Piperazine phenothiazine derivatives for treating spasticity

Abstract

The present invention relates to piperazine phenothiazine derivatives useful as therapeutic agents for treating spasticity, particularly following an ischemia or traumatic injury, or compression syndrome. The invention further relates to a pharmaceutical composition comprising a compound of the invention for treating spasticity.

Claims

1. A method for treating spasticity, comprising administering in a patient in need of such treatment an effective amount of a compound of formula (I): ##STR00004## wherein: A represents a linear propyl chain; R.sub.1 represents: a hydrogen atom, a halogen atom, an acyl group COR.sub.7, wherein R.sub.7 represents a (C.sub.1-C.sub.6) alkyl group, a sulfonamide group SO.sub.2NR.sub.8R.sub.9 wherein R.sub.8 and R.sub.9 independently represent a (C.sub.1-C.sub.6) alkyl group, or a (C.sub.1-C.sub.6) alkyl optionally substituted by at least one halogen atom; R.sub.1 represents a hydrogen atom; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 represent a hydrogen atom; and R.sub.6 represents: a (C.sub.1-C.sub.6) alkyl group, optionally substituted by at least one hydroxyl group at the end of the alkyl chain, or one of its pharmaceutically acceptable salts.

2. The method according to claim 1, wherein R.sub.1 represents: a hydrogen atom, a chlorine atom, an acyl group COR.sub.7, wherein R.sub.7 represents a methyl group, a sulfonamide group SO.sub.2NR.sub.8R.sub.9 wherein R.sub.8 and R.sub.9 represent methyl groups, or a (C.sub.1-C.sub.6) alkyl group optionally substituted by at least one fluorine atom.

3. The method according to claim 1, wherein R.sub.6 represents: a methyl or an ethyl group, optionally substituted by at least one hydroxyl group at the end of the alkyl chain.

4. The method according to claim 1, wherein said compound is selected in the group consisting of: 2-[4-[3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]propyl]-piperazin-1-yl]ethanol; 2-[4-[3-(2-chloro-10H-phenothiazin-10-yl) propyl]piperazin-1-yl]ethanol; 2-chloro-10-[3-(4-methyl-1-piperazinyl)propyl]-10H-phenothiazine; 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10H-phenothiazine; 1-[10-[3-[4-(2-hydroxyethyl)piperazin-1-yl]propyl]-10H-phenothiazin-2-yl]ethanone; N,N-dimethyl-10-[3-(4-methylpiperazin-1-yl)propyl]-10H-phenothiazine-2-sulfonamide; and 10-[3-(4-methylpiperazin-1-yl)propyl]-10H-phenothiazine.

5. The method according to claim 1, wherein said compound is selected in the group consisting of: 2-[4-[3-(2-chloro-10H-phenothiazin-10-yl) propyl]piperazin-1-yl]ethanol; and 2-chloro-10-[3-(4-methyl-1-piperazinyl)propyl]-10H-phenothiazine.

6. The method according to claim 1, wherein the treatment of spasticity is the treatment of spasticity following an ischemia or a traumatic injury, or a compression syndrome.

7. The method according to claim 2, wherein the (C.sub.1-C.sub.6) alkyl group is optionally substituted by at least one trifluoromethyl group.

Description

LEGEND TO THE FIGURES

(1) FIG. 1A: Chemical structure of piperazine phenothiazines-derived compounds 1 to 8.

(2) FIG. 1B: Cytotoxicity of piperazine phenothiazines-derived compounds 1 to 8. Cytotoxicity were evaluated with fluorescent viability assay on HEK cells lines incubated 1 h with several concentrations of drugs. TC50: Toxic Concentration for 50% of the population.

(3) FIG. 1C: Comparison of the effect of 8 compounds at 30 M on KCC2 activity. Data are normalized to untreated (i.e. no drug, only DMSO). The effect of each drug was normalized to the maximal effect obtained with the compound 1, perphenazine (100%).

(4) FIG. 1D: Dose response curve obtained for compound 1 (perphenazine) (a) and compound 2 (Prochlorperazine) (b) on HEK Wt and HEK KCC2.s.d. based on three independent experiments (n=3).

(5) FIG. 2A: Detection by western-blot of KCC2 in total cell lysate extract from HEK Wt and HEK KCC2 treated with prochlorperazine (compound 2) or DMSO ().

(6) FIG. 2B: Detection by western-blot of KCC2 in total cell lysate extract from NSC-34 KCC2 treated with prochlorperazine (compound 2) or DMSO ().

(7) FIG. 3 A: Hyperpolarizing shift of EIPSP induced by prochlorperazine (compound 2) in lumbar MNs in neonatal P4-6 rats. E.sub.IPSP (a), V.sub.rest (b) and driving force (E.sub.IPSP-V.sub.rest; (c)) measured from rats before (CTL) and after 20-25 Prochlorperazine (2) (10 M, n=6; *p<0.05 Wilcoxon test).

(8) FIG. 3 B: Prochlorperazine (compound 2) induces a dose-dependent strengthening of reciprocal inhibition in the in vitro spinal cord preparation isolated from P5-7 animals with a spinal cord transection at birth. Error bars represent s.e.m; n=6 in each group.

(9) FIG. 4: Effect of prochlorperazine (compound 2) on spasticity. Rate-dependent depression of the Hoffman (H) reflex evoked in adult rats 4 weeks after SCI, 80 minutes after injection of 10 g/kg of prochlorperazine (2) (i.v.) (n=6) or vehicle (n=5). Error bars represent s.e.m (p<0.05 Mann Whitney test at each frequency). Prochlorperazine (2) reduces spasticity after spinal cord injury.

(10) FIG. 5: Effect on the paw withdrawal thresholds in response to mechanical stimulation with prochlorperazine (compound 2). Prochlorperazine (2) (or placebo) was injected intraperitoneally (2 mg/kg) (FIG. 5 A) or intravenously (50 ng/kg) (FIG. 5 B) to Wistar rats, 21 days after the injury. The pain thresholds were determined before and 10, 40, 70 and 100 minutes after drug injection. Error bars represent s.e.m. Prochlorperazine (2) reduces mechanical hyperalgesia after spinal cord injury.

(11) Further aspects and advantages of the invention will be disclosed in the following experimental section.

EXAMPLES

Example 1: Specific Activation of the Potassium Chloride Co-Transporter KCC2 with Compound 1-8

(12) 1. Materials and Methods

(13) 1.1. Compounds

(14) Chemical structure of compounds 1-8 is illustrated in FIG. 1A. Compounds 1-4, 6 and 8 were purchased from Prestwick Chemical. Compound 5 was purchased from Sigma Aldrich (St Louis, Mo., USA) and compound 7 was purchased from Santa Cruz Biotechnology (Dallas, Tex., USA). All of these compounds are provided in DMSO solution (under argon) and frozen for storage and are supplied at a precise 20 mM concentration.

(15) 1.2. Cellular Model and Cell Culture

(16) Wild-type or KCC2-expressing Human Embryonic Kidney (HEK) 293 cells (provided by Prof. Eric Delpire Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tenn.) were grown up to 80-90% confluence in DMEM/Ham's F-12 (1:1) (Life technologies, Carlsbad, Calif., USA) supplemented with 10% FBS (Life technologies), 50 units/mL penicillin, and 50 pg/mL streptomycin (Life technologies). KCC2-expressing clones were under puromycin selection (20 g/ml, Life technologies).

(17) 1.3. Fluorescence Based Thallium (TI.sup.+) Influx Assay

(18) The FluxOR potassium ion channel assay (Life Technologies Carlsbad, Calif., USA) was performed as outlined in the product information sheet and an earlier published study (Delpire et al., 2009, Proc. Natl. Acad. Sci. USA, 106, 5383-88) to assess KCC2 activity, and adapted to semi-automated high throughput screening. The experiment was performed in the presence of ouabain 200 M and bumetanide 10 M in all buffers to block the Na.sup.+/K.sup.+ pump and NKCC1 cotransporters, respectively. Briefly, cells in suspension were incubated in the loading buffer containing the dye at room temperature for 90 min (1/2000 dilution of FluxOR reagent) at the density of 100.000 cells/75 l, then centrifugated and resuspended in Assay buffer at the same cell density. A number of 100.000 cells (75 l) were manually handled in each well to the 96-wells black-walled, clear-bottom plates (Greiner Bio-One, Monroe, N.C., USA) and 5 l of chemical compounds 1-8 at different concentrations in PBS 1% DMSO (5, 30 and 50 M) were added to the plates using the Biomek NX Laboratory automation workstation (Beckman Coulter, Villepinte, France). After 15 minutes incubation, baseline fluorescent signal was measured with the Polarstar omega microplate reader (490 nm excitation and 520 nm emission, BMG Labtech). Then 20 L per well of 5 thallium stimulus buffer (final concentrations: Tl.sup.+: 2 mM, K.sup.+: 10 mM) was injected and fluorescent signal was read 30 minutes later.

(19) For each plate, 2 columns were dedicated to controls (cell density range from 0.25 to 1.10.sup.5 cells; loading buffer as blank; untreated or treated with NEM (33 M, for 15 min) wild-type and KCC2-expressing HEK293 cells).

(20) 1.4. Cell Viability Assay

(21) After stimulus buffer induced signal acquisition, PrestoBlue Cell Viability Reagent (Life technologies), a cell permeable resazurin-based solution was used according to manufacturer procedure as a cell viability indicator (Excitation/Emission (nm): 535-560/590-615). A fluorescence curve produced using the range of cell numbers was then used to define an equation to calculate the number of cells alive in each well and thereby to evaluate the cytotoxicity of compounds 1-8.

(22) 1.5. Data Analysis

(23) Baseline fluorescent value was subtracted from the fluorescent value measured 30 min after stimulus buffer injection for each well, and this difference was normalized to the number of cells. Compounds displayed selective activity on KCC2-expressing cells with treated cells/untreated cells signal ratio >1.10 and slight effect on wild-type cells (treated cells/untreated cells signal ratio=10.2). Values converted to percentages such that the maximum of ratio is 100%.

(24) 2. Results

(25) The results of FluxOR fluorescent assay for each compound are illustrated in FIG. 1C. () represent control assay (i.e. no drug, only DMSO) and cytotoxic concentrations have been excluded and only data for up to 70% viability of cells in FIG. 1B have been considered. The inventors have demonstrated that all the compounds (1)-(8) enhance KCC2 activity with different efficiencies: the strongest effects were obtained with perphenazine (1) at 30 M (100%) and perazine (7) (55%). No results was obtained with thiethylperazine dimaleate (8) at 30 M as it was cytotoxic at this concentration.

(26) Effects of perphenazine (1) and prochlorperazine (2) on KCC2-expressing HEK cells compared to WT HEK cells across a dose range, chosen on the basis of lack of cytotoxicity, have also been evaluated (FIG. 1D).

(27) The inventors have demonstrated that compounds of the invention, particularly compounds (1) perphenazine and (2) prochlorperazine activate specifically KCC2 in a dose dependant manner.

Example 2: Promoting of KCC2 Expression with Prochlorperazine (2)

(28) 1. Materials and Methods

(29) 1.1. Cellular Model and Cell Culture

(30) Wild-type or KCC2-expressing Human Embryonic Kidney (HEK) 293 cells (provided by Prof. Eric Delpire Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tenn.) were grown up to 80-90% confluence in DMEM/Ham's F-12 (1:1) (Life technologies, Carlsbad, Calif., USA) supplemented with 10% FBS (Life technologies), 50 units/mL penicillin, and 50 pg/mL streptomycin (Life technologies). KCC2-expressing clones were under puromycin selection (20 g/ml, Life technologies).

(31) The NSC-34 motor neuron cell line (CELutions Biosystem Inc, Ontario) which does not express KCC2 endogenously, were infected with lentivirus derived from the human immunodeficiency virus-1 (HIV-1) encoding KCC2 and cultures several week after clonal isolation to establish the NSC-34 KCC2 stable cell line. NSC-34 wild type and KCC2 were cultured in DMEM (Life technologies, Carlsbad, Calif., USA) supplemented with 10% FBS (Life technologies), 50 units/mL penicillin, and 50 pg/mL streptomycin (Life technologies).

(32) 1.2 Western Blot

(33) Wild-type and KCC2-expressing HEK293 cells or NSC-34 were harvested and homogenised in lysis buffer (PBS containing 1% Igepal CA-630, 0.1% SDS, 10 mM sodium pyrophosphate, 10 mM NaF, 10 mM NaVO4, 10 mM iodoacetamide and cocktail protease inhibitors and centrifugated at 18000 g for 30 minutes at 4 C. Protein concentration in supernatants was determined using DC protein assay (Bio-rad). Same amounts of total proteins were separated in 6% or 7% SDS PAGE and transferred onto PVDF membrane. Once blocked in Tris Buffer Saline 0.05% Tween 5% non-fat dry milk, membranes were probes over night at 4 C. with KCC2 antibody (1/1000 dilution, Merck-Millipore, Billerica, Mass., USA), or anti-phospho-serine940 KCC2 (1/1000 dilution, PhosphoSolutions) or anti-Actin antibody (1/500 dilution, Sigma Aldrich). Anti-rabbit secondary antibody HRP conjugated was used for detection in chemiluminescent system (Thermo Scientific, Waltham, Mass., USA). Signal intensity was measured with the image analysis software Image lab (Bio-Rad, Hercules, Calif., USA).

(34) 2. Results

(35) Prochlorperazine (2) effect (10 M) on the total expression of KCC2 in HEK KCC2 and NSC-34 KCC2 cell lines has been tested by means of western blot analysis (FIGS. 2 A and 2B, respectively).

(36) In the whole cell lysate, the inventors have shown that both the total KCC2 protein level (monomers+oligomers) and phosphorylated KCC2 on serine 940 increased after 30 min of cell treatment with prochlorperazine (2). According to these results, the inventors have demonstrated that compound (2) up-regulates the expression of the KCC2 protein in the HEK cells used for the screening but also in NSC34 cell line which present more neuronal characteristics.

Example 3: Electrophysiological Recordings and In Vivo Tests with Prochlorperazine

(37) 1. Materials and Methods

(38) 1.1. Animals

(39) Neonatal and adult (150-250 g) female Wistar rats (Charles River, Burlington Mass. USA) were used. Animals were housed in a temperature-controlled animal care facility with a 12 h light-dark cycle. We made all efforts to minimize animal suffering and the number of animals used. Neonates were anesthetized by hypothermia. We performed experiments in accordance with French regulations (Ministry of Food, Agriculture and Fisheries, Division of Health and Protection of Animals). The local Direction of Veterinary Services and Ethical Committee (Marseille, Provence) delivered the appropriate licenses and approved the protocols, respectively.

(40) 1.2. Intracellular Recordings

(41) Spinal cords isolated from neonatal rats on post natal day 4 or 5 were dissected together with spinal roots. Briefly, after decapitation and evisceration, the spinal cord was exposed by dorsal laminectomy and acute removal of the dura in a cold artificial cerebrospinal fluid (ACSF; containing (in mM): 130 NaCl, 4 KCl, 3.75 CaCl.sub.2, 1.3 MgSO.sub.4, 0.58 NaH.sub.2PO.sub.4, 25 NaHCO.sub.3 and 10 glucose (all compounds were from Sigma); oxygenated with 95% O.sub.2/5% CO.sub.2, pH=7.4). The cord, from sacral segments up to T8, was then removed from the vertebral column together with peripheral roots. The preparation was then transferred to the recording chamber where it was pinned down, ventral side up, in sylgard (Dow-Corning; USA)-covered recording chamber, and continuously perfused with the ACSF solution. After removing the pia, we recorded lumbar motoneurons (MNs) intracellularly using glass microelectrodes filled with 2 M K-acetate (70 to 100-M resistance). We recorded intracellular potentials in the discontinuous current-clamp (DCC) mode (Axoclamp 2B amplifier; Digidata 1200 interface; pClamp9 software; Axon Instruments, Sunnyvale, Calif., USA). We used glass suction electrode to stimulate the ipsilateral ventral funiculus 2 to 3 segments rostral to the recorded one. Such stimulations induced GABA.sub.A- and Gly-mediated Inhibitory Postsynaptic Potentials (IPSPs) in the presence of DL-2-amino-5-phosphonovaleric acid (DL-APV, 50 M) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 M) (Bos et al., 2013; Boulenguez et al., 2010; Jean-Xavier et al., 2006). We recorded IPSPs at various holding potentials (500-ms-long current pulses) and collected at least 20 values for each MN. We measured and plotted amplitudes of IPSPs against holding potentials and obtained E.sub.IPSP from the regression line.

(42) 1.3. Extracellular Recordings and Evaluation of Reciprocal Inhibition

(43) Neonatal Spinal Cord Injury and Preparations for In Vitro Electrophysiology

(44) Rats were deeply anesthetized by hypothermia at birth. A dorsal midline skin incision was made over the thoracic vertebra and the overlying fascia and muscles were retracted to expose the dorsal surface of the vertebrae. After a partial laminectomy, the spinal cord was completely transected at the T8 thoracic level with scissors. The lesion cavity was then filled with sterile absorbable local hemostat Surgicoll (Medical Biomaterial Products; Neustadt-Glewe, Germany). The skin incision was closed with sutures (PDSII 6.0, Ethicon; Johnson and Johnson; Brussels, Belgium) and covered by Steri-Strips (3M Health Care; St. Paul, Minn.). The whole surgical procedure took less tan 10 min after anesthesia. Sham-operated rats were treated in the same way except 5 the spinal cord transection. Following surgery, the neonates recovered 45 min in a warm environment maintaining the temperature at 351 C. Wounds were then cleaned and rats were kept in a warm environment for 40 min before returning to the nest.

(45) Spinal cords isolated from neonatal rats were prepared as described in section 1.2 until recording.

(46) In Vitro Extracellular Recordings and Evaluation of Reciprocal Inhibition

(47) Extracellular electrophysiological signals of lumbar VRs and DRs responses were recorded by contact stainless steel electrodes insulated from the bath with vaseline. Data were acquired through an AC coupled amplifier (bandwidth: 70 to 3 kHz) and a Digidata 1440A interface using the Clampex 10.2 software (Molecular Devices; Sunnyvale, Calif., USA).

(48) Single pulses were delivered to the DR to evoke responses in the homonymous VR. We stimulated at 2-3 times the threshold (T) intensity that evoked an incoming volley in the DR. The current pulses (0.3 ms duration) required to elicit the maximal response varied among preparations (from 0.6 to 1.2 V). Stimulations were delivered every 20 s to avoid fatigue and synaptic depression described in this preparation (Lev-Tov and Pinco, 1992). Test and conditioning stimulations were delivered to the L5 and L3 DRs, respectively. Delays between the two stimulations ranged from 0 to 40 ms. Each delay was tested at least two times in the following way: 5 controls (test only) followed by five paired (conditioning+test) stimulations. The order in which delays were tested was randomized from series to series to prevent possible order-dependent effects. Data analysis was performed offline and consisted in measuring the peak-to-antipeak amplitude of the L5 monosynaptic reflex (Clampfit 10.2 software). To consider only the monosynaptic component, we restricted measurements to the first 3 ms after the response onset (Kudo and Yamada, 1987).

(49) 1.4. Assessment of Spasticity Following Spinal Cord Injury

(50) Surgery:

(51) Thoracic spinal section in rats was used as a model of SCI. Adult female Wistar Rats (225/250 g Charles River) were anaesthetized intraperitoneally with 50 mg/kg ketamine (Imalgen, Merial, Duluth, Ga., USA) and 0.25 mg/kg Medetomidine (Domitor, Janssen Pharmaceutica, Beerse, Belgium). The antibiotic Amoxycyline (Duphamox LA, Pfizer, 150 mg/kg) was injected subcutaneously before the surgery. The skin was cut longitudinally over T8-T10 vertebra and a local anesthetic (2% Procaine hydrochloride, Pharmy H, Saint-Germain-en-Laye, France) was injected intramuscularly before cuting the paravertebral muscles. A laminectomy was performed at vertebral segment T9. The spinal cord was transected with microscissors at the level of the T8 spinal segment. Finally, paravertebral muscles and skin were sutured and rats were treated with buprenorphine for analgesia (1 injection before awakening from anaesthesia and 4 more injections over the next 48 h period). The rats weight, temperature and water intake were checked and their bladder was emptied manually twice a day until recovery of autonomy.

(52) In Vivo Electrophysiological Recordings and Treatment with Prochlorperazine (2).

(53) At day 29 post lesion, the H reflex in the rats under ketamine anesthesia (100 mg/kg, i.p.) were measured using a pair of stainless steel needle electrodes transcutaneously inserted into the vicinity of the tibial nerve stimulation. The recording electrode was placed into the flexor digitorum muscle beneath the ankle and the reference electrode s. c. into the foot.

(54) The H reflex was measured three times at frequencies of 0.2 Hz 1 Hz, 2 Hz and 5 Hz to have their baseline values. Then, rats were treated with either prochlorperazine di-maleate (2) (10 g/kg i.v. in 0.1% DMSO, 0.9% NaCl, n=6) or its vehicle (0.1% DMSO, 0.9% NaCl, n=5). The H reflex was measured each 20 minutes, five times at frequencies of 0.2 Hz 1 Hz, 2 Hz and 5 Hz

(55) 1.5. Behavioural Testing Following Spinal Cord Injury

(56) Surgery:

(57) Thoracic spinal unilateral hemisection in rats was used as a model of SCI. Adult female Wistar Rats (Charles River) were anaesthetized intraperitoneally with 50 mg/kg ketamine (Imalgen, Merial, Duluth, Ga., USA) and 0.25 mg/kg Medetomidine (Domitor, Janssen Pharmaceutica, Beerse, Belgium). The antibiotic Amoxycyline (Duphamox LA, Pfizer, 150 mg/kg) was injected subcutaneously before the surgery. The skin was cut longitudinally over T8-T10 vertebra and a local anesthetic (2% Procaine hydrochloride, Pharmy H, Saint-Germain-en-Laye, France) was injected intramuscularly before cuting the paravertebral muscles. A laminectomy was performed at vertebral segment T9. The spinal cord was hemisected on the left side with microscissors at the level of the T8 spinal segment. Finally, paravertebral muscles and skin were sutured and rats were treated with buprenorphine for analgesia (1 injection before awakening from anaesthesia and 2 more injections over the next 24 h period). Aspirin was diluted in their water bottles for 3 days (aspegic, 200 mg in 150 ml). The rats weight, temperature and water intake were checked and their bladder was emptied manually twice a day until recovery of autonomy.

(58) Treatment with Prochlorperazine (2).

(59) At day 21 post lesion, the rats were tested once with Von Frey hair and plantar test in order to have their baseline values. Five min later they were treated with either prochlorperazine di-maleate (2) (2 mg/kg ip in 0.9% NaCl, or 50 ng/kg iv in 0.9% NaCl, 0.1% DMSO) or its vehicle The experimenters were blind to the treatment that the animals received. Then the effects of the drug was measured alternatively on mechanical and thermal hyperalgesia every 15 min, that is at 10, 30, 40, and 100 min after injection, respectively.

(60) Von Frey test:

(61) The plantar surface of the left, then right hind paws were probed using von Frey monofilaments Bioseb, Paris, France) which apply different calibrated forces when they bent. The test started by applying a Von Frey hair of 8 g for 3 sec, and the 50% withdrawal thresholds of each hindlimb in response to tactile stimulation were measured using the up and down method (Chaplan et al., 1994).

(62) 2. Results

(63) 2.1 Hyperpolarization of EIPSP with Prochlorperazine (2) (Test In Vitro)

(64) In the in vitro spinal cord preparation, isolated from neonatal rat, a way to investigate KCC2 function is provided by recording motoneurons (MNs) intracellularly and measuring the chloride equilibrium potential that is given by the reversal potential of inhibitory synaptic potentials (E.sub.IPSP). The more hyperpolarized the value of E.sub.IPSP, the lower the intracellular concentration of chloride ions and, as a result, the stronger the KCC2 function. The inventors have examined the effect of 10 M Prochlorperazine (2) on E.sub.IPSP of lumbar MNs in P4-6 rats (FIG. 3). It hyperpolarized E.sub.IPSP within 5-10 min and measurements were done 20-25 min after the onset of Prochlorperazine (2) application. E.sub.IPSP was significantly more hyperpolarized when MNs were recorded in the presence of Prochlorperazine (2) compared to control conditions (mean of 3.3 mV; n=6; FIG. 3A; p<0.05, Wicoxon test). Prochlorperazine (2) has no significant impact on the resting membrane potential (V.sub.rest; n=6; FIG. 3B; p>0.05, Wicoxon test). Therefore, the driving force increased significantly (mean of 3.5 mV; n=6; FIG. 3C; p<0.05, Wicoxon test). These results demonstrate that compound (2) is able to strengthen post-synaptic inhibition in the spinal cord by reducing the intracellular concentration of chloride ions as a result of an increased activity of KCC2.

(65) 2.2. Effect of Prochlorperazine (2) on Reciprocal Inhibition

(66) The inventors have examined the effect of 5, 10 and 20 M Prochlorperazine (2) on reciprocal inhibition of lumbar MNs (L3 and L5 segments) projecting to the limb flexor and extensor muscles, respectively (Nicolopoulos-Stournaras and Iles, 1983) in P5-7 rats (FIG. 3D). The spinal cord was transected on the day of birth, a protocol that reduces the strength of post-synapic inhibition (compare SCI with intact). Measurements were done 20 min after the onset of Prochlorperazine (2) application. Prochlorperazine (2) significant increased the strength of reciprocal inhibition in a dose-dependent manner, so that an inhibition comparable to an intact spinal cord was observed with Prochlorperazine (2) at 200 M (n=6; FIG. 3D; ANOVA).

(67) 2.3. Reduction of Spasticity by Prochlorperazine (2)

(68) The Hoffmann reflex (H-reflex), a monosynaptic reflex mediated through the spinal cord, in neurologically intact and spastic individual record during electromyograms.

(69) The H-reflex is commonly used to assess primary (type Ia) afferents-mediated motoneuronal excitability in individuals suffering from spasticity. Electromyograms typically show two responses, an initial M wave resulting from the direct activation of motor axons and a delayed H wave resulting from the monosynaptic activation of motoneurons by Ia afferents. The H wave magnitude is normally attenuated by repeated activations at frequencies higher than 0.2 Hz, with a more than 80% reduction at 5 Hz in rats. The H reflex is progressively increased in individuals with SCI, and this effect is a reliable correlate of the development of spasticity. Prochlorperazine (2) decreases the H reflex by 30% at the different frequencies (Mann Whitney test p=0.0043, p=0.0303, p=0.0173, at 1, 2 and 5 Hz, respectively) 80 min after injection of 10 g/kg, i.v. These data demonstrate that prochlorperazine (2) is a good candidate to treat spasticity after spinal cord injury (FIG. 4).

(70) 2.4. Reduction of Hyperlagesia by Prochlorperazine (2)

(71) The Von Frey test is classically used to evaluate the effect of any treatment onto neuropathic pain, including that after spinal cord injury. This test measures the threshold of a mechanical stimulation that will induce paw withdrawal. This threshold is considerably reduced after spinal cord injury and reflects the hyperalgesia, a component of chronic pain. Results of Von Frey test showed that the acute administration of prochlorperazine (2) either intraperitoneally or intravenously temporarily reduces mechanical hyperalgesia in animals three weeks post-injury (FIG. 5). Effects were significant 40 minutes after the injection started.

CONCLUSION

(72) The inventors have demonstrated that compounds of the invention are able on a cell model to boost potassium/chloride transport (as revealed by the FluxOR assay) and KCC2 cell expression.

(73) The transfer to neurons in the spinal cord of neonatal mammals revealed that compounds of the invention are able to reduce the intracellular concentration of chloride ions (revealed by the hyperpolarizing shift of the chloride equilibrium potential), likely as a result of an up-regulation of KCC2 expression/function.

(74) The translation to adult mammals in pathological conditions such as a spinal cord injury demonstrated that compounds of the invention are able to reduce spasticity and chronic neuropathic pain, likely by restoring endogenous inhibition, as an expected result of an upregulation of KCC2 expression/function.

(75) These results confirm that KCC2 is a druggable target for the development of new therapeutic strategies to treat neuropathic pain and spasticity associated with trauma or compression syndromes (such as for instance persistent pain caused by disc herniation-induced nerve compression).

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