This document summarizes a study comparing a new antivenom produced by ICP for Papuan taipan snake bites to the existing CSL antivenom. In a phase I clinical trial, 18 patients receiving bites from Papuan taipans were randomly assigned to receive either antivenom. Results showed no significant differences in safety or effectiveness between the antivenoms as measured by resolution of coagulopathy and development of neurotoxicity. Based on equivalent performance, the new antivenom was deemed suitable for further evaluation in a larger phase II clinical trial.
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Killer in the Kunai by Dr. David Williams
1.
2.
3.
4. “”The Papuan black snake would appear to be naturally more
aggressive than Slater suggests…”
Charles H. Campbell (1966)
5.
6.
7.
8. • Shy species and retreats quickly if
encountered, relies on bluff as next
line of defence, bites as last resort
• Diet always assumed to be frogs,
leading to long-held belief that the
scarcity of the snake was due to the
impact of the poisonous marine
toad (Bufo marinus)
• Average of 1.2 metres, reaching
maximum of 2.1-2.2 metres
• Until 2006 no live or dead
specimens had been found in
Central Province since 1992
• 6 specimens recorded since then in
25 October 2014 three locations. 8
9.
10.
11.
12.
13.
14. PPoorrtt MMoorreessbbyy
NNaappaa NNaappaa
GGeemmoo IIssllaanndd
Roku Bay
HHaaiiddaannaa IIssllaanndd
FFAAIIRRFFAAXX
HHAARRBBOOUURR
BBooeerraa
PPoorreebbaaddaa
15.
16.
17. “The Papuan black snake … is the
commonest snake encountered in
Papua, bites from this snake being
responsible for more hospital
admissions than any other snake.”
Charles H. Campbell (1967)
18.
19. ““ The commonest poisonous snake in the area
defined would be the Papuan whip-snake
papuensis Demansia psammophis papuensis…” …”
20.
21.
22. • Very common snake in the savanna
grasslands of southern PNG
• Extremely dangerous if approached
• Alert, shy and nervous snake that is
active by day (rarely at dusk)
• Very fast-moving and will retreat
from people if given the opportunity
but may bite multiple times if it is
cornered, stepped on, or handled
• Averages 1.8-2.0 metres, but may
reach a length of 2.6-3.4 metres
• Venom causes bleeding, irreversible
neurotoxicity, myocardial damage
and (occasionally) renal failure
• Without early antivenom more than
67% of patients require intubation.
23. • Papuan taipans are very adaptable
and are common in urban areas, in
plantations, and in village gardens
• Deforestation increases their habitat
• Reproduce annually with up to two
clutches (of 16-22 eggs) possible
• Fast growing: may reach 1.5 metres
in less than 1 year, and attain sexual
maturity in 2-3 years
• Capable of rapid population growth
under favourable conditions
• Expansion of oil palm plantations
into Central Province will increase
the number of bites greatly
• Preventative measures must be
combined with definitive treatment
24.
25.
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30.
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32.
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35.
36. Normal neuromuscular junction in rat soleus muscle prior
to subcutaneous injection with taipoxin
Rat soleus muscle NMJ one hour after subcutaneous
injection with 2 ug taipoxin into the anterolateral aspect
of hind limb.
Please note: Depletion of synaptic vesicles and loss of cristae in damaged mitochondria
(arrows)
From Harris et al (2000)
37. Neuromuscular junction in rat soleus muscle 24 hours
after subcutaneous injection with taipoxin
Neuromuscular junction in rat soleus muscle showing
complete destruction of the nerve terminal
Please note: Damaged mitochondria (heavy arrows) and clathrin-coated Ω-shaped
indentations on nerve terminal membrane
From Harris et al (2000)
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44.
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47.
48.
49. “You’ll never be able to do it, it’s simply too costly, and elapid snake venoms
are not immunogenic enough”
50. • PNG currently relies on expensive
Australian-made antivenoms
• Four land snake antivenoms are
available:
– Polyvalent (any species)
– Taipan (for Oxyuranus scutellatus)
– Death adder for Acanthophis spp.)
– Black Snake (for Pseudechis spp.)
• Costs of all antivenoms have risen
greatly over last 20 years
• Key to successful antivenom therapy is
to administer the appropriate
antivenom in a therapeutic dose as
early as possible
• Current supply is unsustainable so we
set out to develop an alternative
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
1991 1993 1995 1997 1999 2001 2003 2005 2007
Year
Unit price (Kina)
Blacksnake
Death adder
Polyvalent
Taipan
51. • Five key criteria were deemed important in developing a new
taipan antivenom for use in PNG:
– Need for achieve equivalent potency, efficacy and safety (compared to
currently available CSL products).
– High stability under tropical transport and storage conditions, including
increased resistance to biological deterioration or loss of potency with
extended exposure to ambient temperature.
– Final production cost under US$250 per vial.
– Involvement of PNG scientists in the production of venoms, with phased
technology transfer opportunities and infrastructure development that could
eventually enable local antivenom production.
– Compliance with the new WHO Guidelines for the Production, Control and
Regulation of Snake Antivenom Immunoglobulins (2009).
• Our aim was to produce an antivenom ready for assessment
through the WHO Prequalification of Essential Medicines Program.
52. • Serpentarium established at SMHS at
University of PNG in 2005 following
DEC and ethics approval.
• Operation governed by an MoA
between University of PNG and the
University of Melbourne
• Funded by AVRU and UPNG
• Capacity to maintain 30 adult taipans
and up to 50 snakes from other
species.
• Enables collection of venom for
antivenom production and for basic
research at SMHS.
• Local personnel responsible for the
maintenance of the animal collection
53. • Specimens collected in Milne Bay and
Central Provinces
• Maintained in individual terrariums
• Venoms collected only from healthy
adult snakes of both sexes
• Minimum 3 weeks between each
‘milking’
• Venom from 12 snakes was pooled
and frozen at -80C
• Exported to University of Melbourne
for lyophilisation
• Pool of 2 grams supplied to Instituto
Clodomiro Picado for immunization
of the first group of three horses
• Now have own laboratory on-site
54.
55. • Established in 1970 and operated
since 1972 as an Institute within the
Universidad de Costa Rica.
• Focus is on production of antivenom
against venom of Central American
snakes, and basic venom research
relating to public health.
• Produce whole IgG antivenoms from
antibodies raised in horses.
• Recently produced a new low cost
antivenom for West Africa.
• ICP were very interested in using
their technology to attempt to raise
an antivenom to Papuan taipan
venom for use in treating snake bite
in Papua New Guinea.
56. • Three adult horses were immunized
with venom from O. scutellatus.
• Venom injections were administered
subcutaneously.
• Primary immunization was carried
out using Freund’s complete and
incomplete adjuvants.
• Subsequent injections were given
using venom dissolved in saline
solution.
• No evident adverse clinical effects
were observed in the horses.
• Animals were bled once adequate
antibody titres were obtained.
57. Criteria CSL Taipanc ICP Taipan*d
Protein (g/L) 144.6 ± 0.4 45.9 ± 0.9
Phenol (g/L) 2.12 ± 0.03 1.6 ± 0.04
pH 6.4 7.2
Caprylic acid (mg/L) - 31 ± 1
Monomer content (%)a 93.0 ± 0.04 93.0 ± 1.0
Turbidity (NTU)b 23 25
* Specifications meet the relevant recommendations of new WHO Guidelines for Production, Control and Regulation of Snake Antivenom Immunoglobulins
a Monomer content is expressed as the percentage of antivenom protein present in either IgG or F(ab’)2 monomers, as analysed by gel filtration
b Turbidity is expressed as Nephelometric Turbidity Units (see details in Materials and Methods)
c Batch B0548-06301 CSL Expiry: March 2012
d Batch 4511209 ICP Expiry: November 2012
58. The i.p. LD50 of Papuan taipan venom (0.04 μg/16-18 gram mouse = 2.35 μg/kg) is more potent than was
previously thought to be the case (NB: Australian taipans have i.p. LD50 of 9.0 μg/kg).
Effect Measured Venom Activity
ICP IgG taipan antivenom
(Batch 4511209: 40mL)
CSL F(ab’)2 taipan antivenom
(Batch B0548-06301: 33mL)
mg venom/mL
antivenom
Neutralising
Units
mg venom/mL
antivenom
Neutralising
Units
Lethality
(i.p. LD50) 0.04 ± 0.01 μg
4.50
(3.18-6.41)
18,000
5.65
(3.89-8.77)
18,645
Lethality
(i.v. LD50) 0.08 ± 0.01 μg
4.35
(3.05-5.29)
17,400
5.81
(4.08-7.04)
19,173
Coagulant (MCC)
without Ca2+
0.76 ± 0.20
μg/mL
2.43 ± 0.29 0.84 ± 0.04
Coagulant (MCC)
with Ca2+
0.33 ± 0.13
μg/mL
2.37 ± 0.08 0.45 ± 0.17
Myotoxic (MMD) 1 μg 4.0 4.0
PLA2
(μEq/mg/min)
297 ± 7 1.47 ± 0.29 1.10 ± 0.38
63. • We conducted a successful Phase I dose-finding & safety study
involving 18 patients in 2013-2014 and have now commenced a
larger Phase II clinical trial that will involve 86 patients over the next
12-18 months.
• Phase I directly compared the new ICP whole IgG taipan AV with the
current CSL F(ab’)2 taipan AV.
• Minimum requirement for success was equivalent patient survival
and equivalent safety.
• These trials are formally registered with the Australia/New Zealand
Clinical Trials Register (ANZCTR) and all results will be reported
publicly.
• Ethics approvals obtained from UPNG SMHS Human Ethics
Committee, PNG National Medical Research Advisory Committee
(MRAC) and UoM Human Research Ethics Committee (HREC).
• Funding: K250,000 OHE; A$1.4 Million NHMRC Project Grant.
64.
65. • Both antivenoms were relabelled to
blind the researchers, clinicians and
patients to their identities:
– ICP Papuan taipan monovalent
antivenom (Batch #4511109 TALQ);
– bioCSL taipan antivenom (Batch #
0548-06601).
• The first three patients in each
group received a 2 vial dose of
antivenom, while the remaining 6
patients in each group received a 1
vial dose.
66. • Eighteen patients (13M, 5F; mean age=22.4 yrs, range=9-43 yrs)
including 7 children (<16 yrs of age).
• The biting snake was seen by 16 patients and all 18 had a positive
Snake Venom Detection Kit test result indicating the presence of
the taipan venom immunotype
• All 18 patients had a positive 20WBCT and an INR>1.4;
• Only one patient had any evidence of cranial nerve palsy on
presentation (this patient had ptosis on arrival);
• Informed consent was obtained and each patient was randomised
into Group A (Antivenom A) or Group B (Antivenom B) and
received an antivenom dose of either 2 vials or 1 vial according to
a predetermined protocol sequence.
67. • Endpoint 1: evolution of oropharyngeal paralysis:
– No patients reached this endpoint
– 1 patient in each product group developed mild/moderate cranial nerve
palsy (ptosis, ophthalmoplegia) which resolved over 2-3 days.
• Endpoint 2: time to resolution of blood coagulability:
– There were no statistically significant differences in the rates of recovery of
prothrombin time (PT) activated partial thromboplastin time (aPTT),
fibrinogen, blood Factors II, V, VII, VIII, IX or X or D-Dimer.
• Endpoint 3: adverse reactions to administered antivenom:
– No serious adverse drug reactions were observed with either product.
– 1 patient given 2 vials of bioCSL taipan AV developed mild itching to
abdomen & upper chest & IV-bearing arm during infusion, which settled
within 5-10 minutes with routine standard treatment.
68. • Endpoint 1: evolution of oropharyngeal paralysis:
– Oropharyngeal paralysis developed in 2/6 patients given bioCSL taipan AV,
one was intubated at 23 hours post-bite and ventilated for 4 days.
– Mild/moderate cranial nerve palsy post-antivenom in 4/6 patients given
ICP taipan AV, and in 3/6 other patients given bioCSL taipan AV.
• Endpoint 2: time to resolution of blood coagulability:
– There were no statistically significant differences in the rates of recovery of
prothrombin time (PT) activated partial thromboplastin time (aPTT),
fibrinogen, blood Factors II, V, VII, VIII, IX or X or D-Dimer.
• Endpoint 3: adverse reactions to administered antivenom:
– No serious adverse drug reactions were observed with either product.
– 2 patients given 1 vial doses of bioCSL taipan AV developed generalised
itching (without urticaria) and a cough, which settled within 15 minutes
with routine standard treatment.
69.
70. • A higher dose (e.g.: 2 vials) of either antivenom appears more
likely to prevent development of neurotoxicity, but this is not
practical with bioCSL taipan AV due to cost and availability issues;
• There do not appear to be any significant differences in the
clinical outcomes achieved using these two antivenoms at the
doses we evaluated;
• Both products are safe, with only bioCSL taipan AV leading to
mild-moderate events that were managed quickly and routinely,
and no SAE were observed.
• This small-scale exploratory study provides sufficient confidence
in the non-inferiority and safety of the new ICP antivenom to
justify a larger, exact single stage Phase II clinical trial.
Editor's Notes
Established as a clinical research laboratory to support the taipan antivenom trials at PMGH
Based at UPNG School of Medicine & Health Sciences
Construction by Cord & Associates, with independent 28.8 kVa backup power, and VSAT internet link.
Equipped with PNG’s only state-of-the-art Diagnostica Stago STA Compact Haemostasis system for studying coagulation defects caused by snakebites.
Boule Swelab Haematology System, Reflotron enzyme assays and Thermo-Fisher immunoassay facilities as well as basic proteomics equipment and our Mastercycler PCR system.
We are now conducting a Phase I small-scale dose-finding & safety studies to be followed by a larger randomised control trial in 2012-14.
The RCT will directly compare the new ICP whole IgG taipan AV with the current CSL F(ab’)2 taipan AV.
Minimum requirement for success is equivalent patient survival and equivalent safety.
Both studies will be formally registered with the Australia/New Zealand Clinical Trials Register (ANZCTR) and all results will be reported publicly.
Ethics approval is being obtained from both the UPNG SMHS Human Ethics Committee and from the PNG National Medical Research Advisory Committee (MRAC).
Funding: K250,000 OHE; A$1.4 Million NHMRC Project Grant.
Five key criteria were deemed important in developing a new taipan antivenom for use in PNG:
Need for achieve equivalent potency, efficacy and safety (compared to currently available CSL products).
High stability under tropical transport and storage conditions, including increased resistance to biological deterioration or loss of potency with extended exposure to ambient temperature.
Final production cost under US$250 per vial.
Involvement of PNG scientists in the production of venoms, with phased technology transfer opportunities and infrastructure development that could eventually enable local antivenom production.
Compliance with the new WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins (2009).
Our aim was to produce an antivenom ready for assessment through the WHO Prequalification of Essential Medicines Program.
Specimens collected in Milne Bay and Central Provinces
Maintained in individual terrariums
Venoms collected only from healthy adult snakes of both sexes
Minimum 3 weeks between each ‘milking’
Venom from 12 snakes was pooled and frozen at -80C
Exported to University of Melbourne for lyophilisation
Pool of 2 grams supplied to Instituto Clodomiro Picado for immunization of the first group of three horses
Now have own laboratory on-site
The graph shows the increasing cost of antivenoms in PNG from 1992 to 2007. Since 2007 the Health Department has refused to supply data on the costs of antivenom, or the numbers of vials supplied and distributed.
Photos show UPNG Snake Keeper, Mr Jasper Gabugabu at work in the AVRU-UPNG Serpentarium. Training local staff to handle venomous snakes has been an important part of our capacity building process.
Top Photo: Dr Simon Jensen (AVRU-UPNG) hunting taipans in Milne Bay Province.
Lower Left: UPNG Snake Keeper, Mr Jasper Gabugabu with a freshly bagged Papuan taipan
Lower Right: UPNG MMedSci student, Mr Owen Paiva working in the Serpentarium laboratory
Instituto Clodomiro Picado – non-profit Costa Rican antivenom producer based at the Universidad de Costa Rica in San Jose, Costa Rica. A university-based, non-profit antivenom production facility is our model for eventual local antivenom production in PNG.
Top: Horse blood containing antivenom antibodies ready for processing
Bottom: ICP production facility
Top: Immunizing a horse with snake venom at ICP
Bottom: Vials of antivenom coming off the production line
Possible package design
Figure shows the purity of the ICP antivenom
Emphasise that patient mix is approximately representative of normal presentations – eg: 38.9% of trial patients under 16 is within the range of the proportions in other studies (i.e.: 30.1% to 48%). Also emphasise that all of the patients had positive SVDK for taipan venom immunotype.
No difference in the results with either antivenom. The occurrence of a mild reaction in 1/6 bioCSL case is not significant.
No difference in the results with either antivenom. The occurrence of a mild reaction in 2/6 bioCSL cases is not significant.
Emphasise that this study demonstrated support for our hypothesis that “ICP antivenom is non-inferior to bioCSL antivenom” and was safe as well as effective – therefore suitable to take forward into Phase II trial.