killer in the kunai by dr. david williams

“”The Papuan black snake would appear to be naturally more aggressive than

Slater suggests…” Charles H. Campbell (1966)

• 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 three locations.

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Port MoresbyPort MoresbyGemo IslandGemo Island

Roku BayRoku Bay

Haidana IslandHaidana Island

FAIRFAXFAIRFAXHARBOURHARBOUR

Napa NapaNapa Napa

BoeraBoera

PorebadaPorebada

“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)

““The commonest poisonous

The commonest poisonous

snake in the area defined

snake in the area defined

would be the Papuan whip-

would be the Papuan whip-

snake snake Demansia Demansia

psammophis papuensis

psammophis papuensis…”…”

• 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.

• 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

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)

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)

“You’ll never be able to do it, it’s simply too costly, and elapid snake venoms are not

immunogenic enough”

• 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

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Blacksnake

Death adder

Polyvalent

Taipan

• 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.

• 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

• 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

• 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.

• 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.

* 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 filtrationb Turbidity is expressed as Nephelometric Turbidity Units (see details in Materials and Methods)c Batch B0548-06301 CSL Expiry: March 2012d Batch 4511209 ICP Expiry: November 2012

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

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

Crude plasma

CSL TaipanAntivenom

ICP TaipanAntivenom

Whole IgG

F(ab’)2

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.