay).
[0040] For example, the inventors have surprisingly demonstrated
(see Example 5 table 5 that the HTB-14 cells (glioma cells line) do
not express enough regular and constitutive 5HT2CR and cannot be
used as mammal cells line in the method of the present
invention.
[0041] The expression of the editing enzymes ADARs alone is not
sufficient to predict the potential toxicity or side effects of a
test compound or for selecting a therapeutical compound according
to the present invention, or to find new reference compound (as
interferon alpha) for comparing the effect of tested compound on
the alteration of the 5HT2CR edition and to predict the potential
toxicity or side-effects in view of the known alteration and
toxicity/side-effects of these reference compounds (panel of
reference compounds wherein their effects on the alteration of the
5HT2CR edition have been studied on the mammal cell lines used in
the method of the present invention (such as neuroblastoma cell
line (i.e. SH-SY5Y) and wherein the potential toxicity or
side-effect care known) in view of the known alteration and
toxicity/side-effects of these reference compounds.
[0042] Indeed, the quantitative expression of ADARs is not
sufficient to predict their combined effect on the 5HT2C edition.
The ADARs quantification assay is not an activity assay and the
inventors have demonstrated that only the determination of the
distribution of all the 5HT2C isoforms is significant if the real
alteration of the 5HT2C edition. It is the reason why it is
preferable to determine the distribution of all the 5HT2C isoforms
in correlation with the editing enzyme action.
[0043] In a yet more preferred embodiment of the methods of the
present invention in step a), said mammal cells are mammal cells
lines, particularly from human, mouse or rat, having the following
characteristics:
1) having a regular and constitutive expression of the 5HT2CR,
ADAR1 and ADAR2 enzymes, preferably ADAR1a, ADAR1b and ADAR2
enzymes; 2) when said mammal cell is treated by a drug capable to
alter the edition of the 5HT2CR,
[0044] capable of expressing of a significant number of 5HT2CR
edited isoforms,
[0045] preferably capable of expressing at least one 5HT2CR isoform
exhibiting at least the editing site A edited, one 5HT2CR iso form
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 iso
form exhibiting at least the editing site E edited, preferably with
in addition the non edited 5HT2CR iso form, when said mammal cell
is treated by a drug capable to alter the edition of the
5HT2CR,
[0046] more preferably all the 5HT2CR edited and non edited
isoforms are able to be expressed.
[0047] In a particular embodiment, said drug capable to alter the
edition of the 5HT2CR is the interferon alpha when said cell lines
have to be tested and selected for their capacity to exhibit an
alteration of the 5HT2CR edition and to express or not certain
5HT2CR isoforms in presence of such a 5HT2CR drug.
[0048] In a more preferred embodiment of the methods of the present
invention in step a), said mammal cells are mammal cells lines,
particularly from human, mouse or rat, which exhibit a regular and
constitutive expression of the 5HT2CR, ADAR1 and ADAR2 enzymes,
preferably ADAR1a, ADAR1b and ADAR2 enzymes.
[0049] In a yet more preferred embodiment of the methods of the
present invention in step a), said mammal cells are mammal cells
lines, particularly from human, mouse or rat, having the following
characteristics:
a) having a regular and constitutive expression of the 5HT2CR,
ADAR1 and ADAR2 enzymes, preferably ADAR1a, ADAR1b and ADAR2
enzymes; b)--capable of expressing of a significant number of
5HT2CR edited isoforms, preferably all of the 5HT2CR edited
isoforms and, when said mammal cell is treated by a drug capable to
alter the edition of the 5HT2CR; and
[0050] wherein the drug capable to alter the edition of the 5HT2CR
is a drug known to present a warning from the Scientific Community
or/and the Food and Drug Administration (FD) about risk of side
effect in some patients having received said drug, such as
psychological or neuropsychiatric side effects, this alteration
which thus can be correlated to this warning.
[0051] In an even more preferred embodiment of the methods of the
present invention in step a), said mammal cells are from a
neuroblastoma cell line, particularly from a human neuroblastoma
cell line.
[0052] In a preferred embodiment of the methods of the present
invention in step a), said mammal cells are from human, mouse or
rat, more preferably said mammal cells are cells from a
neuroblastoma cell line, particularly from a human neuroblastoma
cell line.
[0053] In a more preferred embodiment of the methods of the present
invention in step a), said mammal cells are from the human
neuroblastoma SH-SY5Y cell line, particularly the SH-SY5Y cell line
number EC94030304 from the European Collection of Cell Cultures
(ECACC).
[0054] In a preferred embodiment, in step c) of the methods of the
present invention, the editing profile of each identified iso form
of the 5-HT2CR mRNA and the quantitative expression of said editing
enzymes ADAR1a, ADAR1b and ADAR2 are determined in the same
cellular extract.
[0055] In a preferred embodiment of the methods of the present
invention in step c), when the editing profile of each identified
isoform of the 5-HT2CR 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.
[0056] In a preferred embodiment of the methods of the present
invention in step c), the analysis of the results of the
determination of the editing profile allow to obtain the activity
indexes of these editing enzymes ADAR1a, ADAR1b and ADAR2.
[0057] In a more preferred embodiment, said activity indexes of
these editing enzymes are calculated by a method comprising the
step of):
a) determining the mean proportion (%), preferably .+-.SEM (n 3, 4,
5 and 6), of each identified isoform of the 5-HT2cR mRNA measured
in the cellular RNA, or the mean proportion at least the set of iso
forms corresponding to the editing sites which are chosen to be
associated to the signature in step c) (A, B, C, D and/or E editing
sites), or the mean proportion at least the major isoforms found in
a control and/or a reference treated sample and corresponding to
the editing sites which are chosen to be associated to the
signature in step c) (A, B, C, D and/or E editing sites); b)
optionally, classifying these iso forms in function of the
algebraic delta when compared to the control and, optionally, to a
reference group, preferably to a treated group with a reference
drug; c) determining a signature associated to and significant of
the activity of the editing enzymes, preferably by classification
of the products of the enzymes activities, more preferably by a
method which calculates and correlates the percentage of edition of
at least two, preferably 3 or 4 of the editing sites selecting from
the: A, B, C, D and E editing sites, more preferably by a method
which calculates and correlates the percentage of edition of each
of the editing sites found in each part of the signature; and d)
optionally, classifying the expressed activity by measuring the %
represented by all the isoforms, or by the selected iso forms, for
which have been implicated ADAR 1 action (ADAR I) or ADAR2 (ADAR
II).
[0058] Concerning step a), it can be chosen to evaluate only some
specific editing action, for example to identify the proportion of
products of the enzyme activities in which only the sites C and E
have been found edited, or A and C, A and B, A and B and C, etc.
The non edited isoform (NE) can be also significant and
consequently chosen.
[0059] For example, when a comparison has to be done with a
reference sample which has been treated with a drug, it can be
chosen to evaluate only the specific editing action which have been
demonstrated as being altered by this treatment, compared to
control sample, and thus to identify only the proportion of the
corresponding products of these specific enzyme (enzyme activities
in which only the sites C and E have been found to be altered or A
and C, A and B, A and B and C, etc.).
[0060] In a yet more preferred embodiment, said activity indexes of
these editing enzymes are calculated from the editing isoforms due
to the action of at least ADAR1 alone (ADAR1) and the action of the
ADAR2 (ADAR2).
[0061] In an even more preferred embodiment, said activity indexes
of these editing enzymes are calculated from the editing isoforms
due to the action of ADAR1 alone and to the combined action of ADAR
1 and of ADAR 2 (ADAR1+2) and from the exclusive action of the
ADAR2 (ADAR2).
[0062] In a yet more preferred embodiment, the step a) of the
method for calculating said activity indexes of these editing
enzymes comprises the determination of the mean proportion (%),
preferably .+-.SEM (n 3, 4, 5 and 6), of all the 32 isoforms of the
5-HT2cR mRNA capable of being present in the cellular RNA extract
after contacting the cells with the compound to be tested.
[0063] In a preferred embodiment of the methods of the present
invention in step c), the quantitative expression of said editing
enzymes ADAR1a, ADAR1b and ADAR2 is determined by the measure of
the mRNA expression of said editing enzymes or by the measure of
said editing enzymes protein expressed in the cellular extract.
[0064] In a preferred embodiment of the methods of the present
invention the potential toxicity or side-effects of said test
compound to be determined is the potential risk of drug induced
mood disturbance and suicide, particularly mental disorders,
schizophrenia, depression, depressed suicide or abnormal feeding
behaviour.
[0065] In a preferred embodiment of the methods for screening
and/or selecting potential drug compound of the present invention,
for treating pathology related to the alteration of the mRNA
editing the 5-HTR2C after its administration, are pathologies
selected from the group consisting of mental disorders,
schizophrenia, depression, depressed suicide or abnormal feeding
behaviour.
[0066] In a preferred embodiment of the methods of the present
invention in step b) said mammals cells are cultivated in presence
of the compound to be tested in a medium suitable or convenient for
the culture of said mammal cells, and preferably convenient for the
expression of the 5-HTR2C and the editing enzymes ADAR1 (1a and 1b)
and ADAR2.
[0067] Preferably, in step b) said mammals cells are cultivated in
presence of the compound to be tested for at least the time
necessary to modify the expression of edited isoforms of 5HT2CR
mRNA and/or the ADAR1a, ADAR1b and ADAR2 enzymes expressed, whether
they can be modified by such a compound.
[0068] Preferably in step b) said mammals cells are cultivated in
presence of the compound to be tested for at least 1 hour, more
preferably at least 5, 10, 16, 24 and 48 hours before the step c)
of determining in the same cellular extract the editing profile of
each identified isoform of the 5-HT2CR mRNA and/or the quantitative
expression of said editing enzymes ADAR1a, ADAR1b and ADAR2.
[0069] The methods for determining the editing steady state
includes to determine the profile of each identified isoform of the
5-HT2CR mRNA measured in a cellular RNA extract involving a nested
type PCR allowing the determination of the exact distribution the
ADAR iso forms expressed in a cell extract and to estimate, in the
same extract, the steady state of expression of the editing
enzymes. They can be found also in the PCT Patent application
"Peripherical tissue sample containing cells expressing the 5HTR2C
and/or ADARs as markers of the alteration of the mechanism of the
5HTR2C mRNA editing and its applications" filed on Jun. 13, 2008
under the number PCT/EP2008057519 and published on Dec. 18, 2008
(WO 2008/152146).
[0070] In a preferred embodiment of the methods of the present
invention in step c), the editing profile giving the mean
proportion of each identified iso form of the 5-HT2CR mRNA measured
in the cellular RNA extract is determined by a nested type PCR
comprising two rounds of PCR, and wherein the first round of PCR is
carried out by the following sets of primers:
for mouse or rat mammal cell lines:
TABLE-US-00001 Forward: (SEQ ID NO. 1)
5'-TGTCCCTAGCCATTGCTGATATGC-3', Reverse: (SEQ ID NO. 2)
5'-GCAATCTTCATGATGGCCTTAGTC-3';
for human cell lines:
TABLE-US-00002 Forward: (SEQ ID NO. 1)
5'-TGTCCCTAGCCATTGCTGATATGC-3', Reverse: (SEQ ID NO. 2)
5'-GCAATCTTCATGATGGCCTTAGTC-3';
and wherein the second round of PCR is carried out by the following
set of primers: for mouse or rat cell lines:
TABLE-US-00003 Forward: (SEQ ID NO. 5) 5'-TTTGTGCCCCGTCTGGAT-3',
Reverse: (SEQ ID NO. 6) 5'-GCCTTAGTCCGCGAATTG-3';
and for human cell lines:
TABLE-US-00004 Forward: (SEQ ID NO. 3)
5'-ATGTGCTATTTTCAACAGCGTCCATC-3', Reverse: (SEQ ID NO. 4)
5'-GCAATCTTCATGATGGCCTTA-3'.
[0071] In a preferred embodiment of the method according to the
present invention, the editing rate for each edited and unedited
form of said 5HTR2C mRNA is determined by a method which comprises
the following steps:
[0072] A) extraction of the total RNAs of said mammal cells,
followed, where appropriate, by purification of the mRNAs;
[0073] B) reverse transcription of the RNAs extracted in step A);
and
[0074] C) PCR amplification of the cDNAs obtained in step B) using
at least a pair of primers specific for the 5HTR2C mRNA fragment
containing the edition sites which may be edited, this pair of
primers being chosen so as to be able to amplify all the editing
forms and the unedited form potentially present in the RNA
extract.
[0075] In a preferred embodiment of the method according to the
present invention, the editing rate for each edited and unedited
form of said 5HTR2C mRNA is determined by a method which comprises
the following steps:
[0076] A) extraction of the total RNAs of said mammal cells,
followed, where appropriate, by purification of the mRNAs;
[0077] B) reverse transcription of the RNAs extracted in step A);
and
[0078] C) PCR amplification of the cDNAs obtained in step B) using
at least a pair of primers specific for the 5HTR2C mRNA fragment
containing the edition sites which may be edited, this pair of
primers being chosen so as to be able to amplify all the editing
forms and the unedited form potentially present in the RNA extract,
and wherein the step B) of reverse transcription is carried out by
using an oligonucleotidic primer specific of the 5HTR2C gene.
[0079] In a preferred embodiment of the method according to the
present invention, in step C), the primers used in the PCR
amplification step (in the second round if it is a nested type PCR)
are labelled, preferably labelled with fluorophores.
[0080] In a preferred embodiment of the methods of the present
invention in step c), the editing profile giving the mean
proportion of each identified iso form of the 5-HT2CR mRNA is
determined by an CE-SSCP method capable of providing the editing
profile for each of the edited and unedited separate forms of said
mRNA, said SSCP method being characterized in that it comprises
after the steps A), B) and C) the following steps:
D) where appropriate, purification of the PCR products obtained in
step C); E) where appropriate, quantification of the PCR products
obtained in step D); F) dissociation of the double-stranded cDNAs
to single-stranded cDNAs, in particular by heating followed by
abrupt cooling; G) separation of the single-stranded cDNAs by
capillary electrophoresis; and H) obtaining of the editing profile
by reading of the fluorescence and, where appropriate, acquisition
of the profile data by means of the exploitation system associated
with the fluorescence reader.
[0081] In a preferred embodiment of the methods of the present
invention in step c), the pair of primers specific for the ADAR
mRNA PCR amplification are selected from the group consisting
of:
[0082] for human ADAR1-150 isoform mRNA amplification:
TABLE-US-00005 Forward: (SEQ ID NO. 7)
5'-GCCTCGCGGGCGCAATGAATCC-3', Reverse: (SEQ ID NO. 8)
5'-CTTGCCCTTCTTTGCCAGGGAG-3';
[0083] for human ADAR1-110 isoform mRNA amplification:
TABLE-US-00006 Forward: (SEQ ID NO. 9)
5'-CGAGCCATCATGGAGATGCCCTCC-3', Reverse: (SEQ ID NO. 10)
5'-CATAGCTGCATCCTGCTTGGCCAC-3';
[0084] for human ADAR2 mRNA amplification:
TABLE-US-00007 Forward: (SEQ ID NO. 11)
5'-GCTGCGCAGTCTGCCCTGGCCGC-3', Reverse: (SEQ ID NO. 12)
5'-GTCATGACGACTCCAGCCAGCAC-3';
[0085] for mouse ADAR1-150 isoform mRNA amplification:
TABLE-US-00008 Forward: (SEQ ID NO. 13)
5'-GTCTCAAGGGTTCAGGGGACCC-3', Reverse: (SEQ ID NO. 14)
5'-CTCCTCTAGGGAATTCCTGGATAC-3';
[0086] for mouse ADAR1-110 isoform mRNA amplification:
TABLE-US-00009 Forward: (SEQ ID NO. 15)
5'-TCACGAGTGGGCAGCGTCCGAGG-3', Reverse: (SEQ ID NO. 14)
5'-CTCCTCTAGGGAATTCCTGGATAC-3';
and
[0087] for mouse ADAR2 mRNA amplification:
TABLE-US-00010 Forward: (SEQ ID NO. 16)
5'-GCTGCACAGTCTGCCTTGGCTAC-3', Reverse: (SEQ ID NO. 17)
5'-GCATAAAGAAACCTGAGCAGGGAC-3'.
[0088] In a preferred embodiment of the methods of the present
invention 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 this test compound after its administration in this
animal model can be evaluated, particularly by evaluating the
alteration of the mRNA editing of the 5HTR2C and/or the ADAR
isoforms expressed in total blood and skin sample, or in brain (as
disclosed in the international PCT patent application filed on Jun.
13, 2008 under the number PCT/EP2008057519 and published on Dec.
18, 2008 under the number WO 2008/152146).
[0089] In another aspect, the present invention is directed to a
kit for the determination of the potential toxicity or side-effects
of a test compound after its administration in a patient or for the
selection of a 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 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 set of primers for measuring each
isoform of the 5-HT2CR mRNA which can be present in a RNA extract
of said mammal cells by a CE-SSCP method involving a nested type
PCR comprising two rounds of PCR; and/or c) a set of primers for
measuring by a quantitative Q-PCR the quantitative expression of
the editing enzymes ADAR1a, ADAR1b and ADAR2.
[0090] In a preferred embodiment of the kit of the present
invention, said mammal cells are capable of expressing of a
significant number of edited isoforms of 5HT2CR mRNA demonstrating
that these ADAR1a, ADAR1b and ADAR2 enzymes are active to control
the production of several edited isoforms of the receptor in these
cells.
[0091] More preferably, these mammal cells are able to present at
least the sites A, B and also C and E edited, also more preferred
are mammals cells which can present at least 1, preferably 2, 3, 4,
5, 6, 7, 6, 9, 10, 11 and 12, edited ADAR 1 isoforms selected from
the group consisting of A, AB, ABC, ABCE, ABE, AC, ACE, AE, B, BC,
BCE, BE edited isoform, together with at least 1, preferably 2, 3,
4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14 and 15 isoforms presenting
also the edited D site selecting from the group of the edited
isoforms ABCD, ABCDE, ABD, ABDE, ACD, ACDE, AD, ADE, BCD, BCDE, BD,
BDE, C, CE and E.
[0092] In a preferred embodiment of the kit of the present
invention said mammal cells are from human, mouse or rat, more
preferably said mammal cells are cells from a neuroblastoma cell
line, particularly from a human neuroblastoma cell line.
[0093] In a more preferred embodiment of the kit of the present
invention, said mammal cells are from the human neuroblastoma
SH-SY5Y cell line.
[0094] In a particular aspect, the present invention is directed to
an in vitro method for the determination or for the prediction of
the potential toxicity or side-effects of a interferon alpha
(IFN.alpha.) treatment after its administration in a patient,
particularly for a patient infected by the HCV (Hepatitis C virus),
said method comprising the following steps of:
a) obtaining a biological sample containing mammal white cells,
preferably leucocytes or monocytes cells, from said treated
patient; b) determining in the cellular extract of said biological
sample containing mammal white cells the quantitative expression of
each of the editing enzymes ADAR1a, ADAR1b and ADAR2, and,
optionally the editing profile giving the mean proportion of each
identified isoform of the 5-HT2CR mRNA measured in the cellular RNA
extract; d) comparing the results obtained in step b) between said
cells from said IFN.alpha. treated patient with non treated control
cells or with. IFN.alpha. treated cells prior obtained from the
same patient at the beginning or during the IFN.alpha.
treatment.
[0095] The present invention is also directed to an in vitro method
of predicting the potential toxicity of test compounds or 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), which comprises:
(a) screening compounds on a mammal cell line (preferably those
cell lines having the characteristics depicted for the method of
the present invention), preferably neuroblasto cell line, more
preferably the SH-SY5Y cell line, for their ability to alter the
5HT2CR edition, these compounds being known to have or not toxicity
or side-effects, such as psycho logic or neuropsychologic 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 and, optionally, which
differ with respect to their toxicity or side-effects; (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
and, optionally, the ADARs expression for said test compound; (d)
comparing the edition profile of the 5HT2CR and, optionally, the
ADARs expression for said test compound and for said panel of
references; (e) predicting the potential toxicity of test compounds
or selecting the test compound as potential therapeutical 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 profil edition and, optionally, the ADARs expression, is
the same in vitro cell-based assay depicted for the method of the
present invention above, as in claims 1 to 15.
[0096] The present invention finally comprises a kit according to
the present invention, said kit further comprising a panel of
references as selected in step b) of the above method or/and the
edition profile of the 5HT2CR and, optionally, the ADARs expression
for said panel of references.
[0097] The following examples and also the figures and the legends
hereinafter have been chosen to provide those skilled in the art
with a complete description in order to be able to implement and
use the present invention. These examples are not intended to limit
the scope of what the inventor considers to be its invention, nor
are they intended to show that only the experiments hereinafter
were carried out.
LEGEND TO THE FIGURES
[0098] FIGS. 1A and 1B: Effect of human interferon 24 hours
application on ADAR1a mRNA concentration of SH-SY5Y cells:
determination of EC50%.
[0099] ADAR1a RNA Expression in SH-SY5Y cells (Q-PCR, Applied
TaqMan probes ref: Hs 01020780_ml).
[0100] Reference gene: GAPDH.
[0101] 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.
[0102] FIG. 2A. The .DELTA..DELTA.CTs of the ADAR1a mRNA of treated
dishes (n=8) versus controls (n=8) are plotted versus LOG 10 of the
applied concentrations of INF .alpha.. 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).
[0103] 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).
[0104] 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 .alpha. 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 .DELTA..DELTA.CT 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%.
[0105] FIG. 3: A cell model as a basis to evaluate a possible
psychiatric risk.
[0106] 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).
[0107] 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 .alpha. 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.
[0108] FIG. 5: ADAR1 mRNA and protein expression in
IFN.alpha.-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
[0109] 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.
[0110] 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.times. mix of Antibiotic-Antimycotic Stabilized (Sigma,
ref. A5955) at 37.degree. C. under a humidified atmosphere of 5%
CO2. The day preceeding drug or hIFN.alpha. 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 .mu.M solution of the compound to be tested molecules or
a 1000 IU/ml solution of hIFN.alpha. (PBL biomedical laboratories).
For hIFN.alpha. dose-response experiment cells were incubated with
1, 10, 100, and 1000 IU/ml of hIFN.alpha. solutions. For
hIFN.alpha. time-course experiment cells were treated for 24, 48,
or 72 hours with a 1000 IU/ml solution of hIFN.alpha.. 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
In Vivo Protocol for mIFN.alpha. and Drug Treatment
[0111] For the mIFN.alpha. experiment, 8 males (Balb/cJ mice,
Charles Rivers) were injected i.p route once with 10000 IU of
mIFN.alpha. (PBL biomedical laboratories). Control mice were
injected same route with sterile Phosphate Buffer Saline. 8 hours
after injection animals were sacrificed 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).
[0112] 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 sacrificed 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
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
[0113] 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 (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
[0114] 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 .mu.l of each RNA (between 88 ng and 1.3 .mu.g) were
then treated with 1 unit of DNase I (Invitrogen) for 15 min at room
temperature in a final volume of 10 The reaction was stopped by
adding 1 .mu.l of 25 mM EDTA and then heated for 10 min at
65.degree. C. The reverse transcription of DNAse I-treated RNAs (10
.mu.l) 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 .mu.M.
[0115] A first PCR reaction (final volume 25 .mu.l) resulting in a
250 bp fragment, was then carried out on 1 .mu.l 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 .mu.M) located on exon IV and exon
V of the Human 5-HT2cR cDNA, respectively. After a denaturing step
at 95.degree. C. for 3 min, the PCR was brought to its final point
after 35 cycles (15 s at 95.degree. C.; 30 s at 60.degree. C.; 20 s
at 72.degree. C.), and a final elongation step of 2 min at
72.degree. 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)
[0116] 1 .mu.l 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
.mu.l 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 .mu.M), and 0.2 unit of Platinum Pfx DNA polymerase
(Invitrogen).
[0117] 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 forward 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.
[0118] 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).
[0119] The amplified fragment is 127 bp-long. As for RT-PCR, after
an initial denaturing step of 5 min at 94.degree. C., the
amplification reaction was brought to an end with 35 cycles (15 s
at 94.degree. C.; 30 s at 55.degree. C.; 20 s at 68.degree. C.) and
a final elongation step of 2 min at 68.degree. 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).
[0120] Fluorescent PCR products corresponding to standard isoforms
(1 .mu.l of a 1/100 dilution in DEPC treated water) and samples (1
.mu.l of a 1/30 dilution) diluted in 11 .mu.l of deionized
formamide were added to a mixture of ROX labelled migration
standards (MWG-BIOTECH, AG) (0.5 .mu.l 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.degree. 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.times. 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.degree. 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
[0121] 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.
[0122] 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
S ( t ) = i = 1 N g i R i ( t ) ##EQU00001##
in which R.sub.i(t), with i.epsilon.{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)
SSE = .intg. [ S ( t ) - i = 1 N g i R i ( t ) ] 2 ##EQU00002##
and was controlled by the least square statistical analysis.
[0123] The result of this best fitting was statistically evaluated
after calculation of the r.sup.2 value such as
r 2 = 1 - SSE SSM ##EQU00003##
in which SSM is the Sum of Square about Mean such as
SSM = i = 1 t ( S ( t ) - S _ ) 2 . ##EQU00004##
The maximum theoretical best fit would give an r.sup.2=1.
[0124] 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.
[0125] 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
[0126] 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):
[0127] 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_m1 Mm
00508001_m1 Mm 00507997_m1 Mm 00504621_m1 Mm 00507998_m1 Human
tissues Hs 00968672_m1 Hs 01017596_m1 Hs 01020780_m1 Hs 00210562_m1
And Hs 00968671_m1 SH-SY5Y cells
[0128] 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)=.DELTA.Ct, where
Ct is the threshold cycle number. The mean .DELTA.Ct 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
(.DELTA.Ct (individual treated samples)-.DELTA.Ct (mean of
untreated samples)=.DELTA..DELTA.Ct). Changes in target gene
expression (n-fold) in each sample were calculated by
2.sup.-.DELTA..DELTA.Ct, from which the means and standard
deviations (SD) were derived.
[0129] 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
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)
[0130] The concentration of 1000 IU/ml was chosen for additional
evaluation of the distribution of edited isoforms in controls and
interferon treated cells the result is presented on Table 2.
[0131] Table 2: Analysis of the editing profile obtained after 24
hours of treatment by 1000 IU of human interferon alpha.
[0132] 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.
TABLE-US-00012 TABLE 2 Editing Profile Isoforms Ctls SEM IFN1000UI
SEM NE (INI) 54.6 1.9 37.5 3.5 A (VNI) 29.5 1.3 32.3 2.5 B (MNI)
3.4 0.3 3.5 0.8 C (ISI) 2.5 0.5 2.2 0.4 AB (VNI) 1.8 0.8 2.9 0.9
ACE (VGI) 1.4 0.3 0.8 0.2 D (INV) 1.3 0.2 0.5 0.1 BD (MNV) 1.1 0.1
0.3 0.1 AC (VSI) 0.9 0.6 13.5 3.4 AE (VDI) 0.6 0.4 1.1 0.3 ABD
(VNV) 0.4 0.1 0.2 0.2 ADE (VDV) 0.4 0.0 0.04 0.0 DE (IDV) 0.4 0.0
0.27 0.0 AD (VNV) 0.3 0.2 1.40 0.5 ABE (VDI) 0.2 0.2 0.29 0.1 BE
(MDI) 0.2 0.1 0.76 0.7 CE (IGI) 0.2 0.2 0.13 0.1 ABC (VSI) 0.2 0.1
1.62 0.3 ABCD (VSV) 0.1 0.1 0.03 0.0 CD (ISV) 0.1 0.0 0.19 0.1 BCE
(MGI) 0.1 0.0 0.02 0.0 ABDE (VDV) 0.1 0.1 0.04 0.0 100 100
Signature Isoforms .DELTA. % Student ADE (VDV) -89.2 0.00002 ABCD
(VSV) -82.4 0.21792 BD (MNV) -75.9 0.00002 BCE (MGI) -69.2 0.12449
ABD (VNV) -60.5 0.13164 D (INV) -59.8 0.00221 CE (IGI) -43.9
0.34397 ABDE (VDV) -41.7 0.36296 ACE (VGI) -40.2 0.07315 NE (INI)
-31.3 0.00082 DE (IDV) -28.3 0.06225 C (ISI) -14.2 0.28715 B (MNI)
2.6 0.46177 A (VNI) 9.5 0.17352 ABE (VDI) 25.8 0.40553 AB (VNI)
63.7 0.18693 AE (VDI) 69.2 0.17984 CD (ISV) 123.2 0.24804 BE (MDI)
228.5 0.24235 AD (VNV) 342.7 0.03539 ABC (VSI) 758.7 0.00104 AC
(VSI) 1324.2 0.00235 Ctls SEM IFN1000UI SEM .DELTA. % Student
Global signature NE (INI) 54.6 1.9 37.5 3.5 -31.3 0.0008 Sum of
Delta > 0 37.3 1.9 57.5 3.5 54.2 0.0002 Sum of Delta < 0 8.1
0.4 4.5 0.5 -44.3 0.0001 Edited Sites Delta > 0 A 33.6 2.1 53.6
3.9 59.3 0.0006 B 5.8 0.7 9.1 1.6 56.6 0.0447 C 1.2 0.6 15.8 3.6
1191.6 0.0013 D 0.4 0.2 2.0 0.6 409.8 0.0125 E 1.1 0.6 2.2 0.6 99.4
0.1233 Delta < 0 A 2.4 0.4 1.1 0.4 -54.1 0.0224 B 1.8 0.3 0.5
0.2 -71.3 0.0014 C 4.3 0.6 3.2 0.5 -27.2 0.0793 D 3.9 0.2 1.4 0.3
-64.9 0.0000 E 2.5 0.3 1.3 0.3 -46.9 0.0083 Total A 36.1 2.3 54.7
3.9 51.7 0.0011 B 7.7 0.8 9.6 1.7 25.8 0.1596 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
[0133] 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.
[0134] 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). The asterisk marked (*) area and bold numbers
indicate a significant variation (p < .05). ENZYME ACTIVITY
INDEX (% .DELTA.) Q-PCR (% .DELTA.) ADAR1 ADAR1+ 2 ADAR2 ADAR I
ADAR II NE ADAR1a ADAR1b ADAR2 IFN 24H 48.05* -10.25 -44.75* 41.07*
-19.08 -31.30* 970* 0 68
[0135] 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) ADAR1 ADAR1 + 2 ADAR2 NE ADAR I ADAR II
EC ADAR1a ADAR1b ADAR2 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*
[0136] 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
Choice of a Cell Line for In Vitro Predictive Effect of Molecules
a-Criteria for Selection
[0137] To be eligible for the in vitro screening of molecules the
cell line must validate the main following points: [0138] to be
from Human origin; [0139] to express the 5-HT2cR receptor at a
range allowing a reproducible evaluation of the editing profile in
control conditions; [0140] 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.
[0141] Among ten different cell lines, the SH-SY5Y Human
neuroblastoma cell line was selected as the most interesting when
used in the following conditions.
[0142] 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.times. mix
of Antibiotic-Antimycotic Stabilized (Sigma, ref A5955) at
37.degree. C. under a humidified atmosphere of 5% CO2. The day
preceeding hIFN.alpha. 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 .mu.M solution of the to be tested molecules
or a 1000 IU/ml solution of hIFN.alpha. (PBL biomedical
laboratories). For hIFN.alpha. dose-response experiment, cells were
incubated with 1, 10, 100, 1000, or 10000 IU/ml of hIFN.alpha.
solutions. [For hIFN.alpha. time-course experiment cells were
treated for 24, 48 or 72 hours with a 1000 1 U/ml solution of
hIFN.alpha.. In the case of the 72-hour treatment points, medium
was changed for both controls and hIFN.alpha.-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.
[0143] 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 .mu.l 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.times.15 seconds on ice, rocked for at
least 2 hours at 4.degree. C. and then spun at 100 g for 10 min at
4.degree. C. Insoluble pellets were resuspended in 40 .mu.A of
2.times. 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.degree.
C., 75 .mu.g 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.
[0144] 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.
rcf 94030304) and ATCC (HTB-14, rcf HTB-14). Ccrcbral cortcx 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
.TM. 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_ml), ADAR1 p110 constitutive isoform (Hs 01017596_ml),
ADAR1 p150 inducible isoform (Hs 01020780_ml), 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. RNA Origin RQ
ADAR1a RQ ADAR1b p RQ ADAR2 p RQ 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 cells 1 20.27
<0.0001 4.23 0.000018
[0145] 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 (ADAR1b) 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.
[0146] 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
IFN.alpha.-treated SH-SY5Y cells. SH-SY5Y cells were cultured for
48 hours in presence of IFN.alpha. 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
.TM. 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_m1), ADAR1 p150 inducible isoform (Hs 01020780_m1). 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)
[0147] This experiment clearly demonstrates that in the culture
conditions expressed above the chosen cell line respond to the
induction produced by INF.alpha. can be observed at the enzymatic
protein level with the same specificity and amplitude ratio than
those predicted by the mRNA quantification.
[0148] 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.
TABLE-US-00017 TABLE 8 Comparison of the editing profiles of
5-HT2cR determined in 3 area of the human brain of control subjects
and in the cultured SH- SY5Y cells in control conditions. We can
note that a reproducible editing profile can be observed from
individual dishes of culture (n = 8). We note than the number of
edited isoforms is smaller in the cell line but the mean proportion
of isoforms in a range .gtoreq.1% remains similar indicating a
similar efficiency of the analytical process use for the
quantification. The shadowed cells indicated the NE isoform
proportions and the group of isoforms under a limit of 1%. The
results are expressed as the mean % of the total specific mRNA .+-.
SEM (n = 6 control human subjects and 8 cultured dishes in control
conditions). ##STR00001## ##STR00002## ##STR00003##
##STR00004##
[0149] 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
[0150] 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.
[0151] 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).
[0152] 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.
[0153] 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.
[0154] 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.
[0155] The result of such an analysis is presented on FIG. 3.
[0156] 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 INF.alpha. application.
Example 7
Use of the In Vitro Platform to the Detection of Reference
Molecules Inducing Similar Alteration of the Editing Profile to
that Observed after INF.alpha. Treatment
[0157] 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 .mu.M. 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).
[0158] 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.
[0159] 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.
Sequence CWU 1
1
17124DNAArtificial SequenceDescription of the artificial sequence
sequence derived from the cDNA coding for 5-HT2C receptor
1tgtccctagc cattgctgat atgc 24224DNAArtificial SequenceDescription
of the artificial sequence sequence derived from the cDNA coding
for 5-HT2C receptor 2gcaatcttca tgatggcctt agtc 24326DNAArtificial
SequenceDescription of the artificial sequence sequence derived
from the cDNA coding for 5-HT2C receptor 3atgtgctatt ttcaacagcg
tccatc 26421DNAArtificial SequenceDescription of the artificial
sequence sequence derived from the cDNA coding for 5-HT2C receptor
4gcaatcttca tgatggcctt a 21518DNAArtificial SequenceDescription of
the artificial sequence sequence derived from the cDNA coding for
5-HT2C receptor 5tttgtgcccc gtctggat 18618DNAArtificial
SequenceDescription of the artificial sequence sequence derived
from the cDNA coding for 5-HT2C receptor 6gccttagtcc gcgaattg
18722DNAArtificial SequenceDescription of the artificial sequence
sequence derived from the cDNA coding for human ADAR1 150 isoform
7gcctcgcggg cgcaatgaat cc 22822DNAArtificial SequenceDescription of
the artificial sequence sequence derived from the cDNA coding for
human ADAR1 150 isoform 8cttgcccttc tttgccaggg ag
22924DNAArtificial SequenceDescription of the artificial sequence
sequence derived from the cDNA coding for human ADAR1 110 isoform
9cgagccatca tggagatgcc ctcc 241024DNAArtificial SequenceDescription
of the artificial sequence sequence derived from the cDNA coding
for human ADAR1 110 isoform 10catagctgca tcctgcttgg ccac
241123DNAArtificial SequenceDescription of the artificial sequence
sequence derived from the cDNA coding for human ADAR2 11gctgcgcagt
ctgccctggc cgc 231223DNAArtificial SequenceDescription of the
artificial sequence sequence derived from the cDNA coding for human
ADAR2 12gtcatgacga ctccagccag cac 231322DNAArtificial
SequenceDescription of the artificial sequence sequence derived
from the cDNA coding for murine ADAR1 150 isoform 13gtctcaaggg
ttcaggggac cc 221424DNAArtificial SequenceDescription of the
artificial sequence sequence derived from the cDNA coding for
murine ADAR1 150 isoform 14ctcctctagg gaattcctgg atac
241523DNAArtificial SequenceDescription of the artificial sequence
sequence derived from the cDNA coding for murine ADAR1 110 isoform
15tcacgagtgg gcagcgtccg agg 231623DNAArtificial SequenceDescription
of the artificial sequence sequence derived from the cDNA coding
for murine ADAR2 16gctgcacagt ctgccttggc tac 231724DNAArtificial
SequenceDescription of the artificial sequence sequence derived
from the cDNA coding for murine ADAR2 17gcataaagaa acctgagcag ggac
24
* * * * *