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Acta Médica Costarricense
versión On-line ISSN 0001-6002versión impresa ISSN 0001-6012
Acta méd. costarric vol.54 no.2 San José abr./jun. 2012
Review
Snake
venoms: from research to treatment
Bruno Lomonte
Instituto
Clodomiro Picado, Facultad de
Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Authors’
affiliation:
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de
Costa Rica.
Abstract
The present work offers a brief overview on how the problem of
snakebite has been confronted in Costa Rica, and especially, on how
scientific and technological research has contributed to this goal.
The origins of the antiophidic struggle in the country and the creation
of the Instituto Clodomiro Picado, at the University of Costa Rica in
1970, are briefly summarized. The first stages of its work are
described, as well as the evolution of different research areas on
snakes, venoms and
antivenoms; which have contributed during four decades to the existence
of adequate and sufficient therapeutic resources to tackle snakebites
in Costa Rica and in the Central American region.
Keywords: snake venoms, toxins,
antivenoms, anti-ophidic sera, ophidism
The
world’s situation with regard to this health
problem is not flattering. Although prompt treatment with antivenom
(anti-ophidic serum) has been known for
over 120
years, there is a serious shortage of antidotes in many countries. The
production
of antivenoms is not commercially
attractive for
large pharmaceutical industries, whose priorities are focused on
medicines with
larger markets and on diseases affecting nations with high incomes. On
the
other hand, the efforts of public institutions that oversee health
policy in
countries located in the regions most affected by snakebite, have not
always
managed to solve the problem of production and supply of antivenoms.6,7
In addition, efficacy of antivenoms is geographically restricted, since
their therapeutic coverage is limited to a group of species of venomous
snakes whose toxins share immunological similarities. Therefore, an
antivenom prepared against a snake speciesfrom a particular
geographical area may have little or no neutralizing efficacy in
another region, due to the antigenic variability of venoms from
different species.
The
scarcity of antivenoms
in some regions of the world has been a reason for concern for decades.
It led
in 2009 to the classification of snakebite by the World Health
Organization
(WHO) in the category of neglected tropical diseases. Nevertheless, the
political and economic support to resolve this problem has not improved
significantly.
In
Scientific
legacy of Dr. Clodomiro Picado,
a pioneer of the fight against snakebite in
Clodomiro
Picado Twight
(1887-1944)
is widely known in
Dr. Roger Bolaños and the creation of the Clodomiro
Picado Institute (CPI)
Roger Bolaños Herrera
(1931-2007) was familiar with the subject of venomous snakes from a
very young
age because his father worked with Clodomiro
Picado. Dr. Bolaños
studied microbiology at the
As a
center for the production of antivenoms,
the CPI is a very particular case because it was created within a
public
university, the University of Costa Rica
(UCR). Due to
the fact that it was embedded in a purely academic environment since
its origin
(in contrast to other health-related public institutions), and thanks
to the
vision and spirit instilled by Dr. Bolaños,
the CPI was never envisaged as a mere immunotherapeutic product
factory. It was
conceived as an academic center that would combine production, intense
scientific
and technological research, teaching of undergraduate and graduate
students and
various social extension activities. All of these tasks would be
closely
related. In particular, the “production-research” pair, clearly
mutually beneficial, became a key factor for the development,
consolidation,
competitiveness and growth of the CPI during its four decades of work
on behalf
of national and regional health. As a manager, but at the same time as
an
intellectual leader and researcher, Dr. Bolaños
had the vision required to assign equal importance and interest to
basic
scientific research (for example, the study of the karyotypes
of the snakes found in Costa Rica),11 and technology
research (for
example, the development of an antivenom
of Panamerican coverage against coral
snakes).12 This
attitude is not common in the general Costa Rican culture (even among
certain
academic sectors), which tends to overvalue the immediate applications
of
research at the expense of work that seeks to answer fundamental
scientific questions.
The influence of the media, which encourages such culture, coupled with
the
discourse promoted by government institutions governing science-related
policy,
constitute elements which could undermine the philosophy of harmonious
balance
between science and technology promoted by Dr. Bolaños
in the various research activities that were increasingly developed at
the CPI.
From
research to treatment: the early stages
The basic
principles of antiophidic
therapy, discovered in the late XIX century, have remained unchanged:
the antivenoms are prepared from the
plasma of animals
immunized with one or more venoms, and which develop an adequate
antibody
response to achieve their neutralization.13 Technologically,
progress has being made in the purification of antibodies out of
complete
plasma, when compared to the rudimentary original methods. However, in
essence,
antivenoms are produced following the same
principle
and they become the physician’s fundamental tool to treat patients who
have been victims of an envenoming snakebite. Therefore, they are an
essential
element in the drug inventory of any health system.
During the
early stages of research Dr. Bolaños and
his small group of collaborators, which
formed the initial staff of CPI, conducted research on several
fundamental
questions. This was necessary to structure the knowledge platform that
would
allow them to address the snakebite problem in a comprehensive manner
in order
to implement the production of the first indigenous antivenoms.
On one hand, they investigated which are the species of venomous snakes
found
in
Two types
of antivenom, the polyvalent and the anticoral, simplify the treatment
In some
regions of the world venomous snakes form complex
antigenic groups, making clinical diagnosis and the choice of
appropriate antivenom difficult. However,
in
The viper
venoms (such as “terciopelo”,
rattlesnake, bocaracá, parrotsnake,
etc.) produce evident local manifestations, which can be highly
visible,
including: edema, pain, hemorrhage, dermonecrosis,
myonecrosis, and blisters.18,19 The
intensity of
these signs depends on the amount of venom injected by the bite, and to
some
extent, the aggressor species, as some venoms are more destructive than
others.
However, edema is an almost omnipresent sign in viper bites.20 At the systemic level, the viper venoms induce
other
manifestations, such as coagulation abnormalities (prolongation of
coagulation
time, fibrinogen consumption), thrombocytopenia, other sites bleeding
and,
bruising. It may occur with significant hypotension and renal failure
in the
most severe cases.21-23 Again, there may be exceptions to
one or
more of these manifestations, depending on the aggressor species. For
example,
some viper venoms lack procoagulant
activity and do
not significantly alter the coagulation parameters.24 However,
in
general terms, the viper-induced clinical condition is recognized based
on the
above mentioned characteristics, without identifying the particular
aggressor
species, in order to provide specific treatment with polyvalent antivenom as soon as possible.
In
contrast, envenoming by coral snakes does not
induce significant local manifestations, except for a feeling of
numbness or
mild pain. It is characterized by symptoms of peripheral neurotoxicity,
together with a progressive neuromuscular blockade, that leads to a
characteristic myasthenic syndrome or
flaccid
paralysis.15,25 Bipalpebral
ptosis is a sign of an advanced stage of
envenoming
and there is a significant risk of respiratory failure. The appropriate
antivenom (anticoral)
should be
given promptly to prevent, to the extent possible, the occupation of
the
acetylcholine receptors at the neuromuscular junction by the potent
neurotoxins
of these venoms, among other pharmacological targets.
In recent
years, the wide availability of both types
of antivenoms in Costa Rica and the
cumulative
experience of hospitals that provide attention to the largest number of
snakebite cases have benefited the treatment protocols through an
increasing
uniformity or standardization26 (Table 2). Intradermal
tests that attempt to predict a possible allergic reaction to equine
proteins
(the species in which antivenoms are
produced in
Costa Rica and in most production centers) have been abandoned
internationally
due to their lack of sufficient predictive value.27 However,
once
envenoming in the patient, either by a vipid
or an
elapid, has been established, the urgent administration of the
respective
treatment is performed with all necessary care, in order to address
potential
immediate adverse reactions. These may relate, albeit rarely, to a true
anaphylactic reaction (hypersensitivity type I, mediated by the
pre-existence
in the patient of IgE antibodies against
equine
proteins), or, more commonly, to an anaphylactoid
type reaction, which does not involve IgE,
but the
activation of the complement system and the direct release by the antivenom of several inflammatory mediators.27
Some
clinical studies that have evaluated the antivenoms
produced in the CPI describe a frequency of immediate adverse reactions
close
to 15-25%, which is acceptable for therapeutic products of this nature.28-30
Unlike the
early adverse reactions to anti-ophidic
serum therapy, delayed reactions are more common.
These are mediated by a Type III mechanism of hypersensitivity (serum
sickness), caused by the abundant formation of immune complexes between
equine
proteins and their respective antibodies, that peaks between the first
and
second week after treatment. The frequency of these reactions is more
difficult
to establish, as in most cases they occur when the patients have
abandoned
treatment centers. This condition is generally very benign and self-
limiting
and responds well to treatment with antihistamines and steroids.27
The antivenoms continue to evolve through research
Polyvalent
and anticoral
antivenoms,31 prepared
in the
CPI since its origins and to date, have been improved over the years
through
the hard research work of academic and technical staff. One of the
major changes
was the substitution of the ammonium sulphate
precipitation technique for immunoglobulins
initially
used, with the use of caprylic acid (octanoic acid) for the removal of albumin and
other plasma
proteins. This method, adapted and optimized for equine immunoglobulins,32 allows to obtain an antivenom with a higher purity and with a
physicochemical
profile above that of ammonium sulphate.
Besides, it
provides a higher final performance and lower processing time. The
current antivenoms compare favorably with
the ones produced decades
ago, and continue to experience a series of gradual improvements, which
are
carefully assessed, in areas such as: potency, safety, stability, lyophilization, content of protein aggregates,
and others.
Apart from the technological improvements introduced in the processing
of hyperimmune equine plasma; changes in
immunization
procedures have also been achieved33, which have resulted in
a
better overall condition of the animals, as well as in the introduction
of
various techniques in accordance with modern practices in manufacturing
and
quality control of the final product.
One of the
current challenges for the improvement of antivenoms,
as described below, is to achieve higher
antibody response to snakebite toxins of major clinical relevance but
with low
immunogenicity. The identification of venom components that are poorly
recognized by antivenoms has advanced
significantly
thanks to the introduction of an analytical strategy known as
antivenomics,34,35 based
on modern proteomics
techniques recently introduced by the CPI.
Research
areas on snakebite
Throughout
its four decades of existence, the CPI has
developed a range of research areas around its central objective: to
contribute
to the solution of the snakebite problem in
(a) Epidemiology
of the snakebite accident: Compilations
and statistical
analyzes are performed on the number, characteristics and consequences
of snakebite
envenoming recorded in the country.36-42 Recently, the
epidemiology
of other envenomings has also been
studied, such as
those by African bees.43 A novel aspect in the field of
snakebite
epidemiology is the application of georeferencing
techniques to construct risk maps and seek the strengthening of the
health
facilities with higher incidence, as well as the distribution and
rational use
of the valuable therapeutic resources embodied in antivenoms.
This type of analysis, already undertaken by countries such as
(b) Herpetological
studies and of natural history of snakes: Analysis
are carried out about the national herpetofauna,
its
distribution, ecology, reproductive biology, ontogenetic shape changes,
taxonomy, improved survival in captivity, venom production, and others.46-60
These biological studies are being extended to the main scorpion
species
found in Costa Rica, aiming at the eventual development of an antivenom.
(c) Studies on
clinical and immune response in equines immunized for the production of
antivenom: Progress
has been achieved
on the optimization of the process of equine immunization with snake
venom,
which has led to a reduction in the dose (with the consequent decrease
in the
lesions induced by the venoms) and a rational design of bleeding
schemes to
obtain the hyperimmune plasma as the raw
material of
the process of antivenom development.
These studies
have been based on clinical, haematological,
serological and blood chemistry parameters of equines.33,61-64
At the same time, research is conducted on the effect of new
immunological adjuvants,65 of options such as immunization
with DNA
encoding for toxins66 and of venom combinations on the
equine
antibody response.67 They are aimed at obtaining higher
levels of
antibodies and a more complete coverage of the various antigens
(toxins) in the
venoms.
(d) Studies on
plasma processing techniques and improvement of the final
characteristics of antivenoms: Research
is being conducted
on new techniques of immunoglobulin purification at an industrial scale
and on
alternative ways to process and stabilize these proteins, either in a
solution
or lyophilised.68-71 Also, basic studies are conducted in
this area
aimed at understanding the mechanisms leading to the development of
early
adverse reactions in a percentage of patients treated with antivenoms.72-75
(e) Studies on
quality control and preclinical assessment of the neutralizing capacity
of antivenoms: Controlling
an immunobiological product for
therapeutic use, such as antivenoms, is
complex. Thus, it requires careful
standardization, as well as a preclinical evaluation on animal models,
using
techniques that reliably predict subsequent clinical performance.
Within this
area of research, 76-81 work has been done on the
comparative
properties of antivenoms prepared as whole
IgG (undigested) or their proteolytic
fragments F(ab’)2
and Fab, on animal models.82 The
antivenoms are produced in the CPI under
the first modality
(IgG), since research to date does not show
a
superiority in the use of fragments F(ab’)2
or Fab.82
(f)
Clinical
research studies on antivenoms: During
the
last two decades some clinical trials have been developed to assess
performance
and other characteristics of the antivenoms
produced
in the CPI in patients who have suffered from snakebite envenoming.
Such
studies have been conducted mainly in collaboration with clinical
experts in
Colombia and Nigeria.28-30, 83,84 Although to date clinical
trials
with antivenoms have not been performed in
Costa Rica
(partly due to the unclear regulatory situation that prevails), it
would be
ideal for addressing important questions in the field of serotherapy.
(g)
Studies on the composition of venoms: The complex protein
formation in
venoms of snakes found in
(h)
Isolation, characterization and study of the mechanism of action of
snakebite
toxins: In order to better understand snake venoms and their
pathological
actions in the body, the different toxins are isolated and studied in a
variety
of experimental models that have allowed a deeper understanding of
their
mechanisms of action and of the physiopathology of snakebite envenoming.85,91
(i) Experimental
pathology of snakebite envenoming: The
pathogenesis of the toxic effects in experimental animal models is
researched
by using complete venoms and their isolated components and employing
techniques
of histology, immunohistochemistry, immunofluorescence and inflammatory exudates
proteomics.19,
92.93
(j) Search
for, characterization and assessment of inhibitors with therapeutic
potential
against snake venom: The
possibility of obtaining
purified toxins from the venoms has allowed work on a research area
whose
objective is to find alternative substances to antibodies, whether of a
natural
or synthetic origin, which are also capable of inhibiting their toxic
effects.94-98
These studies have provided some promising compounds as
candidates for an
eventual therapeutic application, which could complement the use of antivenoms. However, this goal depends on the
possibility
of performing controlled clinical trials, once the preclinical research
stage
has been overcome. As mentioned (section f), greater support would be
required
for this purpose.
(k) Development
of new types of antivenoms: Although
still in an early stage, the ICP is undertaking research on the
potential
development of equine antivenoms against
bees and
scorpions. In a first stage, research has been conducted on the
characteristics
of bee envenoming in an experimental model99 and on the
immune
response to their major toxins. Also, the effects of venoms from
scorpions
found in
From
research to treatment: a look at the future
When
viewed in the context of their historical
evolution, the scientific and technological activities carried out in
the CPI
show positive indexes, both in the generation of knowledge (scientific
publications) and in their production aspect (number of antivenom
vials), as shown in Fig.2.
Looking towards the future, in the short and medium
term, and based on indigenous and foreign research, the question to be
asked is
what improvements and changes could be expected in connection with antivenoms, the key therapeutic element to
address the
problem of snakebite. Although it is difficult to predict on issues
involving
scientific and technological aspects, given the sudden and radical
changes that
may occur due to a fundamental discovery or a high impact innovation,
some
suppositions may be outlined. The use of therapeutic biotechnology
products,
such as recombinant human antibodies, or their fragments, such as scFv or others produced in various prokaryotic
or
eukaryotic expression systems, will probably not constitute a viable
option as antivenoms in the short term.
The reasons for this are
related, on the one hand, to the high production costs for the
production of
elements of this nature that fulfill the requirements of an injectable
product at an industrial scale, and on the other, to the complex
composition of
the venoms, which would require the development of a “cocktail” of
recombinant antibodies or fragments. However, the possibility that
antidotes of
this nature can be developed to combat some particular types of
envenoming
should not be ruled out, such as those caused by some snakes of the Elapidae family. This is the case of species
were it is
possible to show that the toxicity of the venom is attributable to one
or two
of the main components. A good candidate for this might be the case of
the redtail coral snake Micrurus mipartitus,
for which there is no antivenom
available in
It is
likely that improvements and changes in antivenoms
resulting from research will be introduced in a
gradual rather than radical way. Some of the issues for which an
evolution in
the short term can be expected are those related to improvements in the
safety
and security of antivenoms, due to the
development of
increasingly efficient methods of processing, purification and final
stabilization of immunoglobulins. At the
same time,
the therapeutic efficacy of the antivenoms
may be
increased through immunization strategies that can overcome the
limitations in
the immunogenicity of some snakebite toxins, which play a key role from
the
clinical standpoint. Also, it is to be expected that the detailed
analyses of
the venomic composition of snakes found in
various
geographical origins and the development of new antigenic formulas on
such
rationales lead to a significant expansion of the antivenoms’
coverage areas. This could contribute to improve the availability of
treatment
in countries or regions where the shortage of antivenoms
has dramatic consequences for the population, which suffers high
mortality and
morbidity indexes. Finally, an introduction to clinical practice of
some
non-immunologic inhibitors that block specific toxins is possible.
However,
this requires a greater interest from the medical community towards
undertaking
controlled clinical studies, despite not having the sponsorship and
support of
large international pharmaceutical companies.
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