Usefulness of the combination of a single dose of neostigmine-atropine plus antivenom on the experimental treatment of the envenomation caused by Micrurus venoms

Abstract

Introduction

Envenomation by Micrurus snakes is relatively infrequent, but constitutes a persistent public health concern throughout the Americas, due to its potentially severe clinical consequences. The venom of these snakes, like that of other elapids, primarily contains postsynaptic toxins (α-neurotoxins, with a three-finger toxin (3-FTx) structure) and some species also produce presynaptic toxins (β-neurotoxins, with a phospholipase A₂ (PLA₂) structure). Given this composition, the potential therapeutic value of combining an acetylcholinesterase inhibitor, neostigmine (N) plus atropine (A) with an anti-Micrurus antivenom (AV) was investigated. The efficacy of this combination therapy was evaluated in comparison to AV alone, using experimental envenomation models involving Micrurus venoms with different proportions of 3-FTxs and PLA₂s.

Methods

Groups of mice were challenged with 1.2 minimal mortal doses of M. pyrrhocryptus (Mp), M. altirostris (Ma), M. surinamensis (Ms), M. nigrocinctus (Mn), or M. fulvius (Mf) venom. After 15 minutes, the animals received a single rescue treatment with either N+A, various doses of anti-Micrurus AV alone, or a combination of AV plus N+A. Survival was assessed at 24 hours. For the venom control groups and those treated with N+A alone, time to death was also recorded. The median effective dose of AV alone, or with the addition of N+A, was estimated for Mp and Ma venoms.

Results

Treatment with the N+A combination alone did not provide effective protection. However, N+A slightly delayed the time to death in animals envenomed with Ms, Ma, and Mp venoms (p < 0.05). The combination of N+A with AV significantly improved survival compared to AV alone in mice challenged with Ma and Mp venoms (p < 0.05). In contrast, higher AV doses were required to achieve similar effects in animals envenomed with Ms venom, while Mn and Mf venoms required the highest AV doses to show some level of protection. The estimated median effective dose of AV in combination with N+A was more potent, enhancing AV potency by 1.6-fold for Mp and up to 3.4-fold for Ma venom.

Conclusion

The survival of mice treated with AV plus N+A was higher in envenomations caused by Ma, Ms, and Mp venoms, which are characterized by prominent α-neurotoxic activity and a lower proportion of PLA₂s. The effect was less evident and only apparent at the highest AV doses for Mn and Mf venoms, which contain higher levels of PLA₂s and exhibit strong experimental myotoxic capacity. The results show that the combined use of N+A and AV enhances the effectiveness of treatment in Micrurus envenomations, especially those involving predominantly postsynaptic neurotoxins, by reducing the AV dose required for recovery.

1. Introduction

Coral snakes (genera Micrurus, Micruroides and Leptomicrurus) represent the most diversified group of snakes in the Americas. They are the only terrestrial American elapids and inhabit a wide range of terrestrial ecosystems —from sea level to elevations of approximately 3,000 m— from cold and hot desert regions to humid and hot tropical areas, and ranging geographically from northern Patagonia to Mexico and the southern USA [13]. The genus Micrurus comprises around 70 species [13,21,58].

The venom of Micrurus snakes induces loss of muscle strength and respiratory paralysis of peripheral origin. These neurotoxic effects are primarily due to the action of α-neurotoxins (with a three-finger toxin (3-FTx) structure), which block acetylcholine receptors at the end plate, in the neuromuscular junction, and/or β-neurotoxins (with a phospholipase A₂ (PLA₂) structure) that inhibit evoked acetylcholine release by the motor nerve endings at the presynaptic level. In some cases, these effects are further exacerbated by the additive action of myotoxic components on muscle fiber membranes [4,6,10,37,47,67,68,75]. Among the various toxic components of Micrurus venoms, the 3-FTx and PLA₂ neurotoxins are considered the most important [35,42].

The specific treatment for Micrurus envenomation is the administration of a corresponding antivenom (AV). Nevertheless, such AVs are scarce, difficult to produce, and currently manufactured by only a limited number of producers [26]. Unlike the broad cross-reactivity and neutralization often observed among AVs targeting viperid venoms in the Americas [5,22,24,61], coral snake AVs generally exhibit limited cross-reactivity [26,27]. This is likely attributable to the qualitative and quantitative differences in the composition of Micrurus venoms [42].

The use of acetylcholinesterase inhibitors (AChEIs), such as neostigmine (N), as an adjunct treatment for envenomations due to coral snake bites is suggested by some South American ministries of health [45,46]. Experimental data support the potential usefulness of this type of compounds in reversing Micrurus venom induced neurotoxicity [12,20,67]. The AChEIs inhibit acetylcholinesterase activity, thereby increasing acetylcholine levels at the neuromuscular junction, where the excess of acetylcholine can competitively displace the α-neurotoxins from nicotinic receptors [4,37,43]. In elapid envenomation, these drugs are typically administered in combination with atropine (A) to mitigate their muscarinic effects [7,37,43]. Although AChEIs are extensively used in the treatment of envenomation by Asian elapids, whose venoms predominantly contain α-neurotoxins [1,4,29,33,36,37,42,48,56,7 3,74,76], clinical data on their efficacy in Micrurus envenomation is not abundant.

In envenomations registered in Brazil, the response to treatment was reported to be favorable when AChEIs were used as an ancillary to AV treatment [10,11]. However, the clinical response to these drugs depends on the presence of α-neurotoxins and the absence or low abundance of β-neurotoxins. Their mechanism of action, as mentioned above, is limited to displacing α-neurotoxins from nicotinic receptors through elevated acetylcholine concentrations at the end-plate [4,37]. In vitro studies have demonstrated the effectiveness of AChEIs against experimental envenomation by M. pyrrhocryptus, M. frontalis and M. lemniscatus venoms [12,67], three venoms that contain α-neurotoxins [15, 51,60]. On the other hand, their efficacy was not observed against venoms with a higher content of β-neurotoxins, such as that of M. corallinus [19,70]. Beyond the foundational studies of Vital Brazil, experimental in vivo research on the use of AChEIs in South American coral snake envenomation is very scarce or lacking entirely [67].

In Argentina eight species of Micrurus have been described: M. pyrrhocryptus, M. altirostris, M. balyocoriphus, M. tricolor, M. frontalis, M. lemniscatus carvalhoi, M. silviae and M. corallinus [26,45]. Among these, M. pyrrhocryptus has the widest distribution and is likely responsible for the majority of coral snakebites in the country [26]. Its venom is the primary immunogen used in the production of the AV currently employed in Argentina [23,26]. Only limited data are available on the venoms of species of Micrurus described in Argentina [12,25,26,27,28,51], nevertheless the presence of α-neurotoxins in the venoms of these species was described [12,19,28,31,51,60]. This supports the potential role of AChEIs as adjunctive therapies in the treatment of envenomations caused by most of these species.

In this study, we evaluated the efficacy of a single dose of an AChEI, the administration of a South-American Micrurus AV alone, and the combination of AChEI plus AV, in experimental murine models of Micrurus envenomation. Rescue experiments (experiments in which the antivenom is applied a determinate time after the injection of a lethal dose of venom) were conducted in M. pyrrhocryptus (Fig. 1), M. altirostris (Fig. 2), M. surinamensis (Fig. 3), M. nigrocinctus and M. fulvius experimental envenomations.

Figure 1.

Micrurus pyrrhocryptus, province of Córdoba, Argentina (photo credit: Adolfo Rafael de Roodt)

Figure 2.

Micrurus altirostris, province of Misiones, Argentina (photo credit: Adolfo Rafael de Roodt)

Figure 3.

Micrurus surinamensis, Letizia, Colombia (photo credit: Juan Silva Haad)

2. Materials and methods

2.1. Venoms

The venom of M. pyrrhocryptus was obtained from specimens collected in various regions of Argentina and kindly provided by Dr. Eduardo Gould (Fundación de Estudios Biológicos, Buenos Aires, Argentina). M. altirostris venom was donated by Dr. Alejandro Urs Vogt (Centro Toxicológico de Misiones, Oberá, Misiones, Argentina). The venom of Colombian M. surinamensis was a gift from Dr. Juan Silva Haad (Leticia, Colombia). M. fulvius venom from Florida, USA, was sourced from the Miami Serpentarium (Florida, USA) and M. nigrocinctus venom from Costa Rica was acquired from the La Nauyaca Serpentarium (Cuernavaca, Mexico). All venoms were obtained by manual extraction, immediately vacuum dried or lyophilized, and stored at -20ºC until use.

2.2. Animals

All experiments were conducted using 18–22 g CF-1 mice obtained from the Animal Facility of the Instituto Nacional de Producción de Biológicos (INPB). The animals were housed under controlled environmental conditions and received rodent chow and filtered water ad libitum. Ethical approval for animal handling was granted by the INPB – ANLIS “Dr. Carlos G. Malbrán”, in agreement with the guidelines of the National Research Council [49,50] and the Federation of European Laboratory Animal Science Associations [30].

2.3. Neostigmine (N) and atropine (A)

Neostigmine (Neostigmina Drawer, Laboratory Drawer) was used in 1 mL ampoules at a concentration of 0.5 mg/mL. Atropine (Klonatropina, Klonal Laboratorios), was used in 1 mL ampoules at a concentration of 1 mg/mL. All drugs were used prior to their expiration date.

2.4. Antivenom

The AV used was Antiveneno Elapídico, produced by the INPB through the immunization of horses with venoms from Argentine Micrurus species, primarily M. pyrrhocryptus [26], batch No. 133, Exp. Date August 31, 2017. The AV is constituted of F(ab´)₂ fragments. Although the efficacy of AVs after their expiration date has been documented [17,52,53,59,63], this AV was tested for its physicochemical properties and neutralizing capacity prior to use, confirming that it retained all characteristics declared by the manufacturer.

2.5. Determination of the median lethal doses, minimal mortal doses and time to death

The lethal potency of the venoms was determined via the subcutaneous (s.c.) route. Groups of mice (n = 5 per dose level) were injected with various amounts of venom diluted in 0.15 M NaCl, in a final volume of 0.3 mL. Mortality was recorded 24 h post-injection. Lethal potency was defined as 1) the median lethal dose (LD₅₀) representing the dose of venom required to kill 50% of the challenged mice within a period of 24 h, and 2) the minimal mortal dose (MMD), the lowest dose of venom that caused 100% mortality in the same period (equivalent to LD100). The venom doses versus mortality plots were analyzed using a sigmoidal dose-response model with a variable slope [14].

Once the MMD was established, groups of mice were injected with 1.2 MMD of each of the venoms, and time to death (from injection to death) was recorded. Group comparisons were performed using the Mann-Whitney test or Student’s t test, as appropriate.

2.3. Dosages of Neostigmine and Atropine

Several doses of N and A were evaluated based on dose ranges previously used in murine models of intoxication and envenomation involving AChE inhibition [18,34,38,41]. The final selected doses were 0.25 μg/g of N and 2.0 μg/g of A. Both drugs were administered via the intraperitoneal (i.p.) route in a final volume of 0.5 ml/mouse, diluted in 0.15 M NaCl. In combination with AV, the same final volume and diluent were used, with appropriate adjustments for AV volume. The doses of A and N were tested alone and in combination, up to threefold higher than the final experimental dose, in groups of five mice to ensure the absence of toxicity. No adverse effects were observed at these levels. Severe signs of intoxication and mortality (2 out of 5 animals challenged) were only observed at fivefold the selected doses.

2.7. Experiments on toxicity reduction

2.7.1 Effect of different doses of AV with or without N+A

2.7.1.1 Experimental groups

Groups of five mice were injected subcutaneously with 1.2 MMD of each venom, diluted in 0.15 M NaCl to a final volume of 0.3 mL. Fifteen min after envenomation, mice received an intraper-itoneal (i.p.) rescue treatment consisting in AV alone (50 or 100 μL) or AV combined with N+A, diluted in 0.15 M NaCl to a final volume of 0.5 mL. Treatment groups were as follows: group 1) N (5 μg) + A (40 μg); group 2) different doses of AV (50 or 100 μL) plus N (5 μg) +A (40 μg); group 3) AV alone (50 or 100 μL); group 4) venom alone diluted in 0.15 M NaCl (positive control).

2.7.1.2 Data Interpretation

The protection provided by each treatment was expressed as the percentage of mice surviving 24 h post-injection. Survival data (the number of deaths and survivors per group) were analyzed using Fisher’s exact test to compare the effectiveness of AV alone versus AV combined with N+A.

2.7.1.3 Relative delay in death with N+A alone

Due to the rather different times to death observed following administration of 1.2 MMD of the various venoms, the time to death was recorded for each animal. To assess the potential benefit of treatment with N+A in delaying or preventing death for each venom tested, the relative delay in death (RDD) was defined as the ratio between the mean time to death in mice treated with N+A and the mean time to death in controls (venom only), according to the formula: RDD = (time to death using N+A) / (time to death in controls).

2.7.1.4 Analysis of survival data

Median survival outcomes from the various experiments were analyzed using linear and non-linear regression models. The median values derived from sigmoidal dose-response analyses were considered Theoretical Median Doses (T-MED). Additionally, the ratios between the T-MED values obtained with and without N+A treatment, were calculated and analyzed.

2.7.2 Effect of N+A on the rescue Median Effective Dose of the AV on M. pyrrhocryptus and M. altirostris venoms

In order to evaluate whether the use of N+A alters the Median Effective Dose (ED₅₀) of the AV against envenomation by M. pyrrhocryptus and M. altirostris venoms, rescue ED₅₀ (r-ED₅₀) values were estimated in ED₅₀ assays with and without N+A co-administration. Groups of 5 mice were s.c. injected in the dorsal region with 1.2 MMD of the venoms in a final volume of 0.3 mL of 0.15 M NaCl. After fifteen minutes, the mice received an i.p. injection of different doses of AV alone or AV plus N+A, in a final volume of 0.5 ml/mouse, in 0.15 M NaCl. Mice were monitored and after 24 h survival deaths were recorded. The rescue median effective dose (r-ED₅₀) was defined as the AV dose that protected 50% of the mice [54] and was estimated using a sigmoidal dose-response model with variable slope [14].

2.8 Statistics

The distribution of the data was assessed using the Kolmogorov-Smirnov test. For comparison of groups, Mann-Whitney or Student’s t test were used. For multiple comparison analysis, ANOVA or Kruskal-Wallis tests with Bonferron's or Dun's adjustment tests were used. Differences in frequencies were analyzed using Fisher’s exact test. The dose-response experiments were analyzed by linear regression or non-linear regression using a sigmoidal dose-response model with variable slope. All statistical analyses were performed using the software Prism 6.0 (GraphPad Inc., CA, USA) for Mac. A p-value ≤ 0.050 was considered statistically significant.

3. Results

3.1. Lethal potencies

The s.c. LD₅₀s, LD₁₀₀s (MMDs) of the venoms used in this study were 31 μg and 45 μg for M. pyrrhocryptus, 26 μg and 30 μg for M. altirostris, 28 μg and 40 μg for M. surinamensis, 27 μg and 57 μg for M. nigrocinctus and, 22 μg and 40 μg for M. fulvius venoms. These doses and the venom masses that represent the 1.2 MMD challenge doses used in this study, are detailed in Table I.

Table I.

Median lethal dose (LD₅₀), minimal mortal dose (MMD or LD₁₀₀) and challenge dose (1.2 MMD) of the

Venin	Doses toxiques
	DL50 s.c. (μg/souris)	DL100 s.c. (DMM) (μg/souris)	Dose de provocation (1,2 DMM) (μg/souris)
M. pyrrhocryptus	31 (28 to 35)*	45	54
M. altirostris	26 (25-27)*	30	36
M. surinamensis	28 (25 to 30)*	40	48
M. nigrocinctus	27 (21 to 35)*	57	68
M. fulvius	22 (19 to 27)*	40	48

les valeurs entre parenthèses correspondent aux intervalles de confiance à 95 %

3.2. Time to death of mice injected with venom with or without treatment with N+A

The venom of M. surinamensis caused the most rapid lethality, with a mean time to death of 25 ± 6 min while M. fulvius venom resulted in the slowest progression to death, with a mean time of 157 ± 35 min (ANOVA-Bonferroni p < 0.025 and p < 0.0001 for M. surinamensis and M. fulvius respectively). The mean time to death for the remaining venoms were 43 ± 6 min, 47 ± 11 min and 54 ± 9 min for M. nigrocinctus, M. pyrrhocryptus and M. altirostris venoms respectively (Table II). The RDD is shown in Figure 4.

Table II.

Times of death caused by the injection of 1.2 MMD of the different venom and with and without the injection of N+A

Venin	Traitement	Time of death (min)				n	p
		Mean ± SD	Mediane	Minimum	Maximum		Statistic
M. pyrrhocryptus	Venin seul	47 ± 11	48	30	75	22	< 0.05 (U = 174,5; t = 2.09)*
	Venin plus N+A	55 ± 15	50	45	114	24
M. altirostris	Venin seul	54 ± 9	54	41	70	17	< 0.001 (U = 20.5)
	Venin plus N+A	76 ± 14	78	51	101	15
M. surinamensis	Venin seul	25 ± 6	24	18	42	18	< 0.001 (U = 0.0)
	Venin plus N+A	60 ± 27	53	43	122	8
M. nigrocinctus	Venin seul	43 ± 6	42	34	56	13	0.69 (U = 76.5)
	Venin plus N+A	43 ± 4	42	36	51	13
M. fulvius	Venin seul	157 ± 35	151	110	217	12	0.29 (U = 53.5)
	Venin plus N+A	132 ± 46	147	59	190	12

n : nombre d'animaux; U : valeur (test de Mann-Whitney); * : test t de Student); dans ce cas (venin de M. pyrrhocryptus), les différences entre les groupes ont été analysées à l'aide des méthodes du test t de Student et du test de Mann-Whitney

Figure 4.

Relative delay in time to death (RDD) following treatment with N+A. Bars represent the ratio between the time to death of mice injected with V and treated with N+A, and the time to death of the controls injected with V alone (RDD = V + N+A/V alone)

The combination of N+A did not protect mice of the challenge dose, but delayed the time of death of M. surinamensis, M. altirostris and M. pyrrhocryptus venoms. Only in the case of M. altirostris the survival of 2 mice out 15 treated with venom and N+A was observed (Table II).

3.3. Survival of mice injected with venom and rescued with AV alone or with AV plus N+A

The combination of the treatment with AV plus N+A resulted in improved outcomes in enven-omations by South American Micrurus species.

3.3.1 M. pyrrhocryptus venom

The addition of N+A to AV resulted in better outcomes than treatment with AV alone (Table III; Appendix 2). Among mice challenged with this venom, survival was higher in the group treated with 100 μL of AV plus N+A (21/25 mice survived with the addition of N+A, compared to 11/25 in the AV-alone group) or using 50 μL of AV (13/25 survivors with N+A vs 7/25 using AV alone). Fisher’s exact test confirmed a statistically significant improvement with 100 μL of AV plus N+A compared to 100 μL of AV alone (p = 0.0072) (Fig. 5; Fig. 6; Appendix 2). Although protection appeared higher with 50 μL AV plus N+A compared to 50 μL of AV alone, in this assay the difference did not reach statistical significance (p = 0.0833). Nevertheless, statistical differences were observed when the protection provided by this dose of AV plus N+A regarding AV alone in ED₅₀ experiments (n: 30 mice) were analyzed (p = 0.0352) (Appendix 3.a). This difference when all data using a dose of 50 μL of AV alone or plus N+A (fixed doses + ED₅₀ assays, n: 80 mice) were analyzed (p = 0.0032) (Appendix 3.b).

Table 3.

Percentual survival of mice challenged with 1.2 MMD of venom and rescued with different fixed doses of AV alone or combined with N+A

Venin	Traitement	Dose AV (μL)	Protection (%)			Nombre d'expériences
			Médiane	Minimum	Maximum
M. pyrrhocryptus	AV alone	50	20	0	60	5
		100	40	20	60	5
		250	100	*	*	1
	AV plus N+A	50	60	0	100	5
		100	80	60	100	5
		250	100	*	*	1
M. altirostris	AV alone	50	60	0	60	5
		100	60	40	80	5
		250	100	*	*	1
	AV plus N+A	50	100	60	100	5
		100	100	80	100	5
		250	100	*	*	1
M. surinamensis	AV alone	50	0	0	0	2
		100	0	0	0	2
		250	20	0	20	3
	AV plus N+A	50	0	0	0	2
		100	20	0	80	5
		250	60	0	100	5
M. nigrocinctus	AV alone	50	0	0	0	3
		100	0	0	0	4
		250	40	*	*	1
	AV plus N+A	50	0	0	0	3
		100	10	0	20	4
		250	40	*	*	1
M. fulvius	AV alone	50	10	0	20	2
		100	0	0	0	3
		200	40	*	*	1
		400	60	*	*	1
	AV plus N+A	50	10	0	20	2
		100	20	0	20	3
		200	40	*	*	1
		400	100	*	*	1

There were used 5 mice by experiment

Figure 5.

General response to the different treatments. Survival and mortality outcomes of mice challenged with 1.2 MMD of venom and treated with either AV alone or AV plus N and A (doses up to 250 μL were included). The bars represent the cumulative results from all experiments. Black portions indicate deceased mice, and white portions indicate surviving mice. Asterisks indicate statistically significant differences (p < 0.05, Fisher’s exact test) comparing survival and mortality rates between mice treated with AV alone and those receiving AV plus N+A

Figure 6.

Response to different doses of AV with or without N+A. Survival and mortality outcomes in mice challenged with 1.2 MMD of venom (V) and treated with different doses of either AV alone or with AV plus N+A were compared using Fisher’s exact test. Bars represent cumulative data per dose. Black portions indicate deceased mice, and white portions indicate surviving mice. Asterisks indicate statistically significant differences (p < 0.05).

3.3.2 M. altirostris venom

The adding of N+A to the treatment with AV improved the survival. Among mice challenged, the use of the combination showed a better outcome (Table III; Appendix 2), when using 100 μL of AV, 16/25 mice survived in the AV-alone group while 23/25 survived in the AV plus N+A group. When the dose of AV was 50μL, the combination of AV plus N+A protected 22/25 while the use of AV alone only protected 12/25 challenge mice. Fisher’s exact test showed that combining either 100 or 50 μL of AV with N+A offered significantly better protection than AV alone (p = 0.0374 and 0.0054, respectively) (Fig. 5; Fig. 6; Appendix 2).

3.3.3 M. surinamensis venom

Overall, AV alone protected just 1 of 15 mice at a dose of 250 μL (1/15), while 100 or 250 μL of AV plus N+A protected 8/25 and 15/25 respectively. No protection was observed with 50 μL of AV plus N+A (0/10) (Table III). Fisher’s exact test showed that the best outcome was achieved using 250 μL of AV plus N+A (p = 0.0009). The use of 100 μL of AV and N+A, although seemingly better, did not differ significantly from AV alone (p = 0.0734) (Fig. 5; Fig. 6; Appendix 2).

3.3.4 M. nigrocinctus venom

No meaningful differences in survival were observed between AV alone and AV combined with N+A; 2/40 versus 5/40 mice survived (n=80) (Table III; Fig. 6). When each dose was analyzed separately using Fisher’s exact test, no statistically significant differences were found (p > 0.4 in all cases) (Fig. 5; Fig. 6; Appendix 2).

3.3.5 M. fulvius venom

No statistically significant differences in survival were observed between treatment with AV alone or AV combined with N+A. When considering the different doses, some degree of protection was observed (Table III), but Fisher’s exact test showed no significant differences (p > 0.4 in all cases) (Fig. 5; Fig. 6; Appendix 2).

3.4. Variation in the Rescue Median Effective dose of AV with or without N+A

The addition of N+A to the AV treatment en-hanced its neutralizing potency against both venoms, although the magnitude of the improvement varied. For M. altirostris venom, the ED₅₀ of AV alone was 55 μL (95% CI: 46–67 μL, R² = 0.9781), which decreased to 16 μL (9–28 μL, R² = 0.7655) when combined with N+A, reflecting a 3.4-fold increase in neutralizing potency. In contrast, for M. pyrrhocryptus venom, the ED₅₀ of AV alone was 94 μL (81–108 μL, R² = 0.7727), and this was reduced to 59 μL (47–74 μL, R^{2 =} 0.8273) with the addition of N+A, corresponding to a 1.6-fold improvement (Fig. 7).

Figure 7.

Median effective dose of AV, administered alone or in combination with N+A, in rescue experiments. AV or AV plus N+A was injected intraperitoneally 15 min after subcutaneous injection of 1.2 minimal mortal doses (MMD) of the respective venoms. The solid line represents the response to AV alone, while the dashed line represents the response to AV plus N+A. In both cases, the addition of N+A reduced the dose of AV required to rescue 50% of challenged mice. A. Against M. pyrrhocryptus venom, the ED₅₀ of AV alone was 94 μL (95% CI: 81 – 108 μL, R² = 0.7727), while the addition of N+A reduced the ED₅₀ to 59 μL (47–74 μL, R² = 0.8273), increasing neutralizing potency by 1.6-fold. B. For M. altirostris venom, the ED₅₀ of AV alone was 55 μL (46–67 μL, R² = 0.9781), reduced to 16 μL (9–28 μL, R² = 0.7655) when combined with N+A, corresponding to a 3.4-fold increase in neutralizing potency.

3.5 Analysis of the doses used and protection

In all cases, treatment improved the outcome of experimental envenomations to varying degrees. However, statistically significant differences were observed only for M. altirostris and M. pyrrhocryptus venoms and for M. surinamensis venom but only using high doses of AV (Fig. 5 and 6). A preliminary analysis using linear and non-linear regression of median protection values against AV doses confirmed this trend (Appendix 4 and 5). The data indicate that the addition of N+A allows for a reduction in the required AV dose to achieve the same level of protection across all venoms tested (Table III; Fig. 5; Fig. 8; Appendix 4 and 5). For the neutralization of M. pyrrhocryptus and M. altirostris venoms in particular, although the ED₅₀ and T-MED values differed by nearly two-fold, their 95% confidence intervals overlapped (Fig. 7; Appendix 6).

Figure 8.

The median survival values from the different rescue experiments are plotted as percentages against the corresponding AV doses. Treatments using AV alone are represented by black rhombi: ♦ and treatments using AV plus N+A by white circles: O. Stars (*) denote data points for which only a single experiment was performed (highest doses).

4. Discussion

Envenomation by coral snakes is uncommon, but when it does occur it constitutes a medical emergency for which the only effective treatment is the administration of specific AV [45,46]. This AV is difficult to produce and is of limited availability, even in countries with local manufacturing capabilities [10,11,23,25,27].

The AChEIs alone or combined with AVs have been used for the treatment of envenomation by other elapids [4,37,48]. Despite that some experimental benefits were observed on envenomation caused by venoms with both pre and postsynaptic neurotoxins [76], in envenomation by other elapids, clinical experience shows absence or low benefits. In envenomations caused by venoms with presynaptic neurotoxins like Bungarus or Oxyuranus species [2,62,65,72] the benefit is lower or absent regarding their use on envenomations with venoms with predominantly postsynaptic neurotoxins like Naja [29,36,56,73,74,76]. In the case of coral snakes, the use of AChEIs is particularly recommended in case of envenomation by snakes whose venom consists exclusively of postsynaptic neurotoxins [9,10,46,67].

As previously mentioned, the use of AChEIs has been proposed as a therapeutic approach for Micrurus envenomations [32,67,69,70,71], nevertheless, some Micrurus venoms, such as those of M. corallinus and M. fulvius, also cause presynaptic neurotoxicity via β-neurotoxins [19,66,67]. The action of these β-neurotoxins, along with certain non-neurotoxic PLA₂s, involves additional mechanisms that can cause lesions in cellular membranes, potentially complicating the course of envenomation [4,6,47]. Due to this significant variability, AChEIs are not always effective and their role may be more appropriate as an adjunctive rather than a primary treatment modality [8,10,11].

As mentioned above, the outcome of mice treated is related with the composition of the venoms, especially by the type of neurotoxins. The addition of N+A might have been expected to yield good results in venoms with higher proportions of α-neurotoxins, such as M. surinamensis (95.4%) [60] and M. altirostris (79.5%) [19]. In the case of M. altirostris the benefits were clear but in the case of M. surinamensis the results may be possibly related to the differential characteristics of this venom [55,64], the non specificity of the antiven-om as observed in previous studies [26,27] and the difficult in neutralize this venom inclusive with specific antivenom [26]. Nonetheless, the use of N+A at high doses improves the effectiveness of the AV against this venom.

However, despite the presence of 3-FTxs in M. nigrocinctus venom (38%) [31] and in M. fulvius venom (24.9%) [44,66], the improvement was lower than that observed on M. pyrrhocryptus venom, with 27%. The limited or absent effectiveness of the combination AV plus N+A against envenomations by M. fulvius and M. nigrocinctus at the doses tested may be due to the presence of myotoxic and neurotoxic PLA₂s in M. fulvius [44,66], over 58% of the venom mass, and myotoxic PLA₂s representing 48% of the venom mass in M. nigrocinctus venom [25,35,44,57,66] (Appendix 7).

In experimental envenomations using M. fulvius venom, good neutralization has been observed using PLA₂ inhibitors [39,40], which reinforces the important role of β-neurotoxicity in this envenomation. On the other hand, despite the high concentration of PLA₂ in the venom of M. nigrocinctus, its β-neurotoxicity does not appear to play a significant role in envenomation [31], unlike its myotoxicity [25,31,57]. The venom of M. pyrrhocryptus has low experimental myotoxicity [12,25], activity not observed in M. surinamensis and M. altirostris venoms [25]. Since N+A does not counteract these toxic mechanisms, this may explain the results obtained in these envenomations in the present study.

In Brazil, the use of AChEIs is suggested as an ancillary intervention when AV obtaining is un-available, delayed, or insufficient, or in patients who do not improve after receiving the highest recommended doses of AV, or who experience delayed recovery or even recurrent systemic neurotoxic manifestations [9,10]. In vitro studies have shown that the neuromuscular blockade induced by various Micrurus species, e.g., M. altirostris, M. laticollaris, M. nigrocinctus, M. pyrrhocryptus and M. spixii, involves interactions with post-synaptic nicotinic receptors. However, reversal by N is typically transitory and often minimal or discrete (generally <30%), offering no sub-stantial long-term protection against progressive neuromuscular blockade. Complete reversal of postsynaptic neurotoxicity has only been achieved with the administration of AV [9,10,11]. Magalhães Camargo et al. demonstrated that treatment with N transiently reversed neuromuscular blockade following 50% inhibition induced by M. pyrrhocryptus venom, but complete and irreversible blockade occurred after 60 min. This may explain the limited protection afforded by N+A alone in one dose and the improved survival observed when combining N+A with AV [1,4, 33,38].

In the envenomation by snakes it is very important the rapid instauration of the specific treatment, but in the case of neurotoxic envenomations this is specially important since the best effectiveness is obtained when the AV avoid the union of the toxins to their targets [3,16]. The same occurs with the use of N, which would provide better results in the beginning of the envenomation. In Brazil there were observed good outcomes in the envenomation by Micururus using the combination of AV plus N+A, when applied 2 hours and 5 hours after the bite but, using two doses of N+A [11]. Nevertheless efforts must be directed to the early use of the N+A.

Unfortunately dose-response studies conducted to compare ED₅₀s of the AVs alone or combined with N+A could only be done for M. pyrrhocryptus and M. altirostris venoms. We did not have enough material to determine ED₅₀s for all venoms in dedicated experiments. We hope to obtain sufficient venom and AV in the future to conduct further studies.

5. Conclusions

In this study, we observed improved outcomes when combined N+A with AV, being the AV used non species specific for 4 of 5 of the venoms studied. For the three South American coral snake venoms tested, this combination allowed the use of lower AV doses compared to AV alone. In these scenarios, the combination may prove useful when increasing AV dosage is not feasible or when other complementary measures, such as respiratory assistance [11], are unavailable.

We did not observe positive outcomes in the cases of M. nigrocinctus and M. fulvius envenomations. However, it should be noted that the AV used was neither specific nor closely related to these venoms, and previous studies have shown low protection with this AV against these venoms [26,27]. Further studies on the combination AV plus N+A (or other AChEIs) using specific or more closely related AVs, or higher doses of paraspecific AVs, may yield more conclusive results on the potential benefit of using AChEIs in these envenomations.

The combination of AChEIs with AVs, and potentially with PLA₂ inhibitors, should be explored further as a strategy to improve existing treatments or to provide alternative therapeutic options for these rare but very serious envenomations, particularly in regions where AV may be scarce or unavailable.

Our findings agree with several observations on the usefulness of the combination N+A in some Elapidae envenomations and although experimental, show the potential help of the combination of N+A plus antivenom in this type of envenomations. Although our study, made in mice, not necessarily can be extrapolated for the therapeutic in humans, the results strongly provide important information on the use of N+A plus antivenom in envenomations by species Micrurus with venoms of different venom composition.

To our knowledge, this is the first in vivo study on the usefulness of the combination of an anti-Micrurus AV with AChEIs in the treatment of envenomation by the southernmost American coral snakes, M. pyrrhocryptus and M. altirostris.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Authors’ contributions

ARdR designed the study, DD, FAMG, MAD and ArdR conducted the fieldwork, ARdR conducted the data analysis, CFD and ARdR supervised the study, ArdR and DD reviewed and edited the final version. All authors read and approved the final manuscript.

Conflicts of interest

The authors declare no conflicts of interest.

Appendixes

Appendix I.

Survival (percentage) of mice challenged with 1.2 MMD of different Micrurus venoms and treated with AV alone or a combination of AV plus N+A

A: M. pyrrhocryptus venom B: M. altirostris venom

C: M. surinamensis venom D: M. nigrocinctus venom E: M. fulvius venom

Bars indicate the median survival for each group of experiments. Each point represents one experiment

Appendix II.

Fisher exact test of the survival of mice challenged with the different venoms and treated with different doses of AV injected alone or in combination with N+A (neostigmine plus atropin).

Appendix III.

A. Survival of mice challenged with M. pyrrhocryptus venom and treated with 50 μL of AV alone or with the addition of N+A. Data from the ED₅₀ studies.

B. Survival of mice challenged with M. pyrrhocryptus venom and treated with 50 μL of AV alone or with the addition of N+A. Data from the ED₅₀ (n=30) studies and from the challenge with fixed doses (n= 50)

Appendix IVa.

Linear regression of the relationship between log transformed AV doses (in μL) and median survival (%) of mice challenged. Solid lines represent the response to AV alone, while the dashed lines represent the response to AV plus N+A. In all cases, the combination of AV with N+A, required lower AV doses than using the AV alone to produce the same effect.

Appendix IVb.

Regression values and p-values for the results presented in 4.a. In the case of M. altirostris and M. surinamensis venoms, the low R2 and p-values are attributable to the high (for M. altirostris) or low (for M. surinamensis) response observed at the AV doses used.

Appendix V.

Non-Linear regression of the relationship between log transformed AV doses (in μL) and median survival (%) of mice challenged. Solid lines represent the response to AV alone, while the dashed lines represent the response to AV plus N+A. In all cases, lower AV doses were required to produce the same effect (Table III).

Appendix VI.

ED50 and T-MED of the AV with or without N+A. In all cases, both the actual values and the theoretical values obtained from the rescue experiments fell within the range of the 95% confidence intervals. ND = not determined

Appendix VII.

Percentage of α-neurotoxins (3-FTx) and phospholipases A₂ (PLA2) of the venoms from Micrurus species used in this study, and their relation. Data of percentage were obtained from proteomic studies (see Discussion).