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

Bloo

d A

ChE

Act

ivity

0.0

0.1

0.2

0.3

0.4

0.5

AChE data by agent X oxime

Group

Bloo

d A

ChE

Act

ivity

0.0

0.5

1.0

1.5

2.0

24 Hr Body Weight Loss

Route of Administration

Gra

ms B

ody

Wei

ght

-60

-40

-20

0

24 Hr Body Weight Loss

Agent

Gra

ms B

ody

Wei

ght

-60

-40

-20

0