1. Results
Intranasal delivery of oximes to organophosphate-exposed rats
Jordan Horrocks
Mentored by Dr. John McDonough
Introduction
Traditionally, the delivery of antidotes to treat nerve agent
intoxication involved intramuscular administration. However, nasal
administration had recently been considered due to it’s ability to rapidly
deliver drugs to the brain. It was believed that nasally, many drugs are
better able to pass through the blood brain barrier (Shih, Skovira,
O’Donnell, & McDonough, 2010), and it was hoped that such would be
the case for oximes. Oximes like MMB4 and 2-PAM (used in this study)
work by binding to the phosphorous part of the nerve agent and
cleaving it from the cholinesterase enzyme, thus restoring it’s function.
Intranasal delivery would be a major improvement in oxime
administration, as they are quaternary in structure and normally do not
penetrate the brain or reactivate inhibited acetylcholinesterase (AChE)
already in the central nervous system (Shih, Skovira, O’Donnell, &
McDonough, 2009). In previous studies, it has been found that the
intranasal delivery works as well, if not better than the intramuscular
route (McDonough, Van Shura, LaMont, McMonagle, & Shih, 2008).
Besides determining the effectiveness of alternative routes of
administration, this study will also seek to determine the effect multiple
nerve agents and oximes have on animals’ EEG, temperature, weight,
and blood AChE levels. As shown in previous studies (Shih, Skovira,
O’Donnell, & McDonough, 2010), both MMB-4 and 2-PAM provide
good reactivation of cholinesterase activity inhibited by the nerve agents
selected –sarin (GB) and cyclosarin (GF).
Conclusions
Materials and Methods
Experiment Set Up
• Eighty eight telemetry- implanted rats were sedated with isoflurane,
before being exposed to a 1XLD50 dose of either sarin or cyclosarin.
• Rats were then given an oxime treatment(either MMB-4 or 2-PAM)
via intramuscular or intranasal injection.
• Rat EEG data was monitored for 24 hours following nerve agent
exposure.
• Rats were then euthanized, and blood and brain tissue were collected
for AChE analysis.
Analysis
• EEG and body temperature data were analyzed for seizure activity in
Neuroscore®.
• All data (body weight change, blood AChE levels, EEG, and
temperature data) was evaluated in Microsoft Excel®.
• Data was then sent run through Sigma-Stat for statistical analysis.
Based upon the results of the data analysis, this project conclusively
proved that the method of administration, be it intramuscular or
intranasal, had no statistically significant impact on the success of the
treatment. Thus, the project's original hypothesis, that intranasal delivery
would increase the success of the treatment compared to intramuscular
administration, was not supported by these data. These results indicated
that the administration method of the treatment had no differential
effect on the ability of the oximes to treat nerve agent exposure. In other
words, intranasal delivery works as well as intramuscular delivery- no
better or worse. These results support previous experiments
(McDonough, Van Shura, LaMont, McMonagle, & Shih, 2008), where
the intranasal route worked similarly to the intramuscular delivery of
various drugs and treatments. Both the blood AChE and the weight
change data underwent statistical analysis to control for any confounding
variables-including but not limited to interactions between the variables
used. More testing is needed to determine the effectiveness of the
intranasal delivery.
McDonough, J., Van Shura, K., LaMont, J., McMonagle, J., & Shih, T. M.
(2008). Comparison of the intramuscular, intranasal or sublingual
routes of midazolam administration for the control of soman-
induced seizures. Basic and Clinical Pharmacology and Toxicology, 104(1),
27-34.
Shih, T. M., Skovira, J., O’Donnell, J., & McDonough, J. (2009). Central
acetylcholinesterase reactivation by oximes improves survival and
terminates seizures following nerve agent intoxication. Advanced Studies
in Biology, 1(3), 155-196.
Shih, T. M., Skovira, J., O’Donnell, J., & McDonough, J. (2010). In vivo
reactivation by oximes of inhibited blood, brain and peripheral tissue
cholinesterase activity following exposure to nerve agents in guinea
pigs. Chemico- Biological Interactions, 1(87), 207-214.
Bibliography
Acknowledgements
I would like to thank Dr. McDonough and all of his team at the US
Army Medical Research Institute for Chemical Defense (USMRICD) for
incredible help in completing this project. I would also like to thank my
faculty advisor Mrs. McDonough. All animal handlings and nerve agent
exposure were performed by Dr. McDonough and staff.
Graph 1: Weight Loss by Agent Graph 2: Weight Loss by Route
Graph 4: AChE, agent by oxime
Based on Graph 1, a comparison of 24 hour average weight loss in rats
exposed to either GB or GF with a Least Squares Mean statistical test showed
that animals treated with cyclosarin had a significantly higher weight loss when
compared with animals exposed to sarin.
According to Graph 2,when the same test referred to in Graph 1 is used to
compare the treatment route, it showed that the method of administration had
no statistically significant impact on weight loss.
Displayed above (Graph 3) is a one- way analysis of variance for the 8
groups observed in the experiment, including controls. The two controls not
exposed to nerve agent are labeled above as control and iso (short for the
sedative used, isoflurane). The aforementioned controls showed statistically
higher levels of AChE in the blood, as is to be expected in healthy animals.
When the controls were removed (Graph 4), the same test found that when
exposed to GF instead of GB, animals had a significant difference in blood
AChE activity, meaning that animals given GF had a lower level of AChE
levels than in animals given GB. However, as in the weight loss analysis,
method of oxime administration had no impact.
Graph 3: AChE, with controls
AChE data agent X oxime, no controls
Group
BloodAChEActivity
0.0
0.1
0.2
0.3
0.4
0.5
AChE data by agent X oxime
Group
BloodAChEActivity
0.0
0.5
1.0
1.5
2.0
24 Hr Body Weight Loss
Route of Administration
GramsBodyWeight
-60
-40
-20
0
24 Hr Body Weight Loss
Agent
GramsBodyWeight
-60
-40
-20
0