The present invention is concerned with infusions comprising a neuroprotectant and a perfluorochemical as a combined preparation for simultaneous, separate or sequential use in the treatment of conditions involving cerebral hypoxia ; and with devices or packs of said infusions with an appropriate independent drive unit suitable for use, e.g. in the ambulance and emergency room.

In US-4,861,785 there are described benzoxazol- and benzothiazolamine derivatives having anti-hypoxic and anti-anoxic activity. In WO-92/ 14,731 some of these benzothiazolamine derivatives were disclosed having useful anti-stroke activity. Injectable formulations of (S)-4-[(2-benzothiazolyl) methy lamino]-α-[(3,4-difluoro- phenoxy )methyl ]- 1 -piperidine-ethanol (generically known as lubeluzole) are disclosed in European Patent application No. 94203422.4 filed 24 November 1994.

Conditions involving cerebral hypoxia comprise stroke, more in particular ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage, and head trauma. The treatment of conditions involving cerebral hypoxia currently consists mainly of neuroprotective and of haematologic therapeutic strategies. As described in Cerebrovasc. Dis. 1995 ; 5 (suppl 1) : 27-30, though there is an increasing belief in the benefit of combined therapies for acute stroke treatment, optimal combinations remain to be determined. Such an optimized combination for the treatment of hypoxia is the subject of the present invention. In particular, it concerns a combination of neuroprotectant agent and a perfluorochemical. The product is adapted to intravenous and intradermal administration by infusion as these represent the most appropriate routes of administration for hypoxic patients.

Hence, the present invention is concerned with a product containing

(a) a composition suitable for intravenous and intradermal administration comprising a pharmaceutically effective amount of a neuroprotectant and a pharmaceutically acceptable earner ; and

(b) a composition suitable for intravenous and intradermal administration comprising a pharmaceutically effective amount of a perfluorochemical and a pharmaceutically acceptable carrier, as a combined preparation for simultaneous, separate or sequential use in the treatment of hypoxia.

Intravenous administration means introduction by way of infusion into a vein, and intradermal administration means introduction by way of infusion into the dermis.

Hereinafter, the amounts of each of the ingredients in the compositions are expressed as percentages by weight based on the total volume of the formulation, unless otherwise indicated.

Neuroprotectants in panicular are 2-aminobenzothiazole derivatives of formula (I)

the pharmaceutically acceptable acid addition salts, the N-oxide forms, and the stereochemically isomeric forms thereof, wherein Rl represents hydrogen or Ci-4 alkyl ; R2 represents hydrogen, hydroxy or trifluoromethoxy ; and

L represents hydrogen or a radical of formula (TJ)

wherein R^ represents hydrogen or fluoro.

Particular neuroprotectants are lubeluzole (R ¹ is methyl; R^ is hydrogen, L is a radical of formula (II) wherein R^ is fluoro and the atom marked * has the (S) configuration), lubeluzole N-oxide hemihydrate (oxidized piperidine nitrogen atom, preferably cis- oriented), 6-hydroxylubeluzole (R^ is hydroxy) or riluzole (Rl and L are both hydrogen, R^ is trifluoromethoxy). The most preferred neuroprotectant is lubeluzole. Lubeluzole is generic to (S)-4-[(2-benzothiazolyl) methylamino]-α-[(3,4-difluoro- phenoxy)methyl]-l-piperidineethanol. Its preparation and properties are described in WO-92/14,731.

Neuroprotectants may also be NMDA receptor antagonists such as aptiganel, eliprodil, selfotel, tirilazad or remacemide or combinations of the abovementioned 2-amino- benzothiazole derivatives of formula (I) therewith.

Pharmaceutically acceptable acid addition salts comprise the therapeutically active, non-toxic salt forms obtained by treating a base form with an acid such as, for example, an inorganic acid, e.g. hydrochloric, hydrobromic, sulfuric, nitric, phosphoric acid ; or an organic acid, e.g. acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic, succinic, maleic, fumaric, tartaric, citric, methanesulfonic. ethanesulfonic, benzene¬ sulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic acid.

The N-oxide forms of the compounds of formula (I) are meant to comprise those compounds wherein one or more of the nitrogen atoms are oxidized, in particular those wherein the piperidine nitrogen is oxidized. In lubeluzole N-oxide, the oxygen atom on the piperidine nitrogen is cis-oriented, i.e. to the same side of the plane defined by the piperidine ring as the 4-substituent.

The adsoφtion of lubeluzole to the walls of the i.v. administration equipment (infusor) can be reduced significantly by maintaining the pH of the solution (a) below 3.6. In this way, an intravenous solution can be prepared having superior physical stability.

The term "physically stability" or "physically stable" as used herein refers to a solution for which less than 10% by weight of active ingredient is adsorbed after passing through an infusion device. Preferably less than 5% by weight of active ingredient is adsorbed.

Generally, the pharmaceutically acceptable carrier for the neuroprotectant lubeluzole is an aqueous solution (a) comprising water ; an isotonizing agen : and acid, base or buffer substances sufficient to adjust the pH of the solution in the range of from 2.5 to 3.6.

In particular, the concentration of lubeluzole in the present solutions (a) may range from 0.005% to 5%, preferably from 0.01% to 1%, more preferably from 0.02% to 0.2% and in particular is about 0.05%.

Further, the present solutions (a) conveniently comprise from 1 to 10% isotonizing agent. The use of glucose as isotonizing agent has the advantage that very clear solutions are obtained. Preferably, glucose is used in a concentration from 2 to 10%, most preferably of about 5%.

The solutions (a) further comprise acid and base substances to maintain the pH of the solution in the range from 2.5 to 3.6, preferably in the range from 3.0 to 3.4, most preferably at about 3.2. Preferably, the pH of the solutions (a) is adjusted by appropriate amounts of hydrochloric acid and sodium hydroxide. The pH may also be

adjusted by buffer systems comprising mixtures of appropπate amounts of an acid such as phosphoric, tartaric or citric acid, and a base, in particular sodium hydroxide.

In order to increase the solubility of lubeluzole in the present formulations, a solubilizer may be used. Conveniently, a cyclodextrin (CD) or a derivative thereof may be used.

Appropriate cyclodextrin derivatives are α-, β-, γ-cyclodextrins or ethers and mixed ethers thereof wherein one or more of the hydroxy groups ofthe anhydroglucose units of the cyclodextrin are substituted with Cι_6alkyl, particularly methyl, ethyl or isopropyl, e.g. randomly methylated β-cyclodextrin; hydroxyCι_6alkyl, particularly hydroxyethyl, hydroxypropyl or hydroxy-butyl; carboxyCi.galkyl, particularly carboxymethyl or carboxyethyl; Ci-6alkyl-carbonyl, particularly acetyl; Cι_6_ikyloxy- carbonylCi .galkyl or carboxyCi-6alkyl-oxyCι_6alkyl, particularly carboxymethoxy- propyl or carboxyethoxy-propyl; Ci-6alkylcarbonyloxyCι_6alkyl, particularly 2-acetyloxypropyl. Especially noteworthy as solubilizers are β-CD, 2,6-dimethyl-β-

CD, randomly methylated β-cyclo-dextrin, 2-hydroxyethyl-β-CD, 2-hydroxyethyl-γ-

CD, 2-hydroxypropyl-γ-CD and (2-carboxymethoxy)propyl-β-CD, and in particular

2-hy droxypropy 1-β-CD .

The term mixed ether denotes cyclodextrin derivatives wherein at least two cyclodextrin hydroxy groups are etherified with different groups such as, for example, hydroxypropyl and hydroxyethyl.

The average molar substitution (M.S.) is used as a measure of the average number of moles of alkoxy units per mole of anhydroglucose. The M.S.value can be determined by vanous analytical techniques such as nuclear magnetic resonance (NMR), mass spectrometry (MS) and infrared spectroscopy (IR). Depending on the technique used, slightly different values may be obtained for one given cyclodextrin derivative. In the cyclodextrin hydroxyalkyl derivatives for use in the compositions according to the present invention the M.S. as determined by mass spectrometry is in the range of 0.125 to 10, in particular of 0.3 to 3, or from 0.3 to 1.5. Preferably the M.S. ranges from about 0.3 to about 0.8, in particular from about 0.35 to about 0.5 and most particularly is about 0.4. M.S. values determined by NMR or IR preferably range from 0.3 to 1, in particular from 0.55 to 0.75.

The average substitution degree (D.S.) refers to the average number of substituted hydroxyls per anhydroglucose unit. The D.S. value can be determined by various analytical techniques such as nuclear magnetic resonance (NMR), mass spectrometry

(MS) and infrared spectroscopy (IR). Depending on the technique used, slightly different values may be obtained for one given cyclodextnn derivative. In the cyclodextrin derivatives for use in the compositions according to the present invention

the D.S. as determined by MS is in the range of 0.125 to 3, in particular of 0.2 to 2 or from 0.2 to 1.5. Preferably the D.S. ranges from about 0.2 to about 0.7, in particular from about 0.35 to about 0.5 and most particularly is about 0.4. D.S. values determined by NMR or IR preferably range from 0.3 to 1 , in particular from 0.55 to 0.75.

More particular β- and γ-cyclodextrin hydroxyalkyl derivatives for use in the compositions according to the present invention are partially substituted cyclodextrin derivatives wherein the average degree of alkyiation at hydroxyl groups of different positions of the anhydroglucose units is about 0% to 20% for the 3 position, 2% to 70% for the 2 position and about 5% to 90% for the 6 position. Preferably the amount of unsubstituted β- or γ-cyclodextrin is less than 5% of the total cyclodextrin content and in particular is less than 1.5%. Another particularly interesting cyclodextrin derivative is randomly methylated β-cyclodextrin. Most preferred cyclodextrin derivatives for use in the present invention are those partially substituted β-cyclodextrin ethers or mixed ethers having hydroxypropyl, hydroxyethyl and in particular 2-hydroxypropyl and/or 2-(l -hydroxypropyl) substituents.

The most preferred cyclodextrin derivative for use in the compositions of the present invention is hydroxypropyl-β-cyclodextrin having a M.S. in the range of from 0.35 to 0.50 and containing less than 1.5% unsubstituted β-cyclodextrin. M.S. values determined by NMR or IR preferably range from 0.55 to 0.75.

In order to minimize the risk of adverse reactions, an intravenous (or intradermal) formulation preferably contains as few ingredients as possible. Therefore, a formulation without a solubilizer such as a cyclodextrin is preferred. It was found that the solubility of lubeluzole in formulations without a solubilizer ranges between about 9.2 mg/ml (pH 2.5) and about 2 mg/1 (pH 3.6). Formulations of pH 3.2 without a solubilizer comprise at maximum about 3 mg/ml dissolved lubeluzole. Further, the neuroprotectant solution (a) preferably does not contain a preservative.

In particular, the present invention relates to neuroprotectant solutions (a) comprising: (i) 0.005 to 5% lubeluzole or a pharmaceutically acceptable addition salt thereof; (ii) 1 to 10% isotonizing agent;

(iii) acid and/or base substances to adjust the pH in the range from 2.5 to 3.6 ; and (iv) water q.s. ad 100%.

Preferably, the invention relates to neuroprotectant solutions (a) comprising: (i) 0.01 to 1% lubeluzole or a pharmaceutically acceptable addition salt thereof;

(ii) 2 to 10% glucose;

(iii) hydrochloric acid and sodium hydroxide to adjust the pH in the range from 3.0 to 3.4 ; and

(iv) water q.s. ad 100%.

Most preferably, the invention relates to neuroprotectant solutions (a) containing approximately :

(i) 0.05% lubeluzole or a pharmaceutically acceptable addition salt thereof; (ii) 5% glucose; (iii) hydrochloric acid and sodium hydroxide to adjust the pH to about 3.2; and (iv) water q.s. ad 100%.

The solutions (a) are sterilized using art- known techniques.

The neuroprotectant solution (a) of the present product is conveniently used in the treatment of patients suffering from acute hypoxia. In general it is contemplated that an effective treatment for acute hypoxia involves administering to the patient an amount of lubeluzole (or 6-hydroxy-lubeluzole) in the range of 10 to 30 ml of solution (a) or from 5 to 15 mg of the active ingredient during the first hour of therapy. During the following 24 hours about 4/3 or 133% of that amount may be administered. That is, one starts with a relatively high initial flow which is then lowered considerably. The maintenance dose may be administered for several consecutive days.

Preferably about 15 ml of solution or about 7.5 mg ofthe active ingredient is administered by infusion during the first hour of therapy, followed by about 20 ml of solution or about 10 mg of active ingredient during the next 24 hours. It is evident that said effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective amount ranges mentioned hereinabove are therefore guidelines only and are not intended to limit the scope or use of the invention to any extent. The subject solutions may conveniently be co- administered with a physiological salt solution according to art-known procedures.

The reperfusion composition (b) of the present product comprises a pharmaceutically effective amount of a perfluorochemical and a pharmaceutically acceptable carrier. Suitable perfluorochemicals are biologically and biochemically inert compounds consisting essentially of carbon and fluorine which are capable of dissolving oxygen and carbon dioxide. These known blood substitutes have a half-life of several hours in the body and thus are ideally suited for acute hypoxia treatment. One of the two

presently approved perfluorochemicals is preferably used. Either the perfluoro¬ chemical is Oxygent® [perflubron], i.e. perfluoro-n-octyl-1 -bromide (n-CgFi7Br), or alternatively, Fluosol®, a (14:6) mixture of perfluorodecalin and perfluoro- triisopropylamine. Each of these perfluoro-chemicals is formulated in a pharmaceutically acceptable surfactant.

Where the perfluorochemical composition (b) is perflubron (Oxygent®), the emulsion comprises :

(v) from 60 % to 100 % (w/v) perfluoro-n-octyl- 1 -bromide (n-C8Fl 7Br) in (vi) egg yolk lecithin.

Where the perfluorochemical composition (b) is Fluosol®, the emulsion comprisies : (vii) 14 % (w/v) perfluorodecalin and (viii) 6 % (w/v) perfluorotriisopropyl amine, in (ix) a mixture of egg yolk phospholipid and a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® F 68).

The solutions (b) are sterilized using art-known techniques.

The reperfusion solution (b) of the present product is conveniently used in the treatment of patients suffeπng from acute hypoxia. In general it is contemplated that an effective treatment for acute hypoxia involves administering to the patient an amount of perfluorochemical in the range of 5 to 100 ml of emulsion (b) during the first hour of therapy. Preferably about 10 to 50 ml of emulsion is administered by infusion during the first hour of therapy. During the next 24 hours, about 10 to 65 ml of emulsion may be administered. It is evident that said effective amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective amount ranges mentioned hereinabove are therefore guidelines only and are not intended to limit the scope or use of the invention to any extent.

The neuroprotectant and reperfusion solutions may conveniently be co-administered with a physiological salt solution following to art-known infusion procedures.

Preferred products according to the present invention are those wherein the neuroprotectant solution (a) and the perfluorochemical emulsion (b) are miscible and - when mixed - form a stable emulsion for up to one day at room temperature. The two solutions can then be stored together, but in separate containers such as vials, pre-filled syringes and the like, and mixed immediately before use. A preferred container

comprises the neuroprotectant solution (a) and the perfluorochemical emulsion (b) separately in a two-chamber container including means to mix both liquids. The two- chamber container ideally is a pre-filled, two-chamber syringe with bypass or similar means (e.g. a breakable seal) allowing mixing of the two separate solutions prior to administration, and which is further adapted for use with infusor devices.

A first specific product is adapted for administering a neuroprotectant solution (a) and a perfluorochemical emulsion (b) during the first hour of therapy. If the neuro¬ protectant is the preferred active ingredient lubeluzole, then the product comprises about 15 ml of solution or about 7.5 mg of the active ingredient. When the perfluorochemical emulsion is Oxygent®, then it comprises about 10 to 50 ml of emulsion.

A second specific product is adapted for administering a neuroprotectant solution (a) and a perfluorochemical emulsion (b) during the 24 hours following the one-hour therapy described in the previous paragraph. When the neuroprotectant is the preferred active ingredient lubeluzole, then the product comprises about 20 ml of solution or about 10 mg of the active ingredient. When the perfluorochemical emulsion is Oxygent®, then it comprises about 10 to 65 ml of emulsion.

Since the product is meant to be administered urgently to patients suffering from hypoxia, that is in the ambulance, emergency room or intense care unit, an infusion device or pack for the treatment of hypoxia comprising the product together with a disposable, independent drive unit is considered to be the most useful presentation of the product according to the present invention. As independent drive units for powering syringes, in particular prefilled syringes, there may be named both gas- operated and vacuum-operated drive units. An interesting gas-operated intradermal drug delivery device permitting delivery of a drug at a slow rate which can be precisely controlled is described in WO-95/13838, corresponding to US-5,527,288. The device comprises a housing with one or more drug reservoirs and a single hollow needle projecting outwards for a sufficient distance so as to penetrate through the stratum comeum and epidermis into the dermis when the housing is pressed against the skin. The device can be of modular design consisting of disposable cartridge units comprising the depletable components (active ingredients, power source) and a reusable drive unit comprising amongst others the housing and the electronic controls.

The present invention evidently also concerns the use of a product as described hereinbefore for the preparation of a medicament for acute hypoxia treatment. Similarly, the present invention relates to a method of treating patients suffering from

hypoxia, comprising administering simultaneously, separately or sequentially to said patients the components of a product as described hereinbefore.

Experimental part Example 1 : Preparation of (-)-[cis] lubeluzole N-oxide hemihydrate.

To a stirred solution of lubeluzole (1 1.6 g ; 27 mmol) in dichloromethane (700 ml), cooled to -10°C, was added m-chloroperbenzoic acid (6.7 g ; 31 mmol). The reaction mixture was stirred for 24 hours, and then washed with an aqueous ammonia solution (2% ; 3 times) and water (3 times). The organis phase was dried on MgSO4, filtered and evaporated, yielding 9.6 of raw material. The product (-)-[cis] lubeluzole N-oxide hemihydrate was purified by recrystallization from methylisopropylketone

20 (mp. 182.8°C) (yield : 4.7 g ; 38.7%) [α] _D = -8.73° (1% in methanol) (comp. 1).

Example 2 : Preparation of 6-hydroxylubeluzole. a) Ethyl 4-(methy lamino)- 1-piperidinecarboxy late (59 mmol), 2-chloro-6-methoxy- benzothiazole (49 mmol) and sodium carbonate (50 mmol) were stirred at 180°C under N2 flow on an oil bath overnight. The mixture was cooled to room temperature. CHCI3 was added. The mixture was stirred on in ultrasonic bath for 10 minutes and then washed twice with water and once with a NaCl 50% solution. The organic layer was separated, dried (MgSO_ø, filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (eluent: CHCI3/ hexane/ CH3OH 50/49/1). The pure fractions were collected and the solvent was evaporated, yielding 16g (93.5%) of ethyl 4-[(6-methoxy-2-benzothiazolyl)-methylamino]-l- piperidinecarboxylate (interm. 1). b) A mixture of intermediate (1) (50.4 mmol) in a solution of hydrobromic acid in water (140ml) was stirred and refluxed overnight. The mixture was cooled, evaporated and the residue was boiled in 2-propanol. After cooling to room temperature, the precipitate was collected by filtration, washed with 2-propanol and with 2,2'-oxybis- propane and was then dried on the air. The precipitate was dissolved in water (200ml) and basified till pH 9.3 with NH4OH. The aqueous layer was decanted, the oily residue was stirred 3 times in a bit water and decanted. The oily residue was boiled in CH3CN (80ml), the precipitate was filtered off at room temperature and dried, yielding

9.1g (71%) of 2-(methyl-4-piperidinylamino)-6-benzothiazolol; mp. 222.9°C (interm. 2). c) A mixture of (+)-(S)-[(3,4-difluorophenoxy)methyl]oxirane (3.22 mmol) and intermediate (2) (3.2 mmol) in 1 -butanol ( 10ml) was stined for 22 hours at 125°C (oil bath). The reaction mixture was cooled to room temperature and the solvent was

evaporated. The residue was purified by column chromatography over silica gel (24xl60mm;eluent: CHCl3/CH3OH/n-hexane 45/10/45). The pure fractions were collected and the solvent was evaporated. The residue (2.458 mmol) was dissolved in 2-propanol (20ml) and converted into the (Z)-2-butenedioic acid salt (1:2) with a solution of (Z)-2-butenedioic acid (4.92 mmol) in 2-propanol (10ml). The mixture was heated for 2 minutes. The crystals were filtered off at room t° and dried. Yielding: 1.471g (67.4%) of (-)-(S)-2-[[l-[3-(3,4-difluoroρhenoxy)-2-hydroxypropyl]-4- piperidinyl]methylamino]-6-benzothiazolol (Z)-2-butenedioate (1:2); mp. 180.2°C;

20 [α] _D = - 8.03° (c = 1% in methanol) (comp. 2).

PharmapQlogica] examples

The useful anti-hypoxic properties of the products of the present invention can be demonstrated in the following test procedure.

Example 3 : Post-Treatment in a Rat Photochemical Stroke Model. Male Wistar rats, weighing 260-280 g, are anesthetized with halothane in a N2O/O2 mixture. The animals are placed in a stereotactic apparatus, the scalp is incised for exposure of the skull surface, and a catheter is inserted into a lateral tail vein. Rose Bengal (30mg/kg; 15 mg/ml in 0.9% NaCl) is infused intravenously for 2 minutes in animals with normal hemodynamics and blood gases. Thereafter, the skull is focally illuminated with cold white light for 5 minutes by means of a fiber-optic bundle inside a 1-mm diameter objective. The light is aimed at the hindlimb area of the right parietal sensorimotor neocortex. Five minutes after infarct induction (i.e. 5 min after light offset), the rats are injected with the product. Neurologic tests, involving limb placing reactions, are conducted on the first two days after infarction at 24-hour intervals after its induction.Tactile forward and sideways placing are tested by lightly contacting the table edge with the dorsal or lateral aspect of a paw (2 tests). Proprioceptive forward and sideways placing involves pushing the paw against the table edge in order to stimulate limb muscles and joints (2 tests). Rats are also put along the edge of an elevated platform in order to assess proprioceptive adduction : a paw is gently pulled down and away from the platform edge, and, upon sudden release, it is checked for retrieval and placing ( 1 test). For each of the 5 tests, placing scores are : 0, no placing; 1, incomplete and/or delayed placing; or 2, immediate, complete placing. For each limb, the summed tactile/proprioceptive placing score, including the platform test, is maximally 10. Results are reported from the deficient hindlimb contralateral to the neocortical infarct. Six rats are used for each dose.