HU206632B - Process for producing complexes of neuronotroph growth hormon and gangliozides and pharmaceutical compositions containing them - Google Patents

Abstract

The invention relates to novel, stable and biologically active complexes of the beta subunits of NGF (nerve growth factors) and natural ganglioides or their semi-synthetic analogues such as carboxylic esters and amides or their physiologically acceptable salts. The invention also encompasses a process for the production of those complexes in which the ingredients of the complex in an aqueous solution are contacted in the presence of a solvent and an inorganic or organic base at a temperature of from room temperature to 50 C. The invention also includes pharmaceutical compositions of said complex with pharmaceutically acceptable carriers.

Description

Scope of the description: 12 pages (including 2 sheets)

EN 206 632 Β

The present invention relates to novel, stable, biologically active complexes formed from the base unit of neuronotrophic growth factor (NGF, nerve growth factor) and from GM, ganglioside or analogue, and to pharmaceutical compositions containing these complexes. These novel complexes are GM, ganglioside or an analogue of ganglioside component, in particular the ganglioside GDla, GDlb or GTlb, or ester of said gangliosides with C 1 -C 8 alkanols or benzyl alcohol, internal ester, C 1-8 alkyl amide. or benzyl amide.

Gangliosides, i.e. glycosphingolipids containing the sialic acid moiety are generally present in mammalian cell membranes and are found in the nerve tissue at the highest concentration. The fact that the gangliosides are located in the outer layer of the plasma membrane suggests that these compounds play an important role in cell recognition, growth and differentiation. It is known that ganglioside compositions and other conjugated glycolipids change on the cell surface during differentiation and cellular maturation. In the normal human brain, four typical gangliosides, namely GM, GDla, GDlb, and GTlb represent about 8090% of all gangliosides.

The ganlizides consist of a bulky hydrophilic siaozyl oligosaccharide moiety and a hydrophobic ceramide moiety that is formed from a sphingoside acylated with fatty acids on the nitrogen atom.

Gangliosides are capable of producing plastic changes in neurons; promote neuritic growth in cell cultures and axonal growth in the central and peripheral nervous system.

It is becoming increasingly evident that not only the development of the nervous system, but also the conditioning of the nervous system is regulated by extracellular signals that affect molecules located on the cell surface.

Although it has only recently begun to study in detail the role of glycolipids on the surface of nerve cells and their effects, it is already apparent that gangliosides are of great importance in regulating both the development of nerve cells and the maintenance of nerve cells. During the formation of nerve cells, gangliosides in the brain undergo significant quantitative and qualitative changes.

It has been reported in the literature that the ingestion of gangliosides in vivo facilitates the recovery of neurons and the inhibition of functional healing of the peripheral nervous system [B. Ceccarelli et al., Adv. Exp. Med. Bioi., 71, 275, Plenum Press, New York 1976; song; A. Gorio et al., Neuroscience, 8, 417 (1983)]. It has also been reported that the administration of gangliosides, particularly known gangliosides such as GM, can alleviate the consequences of central nervous system (CNS) damage [G. Toffano et al., Brain Rés., 261, 163 (1983); A.C. Cuello et al., Dev. Brain, Vol. 376, 373 (1986)]. In connection with the [GM] designation, it is noted that the nomenclature of gangliosides according to Svennerholm can be found in J. Neurochemistry, 10.613 (1963).

Results of various recent studies show that gangliosides can modulate the in vitro activity of the neuronotrophic factor. For example, GM7 exogenous ganglioside has been reported to enhance axon growth in PC-12 consisting of cells stimulated by nerve growth factor (NGF) [S. G. Matta et al., Dev. Brain, Vol. 27, 243 (1986)]; and similarly, GM increases the development of ganglionic neurons from the back of the embryo by NGF-induced and sympathetic ganglia also by NGF [S. D. Skaperés, Int. J. Dev. Neurosci., 3, 187 (1985); S. D. Skaper et al., Dev. Brain, 23, 19 (1985)].

Recent studies have also been carried out to determine whether GM, in vivo, affects the activity of NGF. The results of these studies suggest that NGF neuronotrophic factor is likely to play a leading role in securing cell survival following CNS illness (AC Cuello et al., New Intramembrane Integrative Mechanism, Kjell Kuxe and L. Agnati, 1987) P. 62). An interesting publication is that treatment with GM, not only on dopaminergic and noradrenergic neurons, promotes neuronal survival, but also on cholinergic neurons known to respond to NGF.

The fact that NGF is indispensable for the survival of sympathetic neurons and for the survival of the target nerve network during development is the main proof that this growth factor plays an important role in tissue nutrition in target cells.

An important correlation was observed between NGF levels and the distribution of magnocellular cholinergic neurons. Relatively high levels of NGF - those within the range of values observed in a known target tissue, peripheral sympathetic tissue - have been observed both in the nerve regions of the magnocellular cholinergic neurons and in regions containing their cellular bodies such as the hippocampus. This relationship between the integrity of the basic proenzial cholinergic system and the sensing functions may also be present in the human body. One of the most significant neurodegenerative features of Alzheimer's disease is the strong decrease in magnocellular cholinergic neurons, although other transmission systems also undergo different forms of mutations. Therefore, there is a serious need to examine whether there is a link between the physiopathology and possible cure of NGF and Alzheimer's.

The basis of the experimental research is that it is possible to produce human NGF by biotechnological methods that result in DNA recombination. If you look2

EN 206 632 Β that the improvement observed in the cholinergic system after the experimental damage is concluded, we conclude that NGF had to become much more accessible to these neurons, exogenous use, and \ t by stimulating endogenous production.

With the improvement of biotechnological methods, a growing number of proteins are available as well as peptides of these proteins for possible medical use. There are specific and complex problems with the introduction of these macromolecules into the body. At present, there is only one solution for systemic administration of these pharmacologically active peptides, especially injections for subcutaneous, intramuscular or intracranial injection of NGF. Treatment regimens involving multiple injections daily or weekly have various drawbacks, particularly in the case of protein substitution therapies, including that the patient does not contribute to the treatment, and that treatment is not beneficial from a fanacokinetic point of view.

One of the aims of research in this field is to develop chemical processes that can increase the stability of proteins without altering their biological activity. Various biologically active products have been developed with liposome technology. However, it should be noted that these biological products have different problems, such as the low specificity of the chemicals used to separate the proteins, their special in vivo interaction with the organs, the poor reproducibility, since the properties of the complex compound differ from saras and the proteins. pharmacokinetic unknowns of chemicals used in isolation.

On the one hand, it is possible to produce a stable complex of the two types of compounds which are biologically active and meet the requirements of specificity, stability and bioavailability, taking into account the possibility of the use of the neuronotrophic factors and the in vivo biological activity of the gangliosides. previously mentioned. These properties can be demonstrated in experimental models, both in vitro and in iv delivery conditions

According to the present invention, complexes of the beta subunit of NGF neuronotrophic factor and natural gangliosides or semi-synthetic ganglioside derivatives are prepared by contacting the two components in the form of an aqueous solution at room temperature to provide a slightly alkaline reaction.

Figure 1 shows a patch diagram depicting the immunoreaction of the GM? NGF (beta subunit) complex with affinity purified anti-mouse NGF (beta) unit immunoglobulins. Figure 2 illustrates the effects of GM, beta-NGF complex by showing on day 6 of life the decrease in noradrenaline in the mouse heart caused by vinblastine sulfate (VNB).

The weight ratio of the two components of the complex may vary greatly, but preferably the ratio of neuronotrophic factor to ganglioside is between 1: 100,000 and 1:10. Within these limits, the most important compounds are those in which the ratio of neuronotrophic factor to ganglioside is 1: 1000.

The neuronal growth factor beta subunit (beta-NGF) used in the present invention can be isolated from human or animal tissue, for example, from mammalian tissue, or from cell culture media by known methods. If human beta-NGF produced by recombinant DNA technology is to be used, it is preferable to use preparations made from human placenta or bovine sperm fluid.

However, it should be noted that beta-NGF can also be produced by molecular biological technologies, such as recombinant DNA technology, as disclosed in U.S. Pat. by publishing a European patent application.

The novel complexes of the invention have the same type of biological activity as beta-NGF as can be demonstrated, for example, by in vitro experiments on a PC-12 cell line. The complexes of the present invention can therefore be used for healing in any case where the use of beta-NGF-based formulations is suggested, for example in the case of aging of the nervous system. The new complexes can also be used as a healing or prophylactic drug in cases where the gangliosides present in the new complexes have a particular effect, for example, on immunochemical activity, as seen with the anti-GM compound of the ganglioside complex of beta-NGF GM). polyclonal antibodies.

The biological activity of the novel beta-NGF ganglioside complexes and their derivatives can be demonstrated in laboratory animals as described in Example 1 for the betaNGF-GM complex complex.

Examples of effective semi-synthetic ganglioside analogues include functional derivatives such as esters or amides of the sialic acid carboxyl group, which are, for example, those described in No. 4713,374. United States of America; internal esters, e.g. U.S. Pat. European Patent Application; peracylated ganglioside and analogue derivatives of sialic acid, saccharide and, in some cases, ceramide hydroxyl groups are disclosed in U.S. Pat. U.S. Pat. and other derivatives such as lysogangliosides, i.e. dangylated gangliosides on the sphingosine nitrogen atom, and N-desacetyl gangliosides and N-desacetyl lysogangliosides, the molecules of which are also deacylated by the nitrogen of the neuramin residue; as well as derivatives analogous to gangliosides in which long chain fatty acid moieties, e.g.

There are molecular moieties derived from amino acid or stearic acid - or, optionally, other functional groups, especially polar groups, which are derived from any other aliphatic, aromatic or alicyclic or heterocyclic acid. There are also semi-synthetic analogues which, apart from the "non-natural" acyl groups mentioned in the neuraminic acid nitrogen, have acyl groups that are different from the acetyl group.

Semi-synthetic compounds and their functional derivatives, such as esters, amides, internal esters and peracylated derivatives thereof, can be used to prepare the complexes of the present invention as described in U.S. Pat. and No. 4713374. US Patent Specifications.

Any ganglioside containing a group derived from N-acetyl neuraminic acid (NANA) is capable of forming a complex with the invention according to the invention.

As used herein, "complex" refers to the association between the NANA group of ganglioside and the beta subunit of NGF. This association is the result of the ionic and hydrogen slow interaction between the two compounds. This interaction distinguishes the complex of the invention from the ganglioside and betaNGF blender.

As a starting material for the preparation of the new complexes, gangliosides are preferably selected from the group consisting of the aforementioned gangliosides, which form the major part of the gangliosides of the brain, i.e. GM, A GDla, GDlb and GTlb, as well as the functional derivatives mentioned above and their derivatives. semi-synthetic analogues.

Said gangliosides can also be used as starting materials for the preparation of neuronotropic growth factor beta subunits in the form of acid addition salts in the process of the present invention. In this way, it is also possible to complex the neuronotrophic growth factor beta subunit and the ganglioside or ganglioside derivative. in the form of an acid addition salt; these acid addition salts have the same therapeutic properties and application possibilities as the free, non-salted complexes.

Special mention should be made of complexes derived from the following esters and amides of GM, ganglioside: alcohols with up to 8 carbon atoms, such as methyl alcohol, ethyl alcohol, isopropyl alcohol, butyl alcohol, amyl alcohols, hexyl alcohols, e.g. -hexyl alcohol, aliphatic amides of n-octyl alcohol, aliphatic esters formed, and amines of up to 8 carbon atoms, such as methylamine, ethylamine, propylamine, hexylamine or octylamine.

Complexes of the neuronotrophic growth factor beta subunit with said gangliosides or ganglioside derivatives according to the invention are prepared by the beta subunit of the neuronotrophic growth factor in aqueous solution in the presence of a water-miscible organic solvent in an alkaline medium with GM, ganglioside or analogue such as GDla, GDlb or GTlb with ganglioside or ganglioside ester with C 1 -C 8 alkanols or benzyl alcohol, internal ester, C 1 -C 8 alkyl or benzylamide, or acid addition salt thereof, preferably at room temperature to 50 ° C, and then the complex formed.

The quantitative ratio of the two components, the neuronotrophic growth factor beta subunit and the ganglioside or ganglioside derivative, in the complexing reaction may vary widely; generally, the two components are brought into contact with each other in a weight ratio of 1: 100,000 to 1: 10, preferably 2: 2000 to 1: 500.

Inorganic or organic bases such as ammonium hydroxide, lower aliphatic amines, alkali metal hydroxides, alkali metal carbonates, alkali metal acetate, in particular sodium acetate and the like can be used to alkaline the aqueous medium of the complexing reaction. In alkaline aqueous media, the reaction components are generally soluble; solubility conditions can be improved as needed by adding water-miscible organic solvents such as lower aliphatic alcohols, ketones or sulfoxides to the aqueous medium.

The reaction time is always between a few hours and a few days, but usually between 12 and 24 hours.

The protein / ganglioside derivative can be isolated from the reaction mixture by known methods, for example, by the methods described in Example 1, due to its biological activity and by immunochemical recognition by protein-specific, monoclonal or polyclonal antibodies, or by sedimentation using an ultracentrifuge.

The invention also relates to the preparation of pharmaceutical compositions for the treatment of neurological diseases comprising the complex prepared as described above. These pharmaceutical compositions may be administered orally, rectally, parenterally or topically. They can therefore be assembled in solid or semi-solid form, for example in the form of pills, tablets, gelatin capsules, suppositories or soft gelatin capsules.

For parenteral administration, formulations suitable for intramuscular or subcutaneous administration, infusion, intravenous or intracerebral injection may be used. Against this background, solutions containing active substance, lyophilized active substance, and powders containing one or more pharmaceutically acceptable excipients or diluents compatible with physiological fluids compatible with said physiological fluids may be provided.

Sprays such as creams or externally applicable ointments may be used for topical application.

The compositions of the invention are for human and

EN 206 632 Β may be administered to animals. The compositions preferably contain from 0.01 to 10% by weight of the active ingredient in solutions, sprays, ointments and creams and contain from 1 to 100% by weight, preferably from 5 to 50% by weight, of the active ingredient in solid formulations. The dose to be administered will depend on the individual needs, the desired effect and the chosen route of administration.

With the indications already mentioned, the invention also relates to the therapeutic use of the complexes formed by gangliosides or ganglioside derivatives with the beta subunit of the NGF neutronotrophic growth factor.

For human use, the daily dose is 0.01,100 mg per kilogram of body weight.

By injecting the active ingredient into the human body - under the skin, into the muscle or into the brain - the daily dose is 0.05 mg to 5 mg of active ingredient / kg body weight.

The following examples illustrate the process of the invention. The starting materials used in the examples are known and can be obtained commercially (also from the reporting company).

Example 1

Materials used and methods used

Mouse NGF (beta subunit) was purified by the method described by P. V. Angeletti et al., Natl, Acad. Sci. USA, 64, 787 (1969)]. Skaper et al., Exp. Neurol., 76, 655 (1982)], using "dissociated" fetal cells derived from E8 small chicken nerve root ganglia (DRG E-8). In order to inhibit the biological effect of NGF (beta subunit), an experiment on NGF antibodies against murine NGF is evaluated using said in vitro model.

The immunoreactivity of GM, NGF (beta-subunit) and GM, -NGP (beta-subunit) was measured by immuno-blotting.

The NGF polyclonal antibodies to mouse NGF (beta subunit) were purified by affinity chromatography using 2.5S mouse NGF bound to Sepharose 4B as described by K. Stoeckel et al. Neurochem., 26.1207 (1976)].

GM, polyclonal antibodies against GM, monosialoganglioside were purified by affinity chromatography using GM bound to Sepharose 4B.

Isochor sedimentation of NGF (beta-subunit) and ganglioside complexes is carried out according to B. Ulrich-Bott et al. (Journal of Lipid Research, 25, 1233 (1984)).

mg of GM, monosialoganglioside was dissolved in a 2: 1 mixture of chloroform and methanol and the solvent was evaporated under a nitrogen atmosphere. GM, monosialoganglioside was resuspended for one hour in 50 mM ammonium hydroxide solution (pH 8.5), then the concentration of GM, monosialoganglioside was adjusted to 10 mg / ml, and highly purified mouse NGF beta subunit was dissolved. In a 50 mM sodium acetate solution and from this solution 1: 1000 beta-NGF: GM was added to a weight ratio of GM to this solution. The pH of 8.5 is incubated at room temperature with stirring overnight while the formation of the complex takes place. Performing the same operation at 4 ° C is the same result.

The resulting beta-NGF-GM biological complex was separated by anion-exchange chromatography on a mono Q column from unreacted betaNGF; equilibrium with 50 mM ammonium hydroxide at pH 8.5. Elution was carried out with ammonium hydroxide solution (50 mM, pH 8.5), containing ammonium acetate at 0-1.0 mol / l, at a flow rate of 1 ml / min. Unreacted betaNGF is eluted with ammonium hydroxide solution without ammonium acetate at a concentration of 0.15-0.25 mol / l ammonium acetate in the period between 15 and 18 minutes. In this experimental complex, 12 µg of the NGF beta subunit binds to 1 mg GM, monosialoganglioside.

The chemical, immunochemical, and biological properties of the resulting beta-NGF-GM complex are evaluated in vitro and include:

a) Immunofiltration assay of purified beta-NGF-GM complex shows that the complex retains its immunoreactivity against polyclonal antibodies against purified mouse NGF (beta subunit) (immunoblot diagram in Figure I).

b) The purified beta-NGF-GM complex exhibits biological activity of DRG-E8.

c) The biological activity of the beta-NGF-GM complex is inhibited by polyclonal antibodies purified by affinity chromatography at a concentration of 60 pg / ml and a monoclonal antibody at a concentration of 500 pg / ml. The biological activity of beta-NGF without GM is inhibited by the affinity chromatography polyclonal antibodies at a concentration of 30 µg / ml and monoclonal antibodies at 100 µg / ml.

d) Using the immunoblot technique, the same band that is associated with the biologically active beta-NGF-GM complex refers to reactivity against polyclonal antibodies produced against NGF (beta subunit) and GM monosialoganglioside.

e) beta-NGF, GM, DRG-E8 on the evaluated biological activity is retained in the beta-NGF, GM, incubating after both low pH buffer, both divalent ions - the presence of - for example, 40 mmol / 1 CaCl _second

f) The stability of the beta-NGF-GM complex remains in the presence of human serum at 37 ° C for 96 hours.

g) The biological activity of the beta-NGF-GM complex remains after digestion with proteolytic enzyme. (This digestion is carried out, for example, with 2% pepsin for 16 hours at pH 2.0 at room temperature.) Under the same conditions, the proteolytic enzyme inhibits the effect of GM-free beta-NGF.

h) The biological activity of the beta-NGF-GM complex remains at different temperatures, e.g.

EN 206 632 Β on, after storage at 37 ° C and 45 ° C. Under the same storage conditions, GM-monosialoganglioside-free beta-NGF completely loses its biological activity.

j) By evaluating isochor sedimentation, the beta-NGFGM complex is settled at a density of 1.298 g / cm ³ .

Figure 1 shows the GM, beta-NGF complex, and similarly produced asia-GM, beta-NGF complex (affinity chromatography purified anti-mouse NGF-beta subunit after reaction with immunoglobulins); Mouse-NGF-Beta subunit immunoprecipitate diagram:

Measurement A: GM Beta-NGF Complex Prior to Chromatography Measurement B: Asialo-GM, Beta-NGF Complex C Measurement: Mouse NGF, Beta Subunit, Dimer

GMj and beta-NGF were incubated as described above for two hours at room temperature. Incubation of asialo-GM and NGF is performed under the same conditions.

The biological activity of the beta-NGF-GM1 complex was evaluated in vivo in animals in which the biological activity of betaNGF was printed with vinblastine. Newborn SpragueDawley mice of both sexes were used for the studies. All systemic injections were administered under the buoy, at a dose of 0.01 ml / g body weight. The vinblastine sulphate solution (VNB) is prepared from the salt and obtained from each mouse on the third day of life at a dose of 0.15 mg / kg. VNB-treated mice are divided into three groups: the first group saline, the second group beta-NGF, e.g., 0.1 mg / kg beta-NGF, and the third group beta-NGF-GM, e.g. 0.1 mg / kg betaNGF and 30 mg / kg GM, monosialoganglioside. Animals are killed on the sixth day of their lives. Their hearts are quickly removed, frozen in dry ice and stored at -80 ° C until prepared for use.

The amount of noradrenaline in the heart is determined. As shown in Figure 2, VNB administration significantly reduces noradrenaline in the heart (Group 1) and has a significant antagonistic effect when both GM-free beta-NGF (Group 2) is administered and beta-NGF-1 is present. GM, Complex (Group 3). In the figure, the figures show the degree of effect on each group.

In addition, these results show that beta-NGF-GM, from a complex biological point of view, has an in vivo effect on the sympathetic nervous system and is more biologically active than beta-NGF.

Example 2

The NGF-ganglioside complex is composed of GM, monosialoganglioside and human NGF (beta subunit) produced by recombinant DNA technology exactly as described in Example 1. The in vitro and in vivo properties correspond to those of Example 1.

Example 3

The complex between GDla ganglioside and gonads recovered and purified from the upper jaw gland was prepared under the experimental conditions described in Example 1.

The GDla-beta-NGF complex retains its biological activity on DRG-E8. By means of isochor sedimentation it is found that this complex settles at a density of 1,3496 g / cm ³ .

Example 4

The complex between GD1b ganglioside and gonads recovered and purified from the upper jaw is prepared under the experimental conditions described in Example 1.

The GDlb-beta-NGF complex retains its biological activity on DRG-E8. By isochor sedimentation, we find that this complex settles at a density of 1.3596 g / cm ³ .

Example 5

The complex between GT1b ganglioside and gonads recovered and purified from the upper jaw gland was prepared under the experimental conditions described in Example 1.

The GTlb-beta-NGF complex retains its biological activity on DRG-E8. By isochor sedimentation, we find that this complex settles at a density of 1.3700 g / cm ³ .

Example 6

The complex between GM, its internal ester and the NGF-beta subunit was prepared under the experimental conditions described in Example 1.

Example 7

The complex between GM, methyl ester and NGF-beta subunit was prepared under the experimental conditions described in Example 1.

Example 8

The complex formed between GM, ethyl ester and NGF-beta subunit was prepared under the experimental conditions described in Example 1.

Example 9

The complex formed between GM, isopropyl ester and NGF-beta subunit was prepared under the experimental conditions described in Example 1.

Example 10

A complex formed between GM, tertiary-bulyl ester and NGF-beta subunit was prepared under the experimental conditions described in Example 1.

//. example

The complex between GM, benzyl ester and NGF-beta subunit was prepared under the experimental conditions described in Example 1.

EN 206 632 Β

Example 72

GM! a complex formed between the methyl amide derivative and the NGF-beta alpha under the experimental conditions described in Example 1 above.

Example 13

GM | a complex formed between the ethyl amide derivative and the NGF-beta subunit under the experimental conditions described in Example 1 above.

Example 14

The complex formed between the benzyl amide derivative of GMi and the NGF-beta-allyl was prepared under the experimental conditions described in Example 1.

Example 75

The complex formed between the GM] isopropylamide derivative and the NGF beta-alpha unit was prepared under the experimental conditions described in Example 1.

All complexes produced using the various ganglioside derivatives and the beta-unit of the nerve growth factor retained its biological activity on DRG-E8 cultures.

Pharmaceutical compositions containing the ganglioside-beta-NGF complexes are prepared in a known manner from an effective amount of a complex active ingredient optionally admixed with a physiologically acceptable carrier. Suitable carriers for this purpose, including other proteins, and their formulation are described in the literature, e.g., "Remington's Pharmaceutical Sciences" (Mack Publishing Company, Easton, Pennsylvania, USA, 1985). The described carriers include injectable so-called "deposit" formulations.

On this basis, the pharmaceutical compositions may include, but are not limited to, GM, beta-NGF complex solutions, or freeze-dried GM4 beta-NGF complex powders in association with one or more pharmaceutically acceptable carriers or diluents. buffered and in the same osmotic pressure as the physiological fluid. The formulation of the various GM4 beta-NGF complexes can be considered as examples of pharmaceutical compositions containing complexes produced from ganglioside derivatives and beta-NGF. Formulation depends on the biological activity to be achieved and the route of administration.

In Table I, for purposes of illustration, and not by way of limitation, reference is made to recipes for preparing solutions for the treatment of neurological disorders. For freeze-dried formulations, carrier binders such as mannitol or glycine can be used, and suitable buffered solutions of the desired volume can be used to obtain the appropriately buffered isotonic solutions of the desired pH.

Similar solutions may be used to prepare pharmaceutical compositions containing GM] betaNGF complex in isotonic solutions of the desired volume. Also included are the use of phosphate or citrate buffered saline solutions of appropriate concentration to always obtain isotonic pharmaceutical compositions at the desired pH, such as neutral.

A Π. An example of a composition of Table 1 is pharmaceutical compositions for use in the treatment of neurological disorders.

AII. 4 and 5 in Table 2. formulations are prepared in twin vials each containing a single dose. The first ampoule contains the active ingredient of the composition in admixture with from about 0.01% to about 50% of the physiologically acceptable excipient such as glycine or mannitol. The second ampoule contains the solvent prepared with an appropriate volume of phosphate or citrate buffered saline. The contents of the two ampoules should be mixed immediately before use. The freeze-dried active ingredient dissolves rapidly, resulting in an injectable solution. A Π. Table 1 also shows an example of a pharmaceutical preparation suitable for subcutaneous injection (composition 5).

The pharmaceutical compositions of the complexes of the present invention for use in the manufacture of medicaments or diluents may be formulated as solid, semi-solid, liquid or gaseous, e.g., tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalation preparations, aerosols, for oral or parenteral administration. In the following, methods and excipients will be described by way of example and not by way of limitation.

In the case of preparation of inhaled or aerosol formulations, the compound of the present invention may be filled in liquid or fine powder form with a gas or liquid propellant into an aerosol container, if necessary with conventional excipients such as wetting agents. The active ingredients may also be used in the form of non-pressurized formulations such as spray or spray structures.

The pharmaceutical compositions of the present invention may also be suppositories for rectal administration with lipophilic binders such as water-soluble, self-emulsifying excipients such as glycoselatine or the like. In these formulations, the GM4 beta-NGF complex may be present at 0.0011% by weight of the total binder. In addition, suppositories may also contain an appropriate amount of acetylsalicylate.

The III. Table 1 - only suppositories of suppositories for treating neurological disorders are provided by way of illustration only.

Dosage and frequency of administration of pharmaceutical compositions containing the GM _t- beta-NGF complex are determined by the desired effect and the route of administration as determined by clinical trials. For example, the dosage and frequency of administration are similar

20 may be, if not necessarily, in all cases, doses and frequencies that are customary for other neuronotrophic agents.

Table I

Examples of formulations for use as injectable solutions

The name of the materials Quantity of materials No. 1 preparation (2 ml ampoule) GM, beta-NGF complex -GM] monosialoganglioside 2 mg - beta-NGF 1 pg (8000 BU) NaCl 16 mg pH 7 citrate buffer with distilled water the amount required for filling into 2 ml No. 2 preparation (2 ml ampoule) GM] beta-N GF complex -GM] monosialoganglioside 5 mg - beta NGF 10PG (8000BU) NaCl 16 mg pH 7 citrate buffer with distilled water the amount required for filling into 2 ml

The definition of the biological unit (BU) can be found in the following literature: Fenton, E.L., Expl. Cell Rés., 59, 383 (1970).

II. table:

Examples of two-component pharmaceutical compositions

The name of the materials Quantity of materials No. 1 preparation a) component (2 ml ampoule) - freeze-dried active ingredient -GM] monosialoganglioside 1.5 mg - beta-NGF 4pg (32000BU) - glycine 30 mg b) component (solvent in 2 ml ampoule) - NaCl 16 mg - citrate buffer at pH 7 with distilled water the amount required for filling into 2 ml No. 2 preparation a) component (2 ml ampoule)

The name of the materials Quantity of materials - freeze-dried active ingredient -GM] monosialoganglioside 1.5 mg - beta-NGF 4 pg (32,000 BU) - mannitol 40 mg b) component (solvent in 2 ml ampoule) - NaCl 16 mg - citrate buffer prepared with distilled water the amount required for filling into 2 ml No. 3 preparation Component a) (3 ml ampoule) - freeze-dried active ingredient -GM] monosialoganglioside 3 mg - beta-NGF 10 pg (80000 BU) glycine 45 mg b) component (solvent in 3 ml ampoule) - NaCl 24 mg - citrate buffer prepared with distilled water in the amount required for filling up to 3 ml No. 4 preparation Component a) (3 ml ampoule) - freeze-dried active ingredient -MM, monosialoganglioside 3 mg - beta-NGF 10 pg (80000 BU) - mannitol 60 mg b) component (solvent in 3 ml ampoule) - NaCl 24 mg - citrate buffer prepared with distilled water in the amount required for filling into 3 ml No. 5 preparation for injection under the skin) a) component (2 ml ampoule) - freeze-dried active ingredient

EN 206 632 Β

The name of the materials Quantity of materials -GM! monosialoganglioside 1.5 mg beta-NGF 5 pg (40000 BU) - glycine 30 mg b) component (solvent in 2 ml ampoule) - NaCl 16 mg - citrate buffer prepared with distilled water the amount required for filling into 2 ml

Table 111

Examples of rectal administration for pharmaceutical preparations in the form of suppositories

The name of the materials Quantity of materials No. 1 preparation - GMj-beta-NGF Complex - GMj monosialoganglioside 3 mg beta-NGF 10 pg (80000 BU) - Cocoa butter 2.5 g No. 2 preparation - GMj-beta-NGF complex - GM] monosialoganglioside 3 mg beta-NGF 10 pg (80000 BU) - Carbowax 1540 1.75 g - Carbowax 6000 0.75 g No. 3 preparation - GMpbeta-NGF complex -GM] monosialoganglioside 3 mg beta-NGF 10 pg (80000 BU) -Twenn 61 2.125 g - lanolin 0.25 g No. 4 preparation - GM _r beta-NGF complex - GMj monosialoganglioside 3 mg beta-NGF 10 pg (80000 BU) - glycerol 1.5 g -water 0.75 g - gelatin 0.25 g

The present invention as described herein may, of course, be implemented in many ways, in addition to the solutions described in the Examples. Modified solutions relative to the examples cannot be considered independent of the invention and the subject of the invention. All modifications that are appreciated by those skilled in the art will form part of the present invention.

Claims (10)

PATENT CLAIMS 1. Procedure from the beta subunit of neuronotrophic growth factor (NGF, nerve growth factor) and GM | ganglioside or analogs thereof, such as the ganglioside GDla, GDlb and GTlb, and its ester, inner ester, C1-C8-alkyl amide or benzyl amide with C1-C8 alkanols or benzyl alcohol, hereinafter referred to as "ganglioside component" - stationary complexes thereof, characterized in that the neuronotróf growth factor beta subunit in an aqueous solution - optionally water-miscible organic solvent - an alkaline medium GM _r ganglioside or one of its, analog, ester, amide or acid addition salt thereof the meanings as referred contacting 50C and the resulting complex is isolated. Method according to claim 1, characterized in that the neuronotrophic growth factor beta subunit and the ganglioside component are used in a weight ratio of from 1: 100000 to 1:10, preferably from 1: 2000 to 1: 500. 3. The method of claim 1 or 2, wherein the ganglioside component is used as GM _t, GDla, GDlb and GTlb ganglioside. The process according to claim 1 or 2, wherein the ganglioside component is an ester of the GM1 ganglioside with an aliphatic alcohol having up to 8 carbon atoms. The process according to claim 1 or 2, wherein the ganglioside component is an alkyl amide of the ganglioside GM 1 having up to 8 carbon atoms. 6. The process according to claim 1 or 2, wherein the ganglioside component is the ganglioside inner ester of GM1. The method of claim 1 or 2, wherein the neuronotrophic growth factor beta subunit is a human placental tissue derived and purified nerve growth factor beta subunit. 8. The method according to claim 1 or 2, wherein the neuronotrophic growth factor beta subunit is a bovine sperm fluid and purified nerve growth factor beta subunit. The process according to claim 1 or 2, wherein sodium acetate is used to adjust the solution of the reaction components to a basic pH range. 10. A process for the preparation of a pharmaceutical composition comprising the step of combining a complex of a neuronotrophic growth factor-beta subunit and a ganglioside component prepared according to claim 1 with a pharmaceutically acceptable carrier and / or other pharmaceutical excipient.