EP0607201A1

EP0607201A1 - Composition comprising s-oxiracetame for use as nootropic

Info

Publication number: EP0607201A1
Application number: EP92920534A
Authority: EP
Inventors: Laura Smithkline Beecham Farmaceutici Chiodini; Giancarlo Florence University Pepeu
Original assignee: Smithkline Beecham Farmaceutici SpA
Current assignee: GlaxoSmithKline SpA
Priority date: 1991-10-08
Filing date: 1992-10-03
Publication date: 1994-07-27
Also published as: WO1993006826A1; AU2645692A; GB9121289D0

Abstract

Composition pharmaceutique comprenant de l'oxiracétame sous la forme de son énantiomère S et pratiquement dépourvu de son énantiomère R, ou sel pharmaceutiquement acceptable, ou solvate pharmaceutiquement acceptable de cet élément, et utilisation d'une telle composition en thérapie.Pharmaceutical composition comprising oxiracetam in the form of its S enantiomer and substantially free of its R enantiomer, or pharmaceutically acceptable salt, or pharmaceutically acceptable solvate of this element, and use of such a composition in therapy.

Description

C0MP0SITI0N COMPRISING S-OXIRACETAME FOR USE AS NOOTROPIC

The present invention relates to a pharmaceutical composition, in particular to composition of the S-stereoisomer of oxiracetam and to the therapeutic use of such composition.

It is well known that significant impairment of cognitive functions is the more evident and debilitating symptom of Alzheimer disease, senile dementia of Alzheimer type and multinfarctual dementia. In addition it must be taken into account that the population of the world is becoming older. By the end of the century it is estimated that the population of the western countries aged 65 and over will have doubled. People over the age of 80 represent the fastest growing segment of the population (US Department of Health and Human Services, 1984, Publication no. ADM 84-1323, Washington DC). Furthermore an important portion of this age group suffers serious deteriorations in mental capacity, the first sign of which is progressive memory loss (T. Crook et al. 1986, Developmental Neuropsychology 2 (4), 261-276). Current therapies are not completely satisfactory and there is a medical need for new drugs effective in relieving cognitive decline or in slowing down its progression.

It is well known that two enantiomers may exhibit different pharmacological effects as well as different pharmacokinetic and toxicology (W.H. De Camp, 1989, Chirality, 1, 2-6). Some times only one enantiomer is responsible for the main pharmacological action (e.g.: beta- blockers).

Oxiracetam is usefully used in alleviating memory loss in senile dementia of different types and possesses an asymmetric carbon atom in the position 4 of the ring. Since oxiracetam is structurally related to 4-amino- 3-hydroxybutyric acid (GABOB) and to its betaine derivative, carnitine (Vitamine BT), and it is well known that these amino acids are more active (E. Roberts et al. 1981, J. Neurosci., 1, 132-140; LB. Fritz 1963, Adv. Res., 1, 283) in the absolute configuration R, we prepared both R and S forms of oxiracetam for their evaluation on tests predictive for activity on learning and memory.

It has now been surprisingly discovered that the S- form of oxiracetam (I) is particularly useful as a nootropic agent.

S-oxiracetam, maybe prepared using a stereospecific analogue, specifically for the S-oxiracetam, of the methods for preparing oxiracetam disclosed in US 4,173,569 and II Farmaco, Ed. Sci., 39 (1984) starting from S-4-amino-3-hydroxybutyric acid (S-GABOB). These methods are incorporated herein by reference: The final step of the process may be represented schematically as follows:

E I

wherein, R is an alkyl radical containing up to 4 carbon atoms or a trichlorophenyl, nitrophenyl or trichloroethyl radical, the asterisk represents a centre of asymmetry of the molecule, which is in the S-form.

The ammonolysis of the compound of formula II may be carried out under conventional ammonolysis conditions, for example the compound of formula II may be treated with ammonium hydroxide, conveniently at ambient temperature.

A suitable preparation of a compound of formula II is represented schematically as follows:

IV

XCH₂COOR b) H

in

wherein R* is a methyl or ethyl radical, X is a bromine, chlorine or iodine atom, M is a sodium, potassium or lithium atom and the asterisk represents a centre of asymmetry, which is in the S-form.

In the practice, steps a, b and c of the above-mentioned process may be carried out without separation of the intermediates: γ-Amino-β- hydroxybutyric acid V is treated under anhydrous conditions in an inert aprotic solvent, such as toluene, acetonitrile, dioxan and xylene, with an excess of a silylating agent at the boiling point of the solvent employed and the cyclised silyloxy derivative IV obtained is reacted with a halide derivative of an aliphatic acid ester XCH2COOR, wherein X and R have the same meanings as above, in an aprotic and preferably polar solvent, such asacetonitrile, dimethylformamide, dioxan, dimethyl sulphoxide or hexamethyl phosphoramide, and then with an alkali metal hydride, such as sodium, potassium or lithium hydride.

The temperature used is not critical for the reaction but is preferably in the range of from 35 to 80°C, optionally with refluxing for a short period of time in order to complete the reaction for obtaining the compound III, from which the silyl protecting group is removed by hydrolysis to give the corresponding 4-hydroxy derivative II.

The silylating agent may be, for example, hexamethyl disilazane, bis- trimethylsilylurea or bis-trimethylsilylacetamide: in practice, the silylating agent is preferably employed in the presence of a small quantity of trimethylchlorosilane.

As an alternative preparation a steroespecific analogue, specifically for the S-stereochemistry, of the methods disclosed in US 4,797,496 may be used to prepare S-oxiracetam. These methods are also incorporated herein by reference: The final step of the US 4,797,496 process provides for an intramolecular cyclisation represented schematically as follows:

OH *

N H ₂

wherein X represents Cι_ o alkyl and the asterisk represents the asymmetric centre, which is in the S-form.

The US 4,797,496 methods start from S-forms of alkyl 3,4- epoxybutanoates VI.

S-VI S-I

The enantiomeric alkyl butanoates VI can be obtained by cyclization of the corresponding halohydiins VTH

OH

^{1 1} COOR < . tX_^/CCOR S - VIII S - VI

Preparation of compound VIII was performed from (S)-beta- hydroxybutyro-lacton [obtained from L-malic acid S. Henrot, M. Larcheveque, Y. Petit, Synth. Comm., 16, 183 (1986) or from D-ascorbic acid according to A. Tanaka, Yamashita, Synthesis, 1987, 5701, by a modification of a method reported by M. Larcheveque, S. Henrot, Tetrahedron Lett., 28, 1781 (1987) using a reagent described by G.A. Olah et al., J. Org. Chem., 48, 3667 (1983).

Cyclization of halohydrins VIII can be obtained either by treatment with silver oxide according to J.D. McClure, J. Org. Chem., 32, 3888 (1967), or especially in the case of X* is iodine, by a novel method entailing the use of an anion exchange resin in carbonate form (these kind of epoxides are very sensitive to base catalyzed rearrangment, and cannot be obtained by action of conventional bases on the halohydrins, see J. D. McClure, loc. cit).

The R-enantiomer of oxiracetam may be prepared by using analogous methods to those described above.

When used in the therapeutic treatment of humans and animals, the compositions of the invention are normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.

Therefore the present invention provides a pharmaceutical composition which comprises oxiracetam in the form of its S-enantiomer, substantially free of its R-enantiomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

The compositions of the invention may be administered in standard manner for the treatment of the indicated diseases, for example orally, parenterally, rectally, transdermally or by transmucosal (for example sub- lingual, or buccal or insufϊlatory) administration.

The compositions of the invention which are active when given orally or via sub-lingual or buccal administration can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, glycerine or water with a flavouring or colouring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be utilised, for example aqueous gums, celluloses, silicates or oils are incorporated in a soft gelatin capsule shell.

Typical parenteral compositions consist of a solution or suspension of the compound of the invention in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycocl, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.

A typical suppository formulation comprises a compound of the invention which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats.

Typical transdermal formulations_^ comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or can be in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet or capsule, so that the patient may administer to himself a single dose.

Each dosage unit for oral administration contains suitably from 0.05 mg/kg to 20 mg/kg, and preferably from 0.1 mg kg to 5 mg/kg, and each dosage unit for parenteral administration contains suitably from 0.05 mg kg to 10 mg kg, of a compound of the invention.

The daily dosage regimen for oral administration is suitably about 0.05 mg/kg to 50 mg kg, more suitably about 0.1 mg kg to 20 mg/kg of a compound of the invention. The active ingredient may be administered from 1 to 6 times daily. The compositions of the invention may be co-administered with other pharmaceutically active compounds, for example in combination, concurrently or sequentially, particularly with other compounds used in the treatment of elderly patients e.g. tranquillisers, diuretics, antihypertensives, vasodilators and inotropic agents.

As stated above S-oxiracetam is a particularly effective nootropic agent: Thus in a further aspect of the present invention, there is provided a composition which comprises oxiracetam in the form of its S-enantiomer, substantially free of its R-enantiomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, for use as a nootropic agent.

The invention also provides the use of a composition which comprises oxiracetam in the form of its S-enantiomer, substantially free of its R- enantiomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament for use as a nootropic agent.

The activity of the S-isomer is demonstrated as follows: it was found that the S enantiomer of oxiracetam is more active than the R, in inducing long term potentiation (LTP) in the rat hippocampal slices "in vitro", in potentiating glutamate stimulated Ca++ uptake in cultured cerebellar granule cells and in reverting the scopolamine induced amnesia in rats.

Hippocampal LTP is widely accepted to be the synaptic basis of learning and memory. Compounds able to improve memory functions such as piracetam (Olpe M.R., 1982 , et al. Eur. J. Pharmacol., 80, 415-419) aniracetam (Satoh M. et al., 1986 Neurosci. Lett., 68, 216-220) and phorbol-esters (Malenka Q.C. et al., 1986, Nature 321, 171-177) are able to potentiate LTP. On the contrary treatments inducing loss of memory as electroconvulsive shock (Anwyl L. et al., 1987, Brain Res. 435, 373-379) aminophosphonovaleric acid (APV) (Lowingen M.D. et al., 1987, Brain Res., 436, 177-183) and benzodiazepines (Satoh M. et al., 1986 Neurosci. Lett., 68, 216-220) inhibit LTP. LTP is an electrophysiological phenomenon peculiar to glutamatergic synapsis and is triggered by postsynaptic stimulation of N-methyl-D-aspartic acid (NMDA) receptors followed by Ca++ channels opening (Smith S.J., 1987, TINS, 10, 142-144). Ca++ entry into the postsynaptic cells triggers a series of intracellular biochemical events that lead to LTP of synaptic signal transmission wich lasts hours or even weeks.

Considering that glutamate, directly involved in LTP, stimulates Ca++ uptake by postsynaptic cells, the evaluation of the effect of the two compounds on Ca++ uptake stimulated by glutamic acid in cultured cerebellar granular cells (glutamatergic neurons) was also consequentely taken into consideration.

To furtherly confirm the results obtained in "in vitro" experiments, the R and S enantiomers of oxiracetam were tested against the amnesia induced by the anticholinergic scopolamine using a one trial passive avoidance test in adult rats, a procedure appropriate to evaluate the specific effect of nootropic drugs on learning and memory (G. Spignoli et al., 1986, Pharmacol. Biochem. and Behav., 27, 491-495).

The results indicate that in all the three tests the S compound is more potent than the R compound and that the S-oxiracetam is the responsible of the great part of the activity previously demonstrated in the racemic oxiracetam. This observation is completely unexpected and is a new important feature of the drug, since S-oxiracetam is structurally related to the less potent or inactive S-forms of Gabob and carnitine.

Hippocampal long term potentiation in vitro

Transverse slices, 400 um thick, prepared from hippocampi removed from adult male Wistar rats were superfused with oxygenated artifical cerebrospinal fluid. (Corradetti R., et al., 1983 J. Neurochem., 41, 1518- 1525). Test pulses (80 us - 0.1 Hz) were delivered through bipolar electrodes positioned in the stratum radiatum and evoked orthodromic potentials were extracellularly recorded from the pyramidal cells of the CAl region. Percentage changes in amplitude of population spike or in excitatory post synaptic potential (e.p.s.p.) were calculated as indexes of synaptic efficiency. The compounds R and S were added to the superfusion medium at concentrations ranging from 10-5 to 10-8 M. Compound S clearly increased the amplitude of the evoked potentials starting from the concentration of 10-7M while the compound R was active only at the concentration of 10-5M as reported in the table 1.

Table 1 - Effects on hippocampal long term potentiation

Calcium uptake in cultured cerebellar neurons

Primary cultures of cerebellar granular cells (glutamatergic neurons) were used (Nicoletti F., et al., 1986, J. Neuroscience, 6, 1905-1911).

Glutamate added to the medium, stimulates dose dependently the 45Ca2+ uptake at concentrations from 10 to 100 uM (Table 2).

Table 2 - Effect of glutamate on ⁴⁵Ca²⁺ uptake in cerebellar granular cell

100 μM concentration of S or R forms of oxiracetam did not modify basal 5C 2+ uptake. In the presence of 5 μM glutamate the R enantiomer is still inactive while the S isomer stimulates dose dependently Ca++ uptake, as evidenced in Table 3.

Table 3 - Effects on glutamate-stimulated 45(3a2+ uptake

Net increase over 5 μM glutamate-stimulated

Antagonism against scopolamine amnesia

S and R enantiomers were checked for their ability to antagonize amnesia induced by the anticholinergic drug scopolamine in a passive avoidance step through test in the rat. (Essmann et al., Pharmacol. Res. Commun., 1973, 5, 295).

Dining the learning session each animal was placed into the lighted compartment of a passive avoidance apparatus and the time required to cross into the dark one was recorded (1st latency time). Once in the dark side each rat received an unavoidable foot shock. Thirty minutes later the animals were again placed into the lighted box and the 2nd latency time was recorded and taken as an index of learning (cut off time was 120 sec).

Scopolamine (0.66 mg/kg sc) administered 60 minutes before the learning session completely abolished the acquisition of the passive avoidance task as indicated by the fall of the 2nd at 1st LT level; furthermore the 1st LT is not affected by scopolamine treatment suggesting that the amnestic effect is quite specific. S enantiomer, administered thirty minutes before scopolamine, significantly protects animals from the disrupting effect of scopolamine at all tested doses of 25-50-100 mg/kg i.p.. R enantiomer, tested in the same experimental conditions and doses, was completely inactive as shown in Table 4.

Table 4 - Comparative evaluation of compounds R and S against learning impairment caused by scopolamine (0.63 mg kg s.c.) in a passive avoidance test in rats, (n = 10 rats/group).

(1) SCOP. = Scopolamine

(2) P < 0.001 versus saline + scopolamine group (one-way ANOVA test)

The invention will now be illustrated in greater detail by the following examples.

Example 1

Isobutyl (S)-3-hydroxy-4-iodobutanoate (VIII)

To a mixture of (S)-4-hydroxytetrahydro-2-furanone (33 g) and anhydrous sodium iodide (97 g) in anhydrous acetonitrile (825 ml), trichloromethylsilane (76 ml) was added dropwise. The reaction mixture was stirred at reflux temperature for 3 hours, then the solvent was evaporated under vacuum. To the residue, isobutanol (825 ml) was added and the resulting mixture was stirred at room temperature for 4 days. The solvent was evaporated under vacuum and the residue was partitioned between dichloromethane (350 ml) and water (150 ml).

The organic layer was washed in sequence with a saturated solution of sodium metabisulfite, saturated solution of sodium bicarbonate and water. After drying over Na2SO4, the solvent was removed under vacuum to afford 87.5 g of the title compound, [alpha]D = -20.3 (c=3, MeOH).

Example 2

Isobutyl (S)-3,4-epoxybutanoate (VI)

A mixture of isobutyl (S)-3-hydroxy-4-iodobutanoate (5.1 g) and silver oxide (2.4 g) in 1,2-dimethoxyethane (20 ml) was refluxed for 1 hour. After cooling, the precipitate was filtered off and the filtrate was evaporated under vacuum. The residue was chromatographed over silica gel (cyclohexane - ethyl acetate 9:1) to yield 2 g of the title compound, [alphajD = -20.3 (c=3, MeOH).

Example 3

Isobutyl (S)-3,4-epoxybutanoate (VI)

To a solution of isobutyl (S)-3-hydroxy-4-iodobutanoate (87 g) in acetonitrile (2.5 1), Amberlyst A-26 resin in carbonate form (prepared from a column of 0.871 of Amberlyst A-26 through which 29 1 of IN sodium carbonate solution was passed, and thoroughly washed with water and then with acetonitrile) was added. The mixture was vigorously stirred for 15 minutes, then the resin was filtered off. The solvent was evaporated under vacuum, the residue was dissolved in dichloromethane and washed with dil. HC1 and water. The organic layer was dried over Na2S04 and evaporated under vacuum. The residue was distilled under vacuum (87- 95°C, 15 mmHg) to give 30.6 g of the title compound, [alphaJD = -20.5 (c=2, MeOH).

Example 4

(S)-4-hydroxy-2-oxo-l-pyrrolidine acetamide (I)

A mixture of 2-isopropyl-4-imidazolidinone (5 g) and isobutyl (S 3,4- epoxybutanoate (6.2 g) in water (15 ml) and acetone (10 ml) was stirred at 70°C for 45 hours. The organic solvent was evaporated under vacuum and the residue was extracted with dichloromethane (25 ml). The organic layer was washed with 1 N hydrochloric acid (5 ml) and water and evaporated to dryness.

The residue was dissolved in dimethylformamide (30 ml) containing water (4.5 ml) and the solution was refluxed for 15 hours under nitrogen. After cooling the solvent was removed under vacuum and the residue was triturated with acetone to afford crude title compound. This was dissolved in water, treated batchwise with ion exchange resins Amberlite IR 120 and Amberlite IRA 68 and charcoal, filtered and evaporated to dryness.

Cristallization of the residue from acetone yielded 2.9g of pure title compound, m.p. 135-136oC, [alphaJD = -37.3 (c=l, water).

The formulation is prepared by mixing together S-oxiracetam and pregelatinized starch. The resulting mixture is wetted with purified water, granulated through a stainless steel screen and dried with warm air. The dried granules are mixed with croscarmellose sodium and magnesium stearate and then compressed into tablets of 430 mg each.

Example 6

Capsules

The formulation is prepared by mixing together S-oxiracetam, corn starch, croscarmellose sodium and magnesium stearate. The resulting mixture is filled into hard gelatine capsules (filled weight: 425 mg each capsule).

Example 7

Injections

S-oxiracetam (g) 250 Water for injections q.s. ad (ml) 1250

The formulation is prepared by dissolving S-oxiracetam in water for injections. The solution is made up to final volume (1250 ml) with water for injections and then filtered through a 0.22 /urn membrane. The filtered solution is filled into glass ampouls (2.5 ml each ampoule) and then the ampoules are heat-sealed. The filled and sealed ampoules are sterilized by pressurized steam at 121oC for 20 minutes. The pH of the injectable solution ranges from 5 to 7.

Purified water q.s. ad (ml) 2500

The formulation is prepared by dissolving S-oxiracetam and sorbitol in purified water, and by dissolving lemon aroma, methyl parahydroxy¬ benzoate and propyl parahydroxybenzoate in propylene glycol. The two solution are mixed and then made up to final volume (2500 ml) with purified water. The solution is filtered and then filled into glass bottles. The pH of the syrup ranges from 4 to 6.

Claims

1. A pharmaceutical composition comprising oxiracetam in the form of its S-enantiomer, substantially free of its R-enantiomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

2. A composition according to claim 1, in unit dosage form.

3. A composition according to claim 1 or claim 2, in tablet or capsule form or in a form adapted for parenteral administration.

4. A composition according to claim 1, for use as a nootropic agent.

5. The use of a composition according to claim 1, for the manufacture of a medicament for use as a nootropic agent