Biochemical Pharmacology xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Review

Snake antivenom: Challenges and alternate approaches

Aswathy Alangode, Karthika Rajan, Bipin G. Nair
⁎

School of Biotechnology, Amrita Vishwa Vidyapeetham, Clappana P.O, Kollam, Kerala 690 525, India

ARTICLE INFO ABSTRACT

Keywords: Snake envenomation is still a serious threat to many countries in the world. The only mainstay treatment de-
Antivenom pends on the administration of animal derived immunoglobulin based antivenom. Significant limitations to these
Snake venom antivenoms are a challenge in the treatment of snake envenomation. Many alternate approaches have been
Peptide inhibitors explored to overcome the limitations of antivenom. Exploring alternate approaches like use of bioactive com-
Phage display
ponents from plant sources, use of peptide and small molecule inhibitors are some aspects taken towards im-
proving the current limitations of antivenom therapy. However, all these alternate approaches also have many
drawbacks which should be improved by more in vitro and in vivo experiments. Here, we review some of the
limitations of current antivenom therapy and developments as well as drawbacks of these alternate treatment
strategies.

1. Introduction to the action of different enzymatic and non-enzymatic toxins present in

Snake envenomation is a serious threat to many developed and
developing countries. Injection of venomous toxins from snakes to the
victim evokes a chain of reactions which unless treated will pose a
threat to the life of the victim. Worldwide, there are approximately
81,000 to 138,000 deaths (Fig. 1) per year due to snake envenomation
[1–3]. Major areas affected by snakebite include South Asia, with India
having a statistics of 50,000 deaths and 85,000 snakebites per year [4].
Significant morbidity and mortality due to envenomation is attributed
the venom. Snake venom is composed of both enzymatic toxins like
snake venom metalloproteases (SVMPs), PLA2, L-amino acid oxidases
(LAAO), serine proteases and non-enzymatic components including
disintegrins, three finger toxins, protease inhibitors and many more
[5,6]. These toxins affect different physiological systems of the victims
thereby leading to death (Fig. 2). In addition, morbidity due to en-
venomation can cause many physical defects like amputation, necrosis
and severe local damage [7].

⁎
Corresponding author.
E-mail address: bipin@am.amrita.edu (B.G. Nair).

https://doi.org/10.1016/j.bcp.2020.114135
Received 30 April 2020; Received in revised form 25 June 2020; Accepted 1 July 2020
0006-2952/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Aswathy Alangode, Karthika Rajan and Bipin G. Nair, Biochemical Pharmacology,
https://doi.org/10.1016/j.bcp.2020.114135
A. Alangode, et al. Biochemical Pharmacology xxx (xxxx) xxxx

polyclonal (raised against the venom of different species of snakes) or

Nomenclature
monoclonal (raised against venom of one snake species) and the venom
batches vary significantly between younger and older snakes, geo-
SVMP Snake venom metalloproteainases
graphical area, sex and age of snakes [10]. This variability in venom
PLA2 phospholipase A2
toxicity and activity is of relevance because it leads to batch variability
LAAO L-amino acid oxidases LPS – lipopolysaccharide
in the antivenom. Furthermore, adverse reactions triggered by animal
VICC venom induced consumption coagulopathy
proteins, such as polyvalent horse antibodies, is a major problem of
LMW low molecular weight
antivenom therapy (Table 1) [11,12] which requires time consuming
allergy testing prior to administration of antivenom therapy, in a si-
tuation where time is of concern [13]. Despite these limitations, the
Current treatment strategy for snakebite is the administration of
current treatment available in the market owes for conventional anti-
antivenom, which are antibodies obtained after immunising animals
venoms that can neutralize the toxins [8,14].
like horses with sub lethal doses of venom [8,9]. Antivenom can be

Fig. 1. Estimated number of worldwide deaths and snake envenoming. Total global estimated incidence rate is 1.8–2.7 million bites with 81,000–138,000 deaths
annually. Major incidence of snakebite estimated is in south and south-east Asia followed by Africa and South America whereas envenomation is lower in Europe and
North America. Adapted from J.M. Gutierrez et al., 2017. Ref. [3].

2
A. Alangode, et al. Biochemical Pharmacology xxx (xxxx) xxxx

Fig. 2. Diagram showing the physiological effects of different snake venom toxins. Snake venoms exert wide range of pharmacological activities on the physiological
system and cause various effects depending on the toxins present in the venom like snake venom metalloproteases (SVMPs), snake venom serine proteases (SVSPs),
phospholipase A2 (PLA2), three finger toxins (3FTX), disintegrins and many more. Viperid and elapids affect different proteins of the coagulation cascade leading to
consumption coagulopathy and can also induce neuromuscular paralysis. Systemic haemorrhage leading to cardiovascular shock is the major effect caused by
viperids. Other effects of envenomations include myotoxicity, local tissue damage and acute kidney injury. Adapted from J.M. Gutierrez et al., 2017. Ref. [3].

Table 1
Adverse reactions of antivenom.
Adverse reactions of antivenom

1. Early IgE mediated anaphylactic reactions 1. Type 1 hypersensitivity reaction [20].

2. IgE is produced against antivenom components due to previous exposure to animal immunoglobulins [16].
3. Mechanism involves mast cell degranulation and release of histamine, prostaglandin and leukotrienes [20].
4. Results in increased vascular permeability, vasodilation, inflammation, smooth muscle contraction, mucuos secretion, local
inflammation, anaphylactic shock and edema [16,20].
Non-IgE mediated anaphylactic 1. Occur in patients who have not been previously exposed to antivenom components [16].
reactions 2. Presence of immunoglobulin aggregates against blood cells in antivenom [20].
3. Complement activation by aggregates results in mast cell degranulation [16].
4. Results in increased vascular permeability, vasodilation, inflammation, rash, urticaria and pain [20].
Pyrogenic 1. Induced by bacterial endotoxins present in antivenom [16].
2. Mechanism involves activation of macrophages by endotoxin and results in production of cytokines such as TNF-alpha, IL-
1β,IL-6 [20].
3. Results in fever, myalgia, headache, rigors, nausea, sweating, chills, increase in heart rate and vasodilation [11]
2. Late Antigen-Antibody mediated 1. Type III hypersensitivity reaction [17].
2. Also called as serum sickness [17].
3. Mechanism involves complement system activation by immune-complexes and leukocyte infiltration [20].
4. Results in mast cell degranulation and release of histamine, prostaglandin, serotonin and leukotrienes [20].
5. Results in fever, myalgia, arthralgia, utricaria, lymphadenopathy and gastrointestinal disorders [16].
6. Rarely develops rash, glomerulonephritis, laryngeal edema, and vasculitis [17]

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Adverse reactions due to antivenom are classified by WHO as - early
(anaphylactic reactions and pyrogenic reactions) which occur within 24
hrs of antivenom administration and late reactions (serum sickness)
which develop between 5 and 24 days of treatment [15]. Anaphylactic
reactions occur within the first hour of antivenom administration to the
patient and are due to foreign nature of the immunoglobulins in anti-
venom. Pyrogens are substances that induce fever. These substances can
most likely be due to the presence of bacterial components in the an-
tivenom preparation. Due to the presence of bacterial components like
LPS, pyrogenic reactions occur within an hour after antivenom ad-
ministration. Pyrogenic reactions are usually characterized by fever,
chills, headache, nausea, sweating and increase in heart rate [16]. In
the case of late adverse reactions, a delayed immune response is in-
duced where there is an increase in IgG mediated antibody response.
This corresponds to type III hypersensitivity reaction due to the pre-
sence of circulating antigen-antibody or immune complexes. Moreover,
this leads to complement activation and triggers inflammatory reaction
due to the presence of these complexes in various tissues, joints and
lymph nodes. Clinical manifestations include rash, fever, malaise, ar-
thralgia and arthritis [17]. Despite these adverse reactions antivenom is
still the current mainstay treatment for snakebite.
Adverse reactions to antivenom are commonly treated by adminis-
tration of adrenaline [18], antihistamine and hydrocortisone before the
administration of antivenom [19]. The drugs that reduce these adverse
reactions are reviewed in detail elsewhere [20], but its efficacy were
questioned [21]. In snake envenomation involving coagulopathy, the
administration of adrenaline may increase the risk of intracerebral
hemorrhgae [22]. In contrast, a report by Tibballs mentions that pa-
tients had very low intra cerebral hemorrhage after snakebite irre-
spective of whether adrenaline was administered or not [18]. But this
study involved a limited number of patients and hence not conclusive.
Whereas another study by De silva et al. demonstrated that low dose of
subcutaneous adrenaline were safe and limit the risk of adverse reac-
tions by 43% in 1hr and 38% upto 48 hr after antivenom infusion,
however promethazine and hydrocortisone did not have any effect at 1
or 48 h [19]. Moreover, a meta-analysis conducted based on three
randomized and four non-randomized control studies with pre-medi-
cation and control groups found that adrenaline pre-medication might
have a beneficial effect against early adverse reactions (EAR) to
antivenom [23]. Hence, adrenaline is the treatment of choice for ad-
verse reactions of antivenom.
In addition to these adverse reactions, antivenoms do not always be
effective against various complications related to snakebite. For in-
stance, envenomation due to viper and elapid bite leads to venom in-
duced consumption coagulopathy (VICC) which is the major outcome of
envenomation by these species [24,25] and administration of anti-
venom may not be effective in treating VICC since it is irreversible
[26–28]. This could be due to the rapid absorption of venom resulting
in faster onset of VICC [29]. Whereas studies have shown that some
antivenoms are effective against VICC induced by Echis envenomations
[30] however other studies show it is less effective against coagulo-
pathy and displays less cross reactivity towards by Echis envenomations
[31]. For an effective evaluation of these effects, randomized placebo
controlled trials are required which is challenging and considered as
unethical [32,33]. In addition, many studies have shown that anti-
venoms show cross reactivity against many species but some variations
in specificity and efficacy can be observed [34–36]. Such variations in
efficacy could be due to minor changes in the structure or surface
chemistry of the toxins.
Furthermore, there are situations where all the venom components
are not targeted by the antivenom. For instance, many studies including
data from our lab [37] have shown that there are antivenoms which do
not recognize the low molecular weight (LMW) protein components of
the venom compared to the higher molecular weight toxins which play
significant role in envenomation [38–40]. Also, differential binding of
antivenom to toxins present in different species of snakes are observed.
Studies have shown that in some species, fibrinogen cleavage was due
to the action of venom serine proteases and in other species by me-
talloproteases. Hence, antivenom generated against species with serine
protease enzymes may not effectively neutralize the metalloprotease
species [41].
To overcome these limitations of antivenom therapy (Table 2), there
is an urgent need to develop new approaches which will serve the
function of antivenom and will provide minimum adverse reactions. A
more detailed understanding about current antivenom therapies and
their safety as well as effectiveness have been covered elsewhere
[42–44].

Table 2
Limitations of antivenom.
Type of antivenom Drawbacks

Polyvalent antivenom animal derived 1. Batch variability [44].

2. Adverse reactions like severe hypersensitivity reactions occur due to immune response generated against proteins in the
serum of immunized animals [11,16,20].
3. Less effective against venom-induced local effects [44].
4. Containing large proportion of irrelevant antibodies
compared to toxin neutralising antibodies [40,44].
5. Complex manufacturing processes that depend on two biological systems (snake and the immunized animal) [44].
6. Antibody molecule has poor distribution to peripheral sites, but a longer elimination half-life [44,99].
7. Not effective in neutralising irreversible symptoms like VICC, pre-synaptic neurotoxicity, cardiovascular collapse and local
necrosis [44].
8. Geographical variation owing to venom collected from different geographical regions, diet and age of snakes does show
variation in venom composition thereby leads to variation in antivenom [10].
Monoclonal 1. Have all limitations of polyclonal antivenom.
2. Targets single toxins (monoclonal), and are not able to neutralize the large number of different toxins in snake venom
[44].
Recombinant antibody fragments (including Fab 1. They have a shorter serum half-life in vivo [44].
and Fab')2

4
A. Alangode, et al.
Fig. 3. Schematic representation of different steps involved in peptide phage
display technology. A synthetic random peptide phage display libraries are
screened against toxin components of snake venom for binding. These bound
peptides are selected and enriched through multiple panning steps. Enriched
peptides are expressed in bacterial host cells. The recombinant peptide obtained
is optimized and used in assays to confirm peptide-venom binding and neu-
tralization. Adapted from E.C. Roncolato et al., 2015. Ref. [96].
Although this review does not exhaustively discuss all the chal-
lenges related to antivenom and alternate treatment modalities for
snake envenomation, we hope to offer few important aspects related to
the advantages and drawbacks of current treatments and the ad-
vantages of developing peptide inhibitors as an alternate to antivenom
therapy and small molecule inhibitors for complications related to
snake envenomation.
2. Need for a better antivenom and alternate therapies
2.1. Natural products
Many studies have been done to evaluate the effects of natural
products for snake envenomation. Traditional medicines derived from
plant sources could provide a promising lead towards venom neu-
tralization [45]. As India is rich in biodiversity with varied plant species
of which medicinally important plants can serve to be good candidate
for effective snakebite treatment [46]. Literature surveys have shown
that several active natural products obtained from plant species like
Aristolochia indica [47,48], Withania somnifera [37,49], Azadirachta in-
dica extract [50] have been successful in reducing inflammation, local
tissue damage and severe pain at the bite site. The active components
present in these plant species acts on particular enzymes present in
venom and neutralize their effects. One such enzyme is snake venom
phospholipase A2.
Several databases are published which contains bioactive molecules
derived from natural products that show activity against snake venom
components [48]. Even though a lot of research is successful in isolating
new bioactive compounds, the toxicity profile of the active components
5
Biochemical Pharmacology xxx (xxxx) xxxx
isolated from plant sources are not validated and time spent in devel-
oping and purifying these components is a drawback [48].
2.2. Peptide inhibitors
The concept of using peptides specifically to bind and neutralize
major toxins present in snake venom is a significant thought towards
achieving a peptide based antivenom therapy. Use of peptide inhibitors
seems to be advantageous as this could be specific to toxin components
present in venom [51]. Studies published shows two classes of in-
hibitors, where one class belongs to endogenous inhibitors which are
inhibitors found in the blood circulation of venomous snakes [43,52].
These inhibitors provides resistance to their own venoms [53]. Most of
the Viperidae family of snakes has metalloproteinases and phospholi-
pase A2 enzymes in their venom in abundance. SVMPs are proteolytic
enzymes that cause local tissue damage and haemorrhage at bite site
[54,55]. PLA2 are responsible for inflammation and severe pain during
envenomation [53,56]. Peptide Inhibitors that can bind with high af-
finity to these toxins and effectively neutralize the activity of these
enzymes can be of much advantage.
Searching for mechanisms involved in natural resistance shown by
snake venoms is a promising lead. Several endogenous phospholipase
and metalloprotease inhibitors are secreted for self-protection of the
snake from its own venom [57–61]. Characterization of these natural
inhibitor mechanisms through proteomics and transcriptomic studies
can provide a deeper understanding of their interactions with the
venom components [62].
Exploring the immunity to snakebite shown by certain animals like
the opossum forms another class of inhibitors based on the natural
predators of snakes. Work published in this regard by Rocha et al.,
found that certain proteins isolated from the serum of opossum species
could neutralize venom from certain snakes [63]. A short peptide called
LTNF peptide (Lethal Toxin Neutralizing Factor) has been purified from
the sera of Didelphis virginiana which inhibited lethality of venom from
Crotalidae, Elapidae, Viperidae and Hydrophiidae [64]. Further studies
lead by Komives et al., has expressed these peptides in E. coli as re-
combinant peptides and were able to completely neutralize C. atrox and
Russell's viper venom [65]. Such recombinant expression in lower
prokaryotes yields much cheaper and good yield of peptides that can be
further used in several studies to inhibit snake venom toxins. Never-
theless, optimization of expression parameters for maximum yield is
one of the limitations regarding such recombinant proteins.
2.3. Small molecule inhibitors
2.3.1. PLA2 inhibitor
As discussed above, low immunogenicity of certain small toxins in
the venom like svPLA2 could be overcome by using small molecule
inhibitors. In this regard, use of varespladib is highly recommended by
many researchers. Varespladib is a potent inhibitor of secretory PLA2
and was used to treat acute coronary syndrome [66]. Since svPLA2 is a
major toxin in the venoms of many snake species, studies showing the
use of varespladib to inhibit svPLA2 toxicity have been shown. For in-
stance, M. fulvius due to the presence of PLA2, manifest neuromuscular
paralysis, intravascular hemolysis and myonecrosis in animals [67–69].
Lewin et al. demonstated that varespladib and orally bioavailable me-
thyl varespladib rescued pigs that were experimentally injected with
the M. fulvius venom [70]. Another study shows that the IC50 of var-
espladib and methyl varespladib for Russell’s viper venom is 0.0006
and 0.02uM respectively. Both showed high inhibitory activity (low
IC50) towards svPLA2 from 28 different snakes from 6 continents [71].
Similarly, varespladib was able to inhibit the PLA2 induced antic-
oagulant activity of African spitting cobras [72]. Haemorrhagic toxicity
of D. acutus and G. Halys were significantly inhibited by varespladib
[73]. Gutierrez showed that varespladib inhibits myotoxicity and cy-
totoxicity exhibited by the PLA2, MjTX II, present in venom of Bothrops
A. Alangode, et al.
moojeni venom using in vivo and in vitro experiments in mice, respec-
tively. Both structural and functional studies of the complex formed by
the venom and the inhibitor have been studied [74]. Mice injected with
O. scutellatus, B. multicinctus, and C. d. terrificus venom were able to
inhibit the neurotic effects by these venom in the presence of var-
espladib and its orally available drug methyl varespladib [75]. In ad-
dition, a study by Zdenek et al. found that coagulotoxic effects of 9
pseudechis species from Australia, due to PLA2 toxins (except P. por-
phyriacus) inhibitory activity towards FVa and or prothrombinase
complex were not neutralised by both BSAV and TSAV, but varespladib
exhibited potent inhibitory activity towards these venoms [76]. A more
comprehensive review has been published on drug development against
svPLA2 for snake envenomation [77].
Eventhough varespladib has shown inhibitory effect on PLA2 of
many snake species, it is still not approved by FDA [70]. More studies
with timing of administration and different doses of venom in animal
models are required. Varespladib alone or in combination with other
inhibitors should be tested in animal models for effectiveness against
important snake venoms.
2.3.2. SVMP inhibitors
Venom metalloproteases are abundant in many viperid venoms and
induce various effects such as hemmorhage, and local damages in-
cluding necrosis and edema [78]. Bothrops asper is a pit viper which is
prevalent in Central America and southern Mexico and induce hem-
morhage, myonecrosis and edema [79–82]. Often these local damages
cannot be prevented by the administration of antivenom. In this regard,
effect of the MMPIs batimastat and marimastat have been studied.
Batimastat has been shown to inhibit local haemorrhage, dermonecrosis
and hemorrhagic effect in lungs due to Bothrops asper envenomation
[83–85]. Similarly, the effect of matrix metalloproteases inhibitors
(MMPI) marimastat on SVMPs has also been studied [86]. When in-
cubated with venom, both inhibited the lethal and hemorrhagic effects
induced by E. ocellatus venom [87].
These studies show that small molecule inhibitors are effective
against local damages caused by the action of different toxins during
envenomation. These small molecule inhibitors could thus improve the
treatment strategy by their administration even before the adminis-
tration of antivenom to minimise the effects of local tissue damages
which are not usually inhibited by antivenom.
3. Phage display library of peptides and antibodies
Research studies report the use of phage display technique in de-
veloping short peptides that are known to bind snake venom toxins.
Snake venom contains a variety of peptide components that can be
isolated and purified [3]. Newer in vitro selection of antibody for use as
antivenom was developed using antibody phage display technology by
applying the filamentous fusion phage [88,89].
An antibody phage display library development involves fusing the
single chain variable fragments (ScFv's) of antibody domain with the
phage coat protein gene III and thus the phage coat protein display
these domains on the surface of phage. When an appropriate antigen
binding site is screened against this antibody library, high affinity ones
can be selected and enriched [51,90]. Besides these conventional an-
tibody libraries, antibody phage display with multivalent and multi-
specific constructs has also been developed [91]. Further studies have
developed monoclonal antibody fragments specific to more than one
toxin in snake venom, thereby providing one monoclonal antibody
targeted against two or more venom toxins [92–94]. A common pro-
blem related to antibodies are it's half-life and stability issues which
leads to dosage irregularities, as the required dose may not reach the
target and thus gets eliminated in the renal system [95,96]. In this re-
gard, a phage display library which contains random synthetic peptides
incorporated into the phage coat proteins, is used to screen for binding
partners against snake venom toxins [97]. These peptides are then
6
Biochemical Pharmacology xxx (xxxx) xxxx
synthesized chemically and used in neutralization assays to check for
venom enzyme neutralization. This involves selection of a particular
venom component or target against which we need to develop peptide
inhibitors, then fractionation of the crude venom to obtain our com-
ponent of interest, and a method to detect the target and finally purify it
to use it as bait for phage display (Fig. 3).
As compared against recently available phage antibody library,
which invokes several disadvantages like the size of the antibody or its
fragments, low immunogenicity of fragments, cost of development,
stability issues [51,88] and less bioavailability, short peptides with
modified structural properties as lead molecules can be of great ad-
vantage. A major concern is the cost of synthesizing the lead peptides
that needs to be addressed. One way it can be surpassed is by producing
it in larger yields in E. coli systems. But more studies are required for
recombinant peptide expression in E. coli cells.
4. Combinatorial therapy
Eventhough, antivenom is the main treatment for systemic effects of
envenomation it is ineffective in treating local damage due to en-
venomation [98,99]. A combination of antivenom along with inhibitors
to different toxins causing local tissue damage may be effective against
completely inhibiting the effects of envenomation. In this regard, work
done by Howes et al. shows that matrix metalloprotease inhibitor
AG3340 inhibits the hemorrhagic activity of whole E. ocelatus venom
[86]. But more studies are required for evaluating the efficacy of drugs
combined with antivenom, its dosages and route of administration.
5. Conclusions
Current antivenom therapy poses many disadvantages like lack of
specificity, hypersensitivity or adverse reactions to animal proteins in-
volved in antivenom production, owing to which alternative therapy for
snake envenomation is of great importance. Exploring varied variety of
available natural products is one among them, wherein bioactive mo-
lecules from plant extracts are used for treating snakebites or to neu-
tralize venom components. Lack of knowledge about active components
of the extracts and effective analysis procedures for active components
are its drawbacks. Another area focuses on developing antibody library
by phage display technique and modifying existing limitations of an-
tibody derived phage library by introducing monoclonal antibodies that
can cross react with many snake venom proteins. Major concerns about
antibody library including the high cost of production, low half -life
stability, low immunogenicity along with developmental issues stand as
a hindrance. In this regard, short peptides that can be generated against
specific venom toxins can be advantageous. Several known peptide
inhibitors are identified against snake venom components but to pro-
duce them in a way that is less expensive and less time consuming is
important. Use of peptide phage display library will be a significant
thought in that direction. Furthermore, the peptides that show affinity
towards venom proteins can be amplified and enriched in E. coli cells
thereby achieving a good yield for further studies. Optimization of
expression conditions and host bacteria environments can further lead
to optimum production in a smaller time frame and cheap production
cost. Similarly, the use of small molecule inhibitors have shown to be
effective against local damages due to envenomation. Developing drugs
using small molecule inhibitors could be of great advantage in the
treatment of snake envenomation. In this regard, multidisciplinary
approaches are required to improve current antivenom therapy.
CRediT authorship contribution statement
Aswathy Alangode: Writing - original draft. Karthika Rajan:
Writing - original draft. Bipin G. Nair: Writing - review & editing.
A. Alangode, et al.
Acknowledgements
We acknowledge Mata Amritanandamayi Devi, Chancellor, Amrita
Vishwa Vidyapeetham for being the inspiration behind the work. We
would like to thank Dr. Jyotsna Nambiar for her help with the manu-
script preparation. Ms. Aswathy Alangode was supported by
Department of Biotechnology, Govt of India (DBT) for JRF/SRF fel-
lowship DBT-JRF/2010-11/119 and Ms. Karthika Rajan is supported by
JRF/SRF fellowship 19/06/2016(i)EU-V from University Grants
Commission. The authors are also thankful to the Department of
Science and Technology (DST), India for their research grant SR/SO/
BB-29/2010. The work was also supported by Institutional funding
(Amrita University Research) grant (BGN).
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