Toxicon 41 (2003) 237–244 www.elsevier.com/locate/toxicon
Intramuscular administration of antivenoms in experimental envenomation by Bothrops asper: comparison between Fab and IgG Fernando Chavesa, Gilbert D. Lorı´aa,b, Adriana Salazara, Jose´ Marı´a Gutie´rreza,* b
a Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica Departamento de Microbiologı´a e Inmunologı´a, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica
Received 4 July 2002; accepted 11 September 2002
Abstract The efficacy of intramuscular (im) administration of sheep Fab and IgG antivenoms was assessed in a mouse experimental model of envenomation by Bothrops asper, in order to test if the more rapid absorption of Fab improves neutralization. Both antivenoms were adjusted to have a similar neutralizing potency in assays involving preincubation of venom and antivenom. Neither antivenom was effective in neutralizing lethality, nor in prolonging the time of death, in mice injected with either 3, 2 or 1.5 LD50s of venom by the intraperitoneal (ip) route, in experiments in which antivenoms were administered im immediately after envenomation. Antivenoms were effective in the neutralization of defibrinating activity, even if treatment was performed 30 min after envenomation, with no differences between IgG and Fab. Regarding neutralization of local effects, i.e. myonecrosis and hemorrhage, im administration of antivenoms at a site distant from the venom-injection site was completely ineffective in reducing the extent of local tissue damage. However, partial neutralization of these effects was achieved if antivenoms were administered im at the same site of venom injection, provided treatment was performed immediately after envenomation. Fab antivenom was slightly more effective than IgG antivenom in the neutralization of myotoxicity under these conditions, although a similar efficacy was observed between these antivenoms regarding neutralization of hemorrhagic effect. Our observations do not evidence major differences in the neutralizing ability of Fab and IgG antivenoms when applied by the im route, and do not support the hypothesis that im administration of Fab antivenoms constitutes an effective alternative to treat B. asper envenomations. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Snake venom; Bothrops asper; Neutralization; IgG antivenom; Fab antivenom
1. Introduction Since the development of serotherapy in 1894 (Bon, 1996), the parenteral administration of horse or sheepderived antivenoms constitutes the cornerstone in the treatment of snakebite envenomations (Warrell, 1992). Antivenoms are highly efficient in the neutralization of some effects associated with these envenomations (World Health Organization, 1981; Warrell, 1992), and are usually administered intravenously (iv) at health centers to achieve maximum effectiveness (World Health Organization, 1981; * Corresponding author. Fax: 506-2920485. E-mail address:
[email protected] (J.M. Gutie´rrez).
Warrell, 1995). However, the access of antivenom in medical facilities is limited or non-existent in many regions of the world (Chippaux, 1998a,b). Moreover, snakebites often occur in remote locations, with limited possibilities of rapid transportation of the patient to health centers. Thus, antivenom administration by the intramuscular (im) route constitutes an alternative to treat envenomations in the field, in circumstances where health facilities are not readily accessible (Warrell, 1995). The effectiveness of im route of antivenom administration is questionable on several grounds: (a) it is associated with a relatively poor bioavailability of ,40%, in the case of F(ab’)2 antivenoms in rabbits (Pe´pin et al., 1995; Pe´pin-Covatta et al., 1996); (b) absorption of IgG molecules
0041-0101/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 2 8 3 - 0
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or F(ab’)2 fragments to the bloodstream is rather slow after im injection, with Tmax values in rabbits of 76 and 48 h, respectively (Pe´pin et al., 1995; Pe´pin-Covatta et al., 1996; Ismail and Abd-Elsalam, 1996); (c) there is a risk of adverse reactions which require treatment with drugs not usually available in field conditions (World Health Organization, 1981); and (d) in the case of envenomations associated with incoagulable blood, such as those inflicted by pit vipers, im injections may cause large hematomas at the site of injection (World Health Organization, 1981; Warrell, 1995). In addition to the pharmacokinetic drawbacks mentioned earlier, experimental pharmacodynamic studies with IgG and F(ab’)2 antivenoms have evidenced their lack of effectiveness in the treatment of local and systemic manifestations of snakebite and scorpion envenomations when administered by the im route (Gutie´rrez et al., 1981; Gutie´rrez et al., 1991; Ismail and Abd-Elsalam, 1996). It has been suggested by various workers that Fab antivenoms may constitute an alternative to confront snakebite envenomations (Sullivan, 1987; Dart and Horowitz, 1996; Landon and Smith, 1996). Due to their reduced molecular mass, Fab fragments have a larger volume of distribution than IgG and F(ab’)2 molecules, and distribute to tissues after iv injection in a more rapid fashion (Covell et al., 1986). Moreover, upon im administration, Fab fragments reach the bloodstream faster than IgG molecules and F(ab’)2 fragments, with a Tmax of 12 h in rabbits (Rivie`re et al., 1998). Such pharmacokinetic advantages raise the possibility that im injection of Fab antivenoms may constitute an alternative to treat snakebite envenomations in the field, in circumstances where accessibility of medical facilities is remote or deficient. The present work assessed the effectiveness of an Fab ovine polyvalent (Crotalinae) antivenom, when administered by the im route, to neutralize local and systemic toxicity induced by Bothrops asper venom in mice. For comparative purposes, a whole IgG ovine antivenom of similar neutralizing potency was included in the study.
2. Materials and methods 2.1. Venom, antivenoms and mice B. asper venom was a pool obtained from more than 40 adult specimens collected in the Pacific region of Costa Rica and kept at the Serpentarium of Instituto Clodomiro Picado. Once obtained, venom was lyophilized and stored at 2 20 8C. IgG and Fab polyvalent (Crotalinae) antivenoms were prepared from the same pool of hyperimmune plasma obtained from sheep immunized with a mixture of the venoms of B. asper, Crotalus durissus durissus and Lachesis stenophrys, following an immunization protocol similar to the one described by Angulo et al. (1997). Plasma fractionation was performed with either caprylic acid alone, or caprylic acid followed by papain digestion, in
order to obtain IgG or Fab antivenoms, respectively, as described by Rojas et al. (1994) and Leo´n et al. (2000). Antivenoms were concentrated to have a similar neutralizing activity against lethal, hemorrhagic and myotoxic effects of B. asper venom, in assays in which venom and antivenom were incubated for 30 min at 37 8C prior to injection in mice, as described by Leo´ n et al. (2000). Finally, antivenoms were formulated, sterilized by filtration through 0.22 mm membranes and bottled in 10 ml vials under sterile conditions. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed under non-reducing conditions using 10% acrylamide gels (Laemmli, 1970). A band of 150 kDa was observed in IgG antivenom, whereas Fab antivenom presented a band of 50 kDa (not shown). CD-1 mice of both sexes were used in all the assays. This study was approved by the Committee for the Use and Care of Animals of the University of Costa Rica. 2.2. Neutralization studies 2.2.1. Neutralization of lethality ‘Rescue’ experiments were performed in groups of four CD-1 mice (16 – 18 g). Animals were ip injected with either 1.5, 2, or 3 median lethal doses (LD50) of B. asper venom (LD50 ¼ 3.70 mg/g). Venom was dissolved in 0.1 ml of phosphate-buffered saline solution, pH 7.2 (PBS). Then, immediately after envenomation, mice received an im injection of 250 ml of either IgG or Fab antivenoms in the left thigh. For comparative purposes, other groups of mice received 250 ml of antivenoms by the iv route immediately after venom injection. Animals were observed for a maximum period of 48 h, and the time of death was recorded. Control mice were injected with PBS instead of antivenom under otherwise identical envenomation conditions. 2.2.2. Neutralization of defibrinating activity CD-1 mice (n ¼ 4; 18 –20 g) were injected ip with 60 mg of B. asper venom, dissolved in 500 ml of PBS. This corresponds to the ‘minimum defibrinating dose’ by the ip route, i.e. the minimum venom dose that induces defibrination in all mice injected. Control animals received an ip injection of 500 ml of PBS. At either 0 or 30 min after venom injection, 100 ml of either IgG antivenom, Fab antivenom or PBS were administered im in the left thigh. Three hours after envenomation, mice were bled by cardiac puncture, under CO2 anesthesia, and blood was placed in dry glass tubes and incubated at room temperature (20– 24 8C). Blood was observed for clot formation at regular time intervals, for a maximum period of observation of 60 min. Defibrination was evidenced by the inability of blood to form a firm clot. 2.2.3. Neutralization of hemorrhagic activity CD-1 mice (n ¼ 4; 18 –20 g) were injected im in the right gastrocnemius with 50 mg of B. asper venom, dissolved in 50 ml of PBS. This dose induces prominent
F. Chaves et al. / Toxicon 41 (2003) 237–244 Table 1 Effects of Fab and IgG sheep polyvalent antivenoms on the time of death of mice in ‘rescue’ experiments in which antivenoms were injected by the im route Treatment
Time of death (min)
Dose of venom: 3 LD50s Venom þ PBS Venom þ Fab antivenom Venom þ IgG antivenom
16 ^ 4 17 ^ 3 20 ^ 3
Dose of venom: 2 LD50s Venom þ PBS Venom þ Fab antivenom Venom þ IgG antivenom
26 ^ 6 32 ^ 6 27 ^ 5
Dose of venom: 1.5 LD50s Venom þ PBS Venom þ Fab antivenom Venom þ IgG antivenom
48 ^ 21 74 ^ 23 63 ^ 31
Groups of four mice (16–18 g) were injected ip with either 3, 2 or 1.5 LD50s of B. asper venom. Immediately after envenomation, mice received an im injection in the thigh of 250 ml of either PBS, Fab antivenom or IgG antivenom. Times of death in minutes were recorded. Results are presented as mean ^ SD (n ¼ 4). No significant prolongation in the time of death was achieved with the antivenoms ( p . 0.05).
local hemorrhage in muscle tissue (Gutie´rrez et al., 1980, 1984; Moreira et al., 1992), and has been used in previous studies dealing with neutralization of B. asper venominduced local tissue damage (Gutie´rrez et al., 1985; Leo´n et al., 2000). At various time intervals after envenomation, some mice were injected with 250 ml of antivenom by the im route in the left, contralateral thigh. Other envenomated mice received 50 ml of antivenom im at the same site of envenomation, i.e. in the right gastrocnemius. A larger volume of antivenom was not injected in the gastrocnemius muscle due to anatomical constraints. Control mice were envenomated as described, and received 50 ml PBS instead of antivenom. Mice were sacrificed by CO2 inhalation 1 h after envenomation and the envenomated gastrocnemius muscles were dissected out and placed into 1.5 ml of Drabkin solution. After 24 h of incubation at 4 8C, the solution was collected, centrifuged at 1000g for 15 min, and the absorbance of the supernatants was recorded at 540 nm as a quantitative index of the hemoglobin present in the tissue (Ownby et al., 1984; Rucavado et al., 2000). 2.2.4. Neutralization of myotoxic activity CD-1 mice (n ¼ 4; 18 – 20 g) were injected im in the right gastrocnemius with 50 mg of B. asper venom, dissolved in 50 ml PBS. This venom dose is known to induce prominent myonecrosis (Gutie´rrez et al., 1980, 1984). At various time intervals after envenomation, some mice received 250 ml of antivenom by the im route in the left, contralateral thigh, whereas other mice injected im, directly in the envenomated right gastrocnemius, with 50 ml
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antivenom. Control mice were envenomated as described and then injected with 50 ml PBS instead of antivenom. Three hours after envenomation, mice were bled from the tail, under CO2 anesthesia. This time interval was chosen for sample collection since it is when highest levels of creatine kinase (CK) activity are attained in blood in these experimental model (Gutie´rrez et al., 1980). Blood was collected into heparinized capillary tubes. After centrifugation, the CK activity of plasma was determined using the Sigma kit No. 47-UV (Sigma Chemical Co., Missouri, USA). CK activity was expressed as U/l, one unit defined as the activity of enzyme that produces 1 mmol of NADH/min under the conditions of the assay. 2.2.5. Statistical analyses The significance of the differences between the means of two groups was determined by the Student’s t test. When more than two groups were analyzed, analysis of variance was performed, followed by Tukey – Kramer test.
3. Results 3.1. Neutralization of lethality Both antivenoms were adjusted as to have the same effective dose 50% (370 ml antivenom/mg venom) in assays involving incubation of venom and antivenom prior to ip administration in mice. Injection of 1.5, 2 or 3 LD50s of B. asper venom by the ip route induced systemic toxicity and death of all mice injected. The time of death was dosedependent (Table 1). IgG and Fab antivenoms failed to neutralize this effect when administered im (Table 1). Lack of neutralization was evidenced not only by the inability to prevent death, but also by the lack of prolongation of the time of death (Table 1). In contrast, 100% survival was observed when 250 ml of either antivenom were administered iv immediately after venom injection. 3.2. Neutralization of defibrinating activity Mice injected with 60 mg venom by the ip route were defibrinated 3 h after venom injection. im administration of Fab and IgG antivenoms, either immediately or 30 min after envenomation, effectively neutralized this activity, since blood samples formed firm clots, with clotting times between 1 and 3 min. Clotting times of samples from control mice injected with PBS instead of venom ranged from 1 to 2 min. 3.3. Neutralization of hemorrhagic activity No neutralization was observed when antivenoms were administered im at the left, non-envenomated hindlimb. Absorbance at 540 nm in samples from control mice injected with venom, and then receiving PBS instead of
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Fig. 1. Neutralization of hemorrhagic activity of B. asper venom by im administration of Fab and IgG antivenoms at the site of venom injection. Venom (50 mg) was injected im in the right gastrocnemius of mice. At various time intervals after envenomation, 50 ml of either Fab or IgG antivenom were injected im at the same site of envenomation. Control mice received PBS instead of antivenom. Mice were sacrificed 1 h after envenomation, and the hemoglobin present in the right gastrocnemius was quantified as described in Section 2. V: Mice injected with venom and treated with PBS instead of antivenom. Results are presented as mean ^ SD (n ¼ 4). *p , 0.05 when compared with hemorrhage induced by venom alone. No significant differences were detected between Fab and IgG treatments at any time interval.
antivenom, was 0.46 ^ 0.08. Samples from mice receiving venom and then Fab antivenom immediately after envenomation had an absorbance of 0.48 ^ 0.07, whereas samples from envenomated mice treated with IgG antivenom had an absorbance of 0.45 ^ 0.01 ( p . 0.05). On the other hand,
a partial neutralization was achieved by both antivenoms when administered im, immediately after envenomation, at the same site of venom injection (Fig. 1). However, neutralization was not achieved when antivenom administration was delayed by 5 or 10 min (Fig. 1). No significant
Fig. 2. Neutralization of myotoxic activity of B. asper venom by im administration of Fab and IgG antivenoms at the site of venom injection. Venom (50 mg) was injected im in the right gastrocnemius of mice. At various time intervals after envenomation, 50 ml of either Fab or IgG antivenom were injected im at the same site of envenomation. Control mice were injected with only PBS or with venom followed by PBS instead of antivenom. Plasma CK activity was determined 3 h after envenomation. Results are presented as mean ^ SD (n ¼ 4). *p , 0.05 when compared with animals injected with venom alone. **p , 0.05 when IgG and Fab treatments were compared.
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differences were observed between IgG and Fab antivenoms in the neutralization of hemorrhage ( p . 0.05). 3.4. Neutralization of myotoxic activity No neutralization was observed when antivenoms were administered im in the thigh of the non-envenomated, contralateral hindlimb. Plasma CK activity of control mice injected with venom and then receiving PBS was 5749 ^ 818 U/l. Mice receiving venom and then Fab antivenom had CK activity of 5405 ^ 430 U/l, and envenomated mice treated with IgG antivenoms had CK activity of 5707 ^ 395 U/l ( p . 0.05). Non-envenomated mice injected only with PBS had CK activity of 230 ^ 18 U/l. When antivenoms were administered im at the same location of venom injection immediately after envenomation, a partial neutralization was achieved in the case of Fab antivenom (Fig. 2). Fab antivenom displayed a higher neutralizing activity than IgG antivenom in mice receiving antivenoms immediately and 3 min after envenomation (Fig. 2).
4. Discussion The ideal characteristics of antivenoms to be used in the treatment of snakebite envenomations are currently the subject of debate. Most manufacturers produce either F(ab’)2 or IgG antivenoms (World Health Organization, 1981; Theakston and Warrell, 1991), although new Fab antivenoms have been introduced and clinically assessed (Meyer et al., 1997; Ariaratnam et al., 1999; Dart et al., 2001). Fab fragments display a pharmacokinetic profile which greatly differs from those of F(ab’)2 fragments and whole IgG molecules. The former have a larger volume of distribution, a more rapid equilibration into extravascular compartments, and a shorter elimination half-life (Covell et al., 1986). Despite some pharmacokinetic advantages of Fab antivenoms, experimental pharmacodynamic studies have not shown an improved neutralizing profile of these antivenoms over F(ab’)2 and IgG antivenoms, when antibodies or their fragments are administered by the iv route after envenomation (Lomonte et al., 1996; Leo´n et al., 1997, 2000, 2001). Since Fab fragments are more readily absorbed than F(ab’)2 and IgG after im injection, reaching highest concentrations in blood at a faster rate (Rivie`re et al., 1998), it was hypothesized that im injection of Fab antivenoms may constitute a valid alternative to treat snakebite envenomations in conditions where antivenom has to be administered in the field. In the experimental model used to evaluate neutralization of lethality, ‘rescue’ experiments were performed, in which venom was injected ip. Although this does not constitute a real route of envenomation in snakebites in humans, it was used for two reasons: (a) it induces a model
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of systemic envenomation which resembles actual snakebites, characterized by cardiovascular and clotting disturbances; and (b) other routes are not useful for this venom in the mouse model. For instance, iv injection of lethal doses often causes sudden death immediately after venom injection, probably due to intravascular coagulation. Moreover, subcutaneous (sc) and im routes require very high doses of B. asper venom to kill mice, thereby inflicting extremely drastic local tissue damage, with results difficult to reproduce in terms of lethality. Thus, the ip route of envenomation was selected for ‘rescue’ experiments with B. asper venom. However, the time of death of mice envenomated by the ip route was relatively short, i.e. ,60 min with the doses utilized. This reduces the time interval when antivenoms can exert their neutralizing effect. It is necessary to develop alternative models of B. asper envenomation where the time of death is extended, in order to better simulate actual envenomation conditions in which toxins reach the vascular compartment at a slower rate. Neither Fab nor IgG antivenoms neutralized lethality, when administered im immediately after envenomation, nor did they reduce the time of death after envenomation with 3, 2 and 1.5 LD50s of B. asper venom. Thus, despite the fact that Fab fragments reach the bloodstream at a faster rate than the larger IgG molecules upon im administration (Pe´pin et al., 1995; Ismail and Abd-Elsalam, 1996; Rivie`re et al., 1998), no improvement in neutralization of lethality was observed with an Fab antivenom in our experimental conditions. In contrast, when the iv route was used, both IgG and Fab antivenoms were effective in preventing lethality. It is concluded that, by the time significant concentrations of Fab and IgG molecules are reached in blood, severe cardiovascular disturbances associated with lethality have already appeared and cannot be reverted by antibodies or their fragments. Leo´n et al. (2001) observed that horse F(ab’)2 and IgG antivenoms were similarly effective in the neutralization of B. asper venom-induced lethality in similar ‘rescue’ assays, and that the iv route was more effective than the im route with both antivenoms. Thus, despite quantitative differences in neutralization between the study of Leo´n et al. (2001) and the present work, both demonstrate that there is no improvement in the neutralization of B. asper venom-induced lethality when F(ab’)2 and Fab fragments are used, as compared with whole IgG molecules, and that the im route is quite ineffective. Such ineffectiveness was also described in experimental envenomations by the coral snake Micrurus nigrocinctus (Gutie´rrez et al., 1991) and the scorpion Leiurus quinquestriatus (Ismail and Abd-Elsalam, 1996). The ip route of envenomation was adequate for the study of defibrination, since it allowed the development of coagulopathies over a more prolonged time span. The im administration of both IgG and Fab antivenoms was effective in the neutralization of defibrination, even when administered 30 min after envenomation. Thus, the pharmacokinetic divergences between IgG and Fab molecules
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do not translate into a different pharmacodynamic behavior concerning the neutralization of lethality and defibrination in this model. Our observations suggest that the im route of antivenom administration might be effective in the neutralization of defibrinating components of B. asper venom, provided antivenom is given rapidly after the onset of envenomation. Regarding neutralization of locally-acting toxins, i.e. myotoxic phospholipases A2, hemorrhagic metalloproteinases and edema-forming toxins, im administration of antivenoms at a site distant from the venom injection site was ineffective to reduce the extent of local tissue damage. Similar findings were reported for a horse IgG antivenom (Gutie´rrez et al., 1981). These findings may be explained on two grounds. (a) Toxins inducing local tissue damage act very rapidly in this experimental model (Gutie´rrez et al., 1980, 1984; Moreira et al., 1992; Chaves et al., 1995). Rapid development of local tissue damage is a characteristic not only of viperid venoms, but also of some elapid venoms, such as Naja nigricollis (Iddon et al., 1987). In the case of B. asper venom, myonecrosis and hemorrhage develop within minutes after envenomation, precluding an effective neutralization by Fab or IgG antivenoms. Even when antivenoms are administered iv immediately after venom injection, neutralization is achieved only to a partial extent (Gutie´rrez et al., 1981, 1985, 1998; Leo´n et al., 1997, 2000). (b) Absorption of Fab and IgG upon im administration is slow enough to preclude a rapid increment in the blood levels of these neutralizing molecules, thereby delaying their access to the tissue, where venom was injected. The efficacy of local administration of antivenom at the site of venom injection was also evaluated. Partial neutralization of hemorrhage and myonecrosis was observed if antivenoms were administered immediately after envenomation. In the case of myonecrosis, Fab antivenom showed a higher efficacy than IgG antivenom. This might be due to the more rapid diffusion of Fab fragments as compared to the larger IgG molecules, allowing them to reach myotoxins in the tissue before they have reached their target in muscle cells. Local intradermal injection of an Fab antivenom was effective in the neutralization of the local necrotizing effect of the venom of the spider Loxosceles deserta, when administered within the first 4 h after venom injection in rabbits (Go´mez et al., 1999). However, the time-course of the development of local tissue damage in B. asper envenomation is much faster than that described for Loxosceles sp envenomation (Gutie´rrez et al., 1984; Moreira et al., 1992; Go´mez et al., 1999). Therefore, the potential usefulness of local administration of antivenom in snakebites is probably restricted to situations where antivenom injection can be performed rapidly after envenomation. The slightly better performance of Fab fragments over IgG molecules in the neutralization of myotoxic activity,
when administered at the site of envenomation, raises the possibility that smaller antibody fragments, such as Fv fragments (Scherrmann, 1994) may be even more effective when injected locally. It was shown that local administration of the low molecular mass metalloproteinase inhibitor batimastat (BB-94) and the chelating agent CaNa2EDTA are highly effective at inhibiting locally-acting metalloproteinases from B. asper venom (Escalante et al., 2000; Rucavado et al., 2000). This can be explained, at least partially, by the high diffusibility and affinity of these inhibitors. Thus, the potential usefulness of local administration of antivenoms made of small antibody fragments with high affinity for locally-acting toxins in the treatment of local effects induced by crotaline snake venoms deserves further consideration. In conclusion, the im route of administration of Fab and IgG sheep antivenoms was ineffective to neutralize lethality and local toxicity of B. asper envenomation in mice, especially when antivenoms were injected at a site distant from the site of venom injection. In contrast, defibrination was readily neutralized by both antivenoms. A partial neutralization of local hemorrhage and myonecrosis was achieved when antivenoms were administered at the site of envenomation rapidly after venom injection. In these conditions, Fab antivenom displayed a slightly higher efficacy than IgG antivenom in the neutralization of myotoxicity. Overall, our results do not support the hypothesis that im administration of Fab fragments is an effective alternative for treating B. asper envenomations.
Acknowledgements The authors thank Dr Guillermo Leo´n for his advice in the preparation of antivenoms, and Rodrigo Chaves, Javier Nu´n˜ez and Marcos Me´ndez for their collaboration in the laboratory work. This study was supported by Vicerrectorı´a de Investigacio´n, Universidad de Costa Rica (projects 74199-220 and 741-A1-027).
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