WO1994009807A1

WO1994009807A1 - Pharmaceutical preparations containing neurotrophic factors and gangliosides for treating nervous system pathologies

Info

Publication number: WO1994009807A1
Application number: PCT/EP1993/002923
Authority: WO
Inventors: Stephen Drake Skaper; Gino Toffano
Original assignee: Fidia S.P.A
Priority date: 1992-10-23
Filing date: 1993-10-22
Publication date: 1994-05-11
Also published as: IT1259176B; ITPD920189A1; ITPD920189A0; AU5336394A

Abstract

Provided is a pharmaceutical composition comprising at least one neurotrophic factor such as ciliary neurotrophic factor at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt thereof such as GM1 or AGF2 at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity. Such compositions can be prepared in the form of delivery vehicles such as microspheres, microcapsules, sponges, tubes, and guide channels comprising hyaluronic acid esters. These pharmaceutical compositions can be used in human and veterinary medicine to treat excitatory amino acid-induced neurotoxicity.

Description

PHARMACEUTICAL PREPARATIONS CONTAINING NEUROTROPHIC FACTORS AND GANGLIOSIDES FOR TREATING NERVOUS SYSTEM PATHOLOGIES

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to pharmaceutical preparations containing neurotrophic factors such as ciliary neurotrophic factor (CNTF) and gangliosides such as monosialoganglioside G 1 or its derivatives (e.g., the internal ester AGF2, "siagoside") , for the treatment of various pathologies affecting the nervous system.

In particular, this invention relates to the interactive effect of GM1 ganglioside (or its inner ester form, AGF2) with the ciliary neurotrophic factor (CNTF) in promoting a neuroprotective action against excitatory amino acid (EAA) -induced neurotoxicity. The interaction of gangliosides with neurotrophic factors has been observed in the presence of very low concentrations of each, while each compound per se at these low concentrations exhibits no neuroprotective activity. Description of Related Art

A. Ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a multifunctional protein with various biological activities.

It has been demonstrated that CNTF, originally identified by its ability to support the survival of chicken embryonic parasympathetic ciliary ganglion neurons (Barbin et al. J. Neurochem. 43: 1468-1478, 1984) , is also able to promote the survival of both sensory and sympathetic neurons (Heymanns et al . PNAS 84: 7758-7762; Lin et al . J. Biol . Chem. 265: 8942-8947, 1990) , as well as the differentiation of sympathetic neuroblastomas and glial cells (Saadat et al . J. Cell Biol. 108, 1807-1816, 1989; Lillien et al . Neuron 1: 485-494, 1988) .

Recent evidence demonstrates that CNTF promotes spinal motor neuron survival in vitro (Arakawa et al. J. Neurosci. 3507-3515, 1990) and is able to rescue motor neurons from naturally occurring cell death in developing chicken embryos (Oppenheim et al. Science 251, 1616-1618, 1991) . It has also been demonstrated that local administration of CNTF _in vivo to the proximal stump of a lesioned facial nerve prevents the complete loss of axotomized motor neurons in the postnatal rat (Sendtner et al. Nature 345:440-441,

1990) , and the degeneration of axotomized neurons in the adult rat brain (Hagg et al . Neuron 8, 145-158,, 1992) .

Lastly, the trophic effects of CNTF have also been demonstrated in the hippocampal neurons. In particular, it has been observed that CNTF promotes the survival of specific neuronal subpopulations, e.g., cholinergic and GABAergic neurons, and of specific neurons with receptors for the NGF (Ip et al . J. Neurosci. 11: 3124- 3134, 1991; Magal et al . J. Neurosci. Res. 30:560-566, 1991) . The results obtained so far show that CNTF has a remarkably interesting pharmacological profile. This trophic factor represents an especially promising candidate for the prevention and/or functional recovery of an injured nervous system.

The role of neurotrophic factors in repair of the nervous system has been reviewed by Sabel et al, In Vecsei et al, eds. , Neurological Disorders, Novel Experimental and Therapeutic Strategies , Ellis Horwood, New York, pp. 113-180 (1992) .

B. Gangliosides

Gangliosides, complex glycosphingolipids which functionally incorporate into neuronal membranes (Ando Neurochem. Int. 5, 507-537, 1983) , exhibit reparative effects in different models of CNS lesions (Skaper et al. Mol . Neurobiol . 3, 173-199, 1989; Carolei et al . Cerebrovasc. Brain Metab. Rev. 3: 134-157, 1991; Schneider et al. Science 256, 843-846, 1992) .

Based on these results, which were interpreted as being due to a coupling mechanism (neurotrophic and antineurotoxic effect) , gangliosides are not only able to limit long-term degenerative phenomena, but also to limit and/or prevent EAA-induced neurotoxicity. The clinical efficacy of GM1 has been further confirmed both in cerebral ischemic damage (Argentino et al . : "GM-, ganglioside therapy in acute ischemic stroke", Stroke 1989: 20: 1143-1149) and spinal cord injury (Geisler et al . : "Recovery of motor function after spinal-cord injury - a randomized placebo-controlled trial with GM, ganglioside", N. Engl . J. Med. 324: 1829-1838, 1991) .

The therapeutic efficacy of the GM1 inner ester form (AGF2) has been described following acute brain ischemia, at low doses and upon early administration (Cahn et al. : "Influence of monosialoganglioside inner ester on neurologic recovery after global cerebral ischemia in monkeys", Stroke 20, 652-6"56, 1989) . Lastly, it is known that gangliosides, particularly GMl, are able to enhance the trophic effects of the Nerve Growth Factor (NGF) both in vitro (Ferrari et al . Dev. Brain Res. 8: 215-221, 1983; Doherty et al . ___ Neurochem. 44, 1259-1265, 1985) and in vivo (Cuello A. C. et al. PNAS 86: 2056-2060, 1989; Vantini et al. Brain Res. 448, 252-258, 1988) . A similar effect has been shown also for CNTF in .in vitro neuronal cultures of chicken embryonic ciliary ganglia and dorsal root ganglia (Skaper et al . Dev. Brain Res. 29, 19-26; 1985) .

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a pharmaceutical composition, comprising at least one neurotrophic factor at a sub- threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt of any of the foregoing at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity.

Another object of the present invention is to provide a method for treating excitatory amino acid- induced neurotoxicity in a human or animal subject, comprising administering to said subject an anti- excitatory amino acid-induced neurotoxicity amount of said pharmaceutical composition.

Yet another object of the present invention is to provide a method of enhancing the neuroprotective effect of a neurotrophic factor against excitatory amino acid- induced neurotoxicity, comprising administering to a human or animal subject in need thereof a pharmaceutical composition comprising at least one neurotrophic factor at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside derivative,

SUBSTITUTE SHEET semisynthetic ganglioside analogue, or salt of any of the foregoing at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity. Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood^"" that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the present invention will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawings, all of which are given by way of illustration only, and are not limitative of the present invention, in which:

Figure 1 shows the morphological evaluation of the effect of rhCNTF with and without GMl against glutamate- induced excitotoxic damage in pyramidal neuronal cultures: (a,b) , with CNTF and GMl; (c) , with 30 ng/ml of rhCNTF; (d) , with 2 ng/ml of rhCNTF, (e) with 10 μM GMl; (f) , with 2 ng/ml rhCNTF + 10 μM GMl.

Figure 2 shows the quantitative evaluation of the effect of rhCNTF with and without GMl against glutamate- induced excitotoxic damage in pyramidal neuronal cultures. Values are means ± S.E. (n= 8, 2 experiments) . * p ( 0.02 vs glutamate alone.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to a d those skilled in the art in practicing the present invention. Even so, the following detailed description of the invention should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

The contents of each of the references cited herein are herein incorporated by reference in their entirety.

EXAMPLE 1 Described below are the characteristics and benefits of the pharmacological association of gangliosides and CNTF, exemplified by in vitro experiments performed on rat embryonic hippocampal neurons with EAA-mediated excitotoxic damage. The predictive utility of the model test system employed in these experiments is described in Skaper et al . J. Neurosci. Res. 33:330-337 (1992) .

These studies provide strong evidence for an interactive role of gangliosides with CNTF in protecting against glutamate-mediated neuronal damage: 10 μM of GMl or AGF2, in the presence of 2 ng/ml of CNTF, enhance the neuroprotective effect (80%) as compared to CNTF alone administered at higher doses (30 ng/ml) .

The experiments were performed as follows.

MATERIALS AND METHODS

Cell cultures

Fetal cell cultures of rat hippocampus (Sprague Dawley) from an 18-day-old embryo were prepared according to Skaper et al . (In Conn, PM, ed. , Methods in Neurosciences, Vol. 2, pp. 17-33, Academic Press, 1990) .

Cells were mechanically removed from the tissue, then triturated and subsequently trypsin-digested (0.08% at 37°C, 30') into Ca²⁺, Mg²⁺-free Hank's balanced salt solution (HBSS) . Dissociated cells were counted and seeded on multiwell (24-well) plates (Falcon) coated with polyornithine (10 μg/ml) . The desired medium, generally

0.5 ml culture medium containing 200 cells/mm², was then gently added to each 24-mm well.

The culture medium was Dulbecco's modified Eagle's medium (Flow, McLean, VA) , containing 2 mM L-glutamine + 5% glucose + 25 mM KC1 + 100 U/ml penicillin and hormone-supplemented (Clos et al . Neurochem. Res. 14, 919-925, 1989) .

Cultures were kept at 37°C in a humidified 5% C0₂~

95% air atmosphere. They contained 95% neuronal cells and 5% glial cells, according to previous characterizations (Mattson et al . J. Neurosci. 9, 3728- 3740, 1989) .

Tested compounds

The following compounds were tested:

-- rhCNTF (synthesized and purified according to the method described by Negro et al. in J__^ Neurosci . Res . 29, 251-260, 1991) ;

-- monosialoganglioside GMl, isolated from bovine brain and commercialized under the name of Sygen ; -- monosialoganglioside GMl inner ester, prepared as described in US patent 4,476,119.

The trophic factor and/or ganglioside were added to 0-5 day-old embryonic cultures _.in vitro (2nd division) . At the 5th division, medium was removed and the cultures washed twice with fresh medium and exposed to 0.5 ml of medium lacking the factor and/or the ganglioside, and supplemented with 200 μM glutamate.

After 24 hrs incubation, the cultures were fixed with 2% glutaraldehyde and examined under the phase contrast microscope. The viable pyramidal neurons were counted in at least 6 fields, in 3 different wells. The neuronal vitality was determined according to methods described in the literature (Cheng et al . Neuron 7, 1031-1041, 1991) .

RESULTS

The results are shown in Fig. 1 and Fig. 2. It can be seen that:

-CNTF (30 ng/ml) is able to. protect hippocampal cells against glutamate-induced neurotoxicity. A lower concentration of CNTF (2 ng/ml) has no neuroprotective effect; -GMl or its inner ester form AGF2 , when tested at the concentration of 10 μM, has no neuroprotective effect against glutamate-induced neurotoxicity on hippocampal cell cultures; -the association of gangliosides (GMl or AGF2, 10 μM) with CNTF (2 ng/ml) is able to markedly protect hippocampal cells . The neuroprotective effect is comparable to that obtained with 15-times higher concentrations (30 ng/ml) of CNTF alone.

Interestingly, this interactive effect of gangliosides with the neurotrophic factor has been observed under specific experimental conditions, i.e., in the presence of very low concentrations of the two agents^', each of which per se had no neuroprotective effect. These findings are of crucial importance, as the ability of these compounds to antagonize the excitotoxic effects induced by excitatory amino acids can be very useful for the prevention and/or functional recovery in different pathophysiological conditions mediated by the action of excitatory amino acids. It is well known that EAAs play a key role in a variety of disorders affecting the nervous system, such as cerebral ischemia, hypoxia, epilepsy, trauma and compressions, dysmetabolism, aging, and neurodegenerative diseases of both chronic and toxic origin, e.g., Alzheimer's disease, Parkinson's disease, and Huntington's disease (J. W. Olney: "Excitotoxic amino acids and neuropsychiatric disorders", Ann. Rev. Pharmacol . Toxicol . 30, 47, 71, 1990; Taylor R. : "A lot of excitement about neurodegeneration" , Science, 252, 1380-1381, 1991; Beal et al . : "Does impairment of energy metabolism result in excitotoxic neuronal illnesses?", Ann. Neurol. 31, 119-130, 1992; Choi et al. "The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death", Ann. Rev. Neurosci. 13, 171-182, 1990; Faden et al. "The role of excitotoxic amino acids and NMDA receptor in traumatic brain injury", Science 244, 798- 800, 1989; Meldrum et al . "Excitatory amino acid neurotoxicity and neurodegenerative disease", Trends Pharm. Sci. 11, 379-387, 1990) .

EXAMPLE 2

PHARMACEUTICAL PREPARATIONS

The present invention relates to the therapeutic use of neurotrophic factors such as the ciliary neurotrophic factor (CNTF) and their administration via pharmaceutical preparations containing these factors in association with other pharmacologically active substances. The ciliary neurotrophic factor (CNTF) is useful in the maintenance and prevention of nervous function loss and in the treatment of this loss in both acute and chronic pathological conditions (cerebrovascular, infectious, inflammatory, compressive and metabolic deficiencies) , and in chronic or neuro¬ degenerative conditions, or in the treatment of age- related neuropathological conditions. The pharmaceutical preparations described below should prove useful in the treatment of various neurological disorders, including peripheral neuropathies, stroke, and spinal cord injury.

The pharmaceutical preparations may contain, as active ingredients, one or more associations of neurotrophic factors such as CNTF, NGF, or BDNF with a natural ganglioside (or one of its derivatives or semisynthetic analogues or salts) , or associations of these and other trophic factors and gangliosides with polysaccharides (either natural, chemically modified or manufactured as biomaterials) .

Monosialoganglioside GMl and its sodium salt can be prepared as described in U.S. Patent 4,940,694 and PCT applications Q 91/07417 and WO 92/20695; monosialoganglioside GMl inner ester (AGF2, "siagoside") can be prepared as described in U.S. Patent 4,476,119 and EP 0 072 722. Ganglioside derivatives AGF1 (inner ester ganglioside mixture) , AGF4, GMl ethyl ester, and GMl methyl ester can be prepared as described in EP 0 072 722 and EP 0 167 449. The semisynthetic ganglioside analogues Liga 4, Liga 20, and Liga 66 can be prepared as described in EP 0 410 881, EP 0 373 039, and EP 0 410 883, respectively.

Polysaccharides useful in the present compositions include hyaluronic acid esters. These can be prepared as described in U.S. Patent 4,851,521, and can be employed to produce, for example, microspheres or microcapsules to vehicle the present pharmaceutical preparations of neurotrophic factors + gangliosides . Other types of biomedical delivery devices useful in the present invention include sponges, tubes, and guide channels. Use of the total and partial ethyl and benzyl esters of hyaluronic acid to prepare such devices is especially desirable.

The pharmaceutical preparations of the present invention can be administered by different routes: oral, topical, rectal, parenteral, local, inhalatory or intracerebral. They can therefore be in solid or semisolid form, e.g., pills, tablets, creams, gelatin capsules, suppositories, soft gelatin capsules, microspheres, microcapsules, gels, sponges, tubes, or guide channels. For parenteral and intracerebral administration, the forms adequate for intramuscular or subcutaneous administration, or for infusion, intravenous or intracerebral injection can be considered. These can be prepared as solutions or lyophilized forms of the active ingredients, mixed together with one or more excipients or solvents, suitable for these uses and osmolarity- compatible with the physiological fluids.

For local administration, preparations in the form of creams or unguents for topical use can be employed; for inhalatory administration, preparations in the form of sprays, e.g., nasal sprays, can be employed.

The preparations of the present invention can be administered both to humans and animals. They contain preferably 0.01% to 10% of the active ingredient in the case of solutions, sprays, unguents and creams, and 1% to 100%, preferably 5% to 50%, of the active ingredient in the case of solid preparations. Dosage depends on individual requirements, on the desired effect, and the route of administration, but the daily dose to be administered in humans by subcutaneous, intramuscular or intracerebral routes is generally between 0.05 μg and 5 μg of the active ingredient per kg of body weight. For parenteral, intradermic, and subcutaneous administration, the ratio of components can be about 0.5 to about 50 mg (about 0.3 μM to about 6 μM) /kg of ganglioside such as GMl or ganglioside derivative, etc., plus about 0.1 μg to about 5 μg/kg of neurotrophic factor such as CNTF. For parenteral and subcutaneous administration, the dose can be about 10 mg/kg of GMl or ganglioside derivative, etc., plus about 1 μg/kg of neurotrophic factor; for intracerebral administration, the dose can be about 4 mg/kg of GMl or ganglioside derivative, etc., plus about 0.4 μg/kg of neurotrophic factor; for oral administration, the dose can be about 20 mg/kg of ganglioside derivative, plus about 2 μg/kg of neurotrophic factor; for intradermic administration, the dose can be about 15 mg/kg of GMl or ganglioside derivative, etc., plus about 1.5 μg/kg of neurotrophic factor; for topical administration, the dose can be about 30 mg/kg of ganglioside derivative, plus about 3 μg/kg of neurotrophic factor; and for rectal administration, the dose can be about 15 mg/kg of GMl or ganglioside derivative, etc., plus about 1.5 μg/kg of neurotrophic factor. In addition, the present invention relates to the therapeutic use of pharmaceutical preparations containing gangliosides and their derivatives in association with neurotrophic factors and polysaccharides such as hyaluronic acid esters as a biomedical device for the treatment of the various pathologies described above.

The formulation of the pharmaceutical preparations containing solutions of neurotrophic factor(s) along with gangliosides (or with hyaluronic acid and related substances) or one of their salts, includes known methods for preparations acceptable from a pharmaceutical point of view, which can be administered to patients, and which allow a suitable quantity of the neurotrophic factor to be combined with a pharmaceutically acceptable vehicle.

Suitable vehicles and their formulation containing other proteins are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA, 1985) . Such vehicles include injectable "depots".

Based on these observations, the pharmaceutical preparation includes, albeit not exclusively, solutions of a neurotrophic factor such as CNTF or its lyophilized powder in association with one or more vehicles or pharmaceutically acceptable solvents, and contained in buffer solutions having a suitable pH, and osmolarity- compatible with the physiological fluids. In the case of lyophilized preparations, supporting excipients such as mannitol or glycine among others, and suitable buffer solutions of the desired volume, will be supplied, in order to obtain adequate isotonic buffer solutions with the desired pH. Similar solutions can be used for the pharmaceutical preparation of neurotrophic factors obtained via recombinant> DNA techniques in isotonic solutions of the desired volume, and these include, albeit not exclusively, the use of physiological buffer solutions with phosphate or citrate at suitable concentrations, so as to obtain each time isotonic pharmaceutical preparations with the desired pH, e.g., neutral pH.

The pharmaceutical preparations also include, without being limited to the same, suppositories for rectal administration with lipophilic excipients, e.g., water-soluble, self-emulsifying excipients such as glycogelatin and others. In such preparations, the CNTF or other neurotrophic factor obtained via recombinant DNA techniques can be present in quantities varying between 0.01% and 1% by weight of the total excipient. The suppositories may contain, among other things, suitable quantities of acetylsalicylate. The pharmaceutical formulations also include microspheres, and hanocapsules for nasal, inhalatory, and intramuscular administration. Such formulations also include particular forms, according to their uses, such as sponges, tubes, and guide channels.

For purely descriptive and not limitative purposes, described below are examples of pharmaceutical preparations produced according to the present invention. EXAMPLES OF PHARMACEUTICAL PREPARATIONS

Preparation n° 1- One 2-ml ampoule contains: active ingredient 1 μg (5,000 BU) sodium chloride 16 mg monosialotetrahexosylganglioside

GMl sodium salt 7.5 mg citrate buffer pH = 7 in water for injection, q.s. to 2 ml

Preparation n° 2- One 2-ml ampoule contains: active ingredient 1 μg (5,000 BU) sodium chloride 16 mg monosialotetrahexosylganglioside

GMl sodium salt 15 mg citrate buffer pH = 7 in water for injection, q.s. to 2 ml

Preparation n° 3 - One 2-ml ampoule contains: active ingredient 1 μg (5,000 BU) sodium chloride 16 mg monosialotetrahexosylganglioside GMl inner ester 7.5 mg citrate buffer pH = 7 in water for injection, q.s. to 2 ml

Preparation n° 4- One 2-ml ampoule contains: active ingredient 1 μg (5,000 BU) sodium chloride 16 mg monosialotetrahexosylganglioside

GMl inner ester 15 mg citrate buffer pH = 7 in water for injection, q.s. to 2 ml Preparation n° 5 a) One 3-ml ampoule contains: lyophilized active ingredient 1.5 μg (7,500 BU) monosialotetrahexosylganglioside GMl sodium salt 22.5 mg glycine 45 mg b) One 3-ml vial of solvent contains: sodium chloride 24 mg citrate buffer in water for injection q.s., to 3 ml

Preparation n° 6 a) One 3-ml ampoule contains: lyophilized active ingredient 1.5 μg (7,500 BU) monosialotetrahexosylganglioside GMl sodium salt 11 mg glycine 45 mg b) One 3-ml vial of solvent contains: sodium chloride 24 mg citrate buffer in water for injection q.s., to 3 ml

Preparation n° 7 a) One 3-ml ampoule contains: lyophilized active ingredient 1.5 μg (7,500 BU) monosialotetrahexosylganglioside GMl inner ester 22.5 mg glycine 45 mg b) One 3-ml vial of solvent contains: sodium chloride 24 mg citrate buffer in water for injection q.s. to 3 ml Preparation n° 8 a) One 3-ml ampoule contains: lyophilized active ingredient 1.5 μg (7,500 BU) monosialotetrahexosylganglioside GMl inner ester 11 mg glycine 45 mg

■b) One 3-ml vial of solvent contains: sodium chloride 24 mg citrate buffer in water for injection q.s. to 3 ml

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

CLAIMS :

1. A pharmaceutical composition, comprising at least one neurotrophic factor at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt of any of the foregoing at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity.

2. The pharmaceutical composition of claim 1, wherein said neurotrophic factor is a member selected from the group consisting of ciliary neurotrophic factor, nerve growth factor, and brain-derived neurotrophic factor.

3. The pharmaceutical composition of claim 1, wherein said ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt is a member selected from the group consisting of monosialoganglioside GMl, the sodium salt of monosialoganglioside GMl, GMl ethyl ester, GMl methyl ester, AGF1, AGF2, AGF4, Liga 4, Liga 20, and Liga 66.

SUBSTITUTE SHEET

4. The pharmaceutical composition of claim 1, wherein said neurotrophic factor is ciliary neurotrophic factor.

5. The pharmaceutical composition of claim 1, wherein said ganglioside is GMl or AGF2.

6. The pharmaceutical composition of claim 1, wherein said neurotrophic factor is ciliary neurotrophic factor and said ganglioside is GMl or AGF2.

7. The pharmaceutical composition of claim 1, wherein said sub-threshold concentration of said neurotrophic factor is between about 1 ng/ml and about 5 ng/ml .

8. The pharmaceutical composition of claim 1, wherein said sub-threshold concentration of said ganglioside, said ganglioside derivative, said semisynthetic ganglioside analogue, or said salt is between about 5 μM and about 20 μM.

9. The pharmaceutical composition of claim 1, wherein said sub-threshold concentration of said neurotrophic factor is between about 1 ng/ml and about 5 ng/ml, and said sub-threshold concentration of said ganglioside, said ganglioside derivative, said semisynthetic ganglioside analogue, or said salt is between about 5 μM and about 20 μM.

10. The pharmaceutical composition of claim 1, further comprising a polysaccharide.

11. The pharmaceutical composition of claim 10, wherein said polysaccharide is a hyaluronic acid ester.

SUBSTITUTE SHEET

12. The pharmaceutical composition of claim 11, wherein said hyaluronic acid ester is a member selected from the group consisting of a total ethyl ester of hyaluronic acid, a total benzyl ester of hyaluronic acid, a partial ethyl ester of hyaluronic acid, and a partial benzyl ester of hyaluronic acid.

13. The pharmaceutical composition of claim 12, wherein said pharmaceutical composition is in the form of a sponge, a tube, a guide channel, microspheres, or microcapsules.

14. A pharmaceutical composition, comprising ciliary neurotrophic factor at a concentration of 2 ng/ml and monosialoganglioside GMl or AGF2 at a concentration of 10 μM.

15. Use of at least one neurotrophic factor at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt of any of the foregoing at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity to produce a pharmaceutical composition.

16. A method for treating excitatory amino acid- induced neurotoxicity in a human or animal subject, comprising administering to said subject an anti- excitatory amino acid-induced neurotoxicity amount of said pharmaceutical composition of claim 1.

17. The method of claim 16, wherein said method prevents nervous function loss in the central or peripheral nervous system, or maintains nervous function.

SUBSTITUTE SHEET

18. The method of claim 16, wherein said method is employed for the recovery of nervous function in conditions of trauma or for the recovery of nervous function in either an acute or chronic pathological condition.

19. The method of claim 18, wherein said acute pathological condition is an acute neurodegenerative pathological condition.

20. The method of claim 18, wherein said chronic pathological condition is a chronic neurodegenerative pathological condition.

21. The method of claim 16, wherein said method is employed for the treatment of an age-related neuropathological condition.

22. The method of claim 16, wherein said pharmaceutical composition is administered parenterally, intracerebrally, orally, rectally, topically, intradermally, subcutaneously, or via inhalation.

23. The method of claim 16, wherein said neurotrophic factor is ciliary neurotrophic factor, and said ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt is monosialoganglioside GMl or AGF2.

24. A method of enhancing the neuroprotective effect of a neurotrophic factor against excitatory amino acid-induced neurotoxicity, comprising administering to a human or animal subject in need thereof a pharmaceutical composition comprising at least one neurotrophic factor at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity, and at least one ganglioside, ganglioside

SUBSTITUTE SHEET derivative, semisynthetic ganglioside analogue, or salt of any of the foregoing at a sub-threshold concentration ineffective in treating excitatory amino acid-induced neurotoxicity.

25. The method of claim 24, wherein said neurotrophic factor is a member selected from the group consisting of ciliary neurotrophic factor, nerve growth factor, and brain-derived neurotrophic factor, and said ganglioside, ganglioside derivative, semisynthetic ganglioside analogue, or salt is a member selected from the group consisting of monosialoganglioside GMl, the sodium salt of monosialoganglioside GMl, GMl ethyl ester, GMl methyl ester, AFG1, AGF2, AGF4, Liga 4, Liga 20, and Liga ββ .

SUBSTITUTE SHEET