Venom therapy in multiple sclerosis

Neuropharmacology 53 (2007) 353e361 www.elsevier.com/locate/neuropharm

Review

Venom therapy in multiple sclerosis Abbas Mirshafiey* Department of Immunology, School of Public Health, Medical Sciences, University of Tehran, Box 6446, Tehran 14155, Iran Received 22 November 2006; received in revised form 30 April 2007; accepted 3 May 2007

Abstract To date many people with multiple sclerosis (MS) seek complementary and alternative medicines (CAM) to treat their symptoms as an adjunct to conventionally used therapies. Among the common CAM therapies, there is a renewed interest in the therapeutic potential of venoms in MS. The efficacy of this therapeutic method remains unclear. However, venom-based therapy using bee, snakes and scorpions venom and/or sea anemones toxin has been recently developed because current investigations have identified the various components and molecular mechanism of the effects of venoms under in vitro and in vivo conditions. The aim of this review is to describe the recent findings regarding the role of venoms and their components in treatment of MS disease and that whether venom therapy could be recommended as a complementary treatment or not. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Multiple sclerosis; Bee venom; Snake venom; Scorpion venom; Sea anemone toxin; Complementary medicine; Alternative medicine

1. Introduction Multiple sclerosis (MS) is an autoimmune disease associated with chronic inflammatory demyelination of the central nervous system. Due to disease complexity and heterogeneity, its pathogenesis remains unknown and despite extensive research efforts, specific effective treatments have not yet been developed (Alcaro and Papini, 2006; Bar-Or, 2006; Birnbaum, 2006; Goldman et al., 2006). The disease-modifying treatments, IFN-beta and glatiramer acetate, have been widely available over the last decade and have shown a beneficial effect on relapse rate and magnetic resonance imaging parameters of disease activity; however, their effects on disease progression and disability are modest. Thus, the search for alternative therapeutic strategies continues and neurologists are often uncertain how to view the many complementary and alternative medicines (CAM) approaches, used by their patients (Farrell et al., 2005; Yadav and Bourdette, 2006; Carlson and Krahn, 2006; Shinto et al., 2006; Virley, 2005; Stangel et al., 2006; Goodin, 2005; Derwenskus, and Lublin,

* Fax: þ98 21 6646 2267. E-mail address: [email protected] 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.05.002

2006). It is an important issue that 50e75% of MS patients use one or more CAM therapies (Murray, 2006; Huntley, 2006). Among the existing CAM therapies, venom-based therapy has been currently developed using bee venom and/or snakes and scorpion venom in uncontrolled studies and have provided evidence that they may have some benefits (Norton et al., 2004; Hinman et al., 1999; Breland and Currier, 1983; Castro et al., 2005). Regarding the fact that venom therapy has so far been well based on symptomatic treatment and considering to existing appropriate medications for attenuating symptoms in MS, this review undermines the concepts on the magical effects of venom therapy in MS patients. However, the research on venom-based therapy in MS is still exploratory, but considering peoples’ interest and common use of this therapy, further research in this area is clearly warranted (Yadav and Bourdette, 2006; Shinto et al., 2005). The purpose of this review is to assess treatment outcome following venom therapy in multiple sclerosis. 1.1. Bee venom Venom of the honeybee Apis mellifera contains a variety of different low and high molecular weight peptides and proteins

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including melittin, Apamin, adolapin, mast cell degranulating peptide and phospholipase A2 (Eiseman et al., 1982; Kwon et al., 2002; Schmidt, 1982). In addition, it also consists of biologically active amines (histamine, epinephrine) and several other non-peptide components including lipids, carbohydrates and free amino acids (Lariviere and Melzack, 1996). Melittin and phospholipase A2, two major components of bee venom (BV), are generally thought to play an important role in inducing the irritation and allergic reactions associated with the bee stings (Kim et al., 2003). Although the injection of BV has been reported to evoke tonic pain and hyperalgesia (Lariviere and Melzack, 1996), but conflicting evidence in the literature indicates that BV also could exert anti-inflammatory and antinociceptive effects on inflammatory reactions (Lee et al., 2001; Kang et al., 2002; Kwon et al., 2001). In this regard, BV has been traditionally used in oriental medicine to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis (Lee et al., 2001). Moreover, the antiinflammatory property of whole venom of honeybee has been reported in rat model of adjuvant-induced arthritis as experimental model of rheumatoid arthritis and mouse air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leukocyte migration and a significant reduction in concentration of tumor necrosis factor (TNF)alpha. These results suggest that the anti-inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla (Kang et al., 2002; Kwon et al., 2001, 2002, 2003). Furthermore recent reports indicate that BV is also able to inhibit tumor growth and exhibit anti-tumor activity in vitro and in vivo and can be used as a chemotherapeutic agent against malignancy (Liu et al., 2002; Orsolic et al., 2003). Although investigations on the honey BV started almost a hundred years ago, its mechanism of action is still uncertain (Langer, 1897). However, it has been reported that immunotherapy using BV could lead to production of interleukin (IL)-10 and in contrast, it results in a decreased IL-4 and IL-5 and increased interferon-gamma secretion (Jutel et al., 1995; Bellinghausen et al., 1997). In addition, BV can inhibit the activity of pro-inflammatory metaloenzymes, matrix metalloproteinase-2 and -9 and increase interferon beta production based on a time and dose-dependent format and the type of treated cell line (Hamedani et al., 2005). 1.2. Treatment with BV Some recent reports suggest that bee venom may be an effective treatment for patients with MS and/or other autoimmune diseases, such as rheumatoid arthritis (Castro et al., 2005; Kwon et al., 2002; Kang et al., 2002). However patients may be subjected to real risks of serious allergic reactions as well as emotional and economic costs. In a research using a total of nine patients with progressive form of MS, hyperreactivity to bee venom was studied. Although no serious adverse allergic reactions were observed in any of the nine subjects, but four experienced worsening of neurological symptoms, which this could not be ascribed to side effects of the therapy.

Of the remaining five subjects, three felt that the therapy had subjective amelioration of symptoms and two showed objective improvement (Castro et al., 2005). Although this preliminary study suggests its safety, because of the few numbers of studies, there are no definite conclusions regarding BV efficacy and therefore there is a little evidence to support the use of honeybee venom in the treatment of MS. Larger and more carefully conducted multicenter studies will be required to establish efficacy. In contrast, a randomized crossover study based on 24 weeks bee sting therapy for MS using 26 patients with relapsing-remitting or relapsing secondary progressive showed that although, bee sting therapy was well tolerated, and there were no serious adverse events, but it could not reduce the cumulative number of new gadolinium-enhancing lesions which is an important diagnostic determinant in evaluating the process of disease progress. Moreover, there was no significant reduction in relapse rate and improvement of disability, fatigue, and quality of life (Wesselius et al., 2005). Collectively, the different results reported above for bee venom could be dependent on the therapeutic protocols used, type of animal model and/or type of challenged cell line, in addition to potential time and dose-dependent properties. Furthermore side effects associated with BV therapy, as well as immunostimulant properties could essentially limit the efficacy of BV in the treatment of MS (Hamedani et al., 2005; Cerrato, 1998; Fisher, 1986).

1.3. Sea anemones venom In recent years, more than 32 species of sea anemones have been reported to produce lethal cytolytic peptides and proteins. They have been classified into various groups based on structure and functional properties of cytolysins and other biomolecules, such as potassium channel blocker peptide (Anderluh and Macek, 2002; Martinez et al., 2001; Lanigan et al., 2001; Yan et al., 2005; Cristina Pico et al., 2004; Mancheno et al., 2003; Pazos et al., 2003). Stichodactyla helianthus is a sea anemone relatively abundant along Cuban coasts appearing in two morphos with different colors in their tentacles: green or brownish, probably due to their association with algal symbionts. They have been used as a source of potassium channel blocker peptide and sticholysins I and II are the most characterized cytolysins from this anemone (Martinez et al., 2002; Middleton et al., 2003). ShK toxin, a 35-residue polypeptide cross-linked by 3 disulfide bridges isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK adopts a central helix-kink-helix fold, and alanine-scanning and other mutagenesis studies have defined its channel-binding surface. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a synthetic K(v)1.3 inhibitor for blocking this channel (Norton et al., 2004; Lanigan et al., 2001; Middleton et al., 2003; Wulff et al., 2003; Beeton et al., 2001a,b, 2003). In neurons, Kv channels play a crucial role in action potential repolarisation

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and in shaping neuronal excitability. In non-excitable cells, such as T lymphocytes, Kv channels and calcium-activated Kþ channels (KCa channels) determine the driving force for Ca2þ entry. During T cell activation the calcium entry depolarises the cell and increases the cytosolic calcium concentration, which in return activates Kv and KCa channels (Devaux et al., 2004). On the other hand, expression of the two lymphocyte potassium channels, the voltage-gated channel Kv1.3 and the calcium activated channel (KCa), changes during differentiation of human T cells. KCa is a functionally dominant channel in naive and ‘‘early’’ memory T cells, whereas Kv1.3 is crucial for the activation of terminally differentiated effector memory (TEM) T cells. ShK is able to block Kv1.3 at low picomolar concentrations. Because of the involvement of TEM cells in autoimmune processes, Kv1.3 is regarded as a promising target for the treatment of T-cell mediated autoimmune diseases such as multiple sclerosis and the prevention of chronic transplant rejection (Norton et al., 2004). 1.4. Treatment with sea anemone toxin Voltage-gated potassium channels (Kv channels) are ion channels, which their openings provide an outward flow of potassium ions repolarising the cell. It has been analyzed the biophysical and pharmacological properties of five cloned Kþ (Kv) channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv3.1) stably expressed in mammalian cell lines. Kv1.1 is biophysically similar to a Kþ channel in glioma cells and astrocytes, Kv1.3 and Kv3.1 have electrophysiological properties identical to those of the types n and l Kþ channels in T cells, respectively, and Kv1.5 closely resembles a rapidly activating delayed rectifier in the heart (Devaux et al., 2004; Grissmer et al., 1994). The roles of Kv channels in nervous and immune systems have been currently investigated on experimental autoimmune encephalomyelitis (EAE), the experimental model of MS disease. In T-cell line reactive to myelin antigen, Kv1.3 channels are constitutively expressed and their blockade can reduce cytokine production and T cell proliferation (Norton et al., 2004). Blockade of Kv channels alone or in combination with KCa channels by 2 broad-spectrum K channel blockers, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) and/or using sea anemones toxin, ShK and ShKDap(22) improves the symptoms of the disease. These results demonstrate that Kþ channel blockers, such as (Kv1.3 channels in T cell) are immunosuppressive agents with beneficial symptomatic effects in EAE model (Devaux et al., 2004; Judge and Bever, 2006). Thus, ShK and its analogs are currently undergoing further evaluation as leader in the development of new biopharmaceutical agents for the treatment of multiple sclerosis and other T-cell mediated autoimmune disorders (Norton et al., 2004). 1.5. Scorpion venom Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an

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antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume (Drakopoulou et al., 1998). Scorpions venom contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca2(þ)-activated Kþ channels and for dendrotoxin-sensitive voltage-dependent Kþ channels. It has been tested the effects of charybdotoxin and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin, and kaliotoxin from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively on potassium channels (Harvey et al., 1995). Kaliotoxin (a 37 residue polypeptide) displays a sequence homology with other scorpion-derived inhibitors of Ca(2þ)-activated or voltage-gated Kþ channels: 44% homology with charybdotoxin, 52% with noxiustoxin, and 44% with iberiotoxin (Crest et al., 1992; Canto et al., 1999; Fernandez et al., 1994). Kaliotoxin, as blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. Firstly, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Secondly, Kv1.1 is found in paranodal regions of axons in the central nervous system. Blockade of Kv1.3 channels which are expressed on T cell sensitized with myelin basic protein leads to a pronounced reduction of the T cell proliferative response, cytokine production and Ca2þ influx. In the rat, blockade of Kv1.3 inhibits the delayed type hypersensitivity response to myelin basic protein and prevents and treats adoptive experimental autoimmune encephalomyelitis (Beeton et al., 2001a; Devaux et al., 2004). Based on the homologous model of the Kv1.3 potassium channel, the isolation and recognition of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin have been provided (Yu et al., 2004). Maurotoxin (a 34-amino acid polypeptide) is a toxin derived from the Tunisian chactoid scorpion Scorpio maurus palmatus, and it is a member of a new family of toxins that contain four disulfide bridges. It shows 29e68% sequence identity with other Kþ channel toxins and blocks the channels (Kv1.1, Kv1.2 and Kv1.3) in a voltage-dependent manner and despite the unusual disulfide bridge pattern, the mechanism of the action of maurotoxin is similar to those of other K(þ) channel toxins with only three disulfide bridges (Avdonin et al., 2000; Kharrat et al., 1997). In addition, three novel toxins belonging to the scorpion Kþ channel-inhibitor family were purified from the venom of the Chinese scorpion Buthus martensi. They have been characterized as 37-amino acid peptides. One of them shows 81e87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65e70% identity with toxins of the charybdotoxin group. It was proved that these toxins are potent inhibitors of the voltage-gated Kþ channels (Romi-Lebrun et al., 1997). Also, two novel toxins were purified from the venom of the Mexican scorpion, Centruroides limpidus limpidus using an immunoassay based on antibodies raised against noxiustoxin, a known K(þ)-channel-blocker-peptide. These toxins are capable of inhibiting transient K(þ)-currents, in cultured rat cerebellar granule cells (Martin et al., 1994).

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1.6. Treatment with scorpion toxin The therapeutic property of Kaliotoxin was investigated via its immunosuppressive effect and symptomatic influence using EAE, as experimental model for multiple sclerosis. The T cell line used to induce adoptive EAE was myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by Kaliotoxin. Activation was a crucial step for MBP-T cells to become encephalitogenic. Adding the Kaliotoxin during Ag-T cell activation led to a great reduction in the MBP-T cell proliferative response and production of IL-2 and TNF, and Ca(2þ) influx. Furthermore, the use of Kaliotoxin during T cell activation in vitro led to a decreased encephalitogenicity of MBP-T cells. Moreover, injection of Kaliotoxin to Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this neuronal and lymphocyte channels blocker to Lewis rats improved the symptoms of EAE. Collectively, Kaliotoxin could be probably an immunosuppressive agent with beneficial effects on the neurological symptoms of EAE and MS disease (Breland and Currier, 1983; Beeton et al., 2001a; Canto et al., 1999; Fernandez et al., 1994). Also, the Ca2þ-activated Kþ channel of intermediate conductance, such as charybdotoxin (a 37-residue polypeptide with three disulfide bonds) might be a possible target for immune suppression (Jensen et al., 2002). On the other hand, regarding the important role of microglial cells and astrocytes in pathogenesis of MS, microglial cells were cultured from murine neonatal brain. Ramification of isolated microglia could be induced by the application of astrocyte-conditioned medium (ACM). Voltage-gated outward potassium currents (IK) were measured in ramified microglial cells 12e24 h after their treatment with ACM. The effects of the specific Kþ channel blockers charybdotoxin, noxiustoxin and kaliotoxin on IK of ramified microglia were studied. These peptides (toxins) blocked IK in a concentration-dependent manner, while showing a high sensitivity for IK (Eder et al., 1996). Taken together, although biopharmaceutical Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission (Beeton et al., 2001a; Devaux et al., 2004), but available data show that we are still in the stage of experimental investigations.

1.7. Snakes venom Snakes venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. The activators and inhibitors existing in venom which affect on blood coagulation and fibrinolysis systems are of special interest (Gornitskaia et al., 2003). Venom components can be classified according to their action into procoagulants, anticoagulants and fibrinolytic enzymes, neurotoxins, Cardiotoxins (cytotoxins), complement depleting agent, hyaluronidase enzyme, phospholipases and some others (Petretski et al.,

2000; Cho et al., 2001; Iwai et al., 1999; Nose et al., 1994; Maruyama et al., 1990; Marsh and Williams, 2005; Castro et al., 2004; Judge et al., 2002; Zhang and Tu, 2002; Liukmanova et al., 2004; He et al., 2004; Ogay et al., 2005; Pergolizzi et al., 2005; Gatineau et al., 1987; Girish and Kemparaju, 2005; Kock et al., 2004; Newitt et al., 1991). Among the mentioned various components, it is essentially emphasized on fibrinogen depleting agents, dendrotoxin I and complement depleting factor as therapeutic tools in MS disease. It has been shown that the venoms of three snakes can induce defibrinogenation: ancrod from the venom of Calloselasma rhodostoma (formerly known as Agkistrodon rhodostoma), batroxobin from the venom of Bothrops atrox moojeni, and crotalase from the venom of Crotalus adamanteus. The purified fractions of ancrod, batroxobin, and crotalase possess coagulant, proteolytic and esterolytic properties, although their primary mechanism of action is a proteolytic effect on circulating fibrinogen. Ancrod is a biological agent extracted from the venom of the Malayan pit viper that reduces blood fibrinogen levels. This action prolongs blood clot formation and lowers blood viscosity (Bell, 1997; Sherman, 2002; Dempfle et al., 2000, 2001a,b). Ancrod cleaves only the Afibrinopeptides, but not the B-fibrinopeptides, from fibrinogen; this contrasts with thrombin, batroxobin and crotalase action, which cleave both fibrinopeptides A and B (Bell, 1997). Batroxobin, a thrombin-like enzyme of Bothrops atrox moojeni venom specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. Also, Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogenous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snakevenom proteases such as batroxobin (94% identity), and only 30% and 34% identity with human alpha-thrombin and trypsin, respectively (Petretski et al., 2000; You et al., 2004; Watanabe et al., 2002; Wu et al., 2000). Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), which binds to neuronal Kþ channels. This neurotoxin is a powerful potassium channel blocker and a potent inhibitor of various types of voltage-gated Kþ currents (Newitt et al., 1991; Nishio et al., 1998; Lucchesi and Moczydlowski, 1990; Lancelin et al., 1994; Foray et al., 1993; Bidard et al., 1987; Bidard et al., 1989; Rehm et al., 1988). In addition, beta-Bungarotoxin (beta-Butx), a presynaptically active neurotoxin isolated from snake venom, is thought to bind to a subtype of voltage-gated Kþ channels. Also, this neurotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization (Schmidt and Betz, 1989; Herkert et al., 2001). Cobra venom factor (CVF) is a complement activating protein from cobra venom. It is a structural and functional analog of complement component C3. CVF functionally resembles C3b, the activated form of C3. CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase. The CVF depletes plasma from essential

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complement components. Indeed, hypocomplementation and/ or decomplementation by cobra venom factor dose-dependently attenuate inflammatory reactions related to complement system activation (Kock et al., 2004; Brook et al., 2005; Blatteis et al., 2004; Tsuji et al., 1997).

1.8. Treatment with snake venoms In MS lesions, fibrinogen escapes from the blood into brain tissue across a broken down bloodebrain barrier, so that an increased fibrin deposition has also been reported at the site of nerve lesions in patients with MS (Adams et al., 2004). Moreover, it should be noted that absence of fibrin is critical in regeneration of myelin sheath. In an experiment on nerve regeneration in mice lacking fibrin, strikingly, these mice regenerated crushed nerves significantly faster than mice with fibrin. In the absence of fibrin, sheath cells are able to mature more quickly and can more efficiently remyelinate damaged nerves. These results suggest that preventing fibrin deposition may be a means to enhance the nervous system’s regenerative capacities (Akassoglou et al., 2000). The role of coagulationfibrinolysis system in experimental model of MS has been studied using batroxobin, derived from the venom of the South American pit viper Bothrops atrox moojeni. Batroxobin converts circulating fibrinogen into an insoluble form and causes a profound degree of afibrinogenemia. Treatment with batroxobin suppresses clinical signs of cell transferred EAE. Plasma fibrinogen concentration decreases significantly in batroxobintreated rats. Whereas, histological degree of perivascular mononuclear cell infiltration in the spinal cord is not suppressed in batroxobin-treated rats compared to control animals, however, deposition of fibrin around the vessels in the spinal cord is markedly suppressed in rats treated with batroxobin (Iwai et al., 1999; You et al., 2004; Inoue et al., 1996). Moreover, the therapeutic effects of ancrod and batroxobin have been investigated in patients with stroke, deep-vein thrombosis, myocardial infarction, peripheral arterial thrombosis and rheumatoid arthritis (Bell, 1997; Sherman, 2002; Dempfle et al., 2000, 2001a,b; Cai et al., 2002). It has been shown that within minutes of administration of ancrod or batroxobin, there is a significant reduction in plasma fibrinogen levels, and these remain exceedingly low with repeated administration (once or twice daily). The rapid fall in plasma fibrinogen levels is accompanied by a slightly delayed but marked rise in the level of fibrinogen-fibrin degradation products. Plasminogen levels are decreased and blood viscosity is reduced, but formed elements in the circulating blood remain unaltered (Bell, 1997). The venoms that enable to block Kþ channel such as dendrotoxin I, and/or beta-Bungarotoxin could be probably effective as immunosuppressive agents with beneficial symptomatic effects in experimental model of MS (Judge and Bever, 2006; Bidard et al., 1987). On the other side, the effects of decomplementation by CVF on the pathogenesis of inflammation and demyelination in EAE routine model and antibody-mediated demyelinating form of EAE have been verified histologically and immunocytochemically

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(Piddlesden et al., 1991). Depletion of serum complement with CVF suppresses the clinical signs and symptoms of acute inflammatory EAE induced either by immunization with myelin basic protein (MBP) or by the passive transfer of MBP activated spleen cells. Since, complement-dependent mechanisms are involved both in the clinical expression of acute inflammatory lesions and in the pathogenesis of EAE antibody-mediated demyelination (Linington et al., 1989; Hudson et al., 1983; Morariu and Dalmasso, 1978; Levine et al., 1971; Abrahamson, 1971).

2. Discussion The literature data assembled in this article review the role of venom therapy in multiple sclerosis and experimental model of this disease (EAE). An emerging body of literature has recently defined conflicting properties in connection with BV therapy using in vivo and/or in vitro models (Castro et al., 2005; Hamedani et al., 2005; Wesselius et al., 2005; Cerrato, 1998; Fisher, 1986; Nam et al., 2003; Jang et al., 2003; Magnan et al., 2001; Dayan et al., 1983). In one hand, the anti inflammatory and immunosuppressive effects of BV have been reported in MS disease, rheumatoid arthritis and their experimental models (Castro et al., 2005; Shinto et al., 2005; Eiseman et al., 1982; Kwon et al., 2002; Schmidt, 1982; Lee et al., 2001; Kang et al., 2002; Kwon et al., 2001, 2003), based on inhibition of cyclooxygenase-2 expression and the blocking of pro-inflammatory cytokines (TNF-alpha and IL-1 beta) production and its inhibitory effect on synthesis of prostaglandin E-2(PGE-2) (Nam et al., 2003; Jang et al., 2003). On the other hand, it is shown that BV as an immunostimulant agent can decrease IL-4 and IL-5 and increase interferon-gamma secretion which could be effective in wound healing and repair, whereas mentioned mechanism will enhance the progression of autoimmune diseases (Jutel et al., 1995; Hamedani et al., 2005; Magnan et al., 2001; Dayan et al., 1983). Thus there are still some unanswered questions relating to the efficacy of BV therapy which may be determined with more extensive studies. It should be noted that in MS patients, many neurologic signs and symptoms have been attributed to the underlying conduction deficits. The idea that neurologic function might be improved if conduction could be restored in CNS demyelinated axons led to testing potassium (K(þ)) channel blockers as a symptomatic treatment. To date, in addition to 2 broad-spectrum K(þ) channel blockers, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) which have been tested in MS patients, it must be cited that venoms can be used as Potassium channel blockers (Norton et al., 2004; Breland and Currier, 1983; Judge and Bever, 2006). Although both 4-AP and 3,4-DAP produce clear neurological benefits, their use has been limited by their side effects, such as dizziness, numbness, tingling, instability while walking, nausea, vomiting, and abdominal pain. In this connection, sea anemones toxin (ShK and ShK-Dap), scorpions toxin (kaliotoxin, Charybdotoxin and BmK) and snakes venom (dendrotoxin I and beta-Bungarotoxin) have been tested as

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K(þ) channel blockers in the level of experimental models in order to improve the symptoms of the MS disease (Norton et al., 2004; Devaux et al., 2004; Judge and Bever, 2006; Harvey et al., 1995; Bidard et al., 1987; Schmidt and Betz, 1989; Wang and Ji, 2005). Interference in blood coagulation and fibrinolytic system is an other role of snake venom. In MS, in which brain tissue becomes permeable to blood proteins, extravascular fibrin deposition correlates with sites of inflammatory demyelination and axonal damage (Gornitskaia et al., 2003; Maruyama et al., 1990). It has been reported that rats sensitized to neuroantigen and treated with ancrod, a polypeptide derived from the venom of Agkistrodon rhodostoma, develop profound hypofibrinogenemia and show a marked inhibition of fibrin deposition, and often exhibit no paralytic signs whatsoever. In contrast, treatment with ancrod is not demonstrably influenced the monocytes and lymphocytes infiltrates (Paterson, 1976). Also, recent findings suggest that batroxobin suppresses EAE by preventing fibrin deposition, and provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of bloodebrain barrier (BBB), are related prerequisites for the clinical manifestation of EAE (Inoue et al., 1996). In a pharmacological analysis, fibrin depletion using ancrod, in animal model (Tg6074 mice) also delayed the onset of inflammatory demyelination. These results indicate that fibrin regulates the inflammatory response in neuroinflammatory diseases. Design of therapeutic strategies based on fibrin depletion could potentially benefit on the clinical course of demyelinating diseases such as multiple sclerosis (Akassoglou et al., 2004). However, the results have complexity to interpret, thus additional well designed trials are needed to better define the ideal role of ancrod and batroxobin in the management of disease. Although, treatment is well tolerated and serious adverse events are infrequent (Bell, 1997). Here, it must be noted that our problem in venom therapy is the appearance of side effects following the use of these biological agents. Secondly, their application in symptomatic treatment is lain venom therapy in the second lodge of treatment of MS. Side effects of venom-based therapy could be in part due to their non-self polypeptide structure, so that administration of foreign proteins can sensitize the patient and cause hypersensitivity reactions. (1) Stopping investigations in the level of experimental models and lack of necessary data in the field of clinical trial have been limited the application of venoms in treatment of MS. (2) Existing alternative chemical drugs with similar properties for each venom has been reduced the value of venom therapy in MS. Regarding, the most important therapeutic target in MS is to seek an agent that could inhibit demyelinating process, thus venom therapy with role of symptomatic treatment should be considered as second line agents in research and treatment of MS. Collectively, most MS patients use unconventional therapies, usually as complementary measures in addition to the conventional treatment. Only a few adequate clinical trials exist in this field. By definition, the efficacy of these therapies is unproven. Moreover, the possible risks are also largely unknown (Schwarz et al., 2005).

3. Conclusion Although, basic research is providing new insights into the efficacy of venoms for approaching to the treatment of multiple sclerosis, however it is strongly recommended a deep and long research on clinical trials of this traditional procedure in order to approach to a logical decision for choosing this therapeutic method in MS disease.

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