AU2001274195B2 - Use of riluzole or its salts for preventing and treating adrenoleukodystrophy - Google Patents

Abstract

The invention concerns the use or riluzole of one of is pharmaceutically acceptable salts for preventing and treating adrenoleukodystrophy.

Description

IN THE MATTER OF an Australian Application corresponding to PCT Application PCT/FR01/01729 I, Abraham SMITH DipIng, DipDoc, translator to RWS Group plc, of Europa House, Marsham Way, Gerrards Cross, Buckinghamshire, England, do solemnly and sincerely declare that I am conversant with the English and French languages and am a competent translator thereof, and that to the best of my knowledge and belief the following is a true and correct translation of the PCT Application filed under No. PCT/FR01/01729.

Date: 25 November 2002 A. SMITH For and on behalf of RWS Group plc USE OF RILUZOLE OR ITS SALTS FOR THE SPREVENTION AND TREATMENT OF

ADRENOLEUKODYSTROPHY

o The present invention relates to the use of Sriluzole or one of its pharmaceutically acceptable C( salts for the prevention and/or treatment of 0 adrenoleukodystrophy.

C Riluzole (2-amino-6-trifluoromethoxybenzothiazole) is marketed for the treatment of amyotrophic lateral sclerosis (Rilutek®). This compound is also useful as an anticonvulsant, an anxiolytic and a hypnotic (EP 50551), in the treatment of schizophrenia (EP 305276), in the treatment of sleep disorders and of depression (EP 305277), in the treatment of cerebrovascular disorders and as an anesthetic (EP 282971), in the treatment of spinal, cranial and craniospinal traumas (WO 94/13288), as a radio restorative (WO 94/15600), in the treatment of Parkinson's disease (WO 94/15601), in the treatment of neuro-AIDS (WO 94/20103), in the treatment of mitochondrial diseases (WO 95/19170).

X-linked adrenoleukodystrophy (ADL) is the most frequent of the genetic diseases of myelin which is characterized by progressive demyelination of the central nervous system, an adrenal insufficiency and a moderate accumulation (X 3 to 5) of very long chain fatty acids (VLCFA) in most tissues, including the brain and the spinal cord. This accumulation of VLCFA results from a deficiency in their P-oxidation in the peroxisome.

The mechanisms which lead in ALD to a demyelination and a loss of the oligodendrocytes are still not well understood. One of the possibilities is that the accumulation of VLCFAs leads to a destabilization of the myelinic membranes or to a dysfunctioning of the receptors situated at the surface of the oligodendrocytes, making them more sensitive to the signals for programmed cell death (apoptosis). The death of the oligodendrocytes could also result from the production of cytokines (in particular TNF-a) by the activated macrophages which are present in the cerebral inflammatory lesions in ALD.

In a study of the fragmentation of the DNA, by the TUNEL method and the expression of caspase-3, of the oligodendrocytes of the brains of ALD patients exhibit signs of cell death by apoptosis and the intensity of the apoptotic phenomena is correlated with the intensity of the demyelination.

Death of the oligodendrocytes by apoptosis was recently demonstrated in 2 other other murine models of genetic disease of myeline: the twitcher mouse (model of Krabbe's disease) and various models deficient in proteolipid protein, PLP (jimpy mouse, msd mouse, md rat).

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z It has now been found that riluzole, optionally in the form of a pharmaceutically acceptable salt, reduces oligodendrocyte death from apoptosis, induced by kainate and, because of this, can be used in 5 the prevention and/or treatment of adrenoleukodystrophy.

Accordingly the present invention provides the use of riluzole or one of its pharmaceutically acceptable salts in the manufacture of a medicament for the prevention and/or treatment of adrenoleukodystrophy.

The present invention also provides a method of treating or preventing adrenoleukodystrophy in a patient comprising administering to the patient an effective amount of riluzole or one of its pharmaceutically acceptable salts.

The protocol used is the following: Cultures enrinched with oligodendrocytes.

Oligodendrocytes are obtained from primary glial cultures of spinal cords according to the slightly modified method described by Saneto et al.

(Neuchemistry, a practical approach, IRL Press, Oxford- Washington DC, p 27-63 (1987)). Spinal cords from 1-day old Wistar rats are dissected under sterile conditions and separated from the meninges. Five to ten spinal cords are transferred into PBS (phosphate-buffered saline: NaC1 137 mM, KC1 2.68 mM, Na 2 HP0 4 6.45 mM,

KH

2

PO

4 1.47 mM) to which 0.25% of trypsine was added.

The enzymatic treatment is topped by addition of Dubelco's modified Eagle's medium (DMEM) to which of bovine fetal serum was added (FBS). Another dissolution step is carried out by means of a 1 ml P:\OPER\Mal\205\259063 313.doc-09l 1105 O -3A-

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z pipette and the cell suspension is filtered. The cells are collected by centrifugation and plated at a density of 1.5-2 x 106 cells/25 cm 2 of culture medium in I Dubelco's modified Eagle's medium (DMEM) to which 10% of 5 bovine fetal serum is added. After 3 days in vitro, the cultures are fed daily. When a visible monolayer is obtained, the cultures are stirred for 2 hours at 37 0

C

at 250 rpm, the culture medium is removed and the flasks are again stirred for 22 hours. The medium containing the cells is incubated at 37 0 C in petri dishes for 1 hour for the microglia to become attached to the plastic. The oligodendrocytes are collected by centrifugation, plated at a density of 1.6 x 10 6 /cm 2 and maintained in DMEM containing 10% FBS for 1 or 2 days. The medium is then replaced with L15 medium to which sodium bicarbonate (22 mM), conalbumin (0.1 mg/ml), putrescine (0.1 mM), insulin (5 ug/ml), sodium selenite (31 nM), glucose (20 mM), progesterone (21 nM), penicillin (100 IU/ml), streptomycin (100 g/ml) and horse serum The oligodendrocytes become rapidly differentiated within 24 to 48 hours into a mature and immature phenotype. These cultures show a purity of about 95% determined by immunoreactivity to galactocerebroside-C (Gal-C) and to glial fibrillary acidic protein (GFAP).

Kainate in solution in PBS at various concentrations (0.01 mM to 1 mM), riluzole in solution in NaC1 at 0.9% and HC1 at 0.001 N, at various concentrations (0.01 mM to 10 mM) or solvent are then added to these cultures. After 48 hours starting from the treatment, the living oligodendrocytes are counted.

Counting of the cells The cells immunoreactive for Gal-C and exhibiting branched processes longer than the diameters of 2 cells are considered overall as immature or mature oligodendrocytes. The number of Gal-C oligodendrocytes are divided for 2 separate counts of the labelled cells in at least 12 fields of 0.63 mm 2 under a 400x microscope. The values are expressed as the number of cells per cm 2 or as the percentage of the control.

The statistical analyses are carried out using the Student's test (t-test).

The results obtained are the following: Test 1: Effect of kainate on the cultures of oligodendrocytes Exposure of the cultures of oligodendrocytes to concentrations of 0.1 to 1 mM kainate for 48 hours.

Number of oligodendrocytes relative to the control standard deviation Solvent alone 100 17.4 Kainate 0.01 mM 108.2 25.2 0.1 mM 76.3 27.9 mM 25.3 9.3 1 mM 22.9 7.1 These results show that kainate at the doses of 0.1 to 1 mM strongly induces death of the oligodendrocytes through apoptosis.

Test 1: Investigation of the toxic effect of riluzole alone on cultures of oligodendrocytes Exposure of the cultures Number of oligodendrocytes of oligodendrocytes to relative to the control concentrations of 0.01 mM standard deviation to 10 mM riluzole for 48 hours Solvent 100 8.2 Riluzole 0.01 mM 99.6 15.7 0.1 mM 94.3 17.3 1 mM 107.1 mM 86.9 16 These results demonstrate that riluzole at the doses of 0.01 mM to 10 mM has no toxic effect on the survival of the oligodendrocytes and that at the dose of 10 mM, a slight toxic effect is observed.

Test 3: Protective effect of riluzole on oligocyte death induced by kainate Exposure of the oligodendrocytes to kainate at the dose of 1 mM and to riluzole at the doses of 0.01 mM to 10 mM.

Number of oligodendrocytes relative to the control standard deviation Kainate 1000 22.9 7.1 Kainate 1 mM riluzole 0.01 mM 48.7 7 Kainate 1 mM riluzole 0.1 mM 65.2 9.2 Kainate 1 mM riluzole 1 mM 69.4 12 Kainate 1 mM riluzole 10 mM 56.3 6.9 These results demonstrate that riluzole clearly reduces oligodendrocyte death from apoptosis induced by kainate, this being even at the dose where a slight toxic effect of riluzole alone was observed and can thus be used in the prevention and treatment of adrenoleukodystrophy.

As pharmaceutically acceptable salts of Riluzole, there may be mentioned especially the addition salts with inorganic acids such as hydrochloride, sulfate, nitrate, phosphate, or organic acids such as acetate, propionate, succinate, oxalate, benzoate, fumarate, maleate, methane-sulfonate, isethionate, theophyllineacetate, salicylate, phenolphthalinate, methylene-bis-p-oxy-naphthoate or substitution derivatives of these derivatives.

The medicaments consist of at least Riluzole in free form or in the form of an addition salt with a pharmaceutically acceptable acid, in a pure state or in the form of a composition in which it is combined with any other pharmaceutically compatible product, which may be inert or physiologically active. The medicaments according to the invention may be used by the oral, parenteral or rectal route.

As solid compositions for oral administration, tablets, pills, powders, (gelatin capsules, cachets) or granules may be used. In these compositions the active ingredient of the invention is mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica, under an argon stream. These compositions may also comprise substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, a coloring, a coating (sugar-coated tablets) or a glaze.

As liquid compositions for oral administration, there may be used pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil.

These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing products.

The sterile compositions for parenteral administration may preferably be solutions which are aqueous or nonaqueous, suspensions or emulsions. As solvent or vehicle, there may be used water, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, injectable organic esters, for example ethyl oleate or other suitable organic solvents. These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. The sterilization may be carried out in'several ways, for example by aseptisizing filtration, by incorporating sterilizing agents into the composition, by irradiation or by heating. They can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or in any other injectable sterile medium.

The compositions for rectal administration are suppositories or rectal capsules which contain, in addition to the active product, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols.

The doses depend on the desired effect, the duration of the treatment and the route of administration used; they are generally between 50 and 400 mg per day by the oral route for an adult with unit doses ranging from 25 to 200 mg of active substance.

In general, the doctor will determine the appropriate dosage according to the age, weight and all the other factors specific to the subject to be treated.

The following examples illustrate medicaments according to the invention: Example A Tablets containing a 50 mg dose of active product having the following composition are prepared according to the usual technique: Riluzole 50 mg Mannitol 64 mg Microcrystalline cellulose 50 mg Polyvidone excipient 12 mg Sodium carboxymethylstarch 16 mg Talc 4 mg Magnesium stearate 2 mg Anhydrous colloidal silica 2 mg Mixture of methylhydroxypropylcellulose, polyethylene glycol 6000, titanium dioxide (72-3.5-24.5) qs 1 finished film-coated tablet weighing 245 mg Example B Gelatin capsules containing a 50 mg dose of active product having the following composition are prepared according to the usual technique: 50 mg 18 mg 55 mg Colloidal 1 mg Sodium carboxymethylstarch.................. 10 mg 10 mg Magnesium 1 mg Example C An injectable solution containing 10 mg of active product having the following composition is prepared: 10 mg Benzoic 80 mg Benzyl 0.06 cm 3 Sodium 80 mg Ethanol at 0.4 cm 3 Sodium 24 mg Propylene 1.6 cm 3 qs 4 cm 3 P:\OPER\Mlal20(5\2596063 3l.doc-10/l S-12-

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z The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in S 5 Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the p word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (6)

1. Use of riluzole or one of its I pharmaceutically acceptable salts in the manufacture of a 5 medicament for the prevention and/or treatment of adrenoleukodystrophy.

2. Use according to claim 1, wherein the medicament contains 25 mg to 200 mg of riluzole or one of its pharmaceutically acceptable salts.

3. Use according to claim 1 or claim 2 and substantially as hereinbefore described.

4. A method of treating or preventing adrenoleukodystrophy in a patient comprising administering to the patient an effective amount of riluzole or one of its pharmaceutically acceptable salts.

The method according to claim 4 wherein 25 mg to 200 mg of riluzole or one of its pharmaceutically acceptable salts thereof is administered.

6. The method according to claim 4 or claim and substantially as hereinbefore described. DATED this 10 th day of November, 2005 Aventis Pharma S.A. AND Institut National De La Sante Et De La Recherche Medicale By DAVIES COLLISON CAVE Patent Attorneys for the Applicants