Clinical Toxicology (2014), 52, 118–120 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2013.872792
RESEARCH ARTICLE
Trypsin and rosmarinic acid reduce the toxicity of Micrurus fulvius venom in mice J. L. PARKER-COTE,1 D. P. O’ROURKE,2 S. N. MILLER,1 K. L. BREWER,1 M. D. ROSENBAUM,2 and W. J. MEGGS1 1Department
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
2Department
of Emergency Medicine, Brody School of Medicine at East Carolina University, Greenville, NC, USA of Comparative Medicine, Brody School of Medicine at East Carolina University, Greenville, NC, USA
Context. Antivenom is expensive and not always available, so alternative treatments are being investigated. Objective. The efficacy of trypsin or rosmarinic acid (RA) in treating Micrurus fulvius in a murine model is determined. Materials and methods. Design: randomized controlled blinded study. Subjects: Fifty mice (20–30 g). Study groups: Intraperitoneal injections of: 1) 2 mg/kg M. fulvius venom (approximately twice the LD50 for mice; n ⫽ 10); 2) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with RA at a 1:10 ratio (n ⫽ 17); 3) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with 1 mg of trypsin (n ⫽ 17); 4)1 mg trypsin IP without venom (n ⫽ 3); and 5) RA IP without venom (n ⫽ 3). Main outcome: time to toxicity (respiratory distress (⬍ 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self). Statistical analysis: Time to toxicity using Tukey–Kramer HSD; Survival to 4, 6, and 12 h using Chi-square analysis. Results. Onset of toxicity: venom ⫾ saline, 120.3 ⫹ 64.4 min; venom ⫹ rosmarinic acid, 238.1 ⫾ 139.2 min (p ⫽ 0.15 relative to venom ⫹ saline); venom ⫹ trypsin, 319.7 ⫾ 201.0 min (p ⫽ 0.007 relative to venom ⫹ saline). Venom ⫹ trypsin but not venom ⫹ RA survival to 4 h was significant compared to venom ⫹ saline (p ⫽ 0.023). Two mice in the venom ⫹ trypsin group and one mouse in the venom ⫹ RA group survived to 12 h. Mice receiving trypsin without venom or RA without venom survived to 12 h without toxicity. Discussion. This work suggests that trypsin and RA may have efficacy in treatment M. fulvius envenomation. Conclusion. In vitro neutralization of M. Fulvius venom by trypsin justifies progressing to an in vivo model in future studies. Keywords
Snakebites; Rosmarinic acid; Trypsin
Of the Elapidae family, Micrurus genera venom induces neurotoxicity, causing blockage of post-synaptic endplate via alpha neurotoxin.2 In certain species of Micrurus, there is a pre-synaptic inhibition of acetylcholine release at the motor nerve endings. M. fulvius, Eastern Coral Snake, has cardiotoxic and myotoxic effects, which block the endplate receptors and depolarize the muscle fiber membrane.3 Many enzymatic activities have been detected: phospholipase A2, hyaluronidase, phophodiesterase, leucine amino peptidase, L-amino acid dehydrogensase, and acetylcholinesterase.2 Of the enzymatic activities for M. fulvius, phospholipase A2 is more prominent than hyaluronidase and proteolytic activity. Rosmarinic acid (RA), a plant derived phospholipase A2 inhibitor, is found in many plants such as Cordia verbenacea and Plectranthus barbatus. It has been used in combination with antivenom with Bothrops envenomations, showing potentiation of the antivenom’s neutralizing and antimyotoxic effects.4 The suspected mechanism of action of RA is inhibitory function at the venom’s catalytic site and possible other pharmacological activity that is not known. Also, prior studies in the Okinawa habu snake (Trimeresurus flavoviridis) have shown dose-dependence inhibition of hemorrhage by RA against T. flavoviridis: 75–100% inhibition at a concentration of 0.5 mg/ml.5
Introduction Poisonous snakes are found worldwide and account for considerable mortality and morbidity. The definitive treatment is antivenom, which is not always available. In the United States, the FDA-approved antivenom for coral snake bites expired, but the expiration date has been extended.1 Antivenom for exotic species may not be readily available. In developing nations where snakebites can be a major cause of death, antivenom is often not accessible. An effective inexpensive treatment is needed to prevent mortality worldwide. Since snake venoms contain protein toxins, proteolytic enzymes or binding site inhibitors may be efficacious in neutralizing venoms when specific antivenom is not available. This pilot study will determine if in vitro incubation of a proteolytic enzyme is efficacious in preventing toxicity from coral snake Micrurus fulvius (M. fulvius) venom in mice. A phospholipase A2 inhibitor (rosmarinic acid) will also be tested. Received 19 September 2013; accepted 25 November 2013. Address correspondence to William J. Meggs, MD, PhD, Department of Emergency Medicine, Brody School of Medicine at East Carolina University, 600 Moye Boulevard, Greenville, NC 27858, USA. Tel: ⫹ 1-252-744-2954. Fax: ⫹ 1-252-744-3589. E-mail: [email protected]
118
Trypsin and rosmarinic acid for snakebite treatment 119 Trypsin is a proteolytic enzyme that cleaves peptide chains at lysine, arginine, and serine. One prior study investigated trypsin and cobra venom, a member of the Elapid family, and found that all mice survived if trypsin was locally injected within 15 min.6 Another study found that premixing of tiger snake venom and trypsin just prior to injection did not significantly increase the survival rate of mice versus controls.7
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
Materials and methods Design was a randomized controlled blinded study. Setting was a university animal research facility, with approval by the Institutional Animal Care and Use Committee. Subjects were 50 CD-1 mice weighing 20–30 g. Lyophilized 100% pure eastern coral snake (M. fulvius) venom was obtained from Medtoxin Venom Laboratory (Delland, Florida) and reconstituted in sterile water at a concentration of 0.2 mg/mL. Trypsin in powder form rated 95% pure by mass spectroscopy was obtained from SigmaAldrich (St. Louis) and dissolved in normal saline at 10 mg/mL. RA in powder form rated 98% pure using high-performance liquid chromatography was obtained from Sigma–Aldrich (St. Louis) and dissolved in normal saline at a concentration of 2 mg/mL. Mice were given preemptive analgesia with a dose of 0.1 mg of buprenorphine subcutaneously. For each mouse receiving venom ⫹ trypsin, 2 mg/kg of a 0.2 mg/mL venom solution was incubated in vitro for 1 h at room temperature (22°C) with 1 mg of trypsin in 0.1 mL prior to intraperitoneal (IP) injection. For each mouse receiving venom ⫹ RA, 2 mg/mL of RA was incubated in vitro for 1 h with 2 mg/kg of a 0.2 mg/mL venom solution at a 1:10 ratio of venom:RA. An empirical dose of 2 mg/kg of M. fulvius venom was chosen as approximately twice a published LD50 for mice. Mice were randomized to receive the trypsin–venom mixture (n ⫽ 17), the RA–venom mixture (n ⫽ 17), trypsin alone (n ⫽ 3), RA alone (n ⫽ 3), or venom alone (n ⫽ 10). After injection, subjects were continuously observed by direct observation by two blinded observers who are authors for 12 h and assessed for signs of toxicity: respiratory distress (⬍ 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self. Animals were euthanized at the onset of any one of the signs of toxicity, as determined by the blinded observers. Euthanasia was accomplished using isoflurane inhalation followed by cervical dislocation, according to an approved Institutional Animal Care and Use Committee protocol. The 2 mg/kg dose of eastern coral snake (M. fulvius) venom is more than two times the published LD50 for eastern coral snake venom. The concentration of eastern coral snake venom was 0.2 mg/ml. The volume of venom given or preincubated was based on weight of the individual mouse (grams). The trypsin dose was not based on weight of the animal. Each dose of venom per mouse was preincubated with 1 mg of trypsin from a 10 mg/ml solution, which is a volume of 0.1 ml per mouse. The venom to RA concentration was in 1:10 ratio (0.2–2 mg/ml). The amount of RA given is based on the amount of venom given. Copyright © Informa Healthcare USA, Inc. 2014
Comparison of time to onset of toxicity across groups was performed using Tukey–Kramer HSD. The proportion of animals surviving to 4, 6, and 12 h was compared across groups using Chi-square analysis.
Results Times are given as mean ⫾ standard deviation in Fig. 1. Time to onset of toxicity for controls (venom alone) was 120.3 ⫾ 64.4 min. Preincubation of the venom with RA provided a nonsignificant increase in time to onset of toxicity versus controls (238.1 ⫾ 139.2 min.; p ⫽ 0.15). Preincubation with trypsin significantly increased survival time versus controls (319.7 ⫾ 201.0 min, p ⫽ 0.007). Preincubation of venom with trypsin increased the number of animals that survived to 4 h versus controls (p ⫽ 0.023). This effect was not seen with RA. The proportion of animals surviving to 6 and 12 h was similar across groups (p ⫽ 0.12, p ⫽ 0.37, respectively). Two mice in the trypsin ⫹ venom group and one mouse in the RA ⫹ venom group survived to 12 h. All mice that received treatment (trypsin or RA) without venom survived to the end of the 12-h study period. Survival curves for the three groups are given in Fig. 2.
Limitations The major limitation of this study is that the results of an in vitro incubation of venom with trypsin or RA may not extrapolate to an in vivo study. An albumin control was not used because there is no reason to think that albumin would have efficacy in neutralizing venom, as has been demonstrated in a similar study.8 Another limitation is the differences in volume received, but the differences were small so a dilutional effect is improbable.
Discussion The phospholipase A2 inhibitor, RA, was chosen for study because phospholipase A2 is an important constituent of eastern coral snake venom. RA has been shown to have efficacy in other envenomations. It is a plant derivative
Fig. 1. Comparison of time to onset of toxicity for mice receiving intraperitoneal injections of venom incubated with rosmarinic acid, venom incubated with trypsin, and venom alone (colour version of this figure can be found in the online version at www.informahealthcare.com/ctx).
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J. L. Parker-Cote et al. Other potential uses of RA in toxicology include ciguatoxin.12,13 It has also shown to ameliorate liver damage and fibrogenesis in carbon tetrachloride poisoning in mice.14 Other than the studies of trypsin to treat snakebites,6,7 there are no other references to trypsin use as a treatment for poisonings (Ovid search, November 7, 2013).
Declaration of Interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
Fig. 2. Survival curve as decimal percent for mice receiving (a) venom ⫹ saline, (b) venom ⫹ rosmarinic acid, and (c) venom ⫹ trypsin.
found in plants such as Cordia verbenacea and Plectranthus barbatus and has demonstrated efficacy in Bothrops, envenomations in reducing antimyotoxic effects.4 Trypsin was chose for study because it is a potent protease. Two prior studies of the efficacy of trypsin as a treatment for snakebite poisoning gave different results,4,5 as discussed in the Introduction. This difference may relate to known variations in phospholipase A2 between species.9 The present study found that in vitro incubation of trypsin with M. fulvius venom significantly increased the time to toxicity in mice. RA also increased the time to onset of toxicity, but this experiment was not powered for significance. Mice received trypsin alone or RA alone without lethal effects. It can be concluded that both trypsin and RA have the potential to alter the toxic effects of M. fulvius in vivo, potentially neutralizing the venom with efficacy to treat snakebites. In vitro neutralization of M. fulvius venom by trypsin justifies progressing to an in vivo model in future studies of eastern coral snake envenomation. Study of alternatives to antivenom to treat poisonous snake bites is important because antivenom is very expensive to produce and market. Therapy for a crotalid snakebite in the United States can cost tens of thousands of dollars. Antivenom is not readily available in many countries for endogenous snakes where snakebites are a significant cause of morbidity and mortality. In other countries, it may not be available for exotic species. For some venoms, only horse serum is available which poses risks of anaphylaxis, serum sickness, and death. Trypsin is inexpensive and available for as little as $1.57/gram.10 RA is available for $42/gram.11 Whether or not these could become viable options to treat poisonous snakebites in humans requires further study but is an important direction to pursue.
References 1. http://www.fda.gov/biologicsbloodvaccines/safetyavailability/ ucm371055.htm, accessed October 31, 2013. 2. Tanaka G, Furtado M, Portaro F, Sant’Anna O, Tambbourgi D. Diversity of micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization. PLoS Negl Trop Dis 2010; 43:e622. 3. Vital Brazil O. Pharmacology of coral snake venoms. Mem Inst Butantan 1990; 52:32. 4. Ticli FK, Hage L, Cambraia RS, Pereira PS, Magro AJ, Fontes MR, et al. Rosmarinic acid, a new snake venom Phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction. Toxicon 2005; 46:318–327. 5. Aung HT, Nikai T, Niwa M, Takaya Y. Rosmarinic acid in Argusia argentea inhibits snake venom-induced hemorrhage. J Nat Med 2010; 64:482–486. 6. Yü-liang H, Ju-chin T, Yi-ti H, Tz’u-chüan L, Hsing-liang C, Ching-yen L. Experimental studies on curing elapid bite with trypsin. Sci Sin 1975; 18:396–405. 7. Broad AJ, Sutherland SK, Lovering KE, Coulter AR. Trypsin fails as Australian snake bite cure. Med J Aust 1980; 2:388–390. 8. Wisniewski MS, Hill RE, Havey JM, Bogdan GM, Dart RC. Australian tiger snake (Notechis scutatus) and mexican coral snake (Micruris species) antivenoms prevent death from United States coral snake (Micrurus fulvius fulvius) venom in a mouse model. J Toxicol Clin Toxicol 2003; 41:7–10. 9. Doley R, Zhou X, Manjunathga K. Snake venom phospholipase A2 enzymes. In: Mackessy SP, ed. Handbook of Venoms and Toxins of Reptiles. Boca Raton: CRC Press; 2010:173–200. 10. http://www.medixcorp.com/catalog/detail.asp?Item_id ⫽ 3032 . Accessed November 7, 2013. 11. http://www.sigmaaldrich.com/catalog/product/aldrich/536954?lang ⫽ en®ion ⫽ US. Accessed November 7, 2013. 12. Rossi F, Jullian V, Pawlowiez R, Kumar-Roiné S, Haddad M, Darius HT, et al. Protective effect of Heliotropium foertherianum (Boraginaceae) folk remedy and its active compound, rosmarinic acid, against a Pacific ciguatoxin. J Ethnopharmacol 2012; 143:33–40. 13. Braidy N, Matin A, Rossi F, Chinain M, Laurent D, Guillemin GJ. Neuroprotective effects of rosmarinic acid on ciguatoxin in primary human neurons. Neurotox Res 2013. [Epub ahead of print]. 14. Domitrovic´ R, Skoda M, Vasiljev Marchesi V, Cvijanovic´ O, Pernjak Pugel E, Stefan MB. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol 2013; 51:370–378.
Clinical Toxicology vol. 52 no. 2 2014
RESEARCH ARTICLE
Trypsin and rosmarinic acid reduce the toxicity of Micrurus fulvius venom in mice J. L. PARKER-COTE,1 D. P. O’ROURKE,2 S. N. MILLER,1 K. L. BREWER,1 M. D. ROSENBAUM,2 and W. J. MEGGS1 1Department
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
2Department
of Emergency Medicine, Brody School of Medicine at East Carolina University, Greenville, NC, USA of Comparative Medicine, Brody School of Medicine at East Carolina University, Greenville, NC, USA
Context. Antivenom is expensive and not always available, so alternative treatments are being investigated. Objective. The efficacy of trypsin or rosmarinic acid (RA) in treating Micrurus fulvius in a murine model is determined. Materials and methods. Design: randomized controlled blinded study. Subjects: Fifty mice (20–30 g). Study groups: Intraperitoneal injections of: 1) 2 mg/kg M. fulvius venom (approximately twice the LD50 for mice; n ⫽ 10); 2) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with RA at a 1:10 ratio (n ⫽ 17); 3) 2 mg/kg M. fulvius venom incubated in vitro for 1 h prior to injection with 1 mg of trypsin (n ⫽ 17); 4)1 mg trypsin IP without venom (n ⫽ 3); and 5) RA IP without venom (n ⫽ 3). Main outcome: time to toxicity (respiratory distress (⬍ 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self). Statistical analysis: Time to toxicity using Tukey–Kramer HSD; Survival to 4, 6, and 12 h using Chi-square analysis. Results. Onset of toxicity: venom ⫾ saline, 120.3 ⫹ 64.4 min; venom ⫹ rosmarinic acid, 238.1 ⫾ 139.2 min (p ⫽ 0.15 relative to venom ⫹ saline); venom ⫹ trypsin, 319.7 ⫾ 201.0 min (p ⫽ 0.007 relative to venom ⫹ saline). Venom ⫹ trypsin but not venom ⫹ RA survival to 4 h was significant compared to venom ⫹ saline (p ⫽ 0.023). Two mice in the venom ⫹ trypsin group and one mouse in the venom ⫹ RA group survived to 12 h. Mice receiving trypsin without venom or RA without venom survived to 12 h without toxicity. Discussion. This work suggests that trypsin and RA may have efficacy in treatment M. fulvius envenomation. Conclusion. In vitro neutralization of M. Fulvius venom by trypsin justifies progressing to an in vivo model in future studies. Keywords
Snakebites; Rosmarinic acid; Trypsin
Of the Elapidae family, Micrurus genera venom induces neurotoxicity, causing blockage of post-synaptic endplate via alpha neurotoxin.2 In certain species of Micrurus, there is a pre-synaptic inhibition of acetylcholine release at the motor nerve endings. M. fulvius, Eastern Coral Snake, has cardiotoxic and myotoxic effects, which block the endplate receptors and depolarize the muscle fiber membrane.3 Many enzymatic activities have been detected: phospholipase A2, hyaluronidase, phophodiesterase, leucine amino peptidase, L-amino acid dehydrogensase, and acetylcholinesterase.2 Of the enzymatic activities for M. fulvius, phospholipase A2 is more prominent than hyaluronidase and proteolytic activity. Rosmarinic acid (RA), a plant derived phospholipase A2 inhibitor, is found in many plants such as Cordia verbenacea and Plectranthus barbatus. It has been used in combination with antivenom with Bothrops envenomations, showing potentiation of the antivenom’s neutralizing and antimyotoxic effects.4 The suspected mechanism of action of RA is inhibitory function at the venom’s catalytic site and possible other pharmacological activity that is not known. Also, prior studies in the Okinawa habu snake (Trimeresurus flavoviridis) have shown dose-dependence inhibition of hemorrhage by RA against T. flavoviridis: 75–100% inhibition at a concentration of 0.5 mg/ml.5
Introduction Poisonous snakes are found worldwide and account for considerable mortality and morbidity. The definitive treatment is antivenom, which is not always available. In the United States, the FDA-approved antivenom for coral snake bites expired, but the expiration date has been extended.1 Antivenom for exotic species may not be readily available. In developing nations where snakebites can be a major cause of death, antivenom is often not accessible. An effective inexpensive treatment is needed to prevent mortality worldwide. Since snake venoms contain protein toxins, proteolytic enzymes or binding site inhibitors may be efficacious in neutralizing venoms when specific antivenom is not available. This pilot study will determine if in vitro incubation of a proteolytic enzyme is efficacious in preventing toxicity from coral snake Micrurus fulvius (M. fulvius) venom in mice. A phospholipase A2 inhibitor (rosmarinic acid) will also be tested. Received 19 September 2013; accepted 25 November 2013. Address correspondence to William J. Meggs, MD, PhD, Department of Emergency Medicine, Brody School of Medicine at East Carolina University, 600 Moye Boulevard, Greenville, NC 27858, USA. Tel: ⫹ 1-252-744-2954. Fax: ⫹ 1-252-744-3589. E-mail: [email protected]
118
Trypsin and rosmarinic acid for snakebite treatment 119 Trypsin is a proteolytic enzyme that cleaves peptide chains at lysine, arginine, and serine. One prior study investigated trypsin and cobra venom, a member of the Elapid family, and found that all mice survived if trypsin was locally injected within 15 min.6 Another study found that premixing of tiger snake venom and trypsin just prior to injection did not significantly increase the survival rate of mice versus controls.7
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
Materials and methods Design was a randomized controlled blinded study. Setting was a university animal research facility, with approval by the Institutional Animal Care and Use Committee. Subjects were 50 CD-1 mice weighing 20–30 g. Lyophilized 100% pure eastern coral snake (M. fulvius) venom was obtained from Medtoxin Venom Laboratory (Delland, Florida) and reconstituted in sterile water at a concentration of 0.2 mg/mL. Trypsin in powder form rated 95% pure by mass spectroscopy was obtained from SigmaAldrich (St. Louis) and dissolved in normal saline at 10 mg/mL. RA in powder form rated 98% pure using high-performance liquid chromatography was obtained from Sigma–Aldrich (St. Louis) and dissolved in normal saline at a concentration of 2 mg/mL. Mice were given preemptive analgesia with a dose of 0.1 mg of buprenorphine subcutaneously. For each mouse receiving venom ⫹ trypsin, 2 mg/kg of a 0.2 mg/mL venom solution was incubated in vitro for 1 h at room temperature (22°C) with 1 mg of trypsin in 0.1 mL prior to intraperitoneal (IP) injection. For each mouse receiving venom ⫹ RA, 2 mg/mL of RA was incubated in vitro for 1 h with 2 mg/kg of a 0.2 mg/mL venom solution at a 1:10 ratio of venom:RA. An empirical dose of 2 mg/kg of M. fulvius venom was chosen as approximately twice a published LD50 for mice. Mice were randomized to receive the trypsin–venom mixture (n ⫽ 17), the RA–venom mixture (n ⫽ 17), trypsin alone (n ⫽ 3), RA alone (n ⫽ 3), or venom alone (n ⫽ 10). After injection, subjects were continuously observed by direct observation by two blinded observers who are authors for 12 h and assessed for signs of toxicity: respiratory distress (⬍ 25 breaths/min.), loss of spontaneous locomotor activity, or inability to upright self. Animals were euthanized at the onset of any one of the signs of toxicity, as determined by the blinded observers. Euthanasia was accomplished using isoflurane inhalation followed by cervical dislocation, according to an approved Institutional Animal Care and Use Committee protocol. The 2 mg/kg dose of eastern coral snake (M. fulvius) venom is more than two times the published LD50 for eastern coral snake venom. The concentration of eastern coral snake venom was 0.2 mg/ml. The volume of venom given or preincubated was based on weight of the individual mouse (grams). The trypsin dose was not based on weight of the animal. Each dose of venom per mouse was preincubated with 1 mg of trypsin from a 10 mg/ml solution, which is a volume of 0.1 ml per mouse. The venom to RA concentration was in 1:10 ratio (0.2–2 mg/ml). The amount of RA given is based on the amount of venom given. Copyright © Informa Healthcare USA, Inc. 2014
Comparison of time to onset of toxicity across groups was performed using Tukey–Kramer HSD. The proportion of animals surviving to 4, 6, and 12 h was compared across groups using Chi-square analysis.
Results Times are given as mean ⫾ standard deviation in Fig. 1. Time to onset of toxicity for controls (venom alone) was 120.3 ⫾ 64.4 min. Preincubation of the venom with RA provided a nonsignificant increase in time to onset of toxicity versus controls (238.1 ⫾ 139.2 min.; p ⫽ 0.15). Preincubation with trypsin significantly increased survival time versus controls (319.7 ⫾ 201.0 min, p ⫽ 0.007). Preincubation of venom with trypsin increased the number of animals that survived to 4 h versus controls (p ⫽ 0.023). This effect was not seen with RA. The proportion of animals surviving to 6 and 12 h was similar across groups (p ⫽ 0.12, p ⫽ 0.37, respectively). Two mice in the trypsin ⫹ venom group and one mouse in the RA ⫹ venom group survived to 12 h. All mice that received treatment (trypsin or RA) without venom survived to the end of the 12-h study period. Survival curves for the three groups are given in Fig. 2.
Limitations The major limitation of this study is that the results of an in vitro incubation of venom with trypsin or RA may not extrapolate to an in vivo study. An albumin control was not used because there is no reason to think that albumin would have efficacy in neutralizing venom, as has been demonstrated in a similar study.8 Another limitation is the differences in volume received, but the differences were small so a dilutional effect is improbable.
Discussion The phospholipase A2 inhibitor, RA, was chosen for study because phospholipase A2 is an important constituent of eastern coral snake venom. RA has been shown to have efficacy in other envenomations. It is a plant derivative
Fig. 1. Comparison of time to onset of toxicity for mice receiving intraperitoneal injections of venom incubated with rosmarinic acid, venom incubated with trypsin, and venom alone (colour version of this figure can be found in the online version at www.informahealthcare.com/ctx).
120
J. L. Parker-Cote et al. Other potential uses of RA in toxicology include ciguatoxin.12,13 It has also shown to ameliorate liver damage and fibrogenesis in carbon tetrachloride poisoning in mice.14 Other than the studies of trypsin to treat snakebites,6,7 there are no other references to trypsin use as a treatment for poisonings (Ovid search, November 7, 2013).
Declaration of Interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
Clinical Toxicology Downloaded from informahealthcare.com by Rutgers University on 05/31/14 For personal use only.
Fig. 2. Survival curve as decimal percent for mice receiving (a) venom ⫹ saline, (b) venom ⫹ rosmarinic acid, and (c) venom ⫹ trypsin.
found in plants such as Cordia verbenacea and Plectranthus barbatus and has demonstrated efficacy in Bothrops, envenomations in reducing antimyotoxic effects.4 Trypsin was chose for study because it is a potent protease. Two prior studies of the efficacy of trypsin as a treatment for snakebite poisoning gave different results,4,5 as discussed in the Introduction. This difference may relate to known variations in phospholipase A2 between species.9 The present study found that in vitro incubation of trypsin with M. fulvius venom significantly increased the time to toxicity in mice. RA also increased the time to onset of toxicity, but this experiment was not powered for significance. Mice received trypsin alone or RA alone without lethal effects. It can be concluded that both trypsin and RA have the potential to alter the toxic effects of M. fulvius in vivo, potentially neutralizing the venom with efficacy to treat snakebites. In vitro neutralization of M. fulvius venom by trypsin justifies progressing to an in vivo model in future studies of eastern coral snake envenomation. Study of alternatives to antivenom to treat poisonous snake bites is important because antivenom is very expensive to produce and market. Therapy for a crotalid snakebite in the United States can cost tens of thousands of dollars. Antivenom is not readily available in many countries for endogenous snakes where snakebites are a significant cause of morbidity and mortality. In other countries, it may not be available for exotic species. For some venoms, only horse serum is available which poses risks of anaphylaxis, serum sickness, and death. Trypsin is inexpensive and available for as little as $1.57/gram.10 RA is available for $42/gram.11 Whether or not these could become viable options to treat poisonous snakebites in humans requires further study but is an important direction to pursue.
References 1. http://www.fda.gov/biologicsbloodvaccines/safetyavailability/ ucm371055.htm, accessed October 31, 2013. 2. Tanaka G, Furtado M, Portaro F, Sant’Anna O, Tambbourgi D. Diversity of micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization. PLoS Negl Trop Dis 2010; 43:e622. 3. Vital Brazil O. Pharmacology of coral snake venoms. Mem Inst Butantan 1990; 52:32. 4. Ticli FK, Hage L, Cambraia RS, Pereira PS, Magro AJ, Fontes MR, et al. Rosmarinic acid, a new snake venom Phospholipase A2 inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction. Toxicon 2005; 46:318–327. 5. Aung HT, Nikai T, Niwa M, Takaya Y. Rosmarinic acid in Argusia argentea inhibits snake venom-induced hemorrhage. J Nat Med 2010; 64:482–486. 6. Yü-liang H, Ju-chin T, Yi-ti H, Tz’u-chüan L, Hsing-liang C, Ching-yen L. Experimental studies on curing elapid bite with trypsin. Sci Sin 1975; 18:396–405. 7. Broad AJ, Sutherland SK, Lovering KE, Coulter AR. Trypsin fails as Australian snake bite cure. Med J Aust 1980; 2:388–390. 8. Wisniewski MS, Hill RE, Havey JM, Bogdan GM, Dart RC. Australian tiger snake (Notechis scutatus) and mexican coral snake (Micruris species) antivenoms prevent death from United States coral snake (Micrurus fulvius fulvius) venom in a mouse model. J Toxicol Clin Toxicol 2003; 41:7–10. 9. Doley R, Zhou X, Manjunathga K. Snake venom phospholipase A2 enzymes. In: Mackessy SP, ed. Handbook of Venoms and Toxins of Reptiles. Boca Raton: CRC Press; 2010:173–200. 10. http://www.medixcorp.com/catalog/detail.asp?Item_id ⫽ 3032 . Accessed November 7, 2013. 11. http://www.sigmaaldrich.com/catalog/product/aldrich/536954?lang ⫽ en®ion ⫽ US. Accessed November 7, 2013. 12. Rossi F, Jullian V, Pawlowiez R, Kumar-Roiné S, Haddad M, Darius HT, et al. Protective effect of Heliotropium foertherianum (Boraginaceae) folk remedy and its active compound, rosmarinic acid, against a Pacific ciguatoxin. J Ethnopharmacol 2012; 143:33–40. 13. Braidy N, Matin A, Rossi F, Chinain M, Laurent D, Guillemin GJ. Neuroprotective effects of rosmarinic acid on ciguatoxin in primary human neurons. Neurotox Res 2013. [Epub ahead of print]. 14. Domitrovic´ R, Skoda M, Vasiljev Marchesi V, Cvijanovic´ O, Pernjak Pugel E, Stefan MB. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol 2013; 51:370–378.
Clinical Toxicology vol. 52 no. 2 2014
