Use of (1S, 2R) enantiomer of milnacipran for the preparation of a medicine
Abstract
La présente invention concerne l'utilisation d'un mélange d'énantiomères enrichi en énantiomère (1S,2R) du Milnacipran et/ou d'au moins un de ses métabolites, ainsi que leurs sels pharmaceutiquement acceptables, pour la préparation d'un médicament destiné à prévenir ou traiter des pathologies pouvant être soignées par l'inhibition double de la recapture de la sérotonine (5-HT) et de la noradrénaline (NA), tout en limitant les risques de troubles cardiovasculaires et/ou de toxicité organique et/ou tissulaire.
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13 claims: 1 independent, 12 dependent
- 1Use of the (1S, 2R) enantiomer of Milnacipran (Z (±) -2- (amino methyl) -N, N-diethyl-1-phenylcyclopropanecarboxamide), or a pharmaceutically acceptable salt thereof, for the preparation of a drug intended to prevent or treat depression, bipolar syndrome, schizophrenia, generalized anxiety, states of gloom and doldrums, diseases linked to stress, panic attacks, phobia, in particular agoraphobia, disorders obsessive compulsive, conduct disorders, oppositional disorders, post-traumatic stress syndromes, depression of the immune system, fatigue and accompanying pain syndromes, chronic fatigue syndrome, fibromyalgia, and other pathologies of a nature functional somatic, autism, pathologies characterized by lack of attention due to general medical conditions, attention deficit hyperactivity disorder, eating disorders, bulimic neurosis, anorexic neurosis, obesity, psychotic disorders, apathy , migraine, pain, and in particular chronic pain, irritable colon syndrome, cardiovascular diseases, and in particular anxio-depressive syndrome in myocardial infarction or in hypertension, neurodegenerative diseases and associated anxio-depressive syndromes (Alzheimer's disease, Huntington's chorea, Parkinson's disease), urinary incontinence, drug dependence, and in particular anxio-dependence on tobacco, alcohol , narcotics, drugs, analgesics during the withdrawal from these states of dependence, while limiting the risks of cardiovascular disorders linked to the administration of Milnacipran in the form of a racemic mixture.
189 paragraphs in 1 section, as filed
The present invention relates to the use of a mixture of enantiomers enriched in enantiomer of spatial configuration (1S, 2R) of Milnacipran and / or at least one of its metabolites, as well as their pharmaceutically acceptable salts, for the preparation of '' a drug intended to prevent or treat pathologies which can be treated by double inhibition of the reuptake of serotonin (5-HT) and norepinephrine (NA), while limiting the risks of cardiovascular disorders and / or organic and / or tissue toxicity. More particularly, the mixture of enantiomers according to the invention is intended to treat depression, chronic fatigue syndromes and urinary incontinence.
Milnacipran (Z (±) -2- (amino methyl) -N, N-diethyl-1-phenyl cyclopropane carboxamide), molecule synthesized at the PIERRE FABRE MEDICAMENT Research Center (Castres, France), also called TN-912, Dalcipran , Minalcipran, Midalcipran or Midalipran is known as a dual inhibitor of the reuptake of serotonin (5-HT) and norepinephrine (NA). Milnacipran and its preparation process are described in the patent<patcit id="pcit0001" dnum="US4478836A"><text>US No. 4,478,836</text></patcit>. More information on Milnacipran can be found in the twelfth edition of the Merck index, under entry no. 6,281.
Dual inhibitors of serotonin and norepinephrine reuptake correspond to a well-known class of antidepressant agents that selectively inhibit the reuptake of both serotonin and norepinephrine. For example, venlafaxine and duloxetine are also dual inhibitors of serotonin and norepinephrine. Studies have shown that the ratio of inhibition of norepinephrine reuptake to inhibition of serotonin by Milnacipran is approximately 2: 1 (<nplcit id="ncit0001" npl-type="s"><text>Moret et al., 1985 Neuropharmacology 24 (12): 1211-1219</text></nplcit> ; <nplcit id="ncit0002" npl-type="s"><text>Palmier et al., 1989, Eur J Clin Pharmacol 37: 235-238</text></nplcit>).
The patent <patcit id="pcit0002" dnum="US4478836A"><text>US 4,478,836</text></patcit> describes the use of Milnacipran for the treatment of pathologies of the central nervous system, in particular depression. The patent application<patcit id="pcit0003" dnum="WO0126623A"><text>WO01 / 26623</text></patcit> describes the use of Milnacipran in combination with phenylalanine and tyrosine in indications such as the treatment of fatigue, pain-associated syndromes, chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome . The patent application<patcit id="pcit0004" dnum="WO0162236A"><text>WO01 / 62236</text></patcit> describes a composition comprising Milnacipran in combination with one or more anti-muscarinic agents in a large number of indications including depression. Requirement<patcit id="pcit0005" dnum="WO9735574A"><text>WO97 / 35574</text></patcit> describes a pharmaceutical composition containing Milnacipran and idazoxan as a combination product for simultaneous, separate or spread over time to treat depression and its various forms, as well as the pathologies in which antidepressants are used. Milnacipran is also used in an indication for the treatment of urinary incontinence (<patcit id="pcit0006" dnum="FR2759290"><text>FR 2 759 290</text></patcit>).
The Milnacipran molecule has two asymmetric carbons leading to two different spatial configurations (1S, 2R) and (1R, 2S). These spatial configurations not being superimposable, the Milnacipran molecule therefore presents an optical isomerism.
Milnacipran hydrochloride thus exists in the form of two optically active enantiomers: the dextrorotatory enantiomer or alternatively Z- (1S, 2R) -2- (amino methyl) -N, N-diethyl-1-phenyl cyclopropane carboxamide and the levorotatory enantiomer Z- (1R, 2S) -2- (amino methyl) -N, N-diethyl-1-phenyl cyclopropane carboxamide hydrochloride. Milnacipran in its hydrochloride form (also called F2207) is currently marketed (IXEL, PIERRE FABRE MEDICAMENT, France) in the form of a racemic mixture as a serotoninergic-noradrenergic antidepressant drug. F2695 and F2696 respectively denote the (1S, 2R) (dextrorotatory) and (1R, 2S) (levorotatory) enantiomers of Milnacipran hydrochloride (F2207):<chemistry id="chem0001" num="0001"><img file="EP1908461A1_D0001.tif" /></chemistry>
These two enantiomers can be separated and isolated according to methods described in the literature (<nplcit id="ncit0003" npl-type="s"><text>Bonnaud et al., 1985, Journal of Chromatography, Vol. 318: 398-403</text></nplcit> ; <nplcit id="ncit0004" npl-type="s"><text>Shuto et al., Tetrahedron letters, 1996 Vol. 37: 641-644</text></nplcit> ; <nplcit id="ncit0005" npl-type="s"><text>Grard et al., 2000, Electrophoresis 2000 21: 3028-3034</text></nplcit>; <nplcit id="ncit0006" npl-type="s"><text>Doyle and Hu, 2001, Advanced Synthesis and Catalysis, Vol. 343: 299-302</text></nplcit>).
The inventors have now carried out a pharmacokinetic study in humans of the racemate and of the two enantiomers of Milnacipran which implements enantio-selectivity assay methods. They thus demonstrated the absence of racemization of the enantiomers<i>in vivo.</i>
Furthermore, although the racemate has been resolved, no analysis of the pharmacological and toxicological properties of the two enantiomers has been carried out, which uses the modern methods currently available such as cardiovascular measurements by telemetry, or pharmaco-toxico predictive analyzes -Genomics <i>in vitro.</i>
Antidepressants, like any active ingredient, can generate undesirable effects or certain toxicities which arise essentially from the pharmacological properties of these drugs, but also from the dosage, from the individual variability of the patient (genetic polymorphism, organic insufficiency, sex, age) or medicinal interactions . Antidepressants represent the third class of products responsible for intoxication, after hypnotics and tranquilizers (<nplcit id="ncit0007" npl-type="s"><text>Nores et al., 1987 Therapy 42: 555-558</text></nplcit>). The risk of overdose with antidepressants is serious since it can lead to death. Among the causes of acute intoxication by antidepressants, mention should be made of involuntary ingestion by children (especially since certain antidepressants are used for the treatment of enuresis), attempted suicide, involuntary overdose by doctor, associated co-medication in the elderly, age-related physiological and pharmacokinetic changes (heart failure, liver and / or kidney failure, etc.), a slower metabolism of genetic or drug origin (enzyme inhibition). After the children, the elderly therefore constitutes the second population at risk among the treated patients. These people have higher plasma concentrations, linked to reduced renal and / or hepatic clearance, and the risks of poisoning are more serious (<nplcit id="ncit0008" npl-type="s"><text>Meadoer-Woodruff et al., 1988 J. Clim. Psychopharmacol. 8: 28-32</text></nplcit>).
The undesirable side effects, generally mild, observed during treatment with Milnacipran are especially noted during the first or even the first two weeks of treatment and fade away thereafter, alongside the improvement in the depressive episode. The most commonly reported adverse events in monotherapy or when combined with other psychotropic drugs are dizziness, hyper-sweating, anxiety, hot flashes, and dysuria. Some less commonly reported side effects are nausea, vomiting, dry mouth, constipation, tremors, palpitations, restlessness, rashes. It is also known that in patients with a history of cardiovascular disease or simultaneously receiving treatment for the cardiac purpose, the incidence of adverse cardiovascular effects (hypertension, hypotension, orthostatic hypotension, palpitations) may be increased by Milnacipran. In hypertensive patients or patients with heart disease, it is therefore recommended to reinforce clinical monitoring since Milnacipran in the form of a racemic mixture is likely to increase the heart rate. Thus, in rare cases of overdose observed with Milnacipran (at doses of 800 mg to 1 g) in mono-therapy, the main symptoms observed are vomiting, respiratory disorders and tachycardia (<nplcit id="ncit0009" npl-type="b"><text>Vidal Dictionary, 78th edition, 2002</text></nplcit>). Another side effect exceptionally induced by Milnacipran is a high elevation of transaminases which may reflect some hepatic toxicity.
In fact, the populations at risk likely to develop a certain number of adverse clinical manifestations during or following treatment with Milnacipran, are children, the elderly, patients with hepatic and / or renal impairment, patients receiving a treatment inducing organic and / or tissue toxicities, in particular hepatic and / or renal toxicities, patients receiving treatment for cardiac purposes or inducing cardiovascular side effects, patients with a cardiovascular history and / or suffering from cardiovascular disorders, in particular patients with disturbances of cardiac rhythm, blood pressure (hypo- or hypertensive patients) or patients with heart disease.
In an effort to prevent, ever further, the occurrence of possible side effects which may constitute a risk, however small, for the health of patients treated with Milnacipran, the inventors have now surprisingly and unexpectedly discovered that the (1S, 2R) enantiomer of Milnacipran, which exhibits most of the selective inhibition activity of serotonin and noradrenaline re-capture induces fewer cardiovascular side effects and organic and / or tissue toxicity, especially liver toxicity, than the racemic mixture. In particular, the inventors have discovered that administration of the Milnacipran (1S, 2R) enantiomer in dogs causes a lesser increase in heart rate and blood pressure, in particular diastolic blood pressure, than that likely to be caused by the administration of the racemic mixture. The inventors have further discovered that the (1S, 2R) enantiomer of Milnacipran hydrochloride (F2695) has a better toxicogenomic profile than the (1R, 2S) enantiomer of Milnacipran hydrochloride (F2696) primary rat hepatocytes. The inventors have also demonstrated that the (1R, 2S) enantiomer (F2696) has a toxicogenic-genomic profile similar to that obtained with Clomipramine, used as a reference psychotropic drug known for its relative hepatotoxicity.
The subject of the present invention is therefore the use of a mixture of enantiomers of Milnacipran enriched in (1S, 2R) enantiomer, preferably the substantially pure F2695 enantiomer, as well as their pharmaceutically acceptable salts, for the preparation of a drug intended to prevent or treat pathologies or affections which can be treated by the double inhibition of the reuptake of serotonin (5-HT) and norepinephrine (NA), while limiting the risks of cardiovascular disorders and / or while limiting the risks of organic and / or tissue toxicity.
The term “cardiovascular disorders” is understood to mean undesirable cardiovascular side effects of the drug administered as a single therapy or in combination with other active ingredients.
For the purposes of the present invention, the term "side effect" means the foreseeable activity of a medicament in a field other than that for which it is administered, which can be bothersome or undesirable when it limits the use of the medicament .
The term “toxicity” is understood to mean the property of a drug to cause harmful effects at the organic and / or tissue level, in particular at the level of the organs or tissues involved in the metabolism of Milnacipran, in particular the hepatic and / or renal metabolism of Milnacipran, and more particularly during the first passage of Milnacipran in the liver. Preferably, the organic toxicity is the cardiac toxicity and the said tissue toxicity is the hepatic and / or renal toxicity.
In the context of the present invention, the term “while limiting the risks of cardiovascular disorders” or “while limiting the risks of toxicity” means preventing these risks from significantly increasing in a patient following the drug administration.
In the context of the present invention, “(1S, 2R) enantiomer of Milnacipran” denotes the (1S, 2R) enantiomer of Milnacipran, as well as the pharmaceutically acceptable salts thereof. Preferably, it is the (1S, 2R) enantiomer of Milnacipran hydrochloride (F2695). "Milnacipran (1R, 2S) enantiomer" means the (1R, 2S) enantiomer of Milnacipran, as well as the pharmaceutically acceptable salts thereof such as the hydrochloride (F2696). "Racemic mixture" means a 50:50 mixture by weight of (1S, 2R) enantiomer of Milnacipran and (1R, 2S) enantiomer of Milnacipran, as well as the pharmaceutically acceptable salts thereof.
In the context of the present invention, "mixture of enantiomers of Milnacipran enriched in enantiomer (1S, 2R)" means a mixture of enantiomers (1S, 2R) and enantiomer (1R, 2S) of Milnacipran, in which the mass ratio between the (1S, 2R) enantiomer and the (1R, 2S) enantiomer is greater than 1: 1. In the mixture of enantiomers of Milnacipran enriched in enantiomer (1S, 2R), the mass ratio between the enantiomer (1S, 2R) and the enantiomer (1R, 2S) is advantageously greater than or equal to 55:45, more advantageously greater than 60:40, even more advantageously greater than 65:35, even more advantageously greater than 70:30, even more advantageously greater than 75:25, even more advantageously greater than 80:20. In a particularly advantageous embodiment, the mass ratio between the (1S, 2R) enantiomer and the (1R, 2S) enantiomer is greater than 82:18, more advantageously greater than 84:16, even more advantageously greater than 86:14, even more advantageously greater than 88:12, still more advantageously greater than 90:10. In a preferred embodiment, the mass ratio between the (1S, 2R) enantiomer and the (1R, 2S) enantiomer is greater than 91: 9, more preferably greater than 92: 8, even more preferably greater than 93: 7, even more preferably greater than 94: 6, still more preferably greater than 95: 5, still more preferably greater than 96: 4, still more preferably greater than 97: 3, even more preferably greater than 98: 2, even more preferably greater than 99: 1, even more preferably greater than 99.5: 0.5. In a particularly preferred manner, the mixture of enantiomers of Milnacipran enriched in (1S, 2R) enantiomer is substantially pure, that is to say containing approximately 100% by weight of (1S, 2R) enantiomer.
It is also within the scope of the present invention to use the metabolites, preferably the active metabolites <i>in vivo</i> Milnacipran, and their pharmaceutically acceptable salts, such as: ○ Z- (±) phenyl-1-aminomethyl-2-cyclopropane carboxylic acid hydrochloride (F1567):<chemistry id="chem0002" num="0002"><img file="EP1908461A1_D0002.tif" /></chemistry><tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="56mm" /><tbody><row><entry>Molecular weight :</entry><entry>277,7</entry></row><row><entry>Characteristics :</entry><entry>white crystals</entry></row><row><entry>Fusion point :</entry><entry>230 ° C</entry></row><row><entry>Plate chromatography:</entry><entry>support: silica</entry></row><row><entry /><entry>Solvent: Butanol / Ethanol / water (6/2/2)</entry></row><row><entry /><entry>Revelation: Ultra-violet and ninhydrin</entry></row><row><entry /><entry>Rf: 0.6</entry></row></tbody></tgroup></table></tables>○ (±) phenyl-3-methylene-3-4 pyrrolidone-3 (F1612):<chemistry id="chem0003" num="0003"><img file="EP1908461A1_D0003.tif" /></chemistry><tables id="tabl0002" num="0002"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="68mm" /><tbody><row><entry>Molecular weight:</entry><entry>173,2</entry></row><row><entry>Characteristics :</entry><entry>white crystals</entry></row><row><entry>Fusion point :</entry><entry>70 ° C</entry></row><row><entry>Plate chromatography:</entry><entry>support: silica</entry></row><row><entry /><entry>Solvent: Benzene / dioxane / ethanol (90/25/4)</entry></row><row><entry /><entry>Revelation: Ultra-violet and iodine</entry></row><row><entry /><entry>Rf: 0.46</entry></row></tbody></tgroup></table></tables>○ Z (±) - (para-hydroxyphenyl) -1 diethylaminocarbonyl-1 aminomethyl-2 cyclopropane hydrochloride (F2782):<chemistry id="chem0004" num="0004"><img file="EP1908461A1_D0004.tif" /></chemistry><tables id="tabl0003" num="0003"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="65mm" /><tbody><row><entry>Molecular weight :</entry><entry>298,82</entry></row><row><entry>Characteristics :</entry><entry>white crystals</entry></row><row><entry>Fusion point :</entry><entry>250 ° C</entry></row><row><entry>Plate chromatography:</entry><entry>support: silica</entry></row><row><entry /><entry>Solvent: Butanol / Ethanol / water (6/2/2)</entry></row><row><entry /><entry>Revelation: Ultra-violet and iodine - ninhydrin</entry></row><row><entry /><entry>Rf: 0.42</entry></row></tbody></tgroup></table></tables>○ Z (±) -phenyl-1-ethylamino carbonyl-1 aminomethyl-2 cyclopropane acid oxalate (F2800):<chemistry id="chem0005" num="0005"><img file="EP1908461A1_D0005.tif" /></chemistry><tables id="tabl0004" num="0004"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="65mm" /><tbody><row><entry>Molecular weight :</entry><entry>308,33</entry></row><row><entry>Characteristics :</entry><entry>white crystals</entry></row><row><entry>Fusion point:</entry><entry>150 ° C</entry></row><row><entry>Plate chromatography:</entry><entry>support: 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>Revelation: Ultra-violet and ninhydrin</entry></row><row><entry /><entry>Rf: 0.40</entry></row></tbody></tgroup></table></tables>○ Z (±) -phenyl-1 aminocarbonyl-1 aminomethyl-2 cyclopropane hydrochloride (F2941)<chemistry id="chem0006" num="0006"><img file="EP1908461A1_D0006.tif" /></chemistry><tables id="tabl0005" num="0005"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="66mm" /><tbody><row><entry>Molecular weight :</entry><entry>226,74</entry></row><row><entry>Characteristics :</entry><entry>white crystals</entry></row><row><entry>Fusion point :</entry><entry>245 ° C</entry></row><row><entry>Plate chromatography:</entry><entry>support: 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>Revelation: Ultra-violet and ninhydrin</entry></row><row><entry /><entry>Rf: 0.30</entry></row></tbody></tgroup></table></tables>
Like Milnacipran, these metabolites have two asymmetric carbons leading to two different spatial configurations (1S, 2R) and (1R, 2S). As these spatial configurations are not superimposable, these metabolites also exhibit optical isomerism. The ratio of the two enantiomers of the metabolite of Milnacipran in the mixture of enantiomers is as described above for the enantiomers of Milnacipran.
The present invention therefore covers these active metabolites, their enantiomers, as well as their pharmaceutically acceptable salts, as well as their use as a medicament for the treatment of the pathologies described in this patent such as, for example, depression, pain, fibromyalgia and 'urinary incontinence. The metabolites according to the invention are in the form of a racemate or preferably in the form of a mixture of enantiomers enriched in the most active (1S, 2R) enantiomer. Preferably, the active metabolite used comes from the F2695 enantiomer and it is the (1S, 2R) enantiomer of the active metabolite. More preferably, it is the (1S, 2R) enantiomer of Z- (para-hydroxyphenyl) -1 diethylaminocarbonyl-1 aminomethyl-2 cyclopropane hydrochloride (F2782). The term active metabolite is intended to denote a derivative originating from the metabolism of Milnacipran in vitro or in vivo and which has an ability to inhibit the reuptake of serotonin and norepinephrine; preferably it is F2782, F2941, F2800, F1612 and F1567
The present invention therefore also relates to the use of a mixture of enantiomers preferably enriched in (1S, 2R) enantiomer of at least one metabolite of Milnacipran, preferably chosen from F2782, F2941, F2800, F1612, F1567 , as well as their pharmaceutically acceptable salts, for the preparation of a medicament intended to prevent or treat pathologies or affections which can be treated by the double inhibition of the reuptake of serotonin (5-HT) and norepinephrine (NA), while limiting the risks of disorders cardiovascular and / or while limiting organic and / or tissue toxicity, in particular cardiac, hepatic and / or renal toxicity.
It is also within the scope of the present invention to use a mixture of enantiomers of Milnacipran enriched in (1S, 2R) enantiomer, preferably the substantially pure F2695 enantiomer, and at least one of its metabolites, preferably chosen from F2782, F2941, F2800, F1612, F1567, preferably enriched in enantiomer (1S, 2R), for the preparation of a medicament intended to prevent or treat pathologies or affections which can be treated by the double inhibition of the reuptake of serotonin (5-HT) and of noradrenaline (NA), while limiting the risks of disorders cardiovascular and / or while limiting organic and / or tissue toxicity, in particular cardiac, hepatic and renal toxicity.
“Pharmaceutically acceptable salt” designates all the salts which retain the effectiveness and the properties of an active principle and which do not exhibit any side effects. Preferably, they are salts of pharmaceutically acceptable mineral or organic acids. By way of preferred, but not limiting, examples, the halohydrates, such as hydrochloride and hydrobromide, fumarate, maleate, oxalate, citrate, methane sulfonate, glutamate, tartrate, mesylate, and their possible hydrates.
In the sense of the present invention, the term “mixture of enantiomers” means the mixture of enantiomers of Milnacipran enriched in enantiomer (1S, 2R), as well as their pharmaceutically acceptable salts, and / or the mixture of enantiomers of at least one of the metabolites of Milnacipran, preferably enriched in enantiomer (1S, 2R) as well as their pharmaceutically acceptable salts.
The mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, are administered to any type of patient in need of such treatment, whether for therapeutic or prophylactic purposes. For therapeutic purposes, the aim is to eradicate or improve the condition to be treated and / or one or more associated symptom (s). For prophylactic purposes, the aim is to prevent the appearance of the condition to be treated and / or one or more associated symptom (s). However, the mixture of enantiomers according to the invention is more particularly suitable for populations of patients at risk who are likely to develop certain undesirable clinical manifestations during or following treatment with Milnacipran in racemic form. These include children, the elderly, patients with liver and / or kidney failure, patients receiving treatment inducing organic and / or tissue toxicities, liver or kidney, patients receiving cardiac treatment, patients receiving treatment inducing cardiovascular side effects, patients with a cardiovascular history (for example myocardial infarction) and / or suffering from cardiovascular disorders, such as patients with heart rhythm disorders (tachycardia, bradycardia, palpitations), patients with blood pressure disorders (hypo- or hypertensive patients) or patients with heart disease.
Among the multiple pathologies or affections which present as a symptom disorders of the cardiac rhythm and for which the present invention is particularly suitable for the treatment of patients at risk who are affected by it, it is worth mentioning more particularly the tachycardia which corresponds to an acceleration of the rhythm heartbeat (tachycardia is moderate when the pulsations are 80 to 100 per minute, intense when they exceed 100), palpitations, extrasystoles (sporadic, frequent or during myocardial infarction), atrial fibrillation, atrial flutter and tachysystolia, bradycardia, heart failure, and myocardial infarction.
Among the multiple pathologies which present as a symptom of disorders of arterial pressure and for which the present invention is particularly suitable for the treatment of patients at risk who are affected by it, it is worth mentioning more particularly: hypertension, malignant hypertension, pulmonary hypertension, portal hypertension, essential paroxysmal hypertension, hypotension, orthostatic hypotension, intracranial hypertension.
Advantageously, the cardiovascular disorders whose risks can be limited by the administration of the mixture of enantiomers according to the invention, and preferably by the administration of the substantially pure F2695 enantiomer, are:<ul id="ul0001" list-style="none" compact="compact"><li>➢ the rise in diastolic and / or systolic blood pressure measured in millimeters of mercury (mm Hg); more particularly, it is an increase in diastolic heart pressure, and / or,</li><li>➢ heart rhythm disorders, including an increase in the patient's heart rate.</li></ul>
Systolic blood pressure is the maximum value for blood pressure, and it corresponds to the moment when the first heartbeat is heard at the level of the humeral artery when measuring blood pressure. Systole is the period of the cardiac revolution during which the chambers of the heart contract, thereby causing the ejection of blood. Diastolic blood pressure is the minimum value of blood pressure, which corresponds to the disappearance of heart sounds at the level of the humeral artery when the blood pressure cuff is deflated when measuring blood pressure. The diastole is the period of the cardiac revolution during which the chambers of the heart fill with blood. The rise in systolic and / or diastolic pressure implies the rise in blood pressure which characterizes systemic hypertension (and its variants), the symptoms of which may be the following: headache, fatigue, mild sensory disturbances such as dizziness, ringing in the ears, palpitations, nosebleeds, confusion or drowsiness, cramps, numbness or tingling in the feet and hands. Systemic hypertension (and its variants) can lead to serious, and sometimes fatal, complications: neurological accidents of vascular origin, left ventricular failure, renal failure, ischemic heart disease (myocardial infarction, angina and their variants). According to current recommendations, a patient is considered to have high blood pressure when the blood pressure is greater than 90 mm Hg for diastolic pressure and 140 mm Hg for systolic pressure.
The toxicity, the risks of which can be limited by the administration of the mixture of enantiomers according to the invention is advantageously organic toxicity, in particular cardiac toxicity, and / or tissue toxicity, in particular hepatic and / or renal toxicity. This tissue toxicity can be revealed by the presence of jaundice or by biological markers.
It is also within the scope of the present invention to use the mixture of enantiomers according to the invention in veterinary medicine for the treatment of animals, in particular domestic or farm animals requiring such treatment.
Due to their pharmacological properties, in particular dual inhibitors of the re-capture of serotonin (5-HT) and norepinephrine (NA), the mixture of enantiomers is particularly useful for the preparation of medicaments intended for the preventive treatment and / or curative of many pathologies or affections (syndrome) described below while limiting the risks of cardiovascular disorders and / or while limiting organic and / or tissue toxicity, especially hepatic and / or renal cardiac toxicity.
Among these pathologies or affections, mention should be made of pathologies of the central nervous system as defined in " <nplcit id="ncit0010" npl-type="b"><text>The Diagnostic and Statistical Manual of Mental Disorders -IV (DSM-IV), 1995 American Psychiatric Association </text></nplcit>" By way of illustrative and nonlimiting examples, mention should be made of depression, in particular deep depression, resistant depression, depression in the elderly, psychotic depression, depression induced by interferon treatments, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes linked to a general medical condition, depressive episodes linked to substances acting on mood, bipolar syndrome, schizophrenia, generalized anxiety, moodiness and doldrums, stress-related illnesses, panic attacks, phobia, especially agoraphobia, obsessive-compulsive disorder, conduct disorder, oppositional disorder, post-traumatic stress syndromes, depression of the immune system, fatigue and the accompanying pain syndromes, chronic fatigue syndrome, fibromyalgia, and other pathologies of functional somatic nature, autism, pathologies characterized by a lack of attention due to general medical conditions, attention disorders due to hyperactivity, eating disorders, bulimic neurosis , anorexic neurosis, obesity, psychotic disorders, apathy, migraine, pain, and in particular chronic pain, irritable bowel syndrome, cardiovascular diseases, and in particular anxio-depressive syndrome in myocardial infarction or in hypertension, neurodegenerative diseases and the associated anxio-depressive syndromes (Alzheimer's disease, Huntington's disease, Parkinson's disease), urinary incontinence, in particular urinary incontinence linked to stress and enuresis, drug dependence, and in particular anxio-dependence on tobacco, in particular nicotine, alcohol, narcotic drugs, drugs, to analgesics when weaning from these states of dependence.
More particularly, the subject of the present invention is the use of a mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, for the preparation of a medicament intended for preventing or treating depression or depressed state while limiting the risks of cardiovascular disorders and / or while limiting organic and / or tissue toxicity, in particular hepatic and / or renal toxicity. In the context of the present invention, the term "depression" means a set of symptoms comprising on the one hand a psychic aspect consisting of mood disorders with pessimism, moral pain, ideas of death and suicide, psychic inhibition, and on the other hand, a physical aspect of motor inhibition consisting in particular of a slowing down of the motor, appetite disorders, constipation, sleep disorders and weight regulation. Depression therefore corresponds to a pathological mental state associating a painful modification of the mood and a slowing down of intellectual and motor activity. By "depressive state" is meant a mental state characterized by a decline in the neuropsychic tone, manifested by lassitude, fatigability, discouragement and tendency to pessimism and sometimes accompanied by anxiety.
Also, the subject of the present invention is more particularly the use of a mixture of enantiomers according to the invention, preferably the substantially pure enantiomer F2695, for the preparation of a medicament intended for preventing or treating fibromyalgia and / or chronic fatigue syndrome while limiting the risks of cardiovascular disorders and / or while limiting organic and / or tissue toxicity, in particular liver and / or renal toxicity. Fibromyalgia syndrome is a chronic syndrome characterized by a feeling of pain or burning with morning stiffening mainly affecting the articular and peri-articular fibrous tissues, and by a feeling of deep fatigue. Fibromyalgia has a variety of symptoms. The most common are non-restorative sleep, headaches, digestive disorders, depression, muscle spasms, pain in the face, numbness, etc. Chronic fatigue syndrome is characterized by a state of exhaustion or fatigue. The most common symptoms are weakness, spasms and / or muscle pain, excessive sleep, fever, angina, memory loss and / or concentration problems, insomnia, depression .
Also, the subject of the present invention is more particularly the use of a mixture of enantiomers according to the invention, preferably the substantially pure enantiomer F2695, for the preparation of a medicament intended for preventing or treating pain and in particular the chronic pain while limiting the risks of cardiovascular disorders and / or while limiting organic and / or tissue toxicity, in particular liver and / or renal toxicity. Pain can be associated with various pathologies and / or injuries. It can be acute or chronic. Epidemiological studies have demonstrated the relationships between chronic pain conditions and anxio-depressions. Thus, patients with chronic pain can develop emotional problems that lead to depression, and, in the worst case, suicide attempts. A patient is considered to have chronic pain if they complain of pain for more than six months. Among the chronic pains, it should be mentioned by way of illustrative and nonlimiting example, the pains associated with fibromyalgia and / or originating from fibrous tissues, muscles, tendons, ligaments and other sites, abdominal pain and diarrhea in irritable bowel syndrome, and also lower back pain.
Also, the subject of the present invention is more particularly the use of a mixture of enantiomers according to the invention, preferably the substantially pure enantiomer F2695, for the preparation of a medicament intended for preventing or treating urinary incontinence, in particular urinary incontinence linked to stress and enuresis, while limiting the risks of cardiovascular disorders and / or while limiting organic and / or tissue toxicity, including liver and / or kidney toxicity.
The prophylactic and therapeutic treatment of the above pathologies is carried out by delivering to an animal, preferably man, a therapeutically effective amount of a mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, alone or in combination with at least one other active ingredient. In most cases, it is human, but the treatment is also suitable for animals, especially farm animals (livestock, rodents, poultry, fish, ...) and pets (dogs , cats, rabbits, horses, ...).
The mixture of enantiomers, enriched in enantiomer (1S, 2R), 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 in a time-delayed manner at least one second active compound in the treatment of the pathologies previously mentioned.
Preferably, the present invention also relates to the use as a medicament:<ol id="ol0001" compact="compact" ol-style=""><li>a) of said mixture of enantiomers enriched in the (1S, 2R) enantiomer of Milnacipran and / or of at least one of its metabolites as well as their pharmaceutically acceptable salts, and</li><li>b) at least one active compound chosen from psychotropics, in particular anti-depressants, and anti-muscarinic agents,</li></ol>as combination products for simultaneous, separate or staggered use for the treatment or prevention of depression, in particular deep depression, resistant depression, elderly depression, psychotic depression, treatment-induced depression interferon, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes linked to a general medical condition, depressive episodes linked to substances acting on mood.
By psychotropic is meant a substance of natural or artificial origin capable of modifying mental activity and whose essential action is exerted on the central nervous system and the psyche. Psychotropics are divided into three groups: 1) psycholeptics (hypnotics, neuroleptics and anxiolytics), 2) psychoanaleptics (antidepressants and psychotonics) and 3) psychodysleptics (hallucinogens).
Preferably, said psychotropic drug is an antidepressant. By way of nonlimiting example, the antidepressant is chosen from (i) mono-amine oxidase inhibitors (MAOIs) such as iproniazid, pargyline, selegin, (ii) 5HTID agonists such as sumatriptan, epinephrine and noradrenaline (alpha and beta sympathomimetics) (iii) tricyclic antidepressants, such as imipramine, clomipramine, (iv) selective serotonin reuptake inhibitors (SSRI) such as fluoxetine, (v) selective inhibitors of norepinephrine reuptake, such as for example tandamine, fluparoxan, mirtazapine (vi) inhibitors of reuptake of serotonin and norepinephrine, such as venlafaxine and duloxetine. By way of nonlimiting example, the anti-muscarinic agent is selected from tolterodine, propiverine, oxybutynin, trospium, darifenacin, temerivine, ipratropium.
Preferably, the present invention also relates to the use as a medicament:<ol id="ol0002" compact="compact" ol-style=""><li>a) of said mixture of enantiomers enriched in the (1S, 2R) enantiomer of Milnacipran and / or of at least one of its metabolites as well as their pharmaceutically acceptable salts, and</li><li>b) at least one other active ingredient selected from active compounds inducing organic toxicity and active compounds inducing tissue toxicity, in particular hepatic and / or renal or with one or more active ingredients intended for the treatment of hepatic insufficiency and / or renal, as combination products for simultaneous use, separate or staggered in time for the treatment or prevention of conditions or pathologies which can be treated by double inhibition of the re-capture of serotonin (5-HT) and norepinephrine (NA).</li></ol>
Preferably, the present invention also relates to the use as a medicament:<ol id="ol0003" compact="compact" ol-style=""><li>a) of said mixture of enantiomers enriched in the (1S, 2R) enantiomer of Milnacipran and / or of at least one of its metabolites as well as their pharmaceutically acceptable salts, and</li><li>b) at least one other active ingredient selected from the active compounds inducing cardiovascular side effects and the compounds for cardiac purposes,</li></ol>as combination products for simultaneous, separate or staggered use for the treatment or prevention of conditions or pathologies which can be treated by double inhibition of the re-capture of serotonin (5-HT) and norepinephrine (N / A).
Advantageously, the induced cardiovascular side effects are those previously mentioned, and more particularly hypertension, hypotension, heart rhythm disorders (tachycardia, bradycardia, palpitations).
The present invention also relates to pharmaceutical compositions containing the combination products described above.
In the context of the present invention, the mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, is advantageously administered, without limitation, by the oral, nasal, transdermal, rectal, intestinal, parenteral route. , by intramuscular, subcutaneous or intravenous injection, alone or in combination with other active ingredients, as previously described.
When administered alone, the mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, can be administered <i>per se</i> or in the form of a pharmaceutical composition in which the said mixture of enantiomers or of their pharmaceutically acceptable salts is in association or in mixture with one or more pharmaceutically acceptable carriers, excipients and / or diluents, in particular facilitating bioavailability.
When the mixture of enantiomers according to the invention, and preferably the (1S, 2R) F2695 enantiomer of substantially pure Milnacipran, is administered in combination with other active ingredients, said mixture and the other active ingredients can be formulated as mixed or separately in the same or different form. They can be administered by the same route or a different route.
The pharmaceutical compositions according to the invention can be formulated in conventional ways well known to those skilled in the art using one or more physiologically acceptable carriers comprising excipients, adjuvants and auxiliaries such as, for example, preservatives, stabilizers, wetting or emulsifying agents. The formulation method chosen depends on the desired route of administration.
In the case of administration by injection, an aqueous solution is advantageously used, in particular a physiologically acceptable buffer solution, such as a Hank solution, a Ringer solution or a physiological saline buffer solution. In the case of transdermal or mucosal administration, penetrating agents suitable for the mucosa to be crossed are advantageously used. Such penetrating agents are well known to those skilled in the art. In the case of oral administration, the pharmaceutical compositions according to the invention are advantageously administered in unit or multidose forms of administration in admixture with suitable pharmaceutical carriers known to those skilled in the art. The appropriate unit administration forms include in particular optionally scored tablets, capsules, powders, granules and oral solutions or suspensions, aerosols. Suitable multidose forms of administration include, but are not limited to, oral drops, emulsions and syrups.
During the preparation of tablets, the mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, is formulated with a pharmaceutically acceptable vehicle such as in particular polyvinylpyrrolidone, gal carbopol, polyethylene glycol, gelatin, talc, starch, lactose, magnesium stearate, gum arabic or their analogs. By way of example, the tablet contains the following excipients: dehydrated calcium hydrogen phosphate, calcium carmellose, povidone K30, anhydrous colloidal silica, magnesium stearate, talc. The tablets can optionally be coated, that is to say covered with several layers of various substances such as sucrose, in order to facilitate setting or preservation. The coating can also contain pigments or dyes in order to distinguish and characterize the tablets according to their dosage, for example. The tablets may also have a more or less complex formulation intended to modify the rate of release of the active principle. The release of the active ingredient from said tablet can be accelerated, slowed or delayed depending on the desired absorption. The mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, can thus be prepared in a prolonged release dosage form obtained according to the process described in the patent. <patcit id="pcit0007" dnum="EP939626A"><text>EP 939,626</text></patcit>. This dosage form is in multiparticulate form bringing together a plurality of minigranules and has a certain release profile.<i>in vitro.</i>
The release of the mixture of enantiomers according to the invention can be delayed and / or controlled via the use of an implant or via a transcutaneous release, in particular subcutaneous or intramuscular release, via an intramuscular injection or via a transdermal patch. Said mixture is then formulated with in particular suitable hydrophobic or polymeric substances and ion exchange resins.
The quantity to be administered to the patient of the mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, depends on the conditions to be treated, on the intended effect, in particular a therapeutic or prophylactic effect, on the state of health. and the patient's age, including their cardiovascular history, treatment conditions, and method of drug administration. The effective therapeutic or prophylactic amounts to be administered to a human patient can be determined from animal models or from data known to those skilled in the art when treating depression in humans, for example by the use of a racemic mixture of Milnacipran.
As part of the prophylactic and / or therapeutic treatment of the pathologies mentioned above, and in particular of depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain, the medicament according to the invention is advantageously administered at doses between 0.01 mg and 10 mg / kg of body weight per day in one or more doses, even more advantageously at doses of between 0.05 mg and 5 mg / kg of body weight per day in one or more doses, even more advantageously at doses of between 0.1 mg and 1 mg / kg of body weight per day in one or more takes. In a particularly advantageous manner, the administration of said medicament in doses as defined above is divided into two daily doses, preferably in the form of a capsule. By way of example, the mixture of enantiomers according to the invention, preferably the substantially pure F2695 enantiomer, is administered in the form of a capsule the content of active principle of which is advantageously around 6.75 mg / capsule, 12.5 mg / capsule, 25 mg / capsule, 50 mg / capsule.
Other characteristics, objects and advantages of the invention will emerge from the examples which follow. The invention is not limited to the specific examples mentioned for illustrative purposes only and which should be read with reference to the following figures:
Legend of Figures
<dl id="dl0001"><dt>Figure 1 :</dt><dd>Evolution of the heart rate after a single administration (delta values).<ul id="ul0002" list-style="none" compact="compact"><li>*** p ≤ 0.001 versus deionized water</li><li>** p ≤ 0.01 versus deionized water</li><li>* p ≤ 0.05 versus deionized water</li><li>▲ p ≤ 0.05 versus F2207</li></ul></dd><dt>Figure 2:</dt><dd>Evolution of the heart rate after single administration (absolute values).<ul id="ul0003" list-style="none" compact="compact"><li>*** p ≤ 0.001 versus deionized water</li><li>** p ≤ 0.01 versus deionized water</li><li>* p ≤ 0.05 versus deionized water</li><li>▲ p ≤ 0.05 versus F2207</li></ul></dd><dt>Figure 3:</dt><dd>Effects of the various treatments on the average values of diastolic arterial pressures (average over the 6 hours following the last treatment, after 5 days of consecutive treatment).</dd><dt>Figure 4:</dt><dd>Effects of the various treatments on the average values of systolic arterial pressures (average over the 6 hours following the last treatment, after 5 days of consecutive treatment).</dd><dt>Figure 5:</dt><dd>Schematic representation of the method of calculating the toxicity index. The toxicity index is the sum of all up and down-regulated genes (depending on the induction factor defined by the user).</dd><dt>Figures 6a, 6b, 6c:</dt><dd>MTT test on primary rat hepatocytes. The concentrations are expressed in µM.</dd></dl>
EXAMPLES
<u style="single">EXAMPLE 1</u> :
Pharmacokinetic studies of Milnacipran and its enantiomers
Pharmacokinetic studies of Milnacipran hydrochloride (F2207) and its enantiomers (F2695 and F2696) have been carried out in different animal species and in humans.
In animals, the pharmacokinetics of each enantiomer has been studied following administration of the racemate or a single enantiomer. The plasma levels of the F2695 and F2696 enantiomers are approximately equivalent in the species tested (monkey and rat).
A pharmacokinetic study in humans comprising 12 healthy subjects was carried out by administering the racemate or one of the two enantiomers. It appears that the pharmacokinetic profile of each enantiomer is independent of the fact that they were administered either separately or in the form of a racemate indicating the absence of interactions between the two enantiomers (<b>Table 1</b>).
Table 1: Table of the main pharmacokinetic variables of Milnacipran hydrochloride (F2207) and its two enantiomers F2695 and F2696.
<ul id="ul0004" list-style="none" compact="compact"><li>Cmax: Maximum plasma concentration estimated directly from experimental data</li><li>Tmax: Time to obtain maximum plasma concentration</li><li>AUC<sub>0→∞</sub> : Area under the curve of plasma concentrations as a function of time extrapolated to infinity</li><li>T<sub>1/2</sub> : Terminal half-life of decreased plasma concentrations</li></ul><tables id="tabl0006" num="0006"><table frame="all"><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><thead><row><entry morerows="1" align="center" valign="middle"><b>Administered dose (mg)</b></entry><entry namest="col2" nameend="col3" rowsep="0" align="center" valign="middle"><b>F2207</b></entry><entry rowsep="0" align="center" valign="middle"><b>F2695 (D)</b></entry><entry rowsep="0" align="center" valign="middle"><b>F2696 (L)</b></entry></row><row><entry namest="col2" nameend="col3" align="center" valign="middle"><b>(50 mg)</b></entry><entry align="center" valign="middle"><b>(25 mg)</b></entry><entry align="center" valign="middle"><b>(25 mg)</b></entry></row><row><entry align="center" valign="middle" /><entry align="center" valign="middle"><b>F2695 (D)</b></entry><entry align="center" valign="middle"><b>F2696 (L)</b></entry><entry align="center" valign="middle" /><entry align="center" valign="middle" /></row></thead><tbody><row><entry align="center" valign="middle"><b>Cmax (nmol.l<sup>-1</sup>)</b></entry><entry align="center" valign="middle"><b>214</b></entry><entry align="center" valign="middle"><b>179</b></entry><entry align="center" valign="middle"><b>216</b></entry><entry align="center" valign="middle"><b>212</b></entry></row><row><entry align="center" valign="middle"><b>Tmax (hour)</b></entry><entry align="center" valign="middle"><b>3,42</b></entry><entry align="center" valign="middle"><b>2,87</b></entry><entry align="center" valign="middle"><b>3,08</b></entry><entry align="center" valign="middle"><b>2,21</b></entry></row><row><entry align="center" valign="middle"><b>AUC 0-> °° (nmol.hl<sup>-1</sup>)</b></entry><entry align="center" valign="middle"><b>2896</b></entry><entry align="center" valign="middle"><b>1563</b></entry><entry align="center" valign="middle"><b>2869</b></entry><entry align="center" valign="middle"><b>1543</b></entry></row><row><entry align="center" valign="middle"><b>T ½ (hours)</b></entry><entry align="center" valign="middle"><b>9,28</b></entry><entry align="center" valign="middle"><b>5,75</b></entry><entry align="center" valign="middle"><b>9,38</b></entry><entry align="center" valign="middle"><b>5,58</b></entry></row></tbody></tgroup></table></tables>
These results indicate that no bioconversion of the enantiomers F2695 and F2696) was detected in the analyzed species.
<u style="single">EXAMPLE 2</u> :
Biochemical studies of Milnacipran and its enantiomers
The two enantiomers (F2695 and F2696) of milnacipran hydrochloride (F2207) were studied <i>in vitro</i> on the catches of noradrenaline and serotonin as well as on the binding (binding) of paroxetine in the brain of the rat.
<u style="single">2. 1. MATERIALS AND METHODS</u>
2.1.1. Norepinephrine capture by a homogenate (P<sub>2</sub>) rat hypothalamus.
P2 preparation
Male rats, Sprague-Dawley, of 200 to 300 g are knocked out and decapitated, then the hypothalamus removed quickly. Two hypothalami are homogenized in 4 ml of 0.32 M sucrose at Potter S by 16 round trips at 800 rpm, then centrifuged for 10 min at 1000 g to remove cell debris. The supernatant is centrifuged for 20 min at 10,000 g and the P<sub>2</sub> thus obtained taken up in 4 ml of 0.32 M sucrose and homogenized with Dounce.
Capture ("Uptake")
We use <sup>3</sup>H- (1) -NA: 13 Ci / mmole (Amersham).
The uptake is made in phosphate buffer (containing per liter: 8 g of NaCl, 1.21 g of K<sub>2</sub>HPO<sub>4</sub> and 0.34 g KH<sub>2</sub>PO<sub>4</sub>) pre-oxygenated 30 min before use with a mixture O<sub>2</sub>/CO<sub>2</sub> (95 % / 5 %).
The following are introduced into 5 ml plastic tubes placed in a water bath at 37 ° C.<ul id="ul0005" list-style="dash" compact="compact"><li>100 µl of buffer or inhibitor,</li><li>700 µl of buffer (containing 25 µM pargyline),</li><li>100 µl of P<sub>2</sub>.</li></ul>
After equilibration of the temperature, the reaction starts by adding 100 μl of <sup>3</sup>H-NA, 50 nM final.
10 min exactly after, the reaction is stopped by adding 2.5 ml of ice-cold buffer and filtration on GF / F filters. Then the tube is rinsed once and the filter once with 2.5 ml of ice-cold buffer. The filter is then introduced into a Beckman mini-flask and after adding 3 ml of Instagel scintillating liquid (Packard), the radioactivity is measured in a Tricarb Packard liquid scintillation counter.
The non-specific uptake (NS) is measured in the presence of DMI 10<sup>-5</sup> Mr.
The percentage of inhibition is calculated by the formula: <maths id="math0001" num=""><math display="block"><mfrac><mrow><mo>(</mo><mi>total uptake</mi><mo>-</mo><mi>NS</mi><mo>)</mo><mo>-</mo><mfenced separators=""><mi mathvariant="normal">uptake in the presence</mi><mspace width="1em" /><msup><mi mathvariant="normal">d</mi><mo>′</mo></msup><mo></mo><mi>inhibitor</mi><mo>-</mo><mi>NS</mi></mfenced></mrow><mfenced separators=""><mi>total uptake</mi><mo>-</mo><mi>NS</mi></mfenced></mfrac></math><img file="EP1908461A1_D0007.tif" /></maths> inhibition (4 trials) as a function of the log of the inhibitor concentration.
2.1.2. Serotonin capture
The method was performed according to <nplcit id="ncit0011" npl-type="s"><text>Gray and Whittaker (1962, J. Anat., 96: 79-97</text></nplcit>). After homogenization of the brain tissue in a sucrose solution, the presynaptic terminations detach from the axon and close to form synaptosomes obtained by subcellular fractionation.
Male Sprague-Dawley rats (January) of 180-200 g were used. After sacrificing the animal, the hypothalamus was removed, weighed and homogenized using the Dounce in 0.32 M sucrose at 0 ° C.
This homogenate was centrifuged for 10 min at 1,000 g (2,400 rpm - Hettich, Rotenta). The supernatant was collected and centrifuged for 20 min at 10,000 g (8,000 rpm-Beckam, model J2-21 M: rotor J14). The pellet (called fraction P<sub>2</sub>) was taken up in sucrose at a concentration of 50 mg / ml.
We incubated for 5 min at 37 ° C:<ul id="ul0006" list-style="none" compact="compact"><li>○ 350 µl ice cold buffer (NaCl 136mM, KH<sub>2</sub>PO<sub>4</sub> 2.4mM, K<sub>2</sub>HPO<sub>4</sub> 6.9 mM, pH 7.2) pre-oxygenated 30 min before,</li><li>○ 50 µl of membranes (5 mg / ml final),</li><li>○ 50 µl citalopram (10<sup>-5</sup> M final) for non-specific capture,</li><li>○ 50 µl of <sup>3</sup>H-5-HT (50 nM final) (NEN, France, 28.4 Ci / mmol).</li></ul>
Exactly 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 ice-cold buffer then rinsing with 3 times 2.5 ml).
The radioactivity collected on the filter was measured (Packard Tricarb 4640) by liquid scintillation with Emulsifier-Safe (Packard).
The ICs<sub>50</sub> were determined by plotting the inhibition percentages as a function of the log of the product concentration (6 duplicate concentrations).
2.1.3. Binding of paroxetine
Male Sprague-Dawley rats (January) of 180-200 g were used. The hypothalami of several rats were pooled and homogenized in 5 ml of ice-cold buffer (50 mM Tris-HCL, 120 mM NaCl, 5 mM KCl, pH 7.5) with Dounce, and the homogenate centrifuged at 30,000 g (27 000 rpm - Beckman. L5-50E, rotor T40) for 10 min. The pellet obtained was taken up in 5 ml of buffer and recentrifuged under the same conditions. The new pellet was taken up in the same buffer and finally rehomogenized with 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 the (final) concentration of 0.1 nM, at 20 ° C, in a final volume of 1 ml for 2 h. After 2 h of incubation, the reaction was stopped by vacuum filtration on Whatman GF / F filters pretreated in a 0.05% polyethyleneimine solution 30 min before (predilution with 4 ml of ice-cold buffer then rinsing of the tube with 2 times 4 ml). Radioactivity was measured by liquid scintillation spectrometry (Packard, Tricarb 4640) using the Emulsifier-Safe (Packard) as a scintillating agent.
The specific binding of <sup>3</sup>H-paroxetine was defined as the difference between the total binding and that remaining in the presence of 10 μM of fluoxetine.
The ICs<sub>50</sub> were determined by plotting the inhibition percentages as a function of the log of the product concentration (6 duplicate concentrations).
2.1.4. Products used
<ul id="ul0007" list-style="none" compact="compact"><li>F2207: lot n ° 10-CTN3 Key P118</li><li>F2695: lot n ° PL-I-205</li><li>F2696: lot n ° PL-I-204C.</li></ul>
<u style="single">2.2. RESULTS</u>
The effects of F2207 and its two enantiomers on the uptake of noradrenaline and serotonin and on the binding of paroxetine are represented on a graph with on the ordinate the percentage of inhibition in function (%) and on the abscissa the concentration (M) of the F2207, F2695 or F2696 (data not shown). The values of the inhibition percentages corresponding to each concentration of product, tested in duplicate, are the means of the results of four independent experiments.
IC values<sub>50</sub> of the three products were determined from these curves and are shown in Table 2.<tables id="tabl0007" num="0007"><table frame="all"><title><b>Table 2: Inhibition of catches of <sup>3</sup>H-noradrenaline, <sup>3</sup>H-serotonin and binding <sup>3</sup>H-paroxetine.</b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="43mm" /><colspec colnum="3" colname="col3" colwidth="41mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><thead><row><entry namest="col1" nameend="col4" align="center" valign="middle"><b>IC50 (M)</b></entry></row><row><entry align="center" valign="middle">Compounds</entry><entry namest="col2" nameend="col3" align="center" valign="middle">Capture</entry><entry align="center" valign="middle">Binding <sup>3</sup>H-Paroxetine</entry></row><row><entry align="center" valign="middle" /><entry align="center" valign="middle"><sup>3</sup>H-Noradrenaline</entry><entry align="center" valign="middle"><sup>3</sup>H-Serotonin</entry><entry align="center" valign="middle" /></row></thead><tbody><row><entry align="center" valign="middle"><b>F2695</b></entry><entry align="center" valign="middle">1.5 X 10<sup>-8</sup></entry><entry align="center" valign="middle">4.6 X 10<sup>-8</sup></entry><entry align="center" valign="middle">6.0 X 10<sup>-8</sup></entry></row><row><entry align="center" valign="middle"><b>F2207</b></entry><entry align="center" valign="middle">3.0 X 10<sup>-8</sup></entry><entry align="center" valign="middle">15 X 10<sup>-8</sup></entry><entry align="center" valign="middle">13 X 10<sup>-8</sup></entry></row><row><entry align="center" valign="middle"><b>F2696</b></entry><entry align="center" valign="middle">75 X 10<sup>-8</sup></entry><entry align="center" valign="middle">60 X 10<sup>-8</sup></entry><entry align="center" valign="middle">70 X 10<sup>-8</sup></entry></row></tbody></tgroup></table></tables>
The three compounds are active on these three pharmacological tests, but differences exist:<ul id="ul0008" list-style="dash" compact="compact"><li><u style="single">on the capture of noradrenaline</u> : <ul id="ul0009" list-style="none" compact="compact"><li>F2695 is twice as active as F2207.</li></ul>F2695 is 25 times more active than F2696.</li><li><u style="single">on serotonin capture:</u><ul id="ul0010" list-style="none" compact="compact"><li>F2695 is 3 times more active than F2207.</li><li>F2695 is 12 times more active than F2696.</li></ul></li><li><u style="single">on the binding (binding) of paroxetine</u> : <ul id="ul0011" list-style="none" compact="compact"><li>F2695 is twice as active as F2207.</li><li>F2695 is 10 times more active than F2696.</li></ul></li></ul>
The three compounds are active in these pharmacological tests with, however, less activity for the form (1R, 2S) (F2696) and the racemate (F2207). The form (1S, 2R) of Milnacipran hydrochloride (F2695) is 2 to 3 times more active than F2207.
<u style="single">EXAMPLE NO.3</u>
: Comparative oral activity of the racemic Milnacipran hydrochloride (F2207) and its active enantiomer (1S, 2R) (F2695) on heart rate and blood pressure in the alert dog.
3.1. <u style="single">INTRODUCTION</u>
This study intends to study the effects of F2207 and F2695 a) as a single oral administration on the heart rate (n = 28 dogs), and b) after repeated 5-day oral treatment on systolic arterial pressures and diastolic in dogs (n = 6 dogs).
This study was led to pharmacologically active equidoses of F2207 and F2695 on female animals equipped with implants (Data Sciences International) allowing the acquisition of heart rate and blood pressure parameters by telemetry. These animals were divided for all studies into 3 treatment groups:<ul id="ul0012" list-style="dash" compact="compact"><li>group 1 (control) treated with deionized water,</li><li>group 2 treated with F2207 at a dose of 20 mg / kg / D,</li><li>group 3 treated with F2695 at a dose of 10 mg / kg / day.</li></ul>
3.2. <u style="single">METHODOLOGY</u>
Given the small number of animals simultaneously equipped (maximum 8), the number of recording tracks of the equipment used (8 tracks), and so as to constitute homogeneous treatment groups, the overall assessment was carried out in four studies, each study being divided into three passages (treatment of each animal with each of the three products), separated by a period of washing and resetting of probes ("wash-out"). Each passage itself takes place in two phases:<ul id="ul0013" list-style="dash" compact="compact"><li>a first phase during which all the animals are treated with deionized water for training in restraint and oral treatment with a feeding tube,</li><li>a second phase during which the animals receive their respective treatment (single administration for the heart rate, studies n ° 894/926/935/936; repeated administration five days for the arterial pressure, study n ° 894).</li></ul>
The overall experimental scheme is described in the following table:<tables id="tabl0008" num="0008"><table frame="topbot"><title><b>Table 3: Overall experimental design for the telemetry study of the effects of the racemic Milnacipran hydrochloride (F2207) and its active enantiomer (1S, 2R) F2695 administered orally in conscious dogs.</b></title><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="42mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><thead><row><entry align="center" valign="middle"><b>NUMBER GROUP</b></entry><entry align="center" valign="middle"><b>1</b></entry><entry align="center" valign="middle"><b>2</b></entry><entry align="center" valign="middle"><b>3</b></entry></row></thead><tbody><row><entry align="center" valign="middle">ANIMALS</entry><entry align="center" valign="middle" /><entry align="center" valign="middle" /><entry align="center" valign="middle" /></row><row rowsep="0"><entry align="center" valign="middle">Number</entry><entry align="center" valign="middle">27</entry><entry align="center" valign="middle">28</entry><entry align="center" valign="middle">28</entry></row><row rowsep="0"><entry align="center" valign="middle">Identification</entry><entry align="center" valign="middle">1-2-7-8-13-14</entry><entry align="center" valign="middle">3-4-9-10-15-</entry><entry align="center" valign="middle">5-6-11-12-17</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">(study 894)</entry><entry align="center" valign="middle">16</entry><entry align="center" valign="middle">- 18</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">1 - 2 - 7 - 8 - 13 -</entry><entry align="center" valign="middle">(study 894)</entry><entry align="center" valign="middle">(study 894)</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">14</entry><entry align="center" valign="middle">3-4-9-10-15-</entry><entry align="center" valign="middle">5-6-11-12-17</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">(study 926)</entry><entry align="center" valign="middle">16</entry><entry align="center" valign="middle">- 18</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">1 - 2 - 9 - 10</entry><entry align="center" valign="middle">(study 926)</entry><entry align="center" valign="middle">(study 926)</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">11-17-18-19</entry><entry align="center" valign="middle">3-4-5-12</entry><entry align="center" valign="middle">6-7-8-14</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">(study 935)</entry><entry align="center" valign="middle">13 - 20 - 21 - 22</entry><entry align="center" valign="middle">15-16-23-24</entry></row><row rowsep="0"><entry align="center" valign="middle" /><entry align="center" valign="middle">1-2-9-10</entry><entry align="center" valign="middle">(study 935)</entry><entry align="center" valign="middle">(study 935)</entry></row><row><entry rowsep="0" /><entry rowsep="0">11-17-18-19</entry><entry rowsep="0">3-4-5-12 13 - 20 - 21 - 22</entry><entry rowsep="0">6-7-8-14 15 - 16 - 23 - 24</entry></row><row><entry /><entry>(study 936)</entry><entry>(study 936)</entry><entry>(study 936)</entry></row></tbody></tgroup><tgroup cols="4" colsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="42mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><thead><row><entry>TREATMENT</entry><entry align="center" valign="top" /><entry align="center" valign="top" /><entry align="center" valign="top" /></row></thead><tbody><row rowsep="0"><entry>Identification</entry><entry align="center">Deionized water</entry><entry align="center">F2207</entry><entry align="center">F2695</entry></row><row rowsep="0"><entry>Dose</entry><entry align="center">-</entry><entry align="center">20 mg / kg</entry><entry align="center">10 mg / kg</entry></row><row rowsep="0"><entry>Way</entry><entry namest="col2" nameend="col4" align="center">oral</entry></row><row><entry>Volume</entry><entry namest="col2" nameend="col4" align="center">5 ml / kg</entry></row></tbody></tgroup><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="42mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><tbody><row><entry namest="col1" nameend="col4" align="justify"><i>(n = 27 in the control group, the probe signal from animal No. 18 not having been recorded)</i></entry></row></tbody></tgroup></table></tables>
The effects of the different treatments on the heart rate were analyzed in the four studies, after single administration. The analysis covered the following 13 acquisition times:<ul id="ul0014" list-style="dash" compact="compact"><li>before single treatment,</li><li>every 30 minutes after 6 hours after a single treatment.</li></ul>
The effects of the various treatments on blood pressure were analyzed, in study No. 894 at steady state, on D5, D29 and D33 (last day of effective treatment for each of the passages). The analysis focused on the following acquisition times:<ul id="ul0015" list-style="dash" compact="compact"><li>before treatment,</li><li>every 30 minutes after 6 hours after treatment.</li></ul>
3.3. <u style="single">RESULTS</u>
3.3.1. Regarding heart rate (four pooled studies), a Tukey test was performed on individual frequency deltas, for each of the 12 post-treatment experimental times, versus the pre-treatment value, as well as on the absolute values heart rate at each recording time.
The following were objectified, compared to the control animals receiving deionized water: # when the statistical analysis is carried out on the values of deltas, (<figref idref="f0001">figure 1</figref>) : <ul id="ul0016" list-style="dash" compact="compact"><li>a significant increase in the heart rate from the first ½ hour following a single administration of F2207 (20 mg / kg), persistent increase up to 5.5 hours after treatment (p ≤ 0.001 for all acquisition times, at exception of times 0.5 and 5.5 hours - p ≤ 0.01 - and times 5.0 hours - p ≤ 0.05 - after treatment),</li><li>an increase in heart rate after administration of F2695 which is always lower than that obtained after administration of F2207. In addition, this difference between the effects of F2207 and F2695 is significant (p <0.05) at 1 and 4 hours after administration in favor of F2695.</li><li>an increase in the heart rate which lasts less long under F2695 (1.0 to 4.5 h) than under F2207 (persists until 5.5 h after treatment). # when the statistical analysis is carried out on absolute values of heart rate, this same study highlights (<figref idref="f0001">figure 2</figref>) :</li></ul><ul id="ul0017" list-style="dash" compact="compact"><li>a significant increase in the heart rate from the first hour following a single administration of F2207 (20 mg / kg), persistent increase up to 5.5 hours after treatment (p ≤ 0.001 for all acquisition times from 1.0 to 4.5 hours , with the exception of the time 3.5 hours - p ≤ 0.01; and p ≤ 0.01 for the acquisition time 5.5 hours after treatment),</li><li>an increase in heart rate after administration of F2695 which is always lower than that obtained after administration of F2207. In addition, this difference between the effects of F2207 and F2695 is significant (p <0.05) at 1 and 4 hours after administration in favor of F2695.</li><li>an increase in the heart rate which lasts less long under F2695 (1.0 to 4.5 h) than under F2207 (persists until 5.5 h after treatment).</li></ul>
3.3.2. Regarding blood pressure (a repeat administration study), an average diastolic blood pressure value (<figref idref="f0002">figure 3</figref> and Table 4), as well as an average systolic blood pressure value (<figref idref="f0002">figure 4</figref> and Table 5) were calculated for each dog and for the 6 hours following the last treatment, after 5 consecutive days of administration. These mean pressure values were analyzed by an ANOVA followed by a Tukey test when the latter allowed (data not shown).
The following were objectified:<ul id="ul0018" list-style="dash" compact="compact"><li>a significant increase (p ≤ 0.001) in diastolic blood pressure after repeated 5-day administration of F2207 (20 mg / kg / D) or F2695 (10 mg / kg / D) compared to treatment with deionized water,</li><li>a significant difference (p ≤ 0.05) in the average diastolic blood pressure value after repeated administration 5 days of F2207 (20 mg / kg / D) compared to the average diastolic blood pressure value after repeated administration of F2695 (10 mg / kg / D),</li><li>no significant effect on systolic blood pressure; it can however be noted that the values of PAS after repeated administration 5 days of F2695 are close to the values of PAS following treatment with deionized water.</li></ul>
Individual diastolic and systolic blood pressure data are presented in Tables 4 and 5 respectively.<tables id="tabl0009" num="0009"><table frame="all"><title><b>Table 4: Individual diastolic blood pressure data</b></title><tgroup cols="25"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="10mm" colsep="0" /><colspec colnum="3" colname="col3" colwidth="10mm" colsep="0" /><colspec colnum="4" colname="col4" colwidth="10mm" colsep="0" /><colspec colnum="5" colname="col5" colwidth="10mm" colsep="0" /><colspec colnum="6" colname="col6" colwidth="10mm" colsep="0" /><colspec colnum="7" colname="col7" colwidth="10mm" /><colspec colnum="8" colname="col8" colwidth="10mm" colsep="0" /><colspec colnum="9" colname="col9" colwidth="10mm" /><colspec colnum="10" colname="col10" colwidth="10mm" colsep="0" /><colspec colnum="11" colname="col11" colwidth="10mm" colsep="0" /><colspec colnum="12" colname="col12" colwidth="10mm" colsep="0" /><colspec colnum="13" colname="col13" colwidth="10mm" colsep="0" /><colspec colnum="14" colname="col14" colwidth="10mm" colsep="0" /><colspec colnum="15" colname="col15" colwidth="10mm" /><colspec colnum="16" colname="col16" colwidth="10mm" colsep="0" /><colspec colnum="17" colname="col17" colwidth="10mm" /><colspec colnum="18" colname="col18" colwidth="10mm" colsep="0" /><colspec colnum="19" colname="col19" colwidth="10mm" colsep="0" /><colspec colnum="20" colname="col20" colwidth="10mm" colsep="0" /><colspec colnum="21" colname="col21" colwidth="10mm" colsep="0" /><colspec colnum="22" colname="col22" colwidth="10mm" colsep="0" /><colspec colnum="23" colname="col23" colwidth="10mm" /><colspec colnum="24" colname="col24" colwidth="10mm" colsep="0" /><colspec colnum="25" colname="col25" colwidth="9mm" /><thead><row><entry namest="col1" nameend="col25" rowsep="0" align="left" valign="middle"><b>DIASTOLIC BLOOD PRESSURE, (PAD expressed in mm Hg)</b></entry></row><row><entry namest="col1" nameend="col25" align="left" valign="middle">Individual data after repeated administration 5 consecutive days</entry></row><row><entry align="center" valign="middle"><b>GROUP</b></entry><entry namest="col2" nameend="col9" align="center" valign="middle"><b>1</b></entry><entry namest="col10" nameend="col17" align="center" valign="middle"><b>2</b></entry><entry namest="col18" nameend="col25" align="center" valign="middle"><b>3</b></entry></row><row><entry align="center" valign="middle"><b>TREATMENT</b></entry><entry namest="col2" nameend="col9" align="center" valign="middle"><b>VEHICLE</b></entry><entry namest="col10" nameend="col17" align="center" valign="middle"><b>F2207 (20 mg / kg / J)</b></entry><entry namest="col18" nameend="col25" align="center" valign="middle"><b>F2695 (10 mg / kg / D)</b></entry></row><row><entry align="center" valign="middle"><b>Animal No.</b></entry><entry align="center" valign="middle"><b>1</b></entry><entry align="center" valign="middle"><b>2</b></entry><entry align="center" valign="middle"><b>7</b></entry><entry align="center" valign="middle"><b>8</b></entry><entry align="center" valign="middle"><b>13</b></entry><entry align="center" valign="middle"><b>14</b></entry><entry namest="col8" nameend="col9" align="center" valign="middle"><b>Avg. esm</b></entry><entry align="center" valign="middle"><b>3</b></entry><entry align="center" valign="middle"><b>4</b></entry><entry align="center" valign="middle"><b>9</b></entry><entry align="center" valign="middle"><b>10</b></entry><entry align="center" valign="middle"><b>15</b></entry><entry align="center" valign="middle"><b>16</b></entry><entry namest="col16" nameend="col17" align="center" valign="middle"><b>Avg. esm</b></entry><entry align="center" valign="middle"><b>5</b></entry><entry align="center" valign="middle"><b>6</b></entry><entry align="center" valign="middle"><b>11</b></entry><entry align="center" valign="middle"><b>12</b></entry><entry align="center" valign="middle"><b>17</b></entry><entry align="center" valign="middle"><b>18</b></entry><entry namest="col24" nameend="col25" align="center" valign="middle"><b>Avg. esm</b></entry></row></thead><tbody><row><entry align="center" valign="middle">Time before treatment</entry><entry align="center" valign="middle">79</entry><entry align="center" valign="middle">77</entry><entry align="center" valign="middle">73</entry><entry align="center" valign="middle">77</entry><entry align="center" valign="middle">101</entry><entry align="center" valign="middle">76</entry><entry align="center" valign="middle">81</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">112</entry><entry align="center" valign="middle">89</entry><entry align="center" valign="middle">93</entry><entry align="center" valign="middle">88</entry><entry align="center" valign="middle">86</entry><entry align="center" valign="middle">91</entry><entry align="center" valign="middle">93</entry><entry align="center" valign="middle">4</entry><entry align="center" valign="middle">73</entry><entry align="center" valign="middle">89</entry><entry align="center" valign="middle">80</entry><entry align="center" valign="middle">71</entry><entry align="center" valign="middle">76</entry><entry align="center" valign="middle">78</entry><entry align="center" valign="middle">78</entry><entry align="center" valign="middle">3</entry></row></tbody></tgroup><tgroup cols="25"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="10mm" /><colspec colnum="3" colname="col3" colwidth="10mm" /><colspec colnum="4" colname="col4" colwidth="10mm" /><colspec colnum="5" colname="col5" colwidth="10mm" /><colspec colnum="6" colname="col6" colwidth="10mm" /><colspec colnum="7" colname="col7" colwidth="10mm" /><colspec colnum="8" colname="col8" colwidth="10mm" /><colspec colnum="9" colname="col9" colwidth="10mm" /><colspec colnum="10" colname="col10" colwidth="10mm" /><colspec colnum="11" colname="col11" colwidth="10mm" /><colspec colnum="12" colname="col12" colwidth="10mm" /><colspec colnum="13" colname="col13" colwidth="10mm" /><colspec colnum="14" colname="col14" colwidth="10mm" /><colspec colnum="15" colname="col15" colwidth="10mm" /><colspec colnum="16" colname="col16" colwidth="10mm" /><colspec colnum="17" colname="col17" colwidth="10mm" /><colspec colnum="18" colname="col18" colwidth="10mm" /><colspec colnum="19" colname="col19" colwidth="10mm" /><colspec colnum="20" colname="col20" colwidth="10mm" /><colspec colnum="21" colname="col21" colwidth="10mm" /><colspec colnum="22" colname="col22" colwidth="10mm" /><colspec colnum="23" colname="col23" colwidth="10mm" /><colspec colnum="24" colname="col24" colwidth="10mm" /><colspec colnum="25" colname="col25" colwidth="9mm" /><thead><row><entry namest="col1" nameend="col25" rowsep="0" align="left" valign="middle">Time after treatment (h)</entry></row></thead><tbody><row><entry rowsep="0" align="center" valign="middle">0.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry colsep="0" rowsep="0" align="center" valign="middle">76</entry><entry colsep="0" rowsep="0" align="center" valign="middle">70</entry><entry colsep="0" rowsep="0" align="center" valign="middle">63</entry><entry colsep="0" rowsep="0" align="center" valign="middle">80</entry><entry rowsep="0" align="center" valign="middle">70</entry><entry colsep="0" rowsep="0" align="center" valign="middle">74</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">92</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry rowsep="0" align="center" valign="middle">87</entry><entry colsep="0" rowsep="0" align="center" valign="middle">95</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry colsep="0" rowsep="0" align="center" valign="middle">90</entry><entry colsep="0" rowsep="0" align="center" valign="middle">108</entry><entry rowsep="0" align="center" valign="middle">85</entry><entry colsep="0" rowsep="0" align="center" valign="middle">94</entry><entry rowsep="0" align="center" valign="middle">3</entry></row><row><entry rowsep="0" align="center" valign="middle">1.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">82</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry colsep="0" rowsep="0" align="center" valign="middle">72</entry><entry colsep="0" rowsep="0" align="center" valign="middle">72</entry><entry rowsep="0" align="center" valign="middle">76</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry rowsep="0" align="center" valign="middle">2</entry><entry colsep="0" rowsep="0" align="center" valign="middle">130</entry><entry colsep="0" rowsep="0" align="center" valign="middle">117</entry><entry colsep="0" rowsep="0" align="center" valign="middle">113</entry><entry colsep="0" rowsep="0" align="center" valign="middle">113</entry><entry colsep="0" rowsep="0" align="center" valign="middle">90</entry><entry rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">112</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">112</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry colsep="0" rowsep="0" align="center" valign="middle">87</entry><entry rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">94</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">1.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">102</entry><entry colsep="0" rowsep="0" align="center" valign="middle">81</entry><entry colsep="0" rowsep="0" align="center" valign="middle">79</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">82</entry><entry rowsep="0" align="center" valign="middle">68</entry><entry colsep="0" rowsep="0" align="center" valign="middle">81</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">131</entry><entry colsep="0" rowsep="0" align="center" valign="middle">127</entry><entry colsep="0" rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">100</entry><entry rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">114</entry><entry rowsep="0" align="center" valign="middle">8</entry><entry colsep="0" rowsep="0" align="center" valign="middle">109</entry><entry colsep="0" rowsep="0" align="center" valign="middle">83</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry colsep="0" rowsep="0" align="center" valign="middle">87</entry><entry rowsep="0" align="center" valign="middle">112</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">2.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">83</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">71</entry><entry colsep="0" rowsep="0" align="center" valign="middle">98</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">80</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">123</entry><entry colsep="0" rowsep="0" align="center" valign="middle">113</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry colsep="0" rowsep="0" align="center" valign="middle">107</entry><entry rowsep="0" align="center" valign="middle">109</entry><entry colsep="0" rowsep="0" align="center" valign="middle">107</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">115</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry colsep="0" rowsep="0" align="center" valign="middle">93</entry><entry colsep="0" rowsep="0" align="center" valign="middle">95</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry rowsep="0" align="center" valign="middle">109</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">2.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">85</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry colsep="0" rowsep="0" align="center" valign="middle">85</entry><entry rowsep="0" align="center" valign="middle">79</entry><entry colsep="0" rowsep="0" align="center" valign="middle">81</entry><entry rowsep="0" align="center" valign="middle">2</entry><entry colsep="0" rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">111</entry><entry colsep="0" rowsep="0" align="center" valign="middle">116</entry><entry colsep="0" rowsep="0" align="center" valign="middle">101</entry><entry colsep="0" rowsep="0" align="center" valign="middle">115</entry><entry rowsep="0" align="center" valign="middle">107</entry><entry colsep="0" rowsep="0" align="center" valign="middle">115</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">111</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry colsep="0" rowsep="0" align="center" valign="middle">89</entry><entry colsep="0" rowsep="0" align="center" valign="middle">92</entry><entry rowsep="0" align="center" valign="middle">107</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry rowsep="0" align="center" valign="middle">4</entry></row><row><entry rowsep="0" align="center" valign="middle">3.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">95</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry colsep="0" rowsep="0" align="center" valign="middle">85</entry><entry colsep="0" rowsep="0" align="center" valign="middle">79</entry><entry rowsep="0" align="center" valign="middle">84</entry><entry colsep="0" rowsep="0" align="center" valign="middle">89</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">121</entry><entry colsep="0" rowsep="0" align="center" valign="middle">118</entry><entry colsep="0" rowsep="0" align="center" valign="middle">112</entry><entry colsep="0" rowsep="0" align="center" valign="middle">116</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry rowsep="0" align="center" valign="middle">92</entry><entry colsep="0" rowsep="0" align="center" valign="middle">111</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">104</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">100</entry><entry rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry rowsep="0" align="center" valign="middle">2</entry></row><row><entry rowsep="0" align="center" valign="middle">3.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">83</entry><entry colsep="0" rowsep="0" align="center" valign="middle">72</entry><entry colsep="0" rowsep="0" align="center" valign="middle">78</entry><entry colsep="0" rowsep="0" align="center" valign="middle">73</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry rowsep="0" align="center" valign="middle">65</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">120</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">133</entry><entry colsep="0" rowsep="0" align="center" valign="middle">116</entry><entry colsep="0" rowsep="0" align="center" valign="middle">103</entry><entry rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">114</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">96</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">94</entry><entry colsep="0" rowsep="0" align="center" valign="middle">107</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">4.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">81</entry><entry colsep="0" rowsep="0" align="center" valign="middle">79</entry><entry colsep="0" rowsep="0" align="center" valign="middle">75</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry colsep="0" rowsep="0" align="center" valign="middle">82</entry><entry rowsep="0" align="center" valign="middle">68</entry><entry colsep="0" rowsep="0" align="center" valign="middle">77</entry><entry rowsep="0" align="center" valign="middle">2</entry><entry colsep="0" rowsep="0" align="center" valign="middle">133</entry><entry colsep="0" rowsep="0" align="center" valign="middle">114</entry><entry colsep="0" rowsep="0" align="center" valign="middle">105</entry><entry colsep="0" rowsep="0" align="center" valign="middle">111</entry><entry colsep="0" rowsep="0" align="center" valign="middle">110</entry><entry rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">113</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">125</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry colsep="0" rowsep="0" align="center" valign="middle">108</entry><entry colsep="0" rowsep="0" align="center" valign="middle">80</entry><entry rowsep="0" align="center" valign="middle">109</entry><entry colsep="0" rowsep="0" align="center" valign="middle">102</entry><entry rowsep="0" align="center" valign="middle">6</entry></row><row><entry rowsep="0" align="center" valign="middle">4.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">82</entry><entry colsep="0" rowsep="0" align="center" valign="middle">76</entry><entry colsep="0" rowsep="0" align="center" valign="middle">91</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry colsep="0" rowsep="0" align="center" valign="middle">113</entry><entry rowsep="0" align="center" valign="middle">85</entry><entry colsep="0" rowsep="0" align="center" valign="middle">89</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">110</entry><entry colsep="0" rowsep="0" align="center" valign="middle">126</entry><entry colsep="0" rowsep="0" align="center" valign="middle">109</entry><entry colsep="0" rowsep="0" align="center" valign="middle">104</entry><entry rowsep="0" align="center" valign="middle">108</entry><entry colsep="0" rowsep="0" align="center" valign="middle">115</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">104</entry><entry colsep="0" rowsep="0" align="center" valign="middle">92</entry><entry colsep="0" rowsep="0" align="center" valign="middle">100</entry><entry colsep="0" rowsep="0" align="center" valign="middle">85</entry><entry rowsep="0" align="center" valign="middle">108</entry><entry colsep="0" rowsep="0" align="center" valign="middle">99</entry><entry rowsep="0" align="center" valign="middle">3</entry></row><row><entry rowsep="0" align="center" valign="middle">5.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry colsep="0" rowsep="0" align="center" valign="middle">79</entry><entry colsep="0" rowsep="0" align="center" valign="middle">67</entry><entry colsep="0" rowsep="0" align="center" valign="middle">95</entry><entry colsep="0" rowsep="0" align="center" valign="middle">81</entry><entry rowsep="0" align="center" valign="middle">82</entry><entry colsep="0" rowsep="0" align="center" valign="middle">84</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">116</entry><entry colsep="0" rowsep="0" align="center" valign="middle">120</entry><entry colsep="0" rowsep="0" align="center" valign="middle">98</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry colsep="0" rowsep="0" align="center" valign="middle">97</entry><entry rowsep="0" align="center" valign="middle">105</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">126</entry><entry colsep="0" rowsep="0" align="center" valign="middle">100</entry><entry colsep="0" rowsep="0" align="center" valign="middle">92</entry><entry colsep="0" rowsep="0" align="center" valign="middle">95</entry><entry colsep="0" rowsep="0" align="center" valign="middle">110</entry><entry rowsep="0" align="center" valign="middle">102</entry><entry colsep="0" rowsep="0" align="center" valign="middle">104</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">5.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">94</entry><entry colsep="0" rowsep="0" align="center" valign="middle">80</entry><entry colsep="0" rowsep="0" align="center" valign="middle">70</entry><entry colsep="0" rowsep="0" align="center" valign="middle">ND</entry><entry colsep="0" rowsep="0" align="center" valign="middle">85</entry><entry rowsep="0" align="center" valign="middle">82</entry><entry colsep="0" rowsep="0" align="center" valign="middle">82</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">103</entry><entry colsep="0" rowsep="0" align="center" valign="middle">107</entry><entry colsep="0" rowsep="0" align="center" valign="middle">115</entry><entry colsep="0" rowsep="0" align="center" valign="middle">106</entry><entry colsep="0" rowsep="0" align="center" valign="middle">92</entry><entry rowsep="0" align="center" valign="middle">93</entry><entry colsep="0" rowsep="0" align="center" valign="middle">103</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">88</entry><entry colsep="0" rowsep="0" align="center" valign="middle">86</entry><entry colsep="0" rowsep="0" align="center" valign="middle">105</entry><entry colsep="0" rowsep="0" align="center" valign="middle">98</entry><entry colsep="0" rowsep="0" align="center" valign="middle">89</entry><entry rowsep="0" align="center" valign="middle">99</entry><entry colsep="0" rowsep="0" align="center" valign="middle">94</entry><entry rowsep="0" align="center" valign="middle">3</entry></row><row><entry align="center" valign="middle">6.00</entry><entry colsep="0" align="center" valign="middle">83</entry><entry colsep="0" align="center" valign="middle">74</entry><entry colsep="0" align="center" valign="middle">82</entry><entry colsep="0" align="center" valign="middle">82</entry><entry colsep="0" align="center" valign="middle">78</entry><entry align="center" valign="middle">77</entry><entry colsep="0" align="center" valign="middle">79</entry><entry align="center" valign="middle">1</entry><entry colsep="0" align="center" valign="middle">115</entry><entry colsep="0" align="center" valign="middle">133</entry><entry colsep="0" align="center" valign="middle">120</entry><entry colsep="0" align="center" valign="middle">104</entry><entry colsep="0" align="center" valign="middle">103</entry><entry align="center" valign="middle">104</entry><entry colsep="0" align="center" valign="middle">113</entry><entry align="center" valign="middle">5</entry><entry colsep="0" align="center" valign="middle">101</entry><entry colsep="0" align="center" valign="middle">113</entry><entry colsep="0" align="center" valign="middle">98</entry><entry colsep="0" align="center" valign="middle">105</entry><entry colsep="0" align="center" valign="middle">109</entry><entry align="center" valign="middle">108</entry><entry colsep="0" align="center" valign="middle">106</entry><entry align="center" valign="middle">2</entry></row><row><entry align="center" valign="middle"><b>Average PAD after treatment</b></entry><entry colsep="0" align="center" valign="middle"><b>87</b></entry><entry colsep="0" align="center" valign="middle"><b>79</b></entry><entry colsep="0" align="center" valign="middle"><b>78</b></entry><entry colsep="0" align="center" valign="middle"><b>81</b></entry><entry colsep="0" align="center" valign="middle"><b>83</b></entry><entry align="center" valign="middle"><b>76</b></entry><entry colsep="0" align="center" valign="middle"><b>81</b></entry><entry align="center" valign="middle"><b>2</b></entry><entry colsep="0" align="center" valign="middle"><b>122</b></entry><entry colsep="0" align="center" valign="middle"><b>115</b></entry><entry colsep="0" align="center" valign="middle"><b>114</b></entry><entry colsep="0" align="center" valign="middle"><b>104</b></entry><entry colsep="0" align="center" valign="middle"><b>101</b></entry><entry align="center" valign="middle"><b>101</b></entry><entry colsep="0" align="center" valign="middle"><b>110</b></entry><entry align="center" valign="middle"><b>4</b></entry><entry colsep="0" align="center" valign="middle"><b>107</b></entry><entry colsep="0" align="center" valign="middle"><b>95</b></entry><entry colsep="0" align="center" valign="middle"><b>94</b></entry><entry colsep="0" align="center" valign="middle"><b>98</b></entry><entry colsep="0" align="center" valign="middle"><b>92</b></entry><entry align="center" valign="middle"><b>104</b></entry><entry colsep="0" align="center" valign="middle"><b>98</b></entry><entry align="center" valign="middle"><b>2</b></entry></row></tbody></tgroup><tgroup cols="25" rowsep="0"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="9mm" /><colspec colnum="3" colname="col3" colwidth="7mm" /><colspec colnum="4" colname="col4" colwidth="7mm" /><colspec colnum="5" colname="col5" colwidth="7mm" /><colspec colnum="6" colname="col6" colwidth="9mm" /><colspec colnum="7" colname="col7" colwidth="7mm" /><colspec colnum="8" colname="col8" colwidth="7mm" /><colspec colnum="9" colname="col9" colwidth="8mm" /><colspec colnum="10" colname="col10" colwidth="11mm" /><colspec colnum="11" colname="col11" colwidth="11mm" /><colspec colnum="12" colname="col12" colwidth="11mm" /><colspec colnum="13" colname="col13" colwidth="11mm" /><colspec colnum="14" colname="col14" colwidth="11mm" /><colspec colnum="15" colname="col15" colwidth="11mm" /><colspec colnum="16" colname="col16" colwidth="11mm" /><colspec colnum="17" colname="col17" colwidth="9mm" /><colspec colnum="18" colname="col18" colwidth="11mm" /><colspec colnum="19" colname="col19" colwidth="11mm" /><colspec colnum="20" colname="col20" colwidth="11mm" /><colspec colnum="21" colname="col21" colwidth="11mm" /><colspec colnum="22" colname="col22" colwidth="11mm" /><colspec colnum="23" colname="col23" colwidth="11mm" /><colspec colnum="24" colname="col24" colwidth="11mm" /><colspec colnum="25" colname="col25" colwidth="9mm" /><tbody><row><entry namest="col1" nameend="col25" align="justify">ND: not determined</entry></row></tbody></tgroup></table></tables><tables id="tabl0010" num="0010"><table frame="all"><title><b>Table 5: Individual systolic blood pressure data</b></title><tgroup cols="25"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="10mm" colsep="0" /><colspec colnum="3" colname="col3" colwidth="10mm" colsep="0" /><colspec colnum="4" colname="col4" colwidth="10mm" colsep="0" /><colspec colnum="5" colname="col5" colwidth="10mm" colsep="0" /><colspec colnum="6" colname="col6" colwidth="10mm" colsep="0" /><colspec colnum="7" colname="col7" colwidth="10mm" /><colspec colnum="8" colname="col8" colwidth="10mm" colsep="0" /><colspec colnum="9" colname="col9" colwidth="10mm" /><colspec colnum="10" colname="col10" colwidth="10mm" colsep="0" /><colspec colnum="11" colname="col11" colwidth="10mm" colsep="0" /><colspec colnum="12" colname="col12" colwidth="10mm" colsep="0" /><colspec colnum="13" colname="col13" colwidth="10mm" colsep="0" /><colspec colnum="14" colname="col14" colwidth="10mm" colsep="0" /><colspec colnum="15" colname="col15" colwidth="10mm" /><colspec colnum="16" colname="col16" colwidth="10mm" colsep="0" /><colspec colnum="17" colname="col17" colwidth="10mm" /><colspec colnum="18" colname="col18" colwidth="10mm" colsep="0" /><colspec colnum="19" colname="col19" colwidth="10mm" colsep="0" /><colspec colnum="20" colname="col20" colwidth="10mm" colsep="0" /><colspec colnum="21" colname="col21" colwidth="10mm" colsep="0" /><colspec colnum="22" colname="col22" colwidth="10mm" colsep="0" /><colspec colnum="23" colname="col23" colwidth="10mm" /><colspec colnum="24" colname="col24" colwidth="10mm" colsep="0" /><colspec colnum="25" colname="col25" colwidth="9mm" /><thead><row><entry namest="col1" nameend="col25" align="left" valign="middle"><b>SYSTOLIC BLOOD PRESSURE (NOT expressed in mm Hg)</b></entry></row><row><entry namest="col1" nameend="col25" align="left" valign="middle">Individual data after repeated administration 5 consecutive days</entry></row><row><entry align="center" valign="middle"><b>GROUP</b></entry><entry namest="col2" nameend="col9" align="center" valign="middle"><b>1</b></entry><entry namest="col10" nameend="col17" align="center" valign="middle"><b>2</b></entry><entry namest="col18" nameend="col25" align="center" valign="middle"><b>3</b></entry></row><row><entry align="center" valign="middle"><b>TREATMENT</b></entry><entry namest="col2" nameend="col9" align="center" valign="middle"><b>VEHICLE</b></entry><entry namest="col10" nameend="col17" align="center" valign="middle"><b>F2207 (20 mg / kg / J)</b></entry><entry namest="col18" nameend="col25" align="center" valign="middle"><b>F2695 (10 mg / kg / D)</b></entry></row><row><entry align="center" valign="middle"><b>Animal No.</b></entry><entry align="center" valign="middle"><b>1</b></entry><entry align="center" valign="middle"><b>2</b></entry><entry align="center" valign="middle"><b>7</b></entry><entry align="center" valign="middle"><b>8</b></entry><entry align="center" valign="middle"><b>13</b></entry><entry align="center" valign="middle"><b>14</b></entry><entry namest="col8" nameend="col9" align="center" valign="middle"><b>Avg. esm</b></entry><entry align="center" valign="middle"><b>3</b></entry><entry align="center" valign="middle"><b>4</b></entry><entry align="center" valign="middle"><b>9</b></entry><entry align="center" valign="middle"><b>10</b></entry><entry align="center" valign="middle"><b>15</b></entry><entry align="center" valign="middle"><b>16</b></entry><entry namest="col16" nameend="col17" align="center" valign="middle"><b>Avg. esm</b></entry><entry align="center" valign="middle"><b>5</b></entry><entry align="center" valign="middle"><b>6</b></entry><entry align="center" valign="middle"><b>11</b></entry><entry align="center" valign="middle"><b>12</b></entry><entry align="center" valign="middle"><b>17</b></entry><entry align="center" valign="middle"><b>18</b></entry><entry namest="col24" nameend="col25" align="center" valign="middle"><b>Avg. esm</b></entry></row></thead><tbody><row><entry align="center" valign="middle">Time before treatment</entry><entry align="center" valign="middle">139</entry><entry align="center" valign="middle">141</entry><entry align="center" valign="middle">120</entry><entry align="center" valign="middle">157</entry><entry align="center" valign="middle">172</entry><entry align="center" valign="middle">138</entry><entry align="center" valign="middle">145</entry><entry align="center" valign="middle">7</entry><entry align="center" valign="middle">188</entry><entry align="center" valign="middle">164</entry><entry align="center" valign="middle">176</entry><entry align="center" valign="middle">149</entry><entry align="center" valign="middle">130</entry><entry align="center" valign="middle">169</entry><entry align="center" valign="middle">163</entry><entry align="center" valign="middle">8</entry><entry align="center" valign="middle">136</entry><entry align="center" valign="middle">141</entry><entry align="center" valign="middle">138</entry><entry align="center" valign="middle">130</entry><entry align="center" valign="middle">134</entry><entry align="center" valign="middle">149</entry><entry align="center" valign="middle">138</entry><entry align="center" valign="middle">3</entry></row></tbody></tgroup><tgroup cols="25"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="10mm" /><colspec colnum="3" colname="col3" colwidth="10mm" /><colspec colnum="4" colname="col4" colwidth="10mm" /><colspec colnum="5" colname="col5" colwidth="10mm" /><colspec colnum="6" colname="col6" colwidth="10mm" /><colspec colnum="7" colname="col7" colwidth="10mm" /><colspec colnum="8" colname="col8" colwidth="10mm" /><colspec colnum="9" colname="col9" colwidth="10mm" /><colspec colnum="10" colname="col10" colwidth="10mm" /><colspec colnum="11" colname="col11" colwidth="10mm" /><colspec colnum="12" colname="col12" colwidth="10mm" /><colspec colnum="13" colname="col13" colwidth="10mm" /><colspec colnum="14" colname="col14" colwidth="10mm" /><colspec colnum="15" colname="col15" colwidth="10mm" /><colspec colnum="16" colname="col16" colwidth="10mm" /><colspec colnum="17" colname="col17" colwidth="10mm" /><colspec colnum="18" colname="col18" colwidth="10mm" /><colspec colnum="19" colname="col19" colwidth="10mm" /><colspec colnum="20" colname="col20" colwidth="10mm" /><colspec colnum="21" colname="col21" colwidth="10mm" /><colspec colnum="22" colname="col22" colwidth="10mm" /><colspec colnum="23" colname="col23" colwidth="10mm" /><colspec colnum="24" colname="col24" colwidth="10mm" /><colspec colnum="25" colname="col25" colwidth="9mm" /><thead><row><entry namest="col1" nameend="col25" rowsep="0" align="left" valign="middle">Time after treatment (h)</entry></row></thead><tbody><row><entry rowsep="0" align="center" valign="middle">0.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">132</entry><entry colsep="0" rowsep="0" align="center" valign="middle">119</entry><entry colsep="0" rowsep="0" align="center" valign="middle">131</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry rowsep="0" align="center" valign="middle">138</entry><entry colsep="0" rowsep="0" align="center" valign="middle">134</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">152</entry><entry colsep="0" rowsep="0" align="center" valign="middle">128</entry><entry colsep="0" rowsep="0" align="center" valign="middle">126</entry><entry rowsep="0" align="center" valign="middle">129</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry rowsep="0" align="center" valign="middle">6</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">129</entry><entry colsep="0" rowsep="0" align="center" valign="middle">140</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry rowsep="0" align="center" valign="middle">139</entry><entry colsep="0" rowsep="0" align="center" valign="middle">140</entry><entry rowsep="0" align="center" valign="middle">4</entry></row><row><entry rowsep="0" align="center" valign="middle">1.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">134</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">129</entry><entry colsep="0" rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry rowsep="0" align="center" valign="middle">143</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">180</entry><entry colsep="0" rowsep="0" align="center" valign="middle">167</entry><entry colsep="0" rowsep="0" align="center" valign="middle">157</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry colsep="0" rowsep="0" align="center" valign="middle">126</entry><entry rowsep="0" align="center" valign="middle">130</entry><entry colsep="0" rowsep="0" align="center" valign="middle">152</entry><entry rowsep="0" align="center" valign="middle">9</entry><entry colsep="0" rowsep="0" align="center" valign="middle">159</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">124</entry><entry colsep="0" rowsep="0" align="center" valign="middle">148</entry><entry colsep="0" rowsep="0" align="center" valign="middle">131</entry><entry rowsep="0" align="center" valign="middle">143</entry><entry colsep="0" rowsep="0" align="center" valign="middle">140</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">1.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">186</entry><entry colsep="0" rowsep="0" align="center" valign="middle">181</entry><entry colsep="0" rowsep="0" align="center" valign="middle">189</entry><entry colsep="0" rowsep="0" align="center" valign="middle">129</entry><entry colsep="0" rowsep="0" align="center" valign="middle">136</entry><entry rowsep="0" align="center" valign="middle">138</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry rowsep="0" align="center" valign="middle">12</entry><entry colsep="0" rowsep="0" align="center" valign="middle">164</entry><entry colsep="0" rowsep="0" align="center" valign="middle">119</entry><entry colsep="0" rowsep="0" align="center" valign="middle">138</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">127</entry><entry rowsep="0" align="center" valign="middle">156</entry><entry colsep="0" rowsep="0" align="center" valign="middle">144</entry><entry rowsep="0" align="center" valign="middle">8</entry></row><row><entry rowsep="0" align="center" valign="middle">2.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">138</entry><entry colsep="0" rowsep="0" align="center" valign="middle">136</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">173</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">148</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">171</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry colsep="0" rowsep="0" align="center" valign="middle">146</entry><entry colsep="0" rowsep="0" align="center" valign="middle">122</entry><entry colsep="0" rowsep="0" align="center" valign="middle">140</entry><entry rowsep="0" align="center" valign="middle">163</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry rowsep="0" align="center" valign="middle">7</entry><entry colsep="0" rowsep="0" align="center" valign="middle">168</entry><entry colsep="0" rowsep="0" align="center" valign="middle">125</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry colsep="0" rowsep="0" align="center" valign="middle">127</entry><entry rowsep="0" align="center" valign="middle">156</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry rowsep="0" align="center" valign="middle">7</entry></row><row><entry rowsep="0" align="center" valign="middle">2.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry colsep="0" rowsep="0" align="center" valign="middle">143</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">159</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry rowsep="0" align="center" valign="middle">148</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">195</entry><entry colsep="0" rowsep="0" align="center" valign="middle">168</entry><entry colsep="0" rowsep="0" align="center" valign="middle">168</entry><entry colsep="0" rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry rowsep="0" align="center" valign="middle">161</entry><entry colsep="0" rowsep="0" align="center" valign="middle">165</entry><entry rowsep="0" align="center" valign="middle">7</entry><entry colsep="0" rowsep="0" align="center" valign="middle">165</entry><entry colsep="0" rowsep="0" align="center" valign="middle">124</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry colsep="0" rowsep="0" align="center" valign="middle">134</entry><entry rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">143</entry><entry rowsep="0" align="center" valign="middle">6</entry></row><row><entry rowsep="0" align="center" valign="middle">3.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry colsep="0" rowsep="0" align="center" valign="middle">167</entry><entry colsep="0" rowsep="0" align="center" valign="middle">162</entry><entry colsep="0" rowsep="0" align="center" valign="middle">163</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry rowsep="0" align="center" valign="middle">3</entry><entry colsep="0" rowsep="0" align="center" valign="middle">173</entry><entry colsep="0" rowsep="0" align="center" valign="middle">177</entry><entry colsep="0" rowsep="0" align="center" valign="middle">164</entry><entry colsep="0" rowsep="0" align="center" valign="middle">157</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry rowsep="0" align="center" valign="middle">146</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry rowsep="0" align="center" valign="middle">6</entry><entry colsep="0" rowsep="0" align="center" valign="middle">156</entry><entry colsep="0" rowsep="0" align="center" valign="middle">131</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">157</entry><entry colsep="0" rowsep="0" align="center" valign="middle">147</entry><entry rowsep="0" align="center" valign="middle">4</entry></row><row><entry rowsep="0" align="center" valign="middle">3.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">135</entry><entry colsep="0" rowsep="0" align="center" valign="middle">129</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry colsep="0" rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">143</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">165</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry colsep="0" rowsep="0" align="center" valign="middle">184</entry><entry colsep="0" rowsep="0" align="center" valign="middle">167</entry><entry colsep="0" rowsep="0" align="center" valign="middle">139</entry><entry rowsep="0" align="center" valign="middle">155</entry><entry colsep="0" rowsep="0" align="center" valign="middle">161</entry><entry rowsep="0" align="center" valign="middle">6</entry><entry colsep="0" rowsep="0" align="center" valign="middle">146</entry><entry colsep="0" rowsep="0" align="center" valign="middle">147</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry colsep="0" rowsep="0" align="center" valign="middle">169</entry><entry colsep="0" rowsep="0" align="center" valign="middle">123</entry><entry rowsep="0" align="center" valign="middle">156</entry><entry colsep="0" rowsep="0" align="center" valign="middle">147</entry><entry rowsep="0" align="center" valign="middle">6</entry></row><row><entry rowsep="0" align="center" valign="middle">4.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry colsep="0" rowsep="0" align="center" valign="middle">143</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry colsep="0" rowsep="0" align="center" valign="middle">166</entry><entry colsep="0" rowsep="0" align="center" valign="middle">164</entry><entry rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">180</entry><entry colsep="0" rowsep="0" align="center" valign="middle">157</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry colsep="0" rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry rowsep="0" align="center" valign="middle">153</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">180</entry><entry colsep="0" rowsep="0" align="center" valign="middle">132</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry colsep="0" rowsep="0" align="center" valign="middle">124</entry><entry rowsep="0" align="center" valign="middle">164</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">9</entry></row><row><entry rowsep="0" align="center" valign="middle">4.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">140</entry><entry colsep="0" rowsep="0" align="center" valign="middle">159</entry><entry colsep="0" rowsep="0" align="center" valign="middle">170</entry><entry colsep="0" rowsep="0" align="center" valign="middle">190</entry><entry rowsep="0" align="center" valign="middle">152</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry rowsep="0" align="center" valign="middle">8</entry><entry colsep="0" rowsep="0" align="center" valign="middle">184</entry><entry colsep="0" rowsep="0" align="center" valign="middle">161</entry><entry colsep="0" rowsep="0" align="center" valign="middle">180</entry><entry colsep="0" rowsep="0" align="center" valign="middle">155</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry rowsep="0" align="center" valign="middle">168</entry><entry colsep="0" rowsep="0" align="center" valign="middle">166</entry><entry rowsep="0" align="center" valign="middle">6</entry><entry colsep="0" rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry colsep="0" rowsep="0" align="center" valign="middle">138</entry><entry colsep="0" rowsep="0" align="center" valign="middle">163</entry><entry colsep="0" rowsep="0" align="center" valign="middle">131</entry><entry rowsep="0" align="center" valign="middle">163</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">5</entry></row><row><entry rowsep="0" align="center" valign="middle">5.00</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry colsep="0" rowsep="0" align="center" valign="middle">146</entry><entry colsep="0" rowsep="0" align="center" valign="middle">127</entry><entry colsep="0" rowsep="0" align="center" valign="middle">177</entry><entry colsep="0" rowsep="0" align="center" valign="middle">160</entry><entry rowsep="0" align="center" valign="middle">145</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry rowsep="0" align="center" valign="middle">7</entry><entry colsep="0" rowsep="0" align="center" valign="middle">161</entry><entry colsep="0" rowsep="0" align="center" valign="middle">171</entry><entry colsep="0" rowsep="0" align="center" valign="middle">146</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry colsep="0" rowsep="0" align="center" valign="middle">139</entry><entry rowsep="0" align="center" valign="middle">166</entry><entry colsep="0" rowsep="0" align="center" valign="middle">154</entry><entry rowsep="0" align="center" valign="middle">6</entry><entry colsep="0" rowsep="0" align="center" valign="middle">182</entry><entry colsep="0" rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">137</entry><entry colsep="0" rowsep="0" align="center" valign="middle">150</entry><entry colsep="0" rowsep="0" align="center" valign="middle">162</entry><entry rowsep="0" align="center" valign="middle">158</entry><entry colsep="0" rowsep="0" align="center" valign="middle">156</entry><entry rowsep="0" align="center" valign="middle">6</entry></row><row><entry rowsep="0" align="center" valign="middle">5.50</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry colsep="0" rowsep="0" align="center" valign="middle">149</entry><entry colsep="0" rowsep="0" align="center" valign="middle">132</entry><entry colsep="0" rowsep="0" align="center" valign="middle">ND</entry><entry colsep="0" rowsep="0" align="center" valign="middle">148</entry><entry rowsep="0" align="center" valign="middle">144</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry rowsep="0" align="center" valign="middle">4</entry><entry colsep="0" rowsep="0" align="center" valign="middle">151</entry><entry colsep="0" rowsep="0" align="center" valign="middle">154</entry><entry colsep="0" rowsep="0" align="center" valign="middle">173</entry><entry colsep="0" rowsep="0" align="center" valign="middle">152</entry><entry colsep="0" rowsep="0" align="center" valign="middle">132</entry><entry rowsep="0" align="center" valign="middle">155</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry rowsep="0" align="center" valign="middle">5</entry><entry colsep="0" rowsep="0" align="center" valign="middle">142</entry><entry colsep="0" rowsep="0" align="center" valign="middle">127</entry><entry colsep="0" rowsep="0" align="center" valign="middle">152</entry><entry colsep="0" rowsep="0" align="center" valign="middle">153</entry><entry colsep="0" rowsep="0" align="center" valign="middle">141</entry><entry rowsep="0" align="center" valign="middle">153</entry><entry colsep="0" rowsep="0" align="center" valign="middle">145</entry><entry rowsep="0" align="center" valign="middle">4</entry></row><row><entry align="center" valign="middle">6.00</entry><entry colsep="0" align="center" valign="middle">146</entry><entry colsep="0" align="center" valign="middle">144</entry><entry colsep="0" align="center" valign="middle">151</entry><entry colsep="0" align="center" valign="middle">176</entry><entry colsep="0" align="center" valign="middle">146</entry><entry align="center" valign="middle">143</entry><entry colsep="0" align="center" valign="middle">151</entry><entry align="center" valign="middle">5</entry><entry colsep="0" align="center" valign="middle">158</entry><entry colsep="0" align="center" valign="middle">192</entry><entry colsep="0" align="center" valign="middle">171</entry><entry colsep="0" align="center" valign="middle">154</entry><entry colsep="0" align="center" valign="middle">148</entry><entry align="center" valign="middle">172</entry><entry colsep="0" align="center" valign="middle">166</entry><entry align="center" valign="middle">7</entry><entry colsep="0" align="center" valign="middle">156</entry><entry colsep="0" align="center" valign="middle">170</entry><entry colsep="0" align="center" valign="middle">148</entry><entry colsep="0" align="center" valign="middle">159</entry><entry colsep="0" align="center" valign="middle">160</entry><entry align="center" valign="middle">166</entry><entry colsep="0" align="center" valign="middle">160</entry><entry align="center" valign="middle">3</entry></row><row><entry align="center" valign="middle"><b>NOT average after treatment</b></entry><entry colsep="0" align="center" valign="middle"><b>143</b></entry><entry colsep="0" align="center" valign="middle"><b>145</b></entry><entry colsep="0" align="center" valign="middle"><b>143</b></entry><entry colsep="0" align="center" valign="middle"><b>160</b></entry><entry colsep="0" align="center" valign="middle"><b>155</b></entry><entry align="center" valign="middle"><b>144</b></entry><entry colsep="0" align="center" valign="middle"><b>148</b></entry><entry align="center" valign="middle"><b>3</b></entry><entry colsep="0" align="center" valign="middle"><b>172</b></entry><entry colsep="0" align="center" valign="middle"><b>166</b></entry><entry colsep="0" align="center" valign="middle"><b>165</b></entry><entry colsep="0" align="center" valign="middle"><b>146</b></entry><entry colsep="0" align="center" valign="middle"><b>140</b></entry><entry align="center" valign="middle"><b>153</b></entry><entry colsep="0" align="center" valign="middle"><b>157</b></entry><entry align="center" valign="middle"><b>5</b></entry><entry colsep="0" align="center" valign="middle"><b>159</b></entry><entry colsep="0" align="center" valign="middle"><b>136</b></entry><entry colsep="0" align="center" valign="middle"><b>140</b></entry><entry colsep="0" align="center" valign="middle"><b>152</b></entry><entry colsep="0" align="center" valign="middle"><b>139</b></entry><entry align="center" valign="middle"><b>155</b></entry><entry colsep="0" align="center" valign="middle"><b>147</b></entry><entry align="center" valign="middle"><b>4</b></entry></row></tbody></tgroup><tgroup cols="25" rowsep="0"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="9mm" /><colspec colnum="3" colname="col3" colwidth="9mm" /><colspec colnum="4" colname="col4" colwidth="9mm" /><colspec colnum="5" colname="col5" colwidth="9mm" /><colspec colnum="6" colname="col6" colwidth="9mm" /><colspec colnum="7" colname="col7" colwidth="9mm" /><colspec colnum="8" colname="col8" colwidth="9mm" /><colspec colnum="9" colname="col9" colwidth="7mm" /><colspec colnum="10" colname="col10" colwidth="9mm" /><colspec colnum="11" colname="col11" colwidth="9mm" /><colspec colnum="12" colname="col12" colwidth="9mm" /><colspec colnum="13" colname="col13" colwidth="9mm" /><colspec colnum="14" colname="col14" colwidth="9mm" /><colspec colnum="15" colname="col15" colwidth="10mm" /><colspec colnum="16" colname="col16" colwidth="11mm" /><colspec colnum="17" colname="col17" colwidth="9mm" /><colspec colnum="18" colname="col18" colwidth="11mm" /><colspec colnum="19" colname="col19" colwidth="11mm" /><colspec colnum="20" colname="col20" colwidth="11mm" /><colspec colnum="21" colname="col21" colwidth="11mm" /><colspec colnum="22" colname="col22" colwidth="11mm" /><colspec colnum="23" colname="col23" colwidth="11mm" /><colspec colnum="24" colname="col24" colwidth="11mm" /><colspec colnum="25" colname="col25" colwidth="9mm" /><tbody><row><entry namest="col1" nameend="col25" align="justify">ND: not determined</entry></row></tbody></tgroup></table></tables>
3.4. <u style="single">CONCLUSION</u>
Under the experimental conditions of this evaluation, carried out in four successive studies by oral administration in the vigilant dog fitted with telemetry:<ul id="ul0019" list-style="none" compact="compact"><li>¤ in single administration and compared to the control group (n = 28), the increase in heart rate is clearly significant and lasting with F2207 at a dose of 20 mg / kg / D; it is statistically and clinically less and more transient with F2695 at the pharmacologically equiactive dose of 10 mg / kg / D,</li><li>¤ F2695, at a dose of 10 mg / kg / D, does not cause any statistically significant change in systolic blood pressure averaged over the 6 hours following the last treatment, at steady state after repeated administration 5 days,</li><li>¤ a statistically significant difference is highlighted on the values of the diastolic blood pressure averaged over the 6 hours following the last treatment, at steady state after repeated administration 5 days, between the active enantiomer F2695 (98 ± 2 mm Hg) and the racemic F2207 with pharmacologically active equidoses (110 ± 4 mm Hg).</li></ul>
These differences clearly demonstrate better cardiovascular tolerance of the active enantiomer F2695.
<u style="single">EXAMPLE 4:</u> PHARMACO-TOXICO-GENOMIC PREDICTIVITY TEST <i>IN VITRO.</i>
<u style="single">4.1. MATERIALS AND METHODS</u>
The compounds F2695 and F2696, enantiomers of the racemic molecule F2207, as well as a reference product, clomipramine (coded in test C218) were evaluated during the present study. The two enantiomers F2695 and F2696 were first evaluated in a preliminary cytotoxicity test (MTT test) on primary rat hepatocytes, so as to select the three concentrations to be used in the final test.
After treatment of primary rat hepatocytes in culture, the RNA was extracted so as to generate labeled complementary DNA probes, which were then hybridized on a membrane containing 682 alternative splicing fragments specific for cell stress. Toxicity indices were obtained, for each product tested, by comparing the hybridization profile of the treated cells with that obtained from untreated cells.
<i>4.1.1.</i> Principle and purpose of the study
Safe-Hit is a sensitive, robust, reliable, fast and safe predictive pharmaco-toxicogenomic test that allows the comparison and classification of products, based on the optimized assessment of their toxic potential.
Safe-Hit benefits from a technology, property of EXONHIT (DATAS ™: <i><u style="single">D</u>ifferential <u style="single">AT</u>analysis of <u style="single">T</u>ranscripts with <u style="single">AT</u>alternative <u style="single">S</u>plicing</i>), making it possible to isolate and, therefore, to clone the splicing events resulting from a given biological state, compared to a control condition. This allows the isolation of mRNA isoforms, expressed differently depending on the biological condition.
Safe-Hit allows the classification of molecules within a chemical series, according to a Toxic Index, determined after the following basic steps (systematically implemented in duplicate for each product):<ul id="ul0020" list-style="dash" compact="compact"><li>treatment of cell lines with the different products, at three concentrations deduced from a prior cell toxicology test (MTT test): a reference concentration corresponding to 80% cell viability, a concentration 10 times higher - when possible - and a concentration 10 times lower,</li><li>preparation of total RNA and corresponding radiolabeled cDNA probes,</li><li>hybridization of cDNA probes: Safe-Hit macro-array containing 682 independent clones, corresponding to splicing modifications induced by the overexpression of WTp53 (p53 is the most ubiquitous "mediator" of cellular stress chosen for the development of this methodology ),</li><li>acquisition and determination of the Toxicity Index.</li></ul>
4.1.2. Cells
The cells used for the study (prior MTT cytotoxicity test and main test) are cryopreserved hepatocytes from Sprague-Dawley rats in primoculture (lots Hep 184005 and Hep 184006 - Biopredic), cultured under standard conditions.
<i>4.1.2.1 Culture media</i>
<ul id="ul0021" list-style="dash" compact="compact"><li><i>thawing medium</i> : Leibovitz 15 medium with glutamax 1, supplemented with 100 IU / ml of penicillin, 100 μg / ml of streptomycin and 0.6 M of glucose (batch MIL 210009 -Biopredic),</li><li><i>sowing medium:</i> Williams E medium with glutamax 1, supplemented with 100 IU / ml of penicillin, 100 µg / ml of streptomycin, 4 µg / ml of bovine insulin and 10% v / v of fetal calf serum (lot MIL 260005) - Biopredic ),</li><li><i>incubation medium:</i> Williams E medium with glutamax 1, supplemented with 100 IU / ml of penicillin, 100 µg / ml of streptomycin, 4 µg / ml of bovine insulin and</li></ul>50 µM hydrocortisone hemisuccinate (lot MIL 260009-260007 - Biopredic).
<i>4.1.2.2 Culture conditions</i>
37 ° C, CO2 atmosphere (5%), relative humidity (95%).
<i>4.1.2.3 Culture process</i>
<tables id="tabl0011" num="0011"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="57mm" /><colspec colnum="3" colname="col3" colwidth="63mm" /><thead><row><entry align="center" valign="top" /><entry align="center" valign="top">Cell toxicity test</entry><entry align="center" valign="top">Main study</entry></row></thead><tbody><row><entry align="center" /><entry namest="col2" nameend="col3" align="center">Cells seeded on the day of treatment</entry></row><row><entry align="center">Seeding density</entry><entry align="center">35 000 cells / well (96-well plate)</entry><entry align="center">1.5 million cells per 30 mm plate</entry></row><row><entry align="center">Medium volume</entry><entry align="center">0.1 ml</entry><entry align="center">3 ml</entry></row></tbody></tgroup></table></tables>
4.1.3. Cytotoxicity test
This cytotoxicity test (MTT test) detects living cells through a colorimetric reaction which reveals the integrity of cellular respiration involving the activity of mitochondria. MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide), soluble in water, is transformed by cleavage, under the action of a mitochondrial enzyme from living cells, into an insoluble purple formazan. The formazan is dissolved in an organic solvent and the solution obtained can be measured by spectrophotometry. The absorbance measured is proportional to the number of surviving cells.
The cells are brought into contact for 16 hours with the product to be tested at 5 different concentrations (0 - 1 - 10 - 25 - 50 and 100 μM).
After this exposure phase, a solution of MTT (0.5 mg / ml in the incubation medium for primary hepatocytes) is added for 3 hours. After solubilization of the formazan crystals, the multi-well plates are read by a spectrophotometer at 500 nm in order to determine the percentage of cell viability.
4.1.4 Main pharmaco-toxicogenomic study
The main study is carried out in duplicate, from the crops sown and exposed to each product, in order to increase the consistency between the experiments and validate the results obtained.
<i>4.1.4.1 Cell seeding and treatment</i>
The cells are seeded and cultivated for 16 hours with each product, at the three concentrations chosen from the prior MTT test; two controls (untreated cells, solvent only) are added for the series.
<i>4.1.4.2 Extraction of total RNA and assay</i>
After treatment, the RNA is extracted and analyzed as follows:<ul id="ul0022" list-style="dash" compact="compact"><li>cell collection and centrifugation,</li><li>extraction carried out with a ready-to-use phenol reagent (Trizol - lot 1106266 and 1121067 - Invitrogen) according to the manufacturer's protocol,</li><li>solubilization of RNA in water,</li><li>RNA assay in spectrophotometry (optical density measured at 260, 280 and 300 nm),</li><li>RNA quality verification by Agilent.</li></ul>
<i>4.1.4.3 Preparation of cDNA probes</i>
CDNA probes are prepared by reverse radioactive transcription (alpha dATP <sup>33</sup>P - Amersham). The quantification of radioactive cDNA (Instant Imager - Packard) is carried out to confirm the activity of the probes.
<i>4.1.4.4 Hybridization on Safe-Hit membrane</i>
On the Safe-Hit membranes in precut nylon (Q-BIOgene) are deposited in double 682 DATAS clones (alternative splicing patterns), using Q-Pix equipment (GENETIX). The DNA probes are hybridized overnight on the membranes, then these are washed.
<i>4.1.1.5. Preparation of cDNA probes:</i>
- matrix: 5 µg of total RNA (for each series of treatment and for each concentration), - primer: 100 ng of oligo-dTV oligonucleotide, for the 1st and 2nd hybridization on the rat (lot 12.00, Invitrogen), - main mixture:<ul id="ul0023" list-style="none" compact="compact"><li>10 µl of First Strand 5x Premier buffer (lot 1131226 - Invitrogen)</li><li>1 µl dCTP + dGTP + dTTP 20 mM (lot 1105201 - Invitrogen)</li><li>1 µM ATP 120 µM (lot 1105201 - Invitrogen)</li><li>5 µl Dithiotreitol (DTT) 0.1 M (lot 133609 - Invitrogen)</li><li>1 µl RNase Out 40 U (lot 1113345 - Invitrogen)</li><li>5 µl of α <sup>33</sup>P dATP 3,000Ci / mmol 10 mCi / µl (lot B0239 - Amersham)</li><li>4 µl Superscript II (lot 1137806 - Invitrogen)</li><li>1 µl of glycogen (lot 1129328 - Invitrogen)</li></ul> - <b><i>procedure:</i></b><ul id="ul0024" list-style="none" compact="compact"><li>incubate the RNA and oligo-dTV at 70 ° C for 10 minutes then put in ice. Add 27 µl of MasterMix then incubate at 43 ° C / 1h and then at 50 ° C / 15 minutes. Add 20 µl of water, then 20 µl of 50 mM EDTA, then 4 µl of 10N NaOH. Incubate 20 minutes at 65 ° C then put in ice.</li><li>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 20 minutes at 13,000 rpm at 4 ° C. Resuspend in 200µl of water,</li><li>quantification: Instant Imager, Packard: 1 µl of reaction mixture.</li></ul> - <b><i>Media and buffers</i></b><tables id="tabl0012" num="0012"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><thead><row><entry valign="top"><b><i>Common solutions:</i></b></entry><entry valign="top"><b><i>Wash buffer 1:</i></b></entry></row></thead><tbody><row><entry>20X SSC (Invitrogen)</entry><entry>2X SSC</entry></row><row><entry>50X Denhardt's</entry><entry /></row><row><entry>50 % (w / v) Dextran sulfate (ICN)</entry><entry /></row><row><entry>20 % SDS (v / v) (Quantum biotech.)</entry><entry /></row><row><entry>10 mg / ml salmon sperm DNA</entry><entry /></row><row><entry>(Q-Biogene)</entry><entry /></row></tbody></tgroup><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><thead><row><entry valign="top"><b><i>Pre-hybridization buffer:</i></b></entry><entry valign="top"><b><i>Wash buffer 2:</i></b></entry></row></thead><tbody><row><entry>6X SSC</entry><entry>2X SSC</entry></row><row><entry>10X Denhardt's</entry><entry>0.1% SDS</entry></row><row><entry>10 % Dextran sulfate</entry><entry /></row><row><entry>0.5% SDS</entry><entry /></row><row><entry>H<sub>2</sub>O</entry><entry /></row></tbody></tgroup><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><thead><row><entry valign="top"><b><i>Hybridization buffer:</i></b></entry><entry valign="top"><b><i>Wash buffer 3:</i></b></entry></row></thead><tbody><row><entry>5X SSC</entry><entry>0.5X SSC</entry></row><row><entry>5X Denhardt's</entry><entry>0.1% SDS</entry></row><row><entry>0.1% SDS</entry><entry /></row><row><entry>H<sub>2</sub>O</entry><entry /></row></tbody></tgroup><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="57mm" /><colspec colnum="2" colname="col2" colwidth="37mm" /><thead><row><entry valign="top" /><entry valign="top"><b><i>Wash buffer 4:</i></b></entry></row></thead><tbody><row><entry /><entry>1X SSC</entry></row><row><entry /><entry>0.1% SDS</entry></row></tbody></tgroup></table></tables> - <b><i>Pre-Hybridization:</i></b><ul id="ul0025" list-style="none" compact="compact"><li>Aliquot 5 ml of pre-hybridization buffer into the hybridization tubes, add the corresponding volume of salmon sperm DNA for a final concentration of 100 μg / ml,</li><li>soak the membranes with 5X SSC,</li><li>place the membrane in the hybridization tube and pre-hybridize for 2 hours at 65 ° C.</li></ul> - <b><i>Hybridization:</i></b><ul id="ul0026" list-style="none" compact="compact"><li>Remove the pre-hybridization buffer and rinse with 10-20ml of 5X SSC, remove the 5 X SSC, replace with 5ml of buffer + salmon sperm DNA, denature the RT probes for 5 min at 95 ° C, then place on ice / 1 minute, centrifuge for reconstitution, 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> - <b><i>Washing :</i></b><ul id="ul0027" list-style="none" compact="compact"><li>Rinse the membranes with 10-20 ml of washing buffer 1,</li><li>remove the buffer and replace it with 50 ml of washing buffer 2,</li><li>incubate for 30 min at 55 ° C, then eliminate and replace by washing with buffer 4, incubate for 30 minutes at 55 ° C, then decant the final wash, remove the membranes from the tubes, place on a cassette and let the acquisition take place during 3 hours.</li></ul>
<i>4.1.4.5 Image acquisition and analysis</i>
The membranes are placed on a screen (FX Imaging ScreenK - Bio-rad) for 3 hours. The film is then played using a Personal Molecular Imager FX (Bio-rad). Image analysis is performed using the Safe-Hit Reader Software (COSE).
<i>4.1.4.6 Calculation of the Toxicity Index</i>
All data is transferred to an automatic calculation program which normalizes the different membranes and calculates a Toxicity Index = sum of the number of genes up-regulated and down-regulated by a given compound at a given concentration, compared to the results of controls not treated. The results of the two Safe-Hit analyzes are then compared and combined to assess the potential toxicity of the various compounds tested. Two parameters modifiable by the user are involved in the calculation of the Toxicity Index:<ul id="ul0028" list-style="none" compact="compact"><li>¤ <i><u style="single">the Background Threshold (BT)</u></i> smooths weak signals, close to background noise and not attributable to significant gene expression. It therefore determines the detection threshold;</li><li><u style="single">¤ <i>the Induction Factor (IF)</i></u> is determined as the multiplying factor, versus the control samples, so that the clones are up or down regulated. The value of this parameter is generally 2 or below 2 to obtain relevant results. The progressive increase in the value of the IF selects the clones which are more and more strongly up or down regulated.</li></ul>
The method for calculating the Toxicity Index was developed by comparing the reference profiles (R: untreated cells) with an experimental profile (E) and follows the following steps (see <figref idref="f0003">figure 5</figref>) for a schematic view of the process):<ul id="ul0029" list-style="dash" compact="compact"><li>transformation of all the values obtained into log values,</li><li>calculation of the average of the log values for each of the duplicate tests (M<sub>iR</sub> and M<sub>ie</sub>),</li><li>establishment of a matrix with M<sub>iR</sub> - M<sub>ie</sub> for all signals (= D<sub>i</sub>),</li><li>normalization of M<sub>ie</sub> individual by subtracting from M<sub>ie</sub> the median of the 14 proximal values of Di (= NM<sub>ie</sub>),</li><li>comparison of normalized values with reference values (Ci = NM<sub>ie</sub> - M<sub>IR</sub>),</li><li>exponential transformation of C<sub>i</sub> (= F<sub>i</sub>),</li><li>comparison of F<sub>i</sub> with the Induction Factor chosen by the user:<ul id="ul0030" list-style="none" compact="compact"><li>¤ if F<sub>i</sub> > IF, the gene is considered up-regulated,</li><li>¤ if 1 / IF <F<sub>i</sub> <IF, the gene is expressed without modification,</li><li>¤ if F<sub>i</sub> <1 / IF, the gene is considered down-regulated.</li></ul></li></ul>
<u style="single">4.2. TEST TEST RESULTS</u>
These tests were carried out in triplicate on primary rat hepatocytes exposed for 16 hours.
Clomipramine, referenced C218, has significant toxicity at 100 μM since no viability is observed after exposure of the cells for 16 hours. However, no toxicity is observed at 25 µM. At 50 µM, the viability greater than 80% is entirely compatible with a pharmaco-toxicogenomic study. Compounds F2695 and F2696 show no cytotoxicity in this test, even at the concentration of 100 μM.
To carry out pharmaco-toxicogenomic evaluations, 3 concentrations of the same compound are used: the concentration which makes it possible to obtain 80% viability (C), as well as the concentrations which correspond to (C) x10 and to (C) / 10.
In order to compare the capacity of F2695 and F2696 to produce a score in the test used, the same concentrations were used for each of them: 1 μM, 10 μM and 100 μM. In the case of clomipramine, the concentrations of 1 µM, 10 µM and 50 µM were used. See the<figref idref="f0004">Figures 6a, 6b and 6c</figref>.
<u style="single">4.3 RESULTS ON PRIMARY RAT HEPATOCYTES</u>
The Toxicity Indexes (IT) were determined as described above. In these indexes, only the clones which were found to be modulated with respect to the controls in the two independent experiments were taken into account, only considering the clones whose signal is 2 times greater than the background noise (BT). Two different analyzes were carried out taking two levels of difference (Induction Factor - FI) compared to the untreated situation:<ul id="ul0031" list-style="dash" compact="compact"><li>at least 1.7 times compared to the untreated situation. This factor of 1.7 times represents the lowest value which makes it possible not to obtain an index when comparing two untreated situations.</li><li>at least 2 times compared to the untreated situation. This factor of 2 times takes into account the strongest signals.</li></ul>
4.3.1. Induction factor of 1.7 compared to the untreated situation (Table 6)
<tables id="tabl0013" num="0013"><img file="EP1908461A1_D0008.tif" /></tables><tables id="tabl0014" num="0014"><img file="EP1908461A1_D0009.tif" /></tables>
The following toxicity indexes were obtained:<tables id="tabl0015" num="0015"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="top"><b>F2695</b></entry><entry align="center" valign="top"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">0</entry></row><row><entry align="center">10 µM</entry><entry align="center">0</entry></row><row><entry align="center">100 µM</entry><entry align="center">17</entry></row><row><entry namest="col1" nameend="col2" align="center" /></row></tbody></tgroup><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="top"><b>F2696</b></entry><entry align="center" valign="top"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">2</entry></row><row><entry align="center">10 µM</entry><entry align="center">5</entry></row><row><entry align="center">100 µM</entry><entry align="center">22</entry></row><row><entry namest="col1" nameend="col2" align="center" /></row></tbody></tgroup><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="top"><b>C218</b></entry><entry align="center" valign="top"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">9</entry></row><row><entry align="center">10 µM</entry><entry align="center">15</entry></row><row><entry align="center">50 µM</entry><entry align="center">28</entry></row></tbody></tgroup></table></tables>
The following classification can therefore be established, from the most toxic to the least toxic C218 (clomipramine)> F2696 >>> F2695.
Clomipramine, the reference molecule coded in this C218 test, shows an increase in signatures in relation to the concentrations tested: respectively 9, 15 and 28 signatures at concentrations of 1, 10 and 50 µM (maximum concentration defined in the prior test of cytotoxicity). Logically, all of the signatures occurring at low and medium concentrations are found at higher concentrations.
At concentrations of 1 and 10 µM, F2695 does not induce any signature on the 682 possible stress signatures tested in this trial. At the highest concentration of 100 µM, only two signatures are identified, one of which is common with C218 but of unknown meaning.
F2696 shows an increase in signatures in relation to the concentrations tested: respectively 2, 5 and 22 signatures at concentrations of 1, 10 and 100 µM. All of the signatures occurring at low and medium concentrations are found at higher concentrations. None of the 22 signatures is common with the F2695. On the other hand, the signatures that appear at low and medium concentrations (5 of which 2 are present from the low concentration), are all 5 among the 9 objectified signatures for clomipramine from the low dose of 1 µM. At the high concentration of 100 µM, 10/26 signatures of F2696 are found among the 28 identified with clomipramine at 50 µM.
In terms of quality, the impact on F2696 and clomipramine should be emphasized on the mitochondrial transcripts, in particular at the level of Cox1 and cytochrome b. These signatures are not present with the F2695 (positions G05 / G09 / I01).
4.3.2. Induction factor of 2 compared to the untreated situation (Table 7)
<tables id="tabl0016" num="0016"><img file="EP1908461A1_D0010.tif" /></tables><tables id="tabl0017" num="0017"><img file="EP1908461A1_D0011.tif" /></tables>
The following indexes have been obtained:<tables id="tabl0018" num="0018"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="middle"><b>F2695</b></entry><entry align="center" valign="middle"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">0</entry></row><row><entry align="center">10 µM</entry><entry align="center">0</entry></row><row><entry align="center">100 µM</entry><entry align="center">0</entry></row><row><entry namest="col1" nameend="col2" align="center" /></row></tbody></tgroup><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="middle"><b>C218</b></entry><entry align="center" valign="middle"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">8</entry></row><row><entry align="center">10 µM</entry><entry align="center">13</entry></row><row><entry align="center">50 µM</entry><entry align="center">16</entry></row><row><entry namest="col1" nameend="col2" align="center" /></row></tbody></tgroup><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="16mm" /><colspec colnum="2" colname="col2" colwidth="30mm" /><thead><row><entry align="center" valign="middle"><b>F2696</b></entry><entry align="center" valign="middle"><b>Toxicity index</b></entry></row></thead><tbody><row><entry align="center">1 µM</entry><entry align="center">0</entry></row><row><entry align="center">10 µM</entry><entry align="center">5</entry></row><row><entry align="center">100 µM</entry><entry align="center">16</entry></row></tbody></tgroup></table></tables>
According to these parameters, the following classification can be proposed, from the most toxic to the least toxic: C218 (clomipramine)> F2696 >>>>> F2695.
Considering the clones over or under expressed by a factor of 2, F2695 does not induce any signature, even at the concentration of 100 μM.
The effect of the concentration on the appearance of the signatures is confirmed by the disappearance of the low intensity signatures for the 1 μM F2696, present in the previous analysis with an induction factor of 1.7.
In terms of quality, the impact of F2696 and clomipramine on Cox1 and cytochrome b is also confirmed (positions G05 / G09 / I01).
F2695, the pharmacologically active enantiomer of F2207, has no significant impact in this test, while clomipramine is used as a positive control reference product.
In contrast, F2696, the inactive enantiomer of F2207, presents a signature profile quantitatively and qualitatively close to clomipramine, and does not present any common signature with F2695.
All this shows a better toxicogenomic profile for the active enantiomer F2695, of which, on this experimental model, the safety coefficient is very significantly better than for F2696.
<u style="single">4.4 CONCLUSION</u>
The pharmaco-toxicogenomic studies carried out on molecules F2695 and F2696, enantiomers of F2207 (at concentrations of 10, 50 and 100 µM), and C218 (clomipramine, at concentrations of 1, 10 and 50 µM), from hepatocytes of rat in primoculture, allowed to obtain stress signatures and concentration-dependent toxicity indexes. These studies confirm the ability of the pharmaco-toxicogenomic test to reveal signatures of stress under treatment conditions (concentrations, duration of treatment) which do not cause any toxicity in a standard viability test such as MTT.
Several key facts emerge from this study:<ul id="ul0032" list-style="none" compact="compact"><li>¤ on this model of primary rat hepatocytes, only F2695, the pharmacologically active enantiomer of F2207, does not induce a significant toxicity index;</li><li>¤ F2696, the inactive enantiomer of F2207, and clomipramine, the reference psychotropic drug, induce important indices formed by signatures of stress that are common or very close. In this system, clomipramine, a positive product reference molecule, is the product inducing the most stress signatures, significant indexes being obtained at the lowest concentrations. As such, it is worth mentioning that clomipramine can induce a certain number of undesirable effects in humans such as, for example, tachycardia, orthostatic hypotension, disturbances in conduction or rhythm, and exceptionally hepatitis. In case of accidental overdose with clomipramine, one can observe, among other things, syncope, hematological disorders, severe cardiovascular manifestations.</li></ul>
Without prejudging an identity of a pathophysiological mechanism, it is interesting to note that F2696 presents signatures of stress common or very close to those of clomipramine and also induces undesirable effects such as the cardiovascular disorders previously described.
It is therefore legitimate to suggest that the signatures observed are independent of any antidepressant profile or more generally psychotropic. On the other hand, they must be considered as “stress signatures” (F2696 in particular causes a decrease in expression of a gene involved in protein synthesis and of a translation initiation factor). All this shows a better toxicogenomic profile for the active enantiomer F2695, of which, on this experimental model, the safety coefficient is very significantly better than for F2696.
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Every citation, both waysCites: the store holds 7 of 8
| Document | Relation | Office | Category | Cited during | Relevant claims |
|---|---|---|---|---|---|
| WO0126623A2 | Cites | World Intellectual Property Organization (WIPO) | – | Applicant | – |
| WO0162236A2 | Cites | World Intellectual Property Organization (WIPO) | – | Applicant | – |
| EP0939626A1 | Cites | European Patent Office (EPO) | – | Applicant | – |
| FR2759290A1 | Cites | France | – | Applicant | – |
| FR2759906A1 | Cites | France | X | Search report | 1-5,7-10 |
| US4478836A | Cites | United States of America | – | Applicant | – |
| WO9735574A1 | 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, pages 318 - 325, XP002282456, ISSN: 0022-2623 | Non-patent | – | – | Search report | – |
| DEPREZ D ET AL: "Which bioequivalence study for a racemic drug? Application to milnacipran.", EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, vol. 23, no. 2, 1998, pages 166 - 171, XP008021090, ISSN: 0378-7966 | Non-patent | – | – | Search report | – |
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| 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, April 1998 (1998-04-01), pages 280 - 286, XP008021091, ISSN: 0398-7639 | Non-patent | – | – | Search report | – |
| VIAZZO P ET AL: "Microbiological Transformations 34: Enantioselective Hydrolysis of a Key-Lactone Involved in the Synthesis of the Antidepressant Milnacipran@?", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 37, no. 26, 24 June 1996 (1996-06-24), pages 4519 - 4522, XP004029055, ISSN: 0040-4039 | Non-patent | – | – | Search report | – |
| SHUTO SATOSHI ET AL: "-1-phenyl-2-((S)-1-aminopropyl)-N,N-diethylcyclopropanecarboxamide (PPDC), a new class of NMDA-receptor antagonist: Molecular design by a novel conformational restriction strategy.", JAPANESE JOURNAL OF PHARMACOLOGY, vol. 85, no. 3, March 2001 (2001-03-01), pages 207 - 213, XP008021088, ISSN: 0021-5198 | Non-patent | – | – | Search report | – |
| MORET ET AL., NEUROPHARMACOLOGY, vol. 24, no. 12, 1985, pages 1211 - 1219 | Non-patent | – | – | Applicant | – |
| PALMIER ET AL., EUR J CLIN PHARMACOL, vol. 37, 1989, pages 235 - 238 | Non-patent | – | – | Applicant | – |
| BONNAUD ET AL., JOURNAL OFCHROMATOGRAPHY, vol. 318, 1985, pages 398 - 403 | Non-patent | – | – | Applicant | – |
| SHUTO ET AL., TETRAHEDRON LETTERS, vol. 37, 1996, pages 641 - 644 | Non-patent | – | – | Applicant | – |
| GRARD ET AL., ELECTROPHORESIS, vol. 21, 2000, pages 3028 - 3034 | Non-patent | – | – | Applicant | – |
| DOYLE; HU, ADVANCED SYNTHESIS AND CATALYSIS, vol. 343, 2001, pages 299 - 302 | Non-patent | – | – | Applicant | – |
| NORES ET AL., THÉRAPIE, vol. 42, 1987, pages 555 - 558 | Non-patent | – | – | Applicant | – |
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Numbers
- Publication
- 1908461
- Publication, DOCDB
- 1908461
- Publication, EPODOC
- EP1908461
- Application
- 7123564
- Application, DOCDB
- 07123564
- Application, EPODOC
- EP20070123564
Titles3
- German
- Verwendung des Enantiomers (1S, 2R) von Milnacipran zur Herstellung eines Arzneimittels
- English
- Use of (1S, 2R) enantiomer of milnacipran for the preparation of a medicine
- French
- Utilisation de l' énantiomère (1S, 2R) du milnacipran pour la préparation d' un médicament
Classification
- CPC, 16
- A61K31/135
- A61K31/165
- A61K31/167
- A61K31/195
- A61K31/4015
- A61P13/02
- A61P13/10
- A61P25/00
- A61P25/04
- A61P25/16
- A61P25/22
- A61P25/24
- A61P25/28
- A61P25/30
- A61P3/04
- A61P9/00
- IPC, 7
- A61K31 135
- A61K31 165
- A61K31 167
- A61K31 195
- A61K31 4015
- A61P9 00
- A61P25 00
Designated states2
- Contracting states, 1
- Türkiye
- Extension states, 1
- North Macedonia