USRE43879E

Use of the dextrogyral enantiomer of milnacipran for the preparation of a drug

Claim Score by NHIP

Abstract

The present invention concerns the use of a mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one of its metabolites, as well as their pharmaceutically-acceptable salts, for the preparation of a drug intended to prevent or to treat disorders that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or organ and/or tissue toxicity.

USRE43879E, drawing sheet 1 — Sheet 1 of 7

Term

Term ended

Expired 3 June 2023, 3.3 years ago.

25 claims: 4 independent, 21 dependent

1
A method for treating a patient afflicted with a condition or disorder which may be treated by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, comprising the step of administering to the patient an amount of a mixture of enantiomers of milnacipran hydrochloride (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride), such mixture being substantially pure in the dextrogyral enantiomer, effective for alleviation of the condition or disorder, wherein the administration of said mixture limits the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, relative to administration of racemic milnacipran hydrochloride.
17
Broadest claimClaim Score 57, broad(NHIP)A method for treating a patient afflicted with depression, while limiting the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, comprising the step of administering to the patient an amount of:a) a mixture of enantiomers substantially pure in the dextrogyral enantiomer of milnacipran hydrochloride (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride), wherein the administration of said mixture limits the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, relative to administration of racemic milnacipran hydrochloride, and b) at least one active compound selected from the psychotropics, as associated products for use simultaneously, separately or staggered in time, effective for alleviation of depression.
20
A method for treating a patient afflicted with a condition or disorder which may be treated by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, comprising the step of administering to the patient an amount of:a) a mixture of enantiomers substantially pure in the dextrogyral enantiomer of milnacipran hydrochloride (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride), wherein the administration of said mixture limits the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, relative to administration of racemic milnacipran hydrochloride, and b) at least one other active substance selected from the active compounds that induce organ toxicity and the active compounds that induce cell toxicity, as associated products for use simultaneously, separately or staggered in time, effective for alleviation of the condition or disorder.
22
A method for treating a patient afflicted with a condition or disorder which may be treated, by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake while limiting the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, comprising the step of administering to the patient an amount of:a) a mixture of enantiomers substantially pure in the dextrogyral enantiomer of milnacipran hydrochloride (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride), wherein the administration of said mixture limits the risks of cardiovascular disturbances and/or the risks of organ and/or tissue toxicity, relative to administration of racemic milnacipran hydrochloride, and b) at least one other active substance selected from the active compounds that induce cardiovascular side-effects, as associated products for use simultaneously, separately or staggered in time, effective for alleviation of the condition or disorder.

Independent claims4

217 paragraphs in 2 sections, as filed

0001The present invention concerns the use of a mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one its metabolites, as well as their pharmaceutically-acceptable salts, for the preparation of a drug intended to prevent or to treat disorders that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or of organ and/or tissue toxicity. More specifically, the mixture of enantiomers in accordance with the invention is intended to treat depression, chronic fatigue syndrome and urinary incontinence.

0002Milnacipran (Z(±)-2-(amino methyl)-N,N-diethyl-1-phenyl cyclopropane carboxamide), a molecule synthesised at the PIERRE FABRE MEDICAMENT Research Centre (Castres, France), also called TN-912, dalcipran, minalcipran, midalcipran or midalipran is known to be a dual inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake. Milnacipran and its method of preparation are described in U.S. Pat. No. 4,478,836. Other information relating to milnacipran can be found in the twelfth edition of the Merck Index, as entry n<sup>o </sup>6 281.

0003Dual inhibitors of serotonin (5-HT) and norepinephrine (NE) reuptake correspond to a well-known class of antidepressant agents which selectively inhibit reuptake of both serotonin and norepinephrine. By way of example, venlafaxine and duloxetine are also dual inhibitors of serotonin and norepinephrine. Studies have shown that the ratio of norepinephrine reuptake inhibition to serotonin reuptake inhibition by milnacipran is approximately 2:1 (Moret et al., 1985 Neuropharmacology 24(12): 1211-1219; Palmier et al., 1989, Eur J Clin Pharmacol 37: 235-238).

0004U.S. Pat. No. 4,478,836 describes the use of milnacipran for the treatment of disorders of the central nervous system, in particular depression. Patent application WO01/26623 describes the use of milnacipran in association with phenylalanine and tyrosine in indications such as the treatment of fatigue, syndromes associated with pain, chronic fatigue syndrome, fibromyalgia, and irritable bowel syndrome. Patent application WO01/62236 describes a composition containing milnacipran in association with one or several antimuscarinic agents in a large number of indications including depression. Application WO97/35574 describes a pharmaceutical composition containing milnacipran and idazoxan as an associated product for use simultaneously, separately or staggered in time to treat depression and its various forms, as well as disorders in which antidepressants are used. Milnacipran is also indicated for use in the treatment of urinary incontinence (FR 2 759 290).

0005Milnacipran exists in the form of two optically active enantiomers: the dextrogyral enantiomer or Z-(1S,2R)-2-(amino methyl)-N,N-diethyl-1-phenyl cyclopropane carboxamide and the levogyral enantiomer Z-(1R, 2S)-2-(amino methyl)-N,N-diethyl-1-phenyl cyclopropane carboxamide. In its hydrochloride form, milnacipran (also called F2207) is currently marketed (IXEL, PIERRE FABRE MEDICAMENT, France) in the form of a racemic mixture as a serotoninergic and norepinephrinergic antidepressant agent. F2695 and F2696 designate the dextrogyral and levogyral enantiomers respectively of milnacipran hydrochloride (F2207):

0006<chemistry id="CHEM-US-00001" num="00001"><img file="USRE43879E_D0001.tif" /></chemistry>

0007These two enantiomers can be separated and isolated using procedures described in the literature (Bonnaud et al., 1985, Journal of Chromatography, Vol. 318: 398-403; Shuto et al., Tetrahedron letters, 1996 Vol. 37: 641-644; Grard et al., 2000, Electrophoresis 2000 21: 3028-3034; Doyle et Hu, 2001, Advanced Synthesis and Catalysis, Vol. 343: 299-302).

0008The inventors have now performed a pharmacokinetic study in man on the racemate and on the two enantiomers of milnacipran which uses enantiomer-selective assay methods. They have thus demonstrated the absence of racemisation of the enantiomers in vivo.

0009Furthermore, although the racemate has been resolved, no analysis of the pharmacological and toxicological properties of the two enantiomers has been performed using modern, currently-available methods such as cardiovascular measurements by telemetry, or genomic analyses for predictive pharmacotoxicology in vitro.

0010As with any active substance, antidepressants can induce adverse events or certain toxic effects that essentially derive from the pharmacological properties of these drugs, as well as from the dosage, from individual variations in patients (genetic polymorphism, organ-function insufficiency, sex, age) or from drug interactions. Antidepressants are thus the third most common class of products responsible for intoxication, after hypnotics and tranquillisers (Nores et al., 1987 Thérapie 42: 555-558). The risk of overdose with antidepressants is serious, since it can lead to death. Among the causes of acute intoxication with antidepressants should be mentioned accidental ingestion by children (all the more so since certain antidepressants are used in the treatment of enuresis), suicide attempts, accidental overdosage by physicians, concomitant medications in elderly patients, age-related physiological and pharmacokinetic changes (cardiac insufficiency, hepatic and/or renal insufficiency . . .) and slowing down of metabolism whether genetic in origin or drug-induced (enzyme inhibition). After children, the elderly therefore represent the second at-risk population among patients treated. Elderly persons have higher plasma concentrations, related to reduced renal and/or hepatic clearance, and the risks of intoxication are more serious (Meadoer-Woodruffet al., 1988 J. Clim. Psychopharmacol. 8: 28-32).

0011The adverse side-effects, generally benign, which have been observed during treatment with milnacipran usually occur within the first or the first two weeks of treatment and diminish thereafter, in parallel with improvement in the depressive episode. The most commonly-reported adverse events in single-drug therapy or in association with other psychotropics are dizziness, hypersudation, anxiety, hot flushes and dysuria. Certain less commonly-reported adverse events are nausea, vomiting, dry mouth, constipation, tremor, palpitations, agitation, and cutaneous eruptions. Moreover, it is known that in patients with a history of cardiovascular disease or who concomitantly receive treatment for a cardiac condition, milnacipran can increase the incidence of cardiovascular adverse events (hypertension, hypotension, orthostatic hypotension, palpitations). In patients with high blood pressure or having heart disease, it is therefore recommended to increase medical supervision since milnacipran in the form of a racemic mixture is likely to increase the heart rate. In those rare cases of overdose observed with milnacipran (at doses from 800 mg to 1 g) in single-therapy, the main symptoms observed are vomiting, respiratory disturbances and tachycardia (The Vidal Dictionary, 78th Edition, 2002). Another adverse event occasionally induced by milnacipran is elevated transaminase levels which may reflect a certain hepatic toxicity.

0012The at-risk populations that could potentially develop a certain number of adverse clinical manifestations during or following treatment with milnacipran are children, the elderly, patients with hepatic and/or renal insufficiency, patients receiving treatment that induces organ and/or tissue toxicity, in particular hepatic or renal toxicity, patients receiving treatment for a heart condition or that induces cardiovascular side-effects, patients with a history of cardiovascular disease and/or having cardiovascular disorders, especially those with disorders of cardiac rhythm, of blood pressure (hypo- or hypertensive patients) and patients suffering from heart disease.

0013Concerned to prevent, to an ever-greater extent, the occurrence of possible side-effects that could constitute a danger, however small, to the health of patients treated with milnacipran, the inventors have now discovered that, surprisingly and unexpectedly, the dextrogyral enantiomer of milnacipran, which is essentially responsible for the selective inhibitory activity on serotonin and norepinephrine reuptake, induced fewer side-effects of a cardiovascular nature and less organ and/or tissue toxicity, especially hepatic, than the racemic mixture. In particular, the inventors have discovered that, in dogs, administration of the dextrogyral enantiomer of milnacipran leads to a lesser increase in heart rate and blood pressure, particularly diastolic blood pressure, than that which can be induced by administration of the racemic mixture. Moreover, the inventors have discovered that the dextrogyral enantiomer of milnacipran (F2695) has a better profile of genomic toxicity than the levogyral enantiomer of milnacipran (F2696) in an experimental model using primary rat hepatocytes. The inventors have also demonstrated that the levogyral enantiomer of milnacipran (F2696) has a profile of genomic toxicity similar to that obtained with clomipramine, which is used as a reference psychotropic product known for its relative hepatic toxicity.

0014The object of the present invention is thus the use of a mixture of enantiomers of milnacipran enriched in the dextrogyral enantiomer, preferentially the substantially-pure F2695 enantiomer, as well as with their pharmaceutically-acceptable salts, for the preparation of a drug intended to prevent or to treat disorders or conditions that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or while limiting the risks of organ and/or tissue toxicity.

0015The term “cardiovascular disturbances” is understood to refer to adverse cardiovascular side-effects of the drug administered alone or in association with other active substances.

0016For the purposes of the present invention, the term “side-effect” is understood to mean the foreseeable activity of a drug in an area other than that for which it is administered, that may be bothersome or undesirable when it limits the use of the drug.

0017The term “toxicity” is understood to mean the property of a drug to induce harmful effects on organs or tissue, in particular organs or tissues involved in the metabolism of milnacipran, especially hepatic and/or renal metabolism of milnacipran, and more specifically during the first pass of milnacipran in the liver. Preferentially, organ toxicity is cardiac toxicity and the said tissue toxicity is hepatic and/or renal toxicity.

0018For the purposes of the present invention, the phrases “while limiting the risks of cardiovascular disturbances” or “while limiting the risks of toxicity” is understood to mean the fact of preventing these risks from increasing significantly in a patient following administration of the drug.

0019For the purposes of the present invention, the term “dextrogyral enantiomer of milnacipran” designates the dextrogyral enantiomer of milnacipran, as well as its pharmaceutically-acceptable salts. Preferentially, this is the dextrogyral enantiomer of milnacipran hydrochloride (F2695). “Levogyral enantiomer of milnacipran” designates the levogyral enantiomer of milnacipran, as well as its pharmaceutically-acceptable salts (F2696). “Racemic mixture” designates a 50:50 mixture by weight of the dextrogyral enantiomer of milnacipran and the levogyral enantiomer of milnacipran, as well as their pharmaceutically-acceptable salts.

0020For the purposes of the present invention, “mixture of the enantiomers of milnacipran enriched in the dextrogyral enantiomer” signifies a mixture of the dextrogyral enantiomer and the levogyral enantiomer of milnacipran in which the mass/mass ratio of the dextrogyral enantiomer to the levogyral enantiomer is greater than 1:1. In the mixture of the enantiomers of milnacipran enriched in the dextrogyral enantiomer, the mass/mass ratio of the dextrogyral enantiomer to the levogyral enantiomer is advantageously greater or equal to 55:45, more advantageous when greater than 60:40, yet more advantageous when greater than 65:35, yet more advantageous when greater than 70:30, yet more advantageous when greater than 75:25, yet more advantageous when greater than 80:20. Produced in a particularly advantageous mode, the mass/mass ratio of the dextrogyral enantiomer to the levogyral enantiomer is greater than 82:18, in a more advantageous manner greater than 84:16, in an even more advantageous manner greater than 86:14, in an even more advantageous manner greater than 88:12, in an even more advantageous manner greater than 90:10. Produced in a preferred mode, the mass/mass ratio of the dextrogyral enantiomer to the levogyral enantiomer is greater than 91:9, in a more preferred manner greater than 92:8, in an even more preferred manner greater than 93:7, in an even more preferred manner greater than 94:6, in an even more preferred manner greater than 95:5, in an even more preferred manner greater than 96:4, in an even more preferred manner greater than 97:3, in an even more preferred manner greater than 98:2, in an even more preferred manner greater than 99:1, in an even more preferred manner greater than 99.5:0.5. In a particularly preferred manner, the mixture of enantiomers of milnacipran enriched in the dextrogyral enantiomer is substantially pure, that is to say, containing approximately 100% dextrogyral enantiomers by weight.

0021The use of metabolites also enters into the scope of the present invention, preferentially the metabolites of milnacipran that are active in vivo, in their Z or E form, and their pharmaceutically-acceptable salts, such as: <ul id="ul0001" list-style="none"><li id="ul0001-0001" num="0000"><ul id="ul0002" list-style="none"><li id="ul0002-0001" num="0022">the hydrochloride of Z-phenyl-1 aminomethyl-2 cyclopropane carboxylic acid (F1567):</li></ul></li></ul>

0023<tables id="TABLE-US-00001" num="00001"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="right" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>F1567</entry></row><row><entry><chemistry id="CHEM-US-00002" num="00002"><img file="USRE43879E_D0002.tif" /></chemistry></entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="14pt" align="left" /><colspec colname="1" colwidth="77pt" align="left" /><colspec colname="2" colwidth="126pt" align="left" /><tbody valign="top"><row><entry /><entry>Molecular mass:</entry><entry>277.7</entry></row><row><entry /><entry>Characteristics:</entry><entry>white crystals</entry></row><row><entry /><entry>Melting point:</entry><entry>230° C.</entry></row><row><entry /><entry>Plate chromatography:</entry><entry>medium: silica</entry></row><row><entry /><entry /><entry>Solvent: Butanol/Ethanol/water (6/2/2)</entry></row><row><entry /><entry /><entry>Developer: Ultraviolet and ninhydrine</entry></row><row><entry /><entry /><entry>R<sub>F</sub>: 0.6</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><ul id="ul0003" list-style="none"><li id="ul0003-0001" num="0000"><ul id="ul0004" list-style="none"><li id="ul0004-0001" num="0024">phenyl-3 methylene-3-4 pyrrolidone-3 (F1612):</li></ul></li></ul>

0025<tables id="TABLE-US-00002" num="00002"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="182pt" align="center" /><colspec colname="2" colwidth="35pt" align="right" /><thead><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>F1612</entry></row><row><entry><chemistry id="CHEM-US-00003" num="00003"><img file="USRE43879E_D0003.tif" /></chemistry></entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="84pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>Molecular mass:</entry><entry>173.2</entry></row><row><entry>Characteristics:</entry><entry>white crystals</entry></row><row><entry>Melting point:</entry><entry>70° C.</entry></row><row><entry>Plate chromatography:</entry><entry>medium: silica</entry></row><row><entry /><entry>Solvent: Benzene/dioxane/ethanol (90/25/4)</entry></row><row><entry /><entry>Developer: Ultraviolet and iodine</entry></row><row><entry /><entry>R<sub>F</sub>: 0.46</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><ul id="ul0005" list-style="none"><li id="ul0005-0001" num="0000"><ul id="ul0006" list-style="none"><li id="ul0006-0001" num="0026">the hydrochloride of Z-(para-hydroxyphenyl)-1 diethylaminocarbonyl-1 aminomethyl-2 cyclopropane (F2782):</li></ul></li></ul>

0027<tables id="TABLE-US-00003" num="00003"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="182pt" align="center" /><colspec colname="2" colwidth="35pt" align="right" /><thead><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>F2782</entry></row><row><entry><chemistry id="CHEM-US-00004" num="00004"><img file="USRE43879E_D0004.tif" /></chemistry></entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="147pt" align="left" /><tbody valign="top"><row><entry>Molecular mass:</entry><entry>298.82</entry></row><row><entry>Characteristics:</entry><entry>white crystals</entry></row><row><entry>Melting point:</entry><entry>250° C.</entry></row><row><entry>Plate chromatography:</entry><entry>medium: silica</entry></row><row><entry /><entry>Solvent: Butanol/Ethanol/water (6/2/2)</entry></row><row><entry /><entry>Developer: Ultraviolet and iodine - ninhydrine</entry></row><row><entry /><entry>R<sub>F</sub>: 0.42</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><ul id="ul0007" list-style="none"><li id="ul0007-0001" num="0000"><ul id="ul0008" list-style="none"><li id="ul0008-0001" num="0028">the oxalate acid of Z-phenyl-1-ethylamino carbonyl-1 aminomethyl-2 cyclopropane (F2800):</li></ul></li></ul>

0029<tables id="TABLE-US-00004" num="00004"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="182pt" align="center" /><colspec colname="2" colwidth="35pt" align="right" /><thead><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>F2800</entry></row><row><entry><chemistry id="CHEM-US-00005" num="00005"><img file="USRE43879E_D0005.tif" /></chemistry></entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="84pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>Molecular mass:</entry><entry>308.33</entry></row><row><entry>Characteristics:</entry><entry>white crystals</entry></row><row><entry>Melting point:</entry><entry>150° C.</entry></row><row><entry>Plate chromatography:</entry><entry>medium: silica</entry></row><row><entry /><entry>Solvent: CHCl<sub>3</sub>/methanol/NH<sub>4</sub>OH (90/9/1)</entry></row><row><entry /><entry>Developer: Ultraviolet and ninhydrine</entry></row><row><entry /><entry>R<sub>F</sub>: 0.40</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><ul id="ul0009" list-style="none"><li id="ul0009-0001" num="0000"><ul id="ul0010" list-style="none"><li id="ul0010-0001" num="0030">the hydrochloride of Z-phenyl-1 aminocarbonyl-1 aminomethyl-2 cyclopropane (F2941):</li></ul></li></ul>

0031<tables id="TABLE-US-00005" num="00005"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="182pt" align="center" /><colspec colname="2" colwidth="35pt" align="right" /><thead><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>F2941</entry></row><row><entry><chemistry id="CHEM-US-00006" num="00006"><img file="USRE43879E_D0006.tif" /></chemistry></entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="84pt" align="left" /><colspec colname="2" colwidth="133pt" align="left" /><tbody valign="top"><row><entry>Molecular mass:</entry><entry>226.74</entry></row><row><entry>Characteristics:</entry><entry>white crystals</entry></row><row><entry>Melting point:</entry><entry>245° C.</entry></row><row><entry>Plate chromatography:</entry><entry>medium: silica</entry></row><row><entry /><entry>Solvent: CHCl<sub>3</sub>/methanol/NH<sub>4</sub>OH (80/18/2)</entry></row><row><entry /><entry>Developer: Ultraviolet and ninhydrine</entry></row><row><entry /><entry>R<sub>F</sub>: 0.30</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0032These metabolites have, just as milnacipran has, a chiral centre which confers optical isomerism on these metabolites that exist in the form of dextrogyral and levogyral enantiomers. The racemic ratio of the two enantiomers of the milnacipran metabolite in the mixture of enantiomers is as previously described for the enantiomers of Milnacipran.

0033The present invention covers therefore these active metabolites, as well as their pharmaceutically-acceptable salts, in addition to their use as a drug in the treatment of the disorders described in the present patent such as depression, pain, fibromyalgia and urinary incontinence. The metabolites in accordance with the invention are in the form of racemates or preferentially in the form of a mixture of enantiomers enriched in the most active enantiomer. In a preferable manner, the active metabolite used comes from the F2695 enantiomer and is the dextrogyral enantiomer of the active metabolite. In a more preferable manner, this is the dextrogyral enantiomer of the hydrochloride of Z-(para-hydroxyphenyl)-1 diethylaminocarbonyl-1 aminomethyl-2 cyclopropane (F2782). The term “active metabolite” is understood to designate a derivative of milnacipran metabolised in vitro or in vivo and having the capacity to inhibit reuptake of serotonin and of norepinephrine; preferentially, these are F2782, F2941, F2800, F1612 and F1567. For the purposes of the present invention, the active metabolites in vivo described and claimed for in the present invention include all the enantiomers, the isomers or the tautomers when the component is capable of being present in the form of an enantiomer, an isomer or a tautomer.

0034The object of the present invention is therefore the use of a mixture of enantiomers preferentially enriched in the dextrogyral enantiomer of at least one metabolite of Milnacipran, preferentially chosen among F2782, F2941, F2800, F1612 and F1567, as well as their pharmaceutically-acceptable salts, for the preparation of a drug intended to prevent or to treat disorders or conditions that can be managed by double inhibition of reuptake of serotonin (5-HT) and of norepinephrine (NE), while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular, cardiac, hepatic and/or renal toxicity.

0035The use of a mixture of enantiomers of milnacipran enriched in the dextrogyral enantiomer, preferentially the substantially-pure F2595 enantiomer, and at least one of its active metabolites, preferentially chosen among F2782, F2941, F2800, F1612 and F1567, preferentially enriched in the dextrogyral enantiomer, for the preparation of a drug intended to prevent or to treat disorders or conditions that can be managed by double inhibition of reuptake of serotonin (5-HT) and of norepinephrine (NE), while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular, cardiac, hepatic and/or renal toxicity also enters into the scope of the present invention.

0036“Pharmaceutically-acceptable salts” designates all salts that retain the efficacy and properties of an active substance and that do not cause side-effects. Such salts may be prepared starting from acids or bases, organic or mineral. Preferentially, these are pharmaceutically-acceptable salts of mineral or organic acids. By way of example, but not limited to these, halogen hydrates such as the hydrochloride and the bromohydrate, the fumarate, the maleate, the oxalate, the citrate, the methane sulphonate, the glutamate, the tartrate, the mesylate and their possible hydrates should be mentioned.

0037For the purposes of the present invention, the term “mixture of enantiomers” signifies the mixture of enantiomers of milnacipran enriched in the dextrogyral enantiomer, as well as their pharmaceutically-acceptable salts, and/or the mixture of enantiomers of at least one of the metabolites of milnacipran, preferentially enriched in the dextrogyral enantiomer, as well as their pharmaceutically-acceptable salts.

0038The mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, is administered to all types of patients requiring such treatment, whether it be for therapeutic and/or prophylactic purposes. For therapeutic purposes, the aim is to eradicate or to improve the condition to be treated and/or one or more related symptoms. For prophylactic purposes, the aim is to prevent the appearance of the condition to be treated and/or of one or more related symptoms. Nevertheless, the mixture of enantiomers in accordance with the invention is particularly adapted to populations of at-risk patients who may be likely to develop certain adverse clinical manifestations during or following treatment with milnacipran in the racemic form. These are principally children, the elderly, patients with hepatic and/or renal insufficiency, patients receiving treatment that induces hepatic or renal organ and/or tissue toxicity, patients receiving treatment for a heart condition, patients receiving treatment that induces cardiovascular side-effects, patients with a history of cardiovascular disease (for example, myocardial infarctus) and/or having cardiovascular disorders, such as patients with cardiac rhythm disorders (tachycardia, bradycardia, palpitations), patients with blood pressure disorders (hypo- or hypertensive patients) or patients suffering from heart disease.

0039Among the numerous disorders or conditions that have as symptoms cardiac rhythm disorders and for which the present invention is particularly well-adapted in the treatment of at-risk patients who suffer from them, tachycardia which corresponds to an acceleration of the rhythm of the heart beat (tachycardia is moderate when the heart rate is from 80 to 100 beats per minute, severe when it exceeds 100), palpitations, extrasystoles (sporadic, frequent or during myocardial infarctus), auricular fibrillation, flutter and auricular tachysystole, bradycardia, cardiac insufficiency, and myocardial infarctus should be mentioned.

0040Among the numerous disorders or conditions that have as symptoms blood pressure disorders and for which the present invention is particularly well-adapted in the treatment of at-risk patients who suffer from them, arterial hypertension, malignant arterial hypertension, pulmonary arterial hypertension, portal hypertension, paroxysmal essential hypertension, hypotension, orthostatic hypotension and intra-cranial hypertension should be mentioned.

0041Advantageously, those cardiovascular disorders for which the risks can be limited by the administration of the mixture of enantiomers in accordance with the invention, and preferentially by the administration of the substantially-pure F2695 enantiomer, are as follows: <ul id="ul0011" list-style="none"><li id="ul0011-0001" num="0000"><ul id="ul0012" list-style="none"><li id="ul0012-0001" num="0042"><img file="USRE43879E_D0007.tif" /> elevated diastolic and/or systolic blood pressure measured in millimeters of mercury (mmHg); more specifically, this is an increase in diastolic blood pressure, and/or,</li><li id="ul0012-0002" num="0043"><img file="USRE43879E_D0008.tif" /> cardiac rhythm disorders, in particular, an increase in the patient's heart rate.</li></ul></li></ul>

0044Systolic blood pressure is the maximal value for blood pressure, and it corresponds to the moment when the first heart sound is heard in the humeral artery during measurement of blood pressure. The systole is the interval of the cardiac cycle during which the heart cavities contract, causing expulsion of the blood. Diastolic blood pressure is the minimal value of blood pressure, corresponding to the disappearance of heart sounds in the humeral artery when the cuff of the sphygmomanometer is deflated during measurement of blood pressure. The diastole is the interval of the cardiac cycle during which the heart cavities fill with blood. Elevation of systolic and/or diastolic pressure means increased blood pressure which is characteristic of systemic arterial hypertension (and its variant forms), the symptoms of which may be the following: headache, fatigue, mild sensorial disturbances such as dizziness, buzzing in the ears, palpitations, nosebleed, confusion or drowsiness, cramps, numbness or tingling in the feet and hands. Systemic arterial hypertension (and its variant forms) can lead to serious, indeed fatal, complications: cerebral vascular accidents, left ventricular heart failure, kidney failure, ischemic heart diseases (myocardial infarctus, angor and their variant forms). According to current guidelines, a patient is considered to have arterial hypertension when his/her diastolic blood pressure is above 90 mmHg and his/her systolic blood pressure is above 140 mmHg.

0045The toxicity for which the risks can be limited by the administration of the mixture of enantiomers in accordance with the invention is advantageously organ toxicity, particularly cardiac toxicity, and/or tissue toxicity, in particular hepatic and/or renal toxicity. Tissue toxicity may be revealed by the presence of icterus or by laboratory markers.

0046The use of the mixture of enantiomers in accordance with the invention in veterinary medicine for the treatment of animals, in particular household pets or breeding animals that require such treatment also enters into the scope of the present invention.

0047Because of their pharmacological properties, in particular as dual inhibitors of serotonin (5-HT) and norepinephrine (NE) reuptake, the mixture of enantiomers is especially useful in the preparation of drugs intended for preventive and/or curative treatment of a number of disorders and conditions (syndromes) described hereinafter, while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular cardiac, hepatic and/or renal toxicity.

0048Among these disorders or conditions, disorders of the central nervous system as defined in <<The Diagnostic and Statistical Manual of Mental Disorders—IV (DSM-IV), 1995 American Psychiatric Association>> should be mentioned. By way of example, but not limited to these, the following disorders and conditions should be mentioned: depression, in particular deep depression, resistant depression, depression in the elderly, psychotic depression, depression induced by treatment with interferon, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes related to general health status, depressive episodes related to mood-altering substances, bi-polar disease, schizophrenia, generalised anxiety, morose and marasmic states, stress-related diseases, panic attacks, phobias, in particular agoraphobia, obsessive-compulsive disorders, behavioural disorders, oppositional disorders, post-traumatic stress disorder, depression of the immune system, fatigue and accompanying pain syndromes, chronic fatigue syndrome, fibromyalgia, and other functional somatic disorders, autism, disorders characterised by attention deficit due to general health status, attention disorders due to hyperactivity, eating disorders, neurotic bulimia, neurotic anorexia, obesity, psychotic disorders, apathy, migraine, pain and in particular chronic pain, irritable bowel syndrome, cardiovascular diseases and in particular anxiety-depressive syndrome in myocardial infarctus or in hypertension, neuro-degenerative diseases and related anxiety-depressive syndromes (Alzheimer's disease, Huntington's chorea, Parkinson's disease), urinary incontinence, in particular urinary incontinence related to stress and enuresis, drug addiction and in particular anxiety addiction to tobacco, in particular to nicotine, to alcohol, to narcotics, to drugs, to analgesics used in weaning-off from these addictive states.

0049More specifically, the object of the present invention concerns the use of a mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, for the preparation of a drug intended to treat or to prevent depression or depressive state while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular hepatic and/or renal toxicity. In the context of the present invention, the term “depression” is understood to refer to a constellation of symptoms having, on the one hand, a psychological aspect consisting of mood disorders with pessimism, moral suffering, thoughts of death or suicide, mental inhibition, and on the other hand, a physical aspect of motor deficit, consisting in particular of a slowdown in motor activity, of appetite disturbances, of constipation, of sleep disturbances and of weight-control disturbances. Depression therefore corresponds to pathological psychological state combining a painful mood-alteration and a reduction in mental and motor activity. The term “depressive state” is understood to refer to a mental state characterised by a decline in neuropsychological tonicity, manifesting as lassitude, tendency to fatigue, discouragement and tendency to pessimism sometimes accompanied by anxiety.

0050Furthermore, the object of the present invention concerns more specifically the use of a mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, for the preparation of a drug intended to prevent or to treat fibromyalgia and/or chronic fatigue syndrome while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular hepatic and/or renal toxicity. Fibromyalgia syndrome is a chronic syndrome characterised by a feeling of pain and burning with morning stiffness mainly affecting articular and peri-articular fibrous tissues, and by a feeling of deep fatigue. Fibromyalgia includes a constellation of symptoms. The most frequent are non-restorative sleep, headache, digestive disturbances, depressive state, muscle spasm, facial pain, numbness etc. Chronic fatigue syndrome is characterised by a state of exhaustion or of fatigue. The most common symptoms are a state of weakness, spasms and/or muscle pain, excessive need for sleep, fever, angina, memory loss and/or difficulty concentrating, insomnia, depression.

0051In addition, the object of the present invention concerns more specifically the use of a mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, for the preparation of a drug intended to prevent or to treat pain and in particular chronic pain while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular hepatic and/or renal toxicity. Pain may be associated with various disorders and/or wounds. It may be acute or chronic. Epidemiological studies have demonstrated the relations between states of chronic pain and anxiety depression. Thus, patients suffering from chronic pain may develop emotional problems that lead to depression, and, in the worst cases, to a suicide attempt. A patient is considered to be in chronic pain if he/she complains of suffering for a period of more than six months. Among the various forms of chronic pain, the followed should be mentioned by way of example, but not limited to these: pain associated with fibromyalgia and/or arising in fibrous tissues, muscles, tendons, ligaments and other sites, abdominal pain and diarrhoea in irritable bowel syndrome, as well as lower back pain.

0052In addition, the object of the present invention concerns more specifically the use of a mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, for the preparation of a drug intended to prevent or to treat urinary incontinence and in particular urinary incontinence related to stress and enuresis, while limiting the risks of cardiovascular disturbances and/or while limiting organ and/or tissue toxicity, in particular hepatic and/or renal toxicity.

0053Prophylactic and therapeutic treatment of the above-mentioned disorders is achieved by administering to an animal, preferentially to man, a therapeutically-effective quantity of a mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, alone or in association with at least one other active substance. In most cases, this concerns man, however the treatment is also adapted to animals, in particular breeding animals (livestock, rodents, poultry, fish, . . .) and to domestic animals (dogs, cats, rabbits, horses, . . .).

0054The mixture of enantiomers, enriched in the dextrogyral enantiomer, of milnacipran and/or of at least one of its metabolites, as well as their pharmaceutically-acceptable salts, as previously described, is advantageously administered to patients receiving simultaneously, separately or staggered in time at least one other active compound in the treatment of the above-mentioned disorders.

0055Preferentially, the object of the present invention also includes, for use as a drug: <ul id="ul0013" list-style="none"><li id="ul0013-0001" num="0000"><ul id="ul0014" list-style="none"><li id="ul0014-0001" num="0056">a) the said mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one of its metabolites as well as their pharmaceutically-acceptable salts, and</li><li id="ul0014-0002" num="0057">b) at least one active compound chosen among the psychotropics, in particular antidepressants, and antimuscarinic agents <br /> as associated products for use simultaneously, separately or staggered in time in the treatment or the prevention of depression, in particular deep depression, resistant depression, depression in the elderly, psychotic depression, depression induced by treatment with interferon, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes related to general health status, depressive episodes related to mood-altering substances. </li></ul></li></ul>

0058The term “psychotropic” is understood to designate a substance of natural or artificial origin capable of modifying mental activity and whose action is essentially exerted on the central nervous system and the psychological state. Psychotropics are divided into three groups: 1) psycholeptics (hypnotics, neuroleptics and anxiolytics), 2) psychoanaleptics (antidepressants and psychotonics) and 3) psychodysleptics (hallucinogenics).

0059Preferentially, the said psychotropic is an antidepressant. By way of example, but not limited to these, the antidepressant is chosen among (i) monoamine oxidase inhibitors (MAOIs) such as iproniazid, pargyline, selegiline, (ii) 5HT1D-agonists such as sumatriptan, epinephrine and norepinephrine (alpha and beta sympathomimetics), (iii) tricyclic antidepressants, such as imipramine, clomipramine, (iv) selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, (v) selective norepinephrine reuptake inhibitors, such as for example tandamine, fluparoxan, mirtazapine, (vi) serotonin norepinephrine reuptake inhibitors, such as venlafaxine and duloxetine. By way of example, but not limited to these, the antimuscarinic agent is chosen among tolterodine, propiverine, oxybutynin, trospium, darifenacine, temiverine, ipatropium.

0060Preferably, the object of the present invention also includes for use as a drug: <ul id="ul0015" list-style="none"><li id="ul0015-0001" num="0000"><ul id="ul0016" list-style="none"><li id="ul0016-0001" num="0061">a) the said mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one of its metabolites as well as their pharmaceutically-acceptable salts, and</li><li id="ul0016-0002" num="0062">b) at least one other active substance chosen among the active compounds inducing organ toxicity and the active compounds inducing tissue toxicity, in particular hepatic and/renal toxicity or with one or more active substances intended for treatment of hepatic or renal insufficiency. <br /> as associated products for use simultaneously, separately or staggered in time in the treatment or the prevention of conditions or disorders that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake. </li></ul></li></ul>

0063Preferably, the object of the present invention also includes, for use as a drug: <ul id="ul0017" list-style="none"><li id="ul0017-0001" num="0000"><ul id="ul0018" list-style="none"><li id="ul0018-0001" num="0064">a) the said mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one of its metabolites as well as their pharmaceutically-acceptable salts, and</li><li id="ul0018-0002" num="0065">b) at least one other active substance chosen among active compounds inducing cardiovascular side-effects or compounds given to treat a heart condition. <br /> as associated products for use simultaneously, separately or staggered in time in the treatment or the prevention of conditions or disorders that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake. </li></ul></li></ul>

0066Advantageously, the cardiovascular side-effects induced are those mentioned previously, and more specifically, arterial hypertension, hypotension, cardiac rhythm disorders (tachycardia, bradycardia, palpitations).

0067The object of the present invention also includes pharmaceutical compositions containing the associated products previously described.

0068In the context of the present invention, the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, is advantageously administered, but not in a limited manner, via the oral route, the nasal route, the transdermal, rectal, intestinal or parental route, by intramuscular, subcutaneous or intravenous injection, alone or in association with other active substances, as previously described.

0069When administered alone, the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, may be administered per se or in the form of a pharmaceutical composition in which the said mixture of enantiomers or of their pharmaceutically-acceptable salts, is combined or mixed with one or several media, pharmaceutically-acceptable excipients and/or diluents, particularly to enhance bioavailability.

0070When the mixture of enantiomers in accordance with the invention, and preferentially the substantially-pure dextrogyral F2695 enantiomer of milnacipran is administered in association with other active substances, the said mixture and the other active substances may be formulated as a mixture or separately in an identical or different form. They may be administered via the same or a different route.

0071The pharmaceutical composition in accordance with the invention may be formulated in a conventional manner well-known to the man skill in the art using one or more physiologically-acceptable media including excipients, adjuvants and additives such as for example preservatives, stabilisers, wetting agents or emulsifiers. The method of formulation chosen depends on the desired route of administration.

0072In the context of administration by injection, an aqueous solution is advantageously used, in particular a physiologically-acceptable buffer solution, such as Hank's solution, Ringer's solution or physiological saline solution. In the context of transdermal administration or via the mucous membranes, penetrating agents appropriate to the mucous membrane to be crossed are advantageously used. Such penetrating agents are well known to the man skill in the art. In the context of oral administration, the pharmaceutical compositions in accordance with the invention are advantageously administered in unit-dose or multiple-dose administration forms in mixtures containing appropriate pharmaceutical media known to the man skill in the art. Appropriate unit-dose administration forms include in particular tablets, possibly scored, capsules, powders, granules, oral solutions or suspensions, and aerosols. Appropriate multiple-dose administration forms include in particular drinkable drops, emulsions and syrups.

0073In the preparation of tablets, the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, is formulated with a pharmaceutically-acceptable vehicle such as in particular polyvinylpyrrolidone, carbopol gal, polyethylene glycol, gelatine, talc, starch, lactose, magnesium stearate, gum arabic or their analogues. By way of example, the tablet contains the following excipients: calcium hydrogen phosphate dihydrate, calcium carmellose, povidone K30, anhydrous colloidal silicon dioxide, magnesium stearate, talc. The tablets may also be coated, that is to say, covered with several coats of various substances such as saccharose in order to facilitate swallowing or preservation. The coating may also contain dyes or colorants in order to differentiate and to characterise the tablets with regard to their dosage strength, for example. The tablets may also be presented in a more or less complex formulation intended to modify the rate of release of the active substance. Release of the active substance of the said tablet may be rapid, sustained or delayed depending on the desired absorption. Thus, the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, may be prepared in a pharmaceutical form for sustained release obtained according to the process described in patent EP 939 626. This pharmaceutical form is presented in the form of multiparticles containing a large number of mini-granules and has a certain release profile in vitro.

0074Release of the mixture of enantiomers in accordance with the invention may be delayed and/or controlled by using an implant or by transdermal delivery, in particular subcutaneous or intramuscular, by intramuscular injection or by a transdermal patch. The said mixture is then formulated, in particular, with appropriate hydrophobic or polymeric substances and ion-exchange resins.

0075The quantity of the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, to be administered to the patient depends on the condition to be treated, the desired effect, in particular a therapeutic or prophylactic effect, the health status and age of the patient, in particular his/her medical history of cardiovascular disease, the conditions of treatment and the method of administration of the drug. The quantity required to be administered for effective therapeutic or prophylactic use in a human patient can be determined based on animal models or on data, known to the man skill in the art, obtained during the treatment of depression in man, for example, using a racemic mixture of Milnacipran.

0076In the context of therapeutic and/or prophylactic treatment of the disorders mentioned above, and in particular depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain, the drug in accordance with the invention is advantageously administered at doses from 0.01 mg to 10 mg/kg body weight per day in one or more intakes, more advantageously at doses from 0.05 mg to 5 mg/kg body weight per day in one or more intakes, and even more advantageously at doses from 0.1 mg to 1 mg/kg body weight per day in one or more intakes. In a particularly advantageous manner, administration of the said medicinal product at such doses as those defined above is divided into two daily intakes, preferentially in capsule form. By way of example, the mixture of enantiomers in accordance with the invention, preferentially the substantially-pure F2695 enantiomer, is advantageously administered in the form of a capsule containing approximately 6.75 mg of active substance per capsule, 12.5 mg/capsule, 25 mg/capsule, 50 mg/capsule.

0077Other characteristics, aims and advantages of the inventions will become apparent in the examples that follow. The invention is not limited to these particular examples which are provided simply by way of example and which should be read in comparison with the following figures:

BRIEF DESCRIPTION OF THE DRAWINGS

0078<figref idref="DRAWINGS">FIG. 1</figref>: Effects of various treatments on heart rate following single administration (mean values)

0079<figref idref="DRAWINGS">FIG. 2</figref>: Effects of various treatments on mean values of diastolic blood pressure (mean values over 6 hours following the last intake, after 5 consecutive days of treatment). <ul id="ul0019" list-style="none"><li id="ul0019-0001" num="0000"><ul id="ul0020" list-style="none"><li id="ul0020-0001" num="0080">***: p≦0.001 versus deionised water</li><li id="ul0020-0002" num="0081">**: p≦0.01 versus deionised water</li><li id="ul0020-0003" num="0082"><sub>p</sub>: p≦0.05 versus F2207</li></ul></li></ul>

0083<figref idref="DRAWINGS">FIG. 3</figref>: Effects of various treatments on mean values of systolic blood pressure (mean values over 6 hours following the last intake, after 5 consecutive days of treatment). <ul id="ul0021" list-style="none"><li id="ul0021-0001" num="0000"><ul id="ul0022" list-style="none"><li id="ul0022-0001" num="0084">***: p≦0.001 versus deionised water</li><li id="ul0022-0002" num="0085">**: p≦0.01 versus deionised water</li><li id="ul0022-0003" num="0086"><sub>p</sub>: p<0.05 versus F2207</li></ul></li></ul>

0087<figref idref="DRAWINGS">FIG. 4</figref>: Schematic representation of the method of calculation of the Toxicity Index. The Toxicity Index is the sum of all up- and down-regulated genes (in relation to the Induction Factor defined by the user).

0088<figref idref="DRAWINGS">FIGS. 5A</figref>, <b>5</b>B, <b>5</b>C: MTT assay on primary rat hepatocytes.

0089The concentrations are expressed in μM

EXAMPLES

Example N

Pharmacokinetic Studies on Milnacipran and on its Enantiomers

0090Pharmacokinetic studies on milnacipran (F2207) and on its enantiomers (F2695 and F2696) were performed in various animal species and in man.

0091In animals, the pharmacokinetics of each enantiomer were studied following administration of the racemate or of one single enantiomer. Plasma concentrations of the F2695 and F2696 enantiomers are approximately equivalent in the animal species tested (monkey and rat).

0092A pharmacokinetic study in man involving 12 healthy subjects was performed by administering the racemate or one of the two enantiomers alone. It was shown that the pharmacokinetic profile of each enantiomer is independent of whether it was administered separately or in the form of the racemate, indicating the absence of interaction between the enantiomers (Table 1).

0093<tables id="TABLE-US-00006" num="00006"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 1</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Table of the main pharmacokinetic variables</entry></row><row><entry>of milnacipran (F2207)</entry></row><row><entry>and its two enantiomers, F2695 and F2696.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="77pt" align="center" /><colspec colname="3" colwidth="35pt" align="center" /><colspec colname="4" colwidth="42pt" align="center" /><tbody valign="top"><row><entry /><entry>F2207</entry><entry /><entry /></row><row><entry>Dose</entry><entry>(50 mg)</entry><entry>F2695 (D)</entry><entry>F2696 (L)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="35pt" align="center" /><colspec colname="3" colwidth="42pt" align="center" /><colspec colname="4" colwidth="35pt" align="center" /><colspec colname="5" colwidth="42pt" align="center" /><tbody valign="top"><row><entry>administered (mg)</entry><entry>F2695 (D)</entry><entry>F2696 (L)</entry><entry>(25 mg)</entry><entry>(25 mg)</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="5"><colspec colname="1" colwidth="63pt" align="left" /><colspec colname="2" colwidth="35pt" align="char" char="." /><colspec colname="3" colwidth="42pt" align="char" char="." /><colspec colname="4" colwidth="35pt" align="char" char="." /><colspec colname="5" colwidth="42pt" align="char" char="." /><tbody valign="top"><row><entry>Cmax (nmol · l<sup>−1</sup>)</entry><entry>214</entry><entry>179</entry><entry>216</entry><entry>212</entry></row><row><entry>Tmax (hours)</entry><entry>3.42</entry><entry>2.87</entry><entry>3.08</entry><entry>2.21</entry></row><row><entry>AUC 0-><sup>∞</sup></entry><entry>2896</entry><entry>1563</entry><entry>2869</entry><entry>1543</entry></row><row><entry>(nmol · h · l<sup>−1</sup>)</entry><entry /><entry /><entry /><entry /></row><row><entry>T½(hours)</entry><entry>9.28</entry><entry>5.75</entry><entry>9.38</entry><entry>5.58</entry></row><row><entry namest="1" nameend="5" align="center" rowsep="1" /></row><row><entry namest="1" nameend="5" align="left" id="FOO-00001">Cmax: Maximal plasma concentration directly estimated based on experimental data</entry></row><row><entry namest="1" nameend="5" align="left" id="FOO-00002">Tmax: Time to reach maximal plasma concentration</entry></row><row><entry namest="1" nameend="5" align="left" id="FOO-00003">AUC 0-><sup>∞</sup>: Area under the curve for plasma concentrations in relation to time extrapolated to infinity</entry></row><row><entry namest="1" nameend="5" align="left" id="FOO-00004">T½: Terminal half-life of decrease in plasma concentrations</entry></row></tbody></tgroup></table></tables>

0094These findings indicate that no biotransformation of the F2695 or F2696 enantiomers was detected in the species studied.

Example N

Biochemical Studies of Milnacipran and of its Enantiomers

0095The two enantiomers (F2695 and F2696) of milnacipran (F2207) were studied in vitro on uptake of norepinephrine and serotonin as well as on binding of paroxetine in the rat brain.

0096The presence of an asymmetrical carbon in the chemical structure of milnacipran lead to performance of a chiral study on the molecule. In order to study the various isomeric forms, the two enantiomers, F2695 (Zd) and F2696 (Z1), were separated starting from F2207 in its racemic configuration (Z d1), and subjected to tests on uptake of monoamines, norepinephrine and serotonin, and on paroxetine binding.

00002.1. Materials and Methods

00002.1.1. Norepinephrine Uptake by a Homogenate (P<sub>2</sub>) of Rat Hypothalamus.

0000Preparation of P2

0097Male Sprague-Dawley rats, from 200 to 300 g, were stunned and decapitated, and the hypothalami were rapidly removed. Two hypothalami are homogenised in 4 ml of sucrose 0.32 M on a Potter S by 16 complete passes back and forth at 800 rpm, then centrifuged for 10 min at 1,000 g to eliminate cell debris. The supernate is centrifuged for 20 min at 10,000 g and the P<sub>2 </sub>thus obtained is recovered in 4 ml of sucrose 0.32 M, and homogenised on a Dounce.

0000Uptake

0098<sup>3</sup>H-(1)-NE: 13 Ci/mmol (Amersham) is used.

0099Uptake takes place in a phosphate buffer (containing 8 g of NaCl, 1.21 g of K<sub>2</sub>HPO<sub>4 </sub>and 0.34 g of KH<sub>2</sub>PO<sub>4 </sub>per liter) pre-oxygenated 30 min before use with a mixture of O<sub>2</sub>/CO<sub>2 </sub>(95%/5%).

0100In 5-ml plastic tubes placed in a water bath at 37° C., the following are introduced: <ul id="ul0023" list-style="none"><li id="ul0023-0001" num="0000"><ul id="ul0024" list-style="none"><li id="ul0024-0001" num="0101">100 μl of buffer or inhibitor,</li><li id="ul0024-0002" num="0102">700 μl of buffer (containing 25 μM pargyline),</li><li id="ul0024-0003" num="0103">100 μl of P<sub>2</sub>.</li></ul></li></ul>

0104After temperature balance, the reaction begins by the addition of 100 μl of <sup>3</sup>H-NE, 50 nM final concentration.

0105Exactly 10 min later, the reaction is stopped by adding 2.5 ml of chilled buffer and filtering through GF/F filters. The tube is then rinsed once and the filter once with 2.5 ml of chilled buffer. The filter is then introduced into a Beckman mini-vial and, after adding 3 ml of Instagel (Packard) liquid scintillator, radioactivity is measured with a Tricarb Packard scintillation counter.

0106Non-specific uptake (NS) is measured as the presence of DMI 10<sup>−5 </sup>M.

0107The percentage of inhibition is calculated using the formula:

0108<maths id="MATH-US-00001" num="00001"><math overflow="scroll"><mfrac><mrow><mrow><mo>(</mo><mrow><mrow><mi>total</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>uptake</mi></mrow><mo>-</mo><mi>NS</mi></mrow><mo>)</mo></mrow><mo>-</mo><mrow><mo>(</mo><mrow><mrow><mi>uptake</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>in</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>the</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>presence</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>of</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>inhibitor</mi></mrow><mo>-</mo><mi>NS</mi></mrow><mo>)</mo></mrow></mrow><mrow><mo>(</mo><mrow><mrow><mi>total</mi><mo>⁢</mo><mstyle><mspace width="0.8em" height="0.8ex" /></mstyle><mo>⁢</mo><mi>uptake</mi></mrow><mo>-</mo><mi>NS</mi></mrow><mo>)</mo></mrow></mfrac></math></maths><img file="USRE43879E_D0009.tif" />

0109The IC<sub>50 </sub>is determined graphically on the mean curve of percentage of inhibition (4 assays) in relation to the log of the concentration of inhibitor.

00002.1.2. Serotonin Uptake

0110The method was developed following that of Gray and whittaker (1962, J. Anat., 96: 79-97). After homogenisation of brain tissue in a sucrose solution, the presynaptic terminals break away from the axon and close to form synaptosomes obtained by subcellular fractionation.

0111Male Sprague-Dawley (Janvier) rats weighing 180-200 g were used. After sacrifice of the animal, the hypothalamus was removed, weighed and homogenised on a Dounce in 0.32 M sucrose at 0° C.

0112This homogenate was centrifuged for 10 min at 1,000 g (2,400 rpm-Hettich, Rotenta). The supernate was recovered and centrifuged for 20 min at 10,000 g (8,000 rpm-Beckam, model n<sup>o </sup>J2-21 M: J14 rotor). The residue (called the P<sub>2 </sub>fraction) was recovered in sucrose at a concentration of 50 mg/ml.

0113The following were incubated for 5 min at 37° C.: <ul id="ul0025" list-style="none"><li id="ul0025-0001" num="0000"><ul id="ul0026" list-style="none"><li id="ul0026-0001" num="0114">350 μl of chilled buffer (NaCl 136 mM, KH<sub>2</sub>PO<sub>4 </sub>2.4 mM, K<sub>2</sub>HPO<sub>4 </sub>6.9 mM, pH 7.2) pre-oxygenated 30 min before,</li><li id="ul0026-0002" num="0115">50 μl of membranes (5 mg/ml finally),</li><li id="ul0026-0003" num="0116">50 μl of citalopram (10<sup>−5 </sup>M finally) for non-specific uptake,</li><li id="ul0026-0004" num="0117">50 μof <sup>3</sup>H-5-HT (50 nM finally) (NEN, France, 28.4 Ci/mmol).</li></ul></li></ul>

0118Exactly 5 min after the start of incubation, the reaction was stopped by vacuum filtration on Whatman GF/F filters (predilution with 2.5 ml of chilled buffer then rinsing with 3 times 2.5 ml).

0119The radioactivity collected on the filter was measured (Packard Tricarb 4640) by liquid scintillation with Emulsifier-Safe (Packard).

0120The IC<sub>50 </sub>were determined by transposing the percentages of inhibition onto a graph in relation to the log of the product concentration (6 concentrations in duplicate).

00002.1.3. Paroxetine Binding

0121Male Sprague-Dawley rats (Janvier) weighing 180-200 g were used. The hypothalami of several rats were collected and homogenised in 5 ml of chilled buffer (50 mM Tris-HCL, 120 mM NaCl, 5 mM KCl, pH 7.5) on a Dounce, and the homogenate was centrifuged at 30 000 g (27 000 rpm-Beckman. L5-50E, T40 rotor) for 10 min. The residue obtained was recovered in 5 ml of buffer and re-centrifuged under the same conditions. The new residue was recovered in the same buffer and finally re-homogenised on a Dounce at a tissue concentration of 10 mg/ml. The membrane suspension (100 μl) was incubated with 3H-paroxetine (NEN, France, 28.6 Ci/mmol) at a concentration (final) of 0.1 nM, at 20° C., in a final volume of 1 ml for 2 hr. After 2 hr incubation, the reaction was stopped by vacuum filtration on Whatman GF/F filters pre-treated in a 0.05% solution of polyethylenimine 30 min beforehand (prediluted with 4 ml of chilled buffer, then the tube was rinsed with 2 times 4 ml). Radioactivity was measured by liquid scintillation spectrometry (Packard, Tricarb 4640) using Emulsifier-Safe (Packard) as the scintillating agent.

0122Specific <sup>3</sup>H-paroxetine binding was defined as the difference between total binding and that remaining in the presence of 10 μM of fluoxetine.

0123The IC<sub>50 </sub>were determined by transposing the percentages of inhibition onto a graph in relation to the log of the concentration of the product (6 concentrations in duplicate).

00002.1.4. Products Used

0000<ul id="ul0027" list-style="none"><li id="ul0027-0001" num="0000"><ul id="ul0028" list-style="none"><li id="ul0028-0001" num="0124">F2207: batch no 10-CTN3 Key P118</li><li id="ul0028-0002" num="0125">F2695: batch no PL-1-205</li><li id="ul0028-0003" num="0126">F2696: batch no PL-1-204C. <br /> 2.2. Results </li></ul></li></ul>

0127The effects of F2207 and of its two enantiomers on uptake of norepinephrine and serotonin and on paroxetine binding are shown on a graph with the percentage of inhibition in relation (%) on the ordinate and the concentration (M) of F2207, F2695 and F2696 on the apsis (data not shown). The values for the percentages of inhibition corresponding to each product, tested in duplicate, are mean results of four separate experiments.

0128The values of the IC<sub>50 </sub>for the three products were determined on the basis of these curves and are shown in table 2.

0129<tables id="TABLE-US-00007" num="00007"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 2</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Inhibition of <sup>3</sup>H-norepinephrine and <sup>3</sup>H-serotonin</entry></row><row><entry>uptake and <sup>3</sup>H-paroxetine binding.</entry></row><row><entry>IC50 (M)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="112pt" align="center" /><colspec colname="3" colwidth="63pt" align="center" /><tbody valign="top"><row><entry /><entry>Uptake</entry><entry><sup>3</sup>H-Paroxetine</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="70pt" align="center" /><colspec colname="3" colwidth="42pt" align="center" /><colspec colname="4" colwidth="63pt" align="center" /><tbody valign="top"><row><entry>Compounds</entry><entry><sup>3</sup>H-Norepinephrine</entry><entry><sup>3</sup>H-Serotonin</entry><entry>Binding</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry>F2695</entry><entry>1.5 × 10<sup>−8</sup></entry><entry>4.6 × 10<sup>−8</sup> </entry><entry>6.0 × 10<sup>−8</sup> </entry></row><row><entry>F2207</entry><entry>3.0 × 10<sup>−8</sup></entry><entry>15 × 10<sup>−8</sup></entry><entry>13 × 10<sup>−8</sup></entry></row><row><entry>F2696</entry><entry> 75 × 10<sup>−8</sup></entry><entry>60 × 10<sup>−8</sup></entry><entry>70 × 10<sup>−8</sup></entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0130The three compounds were active in these three pharmacological assays, however differences were present:

0131in norepinephrine uptake:

0000F2695 was two times more active than F2207.

0000F2695 was 25 time more active than F2696.

0132in serotonin uptake:

0000F2695 was 3 times more active than F2207.

0000F2695 was 12 times more active than F2696.

0133in paroxetine binding:

0000F2695 was 2 times more active than F2207.

0000F2695 was 10 times more active than F2696.

0134The three compounds were active in these pharmacological assays with however a lesser activity for the levogyral form (F2696) and the racemate (F2207). The dextrogyral form of milnacipran (F2695) was 2 to 3 times more active than F2207.

Example N

Comparative Activity of Milnacipran (F2207) and of its Active Enantiomer (F2695) by the Oral Route on Heart Rate and Blood Pressure in the Waking Dog

00003.1. Introduction

0135This study was designed to study the effects of F2207 (batch n<sup>o </sup>PHA343) and of F2695 (batch n<sup>o </sup>PL-I-221) a) on heart rate after a single administration by the oral route, and b) on systolic and diastolic blood pressure after repeated administration for 5 days by the oral route in dogs.

0136This study was conducted at equally pharmaceutically-active doses of F2695 in 6 dogs equipped with implants (Data Sciences International) allowing for data on heart rate and blood pressure parameters to be captured by telemetry. The animals were allocated to 3 treatment groups: <ul id="ul0029" list-style="none"><li id="ul0029-0001" num="0000"><ul id="ul0030" list-style="none"><li id="ul0030-0001" num="0137">group 1 (control) treated with deionised water,</li><li id="ul0030-0002" num="0138">group 2 treated with F2207 at a dose of 20 mg/kg/day,</li><li id="ul0030-0003" num="0139">group 3 treated with F2695 at a dose of 10 mg/kg/day. <br /> 3.2. Methodology <br /> Given the small number of equipped animals available, and in order to form 3 treatment groups comprising 6 animals each, the study was carried out in three series separated by a wash-out period with re-initialisation of the probes. Each series comprised 2 phases as follows: </li><li id="ul0030-0004" num="0140">the first phase of 5 days during which all the animals were treated with deionised water in order to adapt them to containment and to oral treatment by gavage with stomach tubing,</li><li id="ul0030-0005" num="0141">the second phase of 5 days during which the animals received their respective treatment.</li></ul></li></ul>

0142The treatment regimen and associated schedule were as follows: <ul id="ul0031" list-style="none"><li id="ul0031-0001" num="0000"><ul id="ul0032" list-style="none"><li id="ul0032-0001" num="0143">16 Sep. 2002 (D-7): reception of animals in the study,</li><li id="ul0032-0002" num="0144">18 to 22 Sep. 2002 (D-5 to D-1): adaptation treatment with deionised water for all the animals,</li><li id="ul0032-0003" num="0145">23 to 27 Sep. 2002 (D1 to D5): 1<sup>st </sup>treatment series: the animals received their respective treatment,</li><li id="ul0032-0004" num="0146">28 Sep. to 1 Oct. 2002 (D6 to D9): wash-out period,</li><li id="ul0032-0005" num="0147">2 to 6 Oct. 2002 (D10 to D14): adaptation treatment with deionised water for all the animals,</li><li id="ul0032-0006" num="0148">7 to 11 Oct. 2002 (D15 to D19): 2<sup>nd </sup>treatment series: the animals received their respective treatment,</li><li id="ul0032-0007" num="0149">12 to 15 Oct. 2002 (D20 to D23): wash-out period,</li><li id="ul0032-0008" num="0150">16 to 20 Oct. 2002 (D24 to D28): adaptation treatment with deionised water for all the animals,</li><li id="ul0032-0009" num="0151">21 to 25 Oct. 2002 (D29 to D33): 3<sup>rd </sup>treatment series: the animals received their respective treatment.</li></ul></li></ul>

0152The allocation of animals to the various groups and the associated treatment are shown in table 3. The overall experimental plan is described in table 4.

0153<tables id="TABLE-US-00008" num="00008"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 3</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Table of allocation of dogs to the various groups and associated</entry></row><row><entry>treatment.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="175pt" align="center" /><tbody valign="top"><row><entry /><entry>Definitive numbering of animals </entry></row><row><entry /><entry>and associated treatment</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="63pt" align="left" /><colspec colname="3" colwidth="70pt" align="left" /><colspec colname="4" colwidth="42pt" align="left" /><tbody valign="top"><row><entry>Provisional</entry><entry>1<sup>st </sup>treatment</entry><entry>2<sup>nd </sup>treatment</entry><entry>3<sup>rd </sup>treatment</entry></row><row><entry>numbering</entry><entry>series</entry><entry>series</entry><entry>series</entry></row><row><entry>of animals</entry><entry>(D1 to D5)</entry><entry>(D15 to D19)</entry><entry>(D29 to D33)</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry>dog n° 102</entry><entry>dog n° 3</entry><entry>dog n° 9</entry><entry>dog n° 13</entry></row><row><entry /><entry>F2207 (20 mg/kg/d)</entry><entry>F2207 (20 mg/kg/d)</entry><entry>deionised</entry></row><row><entry /><entry /><entry /><entry>water</entry></row><row><entry>dog n° 103</entry><entry>dog n° 1</entry><entry>dog n° 11</entry><entry>dog n° 17</entry></row><row><entry /><entry>deionised water</entry><entry>F2695 (10 mg/kg/d)</entry><entry>F2695</entry></row><row><entry /><entry /><entry /><entry>(10 mg/kg/d)</entry></row><row><entry>dog n° 104</entry><entry>dog n° 4</entry><entry>dog n° 8</entry><entry>dog n° 18</entry></row><row><entry /><entry>F2207 (20 mg/kg/d)</entry><entry>deionised water</entry><entry>F2695</entry></row><row><entry /><entry /><entry /><entry>(10 mg/kg/d)</entry></row><row><entry>dog n° 106</entry><entry>dog n° 2</entry><entry>dog n° 12</entry><entry>dog n° 16</entry></row><row><entry /><entry>deionised water</entry><entry>F2695 (10 mg/kg/d)</entry><entry>F2207</entry></row><row><entry /><entry /><entry /><entry>(20 mg/kg/d)</entry></row><row><entry>dog n° 109</entry><entry>dog n° 5</entry><entry>dog n° 7</entry><entry>dog n° 15</entry></row><row><entry /><entry>F2695 (10 mg/kg/d)</entry><entry>deionised water</entry><entry>F2207</entry></row><row><entry /><entry /><entry /><entry>(20 mg/kg/d)</entry></row><row><entry>dog n° 110</entry><entry>dog n° 6</entry><entry>dog n° 10</entry><entry>dog n° 14</entry></row><row><entry /><entry>F2695 (10 mg/kg/d)</entry><entry>F2207 (20 mg/kg/d)</entry><entry>deionised</entry></row><row><entry /><entry /><entry /><entry>water</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry namest="1" nameend="4" align="left" id="FOO-00005">Nota:</entry></row><row><entry namest="1" nameend="4" align="left" id="FOO-00006">according to the initial randomisation scheme, each animal was to receive a different treatment in each series. An error committed on D15 forced us to revise the randomisation. The animal bearing provisional ID n° 102 was in fact presented by mistake and treated with F2207, this animal therefore received treatment with F2207 twice. In order to maintain the same number of animals in each treatment group, the animal bearing provisional ID n° 103 also received the same treatment twice, F2695.</entry></row></tbody></tgroup></table></tables>

0154<tables id="TABLE-US-00009" num="00009"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 4</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Overall experimental plan for the telemetry study on the effects of</entry></row><row><entry>milnacipran and of F2695 administered orally</entry></row><row><entry>for 5 days in conscious dogs.</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry /><entry>GROUP NUMBER</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="49pt" align="left" /><colspec colname="3" colwidth="49pt" align="left" /><colspec colname="4" colwidth="49pt" align="left" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>3</entry></row><row><entry namest="1" nameend="4" align="center" rowsep="1" /></row><row><entry>ANIMALS</entry><entry /><entry /><entry /></row><row><entry>Number</entry><entry>6</entry><entry>6</entry><entry>6</entry></row><row><entry>Identification</entry><entry>1-2-7-8-13-14</entry><entry>3-4-9-10-15-16</entry><entry>5-6-11-12-17-18</entry></row><row><entry>TREATMENT</entry><entry /><entry /><entry /></row><row><entry>Identification</entry><entry>Deionised water</entry><entry>F2207</entry><entry>F2695</entry></row><row><entry>Dose</entry><entry>—</entry><entry>20 mg/kg/day</entry><entry>10 mg/kg/day</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="70pt" align="left" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry>Route</entry><entry>oral</entry></row><row><entry>Volume</entry><entry>5 ml/kg</entry></row><row><entry>Frequency/Duration</entry><entry>daily administration/5 days</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0155The effects of the various treatments on heart rate were analysed after single administration. The analysis concerns the following data-capture times: <ul id="ul0033" list-style="none"><li id="ul0033-0001" num="0000"><ul id="ul0034" list-style="none"><li id="ul0034-0001" num="0156">prior to single administration,</li><li id="ul0034-0002" num="0157">every 30 minutes over 6 hours following single administration.</li></ul></li></ul>

0158The effects of the various treatments on blood pressure were analysed at the steady state, on D5, D29 and D33(final effective day of treatment for each series). The analysis concerns the following data-capture times: <ul id="ul0035" list-style="none"><li id="ul0035-0001" num="0000"><ul id="ul0036" list-style="none"><li id="ul0036-0001" num="0159">prior to treatment,</li><li id="ul0036-0002" num="0160">every 30 minutes over 6 hours following treatment. <br /> 3.3. Results <br /> 3.3.1. With regard to heart rate, a Tukey test was performed for each of the 13 experimental times. </li></ul></li></ul>

0161The following observations were made in comparison with the control animals receiving deionised water (<figref idref="DRAWINGS">FIG. 1</figref>): <ul id="ul0037" list-style="none"><li id="ul0037-0001" num="0000"><ul id="ul0038" list-style="none"><li id="ul0038-0001" num="0162">§ a significant increase in heart rate for half of the experimental times (6/12) after single administration of F2207 (20 mg/kg/day), particularly marked one to two hours after treatment (p≦0.01 at 1.5 and 2.0 hours after treatment), followed by a slow decline, still present and significant however 5.5 hours after treatment (p≦0.01).</li><li id="ul0038-0002" num="0163">§ the absence of any significant increase in heart rate for almost all the experimental times (11/12), after single administration of F2695 (10 mg/kg/day); one weak statistical significance was observed at 1.5 hours (p≦0.05).</li></ul></li></ul>

01643.3.2. With regard to blood pressure, one mean value for diastolic blood pressure (<figref idref="DRAWINGS">FIG. 2</figref> and table 5), as well as one mean value for systolic blood pressure (<figref idref="DRAWINGS">FIG. 3</figref> and table 6) were calculated for each dog and for the 6 hours following the final treatment, after 5 consecutive days of administration. These mean blood pressure values were analysed by ANOVA followed by a Tukey test when ANOVA permitted such a test (data not shown).

0165The following were observed: <ul id="ul0039" list-style="none"><li id="ul0039-0001" num="0000"><ul id="ul0040" list-style="none"><li id="ul0040-0001" num="0166">a significant increase (p<0.001) in diastolic blood pressure after repeated administration of F2207 for 5 days (20 mg/kg/day) or of F2695 (10 mg/kg/day) compared to treatment with deionised water,</li><li id="ul0040-0002" num="0167">a significant difference (p<0.05) in the mean diastolic blood pressure value after repeated administration of F2207 (20 mg/kg/day) for 5 days compared to the mean diastolic blood pressure value after repeated administration of F2695 (10 mg/kg/day)</li><li id="ul0040-0003" num="0168">no significant effect on systolic blood pressure; it should be noted however that the values for sBP after repeated administration of F2695 for 5 days are close to the sBP following treatment with deionised water.</li></ul></li></ul>

0169Individual diastolic and systolic blood pressure data are shown in tables 5 and 6 respectively.

0170<tables id="TABLE-US-00010" num="00010"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="406pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 5</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Individual diastolic blood pressure data</entry></row><row><entry>DIASTOLIC BLOOD PRESSURE (dBP expressed in mmHg)</entry></row><row><entry>Individual data after repeated administration for 5 consecutive days</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="357pt" align="center" /><tbody valign="top"><row><entry /><entry>GROUP</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="119pt" align="center" /><colspec colname="3" colwidth="119pt" align="center" /><colspec colname="4" colwidth="119pt" align="center" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>3</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="357pt" align="center" /><tbody valign="top"><row><entry /><entry>TREATMENT</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="119pt" align="center" /><colspec colname="3" colwidth="119pt" align="center" /><colspec colname="4" colwidth="119pt" align="center" /><tbody valign="top"><row><entry /><entry>VEHICLE</entry><entry>F2207 (20 mg/kg/d)</entry><entry>F2695 (10 mg/kg/d)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="357pt" align="center" /><tbody valign="top"><row><entry /><entry>Animal N°</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="25"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="14pt" align="right" /><colspec colname="3" colwidth="14pt" align="right" /><colspec colname="4" colwidth="14pt" align="right" /><colspec colname="5" colwidth="14pt" align="right" /><colspec colname="6" colwidth="14pt" align="right" /><colspec colname="7" colwidth="14pt" align="right" /><colspec colname="8" colwidth="14pt" align="right" /><colspec colname="9" colwidth="21pt" align="center" /><colspec colname="10" colwidth="14pt" align="right" /><colspec colname="11" colwidth="14pt" align="right" /><colspec colname="12" colwidth="14pt" align="right" /><colspec colname="13" colwidth="14pt" align="right" /><colspec colname="14" colwidth="14pt" align="right" /><colspec colname="15" colwidth="14pt" align="right" /><colspec colname="16" colwidth="14pt" align="right" /><colspec colname="17" colwidth="21pt" align="center" /><colspec colname="18" colwidth="14pt" align="right" /><colspec colname="19" colwidth="14pt" align="right" /><colspec colname="20" colwidth="14pt" align="right" /><colspec colname="21" colwidth="14pt" align="right" /><colspec colname="22" colwidth="14pt" align="right" /><colspec colname="23" colwidth="14pt" align="right" /><colspec colname="24" colwidth="14pt" align="right" /><colspec colname="25" colwidth="21pt" align="center" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>7</entry><entry>8</entry><entry>13</entry><entry>14</entry><entry>M</entry><entry>SEM</entry><entry>3</entry><entry>4</entry><entry>9</entry><entry>10</entry><entry>15</entry><entry>16</entry><entry>M</entry><entry>SEM</entry><entry>5</entry><entry>6</entry><entry>11</entry><entry>12</entry><entry>17</entry><entry>18</entry><entry>M</entry><entry>SEM</entry></row><row><entry namest="1" nameend="25" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="25"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="14pt" align="right" /><colspec colname="3" colwidth="14pt" align="right" /><colspec colname="4" colwidth="14pt" align="right" /><colspec colname="5" colwidth="14pt" align="right" /><colspec colname="6" colwidth="14pt" align="right" /><colspec colname="7" colwidth="14pt" align="right" /><colspec colname="8" colwidth="14pt" align="right" /><colspec colname="9" colwidth="21pt" align="center" /><colspec colname="10" colwidth="14pt" align="right" /><colspec colname="11" colwidth="14pt" align="right" /><colspec colname="12" colwidth="14pt" align="right" /><colspec colname="13" colwidth="14pt" align="right" /><colspec colname="14" colwidth="14pt" align="right" /><colspec colname="15" colwidth="14pt" align="right" /><colspec colname="16" colwidth="14pt" align="right" /><colspec colname="17" colwidth="21pt" align="center" /><colspec colname="18" colwidth="14pt" align="right" /><colspec colname="19" colwidth="14pt" align="right" /><colspec colname="20" colwidth="14pt" align="right" /><colspec colname="21" colwidth="14pt" align="right" /><colspec colname="22" colwidth="14pt" align="right" /><colspec colname="23" colwidth="14pt" align="right" /><colspec colname="24" colwidth="14pt" align="right" /><colspec colname="25" colwidth="21pt" align="center" /><tbody valign="top"><row><entry>Time before</entry><entry>79</entry><entry>77</entry><entry>73</entry><entry>77</entry><entry>101</entry><entry>76</entry><entry>81</entry><entry>4</entry><entry>112</entry><entry>89</entry><entry>93</entry><entry>88</entry><entry>86</entry><entry>91</entry><entry>93</entry><entry>4</entry><entry>73</entry><entry>89</entry><entry>80</entry><entry>71</entry><entry>76</entry><entry>78</entry><entry>78</entry><entry>3</entry></row><row><entry>treatment</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>Time after</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>treatment(h)</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>0.50</entry><entry>84</entry><entry>76</entry><entry>70</entry><entry>63</entry><entry>80</entry><entry>70</entry><entry>74</entry><entry>3</entry><entry>103</entry><entry>106</entry><entry>96</entry><entry>92</entry><entry>88</entry><entry>87</entry><entry>95</entry><entry>3</entry><entry>91</entry><entry>91</entry><entry>99</entry><entry>90</entry><entry>108</entry><entry>85</entry><entry>94</entry><entry>3</entry></row><row><entry>1.00</entry><entry>82</entry><entry>84</entry><entry>77</entry><entry>72</entry><entry>72</entry><entry>76</entry><entry>77</entry><entry>2</entry><entry>130</entry><entry>117</entry><entry>113</entry><entry>113</entry><entry>90</entry><entry>106</entry><entry>112</entry><entry>5</entry><entry>112</entry><entry>96</entry><entry>75</entry><entry>97</entry><entry>87</entry><entry>96</entry><entry>94</entry><entry>5</entry></row><row><entry>1.50</entry><entry>102</entry><entry>81</entry><entry>79</entry><entry>75</entry><entry>82</entry><entry>68</entry><entry>81</entry><entry>5</entry><entry>131</entry><entry>127</entry><entry>137</entry><entry>96</entry><entry>100</entry><entry>91</entry><entry>114</entry><entry>8</entry><entry>109</entry><entry>83</entry><entry>88</entry><entry>97</entry><entry>87</entry><entry>112</entry><entry>96</entry><entry>5</entry></row><row><entry>2.00</entry><entry>83</entry><entry>75</entry><entry>71</entry><entry>98</entry><entry>77</entry><entry>75</entry><entry>80</entry><entry>4</entry><entry>123</entry><entry>113</entry><entry>99</entry><entry>88</entry><entry>107</entry><entry>109</entry><entry>107</entry><entry>5</entry><entry>115</entry><entry>88</entry><entry>93</entry><entry>95</entry><entry>84</entry><entry>109</entry><entry>97</entry><entry>5</entry></row><row><entry>2.50</entry><entry>85</entry><entry>75</entry><entry>75</entry><entry>84</entry><entry>85</entry><entry>79</entry><entry>81</entry><entry>2</entry><entry>137</entry><entry>111</entry><entry>116</entry><entry>101</entry><entry>115</entry><entry>107</entry><entry>115</entry><entry>5</entry><entry>111</entry><entry>88</entry><entry>97</entry><entry>89</entry><entry>92</entry><entry>107</entry><entry>97</entry><entry>4</entry></row><row><entry>3.00</entry><entry>91</entry><entry>95</entry><entry>99</entry><entry>85</entry><entry>79</entry><entry>84</entry><entry>89</entry><entry>3</entry><entry>121</entry><entry>118</entry><entry>112</entry><entry>116</entry><entry>106</entry><entry>92</entry><entry>111</entry><entry>4</entry><entry>104</entry><entry>91</entry><entry>96</entry><entry>96</entry><entry>100</entry><entry>106</entry><entry>99</entry><entry>2</entry></row><row><entry>3.50</entry><entry>83</entry><entry>72</entry><entry>78</entry><entry>73</entry><entry>77</entry><entry>65</entry><entry>75</entry><entry>3</entry><entry>120</entry><entry>106</entry><entry>133</entry><entry>116</entry><entry>103</entry><entry>103</entry><entry>114</entry><entry>5</entry><entry>96</entry><entry>106</entry><entry>94</entry><entry>107</entry><entry>77</entry><entry>103</entry><entry>97</entry><entry>5</entry></row><row><entry>4.00</entry><entry>81</entry><entry>79</entry><entry>75</entry><entry>77</entry><entry>82</entry><entry>68</entry><entry>77</entry><entry>2</entry><entry>133</entry><entry>114</entry><entry>105</entry><entry>111</entry><entry>110</entry><entry>103</entry><entry>113</entry><entry>4</entry><entry>125</entry><entry>91</entry><entry>99</entry><entry>108</entry><entry>80</entry><entry>109</entry><entry>102</entry><entry>6</entry></row><row><entry>4.50</entry><entry>82</entry><entry>76</entry><entry>91</entry><entry>84</entry><entry>113</entry><entry>85</entry><entry>89</entry><entry>5</entry><entry>135</entry><entry>110</entry><entry>126</entry><entry>109</entry><entry>104</entry><entry>108</entry><entry>115</entry><entry>5</entry><entry>103</entry><entry>104</entry><entry>92</entry><entry>100</entry><entry>85</entry><entry>108</entry><entry>99</entry><entry>3</entry></row><row><entry>5.00</entry><entry>97</entry><entry>79</entry><entry>67</entry><entry>95</entry><entry>81</entry><entry>82</entry><entry>84</entry><entry>5</entry><entry>116</entry><entry>120</entry><entry>98</entry><entry>97</entry><entry>97</entry><entry>105</entry><entry>106</entry><entry>4</entry><entry>126</entry><entry>100</entry><entry>92</entry><entry>95</entry><entry>110</entry><entry>102</entry><entry>104</entry><entry>5</entry></row><row><entry>5.50</entry><entry>94</entry><entry>80</entry><entry>70</entry><entry>ND</entry><entry>85</entry><entry>82</entry><entry>82</entry><entry>4</entry><entry>103</entry><entry>107</entry><entry>115</entry><entry>106</entry><entry>92</entry><entry>93</entry><entry>103</entry><entry>4</entry><entry>88</entry><entry>86</entry><entry>105</entry><entry>98</entry><entry>89</entry><entry>99</entry><entry>94</entry><entry>3</entry></row><row><entry>6.00</entry><entry>83</entry><entry>74</entry><entry>82</entry><entry>82</entry><entry>78</entry><entry>77</entry><entry>79</entry><entry>1</entry><entry>115</entry><entry>133</entry><entry>120</entry><entry>104</entry><entry>103</entry><entry>104</entry><entry>113</entry><entry>5</entry><entry>101</entry><entry>113</entry><entry>98</entry><entry>105</entry><entry>109</entry><entry>108</entry><entry>106</entry><entry>2</entry></row><row><entry>Mean dBP after </entry><entry>87</entry><entry>79</entry><entry>78</entry><entry>81</entry><entry>83</entry><entry>76</entry><entry>81</entry><entry>2</entry><entry>122</entry><entry>115</entry><entry>114</entry><entry>104</entry><entry>101</entry><entry>101</entry><entry>110</entry><entry>4</entry><entry>107</entry><entry>95</entry><entry>94</entry><entry>98</entry><entry>92</entry><entry>104</entry><entry>98</entry><entry>2</entry></row><row><entry>treatment</entry></row><row><entry namest="1" nameend="25" align="center" rowsep="1" /></row><row><entry namest="1" nameend="25" align="left" id="FOO-00007">ND: not determined</entry></row></tbody></tgroup></table></tables>

0171<tables id="TABLE-US-00011" num="00011"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="406pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 6</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Individual systolic blood pressure data</entry></row><row><entry>SYSTOLIC BLOOD PRESSURE (sBP expressed en mmHg)</entry></row><row><entry>Individual data after repeated administration for 5 consecutive days</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="357pt" align="center" /><tbody valign="top"><row><entry /><entry>GROUP</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="119pt" align="center" /><colspec colname="3" colwidth="119pt" align="center" /><colspec colname="4" colwidth="119pt" align="center" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>3</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="238pt" align="center" /><colspec colname="3" colwidth="119pt" align="center" /><tbody valign="top"><row><entry /><entry>TREATMENT</entry><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="119pt" align="center" /><colspec colname="3" colwidth="119pt" align="center" /><colspec colname="4" colwidth="119pt" align="center" /><tbody valign="top"><row><entry /><entry>VEHICLE</entry><entry>F2207 (20 mg/kg/d)</entry><entry>F2695 (10 mg/kg/d)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="357pt" align="center" /><tbody valign="top"><row><entry /><entry>Animal N° </entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="25"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="14pt" align="right" /><colspec colname="3" colwidth="14pt" align="right" /><colspec colname="4" colwidth="14pt" align="right" /><colspec colname="5" colwidth="14pt" align="right" /><colspec colname="6" colwidth="14pt" align="right" /><colspec colname="7" colwidth="14pt" align="right" /><colspec colname="8" colwidth="14pt" align="right" /><colspec colname="9" colwidth="21pt" align="center" /><colspec colname="10" colwidth="14pt" align="right" /><colspec colname="11" colwidth="14pt" align="right" /><colspec colname="12" colwidth="14pt" align="right" /><colspec colname="13" colwidth="14pt" align="right" /><colspec colname="14" colwidth="14pt" align="right" /><colspec colname="15" colwidth="14pt" align="right" /><colspec colname="16" colwidth="14pt" align="right" /><colspec colname="17" colwidth="21pt" align="center" /><colspec colname="18" colwidth="14pt" align="right" /><colspec colname="19" colwidth="14pt" align="right" /><colspec colname="20" colwidth="14pt" align="right" /><colspec colname="21" colwidth="14pt" align="right" /><colspec colname="22" colwidth="14pt" align="right" /><colspec colname="23" colwidth="14pt" align="right" /><colspec colname="24" colwidth="14pt" align="right" /><colspec colname="25" colwidth="21pt" align="center" /><tbody valign="top"><row><entry /><entry>1</entry><entry>2</entry><entry>7</entry><entry>8</entry><entry>13</entry><entry>14</entry><entry>M</entry><entry>SEM</entry><entry>3</entry><entry>4</entry><entry>9</entry><entry>10</entry><entry>15</entry><entry>16</entry><entry>M</entry><entry>SEM</entry><entry>5</entry><entry>6</entry><entry>11</entry><entry>12</entry><entry>17</entry><entry>18</entry><entry>M</entry><entry>SEM</entry></row><row><entry namest="1" nameend="25" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="25"><colspec colname="1" colwidth="49pt" align="center" /><colspec colname="2" colwidth="14pt" align="right" /><colspec colname="3" colwidth="14pt" align="right" /><colspec colname="4" colwidth="14pt" align="right" /><colspec colname="5" colwidth="14pt" align="right" /><colspec colname="6" colwidth="14pt" align="right" /><colspec colname="7" colwidth="14pt" align="right" /><colspec colname="8" colwidth="14pt" align="right" /><colspec colname="9" colwidth="21pt" align="center" /><colspec colname="10" colwidth="14pt" align="right" /><colspec colname="11" colwidth="14pt" align="right" /><colspec colname="12" colwidth="14pt" align="right" /><colspec colname="13" colwidth="14pt" align="right" /><colspec colname="14" colwidth="14pt" align="right" /><colspec colname="15" colwidth="14pt" align="right" /><colspec colname="16" colwidth="14pt" align="right" /><colspec colname="17" colwidth="21pt" align="char" char="." /><colspec colname="18" colwidth="14pt" align="right" /><colspec colname="19" colwidth="14pt" align="right" /><colspec colname="20" colwidth="14pt" align="right" /><colspec colname="21" colwidth="14pt" align="right" /><colspec colname="22" colwidth="14pt" align="right" /><colspec colname="23" colwidth="14pt" align="right" /><colspec colname="24" colwidth="14pt" align="right" /><colspec colname="25" colwidth="21pt" align="center" /><tbody valign="top"><row><entry>Time before</entry><entry>139</entry><entry>141</entry><entry>120</entry><entry>157</entry><entry>172</entry><entry>138</entry><entry>145</entry><entry>7</entry><entry>188</entry><entry>164</entry><entry>176</entry><entry>149</entry><entry>130</entry><entry>169</entry><entry>163</entry><entry>8</entry><entry>136</entry><entry>141</entry><entry>138</entry><entry>130</entry><entry>134</entry><entry>149</entry><entry>138</entry><entry>3</entry></row><row><entry>treatment</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>Time after</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>treatment(h)</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /></row><row><entry>0.50</entry><entry>135</entry><entry>132</entry><entry>119</entry><entry>131</entry><entry>149</entry><entry>138</entry><entry>134</entry><entry>4</entry><entry>158</entry><entry>154</entry><entry>152</entry><entry>128</entry><entry>126</entry><entry>129</entry><entry>141</entry><entry>6</entry><entry>135</entry><entry>129</entry><entry>140</entry><entry>135</entry><entry>160</entry><entry>139</entry><entry>140</entry><entry>4</entry></row><row><entry>1.00</entry><entry>134</entry><entry>158</entry><entry>129</entry><entry>144</entry><entry>141</entry><entry>143</entry><entry>142</entry><entry>4</entry><entry>180</entry><entry>167</entry><entry>157</entry><entry>150</entry><entry>126</entry><entry>130</entry><entry>152</entry><entry>9</entry><entry>159</entry><entry>135</entry><entry>124</entry><entry>148</entry><entry>131</entry><entry>143</entry><entry>140</entry><entry>5</entry></row><row><entry>1.50</entry><entry>158</entry><entry>151</entry><entry>145</entry><entry>150</entry><entry>153</entry><entry>137</entry><entry>149</entry><entry>3</entry><entry>186</entry><entry>181</entry><entry>189</entry><entry>129</entry><entry>136</entry><entry>138</entry><entry>160</entry><entry>12</entry><entry>164</entry><entry>119</entry><entry>138</entry><entry>158</entry><entry>127</entry><entry>156</entry><entry>144</entry><entry>8</entry></row><row><entry>2.00</entry><entry>138</entry><entry>136</entry><entry>145</entry><entry>173</entry><entry>151</entry><entry>144</entry><entry>148</entry><entry>5</entry><entry>171</entry><entry>160</entry><entry>146</entry><entry>122</entry><entry>140</entry><entry>163</entry><entry>150</entry><entry>7</entry><entry>168</entry><entry>125</entry><entry>135</entry><entry>141</entry><entry>127</entry><entry>156</entry><entry>142</entry><entry>7</entry></row><row><entry>2.50</entry><entry>142</entry><entry>143</entry><entry>145</entry><entry>159</entry><entry>160</entry><entry>148</entry><entry>150</entry><entry>3</entry><entry>195</entry><entry>168</entry><entry>168</entry><entry>144</entry><entry>153</entry><entry>161</entry><entry>165</entry><entry>7</entry><entry>165</entry><entry>124</entry><entry>142</entry><entry>141</entry><entry>134</entry><entry>154</entry><entry>143</entry><entry>6</entry></row><row><entry>3.00</entry><entry>149</entry><entry>167</entry><entry>162</entry><entry>163</entry><entry>150</entry><entry>154</entry><entry>158</entry><entry>3</entry><entry>173</entry><entry>177</entry><entry>164</entry><entry>157</entry><entry>141</entry><entry>146</entry><entry>160</entry><entry>6</entry><entry>156</entry><entry>131</entry><entry>145</entry><entry>144</entry><entry>151</entry><entry>157</entry><entry>147</entry><entry>4</entry></row><row><entry>3.50</entry><entry>135</entry><entry>129</entry><entry>149</entry><entry>154</entry><entry>153</entry><entry>137</entry><entry>143</entry><entry>4</entry><entry>165</entry><entry>153</entry><entry>184</entry><entry>167</entry><entry>139</entry><entry>155</entry><entry>161</entry><entry>6</entry><entry>146</entry><entry>147</entry><entry>141</entry><entry>169</entry><entry>123</entry><entry>156</entry><entry>147</entry><entry>6</entry></row><row><entry>4.00</entry><entry>142</entry><entry>143</entry><entry>149</entry><entry>166</entry><entry>164</entry><entry>144</entry><entry>151</entry><entry>4</entry><entry>180</entry><entry>157</entry><entry>151</entry><entry>154</entry><entry>150</entry><entry>153</entry><entry>158</entry><entry>5</entry><entry>180</entry><entry>132</entry><entry>145</entry><entry>160</entry><entry>124</entry><entry>164</entry><entry>151</entry><entry>9</entry></row><row><entry>4.50</entry><entry>137</entry><entry>140</entry><entry>159</entry><entry>170</entry><entry>190</entry><entry>152</entry><entry>158</entry><entry>8</entry><entry>184</entry><entry>161</entry><entry>180</entry><entry>155</entry><entry>145</entry><entry>168</entry><entry>166</entry><entry>6</entry><entry>158</entry><entry>151</entry><entry>138</entry><entry>163</entry><entry>131</entry><entry>163</entry><entry>151</entry><entry>5</entry></row><row><entry>5.00</entry><entry>150</entry><entry>146</entry><entry>127</entry><entry>177</entry><entry>160</entry><entry>145</entry><entry>151</entry><entry>7</entry><entry>161</entry><entry>171</entry><entry>146</entry><entry>141</entry><entry>139</entry><entry>166</entry><entry>154</entry><entry>6</entry><entry>182</entry><entry>144</entry><entry>137</entry><entry>150</entry><entry>162</entry><entry>158</entry><entry>156</entry><entry>6</entry></row><row><entry>5.50</entry><entry>153</entry><entry>149</entry><entry>132</entry><entry>ND</entry><entry>148</entry><entry>144</entry><entry>145</entry><entry>4</entry><entry>151</entry><entry>154</entry><entry>173</entry><entry>152</entry><entry>132</entry><entry>155</entry><entry>153</entry><entry>5</entry><entry>142</entry><entry>127</entry><entry>152</entry><entry>153</entry><entry>141</entry><entry>153</entry><entry>145</entry><entry>4</entry></row><row><entry>6.00</entry><entry>146</entry><entry>144</entry><entry>151</entry><entry>176</entry><entry>146</entry><entry>143</entry><entry>151</entry><entry>5</entry><entry>158</entry><entry>192</entry><entry>171</entry><entry>154</entry><entry>148</entry><entry>172</entry><entry>166</entry><entry>7</entry><entry>156</entry><entry>170</entry><entry>148</entry><entry>159</entry><entry>160</entry><entry>166</entry><entry>160</entry><entry>3</entry></row><row><entry>Means BP</entry><entry>143</entry><entry>145</entry><entry>143</entry><entry>160</entry><entry>155</entry><entry>144</entry><entry>148</entry><entry>3</entry><entry>172</entry><entry>166</entry><entry>165</entry><entry>146</entry><entry>140</entry><entry>153</entry><entry>157</entry><entry>5</entry><entry>159</entry><entry>136</entry><entry>140</entry><entry>152</entry><entry>139</entry><entry>155</entry><entry>147</entry><entry>4</entry></row><row><entry>after treatment</entry></row><row><entry namest="1" nameend="25" align="center" rowsep="1" /></row><row><entry namest="1" nameend="25" align="left" id="FOO-00008">ND: not determined</entry></row></tbody></tgroup></table></tables><br /> 3.4. Conclusion

0172Under the experimental conditions of the present study by oral administration in the waking dog equipped with a telemetric device: <ul id="ul0041" list-style="none"><li id="ul0041-0001" num="0000"><ul id="ul0042" list-style="none"><li id="ul0042-0001" num="0173">on single administration and compared to the control group, the increase in heart rate was weakly significant and fleeting with F2695 at a dose of 10 mg/kg/day, clearly significant and lasting with F2207 at the equally pharmaceutically-active dose of 20 mg/kg/day,</li><li id="ul0042-0002" num="0174">F2695, at a dose of 10 mg/kg/day, did not induce any statistically significant change in mean systolic blood pressure over the 6 hours following the final treatment, at the steady state after repeated administration for 5 days,</li><li id="ul0042-0003" num="0175">a statistically significant difference was evidenced in mean diastolic blood pressure over the 6 hours following the final treatment, at the steady state after repeated administration for 5 days, between the active F2695 enantiomer (98±2 mm Hg) and the F2207 racemic at equally pharmaceutically-active doses (110±4 mm Hg).</li></ul></li></ul>

0176These differences clearly demonstrated greater cardiovascular tolerability of the active F2695 enantiomer.

Example 4

Genomic Test of Predictive Toxicology In Vitro

00004.1. Materials and Methods

0177The F2695 and F2696 compounds, enantiomers of the racemic molecule F2207, as well as clomipramine, a reference product, (coded C218 in the study) were assessed in the present study. The two enantiomers, F2695 and F2696, were first assessed in a preliminary cytotoxicity text (MTT-assay) on primary rat hepatocytes, in order to select the three concentrations to be used in the final test.

0178After treatment of the primary rat hepatocytes in culture, the RNA was extracted in order to generate labelled complementary-DNA probes which were then hybridised on a membrane containing 682 alternatively-spliced fragments specific to cell stress. A Toxicity Index was obtained for each of the products by comparing the hybridisation profile of the treated cells with that of the untreated cells.

00004.1.1. Purpose and Aim of the Study

0179Safe-Hit is a genomic test for predictive toxicopharmacology that is sensitive, robust, reliable, rapid and sure, enabling products to be compared and ranked on the basis of optimised assessment of their toxic potential.

0180Safe-Hit uses technology, the property of EXONHIT (DATAS™: Differential Analysis of Transcripts with Alternative Slicing), that permits isolation and, consequently, cloning of splicing events that result from a given biological state, in comparison with a control condition. This allows mRNA isoforms, differentially expressed depending the biological conditions, to be isolated.

0181Safe-Hit allows molecules within a chemical series to be ranked according to a Toxic Index, determined after the following basic steps (systematically performed in duplicate for each product): <ul id="ul0043" list-style="none"><li id="ul0043-0001" num="0000"><ul id="ul0044" list-style="none"><li id="ul0044-0001" num="0182">treatment of the cells lines with the various products at three different concentrations, deduced from a preliminary cytotoxicology test (MTT-assay): a reference concentration corresponding to 80% cell viability, a concentration 10-fold higher—when possible—and a concentration 10-fold lower,</li><li id="ul0044-0002" num="0183">preparation of total RNA and of the corresponding radio-labelled cDNA probes,</li><li id="ul0044-0003" num="0184">hybridisation of the cDNA probes: the Safe-Hit macroarray contains 682 independent clones, corresponding to alterations in gene splicing induced by overexpression of WTp53 (p53 is the most ubiquitous “mediator” of cell stress, chosen for the development of this methodology),</li><li id="ul0044-0004" num="0185">acquisition and determination of the Toxicity Index. <br /> 4.1.2. Cells </li></ul></li></ul>

0186The cells used in the study (preliminary MTT-assay of cytotoxicity and the main test) are cryopreserved hepatocytes from Sprague-Dawley rats in primary culture (batches Hep184005 and Hep184006-Biopredic), cultured under standard conditions.

00004.1.2.1 Culture Medium

0000<ul id="ul0045" list-style="none"><li id="ul0045-0001" num="0000"><ul id="ul0046" list-style="none"><li id="ul0046-0001" num="0187">thawing medium: Leibovitz 15 medium with glutamax 1, to which were added 100 IU/ml of penicillin, 100 μg/ml of streptomycin and 0.6 M of glucose (batch MIL 210009-Biopredic),</li><li id="ul0046-0002" num="0188">seeding medium: Williams E medium with glutamax 1, to which were added 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4 μg/ml of bovine insulin and 10% v/v foetal calf serum (batch MIL 260005)-Biopredic),</li><li id="ul0046-0003" num="0189">incubation medium: Williams E medium with glutamax 1, to which were added 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4 μg/ml of bovine insulin and 50 μM of hydrocortisone hemisuccinate (batch MIL 260009-260007-Biopredic). <br /> 4.1.2.2 Culture Conditions </li></ul></li></ul>

019037° C., CO2 atmosphere (5%), relative humidity (95%).

00004.1.2.3 Culture Procedure

0191<tables id="TABLE-US-00012" num="00012"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="70pt" align="left" /><colspec colname="3" colwidth="91pt" align="left" /><tbody valign="top"><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row><row><entry /><entry>Cell toxicity test</entry><entry>Main study</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="2"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="161pt" align="center" /><tbody valign="top"><row><entry /><entry>Cells were seeded on the day of treatment</entry></row><row><entry namest="1" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="1" colwidth="56pt" align="left" /><colspec colname="2" colwidth="70pt" align="left" /><colspec colname="3" colwidth="91pt" align="left" /><tbody valign="top"><row><entry>Seeding density</entry><entry>35 000 cells/well (96 </entry><entry>1.5 million cells per 30 mm </entry></row><row><entry /><entry>wells per plate)</entry><entry>plate </entry></row><row><entry>Medium volume</entry><entry>0.1 ml</entry><entry>3 ml</entry></row><row><entry namest="1" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables><br /> 4.1.3. Cytotoxicity Test

0192The cytotoxicity test (MTT-assay) detects live cells by use of a calorimetric reaction that reveals the integrity of cell respiration implying activity of the mitochondria. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), soluble in water, is transformed by splitting, under the effect of a mitochondiral enzyme in live cells, into insoluble purple formazan. Formazan is solubilised in an organic solvent and the solution obtained can be measured by spectrophotometry. The absorbance measured is proportional to the number of surviving cells.

0193The cells are put into contact with the product to be tested at 5 different concentrations (0-1-10-25-50 and 100 μM) for 16 hours.

0194After this period of exposure, an MTT solution (0.5 mg/ml in the incubation medium of the primary hepatocytes) is added for 3 hours. After solubilisation of the formazan crystals, the multi-well plates are read with a spectrophotometer at 500 nm in order to determine the percentage of cell viability.

00004.1.4 Main Genomic Pharmacotoxicology Test

0195The main study is performed in duplicate, using seeded cultures exposed to each product in order to enhance consistency between the experiments and to validate the results obtained.

00004.1.4.1 Cell Seeding and Treatment

0196The cells are seeded and cultured for 16 hours with each product, at the three concentrations chosen on the basis of the preliminary MTT-assay; two controls (untreated cells in solvent alone) are added to the series.

00004.1.4.2 Total RNA Extraction and Assay

0197After treatment, the RNA is extracted and analysed as follows: <ul id="ul0047" list-style="none"><li id="ul0047-0001" num="0000"><ul id="ul0048" list-style="none"><li id="ul0048-0001" num="0198">collection of cells and centrifugation,</li><li id="ul0048-0002" num="0199">extraction performed with a ready-to-use phenol reagent (Trizol-batches 1106266 and 1121067-Invitrogen) according to the manufacturer's protocol,</li><li id="ul0048-0003" num="0200">solubilisation of the RNA in water,</li><li id="ul0048-0004" num="0201">RNA assay by spectrophotometry (optical density measured at 260, 280 and 300 mm),</li><li id="ul0048-0005" num="0202">verification of the quality of the RNA using Agilent. <br /> 4.1.4.3 Preparation of the cDNA Probes </li></ul></li></ul>

0203The cDNA probes are prepared by reverse radio-active transcription (alpha dATP <sup>33</sup>P-Amersham). The radio-active cDNA is quantified (Instant Imager-Packard) to ensure that the probes are active.

00004.1.4.4 Hybridisation on the Safe-Hit Membrane

0204The 682 DATAS clones (alternately-spliced patterns) are placed on the Safe-Hit membranes, made of pre-cut nylon (Q-BIOgene), with the aid of a Q-Pix apparatus (GENETIX). The DNA probes are hybridised on the membranes overnight and the membranes are washed.

00004.1.1.5. Preparation of the cDNA Probes:

0000<ul id="ul0049" list-style="none"><li id="ul0049-0001" num="0000"><ul id="ul0050" list-style="none"><li id="ul0050-0001" num="0205">matrix: 5 μg of total RNA (for each treatment series and for each concentration),</li><li id="ul0050-0002" num="0206">primer: 100 ng of oligo-dTV oligonucleotide, for the 1st and 2nd hybridisations in rats (batch 12.00, Invitrogen),</li><li id="ul0050-0003" num="0207">main mixture: <ul id="ul0051" list-style="none"><li id="ul0051-0001" num="0208">10 μl of First Strand 5× Premier buffer (batch 1131226-Invitrogen)</li><li id="ul0051-0002" num="0209">1 μl of dCTP+dGTP+dTTP 20 mM (batch 1105201-Invitrogen)</li><li id="ul0051-0003" num="0210">1 μM of ATP 120 μM (batch 1105201-Invitrogen)</li><li id="ul0051-0004" num="0211">5 μl of Dithiotreitol (DTT) 0.1 M (batch 133609-Invitrogen)</li><li id="ul0051-0005" num="0212">1 μl of Out 40 U RNase (batch 1113345-Invitrogen)</li><li id="ul0051-0006" num="0213">5 μl of <sup>33</sup>P dATP 3 000 Ci/mmol 10 mCi/μl (batch B0239-Amersham)</li><li id="ul0051-0007" num="0214">4 μl of Superscript II (batch 1137806-Invitrogen)</li><li id="ul0051-0008" num="0215">1 μl of glycogen (batch 1129328-Invitrogen)</li></ul></li></ul></li></ul>

0216Procedure:

0217Incubate the RNA and the oligo-dTV at 70° C. for 10 minutes then place it on ice. Add 27 μl of MasterMix and incubate at 43° C. for 1 h than at 50° C. for 15 minutes. Add 20 μl of water, then 20 μl of EDTA 50 mM, then 4 μl of NaOH 10N. Incubate for 20 minutes at 65° C. then place on ice. Quantification: Instant Imager, Packard: 1 μl of reaction mixture, add 8 μl of acetic acid, 100 μl of isopropanol and 1 μl of glycogen (20 μg/μl). Incubate at −20° C. for 20 minutes, centrifuge for 20 minutes at 13000 rpm at 4° C. Reconstitute as a suspension with 200 μl of water, quantification: Instant Imager, Packard: 1 μl of reaction mixture.

0218<tables id="TABLE-US-00013" num="00013"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="center" /><thead><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Media and buffers</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="14pt" align="left" /><colspec colname="1" colwidth="126pt" align="left" /><colspec colname="2" colwidth="77pt" align="left" /><tbody valign="top"><row><entry /><entry>Common solutions:</entry><entry>Washing buffer 1:</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>20X SSC (Invitrogen)</entry><entry>2X SSC</entry></row><row><entry /><entry>50X Denhardt's</entry><entry /></row><row><entry /><entry>50% (w/v) Dextran Sulphate (ICN)</entry><entry /></row><row><entry /><entry>20% SDS (v/v)(Quantum biotech.)</entry><entry /></row><row><entry /><entry>10 mg/ml DNA from salmon sperm</entry><entry /></row><row><entry /><entry>(Q-Biogene)</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>Prehybridisation buffer:</entry><entry>Washing buffer 2:</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>6X SSC</entry><entry>2X SSC</entry></row><row><entry /><entry>10X Denhardt's</entry><entry>0.1% SDS</entry></row><row><entry /><entry>10% Dextran Sulphate</entry><entry /></row><row><entry /><entry>0.5% SDS</entry><entry /></row><row><entry /><entry>H<sub>2</sub>O</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>Hybridisation buffer:</entry><entry>Washing buffer 3:</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry>5X SSC</entry><entry>0.5X SSC</entry></row><row><entry /><entry>5X Denhardt's</entry><entry>0.1% SDS</entry></row><row><entry /><entry>0.1% SDS</entry><entry /></row><row><entry /><entry>H<sub>2</sub>O</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry /><entry>Washing buffer 4:</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row><row><entry /><entry /><entry>1X SSC</entry></row><row><entry /><entry /><entry>0.1% SDS</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0219Prehybridisation: <ul id="ul0052" list-style="none"><li id="ul0052-0001" num="0000"><ul id="ul0053" list-style="none"><li id="ul0053-0001" num="0220">Aliquot 5 ml of prehybridisation buffer in the hybridisation tubes, add the corresponding volume of salmon-sperm DNA for a final concentration of 100 μg/ml, soak the membranes in 5×SSC, place the membrane in the hybridisation tube and prehybridise for 2 hours at 65° C.</li></ul></li></ul>

0221Hybridisation: <ul id="ul0054" list-style="none"><li id="ul0054-0001" num="0000"><ul id="ul0055" list-style="none"><li id="ul0055-0001" num="0222">Remove the prehybridisation buffer and rinse with 10-20 ml of 5×SSC, remove the 5×SSC, replace with 5 ml of buffer+salmon-sperm DNA, denature the RT probes for 5 min at 95° C., then place on ice for 1 minute, centrifuge to reconstitute, then recover the appropriate volume of denatured RT probes in the tube (100,000 to 200,000 cpm/ml), incubate overnight at 55° C.</li></ul></li></ul>

0223Washing: <ul id="ul0056" list-style="none"><li id="ul0056-0001" num="0000"><ul id="ul0057" list-style="none"><li id="ul0057-0001" num="0224">Rinse the membranes with 10-20 ml of washing buffer 1, remove the buffer and replace it with 50 ml of washing buffer 2, incubate for 30 min at 55° C., then remove and replace while washing with buffer 4, incubate for 30 mn at 55° C., then pour off the final washing buffer, remove the membranes from the tubes, place them on a cassette and allow acquisition to continue for 3 hours. <br /> 4.1.4.5 Acquisition and Analysis of the Image </li></ul></li></ul>

0225The membranes are placed on a screen (FX Imaging ScreenK-Bio-rad) for 3 hours. The film is then read using a Personal Molecular Imager FX (Bio-rad). The image is analysed using the Safe-Hit Reader Software (COSE).

00004.1.4.6 Calculation of the Toxicity Index

0226All the data are transferred to an automatic calculation programme that normalises the various membranes and calculates a Toxicity Index, equal to the sum of the number of up- and down-regulated genes for a given compound at a given concentration, in comparison with the results of the untreated controls. The results of the two Safe-Hit analyses are then compared and combined to assess the potential toxicity of the various compounds tested. Two parameters that can be modified by the user are involved in the calculation of the Toxicity Index: <ul id="ul0058" list-style="none"><li id="ul0058-0001" num="0000"><ul id="ul0059" list-style="none"><li id="ul0059-0001" num="0227">Background Threshold (BT) smoothes out weak signals, close to background noise and not attributable to significant gene expression. This therefore determines the threshold of detection;</li><li id="ul0059-0002" num="0228">Induction Factor (IF) is determined as the multiplication factor, versus the control samples, for the clones to be up- or down-regulated. The value of this parameter is usually 2 or less than 2 in order to obtain relevant results. Progressively increasing the IF value selects those clones that are more and more strongly up- or down-regulated.</li></ul></li></ul>

0229The procedure for calculating the Toxicity Index was developed by comparing the reference profiles (R: untreated cells) with an experimental profile (E) and goes through the following steps (see <figref idref="DRAWINGS">FIG. 4</figref> for a schematic overview of the procedure): <ul id="ul0060" list-style="none"><li id="ul0060-0001" num="0000"><ul id="ul0061" list-style="none"><li id="ul0061-0001" num="0230">transformation of all the values obtained into log values,</li><li id="ul0061-0002" num="0231">calculation of the mean log value for each of the duplicate assays (M<sub>iR </sub>et M<sub>iE</sub>),</li><li id="ul0061-0003" num="0232">creation of a matrix with M<sub>iR</sub>-M<sub>iE </sub>for all the signals (=D<sub>i</sub>),</li><li id="ul0061-0004" num="0233">normalisation of the individual M<sub>iE </sub>values by subtracting from M<sub>iE </sub>the median of the 14 proximal values of Di (=NM<sub>iE</sub>),</li><li id="ul0061-0005" num="0234">comparison of the normalised values with the reference values (C<sub>i</sub>=NM<sub>iE</sub>−M<sub>IR</sub>),</li><li id="ul0061-0006" num="0235">exponential transformation of C<sub>i</sub>(=F<sub>i</sub>),</li><li id="ul0061-0007" num="0236">comparison of F<sub>i </sub>with the Induction Factor chosen by the user: <ul id="ul0062" list-style="none"><li id="ul0062-0001" num="0237">if F<sub>i</sub>>IF, the gene is considered to be up-regulated,</li><li id="ul0062-0002" num="0238">if 1/IF<F<sub>i</sub><IF, the gene is considered to be without change,</li><li id="ul0062-0003" num="0239">if F<sub>i</sub><1/IF, the gene is considered to be down-regulated. <br /> 4.2. Results of the MTT-Assay </li></ul></li></ul></li></ul>

0240These assays were performed in triplicate on primary rat hepatocytes exposed for 16 hours.

0241Clomipramine, referred to as C218, showed marked toxicity at 100 μM since no cell viability was observed after exposure of the cells for 16 hours. Conversely, no toxicity was observed at 25 μM. At 50 μM, cell viability greater than 80% is entirely compatible with a genomic pharmacotoxicology study. The F2695 and F2696 compounds show no cytotoxicity in this assay, even at a concentration of 100 μM.

0242To perform the genomic pharmacotoxicological assessments, 3 concentrations of the same compound are used: the concentration which allows for 80% cell viability (C) to be obtained, as well as concentrations corresponding to (C)×10 and to (C)/10.

0243In order to compare the capacity of F2695 and F2696 to yield a score in the assay performed, the same concentrations were used in each test: 1 μM, 10 μM and 100 μM. Concentrations of 1 μM, 10 μM and 50 μM were used for clomipramine. See <figref idref="DRAWINGS">FIGS. 5A</figref>, <b>5</b>B and <b>5</b>C.

00004.3 Results on Primary Rat Hepatocytes

0244Toxicity Indices (TI) were determined as described above. Only those clones which were found to be altered in relation to the control were taken into account in these indices, taking into consideration only those clones whose signal was two times higher than the background threshold (BT). Two separate analyses were performed using two levels of differentiation (Induction Factor-IF) in relation to the untreated controls: <ul id="ul0063" list-style="none"><li id="ul0063-0001" num="0000"><ul id="ul0064" list-style="none"><li id="ul0064-0001" num="0245">a factor of at least 1.7 in relation to the untreated controls. This factor of 1.7 times represents the weakest value that allows an index not to be obtained in relation to the two untreated controls.</li><li id="ul0064-0002" num="0246">a factor of at least 2 in relation to the untreated controls. This factor of 2 times allows the most robust signals to be taken into account. <br /> 4.3.1. Induction Factor of 1.7 in Relation to Untreated Control (Table 7) </li></ul></li></ul>

0247<tables id="TABLE-US-00014" num="00014"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="441pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 7</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Up- and down-regulated clones with primary rat hepatocytes (Induction Factor = 1.7 times)</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="13"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="28pt" align="center" /><colspec colname="3" colwidth="28pt" align="center" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="28pt" align="center" /><colspec colname="7" colwidth="28pt" align="center" /><colspec colname="8" colwidth="28pt" align="center" /><colspec colname="9" colwidth="28pt" align="center" /><colspec colname="10" colwidth="28pt" align="center" /><colspec colname="11" colwidth="28pt" align="center" /><colspec colname="12" colwidth="28pt" align="center" /><colspec colname="13" colwidth="105pt" align="left" /><tbody valign="top"><row><entry /><entry /><entry /><entry>F2695-</entry><entry>F2695- </entry><entry>F2695-</entry><entry>F2696-</entry><entry>F2696-</entry><entry>F2696-</entry><entry>C 218-</entry><entry>C 218-</entry><entry>C 218-</entry><entry /></row><row><entry /><entry /><entry /><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>Up</entry><entry>>1.7</entry><entry>Up</entry><entry /><entry /><entry>1</entry><entry /><entry /><entry>15</entry><entry>2</entry><entry>2</entry><entry>13</entry><entry /></row><row><entry>Down</entry><entry><0.588</entry><entry>Down</entry><entry /><entry /><entry>1</entry><entry>2</entry><entry>5</entry><entry>7</entry><entry>7</entry><entry>13</entry><entry>15</entry><entry /></row><row><entry /><entry /><entry>TI</entry><entry /><entry /><entry>2</entry><entry>2</entry><entry>5</entry><entry>22</entry><entry>9</entry><entry>15</entry><entry>28</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>Pos</entry><entry>nb U</entry><entry>nb D</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Gene</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>A09</entry><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.90</entry><entry>2.23</entry><entry>2.14</entry><entry>H. sapiens mitochondrion, 12S</entry></row><row><entry>A20</entry><entry /><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry>0.56</entry><entry /><entry /><entry /><entry>H. sapiens initiation factor</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>elF-5A gene</entry></row><row><entry>B20</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.14</entry><entry>0.27</entry><entry>H. sapiens chromosome 19,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>BAC CIT-B-191n6</entry></row><row><entry>B22</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.17</entry><entry>0.32</entry><entry>H. sapiens Genomic sequence </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>from 17</entry></row><row><entry>C01</entry><entry>4</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>3.20</entry><entry>1.93</entry><entry>1.82</entry><entry>1.91</entry><entry>H. sapiens mitochondrion, 16S</entry></row><row><entry>E01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.73</entry><entry>H. sapiens mRNA for lipocortin II</entry></row><row><entry>E05</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.22</entry><entry>0.35</entry><entry>H. sapiens DNA sequence from</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>clone 740A11 on chromosome</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Xq22.2-23.</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Contains part of the COL4A5 </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>gene for Collagen Alpha 5 (IV) </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Chain Precursor. Contains GSS1, </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>complete sequence </entry></row><row><entry>E11</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.12</entry><entry /><entry /><entry /><entry>H. sapiens chlordecone reductase</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>homolog liver, mRNA</entry></row><row><entry>E19</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.72</entry><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>E21</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.56</entry><entry>0.58</entry><entry>H. sapiens ribosomal protein S14 </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>gene</entry></row><row><entry>F24</entry><entry /><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.52</entry><entry>H. sapiens LIM homeobox protein</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cofactor (CLIM-1) mRNA</entry></row><row><entry>G01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.04</entry><entry>H. sapiens estrogen receptor-</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>related protein (variant ER from </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>breast cancer) mRNA</entry></row><row><entry>G05</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.02</entry><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>G09</entry><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.09</entry><entry /><entry /><entry>1.76</entry><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome b</entry></row><row><entry>I01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.05</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>I18</entry><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.38</entry><entry /><entry /><entry>1.88</entry><entry>H. sapiens 18S rRNA gene</entry></row><row><entry>L0l</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.05</entry><entry /><entry /><entry /><entry>H. sapiens divalent cation tolerant</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>protein CUTA mRNA</entry></row><row><entry>L22</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.78</entry><entry /><entry /><entry /><entry>H. sapiens mRNA for Lon</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>protease-like protein</entry></row><row><entry>L23</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.75</entry><entry>H. sapiens cDNA NIH_MGC_16 </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>clone IMAGE: 3350241 5′,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>mRNA sequence</entry></row><row><entry>M07</entry><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.25</entry><entry /><entry /><entry>1.75</entry><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>M12</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry>0.21</entry><entry /><entry>0.16</entry><entry>0.39</entry><entry>H. sapiens</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>mRNA; cDNA DKFZp564C1563</entry></row><row><entry>M23</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.95</entry><entry /><entry /><entry /><entry>Sequence 21 from U.S. Pat. No. </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>5,851,764</entry></row><row><entry>P05</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.78</entry><entry /><entry /><entry /><entry>H. sapiens PAC clone</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>DJ404K21 from Xq23</entry></row><row><entry>Q11</entry><entry>2</entry><entry /><entry /><entry /><entry>1.81</entry><entry /><entry /><entry /><entry /><entry /><entry>1.92</entry><entry>unk</entry></row><row><entry>Q24</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.77</entry><entry /><entry /><entry /><entry>H. sapiens 28S ribosomal</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>RNA gene</entry></row><row><entry>S01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.98</entry><entry>Mus muculus TCR beta locus</entry></row><row><entry>T08</entry><entry /><entry>6</entry><entry /><entry /><entry /><entry>0.50</entry><entry>0.22</entry><entry>0.20</entry><entry>0.35</entry><entry>0.14</entry><entry>0.22</entry><entry>H. sapiens mRNA</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>for KIAA1185 protein</entry></row><row><entry>U04</entry><entry /><entry>6</entry><entry /><entry /><entry /><entry>0.57</entry><entry>0.26</entry><entry>0.19</entry><entry>0.48</entry><entry>0.22</entry><entry>0.37</entry><entry>H. sapiens translation initiation</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>factor elF-2alpha mRNA</entry></row><row><entry>V22</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>H. sapiens mRNA for elongation</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>factor 1-alpha (clone CEF4)</entry></row><row><entry>W17</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.96</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion, hypoxia</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>inducible gene-14</entry></row><row><entry>X02</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.29</entry><entry>0.20</entry><entry>0.36</entry><entry>0.24</entry><entry>0.31</entry><entry>unk</entry></row><row><entry>X05</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.15</entry><entry>0.24</entry><entry>H. sapiens microsomal epoxide</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>hydrolase (EPHX) gene</entry></row><row><entry>X06</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.2</entry><entry>0.16</entry><entry>0.23</entry><entry>0.15</entry><entry>0.23</entry><entry>H. sapiens Genomic sequence</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>from 9q34</entry></row><row><entry>X23</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.92</entry><entry /><entry /><entry /><entry>unk</entry></row><row><entry>Y17</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.65</entry><entry /><entry /><entry /><entry>H. sapiens 28S ribosomal RNA </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>gene</entry></row><row><entry>Z13</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.34</entry><entry>0.29</entry><entry>0.27</entry><entry>unk</entry></row><row><entry>Z20</entry><entry /><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.57</entry><entry>Homo sapiens cDNA wc44h09, x1</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>NCI_CGAP_Pr28 clone</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>IMAGE: 2321537 3′ similar to</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>SW: RB24_Mouse P35290</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>RAS_RELATED PROTEIN</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>RAB-24;, mRNA sequence</entry></row><row><entry>AA11</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.38</entry><entry>0.27</entry><entry>0.31</entry><entry>H. sapiens Repeat sequence AluJb</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>fragment inserted into a cDNA </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>coding for an unknown protein</entry></row><row><entry>AA13</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>1.79</entry><entry>H. sapiens 18S rRNA gene</entry></row><row><entry>AC13</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.22</entry><entry>0.16</entry><entry>0.28</entry><entry>0.16</entry><entry>0.28</entry><entry>H. sapiens 7S RNA L gene</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0248The following Toxicity Indices were obtained:

0249<tables id="TABLE-US-00015" num="00015"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><thead><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /><entry>F2695</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1</entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry>10 </entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry>100</entry><entry>μM</entry><entry>17</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry /><entry>F2696</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1</entry><entry>μM</entry><entry>2</entry></row><row><entry /><entry>10 </entry><entry>μM</entry><entry>5</entry></row><row><entry /><entry>100</entry><entry>μM</entry><entry>22</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry /><entry>C218</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1</entry><entry>μM</entry><entry>9</entry></row><row><entry /><entry>10</entry><entry>μM</entry><entry>15</entry></row><row><entry /><entry>50 </entry><entry>μM</entry><entry>28</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0250The following ranking could thus be established, from the most toxic to the least toxic: C218 (clomipramine) >F2696>>>F2695.

0251Clomipramine, the reference molecule, coded C218 in the present study, showed an increasing number of signatures with relation to the concentrations tested: respectively 9, 15 and 28 signatures at concentrations of 1, 10 and 50 μM (maximal concentration defined in the preliminary cytotoxicity test). As one might logically expect, all the signatures that occurred at low and moderate concentrations are also found at the higher concentrations.

0252At concentrations of 1 and 10 μM, F2695 did not induce any of the 682 potential signals of stress tested in the present study. At the highest concentration, 100 μM, only two signatures were detected, one of which was common to both F2695 and C218, but whose signification was unknown.

0253F2696 showed an increasing number of signatures in relation to the concentrations tested: 2, 5 and 22 signatures respectively at concentrations of 1, 10 and 100 μM. All of the signatures that occurred at the low and medium concentrations were detected at the higher concentrations. None of the 22 signatures was shared with F2695. Conversely, all 5 of the signatures that appeared at the low and medium concentration (5 including the 2 which were present at the low concentration) were among the 9 signatures detected with clomipramine starting with the low dose, 1 μM. At the high concentration, 100 μM, 10 of the 26 signatures of F2696 were detected among the 28 signatures identified with clomipramine at 50 μM.

0254From a qualitative standpoint, the impact of F2696 and of clomipramine on mitochondrial transcripts, in particular on Cox1 and on cytochrome b, should be stressed. These signatures are not present with F2695 (G05/G09/I01 positions).

00004.3.2. Induction Factor of 2 in Relation to Untreated Control (Table 8)

0255<tables id="TABLE-US-00016" num="00016"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="441pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE 8</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Up- and down-regulated clones with primary rat hepatocytes (Induction Factor = 2 times)</entry></row><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="13"><colspec colname="1" colwidth="28pt" align="left" /><colspec colname="2" colwidth="28pt" align="left" /><colspec colname="3" colwidth="28pt" align="left" /><colspec colname="4" colwidth="28pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="28pt" align="center" /><colspec colname="7" colwidth="28pt" align="center" /><colspec colname="8" colwidth="28pt" align="center" /><colspec colname="9" colwidth="28pt" align="center" /><colspec colname="10" colwidth="28pt" align="center" /><colspec colname="11" colwidth="28pt" align="center" /><colspec colname="12" colwidth="28pt" align="center" /><colspec colname="13" colwidth="105pt" align="left" /><tbody valign="top"><row><entry /><entry /><entry /><entry>F2695-</entry><entry>F2695-</entry><entry>F2695-</entry><entry>F2696-</entry><entry>F2696-</entry><entry>F2696-</entry><entry>C 218-</entry><entry>C 218-</entry><entry>C 218-</entry><entry /></row><row><entry /><entry /><entry /><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry><entry>1 μM</entry><entry>10 μM</entry><entry>100 μM</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>Up</entry><entry>>1.7</entry><entry>Up</entry><entry /><entry /><entry /><entry /><entry /><entry>10</entry><entry>1</entry><entry>1</entry><entry>4</entry><entry /></row><row><entry>Down</entry><entry><0.588</entry><entry>Down</entry><entry /><entry /><entry /><entry /><entry>5</entry><entry>6</entry><entry>7</entry><entry>12</entry><entry>12</entry><entry /></row><row><entry /><entry /><entry>TI</entry><entry /><entry /><entry /><entry /><entry>5</entry><entry>16</entry><entry>8</entry><entry>13</entry><entry>16</entry><entry /></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>Pos</entry><entry>nb U</entry><entry>nb D</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Gene</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row><row><entry>A09</entry><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.90</entry><entry>2.23</entry><entry>2.14</entry><entry>H. sapiens mitochondrion, 12S</entry></row><row><entry>B20</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.14</entry><entry>0.27</entry><entry>H. sapiens chromosome 19,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>BAC CIT-B-191n6</entry></row><row><entry>B22</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.17</entry><entry>0.32</entry><entry>H. sapiens Genomic sequence </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>from 17</entry></row><row><entry>C01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>3.20</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion, 16S</entry></row><row><entry>E05</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.22</entry><entry>0.35</entry><entry>H. sapiens DNA sequence from</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>clone 740A11 on chromosome</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Xq22.2-23. Contains part of the</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>COL4A5 gene for Collagen Alpha</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>5 (IV) Chain Precursor.</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>Contains GSS1, complete sequence</entry></row><row><entry>E11</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.12</entry><entry /><entry /><entry /><entry>H. sapiens chlordecone reductase</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>homolog liver, mRNA</entry></row><row><entry>G01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.04</entry><entry>H. sapiens estrogen receptor-</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>related protein (variant ER from </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>breast cancer) mRNA</entry></row><row><entry>G05</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.02</entry><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>G09</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.09</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome b</entry></row><row><entry>I01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.05</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>I18</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.38</entry><entry /><entry /><entry /><entry>H. sapiens 18S rRNA gene</entry></row><row><entry>J03</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.12</entry><entry /><entry /><entry /><entry>H. sapiens CLP mRNA</entry></row><row><entry>L01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.05</entry><entry /><entry /><entry /><entry>H. sapiens divalent cation</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>tolerant protein CUTA mRNA</entry></row><row><entry>M07</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.25</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion,</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cytochrome c oxidase subunit 1</entry></row><row><entry>M12</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry>0.21</entry><entry /><entry>0.16</entry><entry>0.39</entry><entry>H. sapiens mRNA;</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cDNA DKFZp564C1563</entry></row><row><entry>S01</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.98</entry><entry>Mus muculus TCR beta locus</entry></row><row><entry>T08</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.22</entry><entry>0.20</entry><entry>0.35</entry><entry>0.14</entry><entry>0.22</entry><entry>H. sapiens mRNA for</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>KIAA1185 protein</entry></row><row><entry>U04</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.26</entry><entry>0.19</entry><entry>0.48</entry><entry>0.22</entry><entry>0.37</entry><entry>H. sapiens translation initiation</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>factor elF-2alpha mRNA</entry></row><row><entry>W17</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.96</entry><entry /><entry /><entry /><entry>H. sapiens mitochondrion, hypoxia</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>inducible gene-14</entry></row><row><entry>X02</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.29</entry><entry>0.20</entry><entry>0.36</entry><entry>0.24</entry><entry>0.31 </entry><entry>unk</entry></row><row><entry>X05</entry><entry /><entry>2</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.15</entry><entry>0.24</entry><entry>H. sapiens microsomal epoxide</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>hydrolase (EPHX) gene</entry></row><row><entry>X06</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.20</entry><entry>0.16</entry><entry>0.23</entry><entry>0.15</entry><entry>0.23</entry><entry>H. sapiens Genomic</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>sequence from 9q34</entry></row><row><entry>Y17</entry><entry>1</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>2.65</entry><entry /><entry /><entry /><entry>H. sapiens 28S ribosomal RNA </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>gene</entry></row><row><entry>Z13</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.34</entry><entry>0.29</entry><entry>0.27 </entry><entry>unk</entry></row><row><entry>AA11</entry><entry /><entry>3</entry><entry /><entry /><entry /><entry /><entry /><entry /><entry>0.38</entry><entry>0.27</entry><entry>0.31</entry><entry>H. sapiens Repeat sequence</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>AluJb fragment inserted into a</entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>cDNA coding for an unknown </entry></row><row><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry /><entry>protein</entry></row><row><entry>AC13</entry><entry /><entry>5</entry><entry /><entry /><entry /><entry /><entry>0.22</entry><entry>0.16</entry><entry>0.28</entry><entry>0.16</entry><entry>0.28</entry><entry>H. sapiens 7S RNA gene</entry></row><row><entry namest="1" nameend="13" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0256The following Toxicity Indices were obtained:

0257<tables id="TABLE-US-00017" num="00017"><table frame="none" colsep="0" rowsep="0"><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><thead><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></thead><tbody valign="top"><row><entry /><entry>F2695</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1</entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry>10 </entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry>100</entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry /><entry>C218</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1 </entry><entry>μM</entry><entry>8</entry></row><row><entry /><entry>10</entry><entry>μM</entry><entry>13</entry></row><row><entry /><entry>50 </entry><entry>μM</entry><entry>16</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="3"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="28pt" align="center" /><colspec colname="2" colwidth="147pt" align="center" /><tbody valign="top"><row><entry /><entry>F2696</entry><entry>Toxicity Index</entry></row><row><entry /><entry namest="offset" nameend="2" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="4"><colspec colname="offset" colwidth="42pt" align="left" /><colspec colname="1" colwidth="14pt" align="right" /><colspec colname="2" colwidth="14pt" align="left" /><colspec colname="3" colwidth="147pt" align="char" char="." /><tbody valign="top"><row><entry /><entry>1</entry><entry>μM</entry><entry>0</entry></row><row><entry /><entry>10 </entry><entry>μM</entry><entry>5</entry></row><row><entry /><entry>100</entry><entry>μM</entry><entry>16</entry></row><row><entry /><entry namest="offset" nameend="3" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0258According to theses parameters, the following ranking could be put forward, from the most toxic to the least toxic: C218 (clomipramine)>F2696>>>>>F2695.

0259With regard to over- and under-expressed clones at a Factor of 2, F2695 did not induce any signatures, even at a concentration of 100 μM.

0260The concentration effect on the occurrence of signatures was confirmed by the fact that the weak signatures with F2696 at 1 μM, which were present in the preceding analysis with the Factor of 1.7, disappear.

0261From a qualitative standpoint, the impact of F2696 and of clomipramine on Cox1 and on cytochrome b was also confirmed (G05/G09/I01 positions).

0262F2695, the pharmacologically-active enantiomer of F2207, was without significant impact in this test, whereas clomipramine is used as positive-control reference product.

0263Conversely, F2696, the pharmacologically-inactive enantiomer of F2207, showed a profile of signatures that is quantitatively and qualitatively close to that of clomipramine, and shows no signatures in common with F2695.

0264All of this is evidence of a superior toxico-genomic profile for the active F2695 enantiomer which, in this experimental model, had a very significantly better safety coefficient that that of F2696.

00004.4 Conclusion

0265The genomic pharmacotoxicology studies performed on the F2695 and F2696 molecules, enantiomers of F2207 (at concentrations of 10, 50 and 100 μM), and on C218 (clomipramine, at concentrations of 1, 10 and 50 μM), using rat hepatocytes in primary culture, yielded concentration-dependent stress signatures and Toxicity Indices. These studies confirm the capacity of the genomic pharmacotoxicology test to reveal stress signatures under treatment conditions (concentrations, duration of treatment) that do not cause any toxicity in a classic cell-viability assay such as MTT-assay.

0266This study brings to light several important facts: <ul id="ul0065" list-style="none"><li id="ul0065-0001" num="0000"><ul id="ul0066" list-style="none"><li id="ul0066-0001" num="0267">in the primary rat hepatocyte model, only F2695, the pharmacologically-active enantiomer of F2207, did not induce a significant Toxicity Index;</li><li id="ul0066-0002" num="0268">F2696, the pharmacologically-inactive enantiomer of F2207, and clomipramine, the reference psychotropic product, induced marked Indices involving very similar or common stress signatures. In this system, clomipramine, the positive-control reference product, induced the highest number of stress signatures, significant indices having been observed starting at the lowest concentrations. On this subject, it is interesting to note that clomipramine can induce a certain number of adverse events in man, such as tachycardia, orthostatic hypotension, cardiac conduction or rhythm disturbances, and exceptionally hepatitis. In case of accidental overdosage with clomipramine, syncope, haematological disturbances and severe cardiovascular manifestations can be observed.</li></ul></li></ul>

0269Without inferring a common physiopathological mechanism, it is interesting to note that F2696 showed very similar or common stress signatures to those of clomipramine and also induces adverse events such as the cardiovascular disturbances previously described.

0270Thus, it is legitimate to suggest that the signatures observed are independent of any antidepressant, or more broadly psychotropic, profile. On the contrary, the signatures should indeed be considered to be “signatures of stress” (F2696 causes in particular reduced expression of a gene involved in protein synthesis and of a translation initiation factor). All of this is evidence of a superior toxico-genomic profile for the active F2695 enantiomer which, in this experimental model, had a very significantly better safety coefficient that that of F2696.

Contents2

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Citations
Document	Relation	Office	Cited during
WO0126623A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO0126623A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO0162236A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO0162236A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO03068211A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO03068211A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
US2002010216A1	Cites	United States of America	Search report
US2003130353A1	Cites	United States of America	Search report
US2003139476A1	Cites	United States of America	Search report
US2003203055A1	Cites	United States of America	Search report
US2003232805A1	Cites	United States of America	Search report
US2004019116A1	Cites	United States of America	Applicant
WO2004030633A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO2004030633A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
US2004034101A1	Cites	United States of America	Search report
US2004122104A1	Cites	United States of America	Search report
US2005032782A1	Cites	United States of America	Search report
US2005096395A1	Cites	United States of America	Search report
FR2759290A1	Cites	France	Applicant
FR2759906A1	Cites	France	Applicant
FR2759906A1	Cites	France	Applicant
US4478836A	Cites	United States of America	Applicant
US5532244A	Cites	United States of America	Search report
US6028070A	Cites	United States of America	Search report
US6184222B1	Cites	United States of America	Search report
US6602911B2	Cites	United States of America	Applicant
US6635675B2	Cites	United States of America	Search report
US6699506B1	Cites	United States of America	Applicant
WO9735574A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO9836744A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO9836744A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
Bonnaud B et al., "1-Aryl-2-(aminomethyl)cyclopropanecarboxylic acid derivatives. A new series of potential antidepressants" Journal of Medicinal Chemistry, American Chemical society. Washington, US, vol. 30, No. 2, 1987, pp. 318-325, XP002282456 ISSN: 0022-2623.	Non-patent	–	Applicant
Deprez et al., "Which bioequivalence study for a racemic drug? Application to milnacipran." European Journal of Drug Metabolism and Pharmacokinetics, vol. 23, No. 2, 1998, pp. 166-171, XP008021090 ISSN: 0378-7966.	Non-patent	–	Applicant
Spencer Caroline M et al., > drugs, vol. 56, No. 3, 1998, pp. 405-427, XP008021105 ISSN: 0012-6667.	Non-patent	–	Applicant
Puozzo C et al., "Pharmacokinetics of milnacipran in renal impairment." European Journal of Drug Metabolism and Pharmacokinetics. Switzerland, 1998 Apr.-Jun. vol. 23, No. 2, pp. 280-286, XP008021091 ISSN: 0398-7639.	Non-patent	–	Applicant
Viazzo at al., "Microbiological Transformations 34: Enantioselective hydrolysis of a key-lactone involved in the synthesis of the antidepressant Milnacipran?" Tetrahedron Letters, Elsevier Science Publisher, Amsterdam, NL, vol. 37, No. 26, Jun. 24, 1996, pp. 4519-4522, XP004029055 ISSN: 0040-4039.	Non-patent	–	Applicant
Shuto Satoshi et al., "1-pheny1-2-((S)-1-aminopropyl)-N,N-diethylcyclopropane-carboxamide (PPDC), a new class of NMDA-receptor antagonist: Molecular design by a novel conformational restriction strategy." Japanese Journal of Pharmacology, vol. 85, No. 3, Mar. 2001, pp. 207-213, XP008021088 ISSN: 0021-5198.	Non-patent	–	Applicant
Shuto Satoshi et al., "Synthesis and biological activity of conformationally restricted analogues of milnacipran: (1S, 2R)-1-phenyl-2-((R)-1-amino-2-propynyl)-N,N-diethylcyclopropane-carboxamide is a novel class of NMDA receptor channel blocker." Journal of Medicinal Chemistry, vol. 41, No. 18, Aug. 27, 1998, pp. 3507-3514, XP002252359 ISSN: 0022-2623.	Non-patent	–	Applicant
Shuto Satoshi et al., "Synthesis and biological activity of conformationally restricted analogs of milnacipran: (1S, 2R)-1-phenyl-2-((S)-1-aminopropyl)-N,N-diethylcyclopropane-carboxamide an efficient noncompetitive N-methyl-D-aspartic acid receptor antagonist" Journal of Medicinal Chemistry, vol. 39, No. 24, Nov. 22, 1996, pp. 4844-4852, XP000857417 ISSN: 0022-2623.	Non-patent	–	Applicant
Hindmarch I: "The enantiomer debate: current status and future directions" Human Psychopharmacology 2001 United Kingdom, vol. 16, No. Suppl.2, 2001, pp. S101-S104, XP008021086, ISSN: 0885-6222.	Non-patent	–	Applicant
Baldwin D. S. : "Unmet needs in the pharmacological management of depression" Human Psychopharmacology 2001 United Kingdom, vol. 16, No. Suppl.2, 2001, pp. S93-S99, XP008021087, ISSN: 0885-6222.	Non-patent	–	Applicant
W. Retz, et al.; European Neuropsychopharmacology, vol. 5-No. 3 (1995) pp. 296-297.	Non-patent	–	Applicant
Caron, et al., Eur. Neuropsychopharmacol, "Acute electrophysiological effects of intravenous milnacipran, a new antidepressant agent", 1993, 3, 493-500.	Non-patent	–	Applicant
Mills, Crit. Care Clin. "Serotonin Syndrome, A Clinical Update", 1997, 13, 763-783.	Non-patent	–	Applicant
Palazidou, et al., "Rapid Reference to Depression", Jul. 2002, 42-59.	Non-patent	–	Applicant
Index Merck No. 6281 (1996).	Non-patent	–	Applicant
Moret et al., 1985 Neuropharmacology 24(12): 1211-1219.	Non-patent	–	Applicant
Bonnaud et al., 1985, Journal of Chromatography, vol. 318: 398-403.	Non-patent	–	Applicant
Shuto et al., Tetrahedron letters, 1996 vol. 37: 641-644.	Non-patent	–	Applicant
Grard et al.,2000, Electrophorasis 2000 21: 3028-3034.	Non-patent	–	Applicant
Doyle et al., 2001, Advanced Synthesis and Catalysis, vol. 343, 299-302.	Non-patent	–	Applicant
Nores et al., 1987 Therapie 42: 555-558.	Non-patent	–	Applicant
Meador-Woodruff et al., 1988 J. Clim. Psychopharmacol. 8: 28-32.	Non-patent	–	Applicant
Dictionnaire Vidal, 78 eme Edition, 2002.	Non-patent	–	Applicant
The Diagnostic and Statistical Manual of Mental Disorders-IV(DSM-IV), 1995 A.P.A.	Non-patent	–	Applicant
Shuto et al., Journal of Med Chem, American Chem. Society, 1996, vo. 39: 4844-4852.	Non-patent	–	Applicant
Artigas, "Selective Serotonin/Noradrenaline Reuptake Inhibitors", CNS Drugs, 1995, 4, 79-89.	Non-patent	–	Applicant
Preskorn, et al., "Other Antidepressants", Antidepressants: Past, Present and Future, 2004, 264-311.	Non-patent	–	Applicant
Preskorn, "Milnacipran: A Dual Norepinephrine and Serotonin Reuptake Pump Inhibitor",Journal of Psychiatric Practice, 2004, 10, 119-126.	Non-patent	–	Applicant
Yoshida, et al., "Elevation of blood pressure induced by high-dose milnacipran", Hum. Psychopharmacol. Clin. Exp., 2002, 17, 431.	Non-patent	–	Applicant
Schorderet, "Effets sur le syst�me cardiovasculaire", Pharmacologie Des Concepts Fondamentaux Aux Applications Therapeutiques, 1992, Chapter 25, pp. 363-364.	Non-patent	–	Applicant
Ener, et al., "Serotonin Syndrome and Other Serotonergic Disorders", Pain Medicine, 2003, 4, 63-74.	Non-patent	–	Applicant
Kolecki, "Isolated Venlafaxine-Induced Serotonin Syndrome",J. Emerg. Med., 1997, 15, 491-493.	Non-patent	–	Applicant
Hansen, et al., "Long-term antidepressive medication-an increased anesthetic risk?", Der Anaesthesist, 1990, 39, 205-210 *Surgical Medline Extract).	Non-patent	–	Applicant
Thase, "Effects of Venlafaxine on Blood Pressure: A Meta-Analysis of Original Data from 3744 Patients", J. Clin. Psychiatry, 1998, 59, 502-508.	Non-patent	–	Applicant
Partridge, et al., "A Depressed Myocardium", Clinical Toxicology, 2000, 38, 453-455.	Non-patent	–	Applicant
Jordan, et al., "Influence of sibutramine on blood pressure: evidence from placebo-controlled trials", Int. J. Obes. Relat. Metab. Disord., 2005, 29, 509-516 (Medline Extract).	Non-patent	–	Applicant
Birkenfeld, et al., "Paradoxical effect of sibutramine of autonomic cardiovascular regulation", Circulation, 2002, 106, 2459-2465 (Medline Extract).	Non-patent	–	Applicant
Sramek, et al., "Efficacy and safety of sibutramine for weight loss in obese patients with hypertension well controlled by beta-adrenergic blocking agents: a placebo-controlled, double-blind, radomiser trial", J. Hum. Hypertens., 2002, 16, 13-19 (Medline Extract).	Non-patent	–	Applicant
Szabadi, et al., "The human pharmacology of reboxetine", Hum. Psychopharmacol., 1998, Suppl. 1, S3-S12 (Excerpta Medica Extract).	Non-patent	–	Applicant
Middleton, et al., "Evidence that imipramine-induced postural hypotension may be centrally mediated", Hum. Psychopharmacol., 1998, 3, 181-190 (Excerpta Medica Extract).	Non-patent	–	Applicant
Robinson, "Antidepressant Psychopharmacology: Current Limitations and Future Directions", Primary Psychiatry, 2003, 10, 43-49.	Non-patent	–	Applicant

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2015-11-03	Letter from FDA or Dept of Agriculture re PTE applicationAGYL	AGYL
2014-10-22	Initial letter Re: PTE Application to regulating agencyPTELT1	PTELT1
2013-09-19	Patent Term Extension Application under 35 USC 156 FiledPTER	PTER
2012-12-25	Recordation of Patent Grant MailedPGM/	PGM/
2012-12-05	Issue Notification MailedAllowedWPIR	WPIR
2012-11-20	Dispatch to FDCD1935	D1935
2012-11-12	Application Is Considered Ready for IssuePILS	PILS
2012-11-02	Issue Fee Payment VerifiedN084	N084
2012-11-02	Issue Fee Payment ReceivedIFEE	IFEE
2012-10-12	Printer Rush- No mailingTCPB	TCPB
2012-10-12	Printer Rush- No mailingTCPB	TCPB
2012-10-11	Pubs Case Remand to TCPUBTC	PUBTC
2012-10-04	Mail Notice of AllowanceAllowedMN/=.	MN/=.
2012-09-21	Notice of Allowance Data Verification CompletedAllowedN/=.	N/=.
2012-09-20	Examiner's Amendment CommunicationEX.A	EX.A
2012-08-31	Date Forwarded to ExaminerFWDX	FWDX
2012-08-29	Oath or Declaration Filed (Including Supplemental)C602	C602
2012-08-29	Response after Non-Final ActionA...	A...
2012-08-28	Mail Interview Summary - Applicant Initiated - ConferenceMEXAC	MEXAC
2012-08-22	Interview Summary- Applicant InitiatedEXIA	EXIA
2012-08-22	Interview Summary - Applicant Initiated - ConferenceEXAC	EXAC
2012-08-02	Mail Non-Final RejectionNon-final rejectionMCTNF	MCTNF
2012-07-17	Non-Final RejectionNon-final rejectionCTNF	CTNF
2012-06-07	Case Docketed to Examiner in GAUDOCK	DOCK
2012-04-03	Notice of Reissue Published in Official GazetteNRE.	NRE.
2012-03-08	Application Is Now CompleteCOMP	COMP
2012-03-08	Application Dispatched from OIPEOIPE	OIPE
2012-03-08	Filing ReceiptFLRCPT.O	FLRCPT.O
2012-03-01	Cleared by OIPE CSRL194	L194
2012-02-27	Information Disclosure Statement consideredIDSC	IDSC
2012-02-27	Information Disclosure Statement (IDS) FiledM844	M844
2012-02-27	Preliminary AmendmentA.PE	A.PE
2012-02-27	Information Disclosure Statement (IDS) FiledWIDS	WIDS
2012-02-27	IFW Scan & PACR Auto Security ReviewSCAN	SCAN
2012-02-27	Initial Exam Team nnIEXX	IEXX

4 legal events, as the office reported them to INPADOC

Over the term

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Events

Legal events, newest first
	Event	Code
2017-07-06	AssignmentAS	AS
2017-06-13	Fee paymentFPAY	FPAY
2013-09-19	Application for a patent term extensionPTEF	PTEF
2013-04-25	Fee paymentFPAY	FPAY

Use of the dextrogyral enantiomer of milnacipran for the preparation of a drug

Claim Score by NHIP

Abstract

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BRIEF DESCRIPTION OF THE DRAWINGS

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Priority claims11

Members63

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