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Home , Dimethoate, Obidoxime, Omethoate, VR (nerve agent)

Threat by nerve agents: Update of diagnostic and therapeutic strategies!

H. Thiermann and F. Worek

Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany

Congress of the European Society of Emergency Hot Topic Lecture

Wednesday 12th September 2018 Glasgow, United Kingdom

Scenarios of an Attack with Agents – Attack on Civilian Population

1 Scenarios of an Attack with Chemical Warfare Agents – Targeted Homicidal Attack

Nerve Agents and other Organophosphorus Compounds

G - Agents

Tabun (GA), (GB), (GF), (GD) (and Analogs) R R

X V - Agents

VX, Russian VX (VR), Chinese VX (CVX) (and Analogs)

OP-

Diethyl-type, Dimethyl-type, hundreds of other compounds!

2 Characteristics of Poisoning by Nerve Agents

Generally assumed: patients exposed up to 5x LD50

Inhalative G-Type Poisoning - Immediate onset of crisis - Short persistence of nerve agent in the body

Percutaneous V-type Nerve Agent Poisoning - Prolonged onset of cholinergic crisis - Persistence of nerve agent over several days in the body

Organophosphorus Compound Threats

© 2017 by InstPharmToxBw Worek et al. Arch Toxicol 2016

3 Effects of Organophosphorus Compounds

Mechanism: - inhibition of AChE - accumulation of ACh - disturbance of cholinergic functions

https://www.stern.de/politik/ausland/syrien--angriff-auf-klinik- mit-mutmasslichen-giftgas-opfern-7398534.html

Cholinesterases – Reason of Confusion

AChE (EC 3.1.1.7) • found mostly in nervous tissue and blood (red blood cells) • primates have the highest activity, rats one tenth and cats roughly 1 % compared to human RBCs Pl-ChE (EC 3.1.1.8) • synthesis in the liver • capable of splitting several cholinesters, incl. , highest activity with butyrylcholine Confusion Often reported: determination of AChE in human plasma with acetylcholine or acetylthiocholine as substrate: in fact activity of BChE was determined! Blum et al. unpublished

4 Clinical Challenge of Nerve Agent Poisoning

Signs and symptoms Nerve agent poisoning Slight Moderate Severe Miosis / lacrimation Local fasciculations Hypersalivation / sweating Nausea / defacation / emiction Bronchocontriction / bronchorrhoe Bradycardia / circulatory depression Respiratory depression Convulsions Coma Worek et al. Arch Toxicol 2016

Effects of Organophosphorus Compounds

Mechanism: - inhibition of AChE - accumulation of ACh - disturbance of cholinergic functions

Life threatening effects: - bronchoconstriction/ bronchorrhoea ( receptors) - central respiratory arrest (muscarine and nicotine receptors) - peripheral respiratory arrest (nicotine receptors)

5 ChE Check Mobile for on-site Confirmation of the Clinical Diagnosis

Acetylthiocholine

AChE BChE

AS-1397

Acetate Thiocholine

+ DTNB

5-Mercapto-2-nitrobenzoate + TNB- Ellman et al. Biochem Pharmacol 1961

OP-SkinDisclosure-Kit

12,5 cm

16,7 cm

Weight: 20 Gramm

6 OP-SkinDisclosure-Kit: How to Use?

Cup with enzyme Cup with chemicals on test tube, on test tube, Sampling Put the sampler vigorous shaking, vigorous shaking, on the skin in the test tube 1 min incubation 1 min incubation read the color

OP-SkinDisclosure-Kit: Score

4 3 2 1 0

Nerve Agent No Nerven Agent present present

Worek et al. Toxicol. Lett 2880: 190 -194, 2017

7 Peculiarities in Treatment of Chemical Agent Poisoning Self – Protection; Working under aggravated conditions; Protection of medical units; Latency period. Decontamination of victims with signs and symptoms

ouvry.com

Tu, 2002

Compare: Thiermann et al. Chem Biol Interact 2013; Rosman et al. Annal Intern Med 2014

Med C- Special Diagnostics/ Reconnaissance / Verification and Analysis

Biomedical verification of exposure to chemical agents ► chemical warfare agents ► degradation products ► metabolites and adducts

8 for Nerve Agent Poisoning

Military doctrine: autoinjectors for self and buddy aid Enhanced first aid and clinical care by medical personnel)

Aggressive atropine dosing Recommended German regimen 2 mg, i.m. followed by consecutive doubling the dose (4 – 8 – 16 - 32 mg) and finally infusion at field hospital Criteria Clear chest on auscultation; heart rate >80 beats/min; pupils no longer pinpoint; dry skin (axilla); systolic blood pressure >80 mmHg

Thiermann et al. Chem Biol Interact, 2013; according to Eddleston et al. Crit Care 2004 and J Toxicol ClinToxicol 2004. For review see: Thiermann et al. Treatment of Nerve Agent poisoning In:Chemical Warfare Toxicology, Volume 2: Management of Poisoning, © The Royal Society of Chemistry 2016

Development of

Synthesis of thousands 2018 of oximes since the Oximes in use or in early 1950ies advanced development

- Monopyridinium oximes 2-PAM 1955 - Bispyridinium oximes TMB-4 1958 - Asymmetric oximes MMB-4 1959 - Substituted pyridinium oximes 1959 - Uncharged oximes HI-6 1968

9 Effects of Obidoxime in a Patient with Poisoning Patient: 56-year old, female

Emergency situation: Unconscious, cardiovascular resuscitation

Clinical course: As the clinical course situation remained critical for about 4 days without obidoxime, the regimen was started

AChE activity of red blood cells Neuromuscular transmission prior to Obidoxime

600 Obidoxime 500 RBC-AChE in vivo 400 RBC-AChE in vitro 300 200 mU/µmol Hb after Obidoxime 100 0 0 12 24 36 48 Hours

Reactions Occurring at AChE with an OP and an

OP enzyme leaving group inhibited enzyme

X + AChE-OH X + ACHE

RNOH H2OH2O

AChE-OH CH3-CH2OH AChE-OH + + +

R ACHE

oxime induced aging spontaneous reactivation reactivation

10 Effects of Obidoxime in a Patient with Parathion Poisoning Patient: 56-year old, female

Emergency situation: Unconscious, cardiovascular resuscitation

Clinical course: As the clinical course situation remained critical for about 4 days without obidoxime, the regimen was started

AChE activity of red blood cells Neuromuscular transmission prior to Obidoxime

600 Obidoxime 500 RBC-AChE in vivo 400 RBC-AChE in vitro 300 200 mU/µmol Hb after Obidoxime 100 0 0 12 24 36 48 Hours

Kinetic-Based Computer Model

ki (1) [E] + [OP] [EP]

1000 ka 150 Toxicokinetics (2) [EP] + H2O [EP'] Pharmacokinetics 100 100 10 ks VX (nM) VX (3) [EP] + H2O [E] + [P] (µM) 6 HI 50 1

0.1 0 KD kr 0 100 200 300 400 500 0 100 200 300 400 500 (4) [EP] + [OX] [EPOX] [E] + [POX] min min

kr * [OX] kobs = KD + [OX]

d[E]  ki * [OP] * [E]  ks * [EP]  kobs * [EP  EPOX] dt d[EP]  ki * [OP] * [E]  ks * [EP]  kobs * [EP  EPOX]  ka * [EP] dt

Worek et al. TAP 2005

11 The Use of Kinetic Constants, Pharmacokinetic and Toxicokinetic Data for Prediction of AChE-Activity in Human Poisoning 20 100 Patient: Obidoxime 80 Paraoxon [nM] A 45-year old, male 15 60 Emergency situation: 10 40 Unconscious, severe signs and 5 symptoms of cholinergic crisis. [µM] Obidoxime 20 1.5 mg of atropine, intubation and initiation of artificial ventilation. 0 0

Clinical course: 400

2 bolus doses of obidoxime together 300 with an atropine infusion at the local hospital. Transfer to the ICU of 200 Technical University, Munich. AChE: measured The patient recovered uneventfully. 100 AChE (mU/µmol Hb) (mU/µmol AChE AChE: calculated 0 Worek et al. TAP 2005 0 24 48 72 96 120 144 Time p.i. (h)

Reactions Occurring Between Phosphoryl-AChE and Oxime During Reactivation

Inhibition kI E-OH + OP  E-OP

Association KD E-OP + Oxime  Oxime-E-OP

Reactivation kr Oxime-E-OP  Oxime-OP + E-OH

12 Which Oximes Should We Use?

Compare: Thiermann et al. Chem Biol Interact 2013 and Tox Lett 1999; modified by using data from Aurbek et al. Toxicolgy 2006; Bartling et al. Toxicolgy 2007 Worek et al. Tox Lett 2011

Translation of Clinical Findings from Human Poisoning to an Animal Model

Poisoning by (oral) and treatment with 2-PAM

100 ACHE in vivo 75 ACHE calc

50 ACHE (%) 25

0 100 200 300 400 500 600 700 800 time (min) 50 80 40

60 30

40 20 2-PAM (µM) 2-PAM

10 (µM) Omethoate 20 2-PAM Omethoate 0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800 time (min) time (min) Worek et al. Chem Biol Interact. 2010

13 Translation of Findings from OP-- Poisoning to Nerve Agent-Poisoning

Male York-Landrace cross swine (about 20 kg)

Poisoning:

3 x LD50 VX, p.c.

Treatment:

HI-6 (12.7 mg/kg) / Atropine sulfate (0.05 mg/kg) i.m., according to signs and symptoms

Field laboratory diagnosis:

AChE activity on-site

Plasma sampling for laboratory analysis:

HI-6, VX

RBC-AChE Activity - Signs and Symptoms of a VX-Poisoned Swine Treatment according to signs and symptoms breathing problems dysrhythmias / hypotension salivation miosis fasciculation / mastication seizure apnoe / manual ventilation

30 % AChE activity On-site analysis On-site analysis of

VX

3x LD50

14 RBC-AChE Activity, HI-6 and VX Concentrations in Plasma of a VX-Poisoned Swine Treatment according to signs and symptoms Treatment when 100 signs and symptoms 80 occurred 60 - salivation / bronchorrhea,

40 - breathing problems AChE (% ) with HI-6 and atropine, i.m., 20 subsequent treatment 0 according to fixed 0 60 120 180 240 300 360 schedule time [min]

100 10000

80 1000 60 100 40 VX [pM] HI 6 [µM] 6 HI 10 20 VX(+) (pM) VX(-) (pM) 0 1

0 60 120 180 240 300 360 0 60 120 180 240 300 360 time [min] time [min]

Is there a Correlation between RBC-AChE- Activity and Clinical Status? A parathion poisoned patient was treated with obidoxime. The status and NMT were monitored during treatment at the ICU.

600 RBC-AChE in vivo Reactivatability

400 86.4 h

mU/µmol Hb 200

0 0 24 48 72 96 Hours 93.7 h 60

40

20 94.6 h Obidoxime (µmo/l) Obidoxime

0 0 24 48 72 96 Hours Eyer et al. Clincal Toxicology 2009

15 Cholinesterase Status

Blood sampling

Blood dilution Plasma 1. AChE 2. BChE activity activity

Incubation of blood dilution with Incubation of plasma with obidoxime (100 µM, 30 min, 37°C) AChE standard (60 min, 37°C)

AChE activity AChE activity 3. Reactivatability 4. Inhibitory activity

ChE status monitor

Developed by Riele and Securetec + scientific support from IPTBw

=

22 cm

8 cm

22 cm

16 Oximes – a Curious Situation

“Hitherto, alkylphosphate poisoning has been treated mainly by atropine, but now atropine is replaced by PAM” Namba & Hiraki: JAMA 166: 1834, 1958

“A high-dose regimen of (…) reduces morbidity and mortality in moderately severe cases of acute organophosphorus-.“ Pawar et al. Lancet 368: 2136, 2006

“Our results question the value of cholinesterase reactivators in treatment of acute OP poisoning.” De Silva et al. Lancet 339: 1136, 1992

“Based on the current available data on human poisoning, oxime was associated with either a null effect or possible harm.” Peter et al. Crit Care Med 34: 502, 2006

“we found no evidence that this [2-PAM] regimen improves survival or reduces need for intubation in patients with organophosphorus poisoning.“ Eddleston et al. PLoS Med 6: e1000104, 2009

Interim Result

Oximes do their expected job in patients

– often, but not always.

What are the reasons behind such failure?

17 Limitations of Oxime Treatment

Oximes effects can be overwhelmed in mega-dose poisoning

600 100 500 80 400 RBC-AChE in vivo RBC-AChE in vitro 60 300 Paraoxon: about 5 µmol/L !

200 40 mU/µmol Hb 100 20 Inhibitory activity (%)activity Inhibitory 0 0 0 96 192 288 384 0 96 192 288 384 Hours Hours

100 60

75 40 50 NMT (%)NMT 20 25 Obidoxime (µM) Obidoxime

0 0 0 96 192 288 384 0 96 192 288 384 Hours Hours

Compare Eyer et al. Toxicol Rev. 2003

Limitations of Oxime Treatment

Oximes can only achieve reactivation prior aging is complete

600 3000 500 RBC-AChE in vivo Pl-ChE 400 RBC-AChE in vitro 2000 300

200 1000 mU/µmol Hb 100 mU/ml Plasma

0 0 0 24 48 72 0 24 48 72 Hours Hours Patient: 40-year old, male

Emergency situation: Diarrhea and vomiting on the evening, dramatic worsening on the next day; increasing dyspnea, but awake and conscious. After admission to the local hospital somnolent, arrhythmia and respiratory insufficiency

Oximes are not effective in poisoning with several organophophorus compounds, e.g. Soman,

18 Reactivation Rate Constants of Oximes with OP-inhibited human AChE

160

140

120 ) -1 100 min 80 -1 (mM

60 r2 k 40

20

0 HI 6 HI VX VR DFP HLö 7 HLö Sarin Pralidoxime Obidoxime Tabun MFPCh Butylsarin Cyclosarin Paraoxon-ethyl Paraoxon-methyl

Worek et al. Biochem Pharmacol. 2004

Alternative Approaches for Therapy of Nerve

O Agent Poisoning F O P Nu Enhanced O elimination by scavengers

Cyclodextrins Schneider C et al. Angew Chem Int Ed Engl ,2016 Kranawetvogl et al. Toxicol Lett, 2015 Bierwisch et al. Toxicol Lett, 2014 Enzymes, e.g. PON1, PTE

Modulation of nicotinic ACh- receptors

Goldsmith et al. Arch Toxicol ,2016 Wille et al. Tox Lett, 2016 Hansen et Taylor, 2007, modified Ashani et al. Chem Biol Interact, 2016

19 Positive Allosteric Modulators (PAMs) – Potential Binding Sites

Identification of the MB327 Orthosteric binding site binding site using docking studies

Allosteric binding site Binding site for positive allosteric modulators (PAMs) Nikotine

Epibatidine MB327

delta Acetylcholine

alpha

polar Carbamoylcholine

lipophilic Binding sites within the channel

Wein et al. 2018, Curr Comput Aided Drug Des; Wien et al. Toxicol Lett, 2018

Conclusion

Oxime therapy can be beneficial if started early, at sufficient doses, for sufficient time. By this regimen neuromuscular function can be improved, artificial respiration probably shortened, and the occurrence of an intermediate syndrome reduced. In scenarios with limited artificial ventilation capacity oximes along with atropine may be indispensable. Elaborated intensive care, if feasible, may be equally able to keep patients alive with completely inhibited AChE.

20 Outlook

Efficient oximes are able to reactivate AChE inhibited by some nerve agents when they are administered fast enough at an appropriate dose. Under specific conditions, in particular in V-type poisoning, oxime treatment has to be maintained for a longer period. At present no oxime available has a broad spectrum against all nerve agents and organophosphorus pesticides. A combination of effective oximes, e.g. obidoxime and HI 6 may cover a greater spectrum of nerve agents. Alternative approaches are necessary for problematic nerve agents, e.g. soman and tabun.

Acknowledgement

T. Zilker D. Kiderlen K. Wanner F. Eyer B. Fichtl T. Wein R. A. Hatz J.E.H. Tattersall N. Felgenhauer P. Eyer S. Rappenglück M. Eichhorn C.M. Timperly R. Pfab M. Radtke M. Lindner A.C. Green H. Friess M. Bird R. Bumm A.L. Mihaljevic

J. Mikler I. Hill K. Niessen T. Seeger D. Tawfik K. Weatherby G. Reiter H. John M. Goldsmith T. Wille M. Koller Y. Ashani G. Heyes G. Engl J.L. Sussman T. Hannig S. Kirchner S. Eckert S. Kämpfer S. Kubik N. Herkert L. Schnitzler M. Zengerle S. Muschik N. Boos A. Bierwisch M. Eddleston C. Scheffel C. Wübbeke C. Schneider

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