OA16854A - Levomilnacipran drug for functional rehabilitation after an acute neurological stroke - Google Patents

Abstract

The present invention relates to the use of levomilnacipran as a drug in functional recovery after a cerebrovascular accident or head trauma. The pharmaceutical compositions containing levomilnacipran are strictly those in which the levomilnacipran/dextromilnacipran mixture does not contain more than 5 wt % of dextromilnacipran, so as to not risk compromising functional recovery due to the alpha-blocking property of the dextromilnacipran.

Description

Levomilnacipran drug for functional réhabilitation after an acute neurological stroke.

DESCRIPTION

There are two types of acute neurological events leading to motor and cognitive déficit: one is of vascular origin i.e. a Cerebrovascular Accident , (stroke) and the other is of traumatic origin i.e. Traumatic Brain Injury.

According to WHO, a Cerebrovascular Accident (CVA) is rapidly developing clinical signs of focal (at times global) disturbance of cérébral function, lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin. The term brain attack is also used or apoplexy. CVA is to be distinguished from a transient ischémie attack (TIA) defined as sudden loss of cérébral or ocular function lasting less than 24 hours assumed to be due to an embolism or vascular thrombosis. CVA is the most frequent type of neurological disease: in Western countries it represents the third cause of death (after coronary diseases and cancers) and the leading cause of handicap acquired at adult âge and the second cause of dementia (Murray CJ, Lopez AD, Mortality by cause for eight régions of the world: Global Burden of Disease Study, Lancet, 1997; 349:1269-1276).

With CVA the vascular problem concerned is either thrombo-embolic (80% of CVAs) due to interrupted blood supply through obstruction of an artery, or hémorrhagie (20% of CVAs) through rupture of an artery. Cérébral thrombosis is most often caused by arteriosclerosis (hardening and inflammation of the vascular wall). Interrupted circulation secondary to arterial thrombosis (obstruction by a blood clôt) is the cause of infarction (death, necrosis of the région concerned)

accompanied by softening of the corresponding territory which is no longer irrigated. Gradually the dead tissue is replaced by conjunctive tissue formed of glial cells. Another cause of infarction is a brain embolism in which an atheroma plaque (fat) may detach itself from a large vessel, or when a blood clôt is formed for example in embolie cardiopathy (myocardial infarction, valvulopathy, arrhythmia through atrial fibrillation) and cornes to obstruct a cérébral artery causing an infarction. Brain hemorrhage may also be due to arteriosclerosis, most often accompanied by arterial hypertension. Brain hemorrhages may also be caused by congénital arterial malformation, infection, brain tumor, or even an upsetting event, an émotion or strenuous effort. Hemorrhage is the origin of hematoma formation which gradually résolves.

CVA diagnosis is firstly clinical. Examination of motor capacities and sensitivity of ail or part of the body directs diagnosis towards the site of the lésions which is confirmed by brain imaging. Diagnosis may give rise to problems in comatose, aphasie or amnésie patients. The seriousness of clinical signs varies from the lack of any notable sign to death within a period of a few days and may inciude motor, coordination and walking disorders, and disorders of sensitivity, speech, visual field, memory and psyché.

Treatment of CVA is started immediately after the event and takes into account the ischémie or hémorrhagie origin, determined by brain imaging using CT brain scans with or without contrast agent, and magnetic résonance imaging (MRI). To treat ischémie CVA the goal is to regulate hydroelectrolytic balance and arterial pressure and to obtain reperfusion of the injured territory using thrombolytic agents such as anti-platelet agents (aspirin) and fibrinolytics (e.g.

rt-PA (recombinant tissue plasminogen activator)) when the CVA is taken in charge less than 4h30 after the first signs. For hémorrhagie CVA, surgery is indicated if it is possible taking into account the topography and volume of the hematoma, the patient's level of consciousness and the acute phase, several months general condition. Recovery, after is very progressive and may take or years. It often requires réhabilitation to treat speech disorders. While motor disorders and/or walking (movernents) and sensory disorders (sensation) can generally be restored intellectuel sequels may be irréversible.

Traumatic brain injury (TBI) is the main cause of death and severe handicap before the âge of 45. The main causes are: road accidents (about 50%), sports accidents, occupational accidents, domestic accidents, attacks, natural disasters and acts of war. There are different types of TBI:

concussion: jarring of the brain subséquent to a violet blow to the brain, whether or not accompanied by initial or temporary loss of consciousness, with no visible radiological lésion to the brain. Return to consciousness spontaneously occurs after a few seconds, minutes or hours after the traumatic event in relation to the extent of the shock and may leave transient memory disorders, even secondary complications: extradural hematoma, sub-dural hematoma, cérébral edema.

brain contusion: in this case, there are anatomical lésions of the brain (hémorrhagie necrosis with edema), not necessarily at the site of impact, which may become complicated with brain edema.

immédiate deep coma: this is the most serious form of concussion. The patient is in a deep, persistent coma after the shock since the dysfunction of the ascending reticular activating System lies at

I

- i deeper depth. Signs of décérébration are possible indicating the presence of diffuse mesencephalic and axonal lésions related to concentric propagation and the concentration of the shock waves towards the centre of the brain (stereotaxic phenomena).

The management of TBI includes the search by brain imaging for surgically curable lésions (hematoma), surgery on opérable lésions or if not intensive care medical treatment in a specialized unit (anti-edema, pulmonary resuscitation etc.). Diuretics are used to reduce brain edema, and mannitol to dehydrate the brain tissue. At times brain edema is extensive enough to initiate brain herniation (engaging of the lower part of the brain underneath the faix cerebri towards the contralatéral cérébral hemisphere, engaging of the lower part of the brain into the foramen magnum) . Meningeal hemorrhage may also be associated with brain contusion, translating as headaches, stiff neck and alertness disorders. Clinical and radiological monitoring is set up after emergency treatment. Prognosis dépends on the extent of the initial lésions, patient âge and general condition before the event. The more the coma is superficial and the younger the patient in good health before the event the greater the chances of recovery. However coma may lead to brain death in some cases.

After the critical period following after the traumatic event and return to consciousness, as is the case with CVA, there is a period of functional recovery which may leave neurological sequels: signs of plegia or paralysis, balance disorders, symbol disorders of aphasia or agnosia type, signs of lésions to the cranial nerves; neuroendocrine disorders: diabètes insipidus, weight loss, fatigue, dizziness, loss of libido and impotency; mental disorders: anxiety after awareness of potentially irréversible sequels, anhedonia. Other conséquences are less frequent: subséquent post-traumatic epilepsy, vascular disorders such as aneurysm rupture or brain arterial thrombosis.

The field of the invention concerns medical action with the goal of improving recovery and functional réhabilitation after an acute neurological event whether a CVA or TBI. Within the context of the invention, improving recovery and functional réhabilitation means accelerating and amplifying the resolving of motor, neurophysiological, cognitive or psychiatrie symptoms whose onset occurred at the time of the neurological event, or one or more of these symptoms. According to the invention, the motor, neurophysiological, cognitive and psychiatrie symptoms include but are not limited to:

- paralysis and plegia, including hemiplegia and tétraplégie ;

- paresthesia or sensitivity disorders;

- coordination disorders, ataxia of the limbs and walking;

- eye movement disorders;

- swallowing disorders;

- speech disorders whether concerning perception and understanding or expression;

- apraxia and disrupted spatial orientation;

- sight disorders and disorders of the visual field;

- pupil anomalies;

- attention disorders;

- memory disorders affecting short-term memory (recent events) or long-term memory (past events;

- perception disorders, essentially visual concerning récognition of objects, images writing or physiognomies;

- disorders of executory functions such as action planning;

- anxiety;

- anhedonia and dépressive symptoms;

- perseverance;

- impulsiveness.

The invention also concerns recovery and functional réhabilitation after a récurrent neurological event occurring after an initial neurological event, caused by the conséquence thereof on postural balance, loss of sight or visuospatial neglect.

Studies in animal and man hâve shown that it is possible to accelerate or amplify functional recovery after an acute neurological event, via the administration of medical treatment immediately or even after a period of a few days to a few months following after the event. In animal models of unilatéral occlusion of the middle cérébral artery, which mimic CVA, and models of focal cortical lésions, which mimic TBI (Goldstein LB. Basic and clinical studies of pharmacological effects on recovery from brain injury, J. Neural Transplant & Plasticity, 1993, 4:175-192;

Feeney DM, de Smet AM, Rai S, Noradrenergic modulation of hemiplegia: facilitation and maintenance of recovery. Restor Neurol & Neurosci, 2004, 22:175-190), medical treatments which proved to be active in the rat and cat are:

- amphétamine, a product which increases extracellular levels of noradrenaline, serotonin and dopamine,

- transplantation of chromaffin cells, which secrete noradrenaline;

- intracérébral infusion of noradrenaline, but not serotonin or dopamine;

- administration of a noradrenaline precursor;

- administration of alpha-adrenergic agonists.

Clinical studies, in small groups of patients, hâve shown the possibility of improving motor recovery after CVA by treatment with amphétamine (Sonde L, Nordstrôm M, Nilsson CG, Lôkk J, Viitanen M, A doubleblind placebo-controlled study of the effects of amphétamine and physiotherapy after stroke. Cerebrovasc Dis, 2001,12:253-257); L-DOPS, a noradrenaline precursor (Nishino K, Sasaki T, Takahashi K, Chiba M, Ito T, The norepinephrine precursor L-threo-3,4dihydroxyphenylserine facilitâtes motor recovery in chronic stroke patients. J. Clin Neurosci, 2001, 8:547550), methylphenidate, an agent that also increases the extracellular rates of noradrenaline, serotonin and dopamine (Tardy J, Pariente J, Leger A, DechamontPalacin S, Gerdelat A, Guiraud V, Conchou F, Albucher JF, Marque P, Franceries X, Cognard C, Rascol 0, Chollet F, Loubinoux I, Methylphenidate modulâtes cérébral post-stroke reorganization, Neuroimage, 2006, 33 :913-922), reboxetine, a sélective inhibitor for the reuptake of noradrenaline (Zitel S, Weiller C, Liepert J, Reboxetine improves motor function in chronic stroke. A pilot study, J. Neurol, 2007, 254:197-201), fluoxetine, a sélective inhibitor for the reuptake of serotonin (Pariente J, Loubinoux I, Carel C, Albucher JF, Leger A, Manelfe C, Rascol O, Chollet F, Fluoxetine modulâtes motor performance and cérébral activation of patients recovering from stroke, Ann Neurol, 2001, 50:718-729). The effect of fluoxetine administered for 3 months was confirmed in a larger double-blind, * ¹ , placebo-controlled clinical study (Chollet F, Tardy J, Albucher JF, Thalamas C, Berard E, Lamy C, Bejot Y, Deltour S, Jaillard A, Niclot P, Guillon B, Moulin T, Marque P, Pariente J, Arnaud C, Loubinoux I, Fluoxetine for motor recovery after acute ischaemic stroke (FLAME): a randomized placebo-controlled trial. Lancet Neurol, 2011, 10:123-30).

It is important to point out that these therapeutic approaches are not intended to restore or protect the injured brain area but to enable the brain which has some plasticity to reorganize its circuits to allow non-injured régions to ensure the functions normally carried out by the injured région, whether motor, neurophysiological or cognitive functions. This was confirmed by functional imagery after CVA and TBI. The ability of the monoamines (noradrenaline, serotonin, dopamine) to promote the functional reorganization of the brain tallies with their known neurotrophic rôle for developing neurons to ensure the différentiation and survival of neurons.

From these data showing the crucial rôle of serotonin and noradrenaline in functional recovery, it is concluded that a médicinal product which produces a rise in extracellular levels of both noradrenaline and serotonin would hâve an advantage over médicinal products which only increase serotonin levels, such as fluoxetine, for treatment after an acute neurological event.

Levomilnacipran is the (IS, 2R) enantiomer of milnacipran (Z (+)-2-aminomethyl)-N,N'-diethyl-1-phenyl cyclopropane carboxamide) described in patents WO 2004/075886 and WO 2009/127737. Milnacipran is an inhibitor of the reuptake of noradrenaline and serotonin having a balanced effect on these two neurotransmitters (Briley M, Prost JF, Moret C, ¹ ,

Preclinical pharmacology of milnacipran. Int Clin Psychopharmacol, 1996 Suppl 4:9-14; Preskorn SH, Milnacipran: a dual norepinephrine and serotonin reuptake pump inhibitor, J Psychiatr Pract, 2004, 10:119-26). Milnacipran is a drug used in dépréssion (Spencer CM and Wilde MI, Milnacipran: a review of its use in dépréssion. Drugs, 1998, 56:405-427) and in fibromyalgie (Owen RT, Milnacipran hydrochloride: its efficacy, safety and tolerability profile in fibromyalgia syndrome. Drugs Today (Barc) 2008, 44:65360) . Patent applications WO2003/039598, W02003/068211,

W02003/077897,

W02004/030633,

W02003/090743,

W02004/045718,

W02004/009069,

WO/2007/038620,

WO2008/019388,

WO2008/021932 and WO2008/147843 also describe the use of milnacipran and its enantiomers in chronic fatigue syndrome, attention déficit with hyperactivity, viscéral pain syndromes, functional somatic syndromes, cognitive and sleep disorders, irritable bowel syndrome, chronic lumbar pain, chronic pelvic pain, interstitial cystitis, non-cardiac chest pain, neuropathie disorder, atypical multiple chemical pain, temporomandibular joint facial pain, tension headache, sensitivities, chronic pain associated with medical treatment or radiotherapy or other indications of chronic pain; in particular these patent applications do not describe the use of milnacipran for the treatment of CVA and TBI.

Levomilnacipran is the isomer deemed to be the active isomer of milnacipran; it has the highest affinity for noradrenaline and serotonin transporters compared with that of the other enantiomer, dextromilnacipran, and blocks the reuptake of noradrenaline and serotonin at lower concentrations than those required by dextromilnacipran (Example 1) . Surprisingly however dextromilnacipran is the most ⁴ ♦ powerful isomer on the alphal-adrenergic receptor in rat or man (Example 1). In addition, dextromilnacipran has alphal-antagonist behavior: it does not activate the recombinant human alphal receptor and antagonizes the effect of adrénaline (Example 2).

Preclinical and clinical data indicate that the alphal-adrenergic receptor plays a crucial rôle in functional recovery after a neurological event. Indeed a single administration of prazosin, a sélective antagonist of the alphal receptor (Hoffman and Lefkowitz, Catecholamines, sympathomimetic drugs and adrenergic receptor antagonists, in Goodman and Gilman's, The Pharmacological Basis of Therapeutics, Hardman JG, Limbird LE, Molinoff P B, Ruddon RW publishers, 9^th édition, 1995, McGraw-Hill, New York, pp. 229) delays functional recovery after unilatéral focal traumatic contusion of the sensorimotor cortex in the rat (Feeney DM and Westerberg VS, Norepinephrine and brain damage: alpha noradrenergic pharmacology alters functional recovery after cortical trauma. Can J Psychology, 1990, 44: 233-252) and précipitâtes the reonset of motor symptoms in the rat up to 6 months after a unilatéral frontal lésion in the rat when motor functional recovery has been effective (Stibick DL and Fennec DM, Enduring vulnerability to transient reinstatement of hemiplegia by prazosin after traumatic brain injury, J. Neurotrauma, 2001, 18:303-312). In healthy volunteers, the administration of prazosin decreases the efficacy of motor training in inducing cérébral plasticity in the cortex, in the absence of any change in cortical-motor excitability (Sawaki L, Werhahn KJ, Barco R, Kopylev L, Cohen LG, Effect of an alphal-adrenergic blocker on plasticity elicited by motor training, Exp Brain Res, 2003, 148:504-508). Training-induced plasticity is assumed to contribute towards motor functional recovery after an acute neurological event. Goldstein et al (The influence of drugs on the recovery of sensorimotor function after stroke, J Neuro Rehab, 1990, 4:137-144) conducted a study in CVA patients and found that those who were prescribed drugs having deleterious effects on functional recovery in experimental animais, which notably included prazosin, had lower motor scores on the Fugl-Meyer scale than those not given these drugs with deleterious effects, over a 30-day prospective study following after inclusion. Ail these preclinical and clinical data show that an alphal-adrenergic antagonist must not be administered to a patient in functional recovery phase after an acute neurological event.

It has therefore surprisingly been discovered that it would be contra-indicated to administer dextromilnacipran, which was found to be an alphaladrenergic antagonist when developing the invention, to a patient in functional recovery after an acute neurological event whether a CVA or TBI. Therefore contrary to the provision made in application W02006/006617, the milnacipran racemate which contains an equal proportion of levomilnacipran and dextromilnacipran, must not be prescribed in the abovementioned clinical situations. On the contrary, substantially pure levomilnacipran or a levomilnacipran/dextromilnacipran mixture containing dextromilnacipran in a proportion not exceeding 5% by weight of the said mixture (see Example 3) should be used during functional recovery after an acute neurological event whether a CVA or TBI.

Patent W02004/075886 claims the use of levomilnacipran to préparé a médication for a variety of pathologies in patients presenting with t , cardiovascular risk, on the basis of the observation that levomilnacipran induces fewer hémodynamie phenomena than the milnacipran racemate in dogs. However, this patent does not disclose the particular activity of dextromilnacipran on the alphal-adrenergic receptor and even less so the use of levomilnacipran for functional recovery after CVA or TBI.

Further, according to the invention, levomilnacipran is used in the form of a pharmaceutically acceptable sait chosen from among the inorganic acid addition salts non-toxic for patients in whom they are administered. The term pharmaceutically acceptable refers to molecular entities and compositions which do not produce any adverse or allergie effect or other undesirable reaction when administered to man or animal. Examples of pharmaceutically acceptable acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, palmitate, lactate, nitrate, acetate, oxalate, valerate, oleate, stéarate, laurate, borate, citrate, benzoate, fumarate, the like

Monkhouse maleate, and tosylate, tartrate, naphthylate salts Bighley 66:1-19). invention phosphate, succinate, (see for example Berge SM, DC, Pharmaceutical salts, 1977, sait however in the présent preferred levomilnacipran hydrochloride.

The invention also concerne a

LD,

The is pharmaceutical composition characterized in that it contains levomilnacipran as active ingrédient and at least one pharmaceutically acceptable excipient. When used herein, the term pharmaceutically acceptable excipient includes any diluent, adjuvant or excipient such as preserving agents, filler agents, disintegrating, wetting, emulsifying, dispersing, antibacterial or antifungal agents, or agents which can delay intestinal and digestive absorption and résorption. The use of these media or vectors is well known to the person skilled in the art.

The pharmaceutical compositions may contain substantially pure levomilnacipran or mixtures of levomilnacipran and dextromilnacipran, provided that the proportion of dextromilnacipran is insufficient for the alphal-adrenergic antagonist activity to be significant and for the patient to be exposed to 10 blocking of the alphal-adrenergic receptor. A simulation of the activity of the levomilnacipran/dextromilnacipran mixtures, confirmed by experimental data, shows that the anti-alphal activity becomes significant with mixtures containing 15 more than 5% of dextromilnacipran {Example 3) . The proportion of dextromilnacipran in a levomilnacipran/dextromilnacipran mixture must not therefore exceed 5% by weight of the said mixture.

The pharmaceutical compositions according to the 20 présent invention can be formulated for administration to mammals, including man. The compositions of the invention can be administered via oral, sublingual, sub-cutaneous, intramuscular, intravenous, transdermal, local or rectal route. In this case, the active 25 ingrédient can be administered in unit administration forms, in a mixture with conventional pharmaceutical carriers, to animal or human beings. The suitable unit administration forms comprise the forms via oral route such as tablets, capsules, powders, granules, each 30 containing a predetermined quantity of levomilnacipran, they also include oral solutions or suspensions in an aqueous liquid or non-aqueous liquid, or an oil/water or water/oil liquid émulsion, sublingual and mouth administration forms, sub-cutaneous or transdermal, 35 topical, intramuscular, intravenous, intra-nasal or

* intraocular administration forms and rectal administration forms. When a solid composition is prepared in tablet form, the levomilnacipran is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnésium stéarate, talc, gum Arabica, silica or the like. It is possible to coat the tablets with sucrose or other suitable materials.

The release of the said active ingrédient can be delayed to obtain sustained release so as to allow the administration of a single daily dose. Said galenic formulation can be obtained following the method described in patent EP 939 626 or any other method.

A capsule préparation is obtained by mixing the active ingrédient with a diluent and pouring the mixture obtained into soft or hard capsules.

A préparation in syrup or élixir form can contain the active ingrédient combined with a sweetener, an antiseptie, and a suitable flavoring agent and coloring agent.

Powders or water-dispersible granules can contain the active ingrédient in a mixture with dispersing or wetting agents, or suspending agents, and also with flavor enhancing or sweetening agents.

For rectal administration, recourse is had to suppositories prepared with binders melting at rectal température e.g. cocoa butter or polyethylene glycols.

For parentéral administration (intravenous, intramuscular, intradermal, sub-cutaneous), intra-nasal or intra-ocular administration, aqueous suspensions are used, isotonie saline solutions or stérile solutions for injection which contain pharmaceutically compatible dispersing and/or wetting agents.

The active ingrédient may also be formulated in the form of microcapsules optionally with one or more additive carriers.

Advantageously the pharmaceutical composition according to the présent invention is intended for administration via oral route.

The dosages of the pharmaceutical compositions containing levomilnacipran in the compositions of the invention are adjusted to obtain a quantity of active substance which is efficient to obtain the desired therapeutic response for a composition particular to the administration route. The chosen dosage level therefore dépends on the desired therapeutic effect, the chosen route of administration, the desired length of treatment, the weight, âge and gender of the patient, the sensitivity of the individual to be treated. As a resuit, the optimal dosage must be determined in relation to parameters deemed to be relevant by specialists in the field.

Preferably the levomilnacipran is administered in pharmaceutically acceptable compositions in which the daily dose of levomilnacipran, expressed as base amount, is between 25 and 200 mg taken in a single administration or several times per day. Further preferably, the pharmaceutical composition allows modified intestinal absorption so that a single administration per day is sufficient.

Example 1

Measurement of the affinity of the two isomers of milnacipran for the noradrenaline and serotonin transporters and for the alphal-adrenergic receptor.

The affinities of levomilnacipran and dextromilnacipran were measured on the binding to the recombinant human transporters of noradrenaline and serotonin, and on the binding to the human recombinant alphal receptor. The inhibition by these two products of the reuptake of noradrenaline [³H] and serotonin [³H] was also measured.

Methods:

Binding to the noradrenaline transporter: binding was measured on membranes of MDCK cells expressing this transporter, purchased from Perkin-Elmer (batch N° 418-165-A), diluted in 50 mM TRIS-HC1 buffer containing 120 mM NaCL and 5 mM KC1 at a concentration of 5 pg proteins, in the presence of 2 mM N-methyl-nisoxetine [³H] and increasing concentrations of levomilnacipran or dextromilnacipran ( 10^-u to ΙΟ^-5 M) . The bound fraction was separated by filtration and washing in cooled TRIS + NaCl + KC1 buffer. Non-specific binding was measured in the presence of 10 μΜ desipramine.

Binding to measured on the serotonin transporter: binding was membranes of MDCK cells expressing this purchased from diluted in 50 transporter,

N° 316-199-A) containing 120 mM NaCl concentration of 5 μg of of 2 nM citalopram of ( 10'¹¹ separated + NaCl

Perkin-Elmer

TRIS-HC1 mM (Batch buffer and nM concentrations dextromilnacipran fraction was cooled TRIS binding was fluoxetine.

measured by +

mM

KC1 at a the presence increasing or to 10° M) . The bound filtration and washing in KC1 buffer. Non-specific of 10 μΜ in and proteins, [³H] levomilnacipran 10’⁵ in the presence

Binding to the recombinant human alphal receptor: binding was measured on membranes of CHO cells (Chinese Hamster Ovary) expressing the human alphalB receptor (Wurch T, Boutet-Robinet EA, Palmier C, Colpaert FC, Pauwels PJ, Constitutive coupling of chimeric dopamine D2/alphalB receptor to the

* phospholipase C pathway: inverse agonism to silent antagonism of neuroleptic drugs, J Pharmacol Exp Ther, 2003, 304:380-390) diluted in 50 mM TRIS-HCI buffer at a concentration of 7.8 pg of proteins, in the presence of 0.1 nM prazosin [³H] and increasing concentrations of levomilnacipran or dextromilnacipran (10^u to ΙΟ^-5 M) . The bound fraction was separated by filtration and washing in cooled TRIS buffer. Non-specific binding was measured in the presence of 10 pM phentolamine. Reuptake of noradrenaline [³H]: CHO-K1 cells were permanently transfected with the gene of the human transporter of noradrenaline by electric pulsing (Biorad gene puiser) and the transfected clones were then selected by incubation in geneticin. To measure reuptake, the transfected cells were cultured in 24well plates then incubated in the presence of pargyline and ascorbate (100 pM) and noradrenaline [³H] (spécifie activity = 40.7 Ci/mole) at a concentration of 10 nM. Reuptake was halted by aspiration and rinsing the medium and the radioactivity captured by the cells was counted by liquid scintillation. The non-specific signal was determined in the presence of 10 pM desipramine.

Reuptake of serotonin [³H]: CHO-K1 cells were transfected permanently with the gene of the human transporter of serotonin by electric pulsing (Biorad gene puiser) and the transfected clones were selected by incubation in geneticin. To measure reuptake, the transfected cells were cultured in 24well plates then incubated in the presence of pargyline and ascorbate (100 pM) and serotonin[³H] (spécifie activity = 32.7 Ci/mole) at a concentration of 10 nM. Reuptake was halted by aspiration and rinsing of the medium and the captured radioactivity by the cells was counted by liquid scintillation. The non-specific signal was determined in the presence of 10 μΜ fluoxetine.

Résulte :

Table 1 gives the values of inhibition constants (Ki) of levomilnacipran and dextromilnacipran for the serotonin and noradrenaline transporters, and of the alphal-adrenergic receptor.

Table 1

	Value of Ki for binding or of IC50 for reuptake (μΜ)
Target	levomilnacipran	dextromilnacipran
Binding to the noradrenaline transporter (NET)	0.091	10.5
Binding to the serotonin transporter (SERT)	0.011	0.32
Inhibition of noradrenaline reuptake [3H]	0.010	0.15
Inhibition of serotonin reuptake [3H]	0.018	0.28
alA adrenergic receptor	110	3.4

Example 2

Measurement of the intrinsic activity of dextromilnacipran on recombinant human alphalA and 15 alphalB receptors.

The intrinsic functional activity of dextromilnacipran was measured on cells expressing the

human alphalA and alphalB receptors to détermine the agonist/antagonist property thereof.

Methods:

CHO-K1 cells having stable expression of the human alphalA receptor or human alphalB receptor were obtained using the described method (Vicentic et al. Biochemistry and pharmacology of epitope-tagged alphala-adrenergic receptor subtype, J Pharm Exp Ther, 2002, 302-58-65). The agonist activity was evaluated by fluorimetry measurement of the intracellular concentration of calcium following a conventional technique using a fluorescent calcium chelator and signal recording with a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Saint-Grégoire, France). As positive reference (-) adrénaline was used, and responses were then normalized to the response of (-)adrénaline at a concentration of 10 μΜ.

Results:

Over a concentration range of 3.10⁷ M to 10^-3 M, dextromilnacipran did not show any agonist activity higher than 10% of the activity of the (-) adrénaline, whether on the alphalA receptor or alphalB receptor. The (-)adrénaline was then incubated in increasing concentrations (3.10^-10 to 3.10⁵ M) in the presence of levomilnacipran or dextromilnacipran at a concentration of 300 μΜ. Figure 1 shows that the concentrationresponse curve of the (-)adrénaline is shifted towards the right by dextromilnacipran by a factor of about 100 for the alphalA sub-type and by about 10 for the alphalB sub-type. For levomilnacipran, the shift is only about 3 for the alphalA sub-type and 2 for the alphalB sub-type. It is concluded that dextromilnacipran is an antagonist for these two subtypes of alphal-adrenergic receptors. Levomilnacipran is also an antagonist of the alphalA and alphalB

much less powerful extent than characteristics of dextromilnacipran, mixtures with of varying has a racemic IR, 2S enantiomer is the most active binding with the binding with the

2R) receptors but to a dextromilnacipran.

Example 3

Pharmacological levomilnacipran and proportions of the enantiomers.

Objectives

Levomilnacipran (enantiomer, IS, distinct pharmacological profile compared with milnacipran (2207) and the other (dextromilnacipran). Levomilnacipran enantiomer on the desired targets: noradrenaline transporter (NET), serotonin transporter (SERT) and the PCP site (NMDA receptor, glutamate System), but is the least active on non-desired targets i.e. on alA and alB adrenergic receptors. We estimated the pharmacological properties of different mixtures (containing varied percentages of dextromilnacipran). First, binding assays were simulated and the apparent inhibition constants of the mixtures for NET, SERT and the alA receptors were calculated. Next, the simulation was experimentally

validated for the	alA receptors for	which	the
variations in levomilnacipran had the most	impact.
Methods
Simulation
The inhibition	constants (value	of Ki)	of
levomilnacipran and	dextromilnacipran on the	main

targets were experimentally determined using conventional binding assays with spécifie radioligands and recombinant human proteins (see Example 1).

For each target, the total of bound radioligand was calculated taking into account the inhibitor effect of each inhibitor. The total of each occupied target is described using the conventional law of mass action:

* (1) R.S. = K_d

B (2) R, li = K_n

RIi (3) R.I₂ = K_i2

RI_Z (4) Bmax = B + RI_X + RI₂ + R where R is the concentration of free binding sites; Bmax is the total concentration of sites; B is the concentration of sites bound to the radioligand; S is the radioligand concentration; K_d is the dissociation constant of the radioligand; i_x is the concentration of inhibitor 1; Kn is the inhibition constant of inhibitor 1; RI_X is the concentration of sites occupied by inhibitor 1; i₂ is the concentration of inhibitor 2; Ki2 is the inhibition constant of inhibitor 2 and RI₂ is the concentration of sites occupied by inhibitor 2.

On the basis of équations (1) to (4):

Bmax.S

B =

	S + Kd	(1 + ix/KiX + I2/Ki2)
The	values	of	B	were calculated	taking into
account	the real	values	of Kn and Ki2 (see	Table above)

causing the proportion of dextromilnacipran to vary by 0.01% to 30% in the mixture, and varying the concentration of the mixture from 0.01 to 52.0 μΜ. The values of K_d and S which were used were similar to the values of the binding assays. A theoretical inhibition

curve was therefore plotted with each combination of values of the different parameters. Then the apparent IC50 value for each curve was calculated by non-linear régression as per the logistic équation:

Y = 100/1 + 10 (i-LogICso) where i is the concentration of the inhibitor (mixture) and the apparent value of K± was derived using the Cheng-Prussoff équation: IC50 - Κι (1 + S/K_d) .

2.2 Binding assays

A sériés of levomilnacipran and dextromilnacipran mixtures with varying proportions of dextromilnacipran was prepared, and each mixture was incubated at varying concentrations with membranes of cells expressing the recombinant human alA receptor and using prazosin[³H] as radioligand. The apparent IC50 value for each curve was calculated by non-linear régression as per the logistic équation:

Y = 100/1 + 10 (i-LogIC₅o) where i is the concentration of the inhibitor (mixture) and the apparent value of Ki was derived using the Cheng-Prussoff équation: IC50 = K± (1 + S/K_d) .

Results

The results are expressed as apparent Ki value as a function of dextromilnacipran percentage (Figure 1).

Figure 2 shows: the apparent Κχ values of simulated assays (A, B and C) and measured values (D) of mixtures of levomilnacipran (F2695) and dextromilnacipran (F2696) with increasing proportions of dextromilnacipran for NET, SERT or alA receptor targets.

The values of Ki for NET and SERT were not too affected by different proportions of dextromilnacipran. On the contrary, the apparent value of Ki for the alA <·« V « receptors, whether for simulation assays or for real assays, was dramatically affected when the percentage of dextromilnacipran was increased, which indicates that the impact on the al receptors is non-negligible.

If it is considered that the impact becomes nonnegligible when the value of Kl drops by half, the maximum percentage of dextromilnacipran is about 5%.

Conclusion

A mixture with a proportion of dextromilnacipran higher than this 5% may not be bioequivalent to substantially pure levomilnacipran, or to a mixture with smaller proportions of dextromilnacipran. The impact on the alA receptors of mixtures with proportions of dextromilnacipran higher than 5% is not negligible and such mixtures should not be used in the treatment of functional recovery after a stroke.

Claims (8)

1. A levomilnacipran/dextromilnacipran mixture containing dextromilnacipran in a proportion not exceeding 5% by weight of the said mixture for use thereof as médicinal product for recovery and functional réhabilitation after an acute neurological event and récurrences thereof.

2. The mixture according to claim 1 for use thereof in patients diagnosed with cérébral vascular accident of ischémie or hémorrhagie origin.

3. The mixture according to claim 1 for use thereof in patients diagnosed with traumatic brain injury.

4. Pharmaceutical compositions comprising at least one pharmaceutically acceptable excipient and a levomilnacipran/dextromilnacipran mixture containing dextromilnacipran in a proportion not exceeding 5% by weight of the said mixture as active ingrédient for use thereof as médicinal product for recovery and functional réhabilitation after an acute neurological event and récurrences thereof.

5. Pharmaceutical compositions according to claim 4 in patients diagnosed with cerebrovascular accident.

6. Pharmaceutical compositions according to claim 4 in patients diagnosed with traumatic brain injury.

7. Pharmaceutical compositions according to any of of the daily claims 4 to 6 characterized in that levomilnacipran is between 50 and 200 mg.

Pharmaceutical compositions according

4 to 7 characterized in that they are in a intestinal absorption form allowing the dosage

8.

claims to one of modified administration of a single dose per day.