Download Report

Basic & Clinical Pharmacology & Toxicology
Doi: 10.1111/bcpt.12398
The Effect of a Polyvalent Antivenom on the Serum Venom
Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom
in Experimentally Envenomed Rabbits
Michelle Khai Khun Yap1, Nget Hong Tan1, Si Mui Sim2, Shin Yee Fung1 and Choo Hock Tan2
CENAR and Department of Molecular Medicine, University of Malaya, Kuala Lumpur, Malaysia and 2Department of Pharmacology, Faculty of
Medicine, University of Malaya, Kuala Lumpur, Malaysia
1
(Received 18 November 2014; Accepted 20 March 2015)
Abstract: The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient
knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this
study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting
cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV,
F(ab0 )2] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence
an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab0 )2
and venom antigens, as the terminal half-life and volume of distribution of the F(ab0 )2 in serum were comparable to that of
venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen
level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an
adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity.
The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses
as clinically indicated in the presence of rebound antigenemia and symptom recurrence.
Venomous snakebite is an important yet neglected public
health threat, especially in tropical and subtropical countries
where the majority of snakebite victims comprises agricultural
workers [1]. It can lead to local and systemic manifestation of
venom toxicity, a condition called envenomation. Snakebite
cases are known to be under-reported worldwide. Nevertheless, it has been estimated that envenomation incidence may
soar as high as 1,800,000 with 94,000 deaths yearly [2]. The
management for snakebite envenomation faces various challenges, one of which is pertaining to the availability of effective antivenom and the optimization of its use.
Antivenom remains to date the only definitive treatment for
envenomation [3,4]. The sustainability of antivenom supply
has been great challenge worldwide with financial constrain
being cited as the major reasons especially in developing
countries [5]. Therefore, the existing therapeutic protocols for
antivenoms should be further optimized on the basis of pharmacokinetics of venoms/toxins, and how antivenom could
alter the clinicopharmacokinetic profile of venoms/toxins,
which is essential for better tailoring of antivenom dosages.
In this context, changes of venom pharmacokinetics induced
by antivenoms should be part of pre-clinical assessment to
study the in vivo time-based neutralization profile of antivenoms in animal models. However, there are only few studies
Author for correspondence: Dr. Michelle Khai Khun Yap, Department
of Molecular Medicine, Faculty of Medicine, University of Malaya,
50603 Kuala Lumpur, Malaysia (fax +603 79674957, e-mail
myapkk1988@gmail.com).
that addressed the effects of antivenom on the pharmacokinetics of venoms in experimentally envenomed animals, limited
to only certain types of venom and antivenom. In the case of
Vipera aspis venom, Riviere et al. [6] reported that the F(ab0 )2
antivenom given at the right dose and via the appropriate
route of administration induced a complete and durable depletion of plasma venom level in envenomed rabbits, while
Pepin-Covatta et al. [7] showed that an enhanced F(ab0 )2 antivenom redistributed the tissue-deposited venom into blood and
caused a decline in the venom’s terminal half-life in rabbits.
Rocha et al. [8] demonstrated in mice a similar redistribution
phenomenon for Bothrops erythromelas venom. Besides, comparison of different forms of antivenom [IgG, F(ab0 )2, Fab] on
their kinetic behaviours optimal for in vivo neutralization has
also been performed using pharmacokinetic model; for
instance, F(ab0 )2 has been indicated to be the most suitable for
treating envenomation by V. aspis [6] and Walterinnesia
aegyptia [9] because of their unique pharmacokinetic
characteristics.
In Asia, pharmacokinetics of several venoms or toxins have
been reported [10–15], but the modifying effect induced by
antivenom on the venom/toxin pharmacokinetics in vivo has
not been well characterized. One of the medically important
venomous snakes in the South-East Asia is the Javan spitting
cobra (Naja sputatrix). Its pharmacokinetic profile in rabbits
has been characterized in our earlier study, including the findings of intramuscular bioavailability (41.7%) that indicated
extent of absorption of the intramuscularly inoculated venom
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
MICHELLE KHAI KHUN YAP ET AL.
2
into the systemic circulation to cause lethality [13]. The
venom, as with several other Asiatic cobra venoms, has been
recently shown to be effectively neutralized by a regional
polyvalent antivenom, Neuro Polyvalent Antivenom (NPAV)
in mouse lethality assay [16]. Neuro Polyvalent Antivenom is
a newly developed polyvalent F(ab0 )2 antivenom (Queen Saovabha Memorial Institute, Bangkok) derived from horses by
immunization against four common Thai elapid venoms: Naja
kaouthia (Thai monocled cobra), Ophiophagus hannah (king
cobra), Bungarus candidus (Malayan krait) and Bungarus fasciatus (banded krait). In this study, we examined the modifying effect of NPAV on the serum venom levels of
N. sputatrix venom in experimentally envenomed rabbits for
potential therapeutic optimization.
Materials and Methods
Ethics statement. All animals were handled according to CIOMS
(Council for International Organisations of Medical Sciences)
guidelines on animal experimentation [17]. The experimental protocol
on the animal study was approved by the Animal Care and Use
Committee, Faculty of Medicine, University of Malaya (Ethics
clearance number: 2013-06-07/MOL/R/FSY).
Venom,
polyvalent
antivenom,
reagents
and
separation
media. Lyophilized N. sputatrix venom was purchased from Latoxan
(Valence, France). NPAV was a gift from Queen Saovabha Memorial
Institute, Thai Red Cross Society, Thailand. The NPAV (lyophilized;
Batch no: 0030208) is a purified F(ab0 )2 obtained from sera of horses
hyperimmunized against a mixture of the venoms of Naja kaouthia
(Thai monocled cobra), Ophiophagus hannah (king cobra), Bungarus
candidus (Malayan krait) and Bungarus fasciatus (branded krait).
Sephadexâ G-25 gel and Protein A affinity column were purchased
from GE Healthcare (Princeton, New Jersey, USA). Goat anti-rabbit
IgG-horseradish peroxidase (HRP) was obtained from Abcam (Cambridge, UK). Ion-exchange media and all other reagents used in this
study were of analytical grade and purchased from Sigma-Aldrich (St.
Louis, CA, USA).
Animals. The animals used in this study (New Zealand white rabbits,
2 kg; and ICR mice, 18–20 g) were supplied by Chenur Supplier
(Selangor, Malaysia). The animals were housed in Laboratory Animal
Centre, Faculty of Medicine, University of Malaya, and received water
and food ad libitum throughout the experiment.
Preparation of antibodies IgG and IgG-HRP against N. sputatrix
venom. The immunoglobulin G (IgG) against N. sputatrix venom was
produced in rabbits as described earlier [13]. Anti-N. sputatrix IgG
was purified by Sephadexâ G-25 gel filtration chromatography
followed by Protein A affinity chromatography [18]. The IgGhorseradish peroxidase conjugate (IgG-HRP) conjugate was prepared
as described by Wisdom [19].
Determination of serum venom antigen levels using double-sandwich
ELISA. Double-sandwich ELISA was conducted as described
previously [20]. It was used to monitor the serum venom antigen
levels after experimental envenomation in individual rabbits. ELISA
microplates were coated with 100 ll of the anti-N. sputatrix IgG
(4 lg/ml) at 4°C overnight. Plates were then incubated with 100 ll of
diluted serum samples (1:20) collected at different time intervals from
the pharmacokinetic study. This was followed by incubation of 100 ll
of anti-N. sputatrix IgG-HRP conjugate (1:400) for 2 hr, and 100 ll
of the substrate o-phenyldiamine dihydrochloride (0.4 mg/ml) was
then added. The reaction was terminated 1 hr later by adding 50 ll of
12.5% (v/v) sulphuric acid, and the absorbance at 492 nm was
determined using Bio-Rad Model 690 microplate reader. A standard
curve was constructed using pre-envenomed sera spiked with varying
dilutions of the venom.
The effect of NPAV on the pharmacokinetics of N. sputatrix venom in
experimentally envenomed rabbits.
Pharmacokinetics of intramuscularly injected N. sputatrix venom. A
sublethal dose of 1 mg of N. sputatrix venom (0.5 mg/kg) dissolved
in 500 ll normal saline was injected intramuscularly into the hind leg
of rabbits (n = 3). This served as the control group (envenomation
without antivenom treatment). Blood samples were collected from the
central ear artery before and at 5, 30 min., 1, 1.5, 2.5, 4.5, 6.5, 12
and 24 hr after venom injection. The collected blood samples were
centrifuged at 3500 9g for 20 min. to obtain the serum which was
kept at 20°C until further analysis. Serum venom antigen levels
were monitored using the double-sandwich ELISA as described
above.
Pharmacokinetics of N. sputatrix venom in the presence of a single
dose NPAV. A sublethal dose of 1 mg N. sputatrix venom (0.5 mg/
kg) was dissolved in 500 ll normal saline and injected
intramuscularly into the hind leg of rabbits (n = 3). One hour after
venom injection, 4 ml of NPAV was infused into the marginal ear
vein of rabbits over 20 min. Blood samples were collected from the
central ear artery before and at 5, 30 min., 1, 1.5, 2.5, 4.5, 6.5, 12 and
24 hr after experimental envenomation. The collected blood samples
were centrifuged at 3500 9g for 20 min. to obtain the serum which
was kept at 20°C for double-sandwich ELISA.
Pharmacokinetics of N. sputatrix venom after repeated dosing of
NPAV. In the second series of experiment, an additional 2 ml of
NPAV was infused intravenously into experimentally envenomed
rabbits 1 hr after the initial 4-ml infusion. Blood samples were
collected before and at 5, 30 min., 1, 1.33, 1.83, 2.33, 2.5, 4.5, 6.5,
12 and 24 hr after experimental envenomation. The collected blood
samples were processed as described above.
Pharmacokinetic analysis. The pharmacokinetic parameters were
determined using the method of feathering [21]. The area under the
curve (AUC) was calculated from 1 hr (post-experimental
envenomation) to the last experimental time-point by trapezoidal
rule.
The initial phase rate constant (a) and terminal phase rate constant
(b) were determined by the method of feathering using the best fit line
obtained for the initial phase and terminal phase, respectively. The initial phase half-life (T1/2a) and terminal phase half-life (T1/2b) were
determined by the formula T1/2a or T1/2b = 0.693/a or b, respectively.
The distribution rate constants for the transfer between central compartment (designated as 1) and peripheral compartment (designated as
2) were calculated from the equations:
k21 ¼ ðAb þ BaÞ=ðA þ BÞ and k12 ¼ a þ b k21 ðab=k21 Þ:
The other important pharmacokinetic parameters were determined as
follows:
Volume of distribution by area, Vd, area = CL/b.
Clearance, CL = dose (Fi.v. or Fi.m.)/AUC0–∞, where Fi.v. is the
intravenous bioavailability which is 1, while Fi.m. is the intramuscular
bioavailability, hence:
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
ANTIVENOM’S EFFECT ON SERUM VENOM LEVELS
Fi:m: ¼
AUCi:m: Dosei:v:
AUCi:v: Dosei:m:
Determination of the median lethal dose (LD50) of N. sputatrix
venom. The intravenous median lethal dose (LD50 i.v.) of the venom
was determined by injecting appropriate dilutions of the venom into
the caudal vein of mice (n = 4, 18–20 g). The intramuscular median
lethal dose (LD50 i.m.) of the venom was determined by injecting
various amounts of the venom into the thigh muscle of mice (n = 4,
18–20 g). The survival ratio was determined after 24 hr. The
LD50(95% confidence interval) was then calculated by the probit
analysis [22].
Neutralization of N. sputatrix venom by NPAV. Neutralization of
N. sputatrix venom by antivenom was carried out with slight
modification from the method described by Ramos-Cerrillo et al. [23],
where the venom was pre-incubated with the antivenom prior to
injection: N. sputatrix venom (2.5 LD50 i.v.) was pre-incubated with
varying dilutions of NPAV (50–200 ll) for 30 min. at 37°C and the
total injected volume was adjusted to 300 ll. The mixture (300 ll)
was then injected slowly into the caudal vein of mice (n = 4). The
control group consists of mice injected with a mixture (300 ll) of
N. sputatrix venom (2.5 LD50 i.v.) dissolved in normal saline. The
number of mice surviving after 24 hr was recorded. Neutralization
capacity of the antivenom was expressed in terms of median effective
dose, ED50 (ll antivenom/2.5 LD50 i.v.), which is defined as the
amount of antivenom required to neutralize the venom at 50%
survival. ED50 was calculated by the probit method [22].
Another series of neutralization experiment for N. sputatrix venom
was carried out by intramuscular injection of N. sputatrix venom (2.5
LD50 i.m.) into mice (n = 4) at caudal thigh muscle, followed by intravenous injection of varying dilutions of NPAV (50–200 ll) 10 min.
later (experiments without pre-incubation of venom and antivenom).
The number of mice surviving after 24 hr was recorded. The control
group consists of mice (n = 4) injected with the same 2.5 LD50 dose
of N. sputatrix venom, followed by intravenous injection of normal
saline 10 min. later. Median effective dose, ED50 (ll antivenom/2.5
LD50 i.m.), was calculated by the probit method [22]. Neutralization
potency of the antivenom, expressed as mg/ml, or the amount of
venom that is completely neutralized by one unit volume of the reconstituted antivenom, was calculated according to Morais et al. [24].
3
0.82 mg of the venom, neutralized by 1 ml of NPAV, respectively.
The effect of NPAV on the serum venom antigen levels of
N. sputatrix venom.
Fig. 1 shows the serum venom antigen–time profile of the
intramuscularly injected N. sputatrix venom (solid line). Part
of the pharmacokinetic parameters were published in an earlier
report [13] with AUC0-∞ of 6193.59 676.79 ng/ml.hr, T1/2b
of 18.86 5.61 hr, Vd, area of 1.88 0.72 l and CL of
67.89 7.05 ml/hr. Serum venom antigen peaked at approximately 1 hr after injection.
Fig. 1 also shows the serum venom antigen–time profile of
the injected N. sputatrix venom when 4 ml of NPAV was
infused intravenously into the rabbit, over 20 min., 1 hr after
the i.m. injection of the venom. There was a sharp decline in
the venom antigen levels immediately after the infusion of the
antivenom, from a peak 300 ng/ml to 80 ng/ml 1 hr after the
antivenom infusion (fig. 1). This was, however, followed by
transient resurgence of the serum venom antigen level to about
90 ng/ml 3 hr later (i.e. at 4th post-injection of the venom),
and decreased gradually thereafter. As a result, the 4 ml of
NPAV only reduced the AUC1–24 h value of the venom antigens by 41.6% (table 1).
In the second series of experiment, an additional 2 ml of
NPAV was infused intravenously 1 hr after the initial infusion
of 4 ml of NPAV. This managed to reduce the serum venom
antigen levels to <50 ng/ml by 5 hr (fig. 1). This was
followed by a steady decline with the mean antigen levels at
subsequent time-points being significantly lower than controls
and rabbits treated with 4 ml NPAV (p < 0.05). The mean
AUC1–24 h value was significantly reduced by 67.1% compared to controls, and by an extra 25.5% compared to the
group treated with 4 ml NPAV (p < 0.05) (table 1).
Discussion
Statistical analysis. Median lethal dose, LD50, of the venoms and
ED50 of antivenom are expressed as mean with 95% confidence
intervals (CI) and were calculated using the probit method [22]. All
data are reported as the mean S.D. or mean (95% CI). The mean
difference between two independent groups was determined by
Student’s t-test, and one-way ANOVA was used to compare mean
differences between two or more independent groups. The level of
significance was set at p = 0.05. The statistical analysis was
conducted using SPSS 20.0 (SPSS Inc., Chicago, IL, USA).
Results
Neutralization of N. sputatrix venom by NPAV.
The intravenous LD50 and intramuscular LD50 of the N. sputatrix venom in mice were determined to be 0.9 lg/g (0.59–
1.36 lg/g, 95% CI) and 1.12 lg/g (0.62–1.64 lg/g, 95% CI),
respectively. The median effective doses (ED50) of NPAV
were found to be 136.72 ll/2.5 LD50 in experiments with preincubation of venom and antivenom, and 136.68 ll/2.5 LD50
in experiments when venom and antivenom were injected
independently. These values are equivalent to 0.65 mg and
In this study, the level of venom antigen in the serum was
measured as a whole, using the anti- N. sputatrix IgG. Our
earlier study using ELISA and immunoblotting methods has
established that the anti- N. sputatrix IgG reacted mainly with
low molecular weight toxins of the venom, which represent
the bulk of venom proteins (>80%) and also the most important toxins, that is the neurotoxins, cardiotoxins and phospholipase A2 [13]. Thus, the present approach represents a good
approximation of the true picture of the serum kinetics of
N. sputatrix venom.
Neuro Polyvalent Antivenom is a polyvalent antivenom
raised against four Thai elapid venoms, one of which is the
venom of Thai monocellate cobra, N. kaouthia. Our neutralization assays using both inoculation with venom–antivenom
pre-mixture and independent injections of the venom and antivenom into mice confirmed that NPAV could effectively
cross-neutralize N. sputatrix venom, with effective dose and
potency results comparable to those reported by Leong et al.
[16]. Interestingly, although the pre-mixture inoculation
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
MICHELLE KHAI KHUN YAP ET AL.
4
Fig. 1. The effects of Neuro Polyvalent Antivenom (NPAV) on the serum concentration–time profile of Naja sputatrix venom. The serum concentration–time profiles of N. sputatrix venom (in semi-logarithmic plot) of three series of experiments are illustrated. The first series is the control
group (solid line) where only the venom (0.5 mg/kg) was injected intramuscularly into rabbits (control data extracted from Yap et al. [13]). In the
second series of experiment (dash line), 4 ml of NPAV was infused into the marginal ear veins of the rabbits 1 hr after venom injection. In the
third series of experiment (dash dot line), 4 ml of NPAV was infused into the marginal ear veins of the rabbits 1 hr after venom injection, followed
by infusion of another 2 ml of NPAV 1 hr later. Insert: Serum concentration–time profile of N. sputatrix venom (in arithmetic scale) during the
first 2.5 hr, with and without NPAV administration. All data shown are mean S.D. (n = 3).
Table 1.
Area under curve (AUC1–24 h) values with and without Neuro Polyvalent Antivenom (NPAV) antivenom immunotherapy.
Antivenom
immunotherapy
Without NPAV
immunotherapy
Infusion of
4 ml of NPAV
Infusion of
4 + 2 ml of NPAV
AUC1–24 h
(ng/ml.h)
Reduction in
AUC1–24 h value (%)
3409.1 1234.7
–
1991.4 140.5
41.6
1118.9 86.7
67.1
Data are expressed as mean S.D. (n = 3, 2 kg each) for each group.
Reduction in AUC1–24 h value is expressed in percentage (%) of the
AUC1–24 h in the absence of NPAV.
method is generally considered not representative of a
post-envenomation treatment, our study has shown nearly
identical efficacy and potency of NPAV used in neutralizing
the lethal effects of N. sputatrix venom tested on both assays.
The pharmacokinetic study further elucidated how NPAV
affects the serum venom antigen levels in animals. While
venom lethality test (and the corresponding lethality protection
test) was conducted in mice reasonably as permitted by the
institutional animal use ethics, rabbits were used in pharmacokinetic modelling as they are larger animals ensuring sufficient
blood collection from the same animal over a several-day
schedule possible.
Considering the fact that serum venom antigens peaked 1 hr
after venom injection [13], NPAV was administered at that
point of time to observe any noticeable venom level depleting
in the animals. The antivenom NPAV was delivered via intravenous route [4] in contrast to intramuscular for two main reasons: (i) intramuscular injection is associated with incomplete
and slow antivenom absorption that hampers the desired antivenom effect for rapid immunoneutralization; and (ii) intramuscular injection causes intense local tissue reaction and can
be disastrous in envenomation associated with coagulopathy.
In this study, the initial NPAV infusion rapidly reduced the
serum venom antigen levels remarkably ~70% within 1 hr,
indicating the capability of NPAV in rapidly forming immunocomplex with the venom antigens in vivo. The subsequent
gradual and transient resurgence of serum venom antigen levels, however, implied that the antivenom at the given dose
and time induced only partial neutralization of the circulating
venom antigens. In fact, the AUC was only reduced by
41.6%, even though the amount of antivenom injected (4 ml,
with a neutralization potency 0.82 mg/ml, that is able to neutralize 3.28 mg of venom) should be more than sufficient to
neutralize the amount of venom injected (1 mg). This phenomenon of resurgence of snake venom antigen levels in
blood, that is rebound venom antigenemia after antivenom
administration, has been observed in both animal [6,8] and
clinical studies of snakebite treatment [25–29]. The redistribution of tissue-bound antigens into the vascular compartment
following a shift in the intercompartmental equilibrium is
induced by the removal of the intravascular venom antigens
[6,30], while the rebound antigenemia exceeds the limit for
antivenom to bind. This phenomenon is in fact not uncommon
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
ANTIVENOM’S EFFECT ON SERUM VENOM LEVELS
in immunotherapy as the similar rebound antigenemia has
been reported in the treatment for digoxin and colchicine poisoning using Fab [31,32]. Clinically, this can result in recurrent toxicities, for example relapsed neuromuscular paralysis
and haemorrhages in envenomation [25,33,34], worse still if
the seemingly recovering patients are weaned of treatment. As
the monitoring of venom antigenemia is not routinely performed clinically, it is hence of upmost importance to optimize antivenom posology and protocol according to different
venoms and antivenom products, in addition to meticulous
syndrome monitoring. The rebound N. sputatrix antigenemia
was perhaps partially contributed by a small amount of venom
continuously absorbed from injection site within first several
hours. Therefore, it directly resulted in the exhaustion of free
F(ab0 )2 in the blood for immunocomplexation that causes
venom resurgence. The terminal half-lives of other reported F
(ab0 )2 antivenom in rabbit, 55 9 hr [6], 49.52 3.07 hr
[35] and 61.4 7 hr [36], indicated that F(ab0 )2 antivenom is
not eliminated earlier than the venom, hence excluding the
half-life mismatch as the cause for the resurgence for venom
antigen level.
Theoretically, an ideal antivenom possesses the ability to
redistribute tissue-bound antigens into the vascular compartment for the circulating antivenom to bind for elimination
[37]. The rebound N. sputatrix venom antigenemia in the
current study was successfully diminished when a ‘4 + 2 ml’
antivenom infusion regimen was applied. Compared with the
4 ml single dosing, the additional 2 ml of antivenom 1 hr later
was sufficient to neutralize the resurged venom antigens and
to reduce further the subsequent antigenemia to significantly
lower levels, resulting in a significant decrease in the total antigenemia (67.1%, indicated by AUC1–24 h) compared with the
untreated group. The result suggests that NPAV at the given
regimen is able to reduce or reverse the toxic effects of
N. sputatrix venom as serum venom antigen level is known to
correlate with the severity of envenomation [38,39]. Overall,
our results support the use of a high initial dose of NPAV, as
recommended by the manufacturer (10 vials, equivalent to
100 ml) to rapidly induce a sharp decline in venom antigen
levels. The finding of rebound antigenemia also implies that
the use of the antivenom product for N. sputatrix envenomation can potentially induce venom redistribution, and supports
the recommendation of giving repeated antivenom doses to
alleviate recurrent symptoms and to enhance the elimination
of toxins from the body [25,40,41].
As the bioavailability of i.m. injected N. sputatrix venom
was reported to be approximately 41.7% [13], it is thus estimated that when 1 mg of the venom was injected into the rabbit (0.5 mg/kg rabbit), about 0.42 mg of the venom was
absorbed into systemic circulation. However, our results indicated that 4 ml and ‘4 + 2 ml’ dosing regimen over the time
course only managed to reduce the total amount of venom in
the circulation by 41.6% (0.17 mg) and 67.1% (0.28 mg) of
venom, respectively, although according to in vivo neutralization assay (venom and antivenom were injected independently
into mice), the ‘4 + 2 ml’ dosing was capable to neutralize
4.92 mg of venom (as indicated by potency = 0.82 mg/ml).
5
This apparent ‘discrepancy’ in the efficacy of antivenom treatment reflects an important point for antivenom assessment and
its result interpretation: the pharmacokinetic study provides
in vivo evidence of antivenom physically neutralizes
the venom antigens (via immunocomplexation), in contrast to
the neutralization test which instead provides measurement of
the capability of antivenom to prevent the impending death
caused by venom. In fact, the animals survived from neutralization test within the observation period might still have persistent, low but sublethal venom levels in the circulation
which could still be harmful and with possibility of symptom
recurrence. Thus, the neutralization potency of antivenoms
determined in mice can be useful for efficacious comparison
between different antivenoms or against different venoms
[16,42]. However, it is not an absolute indicator of its therapeutic capability to deplete venom antigenemia vis-
a-vis complete neutralization of venom toxicity in animals or human
beings.
Therefore, for an optimal treatment outcome using NPAV
in this study, our pharmacokinetic results suggest the necessity
to administer a far larger amount of antivenom in the case of
human envenomation by N. sputatrix venom than was determined by conventional neutralization assay. Furthermore, in
anticipation of recurrent antigenemia, the patients should be
monitored meticulously after antivenom administration. A
repeated and booster dose of antivenom should be given
where indicated clinically as the patients show worsening systemic signs.
Acknowledgements
The authors thank Queen Saovabha Memorial Institute,
Thai Red Cross Society, for supplying the antivenoms. This
work was funded by RM 282-14APR and RG348-15AFR
from University of Malaya, Kuala Lumpur, Malaysia.
Conflict of Interest
The authors disclose that there are no conflict of interests.
References
1 Gutierrez JM, Theakston RDG, Warrell DA. Confronting the
neglected problem of snake bite envenoming: the need for a global
partnership. PLoS Med 2006;3:727–31.
2 Kasturiratne A, Wickremasinghe AR, de Silva N, Gunawardena
NK, Pathmeswaran A, Premaratna R et al. The global burden of
snakebite: a literature analysis and modelling based on regional
estimates of envenoming and deaths. PLoS Med 2008;5:e218.
3 Warrell DA. Snake bite. Lancet 2010;375:77–88.
4 World Health Organisation. WHO Guidelines for the Production
Control and Regulation of Snake Antivenom Immunoglobulins.
WHO Press, World Health Organisation, Geneva, 2010.
5 Brown NI. Consequences of Neglect: analysis of the Sub – Saharan African snake antivenom market and global context. PLoS
Negl Trop Dis 2012;6:e1670.
6 Riviere G, Choumet V, Audebert F, Sabouraud A, Debray M,
Scherrmann JM et al. Effect of antivenom on venom pharmacokinetics in experimentally envenomed rabbits: toward an optimisation
of antivenom therapy. J Pharmacol Exp Ther 1997;281:1–8.
7 Pepin-Covatta S, Lutsch C, Lang J, Scherrmann JM. Preclinical
assessment of immunoreactivity of a new purified equine F(ab’)2
against European viper venom. J Pharm Sci 1998;87:221–5.
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)
6
MICHELLE KHAI KHUN YAP ET AL.
8 Rocha ML, Valencßa RC, Maia MB, Guarnieri MC, Araujo IC,
Araujo DA. Pharmacokinetics of the venom of Bothrops erythromelas labelled with 131I in mice. Toxicon 2008;53:526–9.
9 Ismail M, Abd-Elsalam MA, Al-Ahaidib MS. Pharmacokinetics of
125
I-labelled Walterinnesia aegyptia venom and its specific antivenins: flash absorption and distribution of the venom and its toxin
versus slow absorption and distribution of IgG, F(ab’)2 and F(ab)
of the antivenin. Toxicon 1998;36:83–114.
10 Maung M-T, Khin M-M, Mi M-K, Thein T. Kinetics of envenomation with Russell’s viper (Vipera russelli) venom and of antivenom use in mice. Toxicon 1988;6:373–8.
11 Sim SM, Saremi K, Tan NH, Fung SY. Pharmacokinetics of
Cryptelytrops purpureomaculatus (mangrove pit viper) venom following intravenous and intramuscular injections in rabbits. Int Immunopharmacol 2013;17:997–1001.
12 Tan CH, Sim SM, Gnanathasan CA, Fung SY, Tan NH. Pharmacokinetics of the Sri Lankan hump-nosed pit viper (Hypnale
hypnale) venom following intravenous and intramuscular injections
of the venom into rabbits. Toxicon 2014;79:37–44.
13 Yap MKK, Tan NH, Sim SM, Fung SY. Toxicokinetics of Naja
sputatrix (Javan spitting cobra) venom following intramuscular and
intravenous administrations of the venom into rabbits. Toxicon
2013;68:18–23.
14 Yap MKK, Tan NH, Sim SM, Fung SY, Tan CH. Pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its
major toxins in experimentally envenomed rabbits. PLoS Negl
Trop Dis 2014;8:e2890.
15 Zhao H, Zheng J, Jiang Z. Pharmacokinetics of thrombin-like
enzyme from venom of Agkistrodon halys ussuriensis Emelianov
determined by ELISA in the rat. Toxicon 2001;39:1821–6.
16 Leong PK, Sim SM, Fung SY, Sumana K, Sitprija V, Tan NH.
Cross neutralization of Afro-Asian Cobra and Asian Krait venoms
by a Thai Polyvalent Snake Antivenom (Neuro Polyvalent Snake
Antivenom). PLoS Negl Trop Dis 2012;6:e1672.
17 Howard-Jones NA. A CIOMS ethical code for animal experimentation. WHO Chron 1995;39:51–6.
18 Hudson L, Hay FC. Practical Immunology. Blackwell Scientific
Publications, Palo Alto, 1980.
19 Wisdom GB. Horseradish peroxidase labelling of IgG antibody. In:
Walker JM, (ed.). The Protein Protocols Handbook. Humana Press,
Totowa, New Jersey, 1996;273–4.
20 Tan NH, Lim KK, Jaafar MI. An investigation into the antigenic
cross-reactivity of Ophiophagus Hannah (king cobra) venom neurotoxin, phospholipase A2, haemorrhagin and L-amino acid oxidase
using enzyme linked immunosorbent assay. Toxicon 1993;31:865–
72.
21 Shargel L, Wu-Pong S, Yu ABC. Applied Biopharmaceutics and
Pharmacokinetics, 6th edn. McGraw Hills, New York, 2012.
22 Finney DJ. Probit Analysis. Cambridge University Press, Cambridge, UK, 1952.
23 Ramos-Cerrillo B, de Roodt AR, Chippaux JP, Olguın L, Casasola
A, Guzman G et al. Characterisation of a new polyvalent antivenom (Antivipmynâ Africa) against African vipers and elapids. Toxicon 2008;52:881–8.
24 Morais V, Ifran S, Berasain P, Massaldi H. Antivenoms: potency
or median effective dose, which to use? J Venom Anim Toxins
Incl Trop Dis 2010;16:191–3.
25 Ariaratnam CA, Sj€ostr€om L, Raziek Z, Kularatne SA, Arachchi
RW, Sheriff MH et al. An open, randomised comparative trial of
two antivenoms for the treatment of envenoming by Sri Lankan
Russell’s viper (Daboia russelii russelii). Trans R Soc Trop Med
Hyg 2001;95:74–80.
26 Warrell DA, Looareesuwan S, Theakston RD, Phillips RE, Chanthavanich P, Viravan C et al. Randomised comparative trial of three
monospecific antivenoms for bites by the Malayan pit viper (Calloselasma rhodostoma) in southern Thailand: clinical and laboratory
correlations. Am J Trop Med Hyg 1986;35:1235–47.
27 Khin OL, Aye AM, Tun P, Theingie N, Min N. Russell’s viper
venom levels in serum of snake bite victims in Burma. Trans R
Soc Trop Med Hyg 1984;78:165–8.
28 Seifert SA, Boyer LV, Dart RC, Porter RS, Sjostrom L. Relationship of venom effects to venom antigen and antivenom serum concentrations in a patient with Crotalus atrox envenomation treated
with a Fab antivenom. Ann Emerg Med 1997;30:49–53.
29 Dart RC, Seifert SA, Boyer LV, Clark RF, Hall E, Mckinney P
et al. A randomised multicentre trial of crotalinae polyvalent
immune Fab (ovine) antivenom for the treatment of Crotaline
snakebite in the United States. Arch Intern Med 2001;161:2030–6.
30 Chippaux JP. Snake Venoms and Envenomations (FW Huchzermeyer, Trans.). Krieger Publishing Company, Malabar, FL, 2006.
31 Smith TW, Haber E, Yeatman L, Butler VP Jr. Reversal of
advanced digoxin intoxication with Fab fragments of digoxin specific antibodies. N Engl J Med 1976;294:797–800.
32 Sabourad AE, Urtizberea M, Cano NJ, Grandgeorge M, Rouzioux
JM, Scherrmann JM. Colchicine-specific Fab fragments alter colchicine disposition in rabbits. J Pharmacol Exp Ther
1992;260:1214–9.
33 Wetzel WW, Christy NP. A king cobra bite in New York City.
Toxicon 1989;27:393–5.
34 Ho M, Silamut K, White NJ, Karbwang J, Looareesuwan S, Phillips RE et al. Pharmacokinetics of three commercial antivenoms in
patients envenomed by the Malayan pit viper, Calloselasma rhodostoma, in Thailand. Am J Trop Med Hyg 1990;42:260–6.
35 Pepin-Covatta S, Lutsch C, Grandgeorge M, Lang J, Scherrmann
JM. Immunoreactivity and pharmacokinetics of horse anti-scorpion
venom F(ab’)2-scorpion venom interactions. Toxicol Appl Pharmacol 1996;141:272–7.
36 Bazin-Redureau M, Pepin S, Hong G, Debray M, Scherrmann JM.
Interspecies scaling of clearance and volume of distribution for horse
antivenom F(ab’)2. Toxicol Appl Pharmacol 1998;150:295–300.
37 Gutierrez JM, Leon G, Lomonte B. Pharmacokinetic-pharmacodynamic relationships of immunoglobulin therapy for envenomation.
Clin Pharmacokinet 2003;42:721–41.
38 Barraviera B, Sartori A, Pereira da Silva MF, Kaneno R,
Peracßoli MTS. Use of an ELISA assay to evaluate venom, antivenom, IgG and IgM human antibody levels in serum and cerebrospinal fluid from patients bitten by Crotalus durissus
terrificus in Brazil. J Venom Anim Toxins Incl Trop Dis
1996;2:14–27.
39 Otero R, Gutierrez JM, Nunez V, Robles A, Estrada R, Segura E
et al. A randomised double blind clinical trial of two antivenoms
in patients bitten by Bothrops atrox in Columbia. Trans R Soc
Trop Med Hyg 1996;90:696–700.
40 Seifert SA. Pharmacokinetic analysis of a crotalid Fab antivenom
and theoretical considerations for the prevention of coagulopathic
recurrence. J Toxicol Clin Toxicol 1998;36:526–7.
41 Warrell DA. Guidelines for the Management of Snake-Bites.
World Health Organisation, Regional Office for South-east Asia,
India, 2010.
42 Leong PK, Tan CH, Sim SM, Fung SY, Sumana K, Sitprija V
et al. Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent antivenom (Hemato Polyvalent Snake
Antivenom). Acta Trop 2014;132:7–14.
© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)