Vestibular Dysfunction in Active Duty Personnel Deployed to OEF

1. Vestibular Dysfunction in Active Duty Personnel Deployed to OEF/OIF LCDR Dennis Faix, MC, USN Navy Environmental and Preventive Medicine Unit FIVE

2. Vestibular Dysfunction Investigation 2 BackgroundEvents leading to Investigation

3. Vestibular Dysfunction Investigation 3 Preliminary Steps Initially: 12 cases of �Larium toxicity� Confirmed diagnosis with outside expert Background rate of dizziness/VOR dysfunction Developed case definition Now only ten cases Multiple (ototoxic) exposures Apparent cluster in one small unit 4 cases from 33-member Navy Tactical Dissemination Module (TDM) team

4. Vestibular Dysfunction Investigation 4 Methods Case-control study looking at multiple exposures Surveyed two units stationed at AASAB at same time Investigator-administered telephone survey, 188 questions Navy TDM (n=33, 100%) ? 4 cases Army 286th ADB (n=18, ~10%) ? 2 cases Medical record review of cases (n=9, 75%) Serum mefloquine levels (n=26, 55%) Mefloquine tablet testing (n=3, 43%) Review of clinic mobilization process

5. Vestibular Dysfunction Investigation 5 ResultsDemographics & Medical History Overall there were no clinically significant demographic difference between cases and controls ADB group was younger, active duty No significant difference in medical history Except for PMH of motion sickness Cases tended to be deployed longer than controls, but not significantly

6. Vestibular Dysfunction Investigation 6 ResultsTimeline of Deployment, Mefloquine Use and Symptoms for Cases

7. Vestibular Dysfunction Investigation 7 ResultsCombat Stress Exposures

8. Vestibular Dysfunction Investigation 8 ResultsNeuropsychiatric Symptoms (TDM group)

9. Vestibular Dysfunction Investigation 9 ResultsNeuropsychiatric Symptoms (ADB group)

10. Vestibular Dysfunction Investigation 10 ResultsMalaria Chemoprophylaxis Exposures (TDM group only)

11. Vestibular Dysfunction Investigation 11 ResultsChart review & Tablet Testing Three of 9 records reviewed had documentation of pre-deployment health assessments None had documentation of counseling or screening prior to issuance of mefloquine Post-Deployment Health Assessment in 6 of 9 charts Mefloquine tablets tested by FDA No abnormalities in quality, constitution Tablets held up well to individual issue and deployment to desert environment

12. Vestibular Dysfunction Investigation 12 DiscussionLimitations Small numbers (pilot study) Potential Bias Selection bias Recall bias Misclassification bias Patient X OIC of TDM group while deployed to AASAB Instructed team to seek medical care if they took mefloquine Informed teammates, Senator Feinstein and lay press of �link� with mefloquine Currently suing Lariam manufacturer

13. Vestibular Dysfunction Investigation 13 DiscussionBiological Plausibility Acute neurotoxicity of mefloquine Anxiety, dizziness are known acute side effects, no evidence of long-term effects Disruption of endoplasmic reticulum and gap junction channels reported (Dow, 2003; Cruikshank 2004) Known association between anxiety disorders and vestibular dysfunction Nearly 3 times as many persons with self-reported dizziness had symptoms of anxiety compared to persons without dizziness in large, U.K. study (Yardley, 1998) Individuals with anxiety may exhibit abnormal findings on rotational chair testing (Rolf, 1996) Interaction of effects of mefloquine and combat stress? Cruikshank, S. J., M. Hopperstad, et al. (2004). "Potent block of Cx36 and Cx50 gap junction channels by mefloquine." Proc Natl Acad Sci U S A 101(33): 12364-9. Recently, great interest has been shown in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with targeted connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain regions. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major effects on other gap junctions or membrane channels. We show that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC(50) approximately 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC(50) approximately 1.1 microM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fold higher. To further examine the utility and specificity of this compound, we characterized its effects in acute brain slices. Mefloquine, at 25 microM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side effect was an increase in spontaneous synaptic activity. Mefloquine (25 microM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular properties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50. Dow, G. S., T. H. Hudson, et al. (2003). "The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro." Malar J 2(1): 14. BACKGROUND: There is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis. METHODS: Laser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin. RESULTS: At physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro. CONCLUSIONS: Mefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro. Cruikshank, S. J., M. Hopperstad, et al. (2004). "Potent block of Cx36 and Cx50 gap junction channels by mefloquine." Proc Natl Acad Sci U S A 101(33): 12364-9. Recently, great interest has been shown in understanding the functional roles of specific gap junction proteins (connexins) in brain, lens, retina, and elsewhere. Some progress has been made by studying knockout mice with targeted connexin deletions. For example, such studies have implicated the gap junction protein Cx36 in synchronizing rhythmic activity of neurons in several brain regions. Although knockout strategies are informative, they can be problematic, because compensatory changes sometimes occur during development. Therefore, it would be extremely useful to have pharmacological agents that block specific connexins, without major effects on other gap junctions or membrane channels. We show that mefloquine, an antimalarial drug, is one such agent. It blocked Cx36 channels, expressed in transfected N2A neuroblastoma cells, at low concentrations (IC(50) approximately 300 nM). Mefloquine also blocked channels formed by the lens gap junction protein, Cx50 (IC(50) approximately 1.1 microM). However, other gap junctions (e.g., Cx43, Cx32, and Cx26) were only affected at concentrations 10- to 100-fold higher. To further examine the utility and specificity of this compound, we characterized its effects in acute brain slices. Mefloquine, at 25 microM, blocked gap junctional coupling between interneurons in neocortical slices, with minimal nonspecific actions. At this concentration, the only major side effect was an increase in spontaneous synaptic activity. Mefloquine (25 microM) caused no significant change in evoked excitatory or inhibitory postsynaptic potentials, and intrinsic cellular properties were also mostly unaffected. Thus, mefloquine is expected to be a useful tool to study the functional roles of Cx36 and Cx50. Dow, G. S., T. H. Hudson, et al. (2003). "The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro." Malar J 2(1): 14. BACKGROUND: There is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis. METHODS: Laser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin. RESULTS: At physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro. CONCLUSIONS: Mefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro.

14. Vestibular Dysfunction Investigation 14 Conclusions Cases had evidence of vestibular abnormalities most commonly seen with migraine or anxiety-related dizziness, and not that seen with ototoxicity. The rate of balance disorders and dizziness among these subjects is within the reported normal range in the general population. However, the actual rate for these disorders in an active duty population is unknown. No evidence that mefloquine or any known vestibulotoxic agent was associated with the vestibular dysfunction exhibited in these cases.

15. Vestibular Dysfunction Investigation 15 Conclusions Cases reported higher levels of combat stress and neuropsychiatric symptoms as compared to controls, suggesting that combat-related stressors may be associated with vestibular dysfunction. An interaction between mefloquine and combat stress cannot be ruled out Documentation of pre-deployment health assessments and appropriate mefloquine screening was not found in the medical records reviewed.

16. Vestibular Dysfunction Investigation 16 Recommendations Further study on possible interaction between mefloquine, vestibular dysfunction and combat stress Exploit existing databases (pharmacy, deployment, PDHA, etc) Selected survey of redeploying personnel DOD-wide case finding for a larger case control study Establish baseline dizziness and VOR abnormality rates among active duty personnel Study underway at NMCSD; 10% complete Re-emphasize DOD guidance regarding pre-deployment medical screening Force Health Protection Prescription Products Pre-Deployment Health Assessments

17. Vestibular Dysfunction Investigation 17 Acknowledgements Centers for Disease Control and Prevention CAPT Peter Bloland, USPHS Aaron Mendelsohn, PhD CAPT Monica Parise, USPHS LCDR Snehal Shah, USPHS Navy Environmental and Preventive Medicine Unit FIVE CAPT Dean Bailey, MC, USN LCDR Julie Howe, MC, USN LCDR Andrew Vaughn, MC, USN LCDR Todd Wagner, MC, USN Navy Health Research Center Defense Medical Surveillance System (AMSA) Food and Drug Administration

18. BACKUP SLIDES

19. Vestibular Dysfunction Investigation 19 BackgroundEvents prior to investigation Fort Bragg suicides/homicides Lay press reports AFEB recommendation Keep mefloquine VA letter Blue ribbon panel Mefloquine recall to wholesale level secondary to possible contamination

20. Vestibular Dysfunction Investigation 20 MethodsCase Definition Cases must be DOD Personnel (active duty, reserve, or civilian) deployed as part of Operation Iraqi Freedom or Operation Enduring Freedom since January 1, 2002, and Report new onset of dizziness during or after deployment lasting two weeks or more as a single episode or multiple episodes, and Have no other identifiable etiology including, vestibular neuronitis, Meniere�s disease, head trauma, acoustic neuroma, benign paroxysmal positional vertigo (BPPV), labyrinthitis, or prior ear surgery, and Demonstrate unsteadiness of gait while walking or while standing still on physical exam, and Have VOR asymmetry on Rotational Chair Testing independently confirmed by two subject matter experts.

21. Vestibular Dysfunction Investigation 21 MethodsCase Selection (Tactical Dissemination Module)

22. Vestibular Dysfunction Investigation 22 MethodsCase Selection (Air Defense Battery)

23. Vestibular Dysfunction Investigation 23 MethodsSurvey Deployment history Medical history Medication history Environmental exposure history Symptoms Combat stress history 188 questions total

24. Vestibular Dysfunction Investigation 24 MethodsTablet Analysis 7 individuals reported leftover tablets available 3 individual actually sent tablets in for testing Sent to FDA for analysis