Use of monosialoganglioside GM1 or N-dichloro-acetyl-lyso-GM1 for preventing or reversing neuronal degeneration induced by a long term treatment with L-DOPA in the therapy of Parkinson's disease

Abstract: The present invention refers to the use of ganglioside GM 1 or the derivative thereof, the N-dichloroacetyl lyso GM 1 (LIGA 20) for preventing or reversing neuronal degeneration induced by a long term treatment with L-DOPA in the therapy of Parkinson's disease.

Description: FIELD OF THE INVENTION This invention relates to the use of monosialoganglioside GM 1 or the derivative thereof the N-dichloroacetyl lyso GM 1 (LIGA 20) for preparing a for preventing or reversing neuronal degeneration induced by a long term treatment with L-DOPA in the therapy of Parkinson's disease. TECHNOLOGICAL BACKGROUND Parkinson's disease is a neurodegenerative disease, which has a high rate of incidence (20 persons in 100,000 in the U.S.A.) and generally affects people older than 45-years. Parkinson's disease is characterized by a dopaminergic deficit, which brings about a series of neuronal events resulting in akinesia, muscle rigidity, and tremors. Symptomatology is believed to manifest itself with the loss of at least 85% of the striatal dopaminergic innervation, consequent to the degeneration of the neurons producing DA (dopamine) in the substantia nigra and pars compacta (Kish S.J. et al., The New England J. of Med:, 318, 876, 1988). It is to be stressed that although pharmacological therapy for Parkinson's disease has been studied for over 25 years, the disease is still a critical problem especially due to the slow and progressive degeneration of the dopaminergic system (McGeer P.L. et al., Ann. Neurol., 24, 574, 1988). As known, the therapy based on L-DOPA, associated with peripheral decarboxylase inhibitors (carbidopa or benserazide), with monoamine oxidase inhibitors (Shoulson I. et al.: "Effect of Deprenyl on the progression of disability in early Parkinson's disease", The New England J. of Med., 16, 1364-1371, 1989), as well as the therapy based on long-acting direct dopaminergic agonists (pergolide, cabergoline), in the great majority of early-stage cases considerably improves the clinical picture and, in some cases, provides a total control of symptoms. However, after some years of treatment, i.e. from 2-3 years min. to generally 10 years max. or even more, symptoms - mainly characterized clinically by fluctuation and dyskinesias of various types - appear anew in most patients (80-90%). Said motor fluctuations (in particular on-off phenomena) and hyperkinesias deeply upset the patient who, after years of well-being resulting from the disease being compensated by the therapy, relapses into a decompensated condition that prevents him/her from enjoying an adequate family, social, and work life. With a view to solving the main problem to be faced by Parkinson's disease therapy, i.e. the decompensated phase, present clinical practice uses slow-release compositions based on levodopa associated with benserazide or carbidopa, long-acting direct dopaminergic agonists (pergolide, cabergoline), as well as infusion methods (lisuride and apomorphine subcutaneous infusion). However, no therapy has so far proved to be effective in slowing down or stopping the progression of the disorder, which is at the base of all complications occurring in the advanced phase of the disease. As known, L-DOPA and its hydroxylated metabolite (TOPA) may produce neurotoxic effects and worsen the disabling neurodegenerative pathology progression (Only J.W. et al.: "Excitotoxicity of L-DOPA and 6-OH-dopa: implications for Parkinson's and Huntington's diseases", Exp. Neurol., 108, 268-272, 1990; Rosenberg P.A. et al.: "2,4,5-Trihydroxyphenylalanine in solution forms a non-N-methyl-D-aspartate glutamatergic agonist and neurotoxin", Proc. Natl. Acad. Sci. USA, 88, 4865-4869, 1991; Newcomer T.A. et al.: "Detection of TOPA (6-OH-DOPA) and TOPA quinone by HPLC reveals a spontaneous DOPA to TOPA conversion in aqueous solutions". Excitatory Amino Acids: Excitotoxicity I p. 83). With a view to developing new pharmacological treatments capable of modifying the evolution of Parkinson's disease, by slowing down or inhibiting the progression of the same, several experimental investigations on animals were carried out, especially aimed at identifying the neurobiological mechanisms that in parkinsonism cause cell death, in particular the death of substantia nigral cells. Of great importance is the information obtained by using methylphenyltetrahydropyridine (MPTP), a toxic substance capable of producing a neuropathologic and neuropharmacological picture very similar to that of Parkinson's disease (Langston J.W.: "MPTP and Parkinson's disease", Trends in Neurosciences, 8, 2, 79-83, 1985). MPTP neurotoxicity was attributed to its oxidation, catalysed by monamine oxidase B, to the ionic species MPP + , which is actively taken up by dopaminergic cell terminals and has a inhibitory effect on the mitochondrial oxidation of NADH-dependent substrates. This results in a loss of the substantia nigra and pars compacta dopaminergic neurons as well as of the striatum-innervating dopaminergic fibres, with consequent biochemical and behavioural deficiencies. It is also known that gangliosides, i.e. the complex sialoglycosphingolipids that are present in neuronal membranes (Ando S.: "Gangliosides in the nervous system", Neuroch. Int., 5, 507-537, 1983) improve the neurologic course in the CNS of several experimental models of acute damage. On the basis of said results, GM 1 was clinically applied to treat cerebral ischemic stroke (U.S. 4,940,694 dated 10th July, 1990; Argentino C. et al.: "GM 1 ganglioside therapy in acute ischemic stroke", Stroke, 20, 1143-1149, 1989) and traumatic spinal cord injury (patent application PD 91 000234 dated 23rd December, 1991; Geisler F.H.: "Recovery of motor function after spinal-cord injury - a randomized placebo-controlled trial with GM 1 ganglioside", The New England J. of Med., 324, 1829-1838, 1991). The inner ester derivative of GM 1 (AGF2) and the low-dose and fastacting therapeutic efficacy of same, especially in acute ischemia models, is 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). Furthermore, the pharmacokinetic advantages offered by GM 1 ester derivatives over their precursor, GM 1 , have already been described (Bellato P. et al.: "Disposition of exogenous tritium labelled GM 1 lactone in the rat", Neurochem., pp. 1-6, 1991; EP patent 85401291.1). SUMMARY OF THE INVENTION It has surprisingly been found that the compounds monosialoganglioside (GM 1 ), and N-dichloroacetyl lyso GM 1 can be successfully applied to chronic Parkinson's disease treatment for preventing or reversing the neuronal degeneration induced by long term L-DOPA treatment in the Parkinson's disease therapy. DESCRIPTION OF THE DRAWINGS Figure 1 represents the dose dependent protective effect of LIGA20 against TOPA induced death of cultured cerebellar granule cells. DETAILED DESCRIPTION OF THE INVENTION In fact said compounds produce a neutralizing effect on the neurotoxicity of L-DOPA metabolites such as TOPA. It was also found that said compounds, may be profitably associated with other pharmaceuticals used for the same purpose, such as L-DOPA and BDNF (brain-derived neurotrophic factor). In particular, the experiments reported below showed the protective action of GM 1 , and LIGA 20 on TOPA neurotoxic effect on neuronal cultures. As known, the therapy based on L-DOPA, the natural precursor of dopamine, though providing some beneficial effects on parkinsonian symptomatology, produces negative effects in the long run; actually L-DOPA and its metabolites, e.g. 6-hydroxy-dopa (TOPA) can be toxic also to neurons, thus worsening the disabling neurodegenerative progression of pathology. The tests carried out by the Applicant revealed that gangliosides are effective in preventing and/or inhibiting the neurotoxicity caused by the L-DOPA hydroxylated metabolite. Therefore, the said results are indicative of the great advantage brought about by the application of said products to Parkinson's disease treatment. Patients may thus be treated with a ganglioside + L-DOPA associated therapy offering the advantage of preventing and/or inhibiting the neuronal degeneration observed in parkinsonism and especially in patients treated with dopaminergic pharmaceuticals, such as L-DOPA. Tests were carried out on two neuronal cultures, i.e. of mesencephalic dopaminergic neurons and of cerebellar neurons. Mesencephalic neuron cultures were prepared according to Dal Toso et al. (Dal Toso et al.: "Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I. Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF)", J. Neurosci., 8 (3) 733-745, 1988) from the mesencephalic tegmentum of 14.5-15 days' rat embryos. Neurons (0.75 x 10 6 ) were plated on a poly-L-ornithine substrate in 12-well (25 mm dia. each) clusters. Cultures were used between the fourth and the sixth day without changing the medium. Granular cell cultures were obtained from 8-day rat cerebella and cultured (3 x 10 6 cells) on plates coated with poly-L-lysine substrate according to Skaper et al. (Skaper S.D. et al.: "Culture and use of primary and clonal neural cells", Methods in Neurosciences, Ed. by P.M. Conn, Vol. 2, pp. 17-33, Academic Press, Orlando, 1990). Twenty-four hours later, cytosine arabinoside (10 µM) was added to inhibit non-neuronal proliferation. Cerebellar granule cells were used 10-12 days later without changing the medium. Cells were incubated with TOPA either for 40 minutes (100 µM acute exposure) or for 24 h (10 µM prolonged exposure). Treatment with GM 1 , (100 µM) and LIGA 20 (1-30 µm) was carried out as follows: 2-hour pretreatment before acute incubation with TOPA; cotreatment only with LIGA 20 before acute incubation with TOPA; pretreatment with/without cotreatment before (100 µM for 2 h) and/or during (100 µM for 24 h) prolonged incubation with TOPA. TOPA cytotoxicity was determined by evaluating cellular survival, and more precisely: TH immunofluorescence (specific for tyrosine hydroxylase) in mesencephalic dopaminergic neurons, as described by Berger et al. (Berger B. et al.: "Long-term development of mesencephalic dopaminergic neurons of mouse embryos in dissociated primary cultures: morphological and histochemical characteristics", Neurosci., 7, 193-205. 1982); MTT colorimetric assay of cerebellar neurons, as described by Mosmann (Mosmann T.: "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays", J. Immunol. Meth., 65, 55-63, 1983). Cytotoxicity is expressed as per cent cellular survival. The results of the two tests (in triplicate) were analysed statistically according to Duncan's test. A brief description of same is reported below: GM 1 (100 µM x 24 h) produces a neuroprotective effect on mesencephalic dopaminergic neurons exposed to TOPA toxic levels (10 µM x 24 h coincubation). TH immunopositive neurons remain intact when coincubated with TOPA and GM 1 (Table 2); GM 1 exerts a neuroprotective action on cerebellar neurons either after acute or after prolonged exposure to TOPA (100 µM x 40 minutes or 10 µM x 24 h). In particular, after prolonged incubation, GM 1 is even more effective if cells are subjected to an associated treatment, i.e. pre- and co-treatment (100 µM GM 1 x 2 h in the absence of TOPA + 100 µM GM 1 x 24 h in the presence of 10 µM TOPA) (Table 3). In addition LIGA20 is dose-dependently effective in cotreatment in protecting cerebellar granule cells against the acute toxicity of TOPA (ED 50 ≈ 9 µm). Under these conditions of cotreatment GM 1 , up to 200 µM, is not active (Fig. 3). In Fig. 1 the dose-dependent protective effect of LIGA20 against TOPA-induced death of cultured cerebellar granule cells is represented. Cells were exposed concurrently to 100 µM TOPA and 1-30 µM LIGA20 or 200 µM GM 1 in Locke's solution for 45 minutes (24°C), washed and returned to their original medium. Survival was assessed 24 h later by the MTT method. Means ± S.E.M. (n=9, 3 experiments). TOPA (▭), LIGA 20 (), GM 1 (○). LIGA20 (30 µM) cotreatment produces a neuroprotective effect on mesencephalic dopaminergic neurons exposed to acute toxic levels of TOPA (100 µM x 45 minutes), while GM 1 (200 µM) in only cotreatment is not active (Table 1). Protective effects of LIGA20 and GM 1 against TOPA-induced loss of dopaminergic neurons in cultures of mesencephalic cells Cultures at 5 DIV were incubated with 30 µM LIGA20 or 200 µM GM 1 in Locke's solution containing 100µM TOPA for 45 minutes (22°C), throroughly washed and returned to their original medium. In some cases cells were treated with 200 µM GM 1 for 2 h (37°C) in Locke's solution, washed, and exposed to TOPA as above. All cultures were processed for TH immunocytochemistry 24 h after termination of the exposure to TOPA, and the TH + cells were counted. Means ± S.E.M. (n=8,4 experiments). Control (100%) = sham-washed sister cultures without TOPA. Drug treatment TH + cells (% control) None 5.2 ± 1.1 LIGA20 83.9 ± 4.2 GM 1 5.6 ± 0.7 GM 1 (pretreatment) 79.1 ± 4.0 Effect of GM 1 on the neurotoxicity caused by TOPA to mesencephalic dopaminergic neurons Treatment No. of TH + cells Control 244 t 20 TOPA (10 µM) 62 ± 8 TOPA (10 µM) + GM 1 (100 µM) 187 ± 10 Mesencephalic serum-free cells were cultivated 5 days, then incubated with GM 1 , (100 µM), and TOPA (10 µM) for 24 h. After fixation and immunostaining with tyrosine-hydroxylase antibodies, TH + neurons were counted. The values are an average (± S.D.) of three determinations. Effect of GM 1 on the neurotoxicity caused by TOPA to cerebellar granule cells Culture Treatment Neuronal survival (%) 40-minute exposure: TOPA (100 µM) 22 ± 2.0 TOPA (100 µM) + GM 1 72.4 ± 5.8 (a) 24-h exposure: TOPA (10 µM) 17.1 ± 1.4 TOPA + GM 1 (pretreatment) 57.0 ± 6.5 (a) TOPA + GM 1 (pre- and co-treatment) 75.4 ± 5.5 (b) In vitro granule cells at the 11th day were incubated as indicated above. In the 40-minute exposure to TOPA, GM 1 (100 µM) was used in 2-h pretreatment only. In the 24-hrs exposure to TOPA, GM 1 (100 µM) was used either in 2-h pretreatment only or in 2-h pretreatment and in cotreatment with TOPA. The values are expressed with reference to the corresponding control cultures (= 100%) and are an average (± S.D.) of two determinations, each being carried out in triplicate. (a) = p < 0.01 vs TOPA alone (b) = p < 0.05 vs GM 1 pretreatment group. The study on mesencephalic dopaminergic neurons was performed with associations of GM 1 , with BDNF (brain-derived neurotrophic factor) in the presence or absence of L-DOPA. Associations of GM 1 with BDNF were found to have a synergic effect in protecting mesencephalic dopaminergic neurons against TOPA-induced neurotoxicity. The effect is considerable even at extremely low concentrations of GM 1 , (1-10 µM) with BDNF (1 ng/ml) that is, at concentrations with no neuroprotective activity of their own. In one particular experiment, in which the mesencephalic dopaminergic neurons were placed together with 1 ng/ml of BDNF and 10 µM of GM 1 , the number of neurons which survived the TOPA-induced toxic insult was not significantly different from that obtained using a saturation concentration (50 ng/ml) of BDNF alone. From the results obtained the conclusion was come to that GM 1 and its derivative LIGA 20 protect neuronal cells against the toxicity caused by TOPA: said effect is clearly apparent not only on mesencephalic neurons (dopaminergic and not), but also on other neuronal populations, such as e.g. cerebellar neurons. Said results are predictive of a new therapeutic application of GM 1 and its derivative LIGA 20 in parkinsonian patients treated with the traditional dopaminergic pharmaceuticals, such as L-DOPA. In particular, the GM 1 derivative LIGA20 is more potent and faster-acting than GM 1 and is effective when used only in cotreatment, suggesting a possible oral use of LIGA20. The new ganglioside + L-DOPA and/or BDNF associated therapy offers the great advantage of preventing and/or stopping the neuronal degeneration which is induced also by the neurotoxic effects produced by a long-term L-DOPA treatment. Therefore these compounds may be utilized in an associated therapy with pharmaceuticals or pharmaceutical combinations to assuage the parkinsonian symptomatology and recover dopaminergic functionality. Particularly convenient is the use of said compounds in association with L-DOPA also in association with BNDF or other decarboxylase or monamine oxidase inhibitors. GM 1 and LIGA 20 may be administered to man orally or parenterally, i.e. by intramuscolar or intravenous or subcutaneous injection in the form of pharmaceutical compositions suitable for parenteral or oral administration containing them as the active ingredient in amounts ranging from 10 to 200 mg per single dose. The active ingredient dose 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 30 mg/kg/day; by os, it may range from 0.5 to 150 mg/kg/day. Some examples of pharmaceutical compositions containing the above mentioned active ingredient are given hereinbelow only for illustrative purposes. CASE 1 One 2 ml vial contains monosialoganglioside (GM 1 ) sodium salt 20.00 mg dibasic sodium phosphate 12 H 2 O 6.00 mg monobasic sodium phosphate 2 H 2 O 0.50 mg sodium chloride 16.00 mg water for injection q.s. CASE 2 One 2 ml vial contains monosialoganglioside (GM 1 ) sodium salt 40.00 mg dibasic sodium phosphate 12 H 2 O 6.00 mg monobasic sodium phosphate 2 H 2 O 0.50 mg sodium chloride 16.00 mg water for injection q.s. CASE 3 One 5 ml vial contains monosialoganglioside (GM 1 ) sodium salt 100.00 mg dibasic sodium phosphate 12 H 2 O 15.00 mg monobasic sodium phosphate 2 H 2 O 1.25 mg sodium chloride 40.00 mg water for injection q.s. CASE 4 One 2 ml vial contains N-dichloroacetyl lyso GM 1 5.00 mg sodium chloride 16.00 mg citrate buffer pH 6 in distilled water to a vol. of 2.00 ml

Claims: Claims for the following Contracting States : AT, BE, CH, LI, DE, DK, FR, GB, IE, IT, LU, MC, NL, PT, SE Use of monosialoganglioside GM 1 or of the derivative thereof N-dichloro acetyl lyso GM 1 as the active ingredient for the preparation of pharmaceutical composition for preventing or reversing the neuronal degeneration which is induced by the neurotoxic effect produced by a long term L-DOPA treatment in Parkinson's disease therapy . The use according to claim 1 wherein said pharmaceutical composition is suitable to be orally or parenterally administered and contains from 10 to 200 mg of said active ingredient. The use according to any one of claims 1 and 2 associated with pharmaceuticals or pharmaceutical combinations for the treatment of parkinsonian symptomatology and for the dopaminergic treatment. The use according to claim 3 wherein said pharmaceuticals are selected from the group consisting of L-DOPA, BDNF and combination thereof with mono amino oxydase inhibitors. Claims for the following Contracting State : ES A process for preparing a pharmaceutical composition for preventing or reversing the neuronal degeneration induced by the neurotoxic effect produced by a long term L-DOPA treatment in Parkinson's disease therapy comprising mixing as the active ingredient monosialoganglioside GM1 or the derivative thereof N-dichloro acetyl lyso GM1 with suitable pharmaceutically acceptable excipients and/or diluents. The process according to claim 1 wherein GM1 is admixed with excipients and/or diluents suitable for the oral or parenteral administration in order to obtain a unitary oral or parenteral pharmaceutical composition containing said active ingredient in amounts ranging from 10 to 200 mg. The process according to anyone of claims 1 and 2 wherein said pharmaceutical composition is associated with pharmaceuticals or pharmaceutical combinations for the treatment of parkinsonian symptomatology and for the dopaminergic treatment. The process according to claim 3 wherein said pharmaceuticals are selected from the group consisting of L-DOPA, BDNF and combination thereof with monoamino oxidase inhibitors.

Inventors: ROMEO AURELIO, SKAPER STEPHEN DRAKE, TOFFANO GINO, ROMEO, AURELIO, SKAPER, STEPHEN DRAKE, TOFFANO, GINO
Applicants: FIDIA FARMACEUTICI, FIDIA FARMACEUTICI S.P.A.
Application year: 1993
Main classification: A61K31
IPC codes: A61K38, A61K31