Use of (1s, 2r) enantiomer of milnacipran for the preparation of a medicine
11 claims: 1 independent, 10 dependent
- 10 enantiómero (IS, 2R) do Milnaciprano (Z(±)—2— (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais f armaceuticamente aceitáveis, para utilização como medicamente destinado a prevenir ou a tratar a depressão, os estados depressivos, a fibromialgia, a
- 2Enantiómero (IS, 2R) do Milnaciprano (Z ( + )-2(aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com a reivindicação 1, caracterizado por a dose administrada estar compreendida entre 0,05 mg e 5 mg/kg de massa corporal, por dia, numa ou em várias tomas.
- 3Enantiómero (IS, 2R) do Milnaciprano (Z ( + )-2(aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com a reivindicação 1 ou 2, caracterizado por a dose administrada estar compreendida entre 0,1 mg e 1 mg/kg de massa corporal, por dia, numa ou em várias tomas.
- 4Enantiómero (IS, 2R) do Milnaciprano (Z ( + )-2(aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 3, caracterizado por as perturbações cardiovasculares corresponderem a uma elevação da pressão arterial e/ou a um aumento da frequência cardíaca.
- 5Enantiómero (IS, 2R) do Milnaciprano (Z(+)-2(aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com a reivindicação 4, caracterizado por a elevação da pressão arterial corresponder a uma elevação da pressão arterial diastólica.
- 6Enantiómero (IS, 2R) do Milnaciprano (Z (±)—2 — (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 5, caracterizado por limitar também os riscos de toxicidade orgânica e/ou tissular.
- 7Enantiómero (IS, 2R) do Milnaciprano (Z(±)—2— (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 6, caracterizado por o referido enantiómero (IS, 2R) do Milnaciprano ser o hidrocloreto de Z-(IS, 2R)-2-(aminometil)-N,N-dietil-1fenilciclopropanocarboxamida (F2695).
- 8Enantiómero (IS, 2R) do Milnaciprano (Z(+)—2— (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 7, para o tratamento ou a prevenção da depressão profunda, da depressão resistente, da depressão do idoso, da depressão psicótica, da depressão induzida por tratamentos com interferão, do estado depressivo, da síndrome maníaco-depressiva, dos episódios depressivos sazonais, dos episódios depressivos ligados a uma condição médica geral ou dos episódios depressivos ligados a substâncias que agem sobre o humor.
- 9Enantiómero (IS, 2R) do Milnaciprano (Z ( + )-2(aminometil)-N, N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 7, para o tratamento ou a prevenção da dor crónica.
- 10Enantiómero (IS, 2R) do Milnaciprano (Z(±)—2— (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 9, caracterizado por os antecedentes cardiovasculares e/ou as perturbações cardiovasculares serem escolhidos entre o enfarte do miocárdio, as perturbações do ritmo cardíaco (taquicardia, bradicardia, palpitações), as perturbações da pressão arterial (pacientes hipo ou hipertensos) ou as cardiopatias.
- 11Enantiómero (IS, 2R) do Milnaciprano (Z(±)—2— (aminometil)-N,N-dietil-l-fenilciclopropanocarboxamida), ou um dos seus sais farmaceuticamente aceitáveis, de acordo com qualquer uma das reivindicações 1 a 10, caracterizado por o medicamento conter:a) o referido enantiómero (1S,2R) do Milnaciprano ou um dos seus sais farmaceuticamente aceitáveis, e b) pelo menos um composto activo escolhido entre os psicotrópicos, nomeadamente os anti-depressores, e os agentes antimuscarlnicos, como produtos de combinação para uma utilização simultânea, separada ou escalonada no tempo para o tratamento ou a prevenção da depressão profunda, da depressão resistente, da depressão do idoso, da depressão psicótica, da depressão induzida por tratamentos com interferão, do estado depressivo, da síndrome maníaco-depressiva, dos episódios depressivos sazonais, dos episódios depressivos ligados a uma condição médica geral, dos episódios depressivos ligados a substâncias que agem sobre o humor.
Independent claims11
604 paragraphs in 19 sections, as filed
DESCRIPTION
USE OF MILNACIPRAN ENANTIOMER (IS, 2R) FOR PREPARATION OF A MEDICINAL PRODUCT
The present invention relates to Milnacipran (Z (±) -2- (aminomethyl) -N, N-diethyl-1-phenylcyclopropanecarboxamide) enantiomer (IS, 2R), or one of its pharmaceutically acceptable salts, for its use. as medically intended to prevent or treat depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain in patients with a cardiovascular history and / or cardiovascular disorders, administered at a dose of 0.01 mg to 10 mg / kg body mass per day in one or more doses. More particularly, the enantiomer according to the invention is intended to treat depression, chronic fatigue syndromes and urinary incontinence.
Milnacipran (Z (+) -2- (aminomethyl) -N, N-diethyl-1-phenylcyclopropanecarboxamide), a molecule synthesized at the PIERRE FABRE MEDICAMENT Research Center (Castres, France), also called TN-912, Dalciprano, Minalciprane, Midalciprane, or Midaliprane, is known as a double serotonin (5-HT) and norepinephrine (NA) reuptake inhibitor. Milnacipran and its preparation process are described in US Patent No. 4,478,836. Further information on Milnacipran can be found in the twelfth edition of the Merck index, entry No 6 281.
Serotonin and norepinephrine dual reuptake inhibitors correspond to a well-known class of antidepressant agents that selectively inhibit the simultaneous reuptake of serotonin and norepinephrine. By way of example, venlafaxine and duloxetine are also dual inhibitors of serotonin and norepinephrine. Studies have shown that the ratio of noradrenaline reuptake inhibition to serotonin reuptake inhibition by Milnacipran is about 2: 1 (Moret et al., 1985 Neuropharmacology 24 (12): 1211-1219; Palmier et al., 1989, Eur J Clin Pharmacol 37: 235-238).
US Patent No. 4,478,836 describes the use of Milnacipran for the treatment of central nervous system disorders, especially depression. WOO1 / 26623 describes the use of Milnacipran in combination with phenylalanine and tyrosine in indications such as treatment of fatigue, pain-related syndromes, chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome. . 0 WO100 / 62236 discloses a composition comprising Milnacipran in combination with one or more antimuscarinic agents in a large number of indications, including depression. 0 WO97 / 35574 describes a pharmaceutical composition containing Milnacipran and idazoxane as a combination product for simultaneous, separate or time-stepped use to treat depression and its different forms, as well as the conditions in which antidepressants are used. Milnacipran is also used in an indication for treatment of urinary incontinence (FR 2 759 290).
The Milnacipran molecule has two asymmetric carbons which leads to two different spatial configurations (1S, 2R) and (1R, 2S). Since these spatial configurations are not superimposable, the Milnacipran molecule therefore has an optical isomer.
Milnacipran hydrochloride thus exists as two optically active enantiomers: the dextrorotine or Z- (IS, 2R) -2- (aminomethyl) N, N-diethyl-1-phenylcyclopropanecarboxamide hydrochloride and the levorotine enantiomer hydrochloride. Z- (IR, 2S) -2- (aminomethyl) -N, N-ethyl-1-phenylcyclopropanecarboxamide. 0 Hydrochloride milnacipran (also called F2207) is currently marketable (IXEL, PIERRE FABRE MEDICAMENT, France) as a racemic mixture as a serotonin-noradrenergic antidepressant drug. F2695 and F2696 designate respectively the (1S, 2R) (dextrogyr) and (1R, 2S) (levogyr) enantiomers of the Milnacipran hydrochloride (F2207):
<img file="PT1908461E_D0001.tif" />
These two enantiomers may be separated and isolated according to the procedures described in the literature (Bonnaud et al., 1985, Journal of Chromatography, Vol. 318: 398-403;
Shuto et al., Tetrahedron letters, 1996 Vol. 37: 641-644;
Gerard et al., 2000, Electrophoresis 2000 21: 3028-3034;
Doyle and Hu, 2001, Advanced Synthesis and Catalysis, Vol.343: 299-302).
The inventors have now performed a human pharmacokinetic study of the racemate and the two enantiomers of Milnacipran 3 using enantioselectivity dosage methods. They thus demonstrated the absence of enantiomer racemization in vivo.
On the other hand, although the racemate has been resolved, no analysis of the pharmacological and toxicological properties of the two enantiomers using the currently available modern methods such as cardiovascular telemetry measurements or pharmacokinetic prediction analyzes has been performed. toxicogenomics in vitro.
Antidepressants, like any active ingredient, may give rise to undesirable effects or certain toxicities that derive essentially from the pharmacological properties of these medicinal products, but also from the dosage, individual patient variability (genetic polymorphism, organ failure, sex, age) or drug interactions. . Thus antidepressants represent the third class of products responsible for intoxication after hypnotics and tranquilizers (Nores et al., 1987 Thérapie 42: 555558). The risk of overdose with antidepressants is serious as it may lead to death. Causes of acute antidepressant poisoning include involuntary ingestion by children (especially as certain antidepressants are used for the treatment of enuresia), suicide attempt, involuntary overdose by the physician, associated co-medication in the elderly, age-related physiological and pharmacokinetic changes (heart failure, hepatic and / or renal insufficiency ...), a slow metabolism of genetic or drug origin (enzyme inhibition). After children, the elderly therefore constitute the second risk population among treated patients. Older people have higher plasma concentrations linked to impaired hepatic and / or renal clearance, and the risks of poisoning are more severe in them (Meadoer-Woodruf et al., 1988 J. Clim. Psychopharmacol 8: 28-32).
The undesirable, usually benign side effects seen during treatment with Milnacipran are mainly noticed during the first or even the first two weeks of treatment and then stop, along with an improvement in the depressive episode. The most commonly reported undesirable events in monotherapy or in combination with other psychotropics are dizziness, hypersudation, anxiety, hot flushes and dysuria. Certain less commonly reported undesirable effects are nausea, vomiting, a dry mouth, constipation, tremors, palpitations, agitation, rash. It is also known that in patients with a history of cardiovascular disease or simultaneously receiving cardiac treatment, the incidence of undesirable cardiovascular effects (hypertension, hypotension, ortho-static hypotension, palpitations) may be increased by Milnacipran. In hypertensive or heart-affected patients, it is therefore recommended to strengthen clinical surveillance, as Milnacipran in the form of a racemic mixture is likely to increase heart rate. Thus, in the rare cases of overdose observed such as Milnacipran (at doses of 800 mg to 1 g) in monotherapy, the main symptoms observed are vomiting, respiratory disorders and tachycardia (Vidal Dictionary, 78).<sup>The</sup> edition, 2002). Another exceptional undesirable effect induced by Milnacipran is a large increase in transaminases which may translate into some liver toxicity.
Indeed, at-risk populations likely to develop a number of undesirable clinical manifestations during or following treatment with Milnacipran are hepatic and / or renal insufficiency, patients receiving treatment that induces organic and / or toxicities. including liver and / or renal toxicities, patients receiving cardiac treatment or inducing cardiovascular side effects, patients with a cardiovascular history and / or those with cardiovascular disorders, such as patients with heart rhythm, blood pressure disorders (hypo or hypertensive patients) or patients with heart disease.
In an effort to prevent and anticipate the emergence of side effects that may pose a small risk to the health of patients treated with Milnacipran, the inventors have now surprisingly and unexpectedly discovered that the enantiomer ( 1S, 2R) of Milnacipran, which shows the essential of selective serotonin and norepinephrine reuptake inhibition activity, it induces fewer cardiovascular side effects and organic and / or tissue toxicity, including liver toxicity, than racemic mixture. In particular, the inventors have found that administration of Milnacipran (1S, 2R) enantiomer to the dog causes a lower increase in heart rate and blood pressure, in particular diastolic blood pressure, than is likely to be caused by administration of the mixture. racemic. The inventors also found that the Milnacipran hydrochloride (1S, 2R) enantiomer (F2695) has a better toxicogenic profile than the hydrochloride enantiomer (IR, 2S).
Milnacipran (F2696) in an experimental model of primary rat hepatocytes. The inventors have further demonstrated that the (IR, 2S) (F2696) enantiomer has a toxic profile similar to that obtained with Clomipramine, used as a reference psychotropic known for its relative hepato toxicity.
The present invention therefore relates to Milnacipran (Z (+) -2- (aminomethyl) -N, N-diethyl-1-phenylcyclopropanecarboxamide) enantiomer (IS, 2R) or one of its pharmaceutically acceptable salts for use as a medically designed to prevent or treat depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain in patients with a cardiovascular history and / or cardiovascular disorders, administered at a dose of 0.01 mg to 10 mg / kg body mass per day in one or more doses.
'Cardiovascular disorders' means undesirable cardiovascular side effects of the medicinal product administered alone or in combination with other active ingredients.
For the purposes of the present invention, "side effect" means the foreseeable activity of a medicinal product in a field other than that for which it is administered, which may be cumbersome or undesirable when limiting the use of the medicinal product.
'Toxicity' means the property of a medicinal product to cause harmful side effects at organic and / or tissue level, including at the level of the organs or tissues involved in Milnacipran metabolism, including hepatic and / or renal metabolism Milnacipran, and more particularly on the first passage of Milnacipran to the liver. Preferably, the organic toxicity is cardiac toxicity and said tissue toxicity is hepatic and / or renal toxicity.
In the context of the present invention, "limiting the risks of cardiovascular disorders" or "limiting the risks of toxicity" means preventing such risks from increasing significantly in a patient after administration of the medicinal product.
In the context of the present invention, 'Milnacipran enantiomer (1S, 2R)' means the Milnacipran enantiomer (1S, 2R) as well as its pharmaceutically acceptable salts. Preferably it is the enantiomer (1S, 2R) of Milnacipran hydrochloride (F2695). "Milnacipran (1R, 2S) enantiomer" means the Milnacipran (1R, 2S) enantiomer as well as its pharmaceutically acceptable salts, such as hydrochloride (F2696). "Racemic mixture" means a 50:50 mixture by weight of the Milnacipran (1S, 2R) enantiomer and the Milnacipran (IR, 2S) enantiomer and their pharmaceutically acceptable salts.
There are metabolites, preferably in vivo active metabolites of Milnacipran, and pharmaceutically acceptable salts thereof, such as:
Z - (+) Phenyl-1-aminomethyl-2-cyclopropanecarboxylic acid hydrochloride (F1567):
<img file="PT1908461E_D0002.tif" />
Molecular mass:
Features:
Fusion point.
Plate chromatography:
277.7 white crystals
230 ° C support: silica
Solvent: Butanol / Ethane / Water (6/2/2)
Development: Ultraviolet and ninhydrin Rf: 0.6 a (+) phenyl-3-methylene-3-4-pyrrolidone (F1612):
<img file="PT1908461E_D0003.tif" />
Molecular mass:
Features:
Fusion point.
173.2 crystals
70 ° C
Plate chromatography: stand:
White Silica Solvent
Benzene / dioxane / ethane (90/25/4)
Ultraviolet and iodine Rf: 0.46 Z (±) - (para-hydroxyphenyl) -1-diethyl aminocarbonyl-1-aminomethyl-2-cyclopropane acid hydrochloride (F2782):
<img file="PT1908461E_D0004.tif" />
NH<sub>r</sub>HCi
Molecular mass:
Characteristics:
Fusion point.
Plate chromatography:
298.82 white crystals
250 ° C support: silica
Solvent: Butanol / Ethane / Water (6/2/2)
Ultraviolet and iodine ninhydrin Rf 0.42 Z (+) phenyl-1-ethylaminocarbonyl-1-amino methyl-2-cyclopropane acid oxalate (F2800):
<img file="PT1908461E_D0005.tif" />
Molecular Mass: 308.33
Features: white crystals
Fusion point. 150 ° C
Plate chromatography: support: silica Z (+) phenyl cyclopropane hydrochloride (F2941):
Solvent: CHCl3 / methanol / NH4 OH (90/9/1)
Revelation: Ultraviolet and ninhydrin Rf: 0.40
-1-aminocarbonyl-1-aminomethyl-2-
<img file="PT1908461E_D0006.tif" />
F2W
Molecular mass:
Characteristics:
Fusion point.
Plate chromatography
226.74 white crystals
245 ° C support: silica
Solvent: CHCl3 / methanol / NH4 OH (80/18/2)
Revelation: Ultraviolet and ninhydrin Rf: 0.30
Like Milnacipran, these metabolites have two asymmetric carbons leading to two different spatial configurations (1S, 2R) and (1R, 2S). Since these spatial configurations are not superimposable, these metabolites also have an optical isomer. The ratio of the two 11 enantiomers of the Milnacipran metabolite in the enantiomer mixture is as described above for the Milnacipran enantiomers.
Active metabolite means a derivative derived from the metabolization of Milnacipran in vitro or in vivo and capable of inhibiting the reuptake of serotonin and norepinephrine; preferably these are F2782, F2941, F2800, F1612 and F1567.
"Pharmaceutically acceptable salt" means all salts which retain the efficacy and properties of an active ingredient and which have no side effects. Preferably, they are pharmaceutically acceptable salts of mineral or organic acids. Preferred but not limiting examples will include hydrohalides such as hydrochloride and hydrobromide, fumarate, maleate, oxalate, citrate, methanesulfonate, glutamate, tartrate, mesylate and their possible hydrates.
The enantiomer according to the invention, preferably the substantially pure enantiomer F2695, is administered to any type of patient in need of such treatment, whether for therapeutic and / or prophylactic purposes. For a therapeutic purpose, the aim is to eradicate or ameliorate the condition to be treated and / or one or more associated symptom (s). With a prophylactic objective, the aim is to prevent the onset of the condition to be treated and / or one or more associated symptoms. However, the enantiomer according to the invention is adapted to at-risk patient populations that would be likely to develop certain undesirable clinical manifestations during or following treatment with racemic Milnacipran. These are patients with a cardiovascular history (eg, myocardial infarction) and / or affected by cardiovascular disorders, such as patients with heart rhythm disturbances (tachycardia, bradycardia, palpitations).
Among the multiple conditions or disorders presenting as symptoms of heart rhythm disorders and for which the present invention is particularly suited to the treatment of at-risk patients, it is worth mentioning in particular the tachycardia which corresponds to an acceleration of the heartbeat rhythm. (tachycardia is moderate when pulsations are between 80 to 100 per minute, intense when they exceed 100), palpitations, extrasystoles (sporadic, frequent or in the course of myocardial infarction), atrial fibrillation, atrial flutter and tachycardia, bradycardia, heart failure and myocardial infarction.
Among the multiple conditions presenting as a symptom of blood pressure disorders and for which the present invention is particularly suited to the treatment of the patients affected by them, it is worth mentioning in particular: hypertension; malignant arterial hypertension, pulmonary arterial hypertension, portal hypertension, essential paroxysmal hypertension, hypotension, ortho-static hypotension, intracranial hypertension.
Advantageously, the cardiovascular disorders whose risks may be limited by administration of the enantiomer mixture according to the invention and preferably by administration of the substantially pure F2695 enantiomer are:
> the elevation of diastolic and / or systolic blood pressure measured in millimeters of mercury (mm Hg); more particularly, it is diastolic heart pressure, and / or> heart rhythm disturbances, namely an increase in the patient's heart rate.
Systolic blood pressure is the maximum value of blood pressure and corresponds to the moment when the first heartbeat at the humeral artery level is heard when measuring blood pressure. Systole is the period of the cardiac revolution during which the cavities of the heart contract, thus ejecting the blood. Diastolic blood pressure is the minimum blood pressure corresponding to the disappearance of cardiac noise at the humeral artery level when the tension cuff is emptied, when the blood pressure is measured. Diastole is the period of the cardiac revolution during which the cavities of the heart are filled with blood. Elevation in systolic and / or diastolic pressure implies elevation of blood pressure that characterizes systemic arterial hypertension (and its variants), the symptoms of which may be: headache, fatigue, mild sensory disturbances such as dizziness, tinnitus. ears, palpitations, nosebleeds, confusion and drowsiness, cramps, disturbances of consciousness or tingling in the feet and hands. Systemic arterial hypertension (and its variants) can lead to serious, even sometimes fatal, complications: neurological accidents of vascular origin, left ventricular failure, renal failure, ischemic heart disease 14 (myocardial infarction, angina pectoris and its variants). According to current recommendations, a patient is considered to be affected by systemic arterial hypertension when blood pressure is greater than 90 mm Hg for diastolic pressure and 140 mm Hg for systolic pressure.
Toxicity whose risks may be limited by administration of the enantiomer according to the invention is advantageously organic toxicity, namely cardiac toxicity and / or tissue toxicity, particularly hepatic and / or renal toxicity. This tissue toxicity may be revealed by the presence of ichthy or by biological markers.
It is also within the scope of the present invention to use the enantiomer of the invention in veterinary medicine to treat animals, namely domestic or farm animals in need of such treatment.
In addition to its pharmacological properties, namely dual serotonin (5HT) and noradrenaline (() reuptake inhibitor, the enantiomer according to the invention is particularly useful in the preparation of medicaments for the preventive and / or curative treatment of numerous pathologies. or disorders (syndrome) described above, while limiting the risks of cardiovascular disorders and / or simultaneously limiting organic and / or tissue toxicity, namely liver and / or renal toxicity.
Among these conditions or disorders, it is worth mentioning central nervous system disorders, as defined in The Diagnostic and Statistical Manual of Mental Didorders IV (DSM-IV), 1995 American Psychiatric Association. For illustrative and non-limiting examples, depression, such as deep depression, resistant depression, elderly depression, psychotic depression, interferon treatment-induced depression, depressive state, manic depression, seasonal depressive episodes, depressive episodes linked to a general medical condition, depressive episodes linked to mood-acting substances, bipolar syndrome, schizophrenia, generalized anxiety, sluggishness and doldrums, stress-related illnesses, panic attacks, phobia, including agoraphobia, obsessive compulsive disorders, conduct disorders, oppositional disorders, post-stress syndromes trauma, depression of the immune system, fatigue and accompanying pain symptoms, chronic fatigue syndrome, fibromyalgia and other functional somatic disorders, autism, disorders characterized by inattention due to general medical conditions, attention disorders due to hyperactivity, eating disorders, bulimic nevrosis, anorectic nevrosis, obesity, psychotic disorders, apathy, migraine, pain, and in particular chronic pain, irritable bowel syndrome, cardiovascular disease, and notably anxiety-depressive syndrome in myocardial infarction or hypertension, neurodegenerative diseases and associated anxiety depressive symptoms (Alzheimer's disease, Huntington's chorea, Parkinson's disease), urinary incontinence, including stress-related urinary incontinence and enuresia, drug dependence, and in particular anxiety -dependence on tobacco, in particular nicotine, alcohol, narcotics, drugs, analgesics, when these states of dependence are withdrawn.
More particularly, the present invention relates to the use of the enantiomer according to the invention, preferably substantially pure F2695 enantiomer, for the preparation of a medicament for preventing or treating depression or depressive state while limiting the risks of cardiovascular disorders and / or at the same time limiting the risks of organic and / or tissue toxicity, including hepatic and / or renal toxicity. In the context of the present invention, "depression" means a set of symptoms comprising on the one hand a psychic aspect consisting of mood disturbances with pessimism, moral pain, ideas of death and suicide, psychic inhibition, and on the other an aspect. physical activity of motor inhibition consisting in particular of a motor slowdown, appetite disturbance, constipation, sleep disturbance and weight regulation. Depression thus corresponds to a pathological psychic state, associated with a painful change in mood and a slowdown in intellectual and motor activity. 'Depressive state' means a state of mind characterized by a deterioration of neuropsychic tone, manifested by laxity, propensity to fatigue, discouragement and tendency towards pessimism, and sometimes accompanied by anxiety.
Also, the present invention is more particularly directed to the use of the enantiomer according to the invention, preferably substantially pure F2695 enantiomer, for the preparation of a medicament for preventing or treating fibromyalgia and / or chronic fatigue syndrome. simultaneously limiting the risk of cardiovascular disorders and / or simultaneously limiting organic and / or tissue toxicity, including hepatic and / or renal toxicity. Fibromyalgia syndrome is a chronic syndrome 17 characterized by a feeling of pain or burning with morning stiffness, mainly covering the articular and peri-articular fibrous tissues, and a feeling of deep fatigue. Fibromyalgia has a set of symptoms. The most common are non-restful sleep, headaches, digestive disorders, a depressed state, muscle spasms, facial pain, consciousness disturbances, etc. Chronic fatigue syndrome is characterized by a state of exhaustion or fatigue. The most common symptoms are a state of weakness, muscle spasms and / or pain, a need for excessive sleep, fever, angina, memory loss and / or concentration problems, insomnia, a depression.
Also, the present invention is more particularly directed to the use of the enantiomer according to the invention, preferably the substantially pure F2695 enantiomer, for the preparation of a medicament for preventing or treating pain, and in particular chronic pain, while limiting the risks of cardiovascular disorders and / or simultaneously limiting organic and / or tissue toxicity, including hepatic and / or renal toxicity. Pain may be associated with different pathologies and / or injuries. It can be acute or chronic. Epidemiological studies have demonstrated the relationship between chronic pain states and anxiety depressions. Thus, patients suffering from chronic pain may develop emotional problems that lead to depression and, in the worst case, to a suicide attempt. A patient is considered to have chronic pain if he complains of suffering for more than six months. Chronic pain should include, by way of illustration and not limitation, pain associated with fibromyalgia and / or from fibrous tissues, muscles, tendons, ligaments and other places, abdominal pain and diarrhea in irritable bowel syndrome and low back pain as well.
Also, the present invention is more particularly directed to the use of the enantiomer according to the invention, preferably substantially pure F2695 enantiomer, for the preparation of a medicament for preventing or treating urinary incontinence, namely stress-related urinary incontinence. and enuresia, while limiting the risk of cardiovascular disorders and / or limiting organic and / or tissue toxicity, namely liver and / or renal toxicity.
Prophylactic and therapeutic treatment of the above conditions is provided by providing to an animal, preferably man, a therapeutically effective amount of the enantiomer according to the invention, preferably substantially pure F2695 enantiomer alone or in combination with at least one another active ingredient. In most cases it is human, but the treatment is also adapted to animals, in particular farmed animals (cattle, rodents, birds, fish ...) and pets (dogs, cats, rabbits, horses .. .).
The enantiomer (1S, 2R) of Milnacipran as well as its pharmaceutically acceptable salts, as described above, is advantageously administered to patients who receive simultaneously, separately or in time lag at least a second active compound in the treatment of the aforementioned conditions. .
Preferably, the present invention also relates to use as a medicament:
a) said Milnacipran enantiomer (1S, 2R), as well as pharmaceutically acceptable salts thereof, and
(b) at least one active compound selected from psychotropic agents, namely antidepressants, and antimuscarinic agents, as combination products for simultaneous, separate or time-stepped use for the treatment or prevention of depression, in particular deep depression, elderly depression, psychotic depression, interferon treatment-induced depression, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes linked to a general medical condition, depressive episodes linked to mood-acting substances.
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 (halucinogens).
Preferably said psychotropic is an antidepressant. By way of non-limiting example, the antidepressant is chosen from (i) monoamine oxidase (MAOI) inhibitors such as iproniazid, pargillin, selegin, (ii) 5HTID agonists such as sumatriptan, adrenaline and noradrenaline (alpha and beta sympathetic mimetics), (iii) tricyclic antidepressants such as imipramine, clomipramine, (iv) selective serotonin reuptake inhibitors (SSRI) such as fluoxetine, (v) selective norepinephrine reuptake inhibitors such as for example tandamine, fluparoxane, mirtazapine (vi) serotonin and norepinephrine reuptake inhibitors such as venlafaxine and duloxetine. By way of non-limiting example, the antimuscarinic agent is selected from tolterodine, propiverine, oxybutynin, trospium, darifenacin, temiverine, ipratropium.
More preferably, the present invention also relates to use as a medicament:
(a) said Milnacipran enantiomer (1S, 2R), as well as pharmaceutically acceptable salts thereof, and
(b) at least one other active ingredient selected from the active compounds which induce organ toxicity and the active tissue-inducing compounds, namely hepatic and / or renal, or one or more active ingredients for the treatment of hepatic impairment; / or renal, as combination products for simultaneous use, separated or time-staggered for the treatment or prevention of conditions or conditions that may be treated by double inhibition of serotonin (5-HT) and noradrenaline (NA) reuptake.
More preferably, the present invention also relates to use as a medicament:
a) said Milnacipran enantiomer (1S, 2R), as well as pharmaceutically acceptable salts thereof, and
b) of at least one other active ingredient selected from active compounds which induce cardiovascular side effects and cardiac compounds as combination products for simultaneous, separate or time-stepped use for the treatment or prevention of conditions or disorders. which can be treated by double inhibition of serotonin (5-HT) and noradrenaline (NA) reuptake.
Advantageously, induced cardiovascular side effects are those previously mentioned, and more particularly hypertension, hypotension, heart rhythm disturbances (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 enantiomer according to the invention, preferably substantially pure enantiomer F2695, is advantageously administered, without limitation, orally, nasal, transdermal, rectal, intestinal, parenteral, by intramuscular, subcutaneous injection. -cutaneous or intravenous alone or in combination with other active ingredients as described above.
When administered alone, the enantiomer according to the invention, preferably substantially pure F2695 enantiomer, may be administered per se or in the form of a pharmaceutical composition in which said enantiomer or pharmaceutically acceptable salts thereof are in combination or in combination. mixing with one or more pharmaceutically acceptable carriers, excipients and / or diluents, in particular facilitating bioavailability.
When the enantiomer according to the invention, and preferably substantially pure Milnacipran (1S, 2R) F2695 enantiomer, is administered in combination with other active principles, said enantiomer and other active principles may be formulated in admixture or separately, in an identical or different form. They can be administered by the same or a different route.
The pharmaceutical compositions according to the invention may be formulated in conventional ways well known to the skilled man 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, such as a physiologically acceptable buffer solution such as Hank's solution, Ringer's solution or physiological saline buffer, is advantageously used. In the case of transdermal mucosal administration, penetrating agents appropriate to the mucosa to be traversed are advantageously used. Such penetrating agents are well known to the skilled person. In the case of an oral administration, the pharmaceutical compositions according to the invention are advantageously administered in unit or multidose forms of administration in admixture with suitable pharmaceutically acceptable carriers. Suitable unit dosage forms include, inter alia, optionally slotted tablets, glands, powders, granules and oral solutions or suspensions, aerosols. Suitable multidose forms of administration include drinkable drops, emulsions and syrups.
substantially pharmaceutically magnesium, for example,
In preparing tablets, the enantiomer according to the invention, preferably pure F2695 enantiomer, is formulated with an acceptable carrier such as polyvinylpyrrolidone, carbopol gel, polyethylene glycol, gelatin, talc, starch, lactose. , gum arabic stearate or its analogs. The tablet contains the following excipients:
dehydrated calcium hydrogen phosphate, calcium carmelose, povidone K30, anhydrous colloidal silica, magnesium stearate, talc. The tablets may optionally be coated, i.e. coated with several layers of various substances, such as sucrose, for ease of taking or storage. The coating may also contain pigments or dyes in order to distinguish and characterize tablets according to their dosage, for example. The tablets may furthermore have a more or less complex formulation designed to modify the rapidity of release of the active ingredient. The release of the active ingredient from said tablet may be accelerated, slowed down or delayed depending on the desired absorption. 0 The enantiomer according to the invention, preferably substantially pure F2695 enantiomer, can thus be prepared in a sustained release dosage form obtained by the process described in EP 939 626. This dosage form is in multiparticulate form comprising a plurality of minigranules and has a certain in vitro release profile.
Release of the enantiomer according to the invention may be delayed and / or controlled by the use of an implant or by transcutaneous, notably subcutaneous or intramuscular release, by intramuscular injection or by a transdermal patch. Said enantiomer is then formulated with, in particular, suitable hydrophobic or polymeric substances and ion exchange resins.
The amount of the enantiomer according to the invention to be administered to the patient, preferably the substantially pure F2695 enantiomer, depends on the conditions to be treated, the intended effect, namely a therapeutic or prophylactic effect, the patient's health and age, in particular. cardiovascular history, treatment conditions and method of administration of the medicinal product. Effective therapeutic or prophylactic amounts to be administered to a human patient may be determined from animal models or data known to the skilled person in the treatment of depression in man, for example by use of said Milnacipran enantiomer.
In the context of the prophylactic or therapeutic treatment of the abovementioned conditions, and in particular depression, depressive states, fibromyalgia, chronic fatigue syndrome and pain, the medicament according to the invention is advantageously administered in doses of 0.01 mg and 10 mg / kg body weight per day in one or more doses, even more advantageously at doses of 0.05 mg to 5 mg / kg body mass per day in one or more doses, even more advantageously at doses ranging from 0.1 mg to 1 mg / kg body mass per day in one or more doses. 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 gellet. By way of example, the enantiomer according to the invention, preferably the substantially pure F2695 enantiomer, is administered in the form of a gel whose active ingredient content is advantageously about 6.75 mg / gell, 12.5 mg / g gell, 25 mg / gell, 50 mg / gell.
Other features, objects and advantages of the invention will emerge from the following examples. The invention is not limited to the particular examples mentioned by way of illustration only and which should be read with reference to the following figures:
Legend of the Figures:
Figure 1: Heart rate evolution after single administration (delta values).
*** p <0.001 versus deionized water ** p <0.01 versus deionized water * p <0.05 versus deionized water À p <0.05 versus F2207
Figure 2: Heart rate evolution after single administration (absolute values).
*** ρ <0.001 versus deionized water ** p <0.01 versus deionized water * p <0.05 versus deionized water A p <0.05 versus F2207
Figure 3: Effects of various treatments on mean values of diastolic blood pressures (averages 6 hours after the last treatment, after 5 consecutive days of treatment).
<td>Figure 4:</td><td>Effects of</td><td colspan="2">several</td><td>treatments</td><td>about the</td>
<td></td><td colspan="2">medium values</td><td>in</td><td>pressures</td><td>arterial</td>
<td></td><td>systolic</td><td>(averages</td><td>of</td><td>6 hours after</td><td>the last</td>
<td></td><td>treatment,</td><td>after</td><td> 5</td><td colspan="2">consecutive days of</td>
<td></td><td>treatment)</td><td></td><td></td><td></td><td></td>
<td>Figure 5:</td><td>Representation</td><td>schematic of</td><td>mode</td><td>of calculation</td>
<td></td><td>of the index</td><td>of toxicity.</td><td> 0</td><td>index of</td>
<td></td><td>toxicity is</td><td>the sum of</td><td>all</td><td>the genes</td>
<td></td><td>regulated up</td><td colspan="2">and down (depending on</td><td>factor of</td>
user defined induction).
Figures 6a, 6b, 6c: MTT test in primary rat hepatocytes. Concentrations are expressed in μΜ.
EXAMPLES
EXAMPLE N<sup>The</sup> Pharmacokinetic studies of Milnacipran and its enantiomers
Pharmacokinetic studies of Milnacipran hydrochloride (F2207) and its enantiomers (F2695 and F2696) have been performed in different animal species and in humans.
In animals, the pharmacokinetics of each enantiomer following administration of the racemate or a single enantiomer was studied. Plasma rates of the F2695 and F2696 enantiomers are approximately equivalent in the species tested (monkey and rat).
A pharmacokinetic study was performed in man comprising 12 healthy subjects administering racemate or one of the two enantiomers alone. It is deduced that the pharmacokinetic profile of each enantiomer is independent of whether it was administered separately or as a racemate, indicating the absence of interactions between the two enantiomers (Table 1).
Table 1: Table of the main pharmacokinetic variables of Milnacipran hydrochloride (F2207) and its two enantiomers F2695 and F2696.
<td colspan="5">Cmax: Estimated maximum plasma concentration directly at from the experimental data Tmax: Time to obtain the maximum plasma concentration AUC-> «: Area under the curve of plasma concentrations as a function of time extrapolated to infinity Ti /<sub>2</sub>: Decreasing terminal half-life of plasma concentrations</td>
<td>Administered dose (mg)</td><td colspan="2">F2207 (50 mg)</td><td>F2695 (D) (25 mg)</td><td>F2696 (L) (25 mg)</td>
<td></td><td>F2695 (D)</td><td>F2696 (L)</td><td></td><td></td>
<td>Cmax (nmol.l<sup>-1</sup>)</td><td> 214</td><td> 179</td><td> 216</td><td> 212</td>
<td>Tmax (hours)</td><td> 3,42</td><td> 2,87</td><td> 3,08</td><td> 2,21</td>
<td>oo AUC 0> (nmol .hl<sup>-1</sup>)</td><td> 2896</td><td> 1563</td><td> 2869</td><td> 1543</td>
<td>T% (hours)</td><td> 9,28</td><td> 5,75</td><td> 9,38</td><td> 5,58</td>
These results indicate that no bioconversion of the enantiomers (F2695 and F2696) was detected in the analyzed species.
EXAMPLE N<sup>The</sup> 2: Biochemical Studies of Milnacipran and its Enantiomers
The two enantiomers (F2695 and F2696) of Milnacipran hydrochloride (F2207) in vitro were studied for norepinephrine and serotonin captures, as well as for rat brain paroxetine binding.
2.1. MATERIALS AND METHODS
2.1.1. Noradrenaline uptake by a homogenate (P<sub>2</sub>) of the rat hypothalamus.
P Preparation<sub>2</sub>
Male Sprague-Dawley rats fall asleep and behead from 200 to 300 g, then the hypothalamus is quickly removed. Two hypothalamus are homogenized in 4 ml 0.32 M sucrose in Potter S for 16 complete round trips at 800 rpm, then centrifuged 10 min at 1000 g to remove cell debris. The supernatant is centrifuged 20 min at 10,000 g and the P<sub>2</sub> thus obtained in 4 ml 0.32 M sucrose and homogenize in Dounce.
Uptake
It is used <sup>3</sup>H- (1) -NA: 13 Ci / mmol (Amersham).
Capture in phosphate buffer (containing per liter: 8 g NaCl, 1.21 g K<sub>2</sub>HPO<sub>4</sub> and 0.34 g of KH<sub>2</sub>POWDER<sub>4</sub>) peroxygenated 30 min before use with a mixture O<sub>2</sub>/ CO<sub>2</sub> (95% / 5%).
In 5 ml plastic tubes placed in a water bath at 37 ° C:
100 μΐ buffer or inhibitor,
700 μΐ buffer (containing 25 μΜ pargillin),
100 μΐ of P<sub>2</sub>.
After temperature stabilization, the reaction starts by adding 100 μΐ of <sup>3</sup>H-NA, 50 nM at the end.
Exactly 10 min later, the reaction is quenched by the addition of 2.5 ml of cold buffer and filtered over GF / F filters. Then the tube is rinsed once and the filter once with 2.5 ml of cold buffer. The filter is then introduced into a Beckman vial and, after addition of 3 ml Instagel scintillating liquid (Packard), the radioactivity is measured in a Tricarb Packard liquid scintillation counter.
Nonspecific capture (NS) is measured in the presence of 10 “DMI<sup>5</sup>
M.
The percentage inhibition is calculated by the formula:
(total capture - NS) - (capture in the presence of inhibitor - NS) (total capture - NS)
IC50 was determined graphically over the mean inhibition percent curve (4 assays) as a function of log inhibitor concentration.
2.1.2. Serotonin Capture
The method was performed according to Gray and Whittaker (1962, J. Anat., 96: 79-97). After homogenization of brain tissue in a sucrose solution, the presynaptic endings detach from the axon and close to form synaptosomes obtained by subcellular fractionation.
Male Sprague-Dawley (Janvier) rats of 180,200 g were used. After sacrifice of the animal, the hypothalamus was removed, weighed, homogenized in Dounce in 0.23 M sucrose at 0 ° C.
This homogenate was centrifuged 10 min at 1000 g (2400 rpm Hettich, Rotenta). The supernatant was recovered and this homogenate was centrifuged 20 min at 10,000 g (8,000 rpm - Beckam, model J2-21 M: rotor J14). The residue (called fraction P<sub>2</sub>) in sucrose at a concentration of 50 mg / ml.
Incubated for 5 min at 37 ° C:
o 350 μ gelado of cold buffer (136 mM NaCl, 2.4 mM KH2PO4, 6.9 mM K2HPO4, pH 7.2) peroxygenated 30 min before, 50 μΐ of membranes (5 mg / ml at the end), 50 μ o from citalopram (10_<sup>5</sup> M at the end) for non-specific capture, the 50 μΐ of <sup>3</sup>H-5-HT (50 nM at the end) (NEN, France, 28.4
Ci / mmol).
The reaction was stopped at exactly 5 min after initiation of incubation by vacuum filtration over Whatman GF / F filters (prediluted with 2.5 ml ice-cold buffer, then rinsed with 3 times 2.5 ml).
Radioactivity collected on the filter (Packard Tricarb 4640) was measured by liquid scintillation with Emulsifier-Save (Packard).
CIs were determined<sub>50</sub> graphically applying inhibition percentages as a function of log concentration in product (6 concentrations in duplicate).
2.1.3. Paroxetine Binding
Male Sprague-Dawley (Janvier) rats of 180,200 g were used. The hypothalamus of several rats were pooled and homogenized in 5 ml of cold buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.5) in Dounce and the homogenate centrifuged at 30,000 g. (27,000 rpm - Beckman. L5-50F, T40 rotor). The residue obtained was taken up in 5 ml of buffer and centrifuged again under the same conditions. The new residue was taken up in the same buffer and finally homogenized in Dounce at a tissue concentration of 10 mg / ml. Membrane suspension (100 μΐ) was incubated with 3H-paroxetine (NEN, France, 28.6 Ci / mmol) at 0.1 nM (final) concentration at 20 ° C in a final volume of 1 ml for 2 h. After two hours of incubation, the reaction was quenched by vacuum filtration over pre-treated Whatman GF / F filters in a 0.05% polyethylenimine solution 30 min before (prediluted with 4 ml of cold buffer, then rinse the tube with 2 times 4 ml). Radioactivity was measured by liquid scintillation spectrometry (Packard Tricarb 4640) using Emulsifier-Save (Packard) as scintillating agent.
The specific binding of <sup>3</sup>H-paroxetine was defined as the difference between total binding and what remains in the presence of 10 μΜ of fluoxetine.
IC50's were determined by graphically applying inhibition percentages as a function of log concentration in product (6 concentrations in duplicate).
2.1.4. Products used
<td>F2207:</td><td>lot</td><td>No.</td><td>10-CTN3 Code P118</td>
<td>F2695:</td><td>lot</td><td>No.</td><td>PL-I-205</td>
<td>F2696:</td><td>lot</td><td>No.</td><td>PL-I-204C.</td>
2.2. RESULTS
The effects of F2207 and its two enantiomers on norepinephrine and serotonin uptake and paroxetine binding are plotted with the percentage inhibition (%) as a function of the concentration (M) in abscissa. F2207, F2695 or F2696 (data not shown). Percent inhibition values corresponding to each product concentration, tested in duplicate, are the means of the results of four independent experiments.
CI values were determined<sub>50</sub> the three products from these curves and are shown in table 2.
Table 2. Inhibition of catches of <sup>3</sup>H-noradrenaline, <sup>3</sup>H-serotonin and binding of <sup>3</sup>H-paroxetine.
<td colspan="4">IC50 (M)</td>
<td>Compounds</td><td colspan="2">Catch</td><td>Binding<sup>3</sup>H-Paroxetine</td>
<td></td><td><sup>3</sup>H-Noradrenaline</td><td><sup>3</sup>H-Serotonin</td><td></td>
<td>F2695</td><td>1.5 x ICO<sup>3</sup></td><td>4.6 x 10<sup>-3</sup></td><td>6.0 x 10<sup>-3</sup></td>
<td>F2207</td><td>3.0 x ICO<sup>3</sup></td><td>15 x 10<sup>-3</sup></td><td>13 x 10<sup>-3</sup></td>
<td>F2696</td><td>75x10_<sup>8</sup></td><td>60x10_<sup>8</sup></td><td>70x10_<sup>8</sup></td>
All three compounds are active in these three pharmacological tests, but there are differences:
in noradrenaline capture:
F2695 is 2 times more active than F2207
F2695 is 25 times more active than F2696.
in serotonin capture:
F2695 is 3 times more active than F2207 0 F2695 is 12 times more active than F2696.
paroxetine binding:
F2695 is 2 times more active than F2207 0 F2695 is 10 times more active than F2696.
All three compounds are active in these pharmacological tests with, however, less activity for the form (IR, 2S) (F2696) and racemate (F2207). The hydrochloride form (1S, 2R)
<td>from Minalciprano</td><td>(F2695) is 2 to 3 times</td><td>more</td><td>active that the</td>
<td>F2207.</td><td></td><td></td><td></td>
<td>EXAMPLE # 3:</td><td>Comparative activity</td><td>per</td><td>orally</td>
<td>hydrochloride</td><td>Racemic Minalcipran</td><td colspan="2">(F2207) and its</td>
active (1S, 2R) enantiomer (F2695) on heart rate and blood pressure in the watchful dog.
3.1. INTRODUCTION
This study aims to analyze the effects of F2207 and F2695 a) single oral administration on heart rate (n = 28 dogs) and b) after repeated 5 day oral treatment on systolic and diastolic blood pressures in the dog (n = 6 dogs).
This study was performed on pharmacologically active F2207 and F2695 echidrosis on implant-equipped female animals (Data Sciences International) that allow for the acquisition of heart rate and tension parameters by telemetry. These animals were divided for all studies into 3 treatment groups:
group 1 (control) treated with deionized water, group 2 treated with F2207 at a dose of 20 mg / kg / D, group 3 treated with F2695 at a dose of 10 mg / kg / D.
3.2. METHODOLOGY
Taking into account the small number of animals equipped simultaneously (maximum 8), the number of tracks of registration of the equipment used (8 tracks) and in order to form homogeneous treatment groups, the overall assessment was carried out in four studies. each study is divided into three passages (treatment of each animal with each of the three products), separated by a wash-out period. Each of the passages is itself carried out in two phases:
a first phase during which all animals are treated with deionized water for habituation to oral containment and treatment by a gastric tube, a second phase during which animals receive their treatment (single heart rate administration, studies n 894/926/935/936; repeated administration five days for blood pressure, study No. 894).
The global experimental scheme is described in the following table:
Table 3: Global experimental scheme for the telemetry study of the effects of racemic Milnacipran hydrochloride (F2207) and its active (1S, 2R) enantiomer administered orally to conscious dog
GROUP
<td>NUMBER</td><td> 1</td><td> 2</td><td> 3</td>
<td>ANIMALS</td><td></td><td></td><td></td>
<td>Number</td><td>ST</td><td> 29</td><td>if</td>
<td>Identification</td><td> 1-27-6-16-14.</td><td></td><td>3 · 3. · 1 M 247</td>
<td></td><td>(study 894)</td><td> 16</td><td> -16</td>
<td></td><td>; 2 ? vuv</td><td>(study 894)</td><td>(study 894)</td>
<td></td><td> 14</td><td>3..4..9.1 CM 5- ·</td><td> 5-6-11-12-17</td>
<td></td><td>(study 926)</td><td> 10</td><td> -18</td>
<td></td><td>t-2-âdí!</td><td>(study 926)</td><td>study 926)</td>
<td></td><td> 11-17-16-7^</td><td></td><td> 67-8--1 4</td>
<td></td><td>(study 935)</td><td> 15797:1-22</td><td> 15-10-25-24</td>
<td></td><td> 1-2-9-10</td><td>(study 935)</td><td>(study 935)</td>
<td></td><td></td><td> 3-4-6-12</td><td>S.7-8-14</td>
<td></td><td>(study 936)</td><td> 15-20-51-22</td><td> 15-157 3-24</td>
<td></td><td></td><td>(study 936)</td><td>(study 936)</td>
<td>TREATMENT</td><td>Water</td><td></td><td></td>
<td>Identification</td><td>deionized</td><td> -227</td><td></td>
<td>Dose</td><td></td><td> 20</td><td> 10</td>
<td>Via</td><td></td><td>orste</td><td></td>
<td>Volume</td><td></td><td>Sml / kg</td><td></td>
<td>(n = 27 at</td><td colspan="2">witness group was not registered</td><td>0 sign of</td>
<td></td><td>probe</td><td>of animal No 18)</td><td></td>
In the four studies, the effects of different treatments on heart rate after single administration were analyzed. The analysis covered the following 13 acquisition moments:
- before single treatment,
- every 30 minutes following at 6 hours after the single treatment.
The effects of different treatments on blood pressure in study # 894 at steady state at D5, D29 and D33 (last day of effective treatment for each pass) were analyzed. The analysis covered the following 13 acquisition moments:
- before treatment,
- every 30 minutes following at 6 hours after treatment.
3.3. RESULTS
3.3.1. With regard to heart rate (four grouped studies), a Tukey test was performed on individual frequency deltas for each of the 12 post-treatment experimental times versus pre-treatment value as well as absolute values. heart rate at each time of recording.
Thus, in relation to the control animals receiving deionized water, the following were realized: # when the statistical analysis is performed on delta values, (figure 1):
- a significant increase in heart rate from the first hour after single administration of F2207 (20 mg / kg), persistent increase up to 5.5 hours after treatment (p <0.001 for all acquisition times except moments 0.5 and 5.5 hours - p <0.01 - and moment 5.0 hours - p <0.05 - after treatment).
- an increase in heart rate after administration of F2695 which always remains 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 h after administration in favor of F2695.
- an increase in heart rate lasting less time under F2695 (1, 0 and 4.5 h) than under F2207 (persists until
5.5 h after treatment).
# When statistical analysis is performed on absolute heart rate values, this same study highlights (Figure 2):
- a significant increase in heart rate from the first hour following single administration of F2207 (20 mg / kg), persistent increase up to 5.5 hours after treatment (p <0.001 for the set acquisition time of 1.0 at 4,5 h, except for the moment 3,5 hours - p <0,01 and p <0,01 for the moment of acquisition
5.5 hours after treatment).
- an increase in heart rate after administration of F2695 which always remains lower than after administration of F2207. In addition, this difference between the effects of F2207 and F2695 is significant (p <0.5) at 1 and 4 h after administration in favor of F2695.
- an increase in heart rate lasting less time under F2695 (1.0 and 4.5 h) than under F2207 (persists until
5.5 h after treatment).
3.3.2. With regard to blood pressure (a study with repeated administration), an average value of diastolic blood pressure (figure 3 and table 4) as well as an average value of systolic blood pressure (figure 4 and 39 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 it (data not shown).
They materialized like this:
- a significant (p <0.001) increase in diastolic blood pressure after repeated 5-day administration of F2207 (20 mg / kg / D) or F2695 (10 mg / kg / D) compared to deionized water treatment,
- a significant difference (p <0.05) in mean diastolic blood pressure after repeated administration 5 days of F2207 (20 mg / kg / D) compared to mean mean diastolic blood pressure after repeated administration of F2695 (10 mg / kg). / D),
- no significant effect on systolic blood pressure; It can however be noted that PAS values following repeated administration of F2695 are close to PAS values following treatment with deionized water.
The individual data of diastolic and systolic blood pressures are presented in tables 4 and 5, respectively.
Table 4: Individual Diastolic Blood Pressure Data
Τ 77
<td colspan="4">DIASTOLIC BLOOD PRESSURE (DBP expressed in mm Hg)</td><td></td><td></td><td></td>
<td colspan="6">Individual data after repeated administration 5 consecutive days</td><td></td>
<td>GROUP</td><td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td>
<td>TREATMENT</td><td colspan="2">VEHICLE</td><td colspan="2">F2207 (20 mg / kg / J)</td><td colspan="2">F2695 (10 mg / kg / J)</td>
<td></td><td></td><td>Avg.</td><td></td><td>Avg.</td><td> 5 6 11 12</td><td>Avg.</td>
<td>Animal No.</td><td> 1278 13 14</td><td></td><td> 34 9 10 15 16</td><td></td><td></td><td></td>
<td></td><td></td><td>esm</td><td></td><td>esm</td><td> 17 18</td><td>esm</td>
<td>Tps before</td><td> 79 77 73 77 101 76</td><td> 81 4</td><td> 112 89 93 88 86 91</td><td> 93 4</td><td> 73 89 80 71 76 78</td><td> 78 3</td>
<td>treatment</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>Tps after the</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>treatment (h)</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> 0,50</td><td> 84 76 70 63 80 70</td><td> 74 3</td><td> 103 106 96 92 88 87</td><td> 95 3</td><td> 91 91 99 90 108 85</td><td> 94 3</td>
<td> 1,00</td><td> 82 84 77 72 72 76</td><td> 77 2</td><td> 130 117 113 113 90 106</td><td> 112 5</td><td> 112 96 75 97 87 96</td><td> 94 5</td>
<td> 1,50</td><td> 102 81 79 75 82 68</td><td> 81 5</td><td> 131 127 137 96 100 91</td><td> 114 8</td><td> 109 83 88 97 87 112</td><td> 96 5</td>
<td> 2,00</td><td> 83 75 71 98 77 75</td><td> 80 4</td><td> 123 113 99 88 107 109</td><td> 107 5</td><td> 115 88 93 95 84 109</td><td> 97 5</td>
<td> 2,50</td><td> 85 75 75 84 85 79</td><td> 81 2</td><td> 137 111 116 101 115 107</td><td> 115 5</td><td> 111 88 97 89 92 107</td><td> 97 4</td>
<td> 3,00</td><td> 91 95 99 85 79 84</td><td> 89 3</td><td> 121 118 112 116 106 92</td><td> 111 4</td><td> 104 91 96 96 100 106</td><td> 99 2</td>
<td> 3,50</td><td> 83 72 78 73 77 65</td><td> 75 3</td><td> 120 106 133 116 103 103</td><td> 114 5</td><td> 96 106 94 107 77 103</td><td> 97 5</td>
<td> 4,00</td><td> 81 79 75 77 82 68</td><td> 77 2</td><td> 133 114 105 111 110 103</td><td> 113 4</td><td> 125 91 99 108 80 109</td><td> 102 6</td>
<td> 4,50</td><td> 82 76 91 84 113 85</td><td> 89 5</td><td> 135 110 126 109 104 108</td><td> 115 5</td><td> 103 104 92 100 85 108</td><td> 99 3</td>
<td> 5,00</td><td> 97 79 67 95 81 82</td><td> 84 5</td><td> 116 120 98 97 97 105</td><td> 106 4</td><td> 126 100 92 95 110 102</td><td> 104 5</td>
<td> 5,50</td><td>94 80 70 ND 85 82</td><td> 82 4</td><td> 103 107 115 106 92 93</td><td> 103 4</td><td> 88 86 105 98 89 99</td><td> 94 3</td>
<td> 6,00</td><td> 83 74 82 82 78 77</td><td> 79 1</td><td> 115 133 120 104 103 104</td><td> 113 5</td><td> 101 113 98 105 109 108</td><td> 106 2</td>
<td>PAD average after</td><td> 87 79 78 81 83 76</td><td> 81 2</td><td> 122 115 114 104 104 101</td><td> 110 4</td><td> 107 95 94 98 92 104</td><td> 98 2</td>
<td>treatment</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td colspan="7">ND: not determined</td>
Table 5: Individual Systolic Blood Pressure Data
<td colspan="4">SYSTEMIC BLOOD PRESSURE (PAD expressed in mm Hg)</td><td></td><td></td><td></td>
<td colspan="6">Individual data after repeated administration 5 consecutive days</td><td></td>
<td>GROUP</td><td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td>
<td>TREATMENT</td><td colspan="2">VEHICLE</td><td colspan="2">F2207 (20 mg / kg / D)</td><td colspan="2">F2695 (10 mg / kg / D)</td>
<td></td><td></td><td>Avg.</td><td></td><td>Avg.</td><td></td><td>Avg.</td>
<td>Animal No.</td><td> 1 2 7 8 13 14</td><td></td><td> 3 4 9 10 15 16</td><td></td><td> 56 11 12 17 18</td><td></td>
<td></td><td></td><td>esm</td><td></td><td>esm</td><td></td><td>esm</td>
<td>Tps before</td><td> 139 141 120 157 172 138</td><td> 145 7</td><td> 188 164 176 149 130 169</td><td> 163 8</td><td> 136 141 138 130 134 149</td><td> 138 3</td>
<td>treatment</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>Tps after the</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>treatment</td><td></td><td> 134 4</td><td> 153 154 152 128 126 129</td><td> 141 6</td><td> 135 129 140 135 160 139</td><td> 140 4</td>
<td>(h) 0.50</td><td> 135 132 119 131 149 138</td><td> 142 4</td><td> 180 167 157 150 126 130</td><td> 152 9</td><td> 159 135 124 148 131 143</td><td> 140 5</td>
<td> 1,00</td><td> 134 158 129 144 141 143</td><td> 149 3</td><td> 186 181 189 129 136 138</td><td> 160 12</td><td> 164 119 138 158 127 156</td><td> 144 8</td>
<td> 1,50</td><td> 158 151 145 150 153 137</td><td> 148 5</td><td> 171 160 146 122 140 163</td><td> 150 7</td><td> 168 125 135 141 127 156</td><td> 142 7</td>
<td> 2,00</td><td> 138 136 145 173 151 144</td><td> 150 3</td><td> 195 168 168 144 153 161</td><td> 165 7</td><td> 165 124 142 141 134 154</td><td> 143 6</td>
<td> 2,50</td><td> 142 143 145 159 160 148</td><td> 158 3</td><td> 173 177 164 157 141 146</td><td> 160 6</td><td> 156 131 145 144 151 157</td><td> 147 4</td>
<td> 3,00</td><td> 149 167 162 163 150 154</td><td> 143 4</td><td> 165 153 184 167 139 155</td><td> 161 6</td><td> 146 147 141 169 123 156</td><td> 147 6</td>
<td> 3,50</td><td> 135 129 149 154 153 137</td><td> 151 4</td><td> 180 157 151 154 150 153</td><td> 158 5</td><td> 180 132 145 160 124 164</td><td> 151 9</td>
<td> 4,00</td><td> 142 143 149 166 164 144</td><td> 158 8</td><td> 184 161 180 155 145 168</td><td> 166 6</td><td> 158 151 138 163 131 163</td><td> 151 5</td>
<td> 4,50</td><td> 137 140 159 170 190 152</td><td> 151 7</td><td> 161 171 146 141 139 166</td><td> 154 6</td><td> 182 144 137 150 162 158</td><td> 156 6</td>
<td> 5,00</td><td> 150 146 127 177 160 145</td><td> 145 4</td><td> 151 154 173 152 132 155</td><td> 153 5</td><td> 142 127 152 153 141 153</td><td> 145 4</td>
<td> 5,50</td><td>153 149 132 ND 148 144</td><td> 151 5</td><td> 158 192 171 154 148 172</td><td> 166 7</td><td> 156 170 148 159 160 166</td><td> 160 3</td>
<td> 6,00</td><td> 146 144 151 176 146 143</td><td></td><td></td><td></td><td></td><td></td>
<td>Average SBP</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td>after</td><td> 143 145 143 160 155 144</td><td> 148 3</td><td> 172 166 165 146 140 153</td><td> 157 5</td><td> 159 136 140 152 139 155</td><td> 147 4</td>
<td>treatment</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td colspan="7">ND: not determined</td>
3.4. CONCLUSION
In the experimental conditions of the present evaluation, performed in four successive oral administration studies in the conscious dog with telemetry:
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; is statistically and clinically inferior and more fleeting with F2695 at the pharmacologically equitable dose of 10 mg / kg / D, F2695 at the dose of 10 mg / kg / D causes no statistically significant change in mean systolic blood pressure. within 6 hours of the last treatment, at steady state after repeated administration of 5 days, A statistically significant difference is evidenced in mean diastolic blood pressure values within 6 hours of the last treatment, at steady state after repeated administration of 5 days, between the active enantiomer F2695 (98 ± 2 mm Hg) and the racemic F2207 in equidose. pharmacologically active (110 ± 4 mm Hg).
These differences are clear evidence of improved cardiovascular tolerance of the active enantiomer F2695.
EXAMPLE 4: IN VITRO PHARMACOTOXICOGENOMIC FORECASTING TEST
4.1. MATERIALS AND METHODS
During the present study compounds F2695 and F2696, enantiomers of racemic molecule F2207, as well as a reference product, clomipramine (encoded in assay C218) were evaluated. Both F2695 and F2696 enantiomers were initially evaluated in a preliminary cytotoxicity test (MTT test) on primary rat hepatocytes to select the three concentrations to be used in the definitive test.
After treatment of cultured rat primary hepatocytes, RNA was extracted to generate labeled complementary DNA probes, which were then hybridized to a membrane containing 682 cell stress-specific splicing fragments . Toxicity indices were obtained for each product tested by comparing the hybridization profile of the treated cells as obtained from untreated cells.
4.1.1. Principle and purpose of the study
Safe-Hit is a sensitive, robust, reliable, fast and reliable pharmaco-toxicogenetic prediction test that allows comparison and classification of products based on the optimal assessment of their toxic potential.
Safe-Hit benefits from a technology owned by
TM
EXONHIT (DATAS: Differential Analysis of Transcripts with Alternative Splicing), which allows to isolate and, consequently, clone excision-repair events resulting from a given biological state compared to a control condition. This allows the isolation of differently expressed mRNA isoforms depending on the biological condition.
Safe-Hit allows the classification of molecules within a chemical series as a function of toxic index, determined after the following base steps (systematically performed in duplicate for each product):
- treatment of cell lines with the different products at three concentrations deducted from a previous cell toxicology test (MTT test): a reference concentration with 80% cell viability, a concentration 10 times higher - when this is possible - and 10 times lower concentration,
- preparation of total RNA and corresponding radiolabeled cDNA probes,
- hybridization of cDNA probes: Safe-Hit macro-array containing 682 independent clones, corresponding to WTp53 overexpression-induced excision-repair modifications (p53 is the most ubiquitous "mediator" of cell stress chosen for the development of this methodology) ,
- Acquisition and determination of Toxicity Index.
4.1.2. Cells
The cells used for the study (MTT cytotoxicity pre-test and main test) are cryopreserved rat hepatocytes from the first Sprague-Dawley rat (Hep 184005 and Hep 184006 - Biopredic) cultured under standard conditions.
4.1.2.1. Defrosting medium: Leibovitz 15 glutamax 1 medium added with 100 æg / ml penicillin, 100 pg / ml streptomycin and 0.6 M glucose (lot MIL 210009 Biopredic),
- Insemination medium: Williams E medium with glutamax 1, added with 100 µg / ml penicillin, 100 pg / ml streptomycin, 4 pg / ml bovine insulin and 10% v / v fetal calf serum (lot MIL 260005 - Biopredic),
- incubation medium: glutamax 1 Williams E medium, added with 100 μg / ml penicillin, 100 μg / ml streptomycin, 4 μg / ml bovine insulin, and 50 μΜ hydrocortisone hemisucinate (Lot 260009 260007 - Biopredic).
4.1.2.2 Culture conditions
37 ° C, CO atmosphere<sub>2</sub> (5%), relative hygrometry (95%).
4.1.2.3 Culture Process
<td></td><td>Toxicity test cell phone</td><td>Main study</td>
<td></td><td>Inseminated cells in the</td><td>treatment day</td>
<td>Density of</td><td> 35 000</td><td>1.5 million</td>
<td>insemination</td><td>cells / cavity (96 plates cavities)</td><td>cells per plate 30 mm</td>
<td>Medium Volume</td><td>0.1 ml</td><td>3 ml</td>
4.1.3. Cytotoxicity test
This cytotoxicity test (mtt test) detects living cells thanks to a colorimetric reaction that reveals the integrity of cellular respiration implying mitochondrial activity. MTT (water soluble 3- [4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide) is transformed by cleavage under the action of a living cell mitochondrial enzyme into insoluble purple formazan. 0 Formazan is solubilized in an organic solvent and the obtained solution can be measured by spectrophotometry. The measured absorbance is proportional to the number of surviving cells.
Cells are contacted for 16 hours as the test product at 5 different concentrations (0 - 1- 10 25 - 50 and 100 μΜ).
After this exposure phase, a mtt solution (0.5 mg / ml in primary hepatocyte incubation medium) is added over 3 hours. After solubilization of the formazan crystals, the multiwell plates are read by a spectrophotomer at 500 nm to determine the percent cell viability.
4.1.4 Main pharmaco-toxicogenomic study
The main study is performed in duplicate from the inseminated and exposed cultures with each product in order to increase consistency between experiments and validate the results obtained.
4.1.4.1 Cellular Inseminations and Treatment
Cells are inseminated and cultured for 16 hours with each product at the three concentrations chosen from the previous MTT test; Two controls (untreated cells, solvent alone) are added to the series.
4.1.4.2 Total RNA Extraction and Dosage
After treatment, RNA is extracted and analyzed as follows:
- cell collection and centrifugation,
- extraction with a ready-to-use phenol reagent (Trizol - lot 1106266 and 1121067 - Invitrogen) following the manufacturer's protocol,
- solubilization of RNA in water,
- RNA dosage in spectrophotometry (optical density measured at 260, 280 and 300 nm),
- RNA quality check by Agilent.
4.1.4.3 Preparation of cDNA Probes
CDNA probes are prepared by radioactive reverse transcription (alpha dATP <sup>33</sup>P - Amersham). Radioactive cDNA (Instant Imager - Packard) quantification is performed to confirm probe activity.
4.1.4.4 Safe-Hit Membrane Hybridization
682 clones, in duplicate, DATAS (alternative excision-repair motifs), are deposited on pre-cut nylon Safe-Hit membranes (Q-BIOgene) with the aid of a Q-Pix apparatus (GENETIX). The DNA probes are hybridized overnight on the membranes, then washed.
4.1.1.5 Preparation of cDNA Probes
- matrix: 5 pg total RNA (for each treatment series and for each concentration),
primer: 100 ng oligo-dTV oligonucleotide, pair I<sup>The </sup>and at 2<sup>The</sup> mouse hybridization (lot 12.00, Invitrogen),
- main mix:
μΐ First Strand 5x Premier Buffer (lot 1131226 - Invitrogen) μCT dCTP + dGTP + dTTP 20 mM (lot 1105201 - Invitrogen) μΜ ATP 120 μΜ (lot 1105201 - Invitrogen) Dithiothreitol (DTT) 0.1 M ( lot 133609 - Invitrogen) μΐ of RNase Out 40 U (lot 1113345 - Invitrogen) μΐ of a<sup>33</sup>P dATP 3,000 Ci / mmol 10 mCi / μΙ (lot B0239 - Amersham) Superscript II μΐ (lot 1137806 - Invitrogen) μΐ glycogen (lot 1129328 - Invitrogen)
- procedure:
incubate the RNA and oligo-dTV at 70 ° C for 10 minutes, then ice. Add 27 μΐ of MasterMix, then incubate at 43 ° C / lh at 50 ° C / 15 minutes. Add 20 μΐ of water, then 20 μΐ of 50 M EDTA, then 4 μΐ of 10N NaOH. Incubate 20 minutes at 65 ° C, then put on ice.
Quantification: Instant Imager, Packard: 1 μΐ reaction mixture, add 8 μΐ acetic acid, 100 μΐ isopropanol and 1 μΐ glycogen (20 μς / μΐ). Incubate at -20 ° C for 20 minutes, centrifuge 20 minutes at 13,000 rpm at 4 ° C.
Resuspend in 200 μΐ water, Quantification: Instant Imager, Packard: 1 μΐ reaction mixture.
Media and Caps
Common Solutions Wash Buffer 1:
20X SSC (Invitrogen) 2X SSC
50X Denhardt's
50% (w / v) Dextran Sulphate (ICN)
20% SDS (v / v) (Quantum biotech.) Mg / ml Salmon Sperm DNA (Q-Biogene)
Prehybridization Buffer Wash Buffer 2:
6X SSC (Invitrogen) 2X SSC
10X Denhardt's 0.1% SDS
10% Dextran Sulphate
0.5% SDS H<sub>2</sub>O
Hybridization Buffer
5X SSC
5X Denhardt's
0.1% SDS
Wash Buffer 3:
0.5X SSC 0.1% SDS
H<sub>2</sub>O
Wash Buffer 4:
X SSC
0.1% SDS
- Prehybridization:
Place 5 ml aliquots of prehybridization buffer in the hybridization tubes, add the corresponding volume of salmon sperm DNA to a final concentration of 100 μς / ιηΐ, soak the membranes with 5X SSC, place the membrane in the hybridization tube. and prehybridize 2 hours at 65 ° C.
- Hybridization:
Discard the prehybridization buffer and rinse with 10-20 ml 5X SSC, discard ο 5X SSC, replace with 5 ml buffer + salmon sperm DNA, denature RT probes for 5 min at 95 ° C, then place over ice / 1 min, centrifuge for reconstitution, then recover appropriate volume of denatured RT probes in tube (100,000 to 200,000 cpm / ml), incubate overnight at 55 ° C.
- Washing:
Rinse membranes with 10-20 ml Wash Buffer 1, discard buffer and replace with 50 ml Wash Buffer 2, incubate 30 min at 55 ° C, then discard and replace by washing with Buffer 4, incubate 30 min at 55 ° C, then decant the final wash, remove the membranes from the tubes, place on a cassette and allow to purchase for 3 hours.
4.1.4.5 Image Acquisition and Analysis
Membranes are placed on a screen (FX Imaging ScreenK - Biorad) for 3 hours. The film is then read using a Personal Molecular Imager FX (Bio-rad). Image analysis is performed using Safe-Hit Reader Softwarw (COSE).
4.1.4.6 Calculation of Toxicity Index
All data is transferred to an automatic calculation program that normalizes the different membranes and calculates a Toxicity Index = sum of the number of genes up and down regulated by a given compound at a given concentration compared to the results of untreated controls. The results of the two Safe-Hit analyzes are then compared and combined to assess the toxicity of the various compounds tested. There are two parameters that can be modified by the user in calculating the Toxicity index:
<sup>0</sup> Background Threshold (BT) levels weak signals that are close to background noise and cannot be attributed to significant gene expression. It therefore determines the detection threshold;
<sup>0</sup> Induction Factor (IF) is determined as the multiplier factor versus control samples so that clones are up or down regulated. The value of this parameter is usually 2 or below 2 to get 52 relevant results. Progressively increasing the IF value selects clones that are increasingly tightly regulated up or down.
The Toxicity Index calculation process was developed by comparing the reference profiles (R: untreated cells) with the experimental profile (E) and follows the following steps (see Figure 5 for a schematic view of the process):
<td>transformation of</td><td>all</td><td>the values</td><td>obtained in</td><td>values</td>
<td>log,</td><td></td><td></td><td></td><td></td>
<td>averaging</td><td>From</td><td>log values</td><td>for each</td><td>one of the</td>
<td>duplicate trials</td><td>(M<sub>go</sub></td><td>θ M<sub>ie</sub>),</td><td></td><td></td>
<td>establishment of</td><td>an</td><td>matrix with M<sub>RR</sub></td><td>- M<sub>RE</sub> for</td><td>all the</td>
signs (= Di),
- standardization of M<sub>ie</sub> by subtracting M<sub>ie</sub> the average of the 14 correlated values of Di (= NM<sub>ie</sub>),
- comparison of normalized values with reference values (C<sub>AND</sub> = NM<sub>1 AND</sub> - M<sub>1R</sub>),
- exponential transformation of khan (= Fi),
- Comparison of Fi with the user-chosen Induction Factor:
<sup>0</sup> if Fi> IF, the up-regulated gene is considered, <sup>0</sup> if 1 / IF <Fi> IF, the gene is expressed without modification, <sup>0</sup> if Fi <1 / IF, the down-regulated gene is considered.
4.2. MIT TEST RESULTS
These tests were performed in triplicate on rat primary hepatocytes exposed for 16 hours.
Clomipramine, reference C218, has significant toxicity at 100 μΜ as no viability is observed after cell exposure for 16 hours. In contrast, no toxicity at 25 μΜ is observed. At 50 μΜ, viability greater than 80% is fully compatible with a pharmaco-toxic study. Compounds F2695 and F2696 show no toxicity in this test, even at 100 μΜ concentration.
In order to carry out the pharmaco-toxicogenic assessments, 3 concentrations of the same compound are used: the concentration which gives 80% viability (C) as well as the concentrations corresponding to (C) x10 and (C) / 10.
In order to compare the ability of F2695 and F2696 to produce a test score, the same concentrations were used for each of them: 1 μΜ, 10 μΜ and 100 μΜ. For clomipramine, the concentrations used were 1 μΜ, 10 μΜ and 50 μΜ. See figures 6a, 6b and 6c.
4.3. RESULTS ON PRIMARY MOUSE HEPATOCYTES
Toxicity indices (IT) were determined as described above. In these indices, it was taken into account that the clones were modulated relative to controls in the two independent experiments, considering only those clones whose signal is 2 times higher than background noise (BT). Two different analyzes were performed taking two levels of difference (Induction Factor - IF) from the untreated situation:
- at least 1.7 times in relation to the untreated situation. This factor of 1.7 represents the weakest value that allows the index not to be obtained when comparing two untreated situations.
- at least 2 times in relation to the untreated situation. This factor of 2 allows for the most robust signals to be taken into account.
4.3.1. Induction factor of 1.7 compared to untreated situation (table 6)
Table 6: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 1.7 fold)
Table 6:
'Up and down' regulated cones with primary rat hepatocytes (Induction Factor = 1.7 fold)
6
<td colspan="2" rowspan="4">Up> V<sup>Down</sup> <08</td><td></td><td>F »1</td><td> «10</td><td>FM</td><td>F »1</td><td></td><td>F ^ i M</td><td></td><td>CB</td><td>C2U- 15 ^</td><td rowspan="4"></td>
<td>Up</td><td></td><td></td><td> 1</td><td></td><td></td><td>u K ·</td><td> 2</td><td> 2</td><td> 13</td>
<td>Sun</td><td></td><td></td><td> 1</td><td> 2</td><td>, V</td><td> ?</td><td> 7</td><td> 13</td><td> 1$</td>
<td>Ί1</td><td></td><td></td><td> 2</td><td> 2</td><td>THE' ν '</td><td> 22</td><td> $</td><td>U</td><td> $</td>
<td colspan="13"></td>
<td>Pos</td><td>rsb t</td><td>ibC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Gene</td>
<td></td><td> 3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,90</td><td> 2.23</td><td> 2,14</td><td>H mitochondria, sapiens, 12S</td>
<td>A20</td><td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td>íí, í) È</td><td></td><td></td><td></td><td>H elF-5A initiation factor encoding gene, sapiens</td>
<td></td><td></td><td>JP</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0,27</td><td>H. sapiens chromosome 19, BAC CIT-B-191n6</td>
<td></td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>J7</td><td></td><td>Genomic Sequence of H, sapiens from 17</td>
<td></td><td> 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 3.20</td><td>-III3</td><td>U2</td><td></td><td>H. sapiens Mitochondria, 16S</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>w</td><td>H. sapiens mRNA for lipocortin II</td>
<td></td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0.22</td><td> 0,35</td><td>H AND sequence, clone 740A11 sapiens in the</td>
(Continuation)
Table 6: Up-and-down-regulated cones with rat primary hepatocytes
Induction = 1.7 times)
<td colspan="2" rowspan="4"> >17 «3</td><td></td><td>F »</td><td></td><td>F »</td><td></td><td>FW</td><td>IV</td><td></td><td></td><td>CB · 1V</td><td rowspan="4"></td>
<td>w</td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td> 2</td><td> 2</td><td> 13</td>
<td>Down</td><td></td><td></td><td></td><td> 2</td><td>is</td><td> 7</td><td> 7</td><td> 13</td><td></td>
<td>'T {</td><td></td><td></td><td>w</td><td> 2</td><td> 3</td><td> 22</td><td> 9</td><td></td><td> 23</td>
<td colspan="13"></td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Xq22.2-23. Contains part of gene COL4A5 to High chain precursor Collagen, 5 (IV). Contains GSS1, full sequence,</td>
<td>Eli</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,12</td><td></td><td></td><td></td><td>H. sapiens homologous liver clordecone reductase, mRNA</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1J2</td><td>Mitochondriade H. sapiens, cytochromoc oxidase subunit 1</td>
<td>E21</td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Λ? R ν. ^ Λΐ</td><td> 0,53</td><td>Protein Gene Sl4 sapiens ribosomal</td>
<td></td><td> 1</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>O</td><td>Homeobox LIM protein co-factor (CLIM-I) MRNA</td>
(Continuation)
Table 6: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 1.7 fold)
<td colspan="2" rowspan="4">ok</td><td></td><td></td><td>s"</td><td></td><td>FB-t</td><td></td><td> »</td><td></td><td>CB</td><td>CB</td><td rowspan="4"></td>
<td>Wow)</td><td></td><td></td><td> 1</td><td></td><td></td><td> <0</td><td> 2</td><td></td><td> 13</td>
<td>Djm</td><td></td><td></td><td>-s</td><td> 2</td><td></td><td> /</td><td> ?</td><td>tr</td><td> 15</td>
<td>YOU</td><td></td><td></td><td> 2</td><td> 2</td><td></td><td> 22</td><td> §</td><td> 15</td><td>2S</td>
<td colspan="13"></td>
<td>GÚ1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H estrogen receptor-related protein, breast cancer variant ER sapiens variant) mRNA</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H. sapiens mitochondria, cytochrome coxidase subunit 1</td>
<td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Z *</td><td></td><td></td><td>vs</td><td>H mitochondria, sapiens, cytochrome b</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2.0?</td><td></td><td></td><td></td><td>H. sapiens mitochondria, cytochrome coxidase subunit 1</td>
<td>ίΐδ</td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td>2.0S</td><td></td><td></td><td>VS</td><td>H gene 18S rRNA, sapiens</td>
<td> 101</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>io?</td><td></td><td></td><td></td><td>Divalent cation tolerant CutA protein H mRNA, sapiens</td>
<td>L22</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H mRNA, Lon protease protein sapiens</td>
(Continuation)
Table 6: Up-and-down-regulated cones with rat primary hepatocytes
Induction = 1.7 times)
<td colspan="2" rowspan="4">1 l Dm</td><td></td><td>í®</td><td>3</td><td></td><td>t *</td><td> 11;</td><td></td><td></td><td>W</td><td>tn 'w *</td><td rowspan="4"></td>
<td></td><td></td><td></td><td> 1;</td><td></td><td></td><td></td><td>i · '</td><td></td><td></td>
<td>rw</td><td></td><td></td><td>·' The ''</td><td>V</td><td></td><td></td><td></td><td> • ></td><td></td>
<td>You</td><td></td><td></td><td> ' 1'</td><td> 2'</td><td> 5</td><td>'il</td><td>β</td><td> ( ''</td><td> > '</td>
<td colspan="13"></td>
<td></td><td> ' 1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 11</td><td>CDNA NIH_MGC_16 clone IMAGE: 3350241 H. sapiens 5 'mRNA sequence</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td><sup>?</sup> O</td><td></td><td></td><td>V5</td><td>H mitochondria, sapiens, cytochrome c oxidase subunit 1</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 1.1</td><td></td><td></td><td>s</td><td>H. sapiens mRNA; CDNA DKFZp564Gl563</td>
<td>Bl</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Sequence 21 of US Patent 5851764</td>
<td></td><td> 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td>il</td><td></td><td></td><td></td><td>PEC H Xq23 Clone DJ404K21, sapiens</td>
<td></td><td> 2</td><td></td><td></td><td></td><td>) il '</td><td></td><td></td><td></td><td></td><td></td><td><sup>;</sup> B </td><td>Unknown</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 11?</td><td></td><td></td><td></td><td>28S ribosomal RNA gene from H sapiens</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>LocusTCR betade mus muculos</td>
(Continuation)
Table 6: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 1.7 fold)
<td colspan="2" rowspan="4">Cp> 1.7</td><td></td><td> »1</td><td>Í »1S I IM</td><td>F » W</td><td>"1 í *</td><td>FW</td><td>Wm</td><td>ÍBW</td><td>C2t</td><td>C2> w</td><td rowspan="4"></td>
<td>Up</td><td></td><td></td><td> 1</td><td></td><td></td><td> 13</td><td> 2</td><td> 2</td><td> 13</td>
<td>D «i</td><td></td><td></td><td></td><td> 2</td><td> 5</td><td> 7</td><td> 7</td><td> 17</td><td></td>
<td>η</td><td></td><td></td><td> 2</td><td> 2</td><td> $</td><td> 22</td><td>and</td><td> 1$</td><td> 2?</td>
<td colspan="13"></td>
<td>B</td><td></td><td>V</td><td></td><td></td><td></td><td> 0$</td><td> 0,22</td><td>O</td><td>O</td><td>au</td><td> 0.22</td><td>H. sapiens mRNA for protein KIAA1185</td>
<td>tm</td><td></td><td>and</td><td></td><td></td><td></td><td>0J7</td><td>O</td><td> 0<sub>f</sub>1§</td><td> 0,48</td><td> 0.22</td><td> 0,37</td><td>Translation initiation factor ei F2 high H. sapiens mRNA</td>
<td> ¥22</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H mRNA, 1-high elongation paraffin sapiens (clone GEF4)</td>
<td>m</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H. sapiens mitochondria, gene-14 induced hypoxia</td>
<td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td>O</td><td> 027</td><td>to</td><td> 0,24</td><td>O</td><td>Unknown</td>
<td>B</td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 024</td><td>Gene epoxide hydrolase H microsomal (EPHX) sapiens</td>
<td>B</td><td> 1</td><td> §</td><td></td><td></td><td></td><td></td><td>d</td><td>ÍW</td><td></td><td>X</td><td>(X</td><td>Sequence Genomics of H. sapiens since 9q34</td>
<td>X23</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>w</td><td></td><td></td><td></td><td>Unknown</td>
(Continuation)
Table 6: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 1.7 fold)
<td colspan="2" rowspan="4">Up> 17 Io <08</td><td></td><td>FBH</td><td>FMiO</td><td>F »</td><td>F »i</td><td>F »</td><td>M 1C0<sub>s</sub>ii ^</td><td>W</td><td>» W</td><td>CB</td><td rowspan="4"></td>
<td>Up</td><td></td><td></td><td>Ί</td><td></td><td></td><td> 15</td><td> 2</td><td> 2</td><td> 13</td>
<td></td><td></td><td></td><td></td><td> 2</td><td></td><td>j</td><td></td><td> 13</td><td> 15</td>
<td>You</td><td></td><td></td><td> 2</td><td> 2</td><td> 5</td><td> 22</td><td> £</td><td> 15</td><td><d</td>
<td colspan="13"></td>
<td>Y17</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,&5</td><td></td><td></td><td></td><td>28S Rhibosomal RNA Gene From H. sapiens</td>
<td> $3</td><td></td><td> $</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0, -34</td><td> 070</td><td> 077</td><td>Unknown</td>
<td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td>O</td><td>CDNA clone wc44h09, x1 NGI CGAP_Pr28deH. sapiens PICTURE: 2321537 3 'similar to</td>
<td></td><td>SW: RB24_Mouse P35290 RAS RELATED PROTEIN RAB-24; mRNA sequence</td>
<td>Thousand</td><td></td><td> 3</td><td></td><td></td><td></td><td></td><td></td><td></td><td>O</td><td>Oh</td><td> 01</td><td>H. sapiens AluJb repeating sequence fragment inserted into cDNA coding for an unknown protein</td>
<td>MIS</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>í></td><td>RRNA gene H 18S, sapiens</td>
<td>O</td><td></td><td>s</td><td></td><td></td><td></td><td></td><td>O</td><td>ΰ, ιθ</td><td>O</td><td>ÍW</td><td>O</td><td>H. sapiens L7S RNA Gene</td>
The following toxicity indices were obtained:
<td>F2695</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 0</td>
<td>10 μΜ</td><td> 0</td>
<td>100 μΜ</td><td> 17</td>
<td>F2696</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 2</td>
<td>10 μΜ</td><td> 5</td>
<td>100 μΜ</td><td> 22</td>
<td>C218</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 9</td>
<td>10 μΜ</td><td> 15</td>
<td>50 μΜ</td><td> 28</td>
The following classification can therefore be established from most toxic to least toxic 0218 (clomipramine)> F2696 >>> F2695.
Clomipramine, a reference molecule encoded in the present assay 0218, shows an increase of the characteristic sequences in relation to the tested concentrations: respectively 9, 15 and 28 characteristic sequences at concentrations 1, 10 and 50 μΜ (maximum concentration defined in the previous toxicity test). ). Of course, the totality of the characteristic sequences occurring at low and medium concentrations is at higher concentrations.
At concentrations 1 and 10 μΜ, F2695 does not induce any characteristic sequence in the 682 possible stress characteristic sequences tested in the present assay. At the highest concentration of 100 μΜ, only two characteristic sequences are identified, one of which is common with C218 but of unknown significance.
F2696 shows an increase of the characteristic sequences in relation to the tested concentrations: 2, 5 and 22 characteristic sequences respectively at concentrations 1, 10 and 100 μΜ. The totality of the characteristic sequences occurring at low and medium concentrations is at higher concentrations. None of the 22 characteristic sequences is common with the F2695. In contrast, the characteristic sequences that appear at low and medium concentrations (5 where 2 are present from the low concentration) are all 5 of the 9 characteristic sequences achieved for clomipramine from the low dose of 1 μΜ. At the strong 100 μΜ concentration, there are 10/26 F2696 characteristic sequences among the 28 identified with 50 μΜ clomipramine.
From a qualitative point of view, the impact on mitochondrial transcripts, particularly at Coxl and cytochrome b levels, should be emphasized for F2696 and clomipramine. These characteristic sequences are not present with the F2695 (positions G05 / G09 / I01).
4.3.2. Induction Factor 2 for Untreated Situation (Table 7)
Table 7: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 2-fold)
Table 7:
'Up and down' regulated cones with primary rat hepatocytes (Induction Factor = 2 fold)
<td colspan="2"></td><td></td><td>Wi</td><td>FM</td><td>FM w</td><td>IMF iaM</td><td>FM φ</td><td>FM w</td><td>CB ΙμΜ</td><td>CB · ip</td><td>CS »</td><td rowspan="4"></td>
<td colspan="2" rowspan="3">up> y to «0.388</td><td>Up</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td> 1</td><td> 1</td><td> 4</td>
<td>I'm</td><td></td><td></td><td></td><td></td><td>y</td><td> 6</td><td> 7 ></td><td> 12</td><td> 12</td>
<td>You</td><td></td><td></td><td></td><td></td><td> 6</td><td> 10</td><td>s</td><td> 13</td><td> 10</td>
<td colspan="13"></td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>The</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2<sub>:</sub>,to</td><td> 2<sub>;</sub>23</td><td> 2,14</td><td>Mitochondria of H. sapiens, 12S</td>
<td></td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0,14</td><td> 0,2?</td><td>H. sapiens chromosome 19, BAC CIT-B-19ln6</td>
<td></td><td></td><td>ϋ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0/1?</td><td> 0,32</td><td>Sequence H. sapiens genomics since 17</td>
<td>C01</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>the 23rd</td><td></td><td></td><td></td><td>Mitochondria of H. sapiens, 16S</td>
<td>EGS</td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0,22</td><td> 0.35</td><td>H. sapiens DNA sequence of clone 740A11 in the chromosome Xq22.2-23. Contains part of the COL4A5 qene for the Alpha Collagen 5 (IV) chain precursor . Contains full sequence GSSl.</td>
(Continuation)
Table 7: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 2-fold)
<td colspan="3"></td><td></td><td>FW-</td><td>15th, $</td><td> »:</td><td>FBI w</td><td></td><td>Saw</td><td>CB</td><td>Uncle</td><td></td>
<td>Up</td><td></td><td>Up</td><td></td><td></td><td></td><td></td><td></td><td>w</td><td> 1</td><td></td><td> 4</td><td></td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2, iM</td><td>Clordecone H. sapiens homologous liver reductase</td>
<td>Yl</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,04</td><td>Protein related with the receiver of (variant Breast cancer ER) mRNA</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,02</td><td>C mitochondria xidase subunit 1</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,39</td><td></td><td></td><td></td><td>H. sapiens mitochondria, cytochrome b</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2.05</td><td></td><td></td><td></td><td>Mitochondria of H. sapiens, cytochrome c oxidase subunit 1</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>i)</td><td></td><td></td><td></td><td>H. sapiens Gene 18S rRNA</td>
<td>Mi</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 212</td><td></td><td></td><td></td><td>PLC mRNA from H. sapiens</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2.0$</td><td></td><td></td><td></td><td>CutA Protein tolerant of divalent cation H. sapiens mRNA</td>
L · Η (Continued)
Table 7: Up-and-down-regulated cones with primary rat hepatocytes (Induction Factor = 2-fold)
<td colspan="3"></td><td>FM Ϊ</td><td>FM · 1W</td><td>F » w</td><td>FM: W</td><td>FM KW</td><td>FM-</td><td>1W</td><td>Ç2K- KW</td><td>«00 w</td><td></td>
<td></td><td> >1,?</td><td>Up</td><td></td><td></td><td></td><td></td><td></td><td>K</td><td> 1</td><td> 1</td><td> 4</td><td></td>
<td>MO?</td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>2, 2S</td><td></td><td></td><td></td><td>Mitochondria of H. sapiens, cytochrome c oxidase</td>
<td>M1l</td><td></td><td> $</td><td></td><td></td><td></td><td></td><td></td><td> 021</td><td></td><td> 0/16</td><td>30</td><td>H. sapiens mRNA; DKFZp564Cl563o</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2$</td><td>Locus TCR mus beta muscles</td>
<td>B</td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 022</td><td> 020</td><td> 0.35</td><td>O</td><td> 0.22</td><td>H. sapiens mRNA for protein KIAA1185</td>
<td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 026</td><td>w</td><td>0A8</td><td> 022</td><td> 02?</td><td>Translation initiation factor elF-2alpha mRNA from H. sapiens</td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 2,06</td><td></td><td></td><td></td><td>H. sapiens mitochondria, induced hypoxia gene-14 of</td>
<td>W</td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 0$</td><td></td><td> 0,36</td><td>W</td><td>Ck</td><td>Unknown</td>
<td>X05</td><td></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0,15</td><td> 024</td><td>Gene epoxide hydrolase sapiens microsomal (EPHX)</td>
<td>W</td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td>O</td><td>OJS</td><td>ÍB</td><td>0, K</td><td> 0/23</td><td>H. sapiens Genomic Sequence since 9q34</td>
(Continuation)
Table 7: Up-and-down-regulated cones with primary rat hepatocytes.
Induction = 2 times)
<td colspan="3"></td><td>f »</td><td>F » 10 #</td><td></td><td>Tm</td><td>F » 10 #</td><td>w</td><td>Ç" 1#</td><td> » 10#</td><td>SIM®</td><td></td>
<td>Up</td><td>> u</td><td>Up</td><td></td><td></td><td></td><td></td><td></td><td> 10</td><td> 1</td><td> 1</td><td> - 4</td><td></td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>RNA gene ribosomal H. sapiens 28S</td>
<td></td><td></td><td> $</td><td></td><td></td><td></td><td></td><td></td><td></td><td>O</td><td>O</td><td>O'</td><td>Unknown</td>
<td>A W</td><td></td><td> $</td><td></td><td></td><td></td><td></td><td></td><td></td><td>the</td><td> 0<sub>:</sub>.27</td><td> 0,31</td><td>Repeated sequence ie H. sapiens UuJb fragment inserted into cDNA: haunting a known protein</td>
<td>Acn</td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td>iW</td><td>Yeah</td><td>O</td><td>Oh</td><td>O</td><td>H. sapiens L 7S RNA gene</td>
The following toxicity indices were obtained:
<td>F2695</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 0</td>
<td>10 μΜ</td><td> 0</td>
<td>100 μΜ</td><td> 0</td>
<td>C218</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 8</td>
<td>10 μΜ</td><td> 13</td>
<td>50 μΜ</td><td> 16</td>
<td>F2696</td><td>Toxicity index</td>
<td>1 μΜ</td><td> 0</td>
<td>10 μΜ</td><td> 5</td>
<td>100 μΜ</td><td> 16</td>
According to these parameters, the following classification may be proposed, from most toxic to least toxic C218 (clomipramine)> F2696 >>>>> F2695.
Considering the over and under expressed clones of a factor 2, F2695 does not induce any characteristic sequence (even at 100 μΜ concentration).
The effect of concentration on the emergence of the characteristic sequences is confirmed by the disappearance of the low intensity characteristic sequences for F2696.
CO at 1 μΜ present in the previous analysis with an induction factor 1,7.
Qualitatively, the impact of F2696 and clomipramine on Coxl and cytochrome b is also confirmed (positions G05 / G09 / I01).
F2695, the pharmacologically active enantiomer of F2207, is devoid of significant impact in this test, while clomipracin is used as a positive control reference product.
In contrast, F2695, an inactive F2207 enantiomer, has a quantitative and qualitatively close sequence profile of clomipramine and has no common sequence sequence with F2695.
All of this testifies to a better toxicogenomic profile for the active enantiomer F2695 whose safety coefficient in this experimental model is much significantly better than for F2695.
4.4 CONCLUSION
The pharmaco-toxicogenomic studies performed on F2695 and F2696 molecules, F2207 enantiomers (at concentrations of 10, 50 and 100 μΜ) and C218 (clomipramine at concentrations of 1, 10 and 50 μΜ) from rat hepatocytes in culture allowed stress sequences and concentration-dependent toxicity indices. These studies confirm the ability of the pharmacotoxicogenomic test to reveal characteristic stress sequences under treatment conditions (concentrations, duration of treatment) that do not cause any toxicity in a classic viability test such as with MTT.
This study highlights several important facts:
In this rat primary hepatocyte model, only F2695, the pharmacologically active enantiomer of F2207, does not induce significant toxicity index;
F2696, inactive F2207 enantiomer, and reference psychotropic clomipramine, induce important indices consisting of common or very close stress characteristic sequences. In this system, clomipramine, the reference molecule, positive product, is the product that induces more stress characteristic sequences, obtaining significant indices from the weakest concentrations. As such, it is interesting to mention that clomipramine may induce a number of undesirable effects in man, such as tachycardia, ortho-static hypotension, conduction or rhythm disturbances and, exceptionally, hepatitis. Accidental overdose with clomipramine may include syncope, hematological disorders, severe cardiovascular manifestations.
Without making premature judgments about the identity of the pathophysiological mechanism, it is interesting to note that F2696 has common stress sequences that are 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 state that the observed characteristic sequences are independent of any antidepressant or more generally psychotropic profile. On the contrary, they should be regarded as 'stress-like sequences' (F2696 in particular causes a decrease in expression of a gene involved in protein synthesis and a translational initiation factor). All this testifies to a better toxicogenomic profile for the active enantiomer F2695 whose safety coefficient in this experimental model is much significantly better than for F2696.
Contents19
6 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6
63 members in 27 offices
Priority claims8
| Document | Office | Kind | Date |
|---|---|---|---|
| 0301849 | France | A | |
| 0301849 | France | A | |
| 45357403 | United States of America | A | |
| 45357403 | United States of America | A | |
| 0301849 | – | – | – |
| 453574 | – | – | – |
| FR20030001849 | – | – | – |
| US20030453574 | – | – | – |
Members63
| Document | Office | Kind | |
|---|---|---|---|
| US2004162334A1 | United States of America | A1 | |
| FR2851163A1 | France | A1 | |
| AU2004216452A1 | Australia | A1 | |
| CA2514948A1 | Canada | A1 | |
| WO2004075886A1 | World Intellectual Property Organization (WIPO) | A1 | |
| US2004259953A1 | United States of America | A1 | |
| NO20054228L | Norway | L | |
| KR20050096190A | Republic of Korea | A | |
| MXPA05008652A | Mexico | A | |
| MA27655A1 | Morocco | A1 | |
| EP1601349A1 | European Patent Office (EPO) | A1 | |
| US2006014837A1 | United States of America | A1 | |
| RU2005128548A | Russian Federation | A | |
| BRPI0407256A | Brazil | A | |
| PL378057A1 | Poland | A1 | |
| US7005452B2 | United States of America | B2 | |
| CN1750817A | China | A | |
| HK1079117A1 | Hong Kong, China | A1 | |
| ZA200506566B | South Africa | B | |
| US7074833B2 | United States of America | B2 | |
| JP2006517571A | Japan | A | |
| FR2851163B1 | France | B1 | |
| TNSN05195A1 | Tunisia | A1 | |
| RU2317817C2 | Russian Federation | C2 | |
| EP1908461A1 | European Patent Office (EPO) | A1 | |
| EP1601349B1 | European Patent Office (EPO) | B1 | |
| AT401872T | Austria | T | |
| ATE401872T1 | Austria | T1 | |
| DE602004015257D1 | Germany | D1 | |
| AU2004216452B2 | Australia | B2 | |
| DK1601349T3 | Denmark | T3 | |
| PT1601349E | Portugal | E | |
| SI1601349T1 | Slovenia | T1 | |
| UA84697C2 | Ukraine | C2 | |
| HK1115329A | Hong Kong, China | A | |
| HK1115329A1 | Hong Kong, China | A1 | |
| ES2310715T3 | Spain | T3 | |
| NZ541733A | New Zealand | A | |
| JP2010090143A | Japan | A | |
| IL170117A | Israel | A | |
| IL201038A0 | Israel | A0 | |
| IL201038D0 | Israel | D0 | |
| JP4515446B2 | Japan | B2 | |
| NZ575218A | New Zealand | A | |
| CN1750817B | China | B | |
| EP1908461B1 | European Patent Office (EPO) | B1 | |
| EP2305225A1 | European Patent Office (EPO) | A1 | |
| AT501716T | Austria | T | |
| ATE501716T1 | Austria | T1 | |
| DE602004031893D1 | Germany | D1 | |
| SI1908461T1 | Slovenia | T1 | |
| DK1908461T3 | Denmark | T3 | |
| ES2359441T3 | Spain | T3 | |
| PT1908461EThis record | Portugal | E | |
| EP1908461B9 | European Patent Office (EPO) | B9 | |
| KR101185322B1 | Republic of Korea | B1 | |
| USRE43879E | United States of America | E | |
| CA2514948C | Canada | C | |
| PL402987A1 | Poland | A1 | |
| NO333987B1 | Norway | B1 | |
| JP5431136B2 | Japan | B2 | |
| IL201038A | Israel | A | |
| PL219671B1 | Poland | B1 |
Numbers
- Publication, DOCDB
- 1908461
- Publication, EPODOC
- PT1908461E
- Application
- 7123564
- Application, DOCDB
- 07123564
- Application, EPODOC
- PT20070123564T
Titles2
- English
- USE OF (1S, 2R) ENANTIOMER OF MILNACIPRAN FOR THE PREPARATION OF A MEDICINE
- Portuguese
- UTILIZA??O DO ENANTI?MERO (1S, 2R) DO MILNACIPRANO PARA A PREPARA??O DE UM MEDICAMENTO
Classification
- CPC, 26
- A61K31/135
- A61K31/165
- A61K31/167
- A61K31/195
- A61K31/4015
- A61P1/04
- A61P13/02
- A61P13/10
- A61P21/00
- A61P25/00
- A61P25/04
- A61P25/06
- A61P25/14
- A61P25/16
- A61P25/18
- A61P25/22
- A61P25/24
- A61P25/28
- A61P25/32
- A61P25/34
- A61P25/36
- A61P3/04
- A61P37/04
- A61P43/00
- A61P9/00
- A61P9/12
- IPC, 7
- A61K31 165
- A61K31 135
- A61K31 167
- A61K31 195
- A61K31 4015
- A61P9 00
- A61P25 00
