Neutralizing Capacity of a New Monovalent Anti-Bothrops Atrox Antivenom: Comparison with Two Commercial Antivenoms

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BrazilianNeutralizing Journal capacity of Medical of a new and antivenom Biological againstResearch Bothrops (1997) atrox30: 375-379 375 ISSN 0100-879X

Neutralizing capacity of a new monovalent anti-Bothrops atrox antivenom: comparison with two commercial antivenoms

R. Otero1, V. Núñez1, 1Proyecto de Ofidismo, Facultad de Medicina, J.M. Gutiérrez4, A. Robles4, 2Facultad de Química Farmacéutica, and R. Estrada4, R.G. Osorio2, 3Facultad de Medicina Veterinaria y de Zootecnia, G. Del-Valle3, R. Valderrama1 Universidad de Antioquia, A.A.1226, Medellín, Colombia and C.A. Giraldo1 4Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

Abstract

Correspondence Three horse-derived antivenoms were tested for their ability to neu- Key words R. Otero tralize lethal, hemorrhagic, edema-forming, defibrinating and myotoxic • Bothrops atrox Proyecto de Ofidismo activities induced by the venom of Bothrops atrox from Antioquia and • Snake venom Facultad de Medicina Chocó (Colombia). The following antivenoms were used: a) polyva- • Antivenom Universidad de Antioquia lent (crotaline) antivenom produced by Instituto Clodomiro Picado • Neutralization A.A.1226, Medellín • (Costa Rica), b) monovalent antibothropic antivenom produced by Antioquia Colombia • Chocó Fax: 57-4-263-8282 Instituto Nacional de Salud-INS (Bogotá), and c) a new monovalent anti-B. atrox antivenom produced with the venom of B. atrox from Research supported by the Antioquia and Chocó. The three antivenoms neutralized all toxic Instituto Colombiano para el activities tested albeit with different potencies. The new monovalent Desarrollo de la Ciencia y la anti-B. atrox antivenom showed the highest neutralizing ability against Tecnología Francisco José de edema-forming and defibrinating effects of B. atrox venom (41 ± 2 Caldas (COLCIENCIAS), the University of Antioquia, the and 100 ± 32 µl antivenom/mg venom, respectively), suggesting that Direccion Seccional de Salud de it should be useful in the treatment of B. atrox envenomation in Antioquia and the International Antioquia and Chocó. Foundation for Science (No. F/0883-4). Introduction 5% and sequelae in 6% of the patients (2,3). This is mainly the result of late arrival at the Received February 23, 1996 Antioquia and Chocó are located in the hospital or, in some cases, of an insufficient Accepted December 2, 1996 northwest region of Colombia, a zone with antivenom supply or the high cost of lyo- large reserves of tropical rain forest that have philized products. The venom of B. atrox has allowed the development of an important proteolytic, defibrinating, hemorrhagic, biodiversity, including 104 snake species (1). myotoxic, edema-forming and indirect These regions and the Amazonas have the hemolytic activities, with regional differences highest incidence of snake bites in Colom- in venom activities (4). bia, most of them caused by the abundant These facts prompted us to produce a species Bothrops atrox, with a death rate of monovalent anti-B. atrox antivenom specific

Braz J Med Biol Res 30(3) 1997 376 R. Otero et al.

for the regions of Antioquia and Chocó, and Costa Rica and, b) the monovalent antibo- to compare its neutralizing activity against thropic antivenom of Instituto Nacional de homologous venom with two other crotaline Salud (INS), Santafé de Bogotá, batch antivenoms available in this region. 140992, produced in horses immunized with B. atrox venom from various regions of Co- Material and Methods lombia. These two antivenoms were also produced using similar fractionation proto- Animals, venom and antivenoms cols based on ammonium sulfate precipitation of globulins, without pepsin digestion Webster white mice (18-20 g) were used (5). Phenol (0.25%) was added as a preser- for the in vivo experiments. The venom of B. vative and the antivenoms were tested be- atrox was a pool obtained from 40 speci- fore their expiration dates. mens collected in Antioquia and Chocó, Colombia, lyophilized and stored at -20oC Pharmacological activities of B. atrox venom until the time for use. One adult horse was immunized at Instituto Clodomiro Picado The minimum edema-forming (MED), (Costa Rica) with B. atrox venom from hemorrhagic (MHD) and defibrinating Antioquia and Chocó, using the immuniza- (MDD) doses of B. atrox venom were deter- tion protocol shown in Table 1. At the end of mined by methods described elsewhere (6- the immunization schedule, the horse was 8), and modified by Gutiérrez et al. (9-11).

bled and plasma was fractionated by ammo- Lethality (lethal dose 50%, LD50) was deter- nium sulfate precipitation (5). mined by the Spearman-Karber method (12) For comparative purposes in the neutral- using the ip route. Myonecrosis was evalu- ization experiments, the following two com- ated by plasma creatine kinase (CK) levels mercially available antivenoms were used: and histologically by determining the a) the polyvalent (crotaline) antivenom of myonecrosis index as described by Gutiérrez Instituto Clodomiro Picado (ICP), San José, et al. (13,14) and Lomonte et al. (15). Costa Rica, batch 2460294 LQ, produced in horses by the same immunization schedule Neutralization assays as described above, using a mixture of equal weights of solid venoms of B. asper, Lachesis The neutralization assays were perfomed muta and Crotalus durissus durissus from by incubating a constant amount of solid venom with various dilutions of antivenom,

Table 1 - Immunization schedule for the produc- in order to obtain several antivenom/venom tion of B. atrox monovalent and crotaline polyva- ratios. Incubations were carried out at 37oC lent antivenoms at Instituto Clodomiro Picado. for 30 min and the mixtures were then tested

The venom was injected subcutaneously. in the corresponding assay systems for each pharmacological activity. The doses of ven- Day No. Venom dose (mg) Adjuvant om selected to test each effect were the following: a) edema: 6 MED = 9 µg; b) 0 0.75 Freunds complete hemorrhage: 10 MHD = 16 µg; c) defibrina- 10 1.5 Freunds incomplete tion: 2 MDD = 2.3 µg; d) lethality: 4 LD = 20 3.0 Sodium alginate 50 265 µg; e) myonecrosis: 50 µg. Neutralizing 30 9.0 Sodium alginate ability is reported as effective dose 50% 40 18.0 Sodium alginate (ED50), defined as the µl antivenom/mg ven- 50 30.0 Sodium alginate om ratio that reduces by 50% the activity of 60 45.0 Sodium alginate venom alone. In the case of neutralization of

Braz J Med Biol Res 30(3) 1997 Neutralizing capacity of a new antivenom against Bothrops atrox 377 defibrinating activity, results are reported as solution: 60 ± 36 U/ml) and the myonecrosis effective dose 100% (ED100), defined as the index was 1.0 (control = 0), confirming the µl antivenom/mg venom ratio at which the myotoxic effect of the venom. effect of the venom was completely neutralized. All experiments were repeated on three Neutralization of venom activities different days. Table 2 shows that the three antivenoms Statistical analysis neutralized all pharmacological activities of B. atrox venom studied, albeit with different Data were analyzed statistically by one- potencies. Monovalent antibothropic (INS) way analysis of variance (ANOVA). When and monovalent anti-B. atrox antivenoms the values were significantly different were equally potent in the neutralization of (P<0.05), the differences between pairs of lethality and hemorrhage, whereas the poly- means were analyzed by the Tukey test. valent (ICP) antivenom was less efficient in the neutralization of these effects. Regard- Results ing edema and defibrination, the monovalent anti-B. atrox antivenom showed the high- Pharmacological activities of B. atrox venom est neutralizing ability. No significant differences were observed between the three anti- The lethal dose 50% of B. atrox venom venoms concerning neutralization of myo- injected intraperitoneally into mice was 66.2 necrosis (Table 2). µg (95% confidence limits: 49.5-88.6). The minimum hemorrhagic dose was 1.6 ± 0.6 Discussion µg, the minimum edema-forming dose 1.5 ± 0.3 µg, and the minimum defibrinating dose The parenteral administration of horse- 1.1 ± 0.3 µg. After im injection of 50 µg of or sheep-derived antivenoms constitutes the venom, plasma CK levels increased to 664 ± cornerstone in the treatment of snakebite 116 U/ml (control mice injected with saline envenomation (16). Since venoms present

Table 2 - Neutralization of pharmacological activities of B. atrox venom from Antioquia and Chocó by the three antivenoms.

Neutralization is reported as effective dose 50% for lethal, hemorrhagic, edema-forming and myonecrotic effects and as effective dose 100% for the defibrinating effect (see Material and Methods). For lethality, results are reported as mean and 95% confidence limits (given in parentheses). For the other effects, results are reported as mean ± SD (N = 3). Values with different superscripts (a,b,c) are significantly different (P<0.05) (Tukey test). +In the neutralization of lethality, results are also reported as mg venom neutralized per ml antivenom.

Antivenom Neutralization (µl antivenom/mg venom)

Lethality Hemorrhage Edema Defibrination Myonecrosis

Monovalent anti-B. atrox 143 (96-213)a 71 ± 6a 41 ± 2a 100 ± 32a 110 ± 20a 7.0 (4.7-10.4)+

Monovalent antibothropic (INS) 147 (100-217)a 66 ± 3a 647 ± 176c 327 ± 133b 236 ± 109a 6.8 (4.6-10.0)+

Polyvalent (ICP) 213 (149-303)b 121 ± 18b 395 ± 13b 959 ± 36c 140 ± 22a 4.7 (3.3-6.7)+

Braz J Med Biol Res 30(3) 1997 378 R. Otero et al.

conspicuous biochemical and pharmacolo- cases, in sequelae (2,3). The rapid develop- gical variations between and within species ment of these local effects makes efficient (17-19), it is recommended that antivenoms neutralization by antivenoms difficult (9-11). should be produced in each country with Thus, it is necessary to have antivenoms with venoms obtained from snakes collected in high neutralizing capacity against the toxins that particular region. Otero et al. (4) demon- responsible for these effects in order to reduce strated variations in several pharmacologi- the local tissue damage. cal activities of B. atrox venoms obtained Our results indicate that the three anti- from different regions of Antioquia and venoms were similarly efficient in neutraliz- Chocó. Thus, in order to obtain a specific ing myonecrosis, whereas monovalent anti- antivenom against the venom of B. atrox B. atrox antivenom had the highest neutral- from Antioquia and Chocó, a monovalent izing ability against the edema-forming ef- antivenom was produced and compared with fect. Regarding hemorrhage, monovalent two other crotaline antivenoms available in anti-B. atrox and monovalent antibothropic these regions. (INS) antivenoms were more potent than Our results indicate that the three anti- polyvalent antivenom, although all three neu- venoms were effective in neutralizing the tralized this effect at a low antivenom/ven- most relevant toxic effects of B. atrox venom ratio. om. This suggests that there is a significant In conclusion, the three antivenoms tested immunological cross-reactivity between the are efficient in neutralizing the most relevant venoms of B. atrox from Antioquia and toxic effects induced by the venom of B. Chocó, B. atrox from other regions of Co- atrox from Antioquia and Chocó. The lombia and B. asper from Costa Rica. Cross- monovalent antivenom produced with ven- neutralization of several antivenoms pro- oms from specimens collected in these re- duced in Latin America was demonstrated gions in Colombia showed the highest neu- when they were tested against the venom of tralizing potency. We suggest that this B. atrox from Antioquia and Chocó (20). antivenom may be of high value in the treat- On the other hand, when the effective ment of B. atrox envenomation in Antioquia

doses 50% of these antivenoms were com- and Chocó. A randomized clinical trial is pared, the monovalent anti-B. atrox currently under way in Colombia in order to antivenom produced with venoms from test this hypothesis. Antioquia and Chocó showed the highest neutralizing ability against the venom of B. Acknowledgments atrox from these regions. Local tissue damage (myonecrosis, hem- We thank Oscar Rojas (Universidad de orrhage and edema) is one of the most typical Antioquia) and the staff of the Production and dangerous consequences of B. atrox en- Division (Instituto Clodomiro Picado) for venomation in Colombia (2). These effects collaboration, as well as Patricia García for result in severe lesions and, in a number of typing the manuscript.

Braz J Med Biol Res 30(3) 1997 Neutralizing capacity of a new antivenom against Bothrops atrox 379

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