(T1430-08-50) Developments of Advanced Military Measure of Olfaction (AMMO) as an Indicator of Traumatic Brain Injury (TBI) Through Sense of Smell: Solving Excipient-Active Stability Interactions

Purpose: The purpose of this research is to develop a shelf-stable olfactory identification test kit as a point-of-injury (POI) pre-screening device that can identify post-traumatic deficits in the sense of smell. A common development after a TBI is the loss of smell, which is often overlooked by doctors who do not test for this condition. Our collaborators have shown promising results and argue that the presence of post-traumatic olfactory deficiency should be viewed as a serious prognostic clinical sign. They have identified 7 to 10 odors of interest to develop a smell kit for the clinical evaluation of TBI. The University of Pennsylvania Smell Identification Test (UPSIT) kit currently used by medical personnel to test patients for anosmia is not intended to screen for a TBI and has a limited shelf life of 6 months. UPSIT uses 40 scratch & sniff items to determine if an individual can correctly identify specific odors. The AMMO device, with a proven shelf life of 18 months (24+ month potential) at room temperature, is expected to provide an early detection of concealed neurologic injury in soldiers with acute TBI resulting from combat blast exposure. In developing this Class II medical device, we identified and mitigated an excipient interaction that readily degraded one odorant within days. The formulation of chemical odorant “Natural Gas” exhibited lower smell intensity over time while also producing degradants observable by Gas Chromatography (GC). It was important to maintain using the identical excipients for all odorants, thus stabilizing this formulation was a key motivation versus investigating alternate excipients. Here we examine the potential variables causing the degradation and potential stabilizers that led to the approved device.

Methods: Odorants were formulated under Current Good Manufacturing Practices (CGMP) into crushable vials and kitted with answer option cards. A dye (Excipient B) was added to all formulations to serve two important functions: 1) blinding the end-user from potential color differences between odorants and 2) acting as a positive visual indication that the odorant successfully wicked from the crushable vial up through the device swab. Stability tests were performed on CGMP formulations for six (6) individual chemical odorants after packing. Specifications for smell identification were also developed and used to track chemical stability with the device efficacy as a function of relative odor intensity. GC-FID (Flame Ionization Detector) and (NMR) Nuclear Magnetic Resonance were used as primary techniques for tracking chemical stability and verifying content (assay) specifications. The formulated lot of vials for chemical odorant “Natural Gas” was found to have insufficient smell intensity, prompting an investigation that identified a degradant along with decreased odorant content. After investigating the impact of packaging materials and headspace on this degradation, we aimed to assess individual components with the following investigative formulations: a control with no excipients, 50% quantity of each individual excipients, and 100% quantity of individual excipients. These were then tested by GC-FID over time to observe any effects and trends. After narrowing the degradation interaction to one excipient, three (3) stabilizers were screened to observe relative impacts to the final formulation’s stability.

Results: GC results of the control formulation without excipients showed ~14% loss of the “Natural Gas” odorant over 7 days. This compares well with both formulations containing 50% and 100% of Excipient A, each exhibiting 10 and 12% loss, respectively, at 7 days. However, 7-day odorant loss trended high in formulations with 50% and 100% of Excipient B (dye), exhibiting 73% and 98% loss, respectively. Gas chromatograms for all time points show the appearance and increase of a degradant at relative retention time (RRT) 12.47 minutes, indicating that vapor loss of the odorant was not major contributing factor among formulations. After assessing potential chemical degradation pathways of “Natural Gas” odorant, three (3) potential stabilizers were screened: sodium thiosulfate, sodium sulfite, and citric acid. Only citric acid provided a meaningful stabilizing effect. Further, citric acid was successful in keeping odorant loss around 4% over a 7-day period. This study led to the incorporation of citric acid into the optimized final formulation for the CGMP Class II Device, which has continued to show stable “Natural Gas” odorant at the 3-, 6-, 9-, 12-, and18-month time points.

Conclusion: We successfully identified and mitigated the degradation cause of the “Natural Gas” odorant through thoughtful experimental design and the use of citric acid as a stabilizer. All six (6) odorants showed stability over 18-months as of February 2025, with anticipated 24-month stability at room temperature. This research provides a shelf-stable olfactory identification test kit for identifying post-traumatic deficits in the sense of smell.

Acknowledgements: The views expressed are those of the authors and do not reflect the official guidance or position of the United States Government, the Department of Defense, or the United States Air Force. The authors are contractors working with Air Force Research Laboratory/711th Human Performance Wing and the views expressed do not reflect the official guidance or position of the Southwest Research Institute or Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Imagery in this document are property of the United States Air Force. The author reports no conflict of interest. This research was conducted by Southwest Research Institute under SwRI project # 27767: A.M.M.O. Test Kit for TBI: Production Phase II, final amended approval 03 November 2022. This work was performed in collaboration with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (Award No FA8650-21-2-6250) and the Air Force Research Laboratory (Contract No.: CON000664).

Figure 1. GC overlay showing peaks related to fresh "Nature Gas Odorant" (black) and aged/degraded "Nature Gas Odorant" (blue).

Figure 2. Top: "Nature Gas Odorant" stability over time in investigative formulations, controlling for each excipient. Bottom: "Nature Gas Odorant" stability over time when formulated with various stabilizers.