DE2404416A1 - NEUROTROPICALLY EFFECTIVE AGENT AND METHOD FOR ITS MANUFACTURING - Google Patents

Description

The invention relates to a neurotropically active agent, in particular for the treatment of hitherto at all degenerative neurological diseases that are impossible to treat or difficult to treat, such as amyotrophic lateral- '. Sclerosis and a method of making this agent.

Degenerative neurological diseases result in chronic cases to severe physical disability, such as _e paralysis, and even death. Although the causes of such neurological diseases are not always known, numerous such diseases are the result of certain infections, _{e.g. virus infections o} It is likely that the viruses causing the specific neurological diseases stick to the nerve cell receptors of the motor cells of the central nervous system or they interact of the viruses concerned with the nerve cell receptors. Some experts in the relevant art suspect that these nerve cell receptors represent individual anatomical nerve cell structures. Other experts believe that the receptors are theoretical biophysical terms that describe one of the functions of nerve cells act as

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whether (in the nerve cell) physical receptors would exist, It is further suspected by those skilled in the art that
through virus-related neurological diseases at least
partly due to viral damage to the nerve cell receptors and possible destruction of the nerve cell
are due. In any case, the attacked nerve cells stop working like healthy cells. The nerve cells attacked presumably contain a range of abnormal conditions from partial and consequently reversible damage to complete destruction or neurophagia.

Numerous degenerative neurological diseases progress insidiously and slowly cause one
Harm to the victim. Such diseases are often overlooked in the early stages even by experienced clinicians, whereby the disease can progress to a point at which a weak paralysis, a twitching of the
Muscles or other early symptoms specific to the disease in question can be observed. Of course, at this point in time numerous nerve cells are already damaged and have therefore, as already stated, stopped working normally « Impairment of the pyramidal orbits, which causes a loss of balance and similar disturbances, even to a bulbar paralysis and possibly to death. The prognosis for numerous diseases of this type is indeed extremely unfavorable

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chemotherapy does not cure the effects of the disease but essentially only slows it down.

It would therefore mean a huge step forward to give the specialist a therapy could, with the help of which the progression of such degenerative diseases of the central nervous system halt or even reverse it.

Taking into account the previous statements, the object of the invention was to provide a means for stopping or even to reverse the progression of progressive degenerative diseases of the nervous system that cause the Function of the motor nerve cells from their origin to the neuromuscular connection, such as elements of the central nervous system, namely, axons, nerve medullary sheaths and the like., affect, i.e. a means of combating der amyotrcpMsehen lateral sclerosis, multiple sclerosis, kuru, Polymyositis, certain meningitis, muscular dystrophy, polyomyelitis and the like. The means in question should be safe, effective and capable of being administered over a long period of time. “Furthermore, the invention was still there the object of the invention is to create a method for producing such an agent.

It has now surprisingly been found that certain modified neurotoxins have the ability to appear to adhere to the motor nerve cell receptors or to be assigned to the motor nerve cell receptors in some other way and these receptors to prevent them from sticking or an assignment of pathogenic organisms, viruses or proteins with potentially harmful effects mask or block. The modified neurotoxins come from the poisons of certain species of snakes and are

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by detoxifying the toxic parts of the poisons while maintaining the neurotropic character of the obtained detoxified part of the poisons and the remaining poison parts produced. Usually the poison gets through controlled treatment with oxygen or oxidation detoxifies, although other known detoxification processes except for certain processes for the preparation of Snake sera can be applied. The detoxified snake venom is then stabilized for storage. That Means according to the invention can be made from all snake poisons, which act essentially as neurotoxins and not essentially as hematoxins, are recovered. As but will be explained in more detail later, the agent must be obtained from a poison, at least partially from a poison that penetrates the central nervous system on a large scale, such as that from the snake genus Bungarus is won.

The agent according to the invention is.by controlled detoxification at least partially from a neurotoxic snake venom high penetration capacity (into the central nervous system) existing gifts made. In this connection it should be noted that the venom of most snakes contains somewhat neurotoxic and somewhat hematoxic components. However, the various lengths can be changed essentially in a first group, namely whose poison acts mainly through a disturbance of the blood chemistry, and a second group whose poison is mainly about destruction the nerve cell components work. The former genus / i.e. the haematoxic venomous snakes is also called the common North American cave vipers such as rattlesnake, copper headed snake, and water moccasin snake. The latter group of these Gatbungai, i.e. the neurotoxic venomous snakes, is made by the cobra, paraguda and coral snake,

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represents. However, there are gradual differences between these groups, for example the venom of the North American rattlesnake is almost completely hematoxic, while the venom of the Central American rattlesnake is hematoxic, however contains significant neurotoxic components. on the other hand owns a certain species of the South American rattlesnakes, namely the Crotalus terrificus terrificus, a poison that is extremely neurotoxic. The poison of the Paraguda, for example the blue krait, is, as an extreme case, a neurotoxin so penetrating that the central nervous system is very badly affected and there is hardly the bite of this snake survivor. The term "neurotoxic Snake poison "stands here and in the following for a snake poison, that is primarily, but not exclusively, toxic to nerve cells and minor components.

The venom of snakes contains a variety of chemical compounds, including various enzymes «The exact action of the enzymes in the poison is not yet complete clarified. Indeed, the enzymes cannot play a direct role in the toxic effects of the poison, ”The Enzymes others may have beneficial effects on the snake in exploiting the victim of the bite for food. For example, it is known that the body of an animal killed by snakebite decomposes more quickly than that Body of an animal that died in another way. It has also been shown that by chromatography separated higher molecular compounds of cobra venom are not significantly toxic. In contrast, the low molecular weight compounds of this poison are highly toxic of the specific compounds in snake venom, the agent according to the invention is preferably made from the whole

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Poison, which of course contains the enzymes, is produced. On the other hand, it was also recognized that numerous components of the poison are inert for the intended use in the present case and separated from the active parts of the poison could. The expression "snake venom" here and in the following is preferably the entire venom, but also (only) to understand the toxic portion of the same, this definition also applies to the expression "snake venom neurotoxin".

Without being bound by any particular theory, the agent according to the invention is believed to work in such a way that it does Extent remains attached to the nerve cell receptors (or combines to such an extent with the nerve cell receptors), that the receptors for adherence or attack by neurological bacteria, viruses or Proteins-with potentially harmful punctures, no more are available «, In this way the progression degenerative nervous system diseases stopped. However, it should be clearly pointed out that the means according to of the invention does not have a prophylactic effect and consequently cannot prevent or control a viral infection. the The effect of the agent according to the invention probably corresponds to the effect of a blocking the cell receptors Means with absorption and excretion by the cells, which is why a dosage is required until the penetrating causative agents are lost through the body's immune response or through natural selection as a result of their lack cellular nutritional factors essential for the existence of the invaded bodies or substances, disappear «, consequently a continued treatment with the agent according to the invention over a longer period of time may be necessary be to ensure that the nerve cell receptors continuously resist attack by the

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The body or substances causing the disease are blocked.

The investigations by Lamb and Hunter (Lancet 1:20, 1904) showed, on the basis of histopathological tests with primates killed by the neurotoxic poison of the Indian cobra, that practically all motor nerve cells in the central nervous system were attacked by the poison. The invention was based on the knowledge that such a neurotropic snake venom in a practically non-toxic form also reaches the same broad spectrum of motor nerve cells and blocks or disturbs the invading pathogenic bacteria, viruses or proteins with possibly harmful properties of the agent according to the invention used snake venom consist of a neurotoxic venom of the type described. Since the poison doses required to block the nerve cell receptors are far greater than they would be sufficient to kill the patient quickly, it is of course essential that the poison has been detoxified _o On the other hand, the essential components of the poison must remain intact in order for it to have the desired effect on the nerve cell receptors' and can effectively block the receptors against attack by bacteria, viruses or possibly harmful protein, it is also essential that the components of the poison are not denatured or otherwise impaired during detoxification. According to the invention, a non-denatured but detoxified poison must therefore be used. The non-denatured poison is referred to as "neurotropic"

In order to be able to detoxify the poison under the compelling conditions described, the poison is

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preferably detoxified in the mildest and gentlest possible way. Although different detoxification procedures, e.g. treatment with formaldehyde, fluorescein, dyes, UV light and the like «,, are known, consists of the Detoxification according to the invention, although the specific detoxification measures are not particularly critical, preferably from a mild oxidation or treatment with oxygen at relatively low temperatures. In It is very advantageous to use a modified Boquet detoxification process, as will be explained in more detail serve.

The usefulness of a special detoxification process can be determined in the most convenient way using the classic Semliki Forest virus test. The implementation of this test is known. Here, in short, a chicken embryo fibroblast cell culture on a glass plate with a gelled nutrient solution, such as Hank ¹ shear solution covered with lactalbumin. The cell layer is then inoculated with the Semliki Forest virus, with the number of spots formed indicating the titer of the virus washed, whereupon the cells show little or no staining. If, however, a significant number of stains are noticeable, the detoxification process carried out with the poison in question is too drastic, with unacceptably strong denaturation of the poison having taken place. Of course, almost or no stains should be detectable in this test. Nevertheless, as is readily apparent, few stains are tolerable compared to the number of stains in the non-washed chicken cells. The cells washed with the detoxified poison should have at least one statistically significant

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Inhibition of staining by the virus, e.g. a J5O, especially 50, preferably 70 to 75% inhibition of the stains. As will be explained in more detail later, with the same Test suitable concentrations of the poison detoxified according to the invention are determined. In fact, this one shows Test very nicely the ability of the detoxified poison to prevent the proliferation of viral spots,

In addition to the Semliki Forest virus test, a test of the detoxified poison should be carried out on living animals to ensure the proper conduct of the detox process and the strength or that Establish absence of toxicity. The toxicity of the respective agent can be determined by vaccinating common test animals, such as mice, rats and guinea pigs. These attempts can, if necessary dogs and monkeys are also used. In the laboratory animals There must be essentially no evidence of toxicity.

If the special detoxification procedure is the portrayed Standard (Semliki Forest virus test and testing on living animals) is sufficient, it can be used according to the invention. So must the agent is a detoxified, neurotropically active snake venom derivative. The neurotropic Activity can be determined according to the Semliki Forest virus inhibition test described. The neurotropic affinity is shown in a patient's responsiveness to therapeutic treatment.

As already stated, the poison can be conveniently removed by a modified Boquet method ("Ann. Inst. Pasteur", 66079-396, 1941) detoxify. First, a

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Solution of the poison in a suitable solvent, especially water, prepared. Although the respective Concentration of the poison in the solution is not critical, it can easily be up to about 3% by weight Make a solution. An anti-foaming agent can be added to the solution, as snake venom usually occurs tend to foam in the form of a solution »Any non-toxic inert agent was used as an anti-foaming agent be used. Numerous such agents are known. In particular, you can use this for food Use silicone compounds. An oxygen-forming compound is then added to the resulting solution of snake venom added. But it can also contain an oxygen-containing gas, in particular a gas containing nascent oxygen, simply bubbled through the solution. On the other hand, one can look at certain mechanisms that work in situ cause oxygen formation, e.g. UV light or Pluorescein dyes, serve to generate oxygen from an aqueous solution. . Even more convenient it is, however, chemically pure hydrogen peroxide in the form of a JO ^ igen solution together with a solution Add catalyst to activate the hydrogen peroxide, e.g. copper sulfate. Because the detox is based on the basic Page expires, the pH value of the solution is adjusted with a suitable base, e.g. an alkali metal or alkaline earth metal hydroxide, carbonate and the like, e.g., sodium hydroxide, are adjusted to above 7 and preferably below 10. Further you can also use gaseous ammonia, ammonium hydroxide, a non-toxic amine and to adjust the pH use.

Suitably the solution is made with a conventional buffer, for example an alkali metal phosphate or acetate buffer buffered to a suitable pH. If no buffer

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is used or if left to stand for a long time even if a buffer is used, the pH value may drop. In this case, additional amounts of a base may be required to maintain the appropriate pH
will.

The solution is kept at moderate temperatures, for example at temperatures between about 15 ° and 40 ° C. The upper part of this temperature range, Deho temperatures of about 20 ° to 40 ⁰ C., preferred temperatures outside this range may be indeed observed, but lower temperatures increase the detoxification duration and higher temperatures may cause an unacceptably strong denaturation of the poison. Occasionally or optionally continuously, the mixture can be stirred. Depending on the temperature and the respective poison, the detoxification is completed after up to about 30 days, in particular between 6 and 16 days, the poison having been modified in the manner necessary according to the invention . Shorter or longer detoxification times can then be selected, provided that the requirements of the Semliki-Porest virus test and the test on living animals are met in a suitable manner.

The detoxification reaction can be started by adding a catalyst deactivator, which prevents the further catalytic effect of the catalyst on the hydrogen peroxide, stopped will. There are numerous suitable deactivators, with chemically pure catalase being preferred for this purpose.

Since the modified neurotoxin made from the poison contains ions formed during the detoxification process, Which in view of the intended use is neither desirable

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are still effective, these ions, especially the copper ions of copper sulphate (which as such are somewhat are toxic) from the modified neurotoxin product removed. The ions can be removed by any method. One is particularly suitable for this Usual dialysis with semipermeable membranes. The detoxified solution is merely converted into a semipermeable membrane, E.g. made of cellulose acetate, whereupon the whole thing in a mi ^ a phosphate buffer / sodium chloride solution of a pH 6.8 filled tank is immersed. This leads to a transfer of the unwanted ions from the modified neurotoxin solution into the salt bath. In suitable Such dialysis is carried out for about a day at room temperature or for about 1 hour to 20 days at temperatures of 50 ° to 0 ° C.

The modified neurotoxin is preferably filtered, for example through a series of membranes with graduated pore diameters, in particular a series of membranes with an end membrane with a very small average pore diameter, for example about 0.22 microns, in order to ensure sterility Solution preferably the commercial preservative merthiolate added in a concentration of 1: 10000. Before the final filtration, the pH of the product should preferably also be adjusted to below "J, for example 6.8, by adding a non-toxic acid suitable as a food additive, for example a mineral acid, acetic acid, lactic acid and the like. A suitable buffer can also be used to keep the pH value on the acidic side. The specific pH value is not critical; pH values below 4 are, however, undesirable for certain forms of administration, e.g. subcutaneous injection. Values above 4 and below 7

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are therefore preferred

The normal dosage of the modified neurotoxin according to the invention for a middle-aged male patient of about 79.5 kg body weight is 0.7 to 2 ml of the agent prepared from a 1% strength by weight solution of the snake venom older patients with greater or lesser body weight are adjusted accordingly. Generally speaking, doses of 0.05 to 10 ml, preferably 0.4 to 5 ml, of the agent prepared from a 1% strength by weight solution of the snake venom can be administered. PUr on other than 1 wt _o - # weight solutions of the toxin produced resources are set, the dosages accordingly. Although the modified neurotoxin can only be administered to a patient every two weeks, it is expediently administered at least weekly and preferably every other day or daily, for example every five times a week. The agent according to the invention can be administered orally, subcutaneously, intramuscularly or intravenously. Obviously, the agent loses some of its effectiveness when administered orally. The possibility of shock appears to be greater with intravenous administration. Thus, subcutaneous or intramuscular administration is preferred. If necessary, the solution can be converted into customary tablets, powders, elixirs and solutions in accordance with the desired form of administration. Conventional binders, extenders, diluents, preservatives and the like can be used here. However, a simple physiological saline solution and the like are preferred for making injections.

As already stated, the agent according to the invention is not prophylactic and eliminates or prevents one

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Infection does not cause pathogenic viruses. Consequently, the therapy must be continued for an indefinite period of time. However, continued treatment does not present any major difficulties, since there are no contraindications and since no other drug, except perhaps vitamin B, p, has to be administered. Obviously, however, the vitamin B, g as nerve-active material somewhat disturbs the effect of the agent according to the invention, which is why the vitamin B, _{2 is} preferably not given in conjunction with the agent according to the invention. On the other hand, seem corticosteroids when co-administered with the agents according to the invention a synergistic effect to develop and the effect of the agent according to the invention to intensify "With simultaneous administration of corticosteroids, Z ₀ B, cortisone, the dose of the agent according to the invention lowered will. On the other hand, the patient should also be closely monitored in order to identify undesirable drug activity as quickly as possible.

As already stated, the snake venom from which the agent according to the invention is obtained must consist of a newly toxic snake venom. However, it was also found that a part of the neurotoxic snake venom must be one with broad penetration into the cells of the central nervous system, such as is obtained, for example, from the genus Bungarus. The remainder of the neurotoxic poison can suitably consist of a poison with a more specific ability to penetrate the cells of the central nervous system and can be obtained, for example, from the genus Naja, Z ₀ B. Naja naja, Naja haja, Naja flava, Naja hannah and Naja tripudians. In this context, as already mentioned, certain

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neurotoxic species of the genus Crotalus, e.g. Crotalus terrificus terrificus. The genus Bungarus includes in particular Bungarus multicinctus and faseiatus * The venom of the genus Bungarus has been found to spread across the cells of the central nervous system penetrates, which is why an agent obtained therefrom according to the invention can also be active against nerve cells, that of agents made from other neurotoxic poisons, even other neurotoxic poisons from snakes of the same family, are essentially unaffected.

In the latter context, it was found that the cobra poison is a somewhat selective neurotoxin. An agent obtained from it is able to protect some cells, especially motor cells, against a virus attack, while other cells are not protected as effectively, so that they are caused by viruses or other causes Diseases can progress, although with greatly reduced speed and impairment. The beneficial effects of agents obtained from cobra poison have been described variously (cf., for example, Sanders and co-workers, “Antipoliomyelitis Action of Certain Toxoids, Acta Neuroveg.” Springer-Verlag, Vienna (published on the basis of results obtained at the 6 ₀ Symposium for Vegetative Neurology in Strasbourg on September 29, 1955); Sanders et al. "Neurotoxoid Interference With Two Human Strains of Poliomyelitis in Rhesus Monkeys" Ann. NY Acad. Sci. 58, page 112, 1953; Sanders "Naja flava Neurotoxoid Interference Late In Experimental Poliomyelitis "J. Path Bact 68, pages I267-1271, 1954; Sanders and coworkers" The Role of Naja flava Toxoid and Toxin in Experimental Poliomyelitis, Acta Neurovegitativa "8, Selten 362-371, 1954; Sanders and coworkers" Neurotoxoid Interference in Macacus rhesus Infected Intramus-cularly with

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Poliovirus "Science 127, pp. 594-596, 1958; Sanders and coworkers "Neurotoxoid Interference Principle In Pseudorabies Infection, In Vivo, Inactivation of Aujeszky virus, Proc. 7th Inter Cong, Microbiol. ", P. 293, 1958 and Sanders "Deceleration of Degeneration of Amyotrophic Lateral Sclerosis in Eight Cases" Proc. Pan Am Med Cong. I96O0 All cited references can be used for explanation the invention can be used. See also WoBe Clark "Supplementary Treatment of Herpes Simplex Infections of the Cornea by Neurotropic Toxoids "Preliminary Report, Concilium Ophthalmologicum Acta, XVIII Congress, Brussels 1958 and W.B "Clark" The Use of Sanders Neurotoxoid I (Modified Snake Venom) in the Treatment of Recurrent Herpes Simplex of the Cornea "Progress Report, Southern Medical Journal, Volume 55, No. 9, Pages 94? - 951, September 1962. These two references can also be used to understand the invention. The modified heurotoxins The cobra venom brought considerable benefits, but they were incapable of port-pacing some neurological ones Stop Diseases. Furthermore, the modified neurotoxins produced from the cobra poison alone could not be completely detoxified, as this would lose strength and the desired effect. So could the experiment on living animals never shows a complete detoxification. Of course, there is an administration an incompletely detoxified poisonous snake venom poses a greater or lesser danger. Even with those modified Neurotoxins were administered to the entire detoxified solution. Certain components of the same caused however, undesirable toxic side effects other than those caused by the poison itself.

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As already stated, the agent according to the invention must be made from a poison that at least partially from a poison with a broad penetration spectrum, i.e. from a poison of the genus Bungarus, originate. Although the Bungarus poison is similar to the Naja poison, the two poisons are different with regard to the intensity of the physiological effect and that required for the physiological effect Time. The Naja poison works more intensely and faster. On the other hand, the Bungarus poison is not as selective as the cobra venom and active against a wider range of nerve cells. Polarity is obtained by combining better results from Bungarus poison with Naja poison than with Naja poison alone or Bungarus poison alone, although the Bungarus poison alone, in contrast to the Bungarus poison alone, can be effectively exploited.

Although the ratio of Naja poison to Bungarus poison is not, and in fact only, critical within narrow limits Bungarus poison can be used, one should preferably use a mixture of Naja poison and Bungarus poison in the weight ratio 400: 1 to 8: 1, preferably about 80: 1 to 40: 1, work o

The following examples are intended to illustrate the invention in more detail.

example 1

This example illustrates the neurotropic properties of neurotoxic snake venom, which after a modified Boquet method were detoxified.

In the present case, type I (Brunhilde) and type II (Lansing) poliomyelitis viruses were used.

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Virus was stored as a 1: 5 emulsion in 20 $ rabbit serum in sterile distilled water at a temperature of -5O ° C. The emulsion came from combined cervical and lumbar spinal cord enlargements of intracerebral infected rhesus monkeys with the first signs of paralysis. The Lansing virus was also ^{stored at a temperature of -3O 0} C in a 2O56 mouse brain suspension. The intracerebral LD, - ₀ in mice fluctuated between 10 " * ' ² and 1O "*' ⁵ .

The test animals were Macacus rhesus monkeys and CPW mice used. All virus injections were by intracerebral or intrathural route. The monkeys were Type I virus (Brunhilde virus) used as test virus « In the cerebral technique, diluted virus suspensions were found in the parietal or frontal areas of the brain the monkey injected.

The poisons used came from the poisons of the South African Cobra Naja flava and the South American rattle snake Crotalus terrificus terrificuse The poisons were detoxified using a modified Boquet method. Because of the requirements of the special neurotoxins were here Changes required So it has been shown that higher concentrations of hydrogen peroxide (in the form of a Solution) were more appropriate and that a better detoxification curve could be achieved with the addition of formalin. Further Since a stable neurotoxin was required, it had to be ensured that the detoxification reaction was completed was brought. This was done by adding catalase and changing the weakly alkaline pH of the solution achieved a value of 6.8 at the time of the catalase addition. The change in pH was necessary as the detoxification continues in the alkaline range.

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¹ K) g of dried venom Naja f lava were introduced into a flask and gradually about 38OO _m i admixed with an aqueous phosphate buffer solution of pH 1.6 to 7.8 and a small amount of a commercially available anti-foaming agent. The flask was shaken occasionally until all of the poison was dissolved. At this point, 6 g of NaCl were then added. After adding 2.0 ml of a freshly prepared 1, above CuSOw solution, the flask was allowed to rotate vigorously. While the flask was still rotating, 80 ml of a commercially available 30% HgOg solution and then 8 ml of 28% formalin were slowly added. The contents of the flask were finally transferred to a 4 l flask, whereupon the flask was filled to k 1 with the buffer solution. The whole was finally poured into a 6 l Florence flask, whereupon the flask was plugged with a gauze and cotton stopper covered with wax paper and finally placed in a ½ ° G warm incubator.

The rate of detoxification was predictable, but it was checked daily by testing on live animals. This was done by intravenous indexing of various amounts of the 0.5 ml of physiological saline solution a constant volume of test solution in CFW mice weighing 20 g. If 0.5 ml of the undiluted solution to the death of About 50 $ of the mice carried the solution from the incubator removed and the detoxification process quickly stopped by adding commercial catalase. To ensure, that enough catalase had been added to destroy excess peroxide became a smoldering stick of wood inserted into the neck of the flask. If there was still a free oxygen-releasing reaction taking place, the wooden stick began to burn. In order to ensure that the detoxification reaction is stopped even further, the pH of the solution was set during

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of the detoxification process had been kept at about 7.6 , decreased to about 6.8. It should be pointed out at this point that it may be necessary to add NaOH pellets or a sodium hydroxide solution during the incubation period, as the pH value tends to drop. After the end of the detoxification, chlorotone was added, then pre-filtered through a K 2 aeitz clarifying filter and finally sterilized by filtering the solution through a large S 1 Seitz pilter. Now the agent could be filled into bottles and stored in a refrigerator without changing its effectiveness. When carrying out the measures outlined, the following amounts of substance were used;

Dried Naja flava poison 40.0 g aqueous phosphate buffer solution

(pH value 7.6) 5800 ml Antifoam agents traces NaCl 56 g CuSOh (l, O # Lge solution) 2 ml H ₂ O ₂ (30 # solution) 80 ml Formalin (39 # solution) 8 ml Aqueous phosphate buffer solution to fill up 4000 ml Catalase upon need Chlorotones 8 g

If the modified Boquet method was carried out with Crotalus terrificus terrificus venom, the Detoxification reaction faster. To obtain a Crotalus terrificus terrifious remedy that meets the above requirements was obtained (compared to the measures outlined) with half the volume of hydrogen peroxide and formalin worked. Even with this modification, the partial peroxide detoxification phase was usually within finished in 15 hours. After adding catalase

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then became the Crotalus terrificus terrificus solution transferred back to the incubator at a pH of 7 * 6. The detox process was far slower further from, the results of which could be followed via the mouse test. Within 2 to 3 days a satisfactory product was obtained. The reaction was started by changing the pH to 6.8, Finish filtering and storage of the agent at a temperature of 4 ° C. The experiment on a living animal for verification of the Crotalus terrificus terrificus remedy corresponded to the experiment on living animals with the Naja flava agent was performed.

6 rhesus monkeys were _{inoculated intracerebrally with about 50 0} (paralyzing dose in 50 # of the test animals) Brunhilde virus «, 24 hours later 3 of the test animals received a single injection (3/8 ml / kg body weight) of the agent derived from Naja flava, the the remaining 3 test animals were treated as control animals and received no injections. On the seventh day after infection, either tetraplegia or severe symptoms of paralysis were found in the 3 untreated test animals. The 3 treated test animals showed no signs of disease whatsoever. On the ninth day, on which tetraplegia was detected in all 3 untreated test animals, the disease only began to be detectable in the 3 treated test animals. In the treated group, symptoms of paralysis were evident on the ninth, eleventh and thirteenth days. This meant an average incubation time of 11 days compared to an average incubation of 7 days for the untreated test animals. This represents a drastic immunity test for the person skilled in the art, with the results described without

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further demonstrate the excellent effectiveness of the neurotropic agent «

Similarly, 58 untreated control rhesus monkeys an immunity test were subjected to poliomyelitis, wherein only 1 monkey Survived ₀ In contrast, survived of 272-challenge subject and treated Rhesus monkeys 69, wherein 22 no signs of disease exhibited and ¹ VJ survived in mainly mild paralysiertem condition "Faced this drastic immunity test again showed the pronounced effectiveness of the neurotropic agent according to the invention.

The following Table A shows the results of similar immunity test in which the agent was used in therapeutic basis _β After the test animals infected and arbitrary groups had been selected (it), no test animal was treated and containing no test animal the means to when attempting a A clear occurrence of paralysis was ascertainable until one or more test animals in each group appeared so paralyzed that this could be ascertained without manipulation through the cage. On that day, which was the 5th day after infection, treatment was started in a fixed manner. As can be seen from Table A, nearly $ 50 of the treated test animals showed no stronger signs of paralysis. A comparison of the 45.8 # of animals that survived well in the treated groups with $ 0 in the two control groups shows the excellent effectiveness of the neurotropic agent in the respective groups Illness,

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Table A. Example __

No. No.

middle

Mouse LD ml

R _v (ml means per number of infi- gut survivors

kg body weight
of the monkey ⁺⁺

adorned rhesus monkeys

Monkeys ⁺⁺⁺

number

40 NP 40 NP 44 NP 44NP

0.6 0.6 0.3 0.3

(Comparison animals)

(Comparison animals)

0.4
0.4
0.1
0.2

6 6

6 6 6 6

0 0

3 2

0 0

) 45.8

++) The dose indicated for each agent was taken on the 5th, 6th, 7th, 8th. and 11 “day after injection administered.

+++) "Surviving well" means that the monkey in question showed no signs of poliomyelitis.

ro

CD

In a similar experiment with 99 treated animals and 24 untreated control animals, administration of the agent was not started until 6 days after the intracerebral injection. None of the 24 untreated control animals survived. In the treated group, 17 animals survived either slightly paralyzed or without paralysis. Reference is therefore made specifically to these 125 test animals because the immunity test was extremely tough: 50 PDc ₀ per test animal were injected intracerebrally. In the treated group of test animals, some test animals survived, despite administration of the agent, only at a point in time at which a number of the test animals were already showing symptoms of paralysis.

In a similar series of experiments, 56,000 mice became intracerebral injected with Lansing virus bloom. One group was treated with the agent according to the invention, the other group served as a comparison after treatment with a placebo. When the daily paralysis and death rate was plotted graphically for both the placebo comparison animals and the groups treated with the agent, the comparison animals followed a straight line within the expected limits. In the treated groups was between 4th and 7th day after infection, a deviation from 50 # less infected and dying animals can be determined. the however, both curves met either abruptly or gradually. «The survivors in all groups were finished No difference was found in the 30-day trials, which illustrates the need for long-term treatment,

Example 2

40 g of dried Naja naja and 0.5 g of dried Bungarus multicinctus venom were mixed with 600 ml of an aqueous phosphate 409832/1005

entered buffer solution of pH 7.5, whereupon traces of a commercially available silicone antifoam agent was added and the mixture was stirred to dissolve the toxins. Now, while stirring, 2 ml of a liquid solution of chemically pure copper sulfate were added. After 80 ml of hydrogen peroxide had been added, the solution was poured into a calibrated flask and made up to 4000 ml with the aqueous phosphate buffer solution. Under monitoring of the pH of the solution was then incubated at a temperature of ⁰ C jfj. If necessary, the pH was kept at about 7.5 by adding a 1 η hydroxide solution. Aliquots of the solution were examined daily for toxicity, by adding 0.5 ml of each undiluted solution was intraperitoneally inoculated into 20 g of heavy Muse ₀ After 14 days of detoxification died 20 so inoculated mice within 24 not std. Detoxification was also tested by giving guinea pigs weighing 350 g each with 5 ml of the undiluted solution. No guinea pigs died within 24 hours. The mass of the solution was then mixed with 3 mg of catalase per ml of solution and poured into a cellulose acetate dialysis bag. The dialysis bag specifically used was one with pores 0.22 microns in diameter. The closed dialysis sack was immersed in a dialysis fluid composed of 1 part by weight of Sörenson buffer and 3 parts by weight of 0.9 # NaCl solution? One volume of the solution was dialyzed against 9 volumes of dialysis fluid. The solution was then filtered through clear membranes and finally through a 0.22 micron filter. Sodium ethyl mercurithiosalicylate was added in a concentration of 1/10000 and the pH of the solution was adjusted to 6.8 with 1 n hydrochloric acid. The Semliki-Porest virus test showed good stain inhibition.

+) laid - 26 -

409832/1005

visibly of its sterility and, through an experiment on living animals, of its toxicity and safety examined.

It should be noted that there are several significant differences between the compositions of Examples 1 and 2 exist. The formalin and chlorotone were removed from the agent of example 1, the agent of the example 2 was dialyzed. Furthermore, the final filtration of the agent of Example 2 was carried out over a series of membranes with an end membrane having a pore diameter of 0.22 microns. Finally it was in this case, sodium ethyl mercurithiosalycilate was added in a concentration of 1/10000 parts. Essential however, it is more important that the agent of Example 2 contains the detoxified Bungarus poison and in comparison is non-toxic to the agent of example 1, which has residual toxicity,

Example 3

Example 2 was repeated with 1 g of Bungarus multicinctus /.

example H-

The agents of Examples 1, 2 and 3 became clinical Investigations used «The agent of Example 1 showed the general disruptive effects of the detoxified neurotoxic Snake venom with Naja venom alone. The means of Examples 2 and 3 showed better results,

In order to interpret the therapeutic results achieved with the relevant agents, it was necessary to assess the clinical condition to classify the disease of the patient as to

409832/1 005

Time of treatment at various stages of amjdfcrophic lateral sclerosis were given. It should be noted that the specific clinical picture in its course in the different individuals could differ. This also applies to a disease whose course is the same as that of amyotrophic lateral sclerosis is actually predictable is. The classification in Table 1 shows the severity of the disease in the patients examined.

Table 2 shows the therapeutic responsiveness of the patients classified according to Table 1. A group of 41 patients with amyotrophic lateral sclerosis was presented. In each The case was diagnosed with "amyotrophic lateral sclerosis" Provided by a qualified neurologist and confirmed by a second neurologist or a recognized clinic. The results suggesting useful therapeutic behavior were of a number / up to 12 years surviving patient. In evaluating the survival of the patient it is of course necessary to have that Take into account the severity of the disease at the time of the start of therapy. The course of the disease during the therapy is described in Table 2. In addition to the actual survival, there have been improvements in muscle function without any Reversal of neurological signs achieved. A number of patients don't just have to survive in terms of survival but also with regard to their well-being, to preservation certain punctures and minimizing bulbar symptoms addressed.

Table 2 also shows that in advanced cases of amyotrophic lateral sclerosis, with obvious

- 28 409832/1005

irreversible histopathology, as well as in advanced cases where bulbar symptoms at the beginning of treatment prevailed, therapeutic failures were recorded. On the other hand, consider attention, for example directed to patient no. 7, in whom a high therapeutic Response was detectable. In Table 2, "M-N" stands for "modified neurotoxin".

409832/1005

Table 1
Classification of clinically tested patients

Great

I limited exposure to various muscle groups; usually none obvious or premature pyramidal orbit signs; no bulbar signs; possibly very pronounced fasciculating; low or none Symptoms of the upper motor neuron; Paralysis limited and flaccid;

II one or two limbs paralyzed; no bulbar signs; Pyramidal orbit +

J ^. (usually + but still early); minor and irregular to numerous

ο and frequent fasciculation; minor or no symptoms of the

cd upper motor neuron;
oo

^ω III being affected by the pyramidal trajectory; diffuse disease with paralysis or paralysis _o '

^ and paresis; Fasciculating; Upper motor skills fluctuate, but the Betz cells are ^ ro

_ ^ usually damaged, usually some bulbous symptoms; Walking around is under j>. ^

ο difficulties possible; Eating by yourself is possible; this is ο '

ο an explosive potential with almost certain, rapid decay; j> ·

IV severe, diffuse, flaccid paralysis with some spasm; Pyramidal Orbit and Bulbar - *

Symptoms; Fasciculate +; Walking around and eating by yourself is not possible borrowed, with assistance and with the help of a walker can be a little movement to be possible;

V a) clinically not completely terminal, but may be plus due to its appearance the duration of the affliction are regarded as terminal; Bulbar symptoms fluctuating, usually at least moderately; Tetraplegia +;

b) extreme bulbar symptoms with surprisingly little limb or trunk paralysis; explosive near terminal stage because of the anatomical appearance.

TABEL

Summary of the clinical tests with amyotrophic lateral sclerosis ^ / η / / -ίο suffering patients on therapy with modified neurotoxins

(0.8 to 1.2 ml of the agent was injected every other day)

patient
No, illness
before Thera
pie (Rx) Classification
during the
Therapy (Rx) used
Means of
Example duration
the The
rapie (Rx) 1 11 months III
Bulbar Symptoms + 1 & 2 11 years &
9 months 2
N 50 months III
Bulbar Symptoms - 1 6+ years

24 Months

IH-IV Bulbar Symptoms ++

19 months

III-IV

Bulbar Symptoms +

6 years

months

Remarks:

Minimal pyramidal orbit symptoms; remains ambulatory and bulbar negative; Can still work;

Leg function is good. The puncture of the arms and hands is bad, she however, they are not completely inoperable. Depomedrol was used (only exception), bulbar symptoms questionable; Bilateral pyramidal orbit +; Death from cardiovascular disease;

Rapid progression of the amyotropic ** phic lateral sclerosis with bulb symptoms in 2 years prior to therapy with modified Neurotoxlrieri; The bulbar paralysis occurred during therapy progressed very slowly. Death as a result of bulbar paralysis;

Significant pyramidal path damage; The original clinical prognosis for the lifespan was about 12 months; Lived 52 months; Unambiguous, however temporary func1onel 3 -

m D

TABLE 2 (continued )

12-24 months 111-17

Bulbar Symptoms +

16 months III-I7

Bulbar Symptoms +

erosion during therapy; Improved well-being; Peaceful death as a result of Bulbar paralysis;

Months Irregular administration of modified neurotoxins; The neurologist noted a temporary improvement after one year of "status quo"; Lost contact;

After 8 months of irregular dosing and administration; Temporary reluctance; lost tact Functional improvement during three months and "status quo" after three Months; Modified neurotoxin was no longer administered after δ months;

12-24 months Vb year

5 months

Vb months

Extreme bulbar paralysis at the beginning of therapy with modified neurotoxins; This is the only example of a functional response in such bulbar paralysis; Arms and legs surprisingly good ;; ^l Get well soon for 6 months v> J Minor skeletal muscle degeneration; In about the 7th month of the treatment the improvement ceased;

Tetrafinal bulbar paralysis; The poor and legs possessed a right & utc Function; Death from bulbar paralysis; Therapeutic failure;

TABLE 2 (Port setting )

1/2 month

Vb

10

months

III Bulbar Symptoms -

409832 / 11 12th months III
Bulbar symptoms 100 5 12th months V a & b

Months Terminal Bulbar Paralysis; Bilate

• Real pyramidal trajectory damage; Moderate limb function; Death from BuI bear paralysis; Therapeutic failure;

Year & diffuse amytrophic months progressive lateral sclerosis; Short periods clearer functional improvement, then regression; Discontinuation of therapy with modified neurotoxins after 1 year and 8 months. Death about sixty days later; Presumably amyotrophic Lateral sclerosis, cause unknown;

Year Irregular therapy with modified neurotoxins; The patient ι did not follow the instructions; Pyramidal tract damage; Past CVS / cardiac pulmonary embolism / prostate complications; Bulbar paralysis questionable; Death probably by Bronchial pneumonia; No post mortem;

Months Less than a month 'after starting treatment with modified The neurologist called in confirmed that neurotoxins were significantly better; The voice was firmer; The thumb, which had not been moved for about two years, showed some abduction and adduction; No Relief of paralysis of the feet; ■ Within two months, this patient strengthened,

TABLE 2 (continued)

240U16

4 months

III

Bulbar Symptoms +

26 months

III

Bulbar Symptoms -

O CO OO CO

months

III

Bulbar Symptoms -

especially noticeable in the quadriceps area and in the hands;

Months of diffuse amyotrophic lateral sclerosis, which progressed rapidly from the time of early diagnosis; Despite short sporadic periods Functional improvement was relentless in this patient Amyotrophic lateral sclerosis continued;

Year Relatively slow disease course j The patient was presented after about seven months of therapy with Isoprinosine; Under the influence of Isoprinosine, clear signs of degeneration were detectable. Was xinen in therapy with modified Neuroto- \ jj _t a temporary improvement and / or a status quo determined. After discontinuing the modified neurotoxins. the degeneration progressed rapidly; 2 months later death from amyotrophic lateral sclerosis;

Year The patient responded well to the modified neurotoxins. If therapy has lasted for more than one year, ⁺ slight degeneration, if any. Complications: inflammation of the prostate (old) and fracture of the neck of the knee; Pyramidal tract damage; Cause of death: cardiovascular disease;

⁺ at most

TABLE 2 (continued )

16

months

II - III Bulbar Symptoms -

1 & 2

21 months

17th

years

IV-V bulbar symptoms

2 1/2 months

18th

years

Va & Vb

1 month

19th

year

II Bulbar Symptoms -

5-6 months

Diffuse, rapidly progressing amyotrophic lateral sclerosisj The patient was at the time of recording semi-ambulatory and showed no signs of bulbar paralysis; Slowdown under modified neurotoxin therapy j

Reacted well to modified neurotoxin doses for the first three weeks, then status quo. After the start of neurotoxin therapy, diffuse and serious course ι

Terminal amyotrophic lateral _x sclerosis diagnosed more than 5 years previously; Volume 4 = · recordings show a functional improvement in the treatment (the patient moved better and was able to understand conversations). After discontinuing the modified neurotoxins, the patient died 41 days later in the hospital, probably of bulbar amyotrophic lateral sclerosis;

When the administration of modified neurotxins began, the patient's condition deteriorated rapidly, with the paresis of one leg being replaced by almost complete paralysis of both legs (neurologically). The degeneration has been questionable since the start of therapy with the modified neurotoxins.

TABLE 2 (port setting )

20th

2 years

IH-IV

0983 21 4th until 5 1005 22nd 6th months

Years III - IV

II - III

The rate of degeneration in arayotrophic lateral sclerosis before and after Administration of modified neurotoxins is essential.

Months Initial Proximation: O, Pasci-

shape the right and left hand: 4+; Almost all of the bulbar symptoms; The tongue; showed complete atrophy. Within seven days Proximation was good in both hands. The patient could do buttons at this point unbutton and button; Full use after 12 days of treatment the tongue, no choking;

Months Treatment improved speech

and swallowing and strengthening, the knees. The ability to swallow improved move on. After the first week of treatment, the Pasci, culate to be made to disappear;

Months of treatment resulted in one

significant decrease in leg (muscle) cramps, so that the patient without Help could go. The patient could get dressed and undressed without assistance. Treatment improved her Agonizing movement of the hands. The proximation is compared to 2+ vcr the treatment is now okay;

TABLE 2 (port setting )

23 2 years II - III
Bulbar Symptoms + 24 3 1/2 years II 25th 8th months II O 26th 2 years Va & Vb 9832 / 1005

2-4 weeks

1 month

1 month

2 months

3 years

rv - v

2 months

Improvement in swallowing ability after 3 days of treatment, Der The patient now walks better without shuffling.

During the treatment there was an improvement in the fascicular nerve and the agonizing movement of the hands.

■ Rapid improvement in the right forearm and triceps after 7 days Treatment.

The function of the sternocleidoma gold was initially rated with zero. The function of other neck muscles was initially also rated zero. The patient showed quadriplegia and aphonia, his Tongue was atrophic. After 5 weeks Treatment could make the patient sound. He held his neck upright. ,

After a month of treatment, the breathing, the movement of the dumbbell and the patient's pasciculation got better.

TABLE 2 (continued )

28

2 1/2 years Va month

29

3 years

1 year

Va & Vb

II

CD OO CO

O O Ol

31

2 years

II - III

52

33

2 1/2 years Vb

7-10 months I months

months

1/2 month

1/2 month

months

After treatment, the patient's breathing, tongue movement, and swallowing ability were up improved. There was also a slight improvement in the movement of the legs detectable.

After the treatment, the patient's ability to swallow was improved »

After treatment, the right hand grasping ability and the pinger's stretching ability were improved. The proximation improved from 2+ to good «,

At first the deterioration of the condition could be brought to a standstill, then it occurred no further worsening of this condition.

The bulbar syrntomas improved upon treatment.

After the treatment, the downward movement of the right foot and the fasciculation improved from 4+ to 2+ "

TABLE 2 (continued)

1 1/2 years

Vb

1 1/2 months

35 7 month I - II 3 2 months 36 3 years II 3 2 months

09832- 37 3 1/2 years II - III 3 1 month Q 38 1 year III 3 2 months O
cn

During the treatment there was an improvement in the ability to swallow; The patient, who initially showed aphonia, was able to speak a little after the treatment.

Less strengthening of the legs after the treatment.

The fine movement of the hands improved during the treatment. The proximation improved from 5O # to 1005έ. They also strengthened both arms. Finally, there was also a decrease in fasciculation.

The patient's ability to walk improved during treatment. «

After the treatment, the patient's ability to swallow was greatly improved. Before the treatment the patient could only eat pureed food; after the treatment he could even eat solid foods. The patient's ability to walk changed from shuffling to lifting his feet. Fasciculation has also been significantly improved.

TABLE 2 (port setting )

6 1/2 years

III

4 years

III - IV

1/2 years Va

CD OO CO IO

Months The weakness of the legs made walking unaided impossible. After one month of treatment, the patient was able to get out of bed and the bathroom without assistance to seek out

Months Little improvement in treatment

development, or at least no further deterioration

Months Little improvement in treatment

development, or at least no further deterioration

Claims (1)

4 «means according to claim 1, characterized in that it is at Semliki Forest virus test at least 50% inhibition which causes stains. 5. Composition according to claim 4, characterized in that there is at least 75 # inhibition in the Semliki Forest virus test the stain causes 6. Composition according to claim 1, characterized in that it is non-toxic is. 7. Composition according to claim 1, characterized in that part of the poison comes from Naja Naja and Bungarus multicinctus. 8. Agent nach.Anspruch 2, characterized in that the ratio Naja snake venom to Bungarus snake venom is 400: 1 to 8: 1. 9. Composition according to claim 8, characterized in that the ratio of Naja snake venom to Bungarus snake venom is 80: 1 to 40: 1. 10. A method for producing an agent according to claim 1, characterized in that the snake venom is so long at a pH above 7 and a temperature of 409832/1006 up to 40 ° C until it has become non-toxic and that the product obtained is dialyzed. 409832/1005