US 3888977 A
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United States Patent [191 Sanders June 10, 1975 MODIFIED NEUROTOXIN  Inventor: Murray J. Sanders, 3009 Spanish Trl., Delray Beach, Fla. 33444 22 Filed: Feb. 1, 1973 2! Appl. No.: 328,724
52 us. Cl. 424/98  Int. Cl A61k 27/00  Field of Search 424/98  References Cited OTHER PUBLICATIONS Chem. Abst. (I), Vol. 74, 305l2p, (1971). Chem. Abst. (2), Vol. 84, 20440c, (1966). Chem. Abst. (3), Vol. 59, 12072f, (1963). Chem. Abst. (4), Vol.55, 20198g, (1961).
Chem. Abst. (5), Vol. 55; 6685gh, (1961).
Primary Examiner-Stanley J. Friedman Attorney, Agent, or FirmCushman, Darby & Cushman [5 7 ABSTRACT 22 Claims, N0 Drawings MODIFIED NEUROTOXIN The present invention relates to a composition, a process of production thereof, and a method for treatment of neurological diseases and especially to the treatment of heretofore intractible diseases such as amyotrophic lateral sclerosis.
BACKGROUND OF THE INVENTION Degenerative neurological diseases progress in a chronic manner to severe physical disability, such as paralysis, and even to death. While the cause of such neurological diseases is not always known, many of the diseases are results of specific infections, e.g., viral infections. It is believed that the specific viruses causing the specific neurological diseases attach to or function in conjunction with nerve cell receptors of the motor cells of the central nervous systems. Some workers in the art believe that these nerve cell receptors are discrete anatomical structures of the nerve cell, while others believe that the receptors are theoretical biophysical concepts which describe one of the functions of the nerve cells. Irrespective of theory, it is well known that nerve cells do act in a manner as if physical receptors exist in the nerve cell. It is further believed in the art that viral caused neurological diseases function, at least in part, through viral impairment of the nerve cell receptors and eventual destruction of the nerve cell. In any event, the affected nerve cells cease to function as healthy cells. These affected nerve cells may be considered to include a range of abnormal conditions varying from a partially damaged and therefore reversible state to a totally destroyed or neuronophagic state.
Many of the degenerative neurological diseases progress in a subtle and insidious manner to slowly cause impairment of the victim. Thus, the diseases are often overlooked in the early stages by the skilled clinician and the disease may progress to a point where mild paralysis, or fasciculation (twitching) of the muscles or other early symptoms which are specific to the disease being observed occur. Of course, at this point many nerve cells have been adversely affected and those cells, as noted above, cease to function in the normal manner. While various known chemotherapies can have some beneficial effect in treating such disease, most often the disease proceeds through increased paralysis and involvement of the pyramidal tracts causing loss of balance and like disorders, even to bulbar involvement and eventually death. For many of these diseases, the prognosis is most unfavorable indeed. Among such diseases of the central nervous system are amyotrophic lateral sclerosis, multiple sclerosis, kuru. acute poliomyelitis, etc. Since known chemotherapies. at best, only slow the progress of the disease, these therapies are not cures but only delay, essentially, the effects of the disease. It would, therefore, be of significant benefit to provide a therapy for treating such degenerative diseases of the central nervous system.
OBJECTS OF THE INVENTION In view of the above, it is an object of the invention to provide a composition and therapy for treating progressive degenerative diseases of the nervous system which involve the function of motor nerve cells from their origin to the neuromuscular junction, as well as elements of the central nervous system including axones, nerve myelin sheaths, etc., such as amyotropic lateral sclerosis, multiple sclerosis, kuru, polymyositis,
certain meningitides, muscular dystrophy, polyomyosi-- ties and the like. It is a further object of the invention to provide a composition and therapy for the treatment of diseases of the aforementioned type, which composition and therapy are safe, effective and may be administered over long periods of time. It is a further object of the invention to provide a method of manufacture of the composition of the present invention. Other objects will be apparent from the following disclosure and claims.
BRIEF STATEMENT OF THE INVENTION It has now been discovered that certain modified neurotoxins have the ability to, apparently, attach to or otherwise involve motor nerve cell receptors and mask or block those receptors from attachment or involvement with pathogenic organisms, viruses, or proteins with potentially deleterious functions. The modified neurotoxins are derived from venoms of certain genera of snakes and are prepared by detoxification of the toxic portions of the venoms while maintaining the neurotropic character of the resulting detoxified portion of the venoms and the remainder of the venom components. Conveniently, the venom is detoxified by controlled oxygenation, although any of the known detoxification procedures may be used with the exception of certain methods used to produce antivenom. The detoxified venom is then stabilized for storage. The present composition may be produced from any snake venom which acts, essentially, as a neurotoxin, as opposed to, essentially, a hematoxin. However, as will be more fully explained below, the composition must be derived from venom which is at least in part a broad central nervous system cell penetration venom such as obtained from the genus Bungarus.
DETAILED DESCRIPTION OF THE INVENTION The present composition is prepared by the con-' trolled detoxification of, at least in part, broad penetration neurotoxic venom. In this regard, it should be appreciated that the venom of most snakes have some neurotoxic and some hematoxic components, but the various genera of snakes can be essentially separated into a first group, the venom of which functions, mainly, through interference with blood chemistry and a second group, the venom of which functions, mainly, through destruction of nerve cell components. The former of these genera, i.e., the hematoxic venom snakes, is represented by the common North American pit vipers, such as the rattlesnake, copperhead and water moccasin. The latter of these genera, i.e., the neurotoxic venom snakes, is represented by the cobra, krait and coral snake. However, there are degrees of distinctions between these two groups. For example, the venom of the North American rattlesnake is almost completely hematoxic, while the venom of the Central American rattlesnake is hematoxic, but yet has significant neurotoxic components. On the other hand, certain species of Sout American rattlesnakes, especially the Crotalus terrzflcus terrificus, has venom which is extremely neurotoxic. At the extreme, the venom of the krait, e.g., blue krait, is such a broad penetration neurotoxin that involvement of the central nervous system is extensive and survivors of the bite of that snake are extremely rare. For purposes of the present specification, the term neurotoxic snake venom is defined as snake venom which is toxic to mainly, but not exclusively, the nerve cells and ancillary components as noted above.
The venom of snakes contain a multitude of chemical compounds, including various enzymes. The exact function of enzymes in the venom is not understood and, indeed, the enzymes may not have any direct function in the toxic effect of the venom. The enzymes may have other functions beneficial to the snake in utilizing the victim of its bite for food. For example, it is known that the body of an animal killed by snake bite will decompose more rapidly than that of other deaths. It has also been found that the higher molecular weight compounds of cobra venom, as can be separated by chromatography, are not significantly toxic, while the lower molecular weights are highly toxic. Irrespective of the specific compounds in snake venom, the present composition is preferably produced from whole venom which will, of course, include the enzymes although it is recognized that many of the components of the venom are inert for present purposes and could be separated from the active portions of the venom. Thus, while whole venom is preferred for this specification snake venom is to be construed as the whole venom or the toxic portion thereof. This definition is also inherent in the terms snake venom neurotoxin".
While not being bound by theory, it is believed that the present composition functions by attaching to or involving the nerve cell receptors to such an extent that the receptors are unavailable for attachment or involvement by neurological bacteria, viruses or proteins with potentially deleterious functions, thus halting the progress of degenerative nervous system diseases. It should be clearly understood, however, that the present composition is not prophylactic and will not, therefore, prevent or destroy a viral infection. The effect of the composition is considered to be that of a cell-receptor blocking agent with absorption and excretion by the cells which, therefore, requires dosing until the invading causative agents are lost by the immune response of the body or disappear by way of natural attrition due to lack of cellular nutritional factors essential to maintenance of the invaders existence. Accordingly, continued treatment with the present composition may be required for long periods of time to ensure that the nerve cell receptors are continuously blocked from involvement with the causative agents of the disease.
The studies of Lamb and Hunter (Lancet 1:20, 1904) showed by histopathologic experiments with primates killed by neurotoxic Indian cobra venom that essentially all of the motor nerve cells in the central nervous system were involved by this venom. A basis of the present invention is the discovery that such neurotropic snake venom, in an essentially non-toxic state, also will reach that same broad spectrum of motor nerve cells and block or interfere with invading pathogenic bacteria, viruses or proteins with potentially deleterious functions. Thus, for the foregoing reasons, the snake venom used in producing the present composition must be a neurotoxic venom, as defined above. Further, since the dosages of venom required to block the nerve cell receptors would be far more than sufficient to quickly kill the patient, it is, of course, imperative that the venom be detoxified. On the other hand, since the essential components of the venom must remain intact so that the venom will involve the nerve cell receptors and effectively block the receptors from bacterial, viral or potentially deleterious protein involvement, it is likewise imperative that the detoxification of the venom not denature or otherwise adversely degradate the venom components. Thus, for purposes of the present invention undenatured, but detoxified venom must be used. The undenatured venom is referred to herein as being neurotropic.
In order to accomplish detoxification of the venom in accordance with the foregoing required conditions, the venom is preferably detoxified in the mildest and most gentle manner. While various detoxification procedures are known to the art, such as treatment with formaldehyde, fluorescein dyes, ultraviolet light and the like, it is preferred that gentle oxygenation at relatively low temperatues be practiced, although the particular detoxification procedure is not critical. Quite suitably a modified Boquet detoxification procedure, as explained hereinafter, may be used.
The acceptability of any particular detoxification procedure can be tested, most conveniently, by the classical Semliki Forest virus test. The procedure for this test is well known. Briefly explained, a chick embryo fibroblastic tissue culture of cells on glass is overlaid with a gelled nutrient preparation such as I-Ianks solution with lactalbumin. The Semliki Forest virus is then inoculated on to the sheet of cells and the number of resulting plaques show the titer of the virus. If the particular detoxification procedure practiced with the venom is suitable, then the chick cells can be washed with the detoxified venom prior to inoculation with the test virus and the cells would then show few or no plaques. If however, a substantial number of plaques is observed, then the detoxification procedure practiced with the venom is too severe and unacceptable amounts of denaturization of the venom has occurred. Of course, it is preferred that almost no plaques or no plaques at all be observed in this test, although, as will be readily recognized, few plaques as compared with the plaques of the unwashed chick cells would be acceptable. The cells washed in the detoxified venom should show at least a statistically significant inhibition of plaque formation by the virus, e.g., 30%, especially 50% inhibition of plaques and preferably to inhibition of plaques. As will be also explained hereinafter, suitable concentrations of the present detoxified venom can be determined with this same test. In effect, as can be appreciated, this test shows the ability of the detoxified venom to prevent the proliferation of the viral plaques.
In addition to the Semliki Forest virus test, a bioassay should be made of the detoxified venom to establish the correctness of the detoxification procedure used, as well as the potency and absence of toxicity. Thus, the toxicity of the compositions is tested by inoculation of standard laboratory animals, such as mice, rats and guinea pigs, although dogs and monkeys may be used if desired, and essentially no signs of toxicity should be observed in the experimental animals.
If the particular detoxification procedure meets the foregoing standards, as established by the Semliki Forest virus test and bioassay, it is acceptable for purposes of the present invention. Thus, the composition must be a detoxified, neurotropically active derivative of snake venom, the neurotropic activity being establishable by the above-described Semliki Forest virus inhibition test and the neurotropic affinity being demon strated by patient response to a therapeutic regimen.
As noted above, it is convenient to detoxify the venom by a modification of the known Boquet technique (Ann. Inst. Pasteur 66:379-396, 1941). According to this procedure, a solution of the venom in a suitable solvent. especially water, is prepared. While the particular concentration of venom in the solution is not critical, up to about 3% by weight solution can be conveniently prepared. An antifoam may be added to the solution, since snake venoms, generally, will cause solutions thereof to foam. Any nontoxic inert antifoam may be used, many of which are known in the art and particularly, the food grade silicon compounds. To this solution is added an oxygen-producing compound, although oxygen containing gases, and especially nascent oxygen-containing gases, may be simply bubbled through the solution. Alternately, in situ, oxygen generating mechanisms, such as ultraviolet light or fluorescein dyes may be used to produce oxygen from an aqueous solution. More conveniently, however, CP hydrogen peroxide (30% solution) is added, along with a catalyst for the activation of the hydrogen peroxide, such as copper sulfate. Since detoxification proceeds on the basic side, the pH is adjusted to above 7, but preferably less than 10, with a suitable base such as an alkali metal or alkaline earth hydroxide, carbonate or the like, e.g., sodium hydroxide. Alternately, ammonium gas or ammonium hydroxide or other nontoxic amine or like compound may be used.
Suitably, the solution is buffered at a proper pH with any conventional buffer such as an alkali metal phosphate or acetate buffer. If a buffer is not used, or even with the use ofa buffer after longer periods of time, the pH may tend to drop. Additional amounts of base may be, therefore, required to maintain the proper pH.
The solution is maintained at moderate temperatures e.g., between about and 40C, although the upper part of this range, i.e., from about to 40C is preferred. Temperatures outside of this range may be used, but lower temperatures prolong the period required for detoxification and higher temperatures can cause unacceptable amounts of denaturization of the venom. Occasionally, or continuously if desired, the mixture is stirred. After about up to 30 days, especially between 6 and 16 days, under the foregoing conditions, depending upon the temperature and the particular venom, detoxification will have been accomplished and the venom will have been modified for purposes of the present invention. Shorter or longer times may be used, however, so long as the Semliki Forest virus test and bioassay, discussed above, are adequately met.
The detoxification reaction can then be stopped by adding a catalyst deactivator to prevent further action of the catalyst on the hydrogen peroxide. Many such deactivators for the reaction are known, but catalase (CP) is most convenient for this purpose.
Since the modified neurotoxin produced from the venom will contain ions generated during the detoxification procedure which are neither desired nor effective for the present purposes, it is preferred that these ions be removed from the modified neurotoxin product, especially the copper ions of the copper sulfate since these ions are somewhat toxic per se. The ions may be removed in any desired way but conventional dialysis with semipermeable membranes may be used. Thus, the detoxified solution is simply contained in a semi-permeable membrane, such as cellulose acetate, and the membrane with its contents are submerged in a tank of phosphate buffer sodium chloride solution, pH 6.8, to cause transfer of the undesired ions from the modified neurotoxin solutions of the salt bath. Suitably, this is carried out at room temperature for approximately one day but temperatures from above 0C up to about 50C for 1 hour to 20 days may be used.
The modified neurotoxin is preferably filtered, e.g., through a series of graded pore diameter membranes, particularly a series including a final membrane with a very small average pore diameter, e.g., about 0.22 microns, to insure sterility. Prior to the final filtration, preferably a concentration of l/l0,000 merthiolate is established in the product. Merthiolate is a preservative produced by Eli Lilly and Co. Also prior to final filtration, it is preferred to adjust the pH of the bulk product to less than 7, e.g., 6.8, by the use of food grade nontoxic acids, such as mineral acids, acetic acid, lactic acid and the like. Here again, a suitable buffer can be employed to maintain the pH on the acid side. The particular pH is not critical, but pHs below 4 are, generally, uncomfortable for certain modes of administration, e.g., subcutaneous injection, and a pH above 4 and below 7 is, therefore, preferred.
The normal dosage of the present modified neurotoxin fora middle aged male of approximately lbs. is from 0.7 to 2 ml. of composition produced from a 1% by weight solution of snake venom. The dosages are correspondingly adjusted for younger or older patients of greater or less body weight. Normally speaking, however, dosages between 0.05 and 10 ml. of the composition produced from a 1% by weight solution of venom can be used, although dosages between 0.4 and 3 ml. are preferred. The dosages are also correspondingly adjusted for compositions obtained from other than 1% solution of venom. While a patient may be given the modified neurotoxin as infrequently as every other week, it is preferred that the composition be administered at least weekly, and preferably every other day or daily, e.g., 3 times a week. The composition may be administered orally, subcutaneously, intramuscularly or intravenously but it appears that some of the potency of the composition is lost with oral administration and intravenous administration appears to increase the possibility of a shock to the body. Thus, either subcutaneous or intramuscular injection is preferred. If desired, for any of the foregoing modes of administration the solution may be compounded into conventional forms such as tablets, powders, elixirs, and solutions. In this regard any of the conventional binders, extenders, diluents, preservatives, etc., may be used. For injection, however, a simple physiological saline solution or the like is preferred.
As noted above, the composition is not prophylactic and will not destroy or present infection from the pathogenic viruses. Accordingly, the present therapy must be continued for an indefinite period. However, continued treatment presents no major difficulty since there is no contra-indication of co-administration of the present composition with any other drug, other than possibly Vitamin B It appears that Vitamin B being a neuroactive material, somewhat interferes with the function of the present composition and, therefore, preferably, Vitamin B is not given in conjunction with the present composition. On the other hand, it appears that corticosteroids act synergistically with the present composition and intensify the effect thereof. Accordingly, while corticosteroids, such as cortisone, may be co-administered, the dosage of the present composition may be reduced or the patient should be closely observed in order to detect any undue drug activity.
As discussed above, the venom from which the present composition is produced must be a neurotoxic venom. However, it has been additionally discovered that a portion of the neurotoxic venom must be a broad central nervous system cell penetration venom, such as obtained from the genus Bungarus. The remainder of the neurotoxic venom may suitably be a more specific central nervous system cell penetration venom such as obtained from the genus Naja, such as Naja naja, Naja haja, Naja flava, Naja llalmah and Naja tripudians, although, as noted above, certain neurotoxic species of the genus Crotalus, such as Crotalus terrificus rerrlficus, may be used in this regard. The genus Bungarus is illustrated, especially by Bungarus nullticincrm' and fasciatus. The venom of the genus Bungarus has been found to broadly penetrate the cells of the central nervous system and, therefore, a composition made therefrom can be active toward nerve cells that are essentially unaffected by compositions made from other neurotoxic venoms, even other neurotoxic venoms of snakes from the same family.
In this latter regard, it has been discovered that cobra venom is. a somewhat selective neurotoxin and while a composition made therefrom will protect some cells, especially motor cells, from viral involvement, other cells will not be so effectively protected and viral or other caused diseases can proceed, although at a much reduced rate and involvement. The beneficial effects of cobra venom derived compositions have been described by the present inventor. See for example, Sanders et al: Antipoliomyelitis Action of Certain Toxoids, Acta Neuroveg. Springer Verlag in Wien (Based on data presented at the Sixth Symposium of Vegetative Neurology, Strasbourg, France, September 29, 1955); Sanders M, et al: Neurotoxoid Interference With Two Human Strains of Poliomyelitis in Rhesus Monkeys, Ann. NY Acad. Sci 58: 112, 1953; Sanders M: Naja flava Neurotoxoid Interference Late In Experimental Poliomyelitis, J Path Bact 68: 1267-1271, 1954; Sanders M, et al: The Role of Naja flara Toxoid and Toxin in Experimental Poliomyelitis, Acta Neurovegitativa 8: 362-371, 1954; Sanders M, et al: Neurotoxoid Interference in Macac'us rhesus Infected Intramus-cularly with Poliovirus, Science 127: 594-596, 1958; Sanders M, et al.: Neurotoxoid Interference Principle In Pseudorabies Infection, In Vivo, Inactivation of Aujeszky virus, Proc. 7th Inter Cong Microbiol. p.293, 1958; and Sanders M: Deceleration of Degeneration of Amyotrophic Lateral Sclerosis in Eight Cases. Proc. Pan Am Med Cong. 1960. All of the foregoing references are incorporated herein by reference and all information therein is relied upon for disclosure in this specification. Also see Clark, W. B: Supplementary Treatment of Herpes Simplex lnfections of the Cornea by Neurotropic Toxoids, Preliminary Report, Concilium Ophthalmologicum Acta, XVIII Congress, Brussels, 1958 and Clark, W. B.: The Use of Sanders Neurotoxoid l (Modified Snake Venom) in the Treatment of Recurrent Herpes Simplex of the Cornea: Progress Report, Southern Medical Journal Vol. 55. No. 9. p 947-951, September 1962, both of which are incorporated herein by reference. The modified neurotoxins of the cobra venom provided substantial benefit but were not capable of completely halting the advancement of many neurological diseases. Further, the modified neurotoxins prepared from the cobra venom, alone, could not be completely detoxified since the potency and desired effect were thereby lost. Thus, the bioassay could never show complete detoxification and there is, of course, a risk in administration ofa poisonous snake venom which has not been fully detoxified. Also, in those modified neurotoxins, the entire detoxified solution was administered and certain components thereof produced undesired side effects in terms of toxicity, other than that of the venom.
As mentioned above, the present composition must be prepared from venom which is at least in part derived from a broad penetration venom, i.e., from the genus Bungarus. While Bungarus venom is similar to Naja venom, the venoms differ with regard to the intensity of the physiological effect and with regard to the time required for the physiological effect. The Naja venom acts more intensely and quickly. On the other hand, Bungarus venom is not so selective as cobra venom and is active toward a wider range of nerve cells. Accordingly, the combination of Bungarus venom and Naja venom gives superior results as opposed to Naja venom alone or Bungarus venom alone, although Bungarus venom alone can be effectively used, as opposed to Naja venom alone, as discussed above.
While the ratio of Naja venom to Bungarus venom is not narrowly critical and indeed all Bungarus venom could be used, it is preferred that the ratio of Naja venom to Bungarus venom be between about 400:1 to 8:1, especially from about to 40:1, on a weight basis.
The invention will be illustrated by way of the following examples but the invention is not limited thereto but is fully applicable to the foregoing disclosure.
EXAMPLE 1 The following example will illustrate the neurotropic character of neurotoxic snake venoms which are detoxified by a modified Boquet procedure.
Type I (Brunhilde) and type II (Lansing) poliomyelitis viruses were used for this example. The Brunhilde virus was stored at 30C as a 1:5 emulsion in 20% rabbit serum, in non-pyrogenic distilled water. The emulsion was derived from pooled cervical and lumbar spinal cord enlargements of intracerebrally infected rhesus monkeys showing initial signs of paralysis. The Lansing type virus was also stored at 30C as 20% mouse brain pools. Mouse intracerebral LD 0s varied between 10*- and 10 MUCHCUS rhesus and CFW mice were used as the experimental hosts. All virus injections were via intracerebral or intrathalamic routes. In monkeys. the test virus was Type I (Brunhilde). The cerebral technique was carried out by injecting dilutions of virus pools into the parietal or frontal brain areas of the monkey.
The venoms used were derived from the venoms of the South African cobra, Naja flava, and from the South American rattlesnake, Crotalus terrificus terrificus. The venoms were detoxified by a modified Boquet method. Changes were necessary because of the requirements of the particular neurotoxins. Higher concentrations of hydrogen peroxide (Superoxol-30% hydrogen peroxide) were found moredesirable and the addition of 0.2% formalin was found to be useful for obtaining an improved curve of detoxification. Also.
because stability of the neurotoxin was required, it was necessary to make certain the detoxification reaction was brought to a conclusion. This was accomplished by the addition of catalase, and by changing the slightly alkaline pH of the solution to pH 6.8 at the time catalase was added. The change of pH is necessary since detoxification continues to occur within the alkaline range.
40 grams of desiccated Naja fluvu are placed in a flask and about 3,800 ml of phosphate buffered aqueous solution, pH 7.6-7.8, are gradually added to the flask, along with a small amount of Dow-Corning antifoam. The flask is shaken occasionally until all the venom is in solution. 36 grams of NaCl are added at this time. 2.0 ml of a freshly made 1.0% solution of CuSO are added and the flask is rotated vigorously. Eighty ml of Superoxol (30% H are then slowly added while still rotating the flask. Eight ml of 38% formalin are then added while still rotating the flask. The contents are poured into a 4-liter volumetric flask and the buffered solution is added to the volume line of the flask. The completed preparation is poured into a 6-liter Florence flask, stoppered with a gauze and cotton stopper which is covered with wax paper, and placed in a 37C incubator.
The rate of detoxification is predictable but is checked by daily bioassay. This is done by injecting various amounts of the test solution, brought to a constant volume of 0.5 ml with saline solution, into 20 gram CFW mice. via the intravenous route. When 0.5 ml of the undiluted solution produces death in approximately 50% of the mice, the solution is taken from the incubator and the detoxification process is brought to a quick stop by the addition of catalase (Armour 10). To make certain that sufficient catalase has been added to destroy the excess peroxide, a smoldering wooden applicator stick is inserted into the neck of the flask. In the presence of a continuing reaction where free oxygen is given off, the wooden applicator stick will flame. To further insure the cessation of detoxification, the pH of the solution maintained at about 7.6 throughout the detoxification procedure, is reduced to about 6.8. It should be noted that it may be necessary to add pellets or solution of NaOH during the incubation period, since the pH tends to drop. When the detoxification procedure has been brought to an end, chlorotone is added, preliminary filtration is done through a K-2 Seitz clarifying filter, and finally sterilization is accomplished by passing the solution through a large S-l Seitz filter. At that point the composition may be bottled and placed in a refrigerator without potency changes. The proportions. as discussed above, are:
Catalase as required Chlorotone 8 g When the modified method of Boquet is applied to the Cram/us terrificus terrzfz'cus (Ctt) venom, detoxification is more rapid. To obtain a Ctt composition which will meet the above requirements, the formula given above is altered by reducing in half the volume of hydrogen peroxide and formalin. Even with this modified formula, the peroxide partial detoxification phase is brought to a close usually within 15 hours. Then, after adding catalase, the Ctt solution is returned to the incubator at a pH of 7.6. The detoxification process continues more slowly and the results can be controlled by mouse test. Within 2 to 3 days a satisfactory product is obtained, and the reaction can be concluded by changing the pH to 6.8, filtering and storing the composition at 4C. The bioassay procedure for testing the Ctt composition is similar to that used for Naju flava composition.
Six rhesus monkeys were injected intracerebrally with approximately 50 PD s (paralyzing dose of 50% of test animals) of Brunhilde virus. Twenty-four hours later, 3 of the animals received a single injection of the Naja flava derived composition (3/8 ml/Kg.), whereas the remaining 3 animals were treated as controls and given no injections. On the 7th day post-infection, the three untreated animals were either quadriplegic or heavily involved with paralysis, whereas the three treated animals showed no signs whatsoever of the disease. By the 9th day, when all three untreated animals were quadriplegic, the disease was only beginning to be manifested in the three treated animals. In the treated group, paralysis appeared on the 9th, 11th and 13th days, with an average incubation of l 1 days, as against a 7day average for the untreated animals. As can be appreciated, this was a most severe challenge and the results show the marked effect of the neurotropic composition.
In a similar manner, 58 untreated control rhesus monkeys were challenged with poliomyelitis and only one survived. By contrast, of 272 challenged and treated rhesus monkeys, 69 survived, 22 of them showing no sign of the disease, and 47 being, for the most part, lightly paralyzed survivors. Here again, the marked effect of the neurotropic composition in the face of this severe challenge is demonstrated.
Table A shows the results of similar challenges when the composition is used on a therapeutic basis. After the animals were infected and groups were determined by random selection, no animal was handled or received the composition until there was a gross appearance of paralysis in the experiment, i.e., when one or more animals in any group appeared to be paralyzed to a degree visible through the cage without manipulation.
Desiccatcd .\'ujufluru g Phosphate buffered aqueous On that day, wh ch was the 5th day after Infection, solution (pH 7.6) 3800 ml treatment was mitlated on a predetermined basis. As 2 2 Tragg g can be seen from Table A, almost 50% of the treated CiiSO, r 1.0% i 3 m1 animals showed no gross signs of paralysis. Comparison 3,0 r g of the 45.8% O.K. survivors in the treated groups with orma in 9 2) m 0 Phosphate buffered aqueous 0% m the two control groups, reveals a marked effect solution us 4000 ml of the neurotropic composition on the disease.
TABLE A Composition RJ. (ml Composition Survivors*** Exp. No. per Kg monkey No Mouse LD bod \\veight)** No rhesus ml infected No "/1 None (controls) 6 O 0 None (controls) 6 (l 0 1 1 12 TABLE A Continued Composition 0.1x.
R (ml Composition Sur\'i\'ors*** Exp. No. per Kg monkey No Mouse LD hodyueight) No rhesus ml infected No i 3 40 NF 0.6 0.4 6 2 4 40 NF 06 0.4 6 4 45.8 5 44 NF 0.3 0. l 6 3 6 44 NF 03 0.2 6 2 H 'l he dose indicated for each composition was administered on the 5th. 6th. 7th. 8th and l lth days after injection.
** OK. Monkey showed no sign of polyiomyclitis.
In a similar experiment involving 99 treated animals and 24 untreated controls, the composition administration was not initiated until the 6th day after the intracerebral injection. None of the 24 untreated controls survived. In the treated group, 17 animals survived, either lightly paralyzed or without paralysis. Specific reference is made of these 123 animals because the chal lenge was extremely severe, consisting of intracerebrally injection of 50 PD per animal. Survivors occurred in the treated group even when the composition was administered at a time when paralysis was present in a number of the animals.
In a similar series of experiments 56,100 mice were injected intracerebrally with pools of Lansing virus. One group was treated with the composition and another group was treated with a placebo as a control. When the daily rate of paralysis and death was ploted for both placebo-controls and composition-treated groups, the controls followed the straight line pattern within the expected limits. In the case of the treated groups, a deviation of as much as 50% less involved and dying animals was noted between the 4th and 7th days post-infection. The two curves, however, met either abruptly or gradually. There was no difference in survivors in either of the groups at the end of the 30-day experiments which illustrates the requirement for continued treatment.
EXAMPLE 2 40 grams of desiccated Naja naja venom and 0.5 grams of desiccated Bungarus multicinctus venom are added to 3,600 ml of phosphate buffered aqueous solution at a pH of 7.5. A trace amount of silicone antifoam (Dow-Corning) is added and the mixture is stirred to dissolve the venom. 2 ml of 1% CP solution of copper sulfate is added with stirring. 80 ml of 30% hydrogen peroxide is added to the solution. The solution is placed in a volumetric flask and the phosphate buffered aqueous solution is added to make 4,000 ml. The solution is incubated at 37C, and the pH ismonitored. The pH is maintained at about 7.5 by the addition of one normal sodium hydroxide solution as required. Aliquots of the solution are tested daily for toxicity by inoculating 0.5 ml of undiluted solution intraperitoneally per mouse in 20 gram mice. At the end of 14 days of detoxification 20 mice showed no deaths in 24 hours at this level of inoculation. Also the detoxification is tested by giving a dose of 5 ml to 350 gm guinea pigs and no deaths occurred in 24 hours. The bulk solution is then mixed with 3 mg of catalase per ml of solution and placed in a cellulose acetate dialysis bag. The particular bag is a APD millipore of 0.22 microns. The closed bag is placed in a dialyzing fluid of 1 part by weight of Sorensons buffer and 3 parts by weight of 0.9% NaCl.
One volume of solution 15 dialyzed against nine volumes of dialyzing fluid. Finally. the solution was filtered through clarifying membranes and a final 0.22 micron filter and 1/10,000 concentration of merthiolate was established and the pH was adjusted to 6.8 with 1N hydrochloric acid. The Semliki Forest virus test showed adequate plaque inhibition. The final product was tested for sterility and bioassayed for lack of toxicity and safety.
It should be noted that there are several important differences between the compositions of Examples 1 and 2. The formalin and chlorotone were deleted from the Example 1 composition and the composition of Example 2 was dialyzed. Also, the final filtration of the Example 2 composition was accomplished through a series of membranes with its final membrane having an APD Millipore of 0.22 microns, and merthiolate was added to a concentration of l/l0,000 parts. More significantly, the composition of Example 2 contains the detoxified Bungarus venom and is atoxic as compared to the composition of Example 1 which had residual toxicity.
EXAMPLE 3 Example 2 was repeated except that 1 gram of Bungarus multicinctus was used.
EXAMPLE 4 The compositions of Examples 1, 2 and 3 were used in clinical studies. The composition of Example 1 shows the general interference effects of the detoxified neurotoxic snake venom with Naja venom alone. The compositions of Examples 2 and 3 show improved results.
In order to interpret the results of the therapy with the compositions, it was necessary to classify the clinical state of deterioration of the patients, since varying degrees of advancement of amyotrophic lateral sclerosis (ALS) was present at the time of treatment. It is also noted that the specific disease may vary in its course within different individuals. This is true even in a disease, generally, as predictable as ALS. The'classification in Table 1 represents the degree of illness in the patients in the study.
In Table 2, the therapeutic responses of the patients classified on the basis of Table l are noted. A group of 41 ALS patients is presented. In each case a diagnosis of ALS has been made by a qualified neurologist and has been confirmed by a second neurologist or a recognized clinic. Data suggestive of a useful therapeutic ap proach are derived from a number of patient survivals of up to 12 years. In evaluating patient survival it is, of course, necessary to consider the severity of the disease at the time the therapy was initiated. The course of disease during therapy is described in Table 2. in addition to survival there have been improvements in muscle function, without reversal of neurologic signs. A significant number of patients have responded, not only in regard to survival, but in their state of comfort, maintenance of function, and minimization of bulbar symptoms.
From Table 2 it may also be noted that therapeutic failures occurred in advanced cases of ALS with appar- 10 or Example 3.
TABLE 1 Classification of Patients Clinical Involvement CLASS 1 Limited involvement of several muscle groups. Usually no apparent or early pyramidal tract signv No bulhar signs. Fasciculations may be prominent. Little or no upper motor neuron involvement. Paralysis limited and flaccid.
ll One or two limbs paralyzed. No bulhar signs. Pyramidal tract i (usually -lbut still early). Fasciculations vary from few and infrequent to many and frequently occurring. Little or no upper motor neurone involvement.
lll Pyramidal tract involvement. Diffuse disease with paralysis and paresis. Fasciculations.
L'pper motor varies but Betz cells usually affected. Usually some bulhar involvement. May ambulate with difficulty. May feed himself. This is an explosive potential with almost certain. rapid deterioration.
l\ Severe. diffuse. flaccid paralysis with some spasticity. Pyramidal tract and bulhar imolvement. Fasciculations 2; Cannot amhulate nor feed himself but may move a little when supported and with aid of a walker.
\' (a) Clinically not quite terminal but on basis of involvement plus duration of involvement may he considered terminal. Bulbar involvement varies. usually at least moderate. Quadriplegia (b) Extreme bulhar involvement with surprisingly little limb or trunk paralysis. Explosively near terminal stage because of anatomical involvement.
TABLE 2 Summary of ALS Patients Under MN Therapy (Treatment by injection of .8 to L2 ml of composition every other day) Patient lllness Prior C lassilication Example Duration Comments Number to MN Rs at time of MN M-N Used of M-N Rx (months Rx or years) l l l months Ill 1 8; 2 l 1 yrs is; Minimal pyramidal tract involvement.
Bulhar 9 months Remains ambulatory & bulhar negative Can still work 2 30 months Ill 1 6+ years Function good in legs. Arms and hands Bulhar poor but not useless. Depomedrol used (the single exception). Bulhar questionable. Bilateral pyramidal tract Death due to cardiovascular accident.
3 24 months lll-lV l 6 years Rapid progression of ALS including hulbar Bulbar -llinvolvement in 2 years prior to MN therapy. Bulhar continued to advance under MN but very slowly. Bulbar death 4 19 months lll-l\' 1 l3 months Marked pyramidal tract involvement.
Bulhar lnitial clinical prognosis for life about l2 mos. Lived 32 months. Definite but temporary functional improvement under MN therapy. increased comfort. Bulbar death tranquil.
5 lZ-24 months llll\' l lo months Irregular use of MN. Neurologist Bulhar states temporary improvement. then status quo" after one year. Contact lost.
6 lo months lll-lV 1 Lost contact Irregular dosage and schedule.
Bulhar after 8 Temporary objection functional months improvement three months and status quo after three months. No MN given after 8 months.
7 12-24 months Vh l l year Bulhar involvement extreme \vhen MN initiated. This is only example functional response with this degree of bulhar involvement. Arms 62 legs surprisingly good. Good improvement for 6 months little skeletal muscle degeneration. About 7th months of Rs improvement stopped;
Patient Number Illness Prior to M-l\' Rx (months or years) 5 months 5 V2 months 15 months I 2 months 36 months 4 months 26 months I 3-16 months I 4 months 2 years 2 years I year 2 years Classification at time of M-N Rx III Bulbar Ill Bulbar III Bulbar III Bulbar Ill Bulbar ll lll Bulbar IV V Bulbar -llll Bulbar TABLE 2 Continued Summary of ALS Patients Under M-N Therapy (Treatment by injection of .8 to 1.2 ml of composition every other day) Example M-N L'sed Duration of MN Rx 4 months 2 months I year 8.: 8 months 1 year 4 months 19 months I year 1 year 2 I months 2% mos.
l month 5-6 months 3 months Comments Terminal bulbar. Arms & legs quite good function. Bulhar death. Therapeutic failure.
Terminal bulbar. Bilateral pyramidal involvement. Fair limb function. Bulhar death. Therapeutic failure.
Diffuse progressive ALS. Short periods of definite functional improvement and then regression. M-N stoppped after I year and 8 months. Death about 60 days later. presumably ALS cause not known.
MN irregular patient did not follow directions. Pyramidal tract involved. History of CVS heart pulmonary embolism prostatic complications. Bulbar questionable Death probably bronchopneumonia No post-mortem.
Diffuse ALS rapidly progressing from time in early diagnosis. In spite of sporadic brief periods of functional improvement. this patient continued on the inexorable course of ALS.
Relativelv slow disease process.
Patient s seen after about 7 months of experimental Rx with lsoprinosine.
Had definitely degenerated under Isoprinosine. With M-N showed temporary improvement and/or status quo. Off
M-N and degeneration was rapid. ALS death 2 months later.
Patient reacted well to M-N. Little or no degeneration in more than 1 year of Rx. Complications: Prostatitis (old) and fracture of neck of femur pyramidal tract involved. C VA cause of death.
Diffuse. rapidly progressive ALS. Patient is now semi-ambulatory and shows no bulhar signs. Deceleration with this M-N Rx.
Reacted well during first three weeks M-N Rx then status quo. Diffuscly and severely involved when M-N initiated.
Terminal ALS with history of more than 3 years of diagnosis. Tape recording indicates functional improvement treatment (moves better and speech can be understood). M-N stopped and patient after 41 days in hospital. died of probable bulbar ALS.
When M-N initiated. patient had deteriorated rapidly in that paresis of one leg was followed by essentially complete paralysis of both legs (neurological). Since M-N Rx there has been questionable degeneration Rate of ALS degeneration before and after M-N is important.
Initial proximation 0. right hand and left hand faciculations 4+.
Almost all bulhar symptoms. Tongue had complete atrophy. Within one week TABLE 2 Continued Summary of ALS Patients Under M N Therapy (Treatment by injection of .8 to 1.2 ml of composition every other day) Patient Illness Prior Classification Example Duration Comments Number to M N Rx at time of M-N M-N Used of M-N Rx (months Rx or years) of treatment pro.\imation ws good with both hands. Subject now can open and close buttons. After 12 days of treatment. there was complete use of the tongue and no choking.
4 to 5 years lll l\' 3 2 months Treatment improved speech swallowing and strength of knees. The wallowcontinues to imprme. Fa culations were eliminated after the first week of treatment.
0 months ll Ill 3 2 months Treatment produced a marked decrease in leg cramps and allowed walking without a. lattice. Subject can pull slacks down and up without assistance. Treatment improved the fine movement of the hands. Proximation is now okay as compared to 2+ prior to treatment.
13 2 years ll Ill 3 3-4 weeks lmproyement in swallowing alter 3 da \s Bulbar of treatment and SUhJBLl now walks better without shuffling.
24 3: years ll 3 l month Treatment impro\ ed lasciculalions and line mmements ol hands.
8 months ll 3 l month Rapid impro ement in right forearm and triceps after 1 week of treatment 2o 2 years \'a & \'b 2 months Sternocleidomastoid was initially tl.
Other neck muscles were initially U. Subject was quadriplegic. aphonic and the tongue was atrophic. Alter 3 weeks ol treatment. subject made sounds.
Maintained neck in upright position.
27 3 years l\ 3 2 months Within l month after treatment subject's breathing. hand movements and faseiculations were improved 2t 1: years \a 3 1 month After treatment. subjects breathing. tongue movement and swallowing improvement. Also there is slight improvement in leg movements.
2) 3 years \'a & \'b 3 2 months After treatment. subjects swallowing improved.
3() l year ll 3 2 months After treatment, there was improvement in right hand grip and extension offingers. Proximation improved from 2+ to okay.
3l 3 years ll-lll 3 2 /2 months Prior deterioration of condition I has been arrested by treatment and no further degeneration has taken place.
32 2 years \'b 3 1: mos. I Bulhar symptoms have improved with treatment.
3} 7-10 months I 3 2 months After treatment, the right foot drop improved and fascieulatiorts improved from 4+ to 2+.
34 l 1: years \'b 3 1 mos. Swallowing improved with treatment and aphonia improved to some speech.
35 months I ll 3 2 months Slight improvement in strength of legs after treatment 36 3 years ll 3 2 months Treatment improved the line movement of hands. Proximation improved to H105? from 50 The strength in both arms improved and there was a decrease in fascieulation.
37 3: years ll Ill 3 l month Treatment improved the walking of the subject.
38 l year Ill 3 2 months Swallowing was considerably improved Summary of ALS Patients Under M-N Therapy (Treatment by injection of .8 to 1.2 ml of composition every other day) Patient lllness Prior Classification Example Duration Comments Number to M-N Rx at time of M-N M-N Used of M-N Rx (months Rx or years) after treatment. Sub ect can now eat solid food as opposed to only being able to eat purecs prior to treatment. subject's walking changed from a shulTle to lifting up of his feet. Fusciculations significantly improved.
3) ti /2 years ill 3 2 months The weakness of legs made walking without help impossible. Within month of treatment. subject could get out of bed without assistance and use the bathroom.
40 4 years lll l\' 3 2 months Slight improvement with treatment but at least no further deterioration.
-ll 3% years \"a 3 3 months Slight improvement with treatment What is claimed is:
l. A method of treatment of animals suffering from a progressive degenerative neurological disease of motor nerve cell origins to neuromuscular junction, axones and nerve myelin sheaths comprising administering to the animal a disease mitigating amount of detoxified. and neurotropically active modified snake venom neurotoxin composition wherein the said snake venom neurotoxin is derived from the venom of a species of the Bungarus genus or from the venom of a species of the Bungarus genus and the Naja genus, and wherein said composition exhibits at least a inhibition of plaques in the Semliki Forest virus test and a bioassay shows that the composition is atoxic, as demonstrated by essentially no signs of toxicity in inoculation of laboratory animals.
2. The method of claim 1 wherein the said snake venom neurotoxin is derived from the venom of a spe cies of the Bungarus genus and the Naja genus.
3. The method of claim 1 wherein part of the said snake venom neurotoxin is derived from the venom of the species Crotalus terrlfi'cus.
4. The method of claim 1 wherein at least 75% inhibition of the said plaques is exhibited.
5. The method of claim 1 wherein in humans the dosage of the composition is from about 0.05 to 10 ml based on a 1% solution of the said modified neurotoxin per 150 lbs body weight.
6. The method of claim 5 wherein the dosage is from 0.4 to 3 ml.
7. The method of claim 5 wherein the dosage is administered in a frequency of from every other week to daily.
8. The method of claim 7 wherein the dosage is administered at least weekly.
9. The method of claim 7 wherein the dosage is administered at least 3 times a week.
10. The method of claim 7 wherein the administration is by subcutaneous or intramuscular injection.
11. The method of claim 2 wherein part of said snake venom neurotoxin is derived from the venom of Naja naja and Bungarus multicinctus..
but at leust no further deterioration.
12. The method of claim 2 wherein the ratio of Naja venom to Bungarus venom is 400:] to 8:1.
13. The method of claim 12 wherein the ratio is 80zl to 40:1.
14. The method of claim 1 wherein the progressive degenerative neurological disease is selected from amyotropic lateral sclerosis, multiple sclerosis, kuru, polymyositis, meningitides, muscular dystrophy, and polyomyosities.
15. A composition comprising in an administrable form detoxified and neurotropically active modified snake venom neurotoxin where the said snake venom neurotoxin is derived from the venom of a species of the Bungarus genus or from the venom of a species of the Bungarus genus and the Naja genus, and wherein the composition exhibits at least a 30% inhibition of plaques in the Semliki Forest virus test and a bioassay shows that the composition is atoxic, as demonstrated by essentially no signs of toxicity in inoculated laboratory animals.
16. The composition of claim 15 where the said snake venom neurotoxin is derived from the venom of a species of the Bungarus genus and the Naja genus.
17. The composition of claim 15 wherein part of the said snake venom neurotoxin is derived from the venom of the species Crotalus terrificus.
18. The composition of claim 15 wherein at least a inhibition of the said plaques is exhibited.
19. The composition of claim 15 wherein part of said snake venom neurotoxin is derived from the venom of the species Naja naja and the species Bungarus multicinctus.
20. The composition of claim 16 wherein the ratio of Naja venom to Bungarus venom is 400:1 to 8:1.
21. The method of claim 20 wherein the ratio is :1
22. The method of producing the composition of claim 15 comprising oxygenating at a pH of above 7 and a temperature of 15 to 40C the said snake venom until the venom is atoxic and dialyzing the resulting product.