HK1115329B - Use of (1s, 2r) enantiomer of milnacipran for the preparation of a medicine - Google Patents

Description

The present invention relates to the enantiomer (1S,2R) of Milnacipran (Z(±)-2-(amino methyl) -N,N-diethyl-1-phenylecyclopropanecarboxamide), or one of its pharmaceutically acceptable salts, for use as a drug to prevent or treat depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain in patients with a history of cardiovascular disease and/or cardiovascular disorders, administered at a dose of 0.01 mg to 10 mg/kg body weight per day or several times. In particular, the enantiomer of the invention is intended for the treatment of depression, chronic fatigue syndrome and urinary incontinence.

Milnacipran (Z (±)-2-(amino methyl) -N,N-diethyl-1-phenyl cyclopropane carboxamide), a molecule synthesized at the Pierre Fable Drug Research Centre (Castres, France), also known as TN-912, Dalcipran, Minalcipran, Midalcipran or Midalipran is known as a dual inhibitor of serotonin (5-HT) and noradrenaline (NA) reuptake. Milnacipran and its preparation process are described in US Patent No. 4,478,836.

Dual serotonin and norepinephrine reuptake inhibitors are a well-known class of antidepressant agents that selectively inhibit both serotonin and norepinephrine reuptake. For example, venlafaxine and duloxetine are also dual serotonin and norepinephrine reuptake inhibitors. Studies have shown that the ratio of norepinephrine reuptake inhibition to milnacipran serotonin inhibition is approximately 2:1 (Moret et al., 1985 Neuropharmacology 2412): 1211-1219; Palmier et al., 1989, J Eurcolin Pharma: 375-238; C 238).

US Patent 4,478,836 describes the use of Milnacipran for the treatment of central nervous system disorders, including depression. Patent application WO01/26623 describes the use of Milnacipran in combination with phenylalanine and tyrosine in indications such as the treatment of fatigue, pain-associated syndromes, chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome. Patent application WO01/62236 describes a composition containing Milnacipran in combination with one or more anti-muscarinic agents in a large number of indications including depression. Application WO97/35574 describes a pharmaceutical composition containing Milnacipran and Milnacipran used as a combination product for the treatment of depression, as well as a combination of the two forms of Milnacipran (FR 299 and Milnacipran) which are used in the treatment of depression, and are also used in the treatment of urine disorders.

Milnacipran has two asymmetrical carbons leading to two different spatial configurations (1S,2R) and (1R,2S).

Milnacipran hydrochloride exists as two optically active enantiomers: the dextrogyre enantiomer or Z-(1S,2R)-2-(amino methyl) -carboxamide hydrochloride and the levogyre enantiomer Z-(1R, 2S)-2-(amino methyl) -N, N-diethyl-1-cyclopropane carboxamide hydrochloride. Milnacipran in its hydrochloride form (currently called F2207) is marketed (IXEL, PIERRE FABRE MEDICAMENT, France) as a melangeemic drug as a serotonin-suppressant. The drug names are F26 and F2695 (S2696 and F2202R) (S2202R) (S2202R) and Milnacipran hydrochloride (S2202R) (S2202R) respectively.

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

The inventors have now conducted a human pharmacokinetic study of the racemate and the two enantiomers of milnacipran using enantio-selectivity dosing methods, demonstrating the absence of racemisation of the enantiomers in vivo.

In addition, although racemate was resolved, no analysis of the pharmacological and toxicological properties of the two enantiomers was performed using currently available modern methods such as cardiovascular telemetry measurements or in vitro pharmacotoxicogenomic predictive analyses.

Antidepressants, like any active ingredient, can cause undesirable effects or certain toxicities which are mainly due to the pharmacological properties of these drugs, but also to the dosage, individual variability of the patient (genetic polymorphism, organ failure, sex, age) or drug interactions.The elderly are therefore the second most at risk population among the treated patients after children. These people have higher plasma concentrations, associated with reduced renal and/or hepatic clearance, and have a higher risk of intoxication (Meadoer-Woodruff et al., 1988 J. Clim. Psychopharmacol. 8 : 28-32).

The most commonly reported adverse events with Milnacipran are dizziness, hyper-sweating, anxiety, hot flashes, and dysuria. Some less commonly reported adverse events are nausea, vomiting, dry mouth, constipation, trembling, agitation, rash, skin rashes. It is also known that in patients with cardiovascular disease or receiving palliative care,In patients with hypertension or heart disease, it is therefore recommended that clinical surveillance be increased as Milnacipran in the racemic mixture form may increase heart rate. Thus, in rare cases of overdose with Milnacipran (at doses of 800 mg to 1 g) as monotherapy, the main symptoms are vomiting, respiratory distress and tachycardia (Vidal Dictionary, 78th edition, 2002).

In fact, the populations at risk of developing a number of clinical adverse events during or as a result of Milnacipran treatment are children, the elderly, patients with hepatic and/ or renal impairment, patients receiving treatment that induces organ and/ or tissue toxicities, including hepatic and/ or renal toxicities, patients receiving cardiac therapy or inducing cardiovascular side effects, patients with a history of cardiovascular disease and/ or cardiovascular disorders, including patients with heart rhythm, blood pressure disorders (hypo- or hypertensive patients) or cardiopathies.

In order to prevent the occurrence of possible side effects which may pose a risk, however small, to the health of patients treated with Milnacipran, the inventors have now discovered in a surprising and unexpected way that the enantiomer (1S,2R) of Milnacipran, which has the main activity of selective inhibition of serotonin and noradrenaline re-uptake, induces fewer cardiovascular and organotoxic and/or tissue-related side effects, particularly hepatic, than the racemic mixture. In particular, the inventors have shown that the use of the enantiomer (1S,2R) of Milnacipran with the most frequent use of the enantiomer (1S,2R) has a similar effect on the blood pressure profile of the drug Milnacipran (R2R926), in addition to its potential to increase the blood pressure.

The present invention therefore concerns the enantiomer (1S,2R) of Milnacipran (Z(±)-2-(amino methyl) -N,N-diethyl-1-phenylecyclopropanecarboxamide), or one of its pharmaceutically acceptable salts, for use as a drug for the prevention or treatment of depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain in patients with a history of cardiovascular disease and/or cardiovascular disorders, administered at a dose of 0.01 mg to 10 mg/kg body weight per day in one or more doses.

Cardiovascular disorders are undesirable cardiovascular side effects of the drug given alone or in combination with other active substances.

For the purposes of the present invention, a "side effect" is the predictable activity of a drug in a domain other than that for which it is administered, which may be bothersome or undesirable when it limits the use of the drug.

toxicity means the property of a medicinal product to cause harmful effects at the organic and/or tissue level, in particular at the level of the organs or tissues involved in the metabolism of Milnacipran, in particular the hepatic and/or renal metabolism of Milnacipran, and more particularly at the first passage of Milnacipran to the hepatic level.

For the purposes of this invention, while limiting the risk of cardiovascular disorders or while limiting the risk of toxicity means preventing these risks from increasing significantly in a patient following administration of the drug.

For the purposes of this invention, enantiomer (1S,2R) of Milnacipran means the enantiomer (1S,2R) of Milnacipran, and pharmaceutically acceptable salts thereof. Preferably, this is the enantiomer (1S,2R) of Milnacipran hydrochloride (F2695). Enantiomer (1R,2S) of Milnacipran means the enantiomer (1R,2S) of Milnacipran, and pharmaceutically acceptable salts thereof such as hydrochloride (F2696). Melancholy of racemic means a 50:50 mixture by weight of anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-anti-

There are metabolites, preferably in vivo active metabolites of Milnacipran, and pharmaceutically acceptable salts thereof, such as: - What? o hydrochloride of Z- ((±) phenyle-1 aminomethyl-2 cyclopropane carboxylic acid (F1567): - What?

Masse moléculaire :	277,7
Caractéristiques :	cristaux blancs
Point de fusion:	230 °C
Chromatographie sur plaque :	support : silice
Solvant : Butanol/Ethanol/eau (6/2/2)
Révélation : Ultra-violet et ninhydrine
Rf : 0,6

- What? o (±) phenyle-3-methylene-3-4 pyrrolidone-3 (F1612): - What?

Masse moléculaire :	173,2
Caractéristiques :	cristaux blancs
Point de fusion :	70 °C
Chromatographie sur plaque :	support : silice
Solvant : Benzène/dioxane/éthanol (90/25/4)
Révélation : Ultra-violet et iode
Rf : 0,46

- What? o Z-hydroxyphenyl-1 diethylaminocarbonyl-1 aminomethyl-2 cyclopropane hydrochloride (F2782):

Masse moléculaire :	298,82
Caractéristiques :	cristaux blancs
Point de fusion :	250 °C
Chromatographie sur plaque :	support : silice
Solvant : Butanol/Ethanol/eau (6/2/2)
Révélation : Ultra-violet et iode - ninhydrine
Rf : 0,42

- What? o Z(±) -phenyl-1-ethylamino-carbonyl-1 aminomethyl-2 cyclopropane acid oxalate (F2800): - What?

Masse moléculaire :	308,33
Caractéristiques :	cristaux blancs
Point de fusion:	150 °C
Chromatographie sur plaque :	support : silice
Révélation : Ultra-violet et ninhydrine
Rf : 0,40

- What? o Z(±) -phenyle-1 aminocarbonyl-1 aminomethyl-2 cyclopropane hydrochloride (F2941) - What?

Masse moléculaire :	226,74
Caractéristiques :	cristaux blancs
Point de fusion :	245 °C
Chromatographie sur plaque :	support : silice
Révélation : Ultra-violet et ninhydrine
Rf : 0,30

Like milnacipran, these metabolites have two asymmetrical carbons leading to two different spatial configurations (1S,2R) and (1R,2S) These spatial configurations are non-overlapping, these metabolites also exhibit optical isomerism. The ratio of the two enantiomers of the milnacipran metabolite in the enantiomer mixture is as described earlier for the enantiomers of milnacipran.

Active metabolite means a derivative obtained from the metabolism of milnacipran in vitro or in vivo and which has the ability to inhibit serotonin and norepinephrine reuptake; preferably these are F2782, F2941, F2800, F1612 and F1567

Pharmaceutically acceptable salt means any salt that retains the efficacy and properties of an active ingredient and does not have any side effects. Preferably, these are salts of pharmaceutically acceptable mineral or organic acids. Preferred but not limited examples include halogen hydrates, such as hydrochloride and bromide, fumarate, maleate, oxalate, citrate, methane sulfonate, glutamate, tartrate, mesylate, and their possible hydrates.

The enantiomer of the invention, preferably the substantially pure enantiomer F2695, is used in any type of patient requiring such treatment, whether for therapeutic and/or prophylactic purposes. In a therapeutic purpose, the goal is to eradicate or improve the condition to be treated and/or one or more associated symptoms. In a prophylactic purpose, the goal is to prevent the onset of the condition to be treated and/or one or more associated symptoms. However, the neomer of the invention is suitable for patients at risk of developing undesirable or unpleasant symptoms during treatment with certain types of cardiovascular disorders (e.g. patients with myocardial infarction and/or palpitations).

Among the many conditions or conditions which are symptoms of cardiac arrhythmia and for which the present invention is particularly suitable for the treatment of patients at risk, the most notable are tachycardia, which is an acceleration of the heart rate (tachycardia is moderate when the pulse rate is 80 to 100 beats per minute, intense when it exceeds 100), palpitations, extrasystolic (sporadic, frequent or during myocardial infarction), atrial fibrillation, atrial flutter and tachysstolia, bradycardia, heart failure, and myocardial infarction.

Among the many conditions which are symptomatic of blood pressure disorders and for which the present invention is particularly suitable for the treatment of patients at risk who are affected by them, the following are particularly noteworthy: hypertension, malignant hypertension, pulmonary hypertension, portal hypertension, essential paroxysmal hypertension, hypotension, orthostatic hypotension, intracranial hypertension.

The cardiovascular disorders whose risks may be reduced by administration of the enantiomer mixture of the invention, and preferably by administration of the substantially pure enantiomer F2695, are: elevation in diastolic and/or systolic blood pressure measured in millimetres of mercury (mm Hg); in particular, increased diastolic heart pressure, and/or heart rhythm disturbances, including an increase in the patient's heart rate.

Systolic blood pressure is the maximum value of blood pressure, and it corresponds to the time when the first heartbeats are heard in the humeral artery when blood pressure is measured. Systole is the period of heart revolution during which the heart cavities contract, causing blood to be ejected. Diastolic blood pressure is the minimum value of blood pressure, which corresponds to the disappearance of heart sounds in the humeral artery when the tension arm is deflated when blood pressure is measured. Diastole is the period of heart revolution during which the heart cavities fill with blood.Increased systolic and/or diastolic blood pressure involves an increase in blood pressure that is characteristic of systemic hypertension (and its variants) with symptoms that may include: headache, fatigue, mild sensory disturbances such as dizziness, ringing in the ears, palpitations, nosebleeds, confusion or drowsiness, cramps, numbness or tingling in the feet and hands. Systemic hypertension (and its variants) can lead to serious complications, sometimes fatal: neurological accidents of origin, left ventricular failure, renal failure, cardiopathic myocardial infarction (ischial artery and variants thereof).Current guidelines consider a patient to have high blood pressure when the blood pressure is greater than 90 mm Hg for diastolic pressure and 140 mm Hg for systolic pressure.

The toxicity risk-limiting agents of the enantiomer of the invention are organic toxicity, particularly cardiac toxicity, and/or tissue toxicity, particularly hepatic and/or renal toxicity, which may be detected by the presence of jaundice or by biological markers.

It is also within the scope of the present invention to use the enantiomer of the invention in veterinary medicine for the treatment of animals, including domestic or livestock animals requiring such treatment.

Due to their pharmacological properties, including dual inhibitors of serotonin (5-HT) and norepinephrine (NA) reuptake, the enantiomer of the invention is particularly useful in the preparation of medicinal products for the preventive and/or curative treatment of many of the pathologies or conditions (syndromes) described below while limiting the risks of cardiovascular disorders and/or while limiting organic and/or tissue toxicity, including cardiac, hepatic and/or renal toxicity.

These conditions or conditions include central nervous system disorders as defined in The Diagnostic and Statistical Manual of Mental Disorders -IV (DSM-IV), 1995 American Psychiatric Association . Examples, which are not limited to illustrative examples, include depression, including major depression, resistant depression, elderly depression, psychotic depression, interferon-induced depression, depressive disorder, manic-depressive syndrome, seizure-related depressive episodes, depressive episodes related to a general medical condition, mood-altering substance-related depressive episodes, bipolar disorder,schizophrenia, generalized anxiety, moody and moody feelings, stress-related diseases, panic attacks, phobia, including agoraphobia, obsessive-compulsive disorder, conduct disorder, opposition disorder, post-traumatic stress disorder, depression of the immune system, fatigue and associated pain disorders, chronic fatigue syndrome, fibromyalgia, and other conditions of a somatic functional nature, autism, conditions characterised by a lack of attention due to general medical problems, conditions of attention due to impairment, eating disorders, neurosis, anorexia nervosa,obesity, psychotic disorders, apathy, migraine, pain, including chronic pain, irritable bowel syndrome, cardiovascular diseases, including anxiety-depressive syndrome in myocardial infarction or hypertension, neurodegenerative diseases and associated anxiety-depressive syndromes (Alzheimer's disease, Huntington's disease, Parkinson's disease), urinary incontinence, including stress-related urinary incontinence and enuresis, drug dependence, and anxiety-dependence on tobacco, including nicotine, alcohol, narcotics, including painkillers, during these states of dependence.

In particular, the present invention is intended to use the enantiomer of the invention, preferably the substantially pure enantiomer F2695, for the preparation of a drug intended to prevent or treat depression or depressive disorder while limiting the risk of cardiovascular disorders and/or while limiting organic and/or tissue toxicity, including hepatic and/or renal toxicity. In the context of the present invention, depression is understood to be a set of symptoms comprising a psychological aspect consisting of mood disorders with a tendency to pessimism, moral anxiety, mental illness and suicide, psychological inhibition, and a pathological aspect consisting of a physical disorder characterized by a tendency to fatigue, fatigue and fatigue, and therefore a change in mental state, sometimes associated with depression and depression, and a change in the mental state, which is sometimes referred to as a "depression", a mental state of fatigue and fatigue, and a change in mental performance and mental fatigue.

The present invention also specifically addresses the use of the enantiomer of the invention, preferably the substantially pure enantiomer F2695, for the preparation of a drug to prevent or treat fibromyalgia and/or chronic fatigue syndrome while limiting the risk of cardiovascular disorders and/or while limiting organic and/or tissue toxicity, including hepatic and/or renal toxicity. Fibromyalgia syndrome is a chronic syndrome characterized by a sensation of pain or burning sensation, with a substantial amount of redness affecting mainly the fibrous tissues of the joints and the joints, and a feeling of deep fatigue. Fibromyalgia syndrome has a set of symptoms of numbness. The most frequent symptoms are chronic pain and/or weakness of the muscles, loss of focus, fatigue, depression, a need for sleep, excessive headache, fatigue, and/or sleep, and a need for sleep, depression, depression, and/or fatigue, and/or a need for increased concentration.

The present invention also specifically addresses the use of the enantiomer of the invention, preferably the substantially pure enantiomer F2695, for the preparation of a drug to prevent or treat pain, including chronic pain, while limiting the risk of cardiovascular disorders and/or while limiting organic and/or tissue toxicity, including liver and/or renal toxicity. Pain may be associated with various pathologies and/or injuries. It may be acute or chronic. Epidemiological studies have demonstrated the relationship between chronic pain conditions and anxiety disorders.

Also, the present invention is specifically intended for use in the enantiomer of the invention, preferably the substantially pure enantiomer F2695, for the preparation of a drug to prevent or treat urinary incontinence, including stress-related urinary incontinence and bedwetting, while limiting the risk of cardiovascular disorders and/or while limiting organic and/or tissue toxicity, including hepatic and/or renal toxicity.

The prophylactic and therapeutic treatment of the above diseases is carried out by delivering to an animal, preferably man, a therapeutically effective amount of the enantiomer according to the invention, preferably the substantially pure enantiomer F2695, alone or in combination with at least one other active substance. In most cases, this is the case for man, but the treatment is also suitable for animals, in particular livestock (cattle, rodents, poultry, fish, etc.) and pets (dogs, cats, rabbits, horses, etc.).

The enantiomer (1S,2R) of milnacipran and its pharmaceutically acceptable salts, as described above, is advantageously administered to patients receiving simultaneously, separately or over time at least one second active compound in the treatment of the above conditions.

Preferably, the present invention is also intended for use as a medicinal product: (a) the enantiomer (1S,2R) of milnacipran and its pharmaceutically acceptable salts, and (b) at least one active compound selected from psychotropic agents, including antidepressants, and anti-muscarinic agents, as combination products for simultaneous, separate or time-sliced use for the treatment or prevention of depression, including major depression, resistant depression, elderly depression, psychotic depression, interferon-induced depression, depressive disorder, manic-depressive syndrome, seasonal depressive episodes, depressive episodes related to a general medical condition, depressive episodes related to mood-altering substances.

Psychotropic drugs are classified into three groups: (1) psycholeptics (hypnotics, neuroleptics and anxiolytics), (2) psychoanaleptics (antidepressants and psychotonic drugs) and (3) psychodysleptics (hallucinogens).

As a non-limiting example, the antidepressant is chosen from (i) monoamine oxidase inhibitors (MAOIs) such as iproniazide, pargyline, selgin, (ii) 5HT1D agonists such as sumatriptan, adrenaline and noradrenaline (sympathetic alpha and beta mimetics) (iii) tricyclic antidepressants such as imipramine, clomipramine, (iv) selective serotonin reuptake inhibitors (SSRI) such as fluoxetine, (v) non-selective serotonin reuptake inhibitors such as fluoxetine, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline, tetracycline,

Preferably, the present invention is also intended for use as a medicinal product: (b) at least one other active substance selected from active substances inducing organic toxicity and active substances inducing tissue toxicity, including hepatic and/or renal toxicity or with one or more active substances intended for the treatment of hepatic and/or renal impairment, as combination products for simultaneous, separate or time-staged use for the treatment or prevention of conditions or pathologies which can be treated by inhibition of the reuptake of serotonin (5-HT) and norepinephrine (NA).

Preferably, the present invention is also intended for use as a medicinal product: (a) the enantiomer (1S,2R) of milnacipran and its pharmaceutically acceptable salts, and (b) at least one other active substance selected from the active compounds inducing cardiovascular side effects and the compounds targeting the heart, as combination products for simultaneous, separate or time-sliced use for the treatment or prevention of conditions or pathologies that can be cured by the double inhibition of serotonin (5-HT) and norepinephrine (NA) re-uptake.

The advantages of induced cardiovascular side effects are those mentioned above, and in particular high blood pressure, hypotension, heart rhythm disturbances (tachycardia, bradycardia, palpitations).

The present invention also applies to pharmaceutical formulations containing the combination products described above.

For the purpose of the present invention, the enantiomer of the invention, preferably the substantially pure enantiomer F2695, is administered advantageously, without limitation, orally, nasally, transdermally, rectally, intestinal, parenterally, by intramuscular, subcutaneous or intravenous injection, alone or in combination with other active substances as described above.

When administered alone, the enantiomer of the invention, preferably the substantially pure enantiomer F2695, may be administered alone or as a pharmaceutical composition in which the enantiomer or its pharmaceutically acceptable salts are in combination or blend with one or more pharmaceutically acceptable media, excipients and/or diluents, facilitating, inter alia, bioavailability.

When the enantiomer of the invention, and preferably the enantiomer (1S,2R) F2695 of milnacipran in substantially pure form, is administered in combination with other active substances, the enantiomer and the other active substances may be formulated in combination or separately in the same or different form, or may be administered by the same or different routes.

The pharmaceutical compositions of the invention may be formulated in conventional ways well known to the art using one or more physiologically acceptable media including excipients, adjuvants and auxiliaries such as preservatives, stabilizers, wetting agents or emulsifiers.

In the case of injection, an aqueous solution, including a physiologically acceptable buffer solution, such as a Hank solution, Ringer solution or a physiological saline buffer solution, is preferable; in the case of transdermal or mucosal administration, suitable penetrant agents are preferable for the mucous membrane to be passed through; such penetrant agents are well known to the user; in the case of oral administration, the pharmaceutical formulations of the invention are preferable for use in unit or multidose oral forms with adequate pharmaceutical media; in the case of transdermal or mucosal administration, suitable penetrant agents are preferable for the mucous membrane to be passed through; in the case of oral administration, the pharmaceutical formulations are preferable for use in unit or multidose oral forms with adequate pharmaceutical media; in the case of oral administration, the known unit forms of administration include the appropriate oral forms, including the appropriate serum, powder, granules, or suspensions, and the appropriate syrups.

In the preparation of tablets, the enantiomer of the invention, preferably the substantially pure enantiomer F2695, is formulated with a pharmaceutically acceptable vehicle such as polyvinylpyrrolidone, carbopol gal, polyethylene glycol, gelatine, talc, starch, lactose, magnesium stearate, gum arabic or their analogues. For example, the tablet contains the following excipients: dehydrated calcium hydrogen phosphate, calcium carmellose, povidone K30, colloidal anhydrous resin, magnesium stearate, talc. The tablets may be taken in various bags, i.e. covered with various substances such as sack-like or sack-like substances, to facilitate the preservation of the silicone.The coating may also contain pigments or dyes to distinguish and characterize tablets according to their dosage, for example. The tablets may also have a more or less complex formulation to change the rate of release of the active substance. The release of the active substance in the tablet may be accelerated, slowed or delayed depending on the desired absorption. The enantiomer of the invention, preferably the substantially pure enantiomer F2695, can thus be prepared in an extended-release galenic form obtained by the process described in EP 62396. This galenic form is multiparticle form with a plurality of mini-granules and has a certain in vitro release profile.

The release of the enantiomer of the invention may be delayed and/or controlled by the use of an implant or by transcutaneous release, including subcutaneous or intramuscular, by intramuscular injection or by transdermal patch.

The amount of the enantiomer of the invention to be administered to the patient, preferably the substantially pure enantiomer F2695, depends on the condition to be treated, the intended effect, including a therapeutic or prophylactic effect, the patient's health status and age, including cardiovascular history, treatment conditions and the method of administration of the drug. Effective therapeutic or prophylactic amounts to be administered to a human patient can be determined from model animals or data known to the professional in the treatment of depression in humans, for example by the use of Milnaci enantiomer.

In the prophylactic and/or therapeutic treatment of the above conditions, including depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain, the medicinal product of the invention is best administered at doses of between 0.01 and 10 mg/kg body weight per day in one or more doses, more favourably at doses of between 0.05 and 5 mg/kg body weight per day in one or more doses, more favourably at doses of between 0.1 and 1 mg/kg body weight per day in one or more doses. In a particularly advantageous way, the administration of the drug in such large doses as defined above is in principle administered twice daily, preferably as a single dose.

The following examples illustrate other features, purposes and advantages of the invention, and are not limited to the particular examples mentioned for illustrative purposes only, which must be read in conjunction with the following figures:

The legend of the figures

Figure 1:Evolution of heart rate after single administration (delta values). *** p ≤ 0.001 compared to deionised water ** p ≤ 0.01 compared to deionised water * p ≤ 0.05 compared to deionized water ▲p ≤ 0.05 versus F2207Figure 2:Evolution of heart rate after single administration (absolute values). *** p ≤ 0.001 compared to deionised water ** p ≤ 0.01 compared to deionised water * p ≤ 0.05 compared to deionized water ▲ p ≤ 0.05 vs F2207Figure 3:Effects of different treatments on mean diastolic blood pressure values (mean 6 hours after the last treatment, after 5 consecutive treatment days).Figure 4:Effects of different treatments on mean systolic blood pressure values (mean 6 hours after the last treatment, after 5 consecutive treatment days).Figure 5:Schematic representation of how the toxicity index is calculated.The toxicity index is the sum of all up and down-regulated genes (as a function of the user-defined induction factor).Figures 6a, 6b, 6c:MTT on primary hepatocytes in rats.MTT concentrations are expressed in μM.

Examples EXAMPLE 1: 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 were studied following administration of racemate or a single enantiomer.

A human pharmacokinetic study of 12 healthy subjects was conducted with either racemate or one of the two enantiomers, and the pharmacokinetic profile of each enantiomer was independent of whether it was administered separately or as racemate, indicating no interaction between the two enantiomers (Table 1).

These results indicate that no bioconversion of the enantiomers F2695 and F2696 was detected in the analysed species.

EXAMPLE 2: Biochemical studies of milnacipran and its enantiomers

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

2.1. MATERIALS and methods 2.1.1. capture of noradrenaline by a homogeneous (P2) rat hypothalamus. Preparation of the P2

Two male Sprague-Dawley rats, 200-300 g, were knocked unconscious and decapitated, and the hypothalamus quickly removed. Two hypothalames were homogenised in 4 ml of sucrose 0.32 M at the Potter S for 16 complete round trips at 800 rpm, then centrifuged 10 mn at 1000 g to remove cellular debris. The supernatant was centrifuged 20 mn at 10,000 g and the resulting P2 was taken up in 4 ml of sucrose 0.32 M and homogenised at the Dounce.

Catch ( Uptake )

The 3H-(1)-NA is used: 13 Ci/mmole (Amersham).

The uptake is carried out in buffered phosphate (containing per litre: 8 g NaCl, 1.21 g K2HPO4 and 0.34 g KH2PO4) pre-oxygenated 30 min before use by an O2/CO2 mixture (95% / 5%).

In 5 ml plastic tubes placed in the water bath at 37°C: 100 μl of buffer or inhibitor,700 μl of buffer (containing pargyline 25 μM),100 μl of P2.

After the temperature is balanced, the reaction is started by adding 100 μl of 3H-NA, 50 nM at the end.

After exactly 10 min, the reaction is stopped by adding 2.5 ml of iced buffer and filtering on GF/F filters. Then the tube is rinsed once and filtered once with 2.5 ml of iced buffer. The filter is then inserted into a Beckman mini-vial and after adding 3 ml of Instagel (Packard) liquid, the radioactivity is measured in a Tricarb Packard liquid scintillation meter.

The non-specific uptake (NS) is measured in the presence of a 10-5 M DMI.

The percentage of inhibition is calculated by the formula: $\frac{\left(\mathrm{uptake\; total},-,\mathrm{NS}\right)-\left(\mathrm{uptake\; en\; présence\; dʹinhibiteur},-,\mathrm{NS}\right)}{\left(\mathrm{uptake\; total},-,\mathrm{NS}\right)}$

The IC50 is determined graphically on the mean curve of the inhibition percentages (4 tests) as a function of the log of the inhibitor concentration.

The concentration of serotonin in the blood is

The method was based on Gray and Whittaker (1962, J. Anat., 96: 79-97). After homogenization of brain tissue in a sucrose solution, the presynaptic terminations detach from the axon and close to form synaptosomes obtained by subcellular fractionation.

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

This homogeneous product was centrifuged 10 minutes at 1 000 g (2 400 rpm - Hettich, Rotenta). The surfactant was recovered and centrifuged 20 minutes at 10 000 g (8 000 rpm - Beckam, model J2-21 M: rotor J14). The coulotte (called fraction P2) was recovered in sucrose at a concentration of 50 mg/ml.

We incubated for five minutes at 100 degrees . The test method is based on the following assumptions: o 350 μl of iced buffer (NaCl 136 mM, KH2PO4 2.4 mM, K2HPO4 6.9 mM, pH 7.2) preoxygenated 30 min before,o 50 μl of membranes (5 mg/ml final),o 50 μl of citalopram (10-5 M final) for nonspecific capture,o 50 μl of 3H-5-HT (50 nM final) (NEN, France, 28.4 Ci/mmol).

Exactly 5 minutes after the start of incubation, the reaction was stopped by vacuum filtration on Whatman GF/F filters (pre-dilutive with 2.5 ml of iced buffer and then rinse with 2.5 ml 3 times).

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

IC50 was determined by graphically plotting the inhibition percentages as a function of the log of product concentration (6 duplicate concentrations).

The use of paroxetine in the production of paroxetine is not recommended.

The hypothalamus of several rats was collected and homogenised in 5 ml of iced buffer (50 mM Tris-HCL, 120 mM NaCl, 5 mM KCI, pH 7.5) at Dounce, and the homogenate centrifuged at 30,000 g (27 000 t/mn - Beckman .L5-50E, rotor T40) for 10 min. The resulting buffer was collected in 5 ml of buffer and recryptrifugable under the same conditions. The new buffer was collected in the same buffer and finally homogenised at the vacuum end at a tissue concentration of 10 ml/ml. The buffer membrane suspension (100 μl) was dissolved with 3 mg H-NFEN-FEN (Fenicamphetamine, dissolved in a 0.6 ml solution of Glycine, 28 ml/ml) during the final dilution by means of a filtration tube at 2 °C. The buffer was dissolved with 3 mg of Tricycline (Fenicamphetamine, dissolved in a 0.6 ml solution of Glycine, dissolved in a 4 ml of Glycine, dissolved in a 2 ml of Glycine, dissolved in a 2 ml of Glycine, dissolved in a 2 ml of Glycine, dissolved in a 2 ml of Glycine (Packman) at 2 ml/ml (Packman) during the final dilution.

Specific binding of 3H-paroxetine was defined as the difference between total and residual binding in the presence of 10 μM of fluoxetine.

IC50 was determined by graphically plotting the inhibition percentages as a function of the log of product concentration (6 duplicate concentrations).

2.1.4 Products used

F2207: Lot No 10-CTN3 Key P118 is not included F2695: Lot number PL-I-205 F2696 : lot number PL-I-204C.

2.2. results of the study

The effects of F2207 and its two enantiomers on noradrenaline and serotonin uptake and paroxetine binding are plotted with the percentage of inhibition in order (%) and the concentration (M) of F2207, F2695 or F2696 in abscissa (data not shown).

The IC50 values for the three products were determined from these curves and are shown in Table 2. - What?

Composés	Capture

The three compounds are active in these three pharmacological tests, but there are differences: on the capture of norepinephrine: F2695 is twice as active as F2207. F2695 is 25 times more active than F2696 in serotonin capture: F2695 is three times more active than F2207. F2695 is 12 times more active than F2696 in paroxetine binding: F2695 is twice as active as F2207. F2695 is 10 times more active than F2696.

The three compounds are active in these pharmacological tests with however less activity for the form (1R,2S) (F2696) and the racemate (F2207).

EXAMPLE 3: Comparative oral activity of racemic milnacipran hydrochloride (F2207) and its active enantiomer (1S, 2R) (F2695) on heart rate and blood pressure in the watchful dog.

The Commission has already made a number of proposals.

This study aims to investigate the effects of F2207 and F2695 (a) on single oral heart rate (n = 28 dogs), and (b) after repeated oral treatment of 5 days on systolic and diastolic blood pressures in dogs (n = 6 dogs).

This study was conducted with pharmacologically active equidae of F2207 and F2695 in female animals equipped with implants (Data Sciences International) for the acquisition of heart rate and blood pressure parameters by telemetry. group 1 (control) treated with deionised water,group 2 treated with F2207 at 20 mg/kg/dose,group 3 treated with F2695 at 10 mg/kg/dose.

The Commission has already adopted a proposal for a directive on the

Given the small number of animals equipped at the same time (maximum 8), the number of recording tracks of the equipment used (8 tracks), and in order to establish homogeneous treatment groups, the overall evaluation was carried out in four studies, each study being divided into three steps (treatment of each animal with each of the three products), separated by a period of washing and reset of probes ( wash-out ). a first phase during which all animals are treated with deionized water for habituation to containment and oral treatment by a feeding tube,a second phase during which the animals receive their respective treatment (single administration for heart rate, studies No 894 / 926 / 935 / 936; repeated administration for five days for blood pressure, study No 894).

The overall experimental design is described in the following table: - What?

Nombre	27	28	28
Identification	1-2-7-8-13-14	3-4-9-10-15-	5-6-11-12-17
(étude 894)	16	-18
1-2-7-8-13-	(étude 894)	(étude 894)
14	3-4-9-10-15-	5-6-11-12-17
(étude 926)	16	-18
1-2-9-10	(étude 926)	(étude 926)
11-17-18-19	3-4-5-12	6-7-8-14
(étude 935)	13-20-21-22	15-16-23-24
1-2-9-10	(étude 935)	(étude 935)
11-17-18-19	3-4-5-12	6-7-8-14
(étude 936)	13-20-21-22	15-16-23-24
	(étude 936)	(étude 936)
Identification	Eau désionisée	F2207	F2695
Dose		20 mg/kg	10 mg/kg
Voie	orale
Volume	5 ml/kg

The effects of different treatments on heart rate were analysed in the four studies, after single administration, and the analysis covered the following 13 acquisition times: Before single treatment, every 30 minutes following 6 hours after single treatment.

The effects of the different treatments on blood pressure were analysed in study 894 at steady state at J5, J29 and J33 (last day of effective treatment for each of the steps). Before treatment, every 30 minutes for 6 hours after treatment.

The results of the study

3.3.1 For heart rate (four pooled studies), a Tukey test was performed on individual heart rate deltas for each of the 12 experimental post-treatment times versus the pre-treatment value and on absolute heart rate values at each recording time.

The following have been objectivised in relation to control animals receiving deionized water: when statistical analysis is carried out on delta values (Figure 1): a significant increase in heart rate within the first 1⁄2 hour after single administration of F2207 (20 mg/ kg), a persistent increase up to 5.5 hours after treatment (p ≤ 0.001 for all acquisition times, except for 0.5 and 5.5 hours - p ≤ 0.01 - and 5.0 hours - p ≤ 0.05 - after treatment), an increase in heart rate after administration of F2695 that is always less than that obtained after administration of F2207.In addition, this difference between the effects of F2207 and F2695 was significant (p< 0.05) at 1 and 4 hours post-treatment in favour of F2695. # when the statistical analysis is carried out on absolute values of heart rate, the same study shows (Figure 2): a significant increase in heart rate within the first hour following single administration of F2207 (20 mg/ kg), persisting up to 5.5 hours after treatment (p ≤ 0.001 for all time-acquisition times of 1.In addition, this difference between the effects of F2207 and F2695 is significant (p< 0.05) at 1 and 4 h after administration in favour of F2695. an increase in heart rate that lasts less under F2695 (1.0 to 4.5 h) than under F2207 (persists for up to 5.5 h post-treatment).

3.3.2 For blood pressure (a repeated administration study), a mean diastolic blood pressure (Figure 3 and Table 4) and a mean systolic blood pressure (Figure 4 and Table 5) were calculated for each dog and for 6 hours after the last treatment, after 5 consecutive days of administration.

The following have been objectivised: a significant (p ≤ 0.001) increase in diastolic blood pressure after 5 days of repeated administration of F2207 (20 mg/ kg/ day) or F2695 (10 mg/ kg/ day) compared to deionised water treatment,a significant (p ≤ 0.05) difference in the mean diastolic blood pressure value following 5 days of repeated administration of F2207 (20 mg/ kg/ day) compared to the mean diastolic blood pressure value following repeated administration of F2695 (10 mg/ kg/ day),no significant effect on systolic blood pressure; however, PAS values following 5 days of repeated administration of F2695 are close to PAS values following de-ionisation.

Individual diastolic and systolic blood pressure data are presented in Tables 4 and 5 respectively.

The Commission has already

In the experimental conditions of this evaluation, conducted in four successive studies by oral administration in the telemetry-equipped watchdog: ¤ on single administration and compared to the control group (n = 28), the increase in heart rate was clearly significant and sustained with F2207 at 20 mg/ kg/ day; it was statistically and clinically less and more transient with F2695 at the pharmacologically equivalent dose of 10 mg/ kg/ day,¤ the F2695 at 10 mg/ kg/ day did not cause any statistically significant change in mean systolic blood pressure over 6 hours following the last treatment, at steady state after repeated administration for 5 days, a statistically significant difference was observed in mean diastolic blood pressure over 6 hours following the last treatment, at steady state between 5 mm Hg, F262 and H2O2 (≥ 4 mm Hg) at 5 days after the last treatment, and at steady state between 5 mm Hg and H2O2 (1 ± 8 mm Hg) at 4 days after the last pharmacologically active dose.

These differences clearly indicate a better cardiovascular tolerance of the active enantiomer F2695.

The following is a list of the substances which are to be used in the preparation of the test chemical: 4.1. materials and methods

The compounds F2695 and F2696, enantiomers of the racemic molecule F2207, and a reference product, clomipramine (coded in test C218) were evaluated in the present study. Both enantiomers F2695 and F2696 were first evaluated in a preliminary cytotoxicity test (MTT test) on rat primary hepatocytes to select the three concentrations to be used in the definitive test.

After treatment of primary rat hepatocytes in culture, RNA was extracted to generate marked complementary DNA probes, which were then hybridized on a membrane containing 682 cell stress specific alternative splicing fragments. Toxicity indices were obtained for each product tested by comparing the hybridation profile of treated cells with that obtained from untreated cells.

4.1.1 Principle and purpose of the study

Safe-Hit is a sensitive, robust, reliable, fast and safe predictive pharmacotoxicogenomic test that allows for the comparison and classification of products based on optimized assessment of their toxic potential.

Safe-Hit is based on EXONHIT (DATASTM: Differential Analysis of Transcripts with Alternative Splicing) technology, which allows the splicing events resulting from a given biological state to be isolated and therefore cloned, compared to a control condition.

Safe-Hit allows the classification of molecules within a chemical series, according to a Toxic Index, determined after the following basic steps (systematically implemented in duplicate for each product): Cell lines with the different products, at three concentrations derived from a prior cell toxicology test (MTT test): a reference concentration corresponding to 80% of cell viability, a concentration 10 times higher - when possible - and a concentration 10 times lower,preparation of the total RNA and corresponding radiolabelled cDNA probes,hybridisation of the cDNA probes: Safe-Hit macro-array containing 682 independent clones, corresponding to splicing changes induced by the overexpression of WTp53 (p53 is the ubiquitous cell stress treatment chosen for the development of this method),acquisition and determination of the Toxicity Index.

4.1.2 Cells

The cells used for the study (primary cytotoxicity MTT test and main test) are cryopreserved Sprague-Dawley rat hepatocytes in primary culture (Hep184005 and Hep184006 - Biopredic lots), cultured under standard conditions.

4.1.2.1 The environment

The test method is based on the following assays: defrosting medium: Leibovitz 15 with glutamate 1 plus 100 IU/ ml penicillin, 100 μg/ ml streptomycin and 0.6 M glucose (lot MIL 210009 -Biopredic),seeding medium: Williams E medium with glutamate 1 plus 100 IU/ ml penicillin, 100 μg/ ml streptomycin, 4 μg/ ml bovine insulin and 10% v/ v foetal veal serum (lot MIL 260005) - Biopredic,incubation medium: Williams E medium with glutamate 1 plus 100 IU/ ml penicillin, 100 μg/ ml streptomycin, 4 μg/ ml insulin and 50 μg of bovine hemicortispheres (lot MIL 260007 - 269-2000).

4.1.2.2 Growing conditions

The temperature of the test chemical is 37°C, atmosphere CO2 (5%), relative humidity (95%).

4.1.2.3 Process of cultivation

	Test de toxicité cellulaire	Etude principale
	Cellules ensemencées le jour du traitement
Densité d'ensemencement	35 000 cellules / puits (plaque 96 puits)	1,5 million de cellules par plaque de 30 mm
Volume de milieu	0,1 ml	3 ml

4.1.3 Test for cytotoxicity

This cytotoxicity test (MTT test) detects living cells through a colorimetric reaction that reveals the integrity of cellular respiration involving mitochondrial activity. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), soluble in water, is transformed by cleavage, under the action of a mitochondrial enzyme of living cells, into a formase for insoluble.

The cells are exposed to the test product for 16 hours at 5 different concentrations (0 - 1 - 10 - 25 - 50 and 100 μM).

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

4.1.4 Main pharmaco-toxicogenomic study The main pharmaco-toxicogenicity study was conducted in the United Kingdom.

The main study shall be carried out in duplicate, on the basis of the seeded and exposed crops to each product, in order to increase consistency between the experiments and validate the results obtained.

4.1.4.1 Cell seeding and treatment

The cells are seeded and cultured for 16 hours with each product at the three concentrations chosen from the MTT pre-test; two controls (untreated cells, solvent alone) are added for the batch.

4.1.4.2 Total RNA extraction and dosing

After treatment, the RNA is extracted and analysed as follows: cell collection and centrifugation,extraction with a ready-to-use phenol reagent (Trizol - batch 1106266 and 1121067 - Invitrogen) according to the manufacturer's protocol,solubilisation of RNA in water,RNA dosing by spectrophotometry (optical density measured at 260, 280 and 300 nm),RNA quality verification by Agilent.

4.1.4.3 Preparation of DNA probes

The cDNA probes are prepared by radioactive reverse transcription (alpha dATP 33P - Amersham).

4.1.4.4 Hybridisation on Safe-Hit membrane

The Safe-Hit pre-cut nylon (Q-BIOgene) membranes are double-deposited with 682 DATAS (alternative splicing patterns) clones, using a Q-Pix (GENETIX) apparatus.

4.1.1.5 Preparation of DNA probes:

Matrix: 5 μg total RNA (for each treatment series and concentration),start: 100 ng of oligonucleotide oligo-dTV, for the first and second hybridisation in the rat (lot 12.00, Invitrogen),main mixture: The following is the list of substances that may be used in the preparation of the product: 10 μl of First Strand 5x Premier (lot 1131226 - Invitrogen) 1 μl of dCTP+dGTP+dTTP 20 mM (lot 1105201 - Invitrogen) 1 μM of ATP 120 μM (lot 1105291 - Invitrogen) 5 μl of Dithiotreitol (DTT) 0,1 M (lot 133609 - Invitrogen) 1 μl of RNase Out 40 U (lot 1113345 - Invitrogen) 5 μl of α 33P dATP 3 000Ci/mmol 10 mCi/μl (lot B0239 - Amersham) II4 μl of Superscript (lot 1137806 - Invitrogen) 1 μl of glycogen (lot 1129328 - Invitrogen)

- procedure:

Incubate the RNA and trace dTV at 70°C for 10 minutes and put in the ice. Add 27 μl of MasterMix and then incubate at 43°C / 1h and then 50°C / 15 minutes. Add 20 μl of water, then 20 μl of EDTA 50 mM, then 4 μl of NaOH ION. Incubate for 20 minutes at 65°C and put in the ice. Quantitatively: Instant Imager, Packard: 1 μl of reaction mixture, add 8 μl of acetic acid, 100 μl of isopropanol and 1 μl of glycogen (20 μg/μl). Incubate at -20°C for 20 minutes, centrifuge for 20 minutes at 13000 t/min at 4°C. Resuspend in 200 μl of water, The test chemical is a chemical that is used to test the reaction.

- Mediums and tampons

20X SSC (Invitrogen)	2X SSC
50X Denhardt's
50 % (w/v) Sulfate de Dextran (ICN)
20 % SDS (v/v)(Quantum biotech.)
10 mg/ml ADN de sperme de saumon
(Q-Biogene)

6X SSC	2X SSC
10X Denhardt's	0,1 % SDS
10 % Sulfate de Dextran
0,5 % SDS

5X SSC	0,5X SSC
5X Denhardt's	0,1 % SDS
0.1 % SDS

1X SSC
0,1 % SDS

- Pre-hybridization:

Allyquate 5 ml of pre-hybridization buffer in the hybridization tubes, add the corresponding volume of salmon semen DNA to a final concentration of 100 μg/ml, soak the membranes with 5X SSC, Place the membrane in the hybridisation tube and pre-hybridise for 2 hours at 65°C.

- Hybridization:

Remove the pre-hybridization pad and rinse with 10-20 ml of 5X SSC, remove the 5X SSC, replace with 5ml of pad + salmon semen DNA, denature the RT probes for 5 min at 95°C, then place on ice /1 min, centrifuge for reconstitution, then collect the appropriate volume of denatured RT probes in the tube (100,000 to 200,000 cpm/ml), incubate overnight at 55°C.

- Washing:

Rinse the membranes with 10-20 ml of washing pad 1, remove the pad and replace it with 50 ml of washing pad 2, incubate 30 min at 55°C, then remove and replace by washing with pad 4, incubate 30 min at 55°C, then decant the final wash, remove the membranes from the tubes, place on a cassette and let the acquisition take place for 3 hours.

4.1.4.5 Image acquisition and analysis

The film is then read using a Personal Molecular Imager FX (Bio-rad) and image analysis is performed using Safe-Hit Reader Software (COSE).

4.1.4.6 Calculation of the toxicity index

All data is transferred to an automatic computation program that normalizes the different membranes and calculates a Toxicity Index = sum of the number of up-regulated and down-regulated genes by a given compound at a given concentration, compared to the results of untreated controls. The results of the two Safe-Hit analyses are then compared and combined to assess the potential toxicity of the various compounds tested. ¤ the Background Threshold (BT) smooths out weak signals close to background noise and not attributable to significant gene expression. It therefore determines the detection threshold;¤ the Induction Factor (IF) is determined as the multiplier factor, versus control samples, for clones to be up or down regulated. The value of this parameter is usually 2 or below 2 to obtain relevant results. The gradual increase in the IF value selects the clones that are increasingly up or down regulated.

The method for calculating the Toxicity Index was developed by comparing the reference profiles (R: untreated cells) with an experimental profile (E) and follows the following steps (see Figure 5 for a schematic view of the process): transformation of all obtained values into log values,computation of the mean of log values for each of the duplicated tests (MiR and MiE),establishment of a matrix with MiR -MiE for all signals (= Di),normalisation of the individual MiE by subtracting from MiE the median of the 14 proximal values of Di (= NMiE),comparison of the normalised values with the reference values (Ci = NMiE - MIR),exponential transformation of Ci (= Fi),comparison of Fi with the Induction Factor chosen by the user: ¤ if Fi > IF, the gene is considered up-regulated,¤ if 1/IF < Fi < IF, the gene is expressed unchanged,¤ if Fi < 1/IF, the gene is considered down-regulated.

4.2. RESULTS of the test at the MTT

These tests were performed in triplets on rat primary hepatocytes exposed for 16 hours.

Clomipramine, referenced C218, has significant toxicity at 100 μM since no viability is observed after cell exposure for 16 hours. However, no toxicity is observed at 25 μM. At 50 μM, viability above 80% is fully compatible with a pharmacotoxicogenomic study.

For pharmaco-toxicogenomic evaluations , 3 concentrations of the same compound are used: the concentration that provides 80% viability (C) and the concentrations that correspond to (C) x10 and (C) / 10.

In order to compare the ability of F2695 and F2696 to produce a score in the test implemented, the same concentrations were used for each of them: 1 μM, 10 μM and 100 μM. For clomipramine, concentrations of 1 μM, 10 μM and 50 μM were used.

4.3 RESULTS on primary hepatocytes in rats

Toxicity indices (IT) were determined as described above. In these indices only clones that were found to be modulated compared to controls in the two independent experiments were considered, only clones with a signal 2 times greater than background noise (BT) were considered. Two different analyses were performed using two levels of difference (Induction Factor - IF) compared to the untreated situation: This factor of 1.7 is the lowest value which allows no index to be obtained when comparing two untreated situations.

4.3.1 Induction factor of 1.7 compared to the untreated situation (Table 6)

Tableau 6: Clones « up- et down-régulés » avec des hépatocytes primaires de rat (Facteur d'Induction = 1,7 fois)

			F2695 -1 µM	F2695 10 µM	F2695- 100 µM	F2696 -1 µM	F2696 10µM	F2696 100µ M	C 218- 1µM	C218- 10µM	C 218- 100µM
Up	>1,7	Up			1			15	2	2	13
Down	<0,58 8	Down			1	2	5	7	7	13	15
		TI			2	2	5	22	9	15	28
Pos	nb U	nb D										Gene
A09								2,90	2,23	2,14	H. sapiens mitochondrion, 12S
A20	1						0,56				H. sapiens initiation factor elF-5A gene
B20	2								0,14	0,27	H. sapiens chromosome 19., BAC CIT-B-191n6
B22	2								0,17	0,32	H. sapiens Genomic sequence from 17
C01							3,20	1,93	1,82	1,91	H. sapiens mitochondrion, 16S
E01										1,73	H. sapiens mRNA for lipocortin II
E05		2								0,22	0,35	H. sapiens DNA sequence from clone 740A11 on chromosome Xq22.2-23. Contains part of the
			COL4A5 gene for Collagen Alpha 5 (IV) Chain Precursor. Contains GSS1, complete sequence
E11							2,12				H. sapiens chlordecone reductase homolog liver, mRNA
E19										1,72	H. sapiens mitochondrion, cytochrome c oxidase subunit 1
E21	2								0,5.6	0,58	H. sapiens ribosomal protein S14 gene
F24	1									0,52	H. sapiens LIM homeobox protein cofactor (CLIM-1) mRNA
G01										2,04	H. sapiens estrogen receptor-related protein (variant ER from breast cancer) mRNA
G05										2,02	H. sapiens mitochondrion, cytochrome c oxidase subunit 1
G09							2,09			1,76	H. sapiens mitochondrion, cytochrome b
I01							2,05				H. sapiens mitochondrion, cytochrome c oxidase subunit 1
I18							2,38			1,88	H. sapiens 18S rRNA gene
L01							2,05				H. sapiens divalent cation tolerant protein CUTA mRNA
L22							1,78				H. sapiens mRNA for Lon protease-like protein
L23											1,75	H. sapiens cDNA NIH_MGC_16 clone IMAGE:3350241 5', mRNA sequence
M07								2,25			1,75	H. sapiens mitochondrion, cytochrome c oxidase subunit 1
M12		3						0,21		0,16	0,39	H. sapiens mRNA; cDNA DKFZp564C1563
M23								1,95				Sequence 21 from patent US 5851764
P05								1,78				H. sapiens PAC clone DJ404K21 from Xq23
Q11					1,81						1,92	unk
Q24								1,77				H. sapiens 28S ribosomal RNA gene
S01											2,98	Mus muculus TCR beta locus
T08		6				0,50	0,22	0,20	0,35	0,14	0,22	H. sapiens mRNA for KIAA 1185 protein
U04		6				0,57	0,26	0,19	0,48	0,22	0,37	H. sapiens translation initiation factor elF-2alpha mRNA
V22												H. sapiens mRNA for elongation, factor 1-alpha (clone CEF4)
W17								2,96				H. sapiens mitochondrion, hypoxia inducible gene-14
X02		5					0,29	0,20	0,36	0,24	0,31	unk
X05		2								0,15	0,24	H. sapiens microsomal epoxide hydrolase (EPHX) gene
X06		5					0,2	0,16	0,23	0,15	0,23	H. sapiens Genomic séquence from 9q34
X23								1,92				unk
Y17								2,65				H. sapiens 28S ribosomal RNA gene
Z13		3							0,34	0,29	0,27	unk
Z20		1									0,57	Homo sapiens cDNA wc44h09,x1 NCI_CGAP-Pr28 clone IMAGE:2321537 3' similar to
											SW:RB24_Mouse P35290 RAS_RELATED PROTEIN RAB-24;, mRNA sequence
AA11		3							0,38	0,27	0,31	H. sapiens Repeat sequence AluJb fragment inserted into a cDNA coding for an unknown protein
AA13											1,79	H. sapiens 18S rRNA gene,
AC13		5					0,22	0,16	0,28	0,16	0,28	H. sapiens 7S RNA L gene

The following toxicity indices were obtained: - What?

1 µM	0
10 µM	0
100 µM	17

1 µM	2
10 µM	5
100 µM	22

1µM	9
10 µM	15
50 µM	28

The following classification can therefore be established, from most toxic to least toxic C218 (clomipramine) > F2696 >>> F2695.

Clomipramine, the reference molecule encoded in this C218 assay, shows an increase in signatures relative to the test concentrations: 9, 15 and 28 signatures at 1, 10 and 50 μM concentrations respectively (maximum concentration defined in the prior cytotoxicity test).

At 1 and 10 μM concentrations, F2695 induces no signature of the 682 possible stress signatures tested in this test. At the highest concentration of 100 μM, only two signatures are identified, one of which is common to C218 but of unknown significance.

F2696 shows an increase in signatures relative to the test concentrations: 2, 5 and 22 signatures at 1, 10 and 100 μM concentrations respectively. All signatures occurring at low and medium concentrations are found at higher concentrations. None of the 22 signatures is common with F2695. However, signatures occurring at low and medium concentrations (5 of which occur at low concentration) are all 5 of the 9 signatures detected for clomipramine at low dose of 1 μM. At high concentration of 100 μM, 10/26 F2696 signatures are found among the 28 identified with clomipramine at 50 μM.

Qualitatively, the impact on mitochondrial transcripts, particularly at Cox1 and cytochrome b levels, is notable for F2696 and clomipramine. These signatures are not present with F2695 (items G05/G09/I01).

4.3.2 Induction factor of 2 compared to the untreated situation (Table 7)

Tableau 7: Clones « up- et down-régulés » avec des hépatocytes primaires de rat (Facteur d'Induction = 2 fois)

			F2695- 1 µM	F2695- 10µM	F2695- 100µM	F2696- 1 µM	F2696- 10µM	F2696- 100µM	C218- 1µM	C 218- 10µM	C218- 100 µM
Up	>1,7	Up						10	1	1	4
Down	<0,588	Down					5	6	7	12	12
		TI					5	16	8	13	16
Pos	nb U	nb D										Gene
A09									2,90	2,23	2,14	H. sapiens mitochondrion, 12S
B20										0,14	0,27	H. sapiens chromosome 19, BAC CIT-B-191n6
B22										0,17	0,32	H. sapiens Genomic sequence from 17
C01								3,20				H. sapiens mitochondrion, 16S
E05										0,22	0,35	H. sapiens DNA sequence from clone 740A11 on chromosome Xq22.2-23. Contains part of the COL4A5 gene for Collagen Alpha 5 (IV) Chain Precursor. Contains GSS1, complet sequence
E11								2,12			2,04	H. sapiens chlordecone reductase homolog liver, mRNA
G01											2,04	H. sapiens estrogen receptor-related protein (variant ER from breast cancer) MRNA
G05											2,02	H. sapiens subunit 1
G09								2,09				mitochondrion, cytochrome c oxidase H. sapiens mitochondrion, cytochrome b
I01								2,05				H. sapiens mitochondrion, cytochrome c oxidase subunit 1
I18								2,38				H. sapiens 18S rRNA gene
J03								2,12				H. sapiens CLP mRNA
L01								2,05				H. sapiens divalent cation tolerant protein CUTA mRNA
M07								2,25				H. sapiens mitochondrion, cytochrome c oxidase subunit 1
M12								0,21		0,16	0,39	H. sapiens mRNA; cDNA DKFZp564C1563
S01											2,98	Mus muculus TCR beta locus
T08							0,22	0,20	0,35	0,14	0,22	H. sapiens mRNA for KIAA1185 protein
U04							0,26	0,19	0,48	0,22	0,37	H. sapiens translation initiation factor elF-2alpha mRNA
W17								2,96				H. sapiens mitochondrion, hypoxia inducible gene-14
X02							0,29	0,20	0,36	0,24	0,31	unk
X05										0,15	0,24	H. sapiens microsomal epoxide hydrolase (EPHX) gene
X06							0,20	0,16	0,23	0,15	0,23	H. sapiens Genomic sequence from 9q34
Y17								2,65				H. sapiens 28S ribosomal RNA gene
Z13									0,34	0,29	0,27	unk
AA11									0,38	0,27	0,31	H. sapiens Repeat sequence AluJb fragment inserted into a cDNA coding for an unknown protein
AC13							0,22	0,16	0,28	0,16	0,28	H. sapiens 7S RNA L gene

The following indices were obtained: - What?

1µM	0
10 µM	0
100 µM	0

1µM	8
10µM	13
30µM	16

1 µM	0
10µM	5
100 µM	16

According to these parameters, the following classification can be proposed, from most toxic to least toxic: C218 (clomipramine) > F2696 >>>>> F2695.

Considering clones over or under-expressed by a factor of 2, F2695 does not induce any signature even at a concentration of 100 μM.

The effect of concentration on the appearance of signatures is confirmed by the disappearance of the low intensity signatures for F2696 at 1 μM, which were present in the previous analysis with an induction factor of 1,7.

Qualitatively, the impact of F2696 and clomipramine on Cox1 and cytochrome b is also confirmed (headings G05/G09/I01).

The pharmacologically active enantiomer F2695, of F2207, had no significant impact in this assay, while clomipramine was used as a positive control reference.

In contrast, F2696, the inactive enantiomer of F2207, has a signature profile that is quantitatively and qualitatively close to clomipramine and has no signature in common with F2695.

This indicates a better toxicogenomic profile for the active enantiomer F2695 whose safety factor is very significantly better than for F2696 in this experimental model.

4.4 CONCLUSIONs and the

Pharmaco-toxicogenomic studies on molecules F2695 and F2696, enantiomers of F2207 (at 10, 50 and 100 μM concentrations) and C218 (clomipramine, at 1, 10 and 50 μM concentrations), from primogenitic rat hepatocytes have yielded concentration-dependent stress signatures and toxicity indices, confirming the ability of the pharmaco-toxicogenomic test to reveal stress signatures under treatment conditions (concentrations, duration of treatment) that do not cause any toxicity in a conventional viability test such as MTT.

Several outstanding facts emerge from this study: ¤ in this model of rat primary hepatocytes, only F2695, the pharmacologically active enantiomer of F2207, does not induce a significant toxicity index;¤ F2696, the inactive enantiomer of F2207, and the reference psychotropic clomipramine induce large indices formed from common or very close stress signatures. In this system, the positive reference molecule clomipramine is the most stress-inducing product, with significant indices obtained at the lowest concentrations. In this respect, it is worth noting that clomipramine can induce a number of undesirable effects in humans such as tachycardia, hypotension, orthostatic or conduction disorders, for example, in the case of cardiovascular accident, and in exceptional cases, on the synthesis of other symptoms, such as oncopathemia.

Without prejudice to an identity of pathophysiological mechanism, it is interesting to note that F2696 has stress signatures common to or very close to those of clomipramine and also induces adverse effects such as the cardiovascular disorders described above.

It is therefore legitimate to argue that the signatures observed are independent of any antidepressant or more broadly psychotropic profile, but should be considered stress signatures (F2696 in particular causes a decrease in expression of a gene involved in protein synthesis and a translation initiation factor).

Claims (11)

1. The (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, for use as a medicament intended for preventing or treating depression, depressive states, fibromyalgia, chronic fatigue syndrome, pain, in patients having a history of cardiovascular disease and/or suffering from cardiovascular disorders, administered at a dose of between 0.01 mg and 10 mg/kg of body weight per day in one or more intakes.
2. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to Claim 1, characterized in that the dose administered is between 0.05 mg and 5 mg/kg of body weight per day in one or more intakes.
3. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to Claim 1 or 2, characterized in that the dose administered is comprised between 0.1 mg and 1 mg/kg of body weight per day in one or more doses.
4. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 3, characterized in that the cardiovascular disorders correspond to an increase in arterial blood pressure and/or an increase in heart rate.
5. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to Claim 4, characterized in that the increase in arterial blood pressure corresponds to an increase in diastolic arterial blood pressure.
6. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 5, while limiting moreover the risk of organ and/or tissue toxicity.
7. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 6, characterized in that said (1S,2R) enantiomer of milnacipran is the hydrochloride of Z-(1S,2R)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide (F2695).
8. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 7, for the treatment or prevention of deep depression, resistant depression, depression in the elderly, psychotic depression, depression induced by treatments with interferon, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes related to general health status, or depressive episodes related to mood-altering substances.
9. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 7, for the treatment or prevention of chronic pain.
10. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 9, characterized in that the history of cardiovascular disease and/or cardiovascular disorders is (are) chosen from myocardial infarction, cardiac rhythm disorders (tachycardia, bradycardia, palpitations), arterial blood pressure disorders (hypo- or hypertensive patients) and cardiopathies.
11. (1S,2R) enantiomer of milnacipran (Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropane-carboxamide) or a pharmaceutically acceptable salt thereof, according to any one of Claims 1 to 10, characterized in that the medicament contains:

    a) said (1S,2R) enantiomer of milnacipran or a pharmaceutically acceptable salt thereof, and

    b) at least one active compound chosen from psychotropic agents, in particular antidepressants, and antimuscarinic agents,

    as combination products for use simultaneously, separately or staggered in time for the treatment or prevention of depression, notably deep depression, resistant depression, depression in the elderly, psychotic depression, depression induced by treatments with interferon, depressive state, manic-depressive syndrome, seasonal depressive episodes, depressive episodes related to general health status, and depressive episodes related to mood-altering substances.