US 20080 107752A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0107752 A1 Reid et al. (43) Pub. Date: May 8, 2008

(54) MODIFIED ELAPD VENOMS AS A6IP3L/00 (2006.01) STMULATORS OF THE IMMUNE (52) U.S. Cl...... 424/542 REACTION (57) ABSTRACT Inventors: (76) Paul F. Reid, Plantation, FL (US); Detoxified cobra Venom and its constituent neurotoxins have Laurence N. Raymond, been reported to have potent antiviral activity. Others have Plantation, FL (US) reported that Snake Venoms were generally immune stimu Correspondence Address: lants. Recent research has revealed more specific details on the effects of detoxified venoms and isolated alpha-neuro Robert J. Van Der Wall toxins on cells of the immune system. Exposure of the Gables One Tower immune cells to these detoxified proteins yields a strong Suite 1275, 1320 South Dixie Highway response in the innate immune reaction that represents the Coral Gables, FL 33146 immune systems initial response to infectious agents. Of particular relevance is the marked increase in the expression (21) Appl. No.: 11/592,896 of genes associated with the production of gamma inter feron, a potent antiviral agent and regulator of the immune (22) Filed: Nov. 6, 2006 response. The ability to induce this strong innate response has significant application to those with weakened immune Publication Classification systems where their ability to fight infection has been (51) Int. C. compromised. It also has the potential application to act as A6 IK 35/58 (2006.01) a method to protect individuals from contagious infectious A6IP37/00 (2006.01) agents as a Substitute for anti-viral vaccines. US 2008/01 07752 A1 May 8, 2008

MODIFIED ELAPD VENOMS AS 1978). This method was patented and published by Sanders STIMULATORS OF THE IMMUNE on several occasions with the last patent expiring in 1994. REACTION Furthermore, several techniques have been developed for modifying neurotoxins. These have included hydrogen per BACKGROUND OF THE INVENTION oxide, oZone, performic acid, iodoacetamide and iodoacetic 0001 1. Field of the Invention acid. Some of these procedures have been published and 0002 The present invention relates to a class of proteins, others patented. Obviously some procedures are easier than and a method for stimulating the immune system, especially others to utilize and the focus for commercial production has to the prevention and treatment of diseases such as viral, been on the simpler methods. bacterial and parasitic infections through the stimulation of 0007 Literature references of interest are: Boquet P; the innate immune system reaction. The composition is Ann. Inst. Pasteur 66:379-396 (1941), Chang C. C., Kawata comprised of a modified elapid venoms containing anticho Y., Sakiyama F. and Hayashi K.; Eur. J. Biochem. 193:L567 linergic alpha-neurotoxins. 572 (1990), Hudson RA, Montgomery IN and Rauch HC. 0003 2. Description of the Prior Art Mol Immunol. (1983) February; 20(2):229-32: Lamb, Gand 0004 Sanders et al. had commenced investigating the Hunter, W. K. The Lancet, 1:20-22; Miller, K., Miller, G. G., application of modified venoms to the treatment of Amyo Sanders, M. and Fellowes, O. N., Biophys et Biophysica trophic Lateral Sclerosis (ALS) in 1953 having employed Acta 496:192-196) (1977); Sanders M. and Fellows O.: poliomyelitis infection in monkeys as a model. Others Cancer Cytology 15:34-40 (1975), Yourist, J. E., Haines, H. antiviral studies had reported inhibition of pseudorabies (a G. and Miller, K. D. (1983) J. Gen Virol. 64, 1475-1481 herpesvirus) and Semliki Forest virus (alpha-Virus). See Sanders’ U.S. Pat. Nos. 3,888,977, 4,126,676, and 4,162, SUMMARY OF THE INVENTION 303. Sanders justified the pursuit of this line of research 0008. It is a principal object of the invention to provide through reference to the studies of Lamb and Hunter (1904) a method for preventing and treating infectious diseases, though it is believed that the original idea was postulated by Such as colds and flu, bacterial and parasitic infections and Haast. See Haast U.S. Pat. Nos. 4,341,762 and 4,741,902. A the like. basis of Sanders invention was the discovery that such 0009. It is a further object of the invention to provide a neurotropic Snake venom, in an essentially non-toxic state, therapy for the relief of diseases of the aforementioned type, also could block or interfere with invading pathogenic which therapy is safe, effective and may be administered bacteria, viruses or proteins with potentially deleterious over long periods of time. functions. Thus, the Snake venom used in producing the 0010. Other objects will be apparent to those skilled in composition was a neurotoxic venom, i.e., causing death the art from the following disclosures and appended claims. through neuromuscular blockade. As the dosages of Venom 0011. The present invention accomplishes the above required to block the nerve cell receptors would have been stated objectives, as well as others, as may be determined by far more than sufficient to quickly kill the patient, it was a fair reading and interpretation of the entire specification. imperative that the venom be detoxified. The detoxified but 0012. In accordance with a principal aspect of the present undenatured venom was referred to as being neurotropic. invention a modified and detoxified cobra Venom and neu The venom was preferably detoxified in the mildest and rotoxins purified therefrom suitably detoxified can prevent most gentle manner. While various detoxification proce the onset and Suppress the continued development of infec dures were known then to the art, such as treatment with formaldehyde, fluorescein dyes, ultraviolet light, ozone or tions in healthy individuals. heat, it was preferred that gentle oxygenation at relatively DETAILED DESCRIPTION OF THE low temperatures be practiced, although the particular PREFERRED EMBODIMENTS detoxification procedure was not defined as critical. Sanders employed a modified Boquet detoxification procedure using 0013 As required, detailed embodiments of the present hydrogen peroxide (Boquet 1941), outlined below. The invention are disclosed herein; however, it is to be under acceptability of any particular detoxification procedure was stood that the disclosed embodiments are merely exemplary tested by the classical Semliki Forest virus test, as taught by of the invention which may be embodied in various forms. Sanders, U.S. Pat. No. 4,162,303. Therefore, specific functional details disclosed herein are 0005. In patents issued to Haast, it was suggested that a not to be interpreted as limiting, but merely as a basis for the combination of neurotoxins and an unknown component of claims to be later appended and as a representative basis for Viperid venom were required. (Sanders did not employ a teaching one skilled in the art to variously employ the Viperid venom component). Haast employed native, present invention in virtually any appropriate circumstance. unmodified venom fractions the administration of which was 0014 Cobra venoms are characterized by their neuro reported to cause quite extensive pain for 1-2 days post toxic activity which is a result of one or more neurotoxins administration resulting often in short therapeutic periods found within the venom. Alpha-cobratoxin is an anticholin even if the reported effects were quite dramatic. Vague ergic neurotoxin (alpha-neurotoxin) found in Some cobra claims to stimulating the immune system were made without Venoms. In their native state they are an antagonist of the a clear mechanism, relating that a strong reaction to the alpha-nicotinic acetylcholine receptor. Other alpha-neuro injection of the venom mixture was indicative of a good toxins have been isolated from related species of Snakes and therapeutic response. fish-eating sea snails (Conus geographus, textilis, imperialis 0006. The production of drug product by Dr. M. Sanders and striatus). Cobratoxin and alpha-bungarotoxin (elapid, was achieved using hydrogen peroxide as the oxidizing krait) have highest affinity for NAchRs containing the alpha agent in addition to other components giving rise to the 1 and 7 subunits (for a review, see Lucas, 1995). The toxicity recipe he employed for over 30 years (Sanders et al., 1975, of these molecules is based upon their relative affinity for the US 2008/01 07752 A1 May 8, 2008

receptor which far exceeds that of acetylcholine. Many stud RNA expression of subunits for both nAchR (a2-a7 and ies (Sanders et al 1953, Miller et al., 1977, Hudson et al., a2-a4) was identified in human PBMCs indicating the pres 1983, Lentz et al., 1987, Donnelly-Roberts and Lentz, 1989, ence of NAchR on the cell surface (Sato et al., 1999). Inhibi Chang et al., 1990) have demonstrated various methods for tion of Concanavalin-A (ConA) induced T cell proliferation the chemical modification of cobratoxin. This is accom was blocked by the NAchRs antagonist mecamylamine and plished by oxidation of the cobratoxin with substances such counter-intuitively by the agonist nicotine (Singh et al., as hydrogen peroxide, formalin and oZone, which results in 2000). Acute nicotine exposure of Con A stimulated mouse an alteration in affinity for the acetylcholine receptor (AchR) splenocytes resulted in decreased production of IL-10 and and a concomitant loss in toxicity. also resulted in increased production of interferon (IFN)- 0015. As taught by Sanders, removal of the toxicity of gamma (Hallquist et al., 2000). Affinity purification T-cell cobratoxin can be achieved by exposure to heat, formalin, receptors using a-BTX confirmed that the NAchR were the hydrogen peroxide, performic acid, oZone or other oxidizing/ same alpha-neurotoxin-sensitive receptors as those found in reducing agents. The result of exposure of cobratoxinto these muscle (Toyabe et al., 1997). Additionally, binding of agents is the modification of amino acids as well as the pos H-nicotine to human neutrophils, monocytes and lympho sible lysis of one or more disulfide bonds. Tu (1973) has cytes (Davies et al., 1982) has been reported. The formation demonstrated that the curaremimetic alpha neurotoxins of of E-rosettes, a function of T cells from peripheral blood, and cobra and krait venoms lose their toxicity upon either oxida a method used for T cell enumeration, is decreased by 30%- tion or reduction and alkylation of the disulfide bonds which 40% by carbamylcholine chloride, a cholinergic antagonist, has been confirmed by Hudson et al. (1983). Loss of toxicity confirming thE expression of NAchRs on at least one subset can be determined by the intraperitoneal injection of excess of human T cells (Mizuno et al., 1982). levels of the modified cobratoxin into mice. In general a 0.5 0018. Therefore, T-cell functions can clearly be influenced ml Volume containing 0.5-1 mg of modified cobratoxin is by anticholinergics including peptide neurotoxins, an impor tested, which represents a minimum of a 4000-fold reduction tant aspect in immunity. Consequently, it was selected to of toxicity. Alternatively, loss of toxicity can be evaluated by assay the effects of MN and MCTX on the transcription of depression of binding of the modified neurotoxin to acetyl genes in purified T-cells by microarray analysis to understand choline receptors (AchR) in vitro. Cobra venoms are a rela the mechanisms associated with immune modulation and tively cheap raw material source whose production can be antiviral activity. Based on prior anti HIV studies, venoms scaled up to meet higher demands. Parenteral, oral and topical from the Cape (Naja nivea) and Thailand (Naja kaouthia) formulations of MN and MCTX have been described previ cobra were chosen in addition to detoxified cobratoxin, iso ously by the above identified authors. lated from the Thailand cobra, to be studied in the micro-array 0016 Cobra venom (MN) and cobratoxin (MCTX) in assay. The data revealed the all these products induced an their oxidized (modified or non-toxic) forms have demon upregulation of the genes encoding cytokines involved in the strated antiviral activities in-vitro and in-vivo against polio innate immune reaction. Cytokines such as IL-2, Il-8, Il-18, virus, pseudorabies virus, Semliki Forest virus, herpes sim Il-23 were upregulated in addition to a major increase in plex 1 virus, HIV and rabies virus, viruses without any genes associated with the expression of interferon gamma obvious similarities instructure or infectious pathway. Native and interferon related proteins over normal unexposed cobratoxin and formaldehyde-treated cobratoxin reportedly T-cells. There were slight differences in the profile of upregu lack this activity (Miller et al., 1977). The mechanism by lation within those associated genes by the different products. which MN or MCTX exert this capacity was not clear as many 0019. It is interesting to note that in most cases MCTX viruses employ a variety cell Surface receptors as portals for induced higher transcription of these immune regulatory entry into the cell prior to replication. Hudson et al. (1983) genes compared to MN possibly due to a dose related effect reported that cobratoxin subjected to alkylation would inhibit (equal doses of each were employed in the studies). MCTX the onset of experimental acute encephalitis (EAE) in guinea upregulated IFN-gamma and Il-23 most whereas MN from pigs, a model for multiple sclerosis. EAE is a T-cell mediated Naja nivea upregulated IL-18 most, which is interesting in autoimmune disease. The study was undertaken as sequence light of this MN's higher antiviral activity. However, it homology was noted between cobratoxin and myelin basic emphasizes the important contribution of alpha-neurotoxins protein (MBP), the protein antigen used to induce the disease. to the activity in MN preparations. Recently, MCTX has been shown to inhibit the replication of 0020. The large and consistent induction of interferon the human immunodeficiency virus (HIV) in peripheral blood gamma provides an explanation for the broad antiviral activ mononuclear cells (PBMC's) suggesting the ability of the ity historically associated with these products. Interferons protein to influence events within immune cells. The mecha alpha and beta were not upregulated to any significant degree nism of action was unclear save to say that there is no direct over control cells. These results are also consistent with clini effect on the virus and there is an event at the cell surface that cal reports by treated subjects of improved health status and renders the cell resistant to viral infection. This conclusion resistance to infections especially of viral origin. The study was drawn from the fact the pretreatment of the immune cells implies that normal individual exposed to these products will with MCTX followed by removal of the drug still results in respond through the increased production of innate immunity reduced viral replication (Yourist et al., 1983). This antiviral cytokines such as interferon gamma and thereby adopt an characteristic is also maintained in cells reportedly devoid of antiviral or heighten immune reactivity to infection. It should acetylcholine receptors such as baby hamster kidney (BHK be noted that similar studies conducted in PMBCs from Sub 21) cells. jects with an autoimmune disease yielded a different expres 0017 Human T lymphocytes are a major source for ace sion profile, which emphasizes the regulatory pathways tylcholine (Ach) (Fujii and Kawashima, 2001; Sato et al., involved in gene expression. Therefore one Supposes that the 1999; Kawashima et al., 1998; Fujii et al., 1996) suggesting a products could be employed within the scope of an infection link between the nervous and immune systems. Messenger control and prevention strategy such as is currently adopted US 2008/01 07752 A1 May 8, 2008 for flu season in elderly, immune Suppressed and infant popu from the test samples. Once background was Subtracted, the lations. In addition, they could be employed in a therapeutic individual data were combined and subjected to statistical role in viral infections or in association with standard treat analysis. The data was reported as the means of the samples ments for bacterial and parasitic infections to effect a more that met 90% confidence (p=0.10). rapid recovery. In viral infections such as influenza the com 0026. The 11 genes that were upregulated most are position would provide advantages over vaccine-based strat reported in Table 1. egies in that it does not have to be virus or subtype specific and would be readily accessible during outbreaks and pandemics. EXAMPLE 2 Moreover the use of these products could be beneficial in subjects suffering from the side effects of cancer therapy Micro-Array Analysis of Human T-cells Exposed to through reduced immune reactivity. Furthermore the upregu Modified Naja kaouthia Venom (MNK) lation of cytokines like IFN-gamma could contribute to the 0027 Method of analysis was as described for Example 1 therapeutic response in cancer patients. and the responses in cytokine production are reported in Table 0021. It is envisioned that a subject may be given MN or 1. MCTX as infrequently as every other week though it is pre ferred that the composition be administered at least biweekly. EXAMPLE 3 The composition may be administered orally, Subcutane ously, intramuscularly or intravenously. Parenterally, either Micro-Array Analysis of Human T-Cells Exposed to subcutaneous or intramuscular injection is preferred. While Modified Cobratoxin (MCTX) the correct formulation with benzalkonium chloride will per 0028 Method of analysis was as described for Example 1 mit oral administration through absorption through the oral and the responses in cytokine production are reported in Table mucosa (preferably Sublingually), this formulation may also 1. permit administration otically. Furthermore transdermal 0029 Cobratoxin represents 15-20% of Naja kaouthia delivery may be effected if formulated in an appropriate venom and so it could be concluded that the venom results cream or lotion base using benzalkonium chloride or propy reflect a cobratoxin low-dose response. However, Naja nivea lene glycol as a permeation enhancer. venom has very low levels of cobratoxin though it still induces the innate immune response strongly and has strong EXAMPLE1 antiviral activity presumably due to the presence of other Micro-Array Analysis of Human T-Cells Exposed to alpha-neurotoxins such as cobrotoxin. Modified Naja nivea Venom (MNN) TABLE 1 0022 Methods: T-cells were collected from waste whole blood collected under IRB-approved protocol. In brief, blood The fold increase in cytokine genes upregulated by MN and was placed in Becton Dickenson Cell-Prep Tubes (CPT) and MCTX over untreated controls. centrifuged to separate PBMC's from RBCs. PBMC's were No. Gene MNN MINK MCTX collected, washed 3X with warm PBS and cultured on plastic 1 Interferon gamma 11.8 6.12 23.1 overnight at 37° C./5% CO, in RPMI-1640 containing pro 2 Interferon inducible protein 7.09 6.16 21.18 tein and cytokine Supplementation to remove macrophages. 3 Macrophage inflammatory protein NA* S.1 12.77 Remaining PBMC's were again washed 3X with PBS and 4 I-23 NA 5.99 1427 mixed with DynaBeads. The T-cells were then isolated by 5 IL-18 5.56 2.1 2.7 6 Il-10 2.06 1.92 3.10 magnetic bead separation. The purified T-cells were washed 7 IL-8 1.99 NA 4.87 3x with warm PBS and cultured for 2 days as described above 8 IL-5 2.1 2.0 0.75 to acclimate the T-cells to culture conditions. T-cell purity 9 Il-2 1.94 3.2 3.06 was confirmed by flow cytometry. 0023 Assay: 5.0E5 T-cells from each sample were *NA = not induced. removed from the culture and divided into 2 parallel cultures, 0030. While the invention has been described, and dis one containing 2.5E5 cells with 10 ug/ml of modified venom closed in various terms or certain embodiments or modifica in 2.5 ml serum/cytokine-free RPMI-1640 and the other con tions which it has assumed in practice, the scope of the inven taining 2.5E5 cells with 2.5 ml serum/cytokine free medium. tion is not intended to be, nor should it be deemed to be, Cells were then placed in culture for 18 hours. Using a limited thereby and such other modifications or embodiments CASYLTTC Cell Analyzer to determine remaining cell con as may be suggested by the teachings herein are particularly centration, 1.25E5 cells were harvested, washed 3X with reserved especially as they fall within the breadth and scope warm PBS-T and centrifuged into a pellet. After removal of of the claims here appended. the final wash Supernatant, the pellet was resuspended in RNA-preservation solution and Snap-frozen in liquid nitro What is claimed is: gen and stored at -130° C. until Micro-array analysis. 1. A method of protecting animals from infections by 0024 Micro-array Analysis: Samples were transported on administering to the animals a detoxified and neurotropically dry ice to the microarray facility and processed per standard active modified venom composition containing alpha neuro protocol as established and published by Affymetrix. Briefly, toxins. RNA is harvested, placed on HG-U133Plus2.0 array chips. 2. The method of claim 1 wherein the detoxified and neu After hybridization, chips were placed into Affymetrix chip rotropically active modified composition is comprised of a reader and analyzed. fraction containing the alpha-neurotoxins. 0025 Statistics: The control samples cultured in parallel 3. The method of claim 1 wherein the alpha-neurotoxins without drug exposure were used to Subtract background are selected from the group consisting of alpha-bungarotoxin, US 2008/01 07752 A1 May 8, 2008

kappa-bungarotoxin, alpha-cobratoxin, alpha-cobrotoxin, 13. The method of claim 11 wherein the alpha-neurotoxins alpha-conotoxins (G1, M1, S1, S1A. ImI), alpha-dendrotoxin are selected from the group consisting of alpha-bungarotoxin, and erabutoxin. kappa-bungarotoxin, alpha-cobratoxin, alpha-cobrotoxin, 4. The method of claim 3 wherein the alpha-neurotoxin alpha-conotoxins (G1, M1, S1, S1A. ImI), alpha-dendrotoxin composition is comprised of alpha-cobratoxin. and erabutoxin. 5. The method of claim 1 wherein the infection is viral in nature. 14. The method of claim 13 wherein the alpha-neurotoxin 6. The method of claim 1 wherein the infection is bacterial composition is comprised of alpha-cobratoxin. in nature. 15. The method of claim 11 wherein the infection is viral in 7. The method of claim 1 wherein the infection is parasitic nature. in nature. 16. The method of claim 11 wherein the infection is bac 8. The method of claim 1 in which administering is com terial in nature. prised of at least one of orally, Subcutaneously, intramuscu 17. The method of claim 11 wherein the infection is para larly, and intravenously. sitic in nature. 9. The method of claim 1 in which administering is pref 18. The method of claim 11 in which administering is erably by one of Subcutaneous and intramuscular injection. comprised of at least one of orally, Subcutaneously, intramus 10. The method of claim 1 in which the animals are cularly, and intravenously. humans. 11. A method of ameliorating infections in animals by 19. The method of claim 11 in which administering is administering to the animals a detoxified and neurotropically preferably by one of Subcutaneous and intramuscular injec active modified venom composition containing alpha neuro tion. toxins. 20. The method of claim 11 in which the animals are 12. The method of claim 11 wherein the detoxified and humans. neurotropically active modified composition is comprised of a fraction containing the alpha-neurotoxins.