Vidatox 30 CH
Descripción
Summary of the Rhopalurus junceus scorpion venom
General Introduction.
Cancer is considered one of the main causes of death worldwide, nowadays. In the treatment of this pathology are used as conventional ones the citostatic medicines (drugs), the radiations and the surgery. However, these treatments present a limited clinical effectiveness, due to, among other causes, the fact that they are not applicable in all the cases and to that the adverse effects that their use may provoke make them impracticable in occasions. This way, the current tendency in the treatment of cancer pursues to obtain a more successful one that increases not only its effectiveness but also that diminishes its adverse effects. In these new therapeutical tendencies are included the treatments that modify the biological answer that increases the immune one , as well as the emergent pharmacological agents which are able to modulate the transduction of signs and the mechanism of apoptosis of the tumoral cells (Konety et al, 2007; Ruggles et al., 2004). In this context, one of the targets that has monopolized the attention in the therapy against cancer has been the ionic channels (Minghua et al, 2011).
The ionic channels, so much those whose opening is regulated by the changes of the potential membrane as those that are open up by the union of specific ligand play an important role at cellular level for their participation in diverse physiological mechanisms such as the transmission of electric signs, genetic expression , hormonal secretion, learning and memory, etc. (Fiske et al., 2006; Roger et al, 2006). However, in certain phytopathological conditions like cancer , as much the expression as the properties of these channels can modify, and under these conditions, they can play a pivotal role in the growth /proliferation, migration and/or invasion of the cancerous cells (Fiske et to the., 2006; Him Guennnec et al 2007; Minghua et al., 2011). This way, the scientific literature of this thematic shows a considerable volume of information indicating the role of the channels of K+ in the proliferation and development of cancer (Brown et al., 2005; Brown et al., 2008) and the overexpression of this channel type in a considerable number of primary tumors (Hemmerleein et al, 2006; Stuhmer et al., 2006). Wide information that sustains the implications of the channels of Na+ in the development of this desease , which is based partly by its regulator role in the proliferation, migration and adhesion of excitable cells is observed.(Diss et al , 2004) and in the overexpression of these channels in several types of tumors, like it is the case of the prostate (Grimes et al., 1995; Laniado et al., 1997) breast cancer (Roger et al ., 2006a; Roger et al, 2004) and in linfoma (Fraser et al., 2004) among others. On the other hand, numerous studies also indicate the participation of the channels of Ca2+, Cl - and those belonging to the
superfamily of the channels TRP, in the development and progression of this desease , playing an important role in the proliferation, migration and the control of cellular volume, as well as in the apoptosis, and the angiogenesis, etc.(Arcangeli et al., 2010). The fact that in the neoplasic cells alteration may occur not only in the expression but also in the functioning of many of these channels and that these changes contribute to the progression of cancer through diverse mechanisms, it opens new perspectives in the treatment of this desease by means of the use of blocking or specific modulators of these channels.
In the search of this new alternative for the antitumoral therapy, the international scientific community has recently directed its attention to the scorpions´s venom. The interest in the study of the potentialities of these venoms as antitumoral agents is sustained in the fact that the studies of characterization and purification of the bioactive components of the scorpion venom indicate the presence of a wide variety of peptides under the molecular weight with specific activities on channels of Na+, K+, Cl - or Ca2+, which promotes a possible antitumoral action of the whole venom for its simultaneous action on several therapeutical targets. In this sense, studies with the venom of several species of scorpions validate this initial hypothesis, finding antitumoral effects in vitro of the venom of Buthus martensis over glioma cells cultivations U251-MG (Wang et al., 2005) and of the black scorpion of India Heterometrus bengalensis on human tumoral lines of leukemia (Das Grupta et al, 2007). In both cases it has been proposed as an action mechanism of fundamental antitumoral action the increase of the apoptosis. Studies performed with the venom of another scorpion, in this case of the Buthidae family, the Leiurus quinquestriatus, have allowed to isolate the clorotoxine, which for its blocking action on the channels of chloride of low conductancy , plays an important role in the control of the gliomas’s growth (DeBin et al, 1991; Lyons et al., 2002). Further studies with this toxin demonstrated that it also has, the capacity to prevent (block) the invasion and dissemination of this type of tumors toward non damage areas of the brain, due to its specific and selective interaction with the protein MMP-2, which increases its potentialities because of its action on the migration and the metastasis (Deshane et al, 2003). Other studies of characterization of the scorpion venom performed with the chinese scorpion Bhutus martensii karsh have allowed the identification of a peptide with a proven antitumoral activity on a sarcoma murino model and the mammary ascitic carcinoma of Erhlich (Yang et al, 2000) and more recently scientifists have found another toxin with antiprolipherative and antitumoral activity that was demonstrated in glioma’s experimental models (Liu et to al, 2000).
In Cuba, the use of the scorpion with therapeutical objectives dates back to the beginning of the last century (Armas , 1974). At the beginning of the 80’s decade it was in initiated, in Guantanamo province, the work with the venom of the Cuban endemic scorpion Rhopalurus junceus (R. junceus), as an antitumoral agent. This property was empirically demonstrated in studies performed by Bordier and col (Bordier, 1994), however, evidences that sustain on scientific bases, the
oral potentialities of this scorpion venom in the treatment of cancer, do not exist , until now.
Basing on these facts and considering the necessity to establish on rational and scientifically documented bases the use of the scorpion venom as an antitumoral agent in humans , our group began the biochemical characterization of the R. junceus venom and the pharmacological and toxicological preclinic evaluation of the venom whose results are presented below.
Biochemical and molecular Introduction.
Study of the R. junceus scorpion venom effect on the cellular viability in normal cellular lines and of tumoral origin.
The R. junceus scorpion venom presents a significant and differential citoxicity in vitro on the tumoral cells of epithelial origin , not presenting any toxicity over the normal tumoral cells neither on the tumorals cells of hematopoyectic origin . This way, while the non tumoral lines were not susceptible to the action of the venom not even to concentrations so high as 1 mg/ml and periods of incubation of up to 72 h, in lines of tumoral origin the cellular viability diminished significantly by appearing this effect to inferior concentrations. However, the venom presents an additional selectivity, since the cellular lines of hematopoyectic origin didn't present sensibility to the venom’s effects , which suggests a selective effect on tumoral cells of epithelial origin.
The venom’s effects on the fragmentation of the DNA, the morphological evaluation and the determination of apoptosis and necrotic cells by means of microscopy of fluorescence in treated cellular cultivations as well as the determination of the caspasas activity 3, 8 and 9 for Western blott, allowed to determine that the cellular death could take place by means of the aoral routetosis’s induction and the necrosis, which can be explained by the complex composition of the venom and by the presence of several components that affect the cellular viability by different mechanisms.
Study of some enzymatic activities of the R. junceus scorpion venom with pharmacological and /or toxicological potential interest.
The R. junceus scorpion venom , showed low proteolitic activity that was only evidenced to concentrations among 500 µg/mL-1000 µg/mL that’s why the venom’s preolitic activity proved to be little relevant.
Further, it didn't show phospholipase A2 activity not even to the concentration of venom maximum assayed (5000 µg/mL) which confirms the absence of phospholipase A2 activity under our experimental conditions.
The R. junceus venom , added to the incubation media to concentrations between 100 µg/mL and 1500 µg/mL, didn't induce hemolysis in the human erythrocytes that’s why in our experimental conditions there is no presence of hemolitic activity.
These discoveries suggest that the presence of the enzymes responsable for these effects is low or its activity is not very relevant inside the components of the R. junceus venom that’s why, its contribution to the pharmacological and/or toxicological effects of the venom possesses limited clinical importance . These facts contribute an important element that could be related, not only with the low toxicity of the R. junceus venom that is observed in mammals but also with the limited citotoxicity observed in the studies of viability in vitro.
Characterization and identification of some components with antitumoral activity in the R. junceus scorpion venom.
The analysis of the electrophoretic profile in SDS-PAGE showed that the R. junceus scorpion venom is a mixture composed by several peptides whose molecular weights are in a range among 70 3KDa. However, this profile revealed that the majority of the venom´s components are grouped in an electrophoretic band that corresponds together with estimated molecular weights below the 14 KDa, which coincides with the peptidic composition commonly described in other scorpion species.
The liquid cromatography of high resolution in Superosa 12 HR10/30 allowed the venom’s division in 4 fractions denominated by the elucidation order as LB01, LB02, LB03 and LB04, which were evaluated in front of cellular lines of tumoral and non tumoral origin. Of these, the fractions LB03 and LB04, composed by the peptides of more low molecular weight, were the ones that showed a greater interest due to their high citotoxicity in front of (Hela and A549) tumoral cells and low toxicity over normal cells (MRC-5 and CHO). However, LB03 presented the most selective action profile in front of the tumoral lines.
By means of the division of LB03 by cromatography of high resolution in reverse phase were collected 8 subfractions that were similarly evaluated in front of the line of tumoral origin Hela and non tumoral CHO. The results of this experience showed that the subfractions 1, 6, 7, 8 were those that presented the greater selective citotoxic effect on the cellular line of tu moral origin Hela.
According to these results these subfractions were subjected to mass espectrometry , from which 5 aminoacidic sequences were obtained that were synthesized, later on. The evaluation of the synthesized peptides in front of the non tumoral line Hacat and the tumoral lines H-29, JIM-T1, HS-762, RWP-1 showed a high citotoxic activity on tumoral cells low toxicity on the normal cells.
The results of the components´s purification obtained by biotechnological method of the R. junceus scorpion venom has shown until now, the presence of 5 peptides of low molecular weight with selective citotoxic activity in front of the tumoral lines , which can be responsable, at least, partially of the venom preferential toxicity over the tumoral cells.
Study of the antitumoral effect of the Cuban R. junceus scorpion venom administered by intraperitoneal and oral via in an experimental model of adenocarcinoma mammary murino.
Our results show for the first time the antitumoral effect of the R. junceus scorpion venom in the experimental model of adenocarcinoma mammary murino F3II.
The administration of the venom by oral route later, to the installation of the tumor to reason of a daily administration during 10 days (0.8 and 3.2 mg/kg) reduces the tumoral progression significantly (tumoral volume) and in agreement with such effects, the time of survival of the tumor implanted animals increases significantly. For both parameters, the dose of 3,2 mg/kg behaved as that of the maximum effect.
The administration by oral route of the venom also showed antitumoral effects . However, by this via , the venom was daily administered during the 24 days later to the implant and to dose between 6 and 50 mg/kg and obtaining reductions in the tumor’s progression comparable to those of the intraperitoneal via . Under this outline, the dose of 12.5 mg/kg behaved as that of the maximum effect, not observing greater effects to the superior assayed doses.
The scorpion venom by both via reduces significantly the appearance of spontaneous metastasis and of experimental metastasis in the used model. By both via the maximum effects were observed to those doses in which the maximum effect on the size of the tumor was produced.
The increase in the dose and in the exposure to the venom orally are logical, taking into account that this route can diminish the systemic bioavailability of the venom active components .Nevertheless, the effectiveness shown by this administration route demonstrates which antitumoral effect of the venom is bioavailable , not depending on the metabolic transformations that its active components can suffer and to the phenomenons of absorption and distribution entailed to this administration route. This way, if we take into account the advantages of this via regarding its security and adherence to the treatment, the antitumoral effects by oral route , represent an advantage for the venom clinical application.
Taking into account the low or practically null toxicity that the venom has presented in the studies of general toxicology performed until now , the oral route worked. We consider that the oral route is the most advisable and attractive to continue with the preclinical studies that endorse the effectiveness of the venom in different carcinoma experimental models.
Beneficial colateral effects.
Inflammation
Diverse studies have established the narrow relationship between cancer and the inflammation. In this sense, although the inflammatory processes are developed as part of the organism´s defense mechanisms in answer to diverse nature stimuli, if the aggressive stimulus is insidious and above all multifactorial, the immune system cannot be able to complete the cycle of aggression-recovery becoming the inflammation in a pathological process. That way, they have been described proinflammatory genes that mediates the apoptosis , the proliferation, the angiogenesis, the invasion and the metastasis (Aggarawal et al., 2006; Rakoff-Nahoum, 2006). Diverse studies argue as the factor of tumoral necrosis(TNFα) in conditions of not regulated secretion promotes several events like the cellular transformation to malignant cells and their proliferation (Balkwill, 2002; Balkwill et al., 2004; Mantovani, 2005), the role of proinflammatory citocines is also described in the angiogenesis and the cellular migration (Aggarwal et al ., 2006) the overexpression of the COX-2 and its implication in the growth and progression of several types of cancers, among those we have ; epithelial cells of breast invasive cancer (Half et al., 2002), esophagus cancer (Zimmmerman et al., 1999), ovary cancer (Aggarawal et al ., 2006), lung carcinoma (Wolff et al ., 1998), pancreatic carcinoma (Tucker et al., 1999), among others. Many authors consider the level of its tumoral overexpression as an index of seriousness and the decrease of survival in several cancer types (Aggarwal et al ., 2006), which is partly linked to their role as promoter and regulator of pro angiogenic factors (Healthy et al ., 1995; Tsuji et al ., 1997). Also the lipoxigenasa -5 other of the enzymes linked to the inflammatory process, has been overexpresed and linked to the progression of this desease in prostate cancer, cerebral tumor and perhaps in skin cancer (Aggarwal et al., 2006).
On the other hand, the tumor when increasing generates an inflammatory process that although it is acute in its nature, it is permanent likewise the tumoral growth . This inflammatory process plays an important role in the tumor´s growth , since the angiogenesis mediates and because it also has an important role in other aspects of the tumoral progression such as the tisular invasion and the metastasis (Opdenakker et al., 2004).
Diverse studies have shown the link of poisoning by scorpions´s stings and the appearance of an inflammatory answer (Petricevich, 2010). In such a sense, the presence of high concentrations of venom after the sting, it can unchain the increase of the citocines´s proinflammatory concentration and the consequent tisular damage. Nevertheless, it has been established that next to the appearance of inflammatory factors other antiinflamatory ones are generated which is a direct consequence of the complex composition of these venoms, for that reason, the balance between both is the one that ultimately, determines the degree and the extension of inflammation and their consequences (Petricevich, 2010).
Taking into account, the antitumoral effects described for the R. junceus venom, the implications that have the inflammatory processes in the development of cancer and the induction of inflammatory answers by the inoculation of high concentrations of scorpion venom, the present working group has as an objective to evaluate the presence or not of anti-inflammatory effects in the dose range in which the antitumoral effects in the models in vivo were observed . To fulfil these objectives and as a first approach to this study, the effects of the venom were investigated by using experimental classical models of inflammation
Evaluation of the anti-inflammatory activity of the R. junceus venom in the model of auricular edema in the mouse´s ear.
The acute intraperitoneal administration of the R. junceus venom evidenced an anti-inflammatory effect in the model of auricular edema of the mouse´s ear. The administration of the venom to a dose of 1, 3 and 5 mg/kg of corporal weight inhibited significantly to all the tested doses the inflammation induced by croto oil in the mouse´s ear being the dose of 3 mg/kg the most effective by producing an average inhibition that was even superior to that of the dexametasone administered as positive control . The anti-inflammatory effects of the venom administered by this route didn't mantain a relationship dose -answer in the range of dose of the present assay.
The oral administration of simple dose of the venom to dose of 5, 10 and 20 mg/kg also has anti-inflammatory effects . However, the reductions of the edema were inferior in all the used doses that those of the dexametasone administered by the same via. It is important to point out that the effects of all the doses were of comparable magnitude, which indicates that neither for this route the effects are dependent of the dose in the dose range and under the used administration outline.
The effects by the intraperitoneal route, are superior to those of the oral one , indicating that the administration via is critical and it plays an important role in the bioavailability of the active components, responsable for the anti-inflammatory effects of the venom.
Evaluation of the anti-inflamatory effect of the R. junceus venom in the inflammation model of cotton pellets in rats.
The oral route administration of the of R. junceus venom showed a moderated inhibition in the formation of the granulomatosic tissue in the granuloma model by the cotton pellets . The reached reduction was of approximately 20% to the doses of 10 and 20 mg/kg.
Evaluation of the R. junceus venom in a model of inflammatory angiogenesis
The intraperitoneal route administration of the R junceus venom to a dose of 3 and 5 mg administered during 5 days to a daily administration showed an inhibition in the formation of the granulomatose tissue . The reached reduction was of approximately 31.7 and 51.3% regarding the group control. Also, the venom reduced the formation of new sanguine vessels in the granulomatose tissue in 39,22% and 69,24%, respectively, that´s why it showed antiangiogenic effects in this experimental model.
Pain
More than the half of all the patients with cancer present intense and uncontrollable pain during the course of the desease (Schmidt et al., 2010), for that reason the treatment of the pain is still the main challenge for the cancer patient and the oncologist.. Although the cancer´s pain etiology is not completely elucidated , the experimental studies and the clinical experience have allowed to figure out some of the implied neuropathologic processes . The pain causing mechanisms in the patient with cancer are several and they can be presented in isolation or combined, for that reason , more than of a receiver type or of a conduction route can be involved.
Evaluation of the R. junceus venom in two nocioception models in mice.
The administration of equivalent dose at 1; 2,5; 5 and 7,5 mg/kg of the R. junceus venom, inhibited significantly in the number of writhing induced by the acetic acid by intraperitoneal route administration regarding the group negative control, what evidences the analgesic properties of the product when being administered by intraperitoneal via in this experimental model.
The oral administration of equivalent dose at 1, 5, 10, 15, 20 and 50 mg/kg of the R. junceus venom also diminished significantly the writhing induced by acetic acid concerning the negative control. The doses of 15, 20 and 50 mg/kg had a similar behavior to that of the aspirin administered to dose of 150 mg/kg. This way, the venom also possesses analgesic effects in this model administered by oral via
The R junceus venom administered by intraperitoneal via to a dose of 2.5, 5 and 7.5 mg/kg, possesses analgesic activity in the model of the hot plate.
Studies of toxicological security.
The Rhopalurus junceus scorpion venom is a natural substance that has a wide pharmacological spectrum demonstrated in experimental models, in which it is included its activity as an antitumoral .From now on , together with the pharmaconidamic effects that support its use, they should be performed assays that evidence its toxicological security. With this purpose , they were carried out different tests established by the international regulations that allowed to evaluate its toxicity , using different treatment protocols.
Acute oral toxicity (OECD 423). In assays of acute toxicity by oral route, mortality was not observed for none of the assayed doses during the whole study and there was an increase of the normal weight of the treated animals regarding to their controls. From the clinical oral point of view , a delay was observed in the locomotion during the first 24 hours (5 mg/kg), until the 11 days (50 mg/kg), and during the whole study of Acute Oral Toxicity (300, 2000 mg/kg). For the four doses it was observed a decrease of the reflexes, sedation and piloerection during the 14 days of the assays . The macroscopic examination during the autopsy for the four doses administered didn't show morphological alterations of the examined organs: heart, lungs, timo, liver, kidneys, stomach, esophagus, intestine, spleen, reproductive organs, bladder, adrenals.
Taking into account the absence of reported mortality in the study of the product, it was located inside the toxicological category " Without classifying" (LD50 >2000 mg/kg).
Acute Intraperitoneal toxicity.It was considered a value of Dose Lethal Media (LD 50) of 16,41 mg/kg for this administration route . During the assay acute toxic signs were observed: marked piloerection, decrease of the moving activity , decrease of the reflexes, sedation and ptosis in all the animals administered by this route as well as the appearance of convulsions before the death of the animals. Significant alterations were not reported in the corporal weight of the treated animals concerning the controls to the doses of 5 and 10 mg/kg intraperitoneally inoculated. Macroscopic damages were not reported at the level of the organs analyzed during the autopsy.
Subcronic toxicological study for 90 days. In the subcronic study for 90 days by oral route in mice to dose of 0,1, 10 and 100 mg/kg, toxic signs neither mortality, biochemical variations nor hematological ones were not observed. A smaller increase of corporal weight was observed in the treated animals with the three doses of venom corresponding with a smaller consumption of foods during the whole study. This effect was reverted in the centinel group used in this assay protocol. The relative weight of the organs was not affected and the hirtopathological analysis didn't show alterations in organs and tissues as consequence of the administration of the test substance.
Acute and chronic irritability in oral mucous . The venom is not irritating on the oral mucous when being administered of acute and chronic form.
Genotoxicity : 2 systems of assays were used, one in vitro, Salmonella/microsome (Ames ) and the assay in vivo of micronucleous induction in mouse bone marrow using different traetments´s protocols (acute, sub-acute, repeated dose of 28 days) and administration routes (oral and intraperitoneal).
The venom in the test of Ames , turned out to be non mutagenic with the strains of Salmonella typhimurium TA98, TA100, TA102 and TA104 when concentrations were assayed of up to 5 mg/plate according to the incorporation method in a standard plate. It was not also observed medullary citotoxicity neither significant alterations in the frequency of micronucleic polychromed erytrocytes in each protocol of induction assay of used micronucleous.
Teratogenesis: The embryotoxic effect of this extract was evaluated by oral via to the doses of 0,1; 10 and 100 mg/kg. As a result it was observed that the venom administered during the period of organogenesis didn't induce maternal toxicity, teratogenic effects , embryoletals neither affected the growth of the embryo in development.
General Introduction.
Cancer is considered one of the main causes of death worldwide, nowadays. In the treatment of this pathology are used as conventional ones the citostatic medicines (drugs), the radiations and the surgery. However, these treatments present a limited clinical effectiveness, due to, among other causes, the fact that they are not applicable in all the cases and to that the adverse effects that their use may provoke make them impracticable in occasions. This way, the current tendency in the treatment of cancer pursues to obtain a more successful one that increases not only its effectiveness but also that diminishes its adverse effects. In these new therapeutical tendencies are included the treatments that modify the biological answer that increases the immune one , as well as the emergent pharmacological agents which are able to modulate the transduction of signs and the mechanism of apoptosis of the tumoral cells (Konety et al, 2007; Ruggles et al., 2004). In this context, one of the targets that has monopolized the attention in the therapy against cancer has been the ionic channels (Minghua et al, 2011).
The ionic channels, so much those whose opening is regulated by the changes of the potential membrane as those that are open up by the union of specific ligand play an important role at cellular level for their participation in diverse physiological mechanisms such as the transmission of electric signs, genetic expression , hormonal secretion, learning and memory, etc. (Fiske et al., 2006; Roger et al, 2006). However, in certain phytopathological conditions like cancer , as much the expression as the properties of these channels can modify, and under these conditions, they can play a pivotal role in the growth /proliferation, migration and/or invasion of the cancerous cells (Fiske et to the., 2006; Him Guennnec et al 2007; Minghua et al., 2011). This way, the scientific literature of this thematic shows a considerable volume of information indicating the role of the channels of K+ in the proliferation and development of cancer (Brown et al., 2005; Brown et al., 2008) and the overexpression of this channel type in a considerable number of primary tumors (Hemmerleein et al, 2006; Stuhmer et al., 2006). Wide information that sustains the implications of the channels of Na+ in the development of this desease , which is based partly by its regulator role in the proliferation, migration and adhesion of excitable cells is observed.(Diss et al , 2004) and in the overexpression of these channels in several types of tumors, like it is the case of the prostate (Grimes et al., 1995; Laniado et al., 1997) breast cancer (Roger et al ., 2006a; Roger et al, 2004) and in linfoma (Fraser et al., 2004) among others. On the other hand, numerous studies also indicate the participation of the channels of Ca2+, Cl - and those belonging to the
superfamily of the channels TRP, in the development and progression of this desease , playing an important role in the proliferation, migration and the control of cellular volume, as well as in the apoptosis, and the angiogenesis, etc.(Arcangeli et al., 2010). The fact that in the neoplasic cells alteration may occur not only in the expression but also in the functioning of many of these channels and that these changes contribute to the progression of cancer through diverse mechanisms, it opens new perspectives in the treatment of this desease by means of the use of blocking or specific modulators of these channels.
In the search of this new alternative for the antitumoral therapy, the international scientific community has recently directed its attention to the scorpions´s venom. The interest in the study of the potentialities of these venoms as antitumoral agents is sustained in the fact that the studies of characterization and purification of the bioactive components of the scorpion venom indicate the presence of a wide variety of peptides under the molecular weight with specific activities on channels of Na+, K+, Cl - or Ca2+, which promotes a possible antitumoral action of the whole venom for its simultaneous action on several therapeutical targets. In this sense, studies with the venom of several species of scorpions validate this initial hypothesis, finding antitumoral effects in vitro of the venom of Buthus martensis over glioma cells cultivations U251-MG (Wang et al., 2005) and of the black scorpion of India Heterometrus bengalensis on human tumoral lines of leukemia (Das Grupta et al, 2007). In both cases it has been proposed as an action mechanism of fundamental antitumoral action the increase of the apoptosis. Studies performed with the venom of another scorpion, in this case of the Buthidae family, the Leiurus quinquestriatus, have allowed to isolate the clorotoxine, which for its blocking action on the channels of chloride of low conductancy , plays an important role in the control of the gliomas’s growth (DeBin et al, 1991; Lyons et al., 2002). Further studies with this toxin demonstrated that it also has, the capacity to prevent (block) the invasion and dissemination of this type of tumors toward non damage areas of the brain, due to its specific and selective interaction with the protein MMP-2, which increases its potentialities because of its action on the migration and the metastasis (Deshane et al, 2003). Other studies of characterization of the scorpion venom performed with the chinese scorpion Bhutus martensii karsh have allowed the identification of a peptide with a proven antitumoral activity on a sarcoma murino model and the mammary ascitic carcinoma of Erhlich (Yang et al, 2000) and more recently scientifists have found another toxin with antiprolipherative and antitumoral activity that was demonstrated in glioma’s experimental models (Liu et to al, 2000).
In Cuba, the use of the scorpion with therapeutical objectives dates back to the beginning of the last century (Armas , 1974). At the beginning of the 80’s decade it was in initiated, in Guantanamo province, the work with the venom of the Cuban endemic scorpion Rhopalurus junceus (R. junceus), as an antitumoral agent. This property was empirically demonstrated in studies performed by Bordier and col (Bordier, 1994), however, evidences that sustain on scientific bases, the
oral potentialities of this scorpion venom in the treatment of cancer, do not exist , until now.
Basing on these facts and considering the necessity to establish on rational and scientifically documented bases the use of the scorpion venom as an antitumoral agent in humans , our group began the biochemical characterization of the R. junceus venom and the pharmacological and toxicological preclinic evaluation of the venom whose results are presented below.
Biochemical and molecular Introduction.
Study of the R. junceus scorpion venom effect on the cellular viability in normal cellular lines and of tumoral origin.
The R. junceus scorpion venom presents a significant and differential citoxicity in vitro on the tumoral cells of epithelial origin , not presenting any toxicity over the normal tumoral cells neither on the tumorals cells of hematopoyectic origin . This way, while the non tumoral lines were not susceptible to the action of the venom not even to concentrations so high as 1 mg/ml and periods of incubation of up to 72 h, in lines of tumoral origin the cellular viability diminished significantly by appearing this effect to inferior concentrations. However, the venom presents an additional selectivity, since the cellular lines of hematopoyectic origin didn't present sensibility to the venom’s effects , which suggests a selective effect on tumoral cells of epithelial origin.
The venom’s effects on the fragmentation of the DNA, the morphological evaluation and the determination of apoptosis and necrotic cells by means of microscopy of fluorescence in treated cellular cultivations as well as the determination of the caspasas activity 3, 8 and 9 for Western blott, allowed to determine that the cellular death could take place by means of the aoral routetosis’s induction and the necrosis, which can be explained by the complex composition of the venom and by the presence of several components that affect the cellular viability by different mechanisms.
Study of some enzymatic activities of the R. junceus scorpion venom with pharmacological and /or toxicological potential interest.
The R. junceus scorpion venom , showed low proteolitic activity that was only evidenced to concentrations among 500 µg/mL-1000 µg/mL that’s why the venom’s preolitic activity proved to be little relevant.
Further, it didn't show phospholipase A2 activity not even to the concentration of venom maximum assayed (5000 µg/mL) which confirms the absence of phospholipase A2 activity under our experimental conditions.
The R. junceus venom , added to the incubation media to concentrations between 100 µg/mL and 1500 µg/mL, didn't induce hemolysis in the human erythrocytes that’s why in our experimental conditions there is no presence of hemolitic activity.
These discoveries suggest that the presence of the enzymes responsable for these effects is low or its activity is not very relevant inside the components of the R. junceus venom that’s why, its contribution to the pharmacological and/or toxicological effects of the venom possesses limited clinical importance . These facts contribute an important element that could be related, not only with the low toxicity of the R. junceus venom that is observed in mammals but also with the limited citotoxicity observed in the studies of viability in vitro.
Characterization and identification of some components with antitumoral activity in the R. junceus scorpion venom.
The analysis of the electrophoretic profile in SDS-PAGE showed that the R. junceus scorpion venom is a mixture composed by several peptides whose molecular weights are in a range among 70 3KDa. However, this profile revealed that the majority of the venom´s components are grouped in an electrophoretic band that corresponds together with estimated molecular weights below the 14 KDa, which coincides with the peptidic composition commonly described in other scorpion species.
The liquid cromatography of high resolution in Superosa 12 HR10/30 allowed the venom’s division in 4 fractions denominated by the elucidation order as LB01, LB02, LB03 and LB04, which were evaluated in front of cellular lines of tumoral and non tumoral origin. Of these, the fractions LB03 and LB04, composed by the peptides of more low molecular weight, were the ones that showed a greater interest due to their high citotoxicity in front of (Hela and A549) tumoral cells and low toxicity over normal cells (MRC-5 and CHO). However, LB03 presented the most selective action profile in front of the tumoral lines.
By means of the division of LB03 by cromatography of high resolution in reverse phase were collected 8 subfractions that were similarly evaluated in front of the line of tumoral origin Hela and non tumoral CHO. The results of this experience showed that the subfractions 1, 6, 7, 8 were those that presented the greater selective citotoxic effect on the cellular line of tu moral origin Hela.
According to these results these subfractions were subjected to mass espectrometry , from which 5 aminoacidic sequences were obtained that were synthesized, later on. The evaluation of the synthesized peptides in front of the non tumoral line Hacat and the tumoral lines H-29, JIM-T1, HS-762, RWP-1 showed a high citotoxic activity on tumoral cells low toxicity on the normal cells.
The results of the components´s purification obtained by biotechnological method of the R. junceus scorpion venom has shown until now, the presence of 5 peptides of low molecular weight with selective citotoxic activity in front of the tumoral lines , which can be responsable, at least, partially of the venom preferential toxicity over the tumoral cells.
Study of the antitumoral effect of the Cuban R. junceus scorpion venom administered by intraperitoneal and oral via in an experimental model of adenocarcinoma mammary murino.
Our results show for the first time the antitumoral effect of the R. junceus scorpion venom in the experimental model of adenocarcinoma mammary murino F3II.
The administration of the venom by oral route later, to the installation of the tumor to reason of a daily administration during 10 days (0.8 and 3.2 mg/kg) reduces the tumoral progression significantly (tumoral volume) and in agreement with such effects, the time of survival of the tumor implanted animals increases significantly. For both parameters, the dose of 3,2 mg/kg behaved as that of the maximum effect.
The administration by oral route of the venom also showed antitumoral effects . However, by this via , the venom was daily administered during the 24 days later to the implant and to dose between 6 and 50 mg/kg and obtaining reductions in the tumor’s progression comparable to those of the intraperitoneal via . Under this outline, the dose of 12.5 mg/kg behaved as that of the maximum effect, not observing greater effects to the superior assayed doses.
The scorpion venom by both via reduces significantly the appearance of spontaneous metastasis and of experimental metastasis in the used model. By both via the maximum effects were observed to those doses in which the maximum effect on the size of the tumor was produced.
The increase in the dose and in the exposure to the venom orally are logical, taking into account that this route can diminish the systemic bioavailability of the venom active components .Nevertheless, the effectiveness shown by this administration route demonstrates which antitumoral effect of the venom is bioavailable , not depending on the metabolic transformations that its active components can suffer and to the phenomenons of absorption and distribution entailed to this administration route. This way, if we take into account the advantages of this via regarding its security and adherence to the treatment, the antitumoral effects by oral route , represent an advantage for the venom clinical application.
Taking into account the low or practically null toxicity that the venom has presented in the studies of general toxicology performed until now , the oral route worked. We consider that the oral route is the most advisable and attractive to continue with the preclinical studies that endorse the effectiveness of the venom in different carcinoma experimental models.
Beneficial colateral effects.
Inflammation
Diverse studies have established the narrow relationship between cancer and the inflammation. In this sense, although the inflammatory processes are developed as part of the organism´s defense mechanisms in answer to diverse nature stimuli, if the aggressive stimulus is insidious and above all multifactorial, the immune system cannot be able to complete the cycle of aggression-recovery becoming the inflammation in a pathological process. That way, they have been described proinflammatory genes that mediates the apoptosis , the proliferation, the angiogenesis, the invasion and the metastasis (Aggarawal et al., 2006; Rakoff-Nahoum, 2006). Diverse studies argue as the factor of tumoral necrosis(TNFα) in conditions of not regulated secretion promotes several events like the cellular transformation to malignant cells and their proliferation (Balkwill, 2002; Balkwill et al., 2004; Mantovani, 2005), the role of proinflammatory citocines is also described in the angiogenesis and the cellular migration (Aggarwal et al ., 2006) the overexpression of the COX-2 and its implication in the growth and progression of several types of cancers, among those we have ; epithelial cells of breast invasive cancer (Half et al., 2002), esophagus cancer (Zimmmerman et al., 1999), ovary cancer (Aggarawal et al ., 2006), lung carcinoma (Wolff et al ., 1998), pancreatic carcinoma (Tucker et al., 1999), among others. Many authors consider the level of its tumoral overexpression as an index of seriousness and the decrease of survival in several cancer types (Aggarwal et al ., 2006), which is partly linked to their role as promoter and regulator of pro angiogenic factors (Healthy et al ., 1995; Tsuji et al ., 1997). Also the lipoxigenasa -5 other of the enzymes linked to the inflammatory process, has been overexpresed and linked to the progression of this desease in prostate cancer, cerebral tumor and perhaps in skin cancer (Aggarwal et al., 2006).
On the other hand, the tumor when increasing generates an inflammatory process that although it is acute in its nature, it is permanent likewise the tumoral growth . This inflammatory process plays an important role in the tumor´s growth , since the angiogenesis mediates and because it also has an important role in other aspects of the tumoral progression such as the tisular invasion and the metastasis (Opdenakker et al., 2004).
Diverse studies have shown the link of poisoning by scorpions´s stings and the appearance of an inflammatory answer (Petricevich, 2010). In such a sense, the presence of high concentrations of venom after the sting, it can unchain the increase of the citocines´s proinflammatory concentration and the consequent tisular damage. Nevertheless, it has been established that next to the appearance of inflammatory factors other antiinflamatory ones are generated which is a direct consequence of the complex composition of these venoms, for that reason, the balance between both is the one that ultimately, determines the degree and the extension of inflammation and their consequences (Petricevich, 2010).
Taking into account, the antitumoral effects described for the R. junceus venom, the implications that have the inflammatory processes in the development of cancer and the induction of inflammatory answers by the inoculation of high concentrations of scorpion venom, the present working group has as an objective to evaluate the presence or not of anti-inflammatory effects in the dose range in which the antitumoral effects in the models in vivo were observed . To fulfil these objectives and as a first approach to this study, the effects of the venom were investigated by using experimental classical models of inflammation
Evaluation of the anti-inflammatory activity of the R. junceus venom in the model of auricular edema in the mouse´s ear.
The acute intraperitoneal administration of the R. junceus venom evidenced an anti-inflammatory effect in the model of auricular edema of the mouse´s ear. The administration of the venom to a dose of 1, 3 and 5 mg/kg of corporal weight inhibited significantly to all the tested doses the inflammation induced by croto oil in the mouse´s ear being the dose of 3 mg/kg the most effective by producing an average inhibition that was even superior to that of the dexametasone administered as positive control . The anti-inflammatory effects of the venom administered by this route didn't mantain a relationship dose -answer in the range of dose of the present assay.
The oral administration of simple dose of the venom to dose of 5, 10 and 20 mg/kg also has anti-inflammatory effects . However, the reductions of the edema were inferior in all the used doses that those of the dexametasone administered by the same via. It is important to point out that the effects of all the doses were of comparable magnitude, which indicates that neither for this route the effects are dependent of the dose in the dose range and under the used administration outline.
The effects by the intraperitoneal route, are superior to those of the oral one , indicating that the administration via is critical and it plays an important role in the bioavailability of the active components, responsable for the anti-inflammatory effects of the venom.
Evaluation of the anti-inflamatory effect of the R. junceus venom in the inflammation model of cotton pellets in rats.
The oral route administration of the of R. junceus venom showed a moderated inhibition in the formation of the granulomatosic tissue in the granuloma model by the cotton pellets . The reached reduction was of approximately 20% to the doses of 10 and 20 mg/kg.
Evaluation of the R. junceus venom in a model of inflammatory angiogenesis
The intraperitoneal route administration of the R junceus venom to a dose of 3 and 5 mg administered during 5 days to a daily administration showed an inhibition in the formation of the granulomatose tissue . The reached reduction was of approximately 31.7 and 51.3% regarding the group control. Also, the venom reduced the formation of new sanguine vessels in the granulomatose tissue in 39,22% and 69,24%, respectively, that´s why it showed antiangiogenic effects in this experimental model.
Pain
More than the half of all the patients with cancer present intense and uncontrollable pain during the course of the desease (Schmidt et al., 2010), for that reason the treatment of the pain is still the main challenge for the cancer patient and the oncologist.. Although the cancer´s pain etiology is not completely elucidated , the experimental studies and the clinical experience have allowed to figure out some of the implied neuropathologic processes . The pain causing mechanisms in the patient with cancer are several and they can be presented in isolation or combined, for that reason , more than of a receiver type or of a conduction route can be involved.
Evaluation of the R. junceus venom in two nocioception models in mice.
The administration of equivalent dose at 1; 2,5; 5 and 7,5 mg/kg of the R. junceus venom, inhibited significantly in the number of writhing induced by the acetic acid by intraperitoneal route administration regarding the group negative control, what evidences the analgesic properties of the product when being administered by intraperitoneal via in this experimental model.
The oral administration of equivalent dose at 1, 5, 10, 15, 20 and 50 mg/kg of the R. junceus venom also diminished significantly the writhing induced by acetic acid concerning the negative control. The doses of 15, 20 and 50 mg/kg had a similar behavior to that of the aspirin administered to dose of 150 mg/kg. This way, the venom also possesses analgesic effects in this model administered by oral via
The R junceus venom administered by intraperitoneal via to a dose of 2.5, 5 and 7.5 mg/kg, possesses analgesic activity in the model of the hot plate.
Studies of toxicological security.
The Rhopalurus junceus scorpion venom is a natural substance that has a wide pharmacological spectrum demonstrated in experimental models, in which it is included its activity as an antitumoral .From now on , together with the pharmaconidamic effects that support its use, they should be performed assays that evidence its toxicological security. With this purpose , they were carried out different tests established by the international regulations that allowed to evaluate its toxicity , using different treatment protocols.
Acute oral toxicity (OECD 423). In assays of acute toxicity by oral route, mortality was not observed for none of the assayed doses during the whole study and there was an increase of the normal weight of the treated animals regarding to their controls. From the clinical oral point of view , a delay was observed in the locomotion during the first 24 hours (5 mg/kg), until the 11 days (50 mg/kg), and during the whole study of Acute Oral Toxicity (300, 2000 mg/kg). For the four doses it was observed a decrease of the reflexes, sedation and piloerection during the 14 days of the assays . The macroscopic examination during the autopsy for the four doses administered didn't show morphological alterations of the examined organs: heart, lungs, timo, liver, kidneys, stomach, esophagus, intestine, spleen, reproductive organs, bladder, adrenals.
Taking into account the absence of reported mortality in the study of the product, it was located inside the toxicological category " Without classifying" (LD50 >2000 mg/kg).
Acute Intraperitoneal toxicity.It was considered a value of Dose Lethal Media (LD 50) of 16,41 mg/kg for this administration route . During the assay acute toxic signs were observed: marked piloerection, decrease of the moving activity , decrease of the reflexes, sedation and ptosis in all the animals administered by this route as well as the appearance of convulsions before the death of the animals. Significant alterations were not reported in the corporal weight of the treated animals concerning the controls to the doses of 5 and 10 mg/kg intraperitoneally inoculated. Macroscopic damages were not reported at the level of the organs analyzed during the autopsy.
Subcronic toxicological study for 90 days. In the subcronic study for 90 days by oral route in mice to dose of 0,1, 10 and 100 mg/kg, toxic signs neither mortality, biochemical variations nor hematological ones were not observed. A smaller increase of corporal weight was observed in the treated animals with the three doses of venom corresponding with a smaller consumption of foods during the whole study. This effect was reverted in the centinel group used in this assay protocol. The relative weight of the organs was not affected and the hirtopathological analysis didn't show alterations in organs and tissues as consequence of the administration of the test substance.
Acute and chronic irritability in oral mucous . The venom is not irritating on the oral mucous when being administered of acute and chronic form.
Genotoxicity : 2 systems of assays were used, one in vitro, Salmonella/microsome (Ames ) and the assay in vivo of micronucleous induction in mouse bone marrow using different traetments´s protocols (acute, sub-acute, repeated dose of 28 days) and administration routes (oral and intraperitoneal).
The venom in the test of Ames , turned out to be non mutagenic with the strains of Salmonella typhimurium TA98, TA100, TA102 and TA104 when concentrations were assayed of up to 5 mg/plate according to the incorporation method in a standard plate. It was not also observed medullary citotoxicity neither significant alterations in the frequency of micronucleic polychromed erytrocytes in each protocol of induction assay of used micronucleous.
Teratogenesis: The embryotoxic effect of this extract was evaluated by oral via to the doses of 0,1; 10 and 100 mg/kg. As a result it was observed that the venom administered during the period of organogenesis didn't induce maternal toxicity, teratogenic effects , embryoletals neither affected the growth of the embryo in development.
