PHARMACEUTICAL COMPOSITIONS CONTAINING MONOSIALOGANGLIOSIDE DERIVATIVES SUITABLE FOR THE TREATMENT OF SPINAL CORD INJURY

Abstract: Pharmaceutical compositions containing monosialoganglioside derivatives suitable for the treatment of spinal cord injury caused by hypoxic-ischaemic or traumatic events, in which said derivatives are selected from the group consisting of the inner ester (AGF2) and the methyl ester (AGF4) of the monosialoganglioside (GM1).

Description: PHARMACEUTICAL COMPOSITIONS CONTAINING MONOSIALOGANGLIOSIDE DERIVATIVES SUITABLE FOR THE TREATMENT OF SPINAL CORD INJURY Technical Field The present invention relates to pharmaceutical compositions containing monosialoganglioside derivatives suitable for the treatment of spinal cord injury. Background art Spinal cord injury induced by ischaemic or traumatic causes is a sudden catastrophic event producing devastating effects on people and, furthermore, a considerable socio-economic impact. The incidence of said injury is high: for example, in the USA over 10,000 people/year sustain spinal cord injury resulting in permanent disablement. Many suffer immediate paralysis and progressive worsening of the medullary pathology. Partial injury may result in temporary paralysis. Spinal cord injury may be defined as primary or secondary. Primary injury arises from tissue destruction by mechanical or ischaemic causes (occurring in the lapse of time between lesion and hospitalization) and involves a practically irreversible neurologic deficit. At the present time there is no possibility of effectively acting on injuries of this type. The secondary injury seems to be the result of the combined action of delayed swelling, additional injuries to the already affected spinal segment, anoxia, reduced perfusion to the impaired medulla tract and/or release of endogenous factors. The investigations carried out so far have pinpointed some molecular bases of spinal injury and recovery, i.e.: a) "neuronal elements injury": as known, secondary injury at least in part results from hypoxic-ischaemic processes damaging neuronal and axonal elements. It is to be stressed that spinal cord traumatic injury seldom causes spinal cord anatomic transection, even in case of total loss of the neurologic clinical function at the caudal portion level. The neurologic deficit is actually induced by a microscopic physical decay of the axons crossing the injury site, by ischaemia or hypoxia followed by local infarction, or by microhaemorrhages or oedema inside the spinal cord at the injury site,- which also affect neuronal impulses transmission; b) "substantia alba tracts injury": as known, clinical deficit after spinal cord injury results also from disorders to the substantia alba, including spinal cord ascending and descending tracts; c) "neurotrophic agents": as known, soluble trophic agents are released by glial cells and improve the survival, the spinal neurons morphofunctional properties and the axonal regeneration; d) "microenvironmental conditions": as known, one of the problems inherent in spinal cord injury recovery is the microenvironment not being "receptive" to neuronal regeneration. This might arise from the regenerating neurons not being capable of re-expressing and maintaining a phenotype adequate for axonal growth and from the presence of a later stage of "inhibitory" proteins on the glial cicatrix surface at the injury site. The foregoing clearly suggests the extreme complexity of spinal pathology as well as the inadequate knowledge of the mechanisms governing the recovery of same . It is also to be stressed that said mechanisms cannot be simply extrapolated from the information acquired so far about traumatic and/or ischaemic brain pathologies . At the present time , only two drugs proved to be effective in improving the neurologic course of spinal cord injured patients , i.e. methylprednisolone and monosialoganglioside GM< . Methylprednisolone is the first drug which proved to be clinically effective , but only in the peracute phase and at high dosage levels (Bracken M . B . et al . : "A randomized , controlled trial of methylprednisolone or naloxone in the treatment of spinal-cord injury: results of the Second National Acute Spinal Cord Injury Study" , New Engl . J . of Med. , 322 , l405-l4ll , 1990) . Monosialoganglioside GM-^ is the second drug which was found significantly to improve the patients ' neurologic functional recovery at one-year distance from injury , on condition that treatment had began within 2 hours from injury, followed by 18-32 i.v. doses of 100 mg/day (Geisler F.H . et al . : "Recovery of motor function after spinal-cord injury - a randomized, placebo-controlled trial with GM j^ ganglioside" , New Engl . J . of Med. , 26, 324 , 1829- 1838; patent application PD 9- 0 023 dated 23rd Dec , 1991) . It is also known that monosialoganglioside GM-^ can produce a neuroprotective effect in the acute phase by limiting the secondary neuronal damage and can improve the functional recovery in the long run. On the basis of said results, GM^ was clinically applied to treat cerebral ischaemic strokes (Argentino C. et al. : "G ^ ganglioside therapy in acute ischemic s roke", Stroke, 20, 1143- 1149. 1989; U.S. patent 4,940,694). The inner ester derivative of GM^ (AGF2) and the small-dose fast- acting therapeutic efficacy of same in acute cerebral eschaemic models are also known (Cahn R. et al.: "Influence of monosialoganglioside inner ester on neurologic recovery after global cerebral ischemia in monkeys", Stroke, 20, 652-656, 1989). Said derivative may be prepared as disclosed in U.S. patent No. 4,476,119 by the Applicant. Furthermore, as disclosed in previous patents by the Applicant (U.S. 4,476,119 and EP 002722), the inner esters of gangliosides are active in the treatment of nervous system disorders, in particular of the peripheral nervous system and of the central nervous system . As claimed by the Applicant in patent applications MI 91 A OO2989 and MI 1 A 002988, the inner ester derivatives of the ganglioside mixture or of the monosialoganglioside GM-^ fraction may be administered by the oral way to treat PNS and CNS disorders. Finally, the outer carboxylic esters of gangliosides and their therapeutic application to treat PNS and CNS disorders - as described above for inner ester derivatives - are also known (EP 85401291.1). In particular, a recent patent application PD 91 A 000101 claims a different therapeutic application of GM-j methyl ester to patients affected by nervous system damages caused by excitatory amino acids (e.g. ischaemia, hypoxia, traumas, compressions, dysmetabolisms, epilepsy, neurodegenerative diseases, and chronic ache). Summary It has been found that surprisingly monosialoganglioside GM^ derivatives, i.e. the inner ester AGF2 and the methyl ester AGF/ j of said monosialoganglioside, can be used as active ingredients for the preparation of pharmaceutical compositions suitable for the therapy of spinal cord injury, caused by hypoxic-ischaemic or traumatic events. The present invention is, therefore, referred to said pharmaceutical compositions and to the therapeutic method envisaging the use of same for the treatment of spinal cord injury. Detailed description of the invention Characteristics and advantages of the claimed pharmaceutical compositions containing monosialoganglioside derivatives suitable for the treatment of spinal cord injury will be illustrated in more detail in the following description. Said derivatives are selected from the group consisting of the monosialoganglioside GM-^ inner ester (AGF2) and the GM-^ methyl ester (AGF 4 ). The experiments reported below show that AGF2 and AGFj, are pharmacologically active in the therapy of spinal cord injury and that AGF2 and AGF^ efficacy is surprisingly higher than that of monosialoganglioside GM^. The experimentation was aimed at reducing the neurologic deficit in rabbit caused by secondary ischaemic spiral cord injury brought on by aortic obstruction. The results obtained are particularly interesting because of the high incidence (up to 50%) of spinal cord injury - i.e. paralysis and paraplegia - in patients having undergone surgical removal of aneurysms in thoracic or thoracicoabdominal aorta segments. In fact, even after 1 to 7 days from the aforesaid operation, which requires thoracic aorta obstruction and complete blood flow discontinuance through the aorta segment affected by the aneurysm, many patients suffer neurological deficit, e.g. paraplegia or paraparesls. Therefore, the possibility of acting pharmacologically - as claimed by the Applicant - in the prevention and/or recovery of neurological damages caused by secondary ischaemic spiral cord injury brought on by aortic obstruction is of major importance. However, said pharmaceutical approach is not only limited to this specific surgical situation but can also be applied to all spinal cord injury events directly brought on by a traumatic or hypoxic- ischaemic lesion. Experiments were carried out on 35 Ne Zealand rabbits (body weight 2-3 kg approx.) , whose spinal cord had been injured surgically by aorta obstruction with consequent formation of thoracicoabdominal aneurysms. The rabbits were anaesthetized i.p. with ketamine (20 mg(kg) and xylazine (2.5 mg/kg) . Then an obstruction system (polyethylene tube) was placed in the aorta region close to distal from the renal artery origin, and enclosed in a subcutaneous pocket. An incision was made above the obstruction to allow the subsequent intervention. Two days after the operation, through the incision, a 21 minutes * aorta obstruction was produced. Two neurosensory electrostimulation electrodes were applied to the skin of the hind legs. The animals were stimulated every five seconds at a lower voltage than required for testing the neurologic functions (motor and sensory) . The spinal cord injury was treated pharmacologically with the following compounds: - monosialoganglioside G ^; - monosialoganglioside GM^ inner ester (AGF2); - monosialoganglioside GM< methyl ester (AGF /j ). The above compounds were dissolved in saline solution and administered i.v. as follows: - 24 h 30 min before aorta obstruction, - 24 h and 48 h after obstruction. Experimentation was carried out on the following animal groups: 1st (n = 11) = saline solution 2nd (n = 8) = GM 1 (20 mg/kg) 3rd (n = 8) = AGF 2 (20 mg/kg) 4th (n = 8) = AGF^ (20 mg/kg) where n is the number of animals utilized for each experimental group . The neurologic functionality (motor and sensory) was assessed according to the Tarlow scale assigning points from 0 (no motor function) to 5 (complete recovery) (Tarlow I.M. : "Spinal cord compression: mechanism of paralysis and treatment", Springfield I: Charles C. Thomas, 1957). In particular, acute motor and sensory functionality (i.e. during obstruction, T = 0) and the subsequent neurologic recovery (expressed as % of paralysed animals) at 24-48-72 hours were assessed. The statistical analysis was carried out by Student's unpaired t- test, Mann-Whitney rank sum and Anova. The results obtained, which are shown on Table 1, represent average values ± standard deviation (s.d.). Table 1 - Effect of AGF2 and AGF4 (compared with GMl) on neurologic recovery in spinal cord injury experimentation conducted on rabbit after aorta obstruction Seconds Paralysis Paralysis Paralysis to acute Seconds to after after after functionality sensory loss 24hrs 48hrs 72hrs motor loss Saline 46.4±2.7 146.7*16.9 63 82 91% Solution (n = 11) AGF 4 8l.0±3-2*** 458.0±25*** 18% 28% 38% (n=8) * p<0.05, **p<0.01, vs saline solution, ***p<0.05 vs GM^ SUBSTITUTE SHEET The results reported in Table 1 show that: - all animals develop neurologic deficit during obstruction. Some recover and some of them are affected by delayed neurologic deficit; - AGF2 and AGFj j considerably delay the acute motor and sensory loss occurring during obstruction; - AGF2 and AGF^ considerably increase the neurologic recovery: after 24 hrs the paralysis incidence is 15% - 18% in treated animals vs 63% in controls and after 7 hours the paralysis incidence is 36% - 38% in treated animals vs 91% in controls; - the effect of AGF 2 and AGF4 is greater than that of monosialoganglioside GM-^ fraction. Therefore, the conclusion may be drawn that the monosialoganglioside derivatives considered herein are effective in preventing and/or recovering neurologic functional disorders, e.g. paralysis, caused by an ischaemic- ype secondary spinal cord injury. In particular, the efficacy of said compounds may be advantageously exploited for preventing and/or recovering acute spinal cord injuries brought on directly by traumatic or hypoxic-ischaemic causes, associated with an acute, subchronic and chronic symptomatology, as well as for preventing and/or recovering the secondary ischaemic spinal cord injury following vascular operations, e.g. the surgical operations to remove thoracic or thoracicoabdominal aneurysms, which must be performed under aorta obstruction conditions. Therefore, monosialoganglioside GMi derivatives, and precisely its inner ester derivative AGF 2 , and its methyl ester derivative AGF/ j can be used as active ingredients for the preparation of pharmaceutical compositions suitable for the treatment of spinal cord injury. Said compositions contain a pharmacologically effective quantity of said derivatives, mixed with pharmacologically acceptable excipients and diluents. The pharmaceutical compositions as per the present invention may have an active ingredient content of r 10 to 200 mg and are prepared as formulations that can be administered to man orally or parenterally, i.e. by intramuscolar or intravenous or subcutaneous injection. The dose of active ingredient to be administered will depend on the desired effects and on the way of administration: by parenteral injection, it may range from 0.1 to 15 mg/kg/day; by os, it may range from 0.5 to 150 mg/kg/day. Pharmaceutical compositions prepared according to this invention are reported below by way of example and not of limitation: CASE 1 One 2 ml ampoule contains - methyl ester AGF/ j 5-00 mg - sodium chloride 16.00 mg - citrate buffer at pH 6 in apyrogenous distilled water q.s. CASE 2 One 2 ml ampoule contains - methyl ester AGF^ 50.00 mg - sodium chloride 16.00 mg - citrate buffer at pH 6 in apyrogenous distilled water q.s. CASE 3 One 4 ml vial contains - methyl ester AGF^ 100.00 mg - sodium chloride 32.00 mg - citrate buffer at pH 6 in apyrogenous distilled water q.s. CASE 4 Pharmaceutical compositions prepared in two vials. The compositions described in this case are prepared in two vials. One vial contains the active ingredient in the form of freeze-dried powder (10% to 90% by wt. ) , mixed with a pharmacologically acceptable exclpient, glyclne or mannitol. The other vial contains the solvent, as sodium chloride solution and a citrate buffer. When the vial of Systems 6, 7. 8, and 9 contains the active ingredient alone, the powder may be obtained either by freeze- drying, using water for injection or another solvent (e.g. tert- butanol) , or by direct partition under aseptic conditions of the sterile powder. System No. 1 a. one 2 ml vial of freeze-dried powder contains - inner ester AGF2 5-00 mg - glycine 30.00 mg b. one 2 ml ampoule of solvent contains - sodium chloride 16.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 2 a. one 3 ~ ~ ~ - ampoule of freeze-dried powder contains - inner ester AGF2 5-00 mg - mannitol 40.00 mg b. one 2 ml ampoule of solvent contains - sodium chloride 16.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 3 a. one 3 nil ampoule of freeze-dried powder contains - inner ester AGF2 50.00 mg " mannitol 20.00 mg b. one 3 nil ampoule of solvent contains - sodium chloride 24.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 4 a. one 5 nil vial of freeze-dried powder contains - inner ester AGF 2 100.00 mg - glycine 50.00 mg b. one 4 ml ampoule of solvent contains - sodium chloride 32.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 5 a. one 5 ml vial of freeze-dried powder contains - inner ester AGF 2 100.00 mg - mannitol 40.00 mg b. one 4 ml ampoule of solvent contains - sodium chloride 32.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 6 a. one vial contains - inner ester AGF 2 100.00 mg b. one 2 ml ampoule of solvent contains - monobasic sodium phosphate 2^0 0.50 mg - dibasic sodium phosphate 12 H2O 6.00 mg - mannitol 80.00 mg - water for injection q.s. System No. 7 a. one vial contains - inner ester AGF 2 100.00 mg b. one 4 ml ampoule of solvent contains - monobasic sodium phosphate 2 -0 1.00 mg - dibasic sodium phosphate 12 H2O 12.00 mg - mannitol 160.00 mg - water for injection q.s. System No. 8 a. one vial contains - methyl ester AGF^ 50.00 mg b. one 2 ml ampoule of solvent contains - monobasic sodium phosphate 2H2O 0.50 mg - dibasic sodium phosphate 12 H2O 6.00 mg - mannitol 80.00 mg - water for injection q.s. System No. 9 a. one vial contains - methyl ester AGF^ 100.00 ml b. one 4 ml ampoule of solvent contains - monobasic sodium phosphate 2H2O 1.00 mg - dibasic sodium phosphate 12 H 2 0 12.00 mg - mannitol 160.00 mg - water for injection q.s. System No. 10 a. one 2 ml vial of freeze-dried powder contains - methyl ester AGF^ 5.00 mg - glycine 30.00 mg b. one 2 ml ampoule of solvent contains - sodium chloride 16.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 11 a. one 5 ml vial of freeze-dried powder contains - methyl ester AGF^ 150.00 mg - glycine 50.00 mg b. one 4 ml ampoule of solvent contains - sodium chloride 32.00 mg - citrate buffer in apyrogenous distilled water q.s. System No. 12 a, one 3 ml vial of freeze-dried powder contains - methyl ester AGF^ 50.00 mg ~ glycine 25.00 mg b. one 3 ml ampoule of solvent contains - sodium chloride 24.00 mg - citrate buffer in apyrogenous distilled water q.s. Excipients: - lactose - microcrystalline cellulose - sodium carboxymethylcellulose - polyvinylpyrrolidone - methacrylic acid copolymer - polyethylene glycol - magnesium stearate - talc CASE 6 One enteric coated tablet contains - inner ester AGF2 Excipients: - hydroxypropyl methylcellulose - lactose - microcrystalline cellulose - methacrylic acid copolymer - polyethylene glycol - magnesium stearate - talc CASE 7 One immediate release tablet contains - inner ester AGF2 Excipients : - lactose - microcrystalline cellulose - sodium carboxymethylcellulose - polyvinylpyrrolidone - magnesium stearate - talc CASE 8 One controlled release tablet contains - inner ester AGF2 Excipients: - hydroxypropyl methylcellulose - lactose - microcrystalline cellulose - magnesium stearate - talc CASE 9 One hard gelatin capsule enclosing enteric granules contains - inner ester AGF2 Excipients: - saccharose - maize starch - polyvinylpyrrolidone - magnesium stearate - methacrylic acid copolymer - polyethylene glycol - talc

Claims: CLAIMS 1 1. Use of monosialoganglioside GM-^ derivatives selected from the 2 group consisting of the inner ester AGF and the methyl ester AGF^ 3 of said monosialoganglioside as active ingredients for the 4 preparation of pharmaceutical compositions suitable for the therapy 5 of spinal cord injury. 1 2. The use according to claim 1 wherein said pharmaceutical 2 compositions contain a therapeutically effective quantity of said 3 active ingredients , mixed with pharmacologically acceptable 4 excipients and diluents . 1 3. Pharmaceutical compositions containing a therapeutically 2 effective quantity of monosialoganglioside GM-^ derivative selected 3 from the group consisting of the inner ester AGF2 and the methyl 4 ester AGF/ j of said monosialoganglioside , mixed with 5 pharmacologically acceptable excipients and diluents, suitable for 6 the therapy of spinal cord injury. 1 4. The compositions according to claim 3 prepared as oral 2 formulations . 1 5 - The compositions according to claim 3 prepared as parenteral 2. formulations. 1 6. Therapeutic method for the treatment of spinal cord injury 2 consisting in the administration of a therapeutically effective 3 quantity of monosialoganglioside GM^ derivative selected from the 4 group consisting of the inner ester AGF2 and the methyl ester AGF/, 5 of said monosialoganglioside. 7- The method according to claim 6 wherein said spinal cord injury is of the acute traumatic type, associated with acute, subchronic and chronic symptomatology. 8. The method according to claim 6 wherein said spinal cord injury is of the acute hypoxic-ischaemic type, associated with acute, subchronic and chronic symptomatology. 9. The method according to claim 6 wherein said spinal cord injury is secondary to surgical operations carried out under aorta obstruction conditions. 10. The method. according to claim 6 wherein said administration is by the oral way. 11. The method according to claim 6 wherein said administration is by the parenteral way.

Inventors: ROMEO AURELIO, TOFFANO GINO, ROMEO, AURELIO, TOFFANO, GINO
Applicants: FIDIA SPA, ROMEO AURELIO, TOFFANO GINO, FIDIA S.P.A.
Application year: 1993
Main classification: A61K31
IPC codes: A61K31