PL165677B1 - Method of obtaining and purifying a mixture of ganglycosides - Google Patents

Abstract

Prepn. of a mixt. of gangliosides comprises (a) subjecting ganglioside-contg. tissue to lipid elimination with acetone to produce an acetone ppte., (b) suspending the acetone ppte. in a first solvent mixt. (e.g. CH2Cl2, MeOH an NaOH) capable of partitioning hydrophobic substances from hydrophilic substances, (c) filtering the partitioning mixt. to obtain a first liquid phase, (d) subjecting the first liquid phase to pptn. (e.g. by adding CaCl2 and acetone) to obtain a first raw material, (e) solubilising the first raw material (using e.g. a mixt. of water, CHCl3 and MeOH) and subjecting the solubilised first raw material to heating at a pH of about 12, (f) subjecting the heated solubilised first raw material to a second partitioning in a second solvent mixt. (e.g. water, CHCl3 and n-butanol) capable of partitioning hydrophobic substances from hydrophilic substances, (g) sepg. the second partitioning mixt. to remove an organic phase and retain an aqs. phase, (h) subjecting the aqs. phase to pptn. (e.g. by adding acetone and NaCl) to produce a second raw material, (j) solubilising the third raw material in a base, e.g. 1N NaOH, (k) neutralising the solubilised third raw material and (l) subjecting the neutralised solubilised third raw material to dialysis through a membrane with a mol. wt. cut off of about 10 kD to produce a ganglioside mixt.. Also claimed is a ganglioside mixt. prepd. by the method.

Description

The subject of the invention is a method of producing a specific ganglioside mixture that selectively eliminates contamination associated with unconventional life-threatening viruses and does not alter the biological and pharmacological properties of the mixture as regards its effects on the central and peripheral nervous systems.

Gangliosides, sialic acid-containing sphingolipids, are components of all mammalian cell membranes and are abundant in nervous tissue / Ando S., Neurochem. Int. 5: 507, 1983 /. The four gangliosides GM1, Gdi _a , GDibi GTib (nomenclature according to L. Svennerholm, J. Neurochem., 10: 613, 1963) account for 80-90% of the total ganglioside content in the mammalian brain. The gangliosides are specifically located in the outer layer of the uveal membrane, which suggests that they play an important role in many biological processes, e.g. as sensors and / or receptors for various molecules and involved in transmitting information across cell membranes (Fishman et al., Science, 194: 906, 1976). For this reason, they play a key role in the regulation of neuronal development and in the repair processes of the central and peripheral nervous systems.

There is extensive documentation that gangliosides are capable of exerting a favorable effect on functional regeneration following damage to the peripheral nervous system / CNS / and the central nervous system / CNS / through the inclusion of specific membrane mechanisms, and as shown in in vitro studies of neuronal cultures, by interaction with neurotrophic factors, / P. Doherty et al., J. Neurochem, 44: 1259, 1985; S. Skaper et al., Molecular Neurobiology, 3: 173,189 /.

In particular, it has been reported that in vivo ganglioside organization facilitates nerve regeneration and functional regeneration in OUS under pathological conditions: positive effects are described in the traumatic neuropathy / B models. Ceccarelii et al. Adv. Exp. Med. Biol. 71: 275, Plenum Press, New York, 1976; A. Gorio et al, Brain Res., 7: 236, 1980; A. Gario et al., Neuroscience, 8: 417, 1983 /, metabolic neuropathies / F. Norido et al., Exp. Neurol., 83: 221,184 (and toxic neuropathies) (F. Di Gregorio et al., Cancer Chemother., Pharmacol, 26:31, 1990).

With respect to the central nervous system, the positive effects of GM1 monosialglioside-induced regeneration in /A.C ischemia models have been widely reported. Cuello et al., Brain Res., 376: 373, 1986; S.E. Karpiak et al., CRC Critical Rev. in Neurobiology, vol. 5, 3rd edition, 1990 /, traumatic pathological lesions / G. Toffano et al., Brain Res., 296: 233, 1984 / and neurotoxic lesions / J. Jchnsson, Dev. Brain Res., 16: 171, 1984) in various neuronal systems of various animal species. Recently, it has been discovered that gangliosides can inhibit glutamate / F induced translocation and activation of protein kinase C. Vaccarino et al., Proc. Nat. Acad. Sei. USA, 84: 8707, 1987- This feature is very important in

In ischemic injury conditions, where excitatory amino acids such as glutamate are reported to play a decisive role and trigger a cascade of processes leading to neuronal death. This mechanism could promote the survival of neurons in the area around the lesion, prevent retrograde degeneration, and accelerate the reparative growth response to local trophic factors.

The results of the experimental studies were sufficiently confirmed by the effects of the clinical application of gangliosides. For over 10 years, gangliosides have been used as therapeutic agents in almost all types of peripheral neuropathy, from forms resulting from mechanical damage to those caused by toxic factors or deficiencies, from infectious and inflammatory conditions to metabolic dysfunctions. These drugs have also been shown to be effective in mono- as well as polyneuropathies, sensorimotor disorders, and pathologies affecting the autonomic nervous system, as is the case with many neuropathies involving the cranial nerves, e.g. Bell's palsy, triangles neuralgia, and herpes zoster. Gangliosides, and in particular monosjaloganglioside, can be widely used in all pathologies associated with severe damage to the central nervous system of the vascular or traumatic type and subsequent to these pathologies (cerebral ischemia, brain and spinal cord injury). The repetitive function of gangliosides within the CNS, proven in studies, also confirms their participation in chronic pathologies of degenerative neuroses such as Parkinson's disease and Alzheimer's disease. The fact that they are endocoids (endogenous drugs), natural components of neuronal membranes, explains their extreme tolerance and the absence, even with prolonged high-dose treatment, of the side effects that are so common in some cases of conventional therapy for peripheral neuropathies.

In general, suitable mixtures of gangliosides, e.g. formulation of the following types: GMi from 18% to 24%. GDi from 36% to 44%, GDi from 12% to 18%, GTi from 16% to 22%, or single ganglioside fractions, especially monosialglioside GMi, exhibit the biological activity described above. These gangliosides, as appropriate mixtures or single fractions, mainly monosyaloganglioside GM1, are extracted from the brains of mammals and hence the determination of their specific biological role and therapeutic application described above in relation to the central and peripheral nervous system is important when using purification methods that ensure the final the product is absolutely pure and free from biological and chemical contamination.

In scientific research it has long been known that mixtures of gangliosides / Tettamanti et al., Biochim can be extracted. e Biophys. Acta, 296: 160,1973; Trams et al., Biochim. e Biophys. Acta, 60: 350, 1962: Bogoch et al., British J. Pharm., 18: 625, 1962; Wiegandt et al., Angew. Chem., 80: 89, 1968; U.S. Patent No. 3,436,413iC.A.61,9851C, 9895d), but none of the above-mentioned methods was developed enough to eliminate and destroy components associated with unconventional viruses. One of the reasons for this was that diseases affecting the species of mammals whose brains were used for extraction were not yet known. Another reason was that reagents for the specific identification of potentially hazardous components were not available, whereas now such reagents have become available through specific methodologies developed on the basis of newly acquired knowledge gathered during the scientific evolution of molecular biology techniques.

Sometimes pathological conditions may arise in which the pathogenic factor or factors cannot be identified. One such pathological condition is bovine spongiform encephalopathy (BSE), first reported in England in 1986. /G. Wells et al., Vet. Record, 419, 1986 /. The name is derived from the appearance of spongy brain tissue in affected animals. During microscopic analysis of the tissue sections, it is found that the lesion is affected by extensive neuronal vacuoles.

All the available evidence suggests that BSE belongs to the group of degenerative encephalopathies of the central nervous system, which are fatal and are caused by a group of unconventional, infectious agents. This group also includes: the endemic form of sheep and goat diarrhea, a chronic wasting disease affecting deer living in reserves, infectious mink encephalopathy and two human diseases: kuru and Creutzfeldt-Jacob disease. The histopathological damage caused by these diseases in the brain is similar in all cases and comparable to that caused by BSE. Many theories have been put forward as to the nature of the etiological factors which are neither bacteria nor viruses, which do not resemble known organisms and are therefore known as unconventional viruses. These viruses are also known as free viruses because of the long incubation periods that go from infection to disease symptoms.

Since several cases were observed in 1986, the disease has spread and has reached epidemic proportions in Great Britain, reaching around 14,000 cattle and affecting around 250-300 head per week. Infected cattle show no signs of disease for several years (incubation period is 4-5 years), and when symptoms show, the condition of the animals rapidly deteriorates and death occurs.

Epidemiological studies conducted by the Central Veterinary Laboratory on behalf of the British Ministry of Agriculture Wilesmith et al., Vet. Record, 123: 638, 1988 / showed that the source of the infection was animal feed produced from processed carcasses of other ruminants and sold in the form of powdered meat or bones. Since it is known that encephalopathy can be widely transmitted to animal species, it seems reasonable to assume that BSE is the result of infection by the etiological agent responsible for endemic diarrhea, transmitted from sheep via contaminated foodstuffs / KL Morgan, Vet. Record, 122: 445, 1988 /.

Based on the results of these studies, the British government banned the sale and supply of animal food products containing animal proteins derived from ruminants by a law that took effect on July 18, 1988.

It is generally believed that many factors combined contributed to the sudden onset of BSE in Great Britain / J. Cherfas. Science, February 1990, 523 /.

First, the number of sheep in the UK increased rapidly in the late 1970s and early 1980s, and in parallel with that, the incidence of endemic diarrhea, a sheep disease reported in Europe for over 250 years / Pattison et al., Vet. Record, 90: 465, 1972). At the same time, in parallel with the gasoline crisis, animal feed plants changed their carcass treatment to a low-temperature method that was probably less effective at killing the highly resistant microbes that cause endemic diarrhea. All but one of the feed manufacturers have given up the use of solvents such as benzene, hexane and trichlorethylene, which are used to remove excess fat from soybeans and the bone nutrient product. The most significant fact is that the final stage of heating the products to remove solvents was consistently skipped: indeed this step required very high temperatures.

Moreover, government policy has encouraged farmers to produce more milk and wean their calves earlier by adopting high-protein diets. These, in turn, were often of poor quality, as food made from meat and bones was cheaper than products such as soybeans and fish, which are more reliable sources of protein. Attempting to answer the question of how disease is transmitted is fundamental to BSE research. The greatest aspect of these experiments is the estimation of the risk of BSE contamination of different species by establishing the boundaries of intraspecific barriers to pathogens. Frasev et al. / Et. Recoed, 123: 472, 1988) showed that the disease could be transmitted from cattle to mice. They vaccinated extracts from the brains of cattle that died due to BsE into the brains of mice that subsequently developed the disease. Later, Barlow et al. / Vet. Record, Feb. 3, 1990 / transferred the disease to mice by feeding them infected brains. This was the first evidence that BSE could be spread through the consumption of contaminated material. No other tissue taken from the affected animals (spleen, spinal cord, lymphoid tissue, milk, etc.) was able to initiate disease in mice.

There is evidence that endemic diarrhea can be passed on to lambs by their mothers, but so far there is no confirmation that BSE is transferable vertically and horizontally within cattle herds.

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The agents that cause subacute infectious encephalopathy are largely resistant to standard decontamination processes. Preliminary data on this issue are largely based on microbial inactivation studies of endemic diarrhea and Creutzfeldt-Jacob disease. The etiological agent of endemic diarrhea is highly resistant to temperature changes. Exposure to temperatures as high as 80 ° C only slightly reduces their infectivity. Higher temperatures, however, significantly reduce infectivity (Hunter et al., J. Gen. Microbiol., 37: 251, 19M). A small amount of infectious virus sometimes survives while the infectious material suspension is heated to 100 ° C for 1 hour or to 118 ° C for 10 minutes.

There has recently been a need to establish new standards for the sterilization of these infectious agents under high steam pressure in autoclaves. The current standards governing autoclave sterilization processes in the United States of Creutzfeldt-Jacobź disease microbial decontamination recommend a temperature of 132 ° C for 1 hour / Rosenberg et al., Annals of Neurology, 19:75, 1986 / and are based on studies performed on brain homogenates containing the microorganisms of endemic diarrhea or Creutzfeldt-Jacob's disease (Bown et al., J. of Infectious Diseases, 153: 1145, 1986). In Great Britain, the current standard for decontamination processes for Creutzfeldt-Jacobś disease recommends autoclaving at 134-138 ° C for 18 minutes, based on studies including conducted by Kimberlin / Kimberlin et al., Journal of Neurogical Science, 59: 355, 1983 /. Unfortunately, the microbes of bovine spongiform encephalopathy are very resistant to both common chemical and physical treatments. Solvents such as benzene, hexane, gasoline, and trichlorethylene have been used as extraction solvents, but little is known about their effect on infectivity. Only a small amount of data is available on the chemical inactivation of infectious microbes, mainly because the studies require huge amounts of animals and a long observation time. Concentrations of 0.3-2.5% sodium hypochlorite greatly reduce infectivity in biological assays, but often do not completely eliminate it (Walker et al., Am. J. Publ. Health 73: 661,1983 /. The data for treatment with sodium hydroxide up to 0.25 N is highly variable; at concentrations above 1N, however, it turns out to be the most effective chemical agent of all the tested. It has been shown that treating 6M-8M with urea also gives very variable results.

The results of research on decontamination in this way show that while most infections are rapidly eradicated by a wide variety of physical and chemical processes, the presence of a small sub-population of resistant infectious microorganisms makes sterilization of contaminated materials extremely difficult in practice.

Once upon a time, BSE was defined as a disease similar to endemic diarrhea. Many important questions were raised about the epidemiological and analytical levels, with the analytical level questions aimed at identifying the relationship between the microorganism and infectivity. Extensive efforts to identify nucleic acids associated with aetiological microbes have been fruitless. The only isolated component that is unambiguously associated with the infectious effect is the scrapie prion protein sialoglycoprotein / Prp ^Sc /.

Genetic research on this protein continues to provide some surprising information. Several DNA probes synthesized according to the N-terminal sequence of the protein made it possible to demonstrate the presence of a single copy chromosomal gene, which shows the same restriction pattern in both the brains of healthy and infected animals. This gene, conserved in a wide variety of species, encodes a protein called the prion cell protein (Prpc) with an actual molecular weight of 33,000-35,000, which is particularly indicative of a clear difference from PrpSc: 1 / Prpc is protease sensitive, while PrpSc is resistant. While PrPc is completely degraded by protease K; PrPSc is hydrolyzed at the N-terminal level into a fragment weighing around 5,000 and gives rise to a protein called PrP27-30. This form is associated with infectivity and is the component most abundantly obtained in infected material preparations. 2 / Both PrP ^c and PrPSc are membrane proteins, but while the former dissolves during detergent treatment, the latter tends to polymerize into fibrous amyloid structures. Similar structures (filaments associated with endemic diarrhea, SAF) have been found in infected brains and are characteristic of this type of infection. Resistance of this protein

The inactivation of infectious agents is unusual: for example, it is sensitive to treatment with concentrated alkaline solutions or exposure to temperatures above 120 ° C, and to a combination of these methods or a combination with various denaturing agents. Consequently, the only diagnostic method available for the unambiguous identification of these spongiform encephalopathies is checking the presence of SAF in infected brain tissues, extraction and immunochemical identification of the PrP27-30 protein, methods applicable only to pathological anatomy.

SAF has been diagnosed in infected bovine brains. A PrP ^Sc homologue was then isolated, and reactivity with anti-SAF serum was demonstrated. Thereafter, the N-terminal sequence of the first 12 amino acids showed 100% homology with sheep PrPSc and difference with mouse, hamster and human PrPSc due to the glycerin insertion. As soon as it was established that BSE is a disease similar to endemic diarrhea, many important questions arose on the epidomyological and analytical level. As for the latter, they concerned the identification of an infectious protein.

The unexpected BSE pogrom and all the aspects so far explained on the basis of the neurological disorders led to serious consideration of the problem. This was especially true of those people who took part in the preparation of products derived from bovine material.

Indeed, this might not be sufficient for the pharmaceutically relevant ingredients or mixtures to be obtained to be usable. It is consequently necessary to develop methods for the production of the parameters referred to by using extraction methods that ensure the elimination of the infectious protein. It is obvious that the extraction of the active fraction in terms of infectivity should at the same time preserve the biological activity of the active ingredients, which is required of the final product.

Another protein potentially dangerous for humans at this level of preparation is the myelin basic protein / MBP /. It is a protein that in humans and most vertebrates has a molecular weight of around 19,500. It occurs in three molecular forms in human myelin, and in mouse myelin 6, it is encoded by a single gene located on chromosome 18 and accounts for approximately 30% of all proteins myelin. Its exact topographic location is not yet known exactly. Its presence was noticed in the cytoplasm of oligodendrocytes, but only at the time of myelination. A presumably similar protein is present in the peripheral nervous system (P1 protein) and the ability of peripheral myelin to induce experimental allergic encephalomyelitis (EAE) in laboratory animals is related to it.

Not all of the MBP molecule is encephalitogenic, but only the part which differs between species: in rabbit the encephalitogenic part is 64-73 amino acid fragment, in Lewis rats 71-85, in guinea pig 113-121, in SJL / J mice 89-169. EAE is a typical autoimmune disease dependent on a cellular response, in fact it is transmitted from one animal to another by infusion of sensitized T cells and not by infusion of serum. Thus, the disease develops due to the introduction of foreign lymphocytes and not the recipient's lymphocytes. Another form of autoimmune disease is experimental allergic neuritis / EAN /. It can be · induced in all higher species · vertebrates by implanting crude peripheral myelin homogenate in Freund's booster. The main antigen responsible for this type of autoimmunity is suspected to be a 12,000 PZ protein present in the peripheral nervous system.

Still another infectious agent in these preparations is the bovine genome in the form of DNA. The possibility of producing biologically active proteins by recombinant DNA technology that could be used as pharmaceutical agents made it necessary to analyze the end products for the presence of DNA sediment belonging to the cell, where the desired protein was expressed. The presence of DNA fragments in pharmaceutical preparations to be used by man raises the problem of the danger of binding these fragments in the genome, with the possibility of uncontrolled

165,677 transfers of genetic information. Although it is still not possible to obtain gangliosides by recombinant DNA technology, it is necessary to apply this type of technology with simultaneous analytical control of the extraction products of crude animal material.

Finally, in order for gangliosides to be used in in vitro and in vivo tests, they should be free from components such as asialogangliosides and glycocerebrosides. These substances in high concentrations can have serious immunological implications and can also lead to erroneous experimental considerations.

The sudden onset of BSE and all other aspects that remain to be clarified in relation to neurological disorders have prompted consideration of this issue, especially by those involved in the manufacture of products derived from bovine material.

The prior processes for obtaining gangliosides as mentioned above require the product to be pharmaceutically acceptable, free from those biological contaminants which were then known to be potentially harmful to health. But, of course, the next onslaught of the aforementioned pathologies within adult cattle herds made it imperative to obtain an active ingredient which, without losing the aforementioned therapeutic properties, is characterized by the absence of an unconventional viral agent; it was obtained by the application of specific processes ensuring the inactivation of these unconventional viral agents and complete elimination of infectivity, and as a result of the use of a specific method to identify such agents. In fact, it may not be enough to use approved raw materials for consumption in order to obtain ingredients or mixtures for the pharmaceutical industry. Obviously, the initial BSE has to be assessed with attention to its biological development in vivo - attention should be paid to the different phases of the process and not to the process as a whole.

Such an in vivo analysis of biological activity is necessary at a time when scientists have not yet reached agreement on the linkage of infection with certain proteins such as PrP27-30- Of course, an extraction process that eliminates infectious activity must leave the biological activity of the active agent intact, which is necessary for use it for therapeutic purposes / 14-24 /. On the one hand, scientific research has developed methods that guarantee that the appropriate ganglioside mixtures or their individual fractions are obtained in forms free from protein, chemical and biological contaminants, and on the other hand, methods that show efficiency in destroying infectivity associated with free viruses, but in practice, the method by which it is possible to obtain them is not known, also on an industrial scale, as is the result, i.e. the product in the required form of a pure, pharmacologically active product free from infectivity associated with pathogenic agents defined as free viruses, is unknown.

The attached figure 1 shows a diagram of the method according to the invention.

Figure 2 is a photograph showing the results obtained from analyzing samples taken in intermediate steps of the method of the invention by Western method and with anti-PrP27-30 antibodies.

Figure 3 is a photograph showing the results of the analysis of samples taken at various stages of the method of the invention on bovine genomic DNA.

Figure 4 is a photograph showing the results of a Western analysis of samples taken in intermediate steps using the method of the invention with anti-MBP antibodies.

Figure 5 is a photograph of the results of silica gel chromatography of a ganglioside mixture prepared by the method of the invention.

Figure 6 is a photograph showing the biological activity of a ganglioside mixture produced by the process of the invention.

Figure 7 is a photograph showing the results of immunochemical analysis of samples taken in intermediate steps of the method of the invention with anti-PrP27-3o antibodies.

The object of the invention is to produce a product characterized by the absence of free viruses, a preferred method which is suitable for industrial production and

A method the inventive nature of which is in the correct sequence of the various extraction phases. A method that during various steps eliminates the infectious contamination associated with free viruses such as bovine spongiform encephalopathy viruses, maintains the therapeutic activity of the resulting mixture.

The product obtained in this way is a defined mixture of gangliosides or individual fractions obtained from bovine brain or parts thereof.

The method according to the invention comprises the following main steps:

a) treatment of bovine brain tissue with acetone to remove lipids, b) preparation of a suspension of acetone precipitate in a mixture of methylene chloride, methanol and sodium hydroxide and kept at 30-35 ° C for at least 3 hours to separate hydrophobic and hydrophytic substances, c / dissolving the precipitate in a mixture of water, chloroform, methanol and sodium hydroxide / pH 12 / by heating at 38-43 ° C for 4 to 8 hours, d / dissolving the precipitate in IN sodium hydroxide at room temperature for at least 1 hour and e / neutralization and dialysis of the solution containing the ganglioside mixture through a membrane cutting off a substance with a molecular weight of 10,000.

For illustrative purposes, preparations from infected bovine brains with spongiform encephalopathy or from raw protein materials obtained from uncontaminated bovine brains to which constant amounts of infected material from the 263K endemic diarrhea strain have been added are presented.

Histological analysis of the ganglioside mixture used for the extraction of the ganglioside mixture according to the invention showed fibrils typical of tissues of infected animals.

Manufacturing examples

Example I. A manufacturing process diagram is shown in FIG. 1 of the drawing.

1000 grams of infected bovine brain, ground and mixed with distilled water in a suspension form, was contacted with 300 to 600 ml of acetone (1: 5 w / v ratio) for approximately 3 hours at room temperature with agitation. The solution was then centrifuged at 6,000 x g and a temperature between 7 ° C and 4 ° C until complete precipitation. The solvent was then removed and 180-350 ml of methylene chloride / methanol / sodium hydroxide was added to the wet powder placed in a suitable glass container and left under magnetic stirring again for at least 3 hours at a temperature between 30 ° C and 35 ° C. . The mixture was finally cooled and then centrifuged for 20 minutes at + 10 ° C under a load of 6000 x g. The liquid phase was filtered through a filter funnel at + 4 ° C. The appropriate amount of calcium chloride and acetone were added to the liquid, left for about 30 minutes with stirring and centrifuged at + 10 ° C under a load of 6000 x g. The pellet (crude material 1) was finally allowed to dry overnight and then under high vacuum for 5 hours. The recovered crude material was resuspended in 10 to 18 ml of water / chloroform / methanol. The pH was adjusted to about 12 with 5N NaOH. It was heated to a temperature in the range of 38 ° C to 43 ° C for 4 to 8 hours and allowed to stir. Finally, after allowing to cool, it was neutralized with 6N HCl and the required amount of water / n-butanol / chloroform was added. It was stirred for 15 to 30 minutes and allowed to stand for 2 to 4 hours. Finally, the lower organic phase was discarded, acetone and sodium chloride were added to the remaining aqueous phase, they were mixed for about 30 minutes and centrifuged for 20 minutes at + 15 ° C under a load of 6000 x g (crude material 2).

The product was dried under high vacuum, resuspended in 6 to 15 ml dry methanol, and then hot for about 2 hours, the solution was stirred from time to time. The suspension was then rapidly centrifuged at 6,000 x g and the supernatant was placed in a freezer for approximately 2 hours. The opalescent white solution was then centrifuged at 0 ° C under a 600 x g overload, and the precipitate was dried under high vacuum. The product was collected in IN sodium hydroxide and allowed to contact the solution for at least 1 hour at room temperature. Ultimately,

The pH of the slurry was adjusted to approximately 9, and the slurry was dialyzed using a membrane having a 10 kd molecular weight cut-off rim against the appropriate volume of distilled water. Sufficient sodium chloride and acetone were added and centrifuged at + 5 ° C, 6000 x g, and then dried under high vacuum (finished product). The sample was absorbed in 10 mM phosphate buffer pH 7.2 and distilled for 30 minutes at + 121 ° C (finished product, sterilized).

Process evaluation:

As explained above, an important aspect of the production method according to the invention is to provide a ganglioside product that is free from undesirable contaminants, especially free from unconventional viruses. In order to evaluate the manufacturing method, samples were tested for possible contamination taken at various stages of the procedure.

The procedure and results of biological / clinical studies are as follows:

A bioassay for epidemic diarrhea.

The animals used in these experiments were golden Syrian hamsters (LVG / Lak). Infection testing was performed on groups of four still suckling, maternal, intracerebral (i.c.) vaccinations using 0.05 ml samples diluted 10 times in sterile phosphate buffer solution. Intracerebral immunization was performed by trained staff using disposable glass syringes with 3/8 inch, 26 gauge, sterile needles.

The final sterilized product, 20-fold concentrated, was used completely as follows:

4.0 ml injected intracerebrally to 40 animals.

3.0 ml diluted 1:20 and injected, undiluted, in 50 intracerebral animals and 22 intramammary animals (i.p.). Volume injected i.p. it was 2.5 ml.

The animals were examined twice a week or more frequently for a period of 12 months for the development of characteristic neurological clinical symptoms. Early symptoms were recorded in each animal, and animals were eliminated when disease was well established. Their brains were split in two, one was fixed in 10% formalin and the other was stored at -70 ° C. Pathological diagnosis was made in all animals that died of suspected causes and in those that showed signs of neurological disorders. At the end of the observation period, all surviving animals were liquidated and their brain pathological diagnosis was made.

Infectious titer was calculated at the final endpoint according to the method of Reed and Munch and expressed as log LD50 / ml.

The following samples were tested using product names as noted in Figure 1 of the figure.

All samples were resuspended in sterile phosphate buffer solution (PBS) using the following volumes calculated to ensure a volume titer homogeneous compared to 16.7% (w / v) in the homogenate used as the starting material:

powdered brain ml 2.0 brain homogenate 16.7% (w / v, undiluted) raw material 1 ml 5.4 undiluted raw material 2 ml 9.7 undiluted finished product ml 14.4 undiluted finished, sterile ml 7.0 concentrated 20 times

product

The results of the biological / clinical tests are presented in Table 1.

BIOLOGICAL DETERMINATION AND CLINICAL EVALUATION

CHORE 0/28 0/35 0/63 STERILIZED END PRODUCT 40-20232 50-20233 (ic) 06 r - CHORE 0/4 \ about 0/4 0/4 | and 0/2 Q / 18 PRODUCT FINAL C- CM CM ABOUT CM 1 here CM OJ ABOUT CM 1 OJ CM CM ABOUT CM AND - WHAT CM at; CM 1 '4 -20231 twenty CHORE 0/3 about 0/4 ______1 0/4 c 0/15 MATERIAL CRUDE 2 4 -20222 WHAT about: CM ABOUT CJ 1 «5 * 'S · in CM about CM Ł <3 * 4 -20225 C £ J CM CM ABOUT CM 1 ABOUT CM CHORE 0/3 ro \ l— 0/1 0/3 0/2 0/3 1/15 <> Μ S ca c W (X »- <æ- · z: <ω Σ 4 -20216 r * r- ’ S2 OJ 1 OO CM ABOUT CM 1 and· 4 -20219 about CM CM <3J CM '1 «! Γ 4 -20221 24 ί ± 3 OC ABOUT * T * about 4/4 \ Ό 3/3 ε / ε - ' 1/4 1/0 15/19 r HOMOGENIZATION CEREBRAL 16.7% 4 -20210 CM O CM 1 M ^- 4 -20212 I 4 -20213 4 -20214 4 -20215 * 3 * OJ A SAMPLE DILUTION 20-fold conc. c 0 N at c about • fH at N ABOUT here 0) • H and » C. «T o r · ⁴ Λ 1 about V 1 ABOUT in> Ł ABOUT IN 1 ABOUT f * 1 ABOUT f-H 8-01 1 e-OI SUM

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All animals showing clinical signs of epidemic diarrhea and all animals still alive at the end of the one-year control period were included in the study. Animals that were killed due to accidents and / or poor health and those that died of causes not related to epidemic diarrhea or that were killed for meat without showing any clinical signs of disease were not included in the study.

The sample column reports the number of animals injected at the start of the experiment and gives a cage number that distinguishes between different samples and dilutions. The sick column gives the number of animals that showed clinical signs of epidemic diarrhea / number of animals injected minus the number of animals that died from causes not related to epidemic diarrhea.

Additional Ratings:

The determination of the PrP protein of epidemic diarrhea is performed using polyclonal antibodies specific to the purified mouse brain protein analog and which cross-react with the bovine PrP diarrhea protein. The Western Blot method was used for the determination. The presence of the PrP protein in diarrhea was assessed by comparison with standards for proteins of different molecular weights.

The determination of the MBP protein was performed with polyclonal antibodies specific to the purified protein analog from bovine brain tissue. The method used for the determination is also the Western Biot method. The presence of MBP was assessed by comparing proteins with different molecular weights to a standard.

Determination of bovine genomic DNA was performed by the DOT BLOT technique on samples taken at different stages of production by methods known to those skilled in the art. In order to consider a sample valid, no specks should be found in the heterelogical DNA deposits. The absence of spots in the samples examined by radioautography shows the absence of bovine genomic DNA.

Figure 2 in the drawing shows the re-results determined by immunochemical analysis using anti-PrP27-30 antibodies for samples derived from some intermediate stages of the ganglioside production and purification process. The number codes of the analyzed samples correspond to the numbers in parentheses given in the purification diagram of figure 1 in the drawing.

Track 1: code sample 1

Track 2: code sample 2

Track 3: code sample 3

Lane 4: code sample 4

Lane 5: code sample 5

Lane 6: finished product

Lane 7: protein with standard molecular weight (downward, the kd values are as follows: 97.4,66.2,45.0, 31.0, 21.5).

The bands marked with an asterisk refer to non-specific cross-reactions that can be attributed to the assay amplification system.

Figure 3 in the figure shows the DOT BLOT analysis of bovine genomic DNA on samples taken from different process transitions.

The intersection of letters and numbers indicates:

Standard curves

1A - 7A beef DNA (1 mg)

IB - 7B bovine DNA (1 ng)

1C-7C bovine DNA (100 pg)

ID - 7D bovine DNA (10 pg)

1E - 7E beef DNA (1 pg)

1F-7F bovine DNA (0 pg)

Test samples

2A - 2F code sample 1

2B code sample 2

2C code sample 3

2D code sample 5

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2E code sample 6

3A raw sample 3

3B p ^^ oc ^ i ^ ltt

3C finished product, steiyliizowjmy

The points of the reference curve and the test samples are given from two determinations.

Figure 4 in the drawing shows the results of the immunochemical analysis using anti-MBP antibodies for samples derived from the various intermediate steps of the production and purification method. The sample codes correspond to the digits marked in the purification diagram of Figure 1 in the drawing.

Track 1: code sample 1

Track 2: code sample 2

Track 3: code sample 3

Lane 4: code sample 4

Lane 5: code sample 5 (raw material 1)

Lane 6: raw sample 2

Track 7: code sample 6

Lane 8: raw code sample 3

Lane 9: finished product

Lane 10: finished product, sterilized

The different forms of MBP proteins identified by the polygonal antibodies used are shown in parentheses.

Figure 5 of the drawing shows the results of silica gel chromatography for the following samples (also according to the method outlined in figure 1 in the figure):

Lane 1: finished product, sterilized

Lane 2: finished product

Lane 3: GTn trisialoganglioside standard

Lane 4: GDib disjaloganglioside pattern

Lane 5: GDu disjaloganglioside pattern

Lane 6: GM1 monosialoganglioside standard

Figure 6 of the drawing is an exemplary photograph showing the biological activity of a ganglioside mixture prepared according to the inventions (arrows indicate sprays) on a peripheral nerve.

Figure 7 of the drawing shows the results of immunochemical analysis with anti-PrP27-3o antibodies for samples taken from some of the intermediate steps of the ganglioside production and purification method. The code of the analyzed samples corresponds to the digits shown in the purification diagram of figure 1 of the drawing.

Lane 1: crude material solution 1 to which was added 1.5 micrograms / ml PrP27-30

Lane 2: raw material code sample 2

Lane 3: code sample 6

Lane 4: raw material code sample 3

Lane 5: finished product

Lane 6: sterilized finished product

Example II. Preparation of a clarified homogenate from infected brains.

Four hamster brains, infected with the 263K strain of epidemic diarrhea, corresponding to a net weight of 3.9 g, were homogenized in 10 ml of distilled water. The volume was then adjusted to 15.5 ml to obtain a homogenate with a concentration of 25% (w / v). The suspension was centrifuged for 40 minutes at 4 ° C and 1800 x g overload: 7.5 ml of the supernatant was recovered, aliquoted and stored at -70 ° C until use.

Preparation of samples ml of infected homogenate, 120 ml of methanol / methylene chloride mixture and 0.71 ml of 5.7N sodium hydroxide were added to 10 g of beef brain acetone powder. This addition was made in order to keep the total volume of the water phase in the solvent constant and to obtain the most suitable operating conditions.

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The final titer of the starting solution was 1% (w / v). The suspension was magnetically stirred for 3 hours at 33 ° C. It was then cooled and centrifuged for 20 minutes at 10 ° C under a load of 6000 x g. The liquid phase was filtered through a Gooch crucible (pore size No. 3) at + 4 ° C in order to avoid evaporation of the solvents; upon completion of this step, 78 ml of the liquid phase were recovered. A 2 mL sample was retained for bioassay. The appropriate volume of calcium chloride and acetone were added to both portions, and after about half an hour of magnetic stirring at room temperature, the samples were centrifuged for 10 minutes at room temperature at an overload of 6000 x g. The precipitate (crude material 1) was dried overnight in a fume hood. and then for 2 hours under high vacuum. One aliquot was stored at -70 ° C for bioassay purposes.

925 mg of crude material 1, recovered from the reaction vessel, was re-suspended in 18.5 ml of chloroform / methanol / water and 0.74 ml of homogenate to obtain a titer of 1% (w / v). The pH was adjusted to approximately 12 (judged with litmus paper) by adding 5N NaOH and the mixture was magnetically stirred for 6 hours at 40 ° C. After cooling to room temperature, the solution was neutralized with 6N HCl and a 250 µl aliquot was saved for bioassay purposes.

Both portions were treated with the appropriate volume of n-butanol / chloroform / water and allowed to stand for 4 hours after stirring for 15 minutes. Eventually, the lower organic phases were discarded. Sodium chloride and acetone were added to the remaining aqueous phases and, after about half an hour of magnetic stirring at room temperature, they were centrifuged for 10 minutes at 15 ° C under a load of 6000 x g (crude material 2).

This product was dried under high vacuum and the sample detached for bioassay was stored at -70 ° C. The remaining material was resuspended in 6.5 ml of anhydrous methanol and left for 2 hours at 50 ° C, with occasional stirring. The suspension was quickly centrifuged at 6,000 × g, and the supernatant was placed in a freezer at -20 ° C for 2 hours. The cold, white, opalescent solution was then centrifuged for 5 minutes at 0 ° C, 6000 x g, and the resulting precipitate was dried under high vacuum. The yield at this point was 6 mg of product. It was resuspended in 500 µl of sodium hydroxide, 460 µl of distilled water and 40 µl of homogenate and left in contact with this solution for 1 hour at room temperature. Finally, the pH of the slurry was adjusted to approximately 9 (estimated by litmus paper) and dialyzed using a membrane having a 10 kd molecular weight cut-off rim for 4 hours against 20,000 volumes of distilled water. The final volume after dialysis was 980 pl. It was divided into two portions of 500 µl and 480 µl respectively; to the first one was added the required volume of sodium chloride and acetone and it was centrifuged for 10 minutes at 5 ° C under a load of 6000 xg. This sample was dried under high vacuum (end product). 20 µl of the homogenate and 50 µl of 10x phosphate buffer solution (PBS) were added to the second portion and sterilized for 30 minutes at 121 ° C (final product, sterilized).

Assessment of the manufacturing method. As described above in Example 1, samples taken at different stages of the process were tested for the potential presence of contaminants.

The samples tested and the results are as follows. All samples were collected in sterile PBS in the following volumes calculated to provide a homogeneous volume comparable to the 1% homogenate (weight / volume) used as starting material:

Brain homogenate 25% (w / v) diluted to 1%

Raw material 1 ml 3.2

Raw material 2ml L0

Finished product γπΙ 0.5

Finished product, n / a 0.5 sterilized

Table 2 shows the results obtained in the bioassays.

undiluted suspension undiluted undiluted undiluted undiluted

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BIOLOGICAL DIAGRAM SCHEME AND CLINICAL EVALUATION

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All animals showing clinical signs of epidemic diarrhea and all animals still alive at the end of the one-year control period were included in the study. Animals removed due to accidents and / or poor health and those that died of causes not related to epidemic diarrhea or that were killed for meat without showing any clinical signs of disease were not included in the study. The sample column gives the number of animals injected at the start of the experiment and gives a cage number that distinguishes between different samples and dilutions. The sick column gives the number of animals that showed clinical signs of epidemic diarrhea / the number of animals injected minus the number of animals that died from causes not related to epidemic diarrhea.

Example III. Biological activity of a mixture of gangliosides.

A series of in vitro experiments were carried out to check whether the ganglioside mixture (obtained according to the above-described method of the invention) had any biological activity qualifying for therapeutic use in the treatment of peripheral nervous system (PNS = CNS) and central nervous system (CNS = CNS) pathologies. In particular, the in vitro activity of ganglysides was investigated to evaluate the neurite formation capacity in cultures of neuroblastoma (N2A) cells (neuroblastoma). These cells, as described in the literature (Denis-Donini et al., Neuronal Development Part II, 323-348, Academic Press, New York 1980; Leon et al., Dev. Neurosci. 5, 108, 1982) can induce under certain conditions, expressing the various functions characteristic of mature neurons, thus allowing a qualitative and quantitative analysis of biochemical parameters correlated with each stage of development.

Thus, the estimates made with this model (% of neurite forming cells, neurite length and relative branching) are important tools when investigating the possible use of a therapeutic drug for the functional recovery of the nervous system.

Materials and methods

Cell cultures

C 1300 cells from mice, N2 A clone, from the American Cell Type Collection (Bethesda, Maryland), were plate cultured at 1000 cells per well (24-position) in the presence of Dulbecco's Modified Eagle's Medium (DMEM) containing P / G (100 U penicillin / ml) and 10% fetal calf serum (FCS from Seromed, batch 4-C04).

The next day, the culture medium was replaced with the same volume of fresh medium containing gangliosides (see below). The cultures were maintained at 37 ° C in a humidified atmosphere containing 5% CO2 (Haerus incubator). Then, after the indicated time (24 hours later), the cultures were fixed with a 2% glutaraldehyde solution.

Preparation of product test solutions

The ganglizide mixture (3 different batches, numbers: 1-2-3) was dissolved in the mixture: chloroform / methanol 2: 1, dried in a nitrogen stream, resuspended in the medium: DMEM + P / G + 10% FCS until the desired concentrations.

Test concentrations: 1 x 10 ^-4 M; 5 x 10 ' ⁵ M and 1 x 10' ⁵ M.

Four different experiments were performed as follows:

- 3 experiments to evaluate the effect of a mixture of ganglosides (batch numbers 1 and 2) at a concentration of 1 x 10 µM;

- 1 experiment to evaluate the dose-response effect at different concentrations (1 x 104, 5 x 10 'and 1 x 10' ⁵ M) of the ganglioside mixture tested (batch no. 3).

Procedure

The medium was withdrawn from the wells and replaced with 350 µl of the mixture: DMEM + P / G + 10% FCS ± test product (at the above-mentioned concentrations).

Control cultures were treated in the same way, but without the addition of a ganglioside mixture. The cultures were then kept in an incubator for 24 hours, after which cells were fixed with a 2% glutaraldehyde solution and observed under a microscope.

Parameters

Summary of morphological examination to estimate:

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- percentage of cells with neurites

- neurite length and relative branching.

Results

As indicated in Table 3, it is clear that the test products are effective (1 x 10 ⁴ M) in inducing the formation of neurites in N2A cells. The performance of the ganglioside mixture is dose-dependent with maximum efficacy using a 1 x M ^ M dose (Table 4).

Table 3 (batch numbers in brackets)

Product % of cells with neurites experience 1 experience 2 experience 3 Control 2 ± 1 1 ± 1 2 ± 2 GA (1) 29 ± 6 27 ± 6 24 ± 5 GA (2) 25 ± 4 25 ± 2 25 + 5

The table shows the effect of a mixture of gangliosides (batches 1, 2) on the formation of neurites in N2A neuroblastoma cells in mice (neuroblastoma). All products were added to the cells at a final concentration of 1 x 10 µM, morphological evaluation was done 24 hours later.

Table 4

Effect of different concentrations of a ganglioside mixture (batch 3) on neurite formation in mouse neuroblastoma N 2A cells (neuroblastoma); dose-response effect

Product Concentration % of cells with neurites Control 3 ± 2 GA (3) 1 x 10- ⁴ 35 ± 6 GA (3) 5 x 10 ' ⁵ 17 ± 5 GA (3) 1 x 10 ' ⁵ 4 ± 2

(batch numbers in parentheses)

Statistical evaluation of the in vitro bioactivity data shows that there is no significant difference between the different batches of the ganglioside mixture (Figure 8 in the figure).

Figure 8 in the drawing shows the overall statistical evaluation of the data obtained for batches 1 and 2. The values reported herein are means of 9 independent tests ± standard deviation.

Moreover, the morphological evaluation of neurites shows that cells infused with the ganglioside mixture present long, significantly branched neurites (i.e., extensive branching).

Conclusions

Therefore, the above-described observations confirm that the analyzed mixture of gangliosides has biological activity, indeed, the obtained product, with a production method that guarantees its special characteristics, can stimulate the formation of neurites in N2A cells. This fact indicates that the product is effective in saving the peripheral and central nervous system. A ganglioside mixture, obtained by the method described above, free of contaminants associated with potentially dangerous unconventional viruses, can also be used for the preparation of individual ganglioside components or mixtures, such as GM1 monosialoganglioside.

In view of the pharmacological properties described above, a mixture of gangliosides can generally be used as a medicine in the treatment of numerous pathologies (caused by causes of different etiopathogenesis) of both the peripheral and the central nervous system. Specific conditions that may be treated include retrobulbar optic neuritis18

165 677 go, paralysis of the oculomotor nerves, tear of the trigeminal nerve, paralysis of the facial nerve and Bell's palsy, Garcin syndrome, radiculitis, traumatic peripheral nerve damage, diabetic and alcohol polyneuritis, 'obstetric and paralytic sciatica, diseases of the motor neuron, amyotrophic lateral sclerosis, spinal muscular atrophy, progressive bulbar palsy, muscle weakness and Lambert Eaton syndrome, muscular dystrophy, impaired transmission of stimuli at the nerve junctions of the CNS and CNS, consciousness deficiencies such as confusion, shock, thrombosis, cerebral embolism, cerebral trauma and spinal.

Administration is usually by injection, intramuscularly, subcutaneously or intravenously, or via the skin, lungs, or orally, preferably in suitably buffered aqueous solutions. The safe storage of the pharmaceutical can be ensured by making it into vials containing the product solutions, possibly together with other auxiliary ingredients as indicated in the pharmacological preparation examples below. For therapeutic or possibly also preventive use using the aforementioned parenteral routes, the dosage preferably varies between 10 mg and 100 mg per day of active ingredient.

For purely descriptive and not restrictive purposes, the following examples of pharmacological kits made according to the invention are provided below.

Example IV. The composition of one vial is as follows:

Active ingredient

- Gangliosides in the form of sodium salts 10.0 mg

- monosialotetr-ahexosyl ganglioside (GMj)

- disialotetrahexosyl ganglioside (GDia)

- disialotetrahexosyl ganglioside (GDib)

- trisialot-ephrahexosylglioside (GTibb

Other ingredients

- dibasic sodium phosphate 12 H2O 6.0 mg

- monobasic sodium phosphate 2 H2O 0.5 mig

- sodium chloride 16.0 mg

- water for injection up to a volume of 2.0 ml!

Example 'V. The composition of one vial is as follows:

Active ingredient

- gangliosides in the form of sodium salts 20.0 mig

- monosialotetrahexosyl ganglioside (GMi)

- disjalotetri ± exosyl ganglioside (GDia)

- disialotetrahexosyl ganglioside (GDib)

- trisialotetrahexosyl ganglioside (GTib)

Other ingredients

- dibasic sodium phosphate 12 H2O 6.0 mg

- monobasic sodium phosphate 2 H2O 0.:5 mg

- sodium chloride H.0 nm

- water for injection up to a volume of 2.0 m3

Example VI. The composition of one vial is as follows:

Active ingredient

- gangliosides as their sodium salts 110.0 mig

- monosialotetrahexosyl ganglioside (GMi)

- disialotetrahexosyl ganglioside (GDu)

- disialotetrahexosyl ganglioside (GDib)

- trisialotetrahexoseioganglioside (GTib)

Other ingredients

- dibasic sodium phosphate 12 H2O 12.0 mg

- monobasic sodium phosphate 2 H2O 1. <0 mg

- sodium chloride 32.0 mg

- water for injections up to a volume of 4.0 ml

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From this description of the production method according to the invention, it is obvious that the same can differ in many ways. Such various variations are not to be viewed as departing from the spirit and scope of the invention, and all such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

FIGURE 1 (1) Frozen brains pulverization fat removal centrifugation

-Liquid phase (2)

V (3) Acetone damp powder, treatment with methylene chloride / methanol / sodium hydroxide (3 hours), centrifugation, filtration

- fixed phase (4)

Liquid phase, precipitation, centrifugation

-b> - Liquid phase drying (5) Raw material 1 solubilization heating with methanol / sodium hydroxide mixture (6 hours) neutralization separation

-s> - Organic phase

Water phase + intermediate phase, precipitation, centrifugation

-s *> Drying liquid

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FIGURE 1 (continued) c * ______...__ n ouiuwy maiciidi z.

solubilization centrifugation

Solid phase (6) cooling, centrifugation

--- liquid phase drying

Crude material 3 solubilization in IN NaOH (1 hour) neutralization dialysis precipitation

-5 »- Liquid phase drying

Finished product Suspension in phosphate buffer heat sterilization in an autoclave

The product is finished, sterilized

FIG. 3

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FIG. 4

FIG.5

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FIG. 6

5 4 3 2 1

FIG. 7% of cells with neurites

50,000

40,000 ..

30,000 ..

20,000.

10,000.

0000

Fig. Θ

Publishing Department of the UP RP. Circulation of 90 copies

Price: PLN 10,000

Claims (17)

Patent claims A method for the preparation and purification of a ganglioside mixture, characterized in that: a) the ganglioside-containing tissue is treated with acetone to remove lipids and produce an acetone precipitate, b) the obtained acetone precipitate is suspended in a first mixture of solvents, which has the property of separating hydrophobic substances and hydrophilic, c / the separated mixture is filtered, and the first crude material is precipitated from the obtained first liquid phase, which e / dissolves and the dissolved heated at a pH of about 12, f / the dissolved heated first raw material is re-separated in the second solvent mixture , which separates hydrophobic and hydrophilic substances, g) a second separating mixture is separated to remove the organic phase and an aqueous phase is left, from which h) the second crude material precipitates and / the second crude material is dissolved and cooled to give a third crude material which j / ro the dissolved third crude material is essentially discharged, and the dissolved neutralized third crude material is dialyzed through a membrane which cuts off a substance with a molecular weight of about 10,000. 2. The method according to p. The process of claim 1, wherein the first solvent mixture is a mixture of methylene chloride, methanol and sodium hydroxide. 3. The method according to p. The process of claim 1 or 2, wherein the first solvent mixture containing acetone precipitate is heated at about 30-35 ° C for about 3 hours. 4. The method according to p. The process of claim 1, wherein the precipitation of the first raw material is carried out by adding calcium chloride and acetone. 5. The method according to p. The process of claim 1, wherein the first raw material is heated to about 38-43 ° C for about 4 to 8 hours. 6. The method according to p. The process of claim 1, wherein the first raw material is dissolved in a mixture of water, chloroform and methanol, and then, after heating, a second solvent mixture consisting of water, chloroform and n-butanol is added. 7. The method according to p. The process of claim 1, wherein the second raw material is precipitated by the addition of acetone and sodium chloride. 8. The method according to p. The process of claim 1, wherein the second raw material is dissolved in methanol. 9. The method according to p. The process of claim 1, wherein the third raw material is dissolved in 1N sodium hydroxide. 10. The method according to p. The method of claim 1, wherein the ganglioside containing tissue is bovine brain tissue. 11. The method according to p. The process of claim 1, characterized in that the produced ganglioside mixture is dried. 12. The method according to p. The process of claim 11, characterized in that the dried end product is suspended in a buffer and sterilized. 13. Method for the preparation of a ganglioside mixture, characterized in that: a) the ganglioside-containing bovine tissue is treated with acetone to remove lipids and produce an acetone precipitate, b) the obtained acetone precipitate is suspended in a first solvent mixture consisting of methylene chloride, methanol and hydroxide sodium, c) the first mixture of solvents containing acetone precipitate is filtered and the first liquid phase is obtained, from which the first crude material is precipitated by adding calcium chloride, e) the first crude material is dissolved in a mixture of water, chloroform and methanol and heated with pH about 12 at about 38-43 ° C for at least about 4-8 hours, f) the heated dissolved first raw material is fed to the second separation mixture consisting of water, n-butanol and chloroform. 165 677 g / this mixture is separated, the organic phase is removed and the aqueous phase is left, from which the second crude material is precipitated by adding acetone and sodium chloride and centrifuged, and / the second crude material is dissolved and heated in methanol, / the dissolved and heated crude material is centrifuged to produce the supernatant, k / the supernatant is cooled, and a third crude material is obtained, which 1 / dissolved in sodium hydroxide in about 1 hour, m / the dissolved third crude material is neutralized and n / neutralized is dialyzed through a membrane which cuts off substances with a molecular weight of about 10,000. 14. The method according to p. The process of claim 13, wherein the third raw material is dissolved in 1N sodium hydroxide. 15. The method according to p. The method of claim 13, wherein the ganglioside containing tissue is bovine brain tissue. 16. The method according to p. A process as claimed in claim 13, characterized in that the ganglioside mixture produced is dried. 17. The method according to p. Process according to claim 16, characterized in that the dried end product is suspended in a buffer and sterilized.