The effects of heat and freeze-thaw cycling on naloxone stability

In North America, opioid-related overdose is the leading cause of death in individuals between the ages of 18 and 35 [1–3]. In 2016, there were 2816 opioid-related deaths in Canada with approximately one third occurring in Ontario [4, 5]. Since 2003, the rate of opioid-related deaths has more than doubled [5]. In response to this crisis, prescription requirements for naloxone, an opioid antagonist used to reverse opioid-overdoses, were modified in 2016 by Health Canada and the National Association of Pharmacy Regulatory Authorities (NAPRA) [6–8]. These changes permitted Canadians to access to THN without a prescription and also expanded the description of who is eligible to receive a naloxone kit [6–8]. Currently, several jurisdictions in Canada, including Alberta, Ontario, Northwest Territories, and Yukon, offer unrestricted access to THN to anyone at risk of an opioid overdose or those in a position to assist in an opioid-related overdose [9].

The availability of THN outside of healthcare settings raises concerns regarding naloxone stability when exposed to conditions outside of the 15–30 °C range as indicated in the product monograph [10]. For example, individuals often inquire about the possibility of THN kit storage within their vehicles for accessibility (personal communication). Storage of THN kits outside or in vehicles may be problematic due to extreme and fluctuating temperatures among the seasons within Canada, which may impact naloxone stability and therefore efficacy. Expiration date and storage conditions are determined by satisfying the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidelines for Stability Testing of New Drug Substances and Products [11]. These guidelines, however, do not address unconventional conditions (e.g., temperature extremes or freeze-thaw cycles). The findings of this study intend to provide healthcare providers, specifically pharmacists, with practical information to relay to individuals with THN kits who may have deviated from the recommended storage conditions.

Previous studies have investigated the consequence of naloxone storage under unconventional conditions. However, they are limited by the fact that they do not reflect conditions that may be encountered if THN kits were left, either intentionally or unintentionally, within a vehicle. For example, although temperatures as high as 70 °C have been evaluated, internal vehicle temperatures can reach up to 80 °C [12, 13]. Several studies also assessed the effect of temperature fluctuations from extreme cold to heat (− 20 °C to 70 °C; − 6 °C to 54 °C; − 20 °C to 45 °C); however, it is unlikely that these conditions would be experienced within the span of a single day, should a THN kit be left within a vehicle [12, 14, 15]. These studies also did not investigate the association of stability with the duration of exposure. Therefore, the objective of this study was to identify how daily exposure to heat (mimicking vehicle storage in the summer months) or freeze-thaw cycles (mimicking vehicle/outdoor storage in the winter months) may impact the stability of naloxone hydrochloride for up to 4 weeks. Findings of this study intend to provide pharmacists with additional counseling points should THN kits be stored outside of the recommended conditions.

In Canada, drug manufacturers must complete stability testing as outlined by the ICH guidelines. Rarely do these guidelines address temperatures exceeding 40 °C or below 5 °C. In addition, research studies evaluating drug stability in extreme environments are few and far in between, especially those addressing the impact of freeze-thaw cycling. Therefore, this study was designed to address conditions that may be encountered by individuals in possession of a THN kit. We assessed the impact of daily exposure to either heat (80 °C for 8 h followed by room temperature for 16 h) or freeze-thaw cycles (− 20 °C for 16 h followed by 4 °C for 8 h) on naloxone hydrochloride stability. Regardless of duration and exposure to either condition, no detectable reduction in naloxone hydrochloride concentration was detected by HPLC, demonstrating drug stability under these conditions.

The stability of naloxone hydrochloride exposed to thermal stresses has been investigated by several studies. However, findings have been variable due to inconsistencies in experimental design, duration of exposure, and methodology. For example, older studies have found that naloxone remains stable under thermal stress whereas newer studies demonstrate heat-dependent degradation [12, 14–16]. In the earliest study, naloxone hydrochloride was stored in a white metal shed to simulate a paramedic vehicle parked during the summer months in Tucson, Arizona for 4 weeks [16]. Temperatures recorded ranged between 26 and 38 °C, which is marginally higher than the recommended storage conditions. These temperatures also fall within the temperature range recommended for stability testing according to the ICH guidelines. Considering these factors, it is not surprising that naloxone hydrochloride did not exhibit any changes in stability. It is, however, important to note that this study was conducted prior to the release of the ICH guidelines. Several years later, Johansen et al. exposed naloxone hydrochloride to − 20 °C, + 70 °C or fluctuating between the two temperatures [12]. Although the study assessed greater thermal extremes, the total exposure time was limited to only 16 h within a 48-h study period. These conditions had no effect on naloxone hydrochloride stability; however, it would have been insightful to study these effects over longer durations of exposure.

In contrast with these findings, newer studies have found that naloxone hydrochloride exhibits temperature-dependent degradation. Gammon et al. exposed naloxone hydrochloride to fluctuating temperatures of − 6 °C and + 54 °C every 12 h, and concentration was measured weekly for up to 4 weeks using HPLC [14]. By the end of 4 weeks, 89.62% of naloxone hydrochloride remained relative to the original concentration. This ~ 10% reduction in concentration was statistically significant. Despite the observed reduction in naloxone concentration, it is likely not clinically significant as the acceptable standards for manufactured and compounded drugs fall between 90 and 110% of the stated concentration [17, 18]. This study also possesses several limitations. First, the study did not contain a control ampoule stored at room temperature. Second, it appears that only one naloxone ampoule was used for the duration of the study. Although a baseline concentration was measured prior to thermal exposures, the remaining contents could have been subject to consequences of humidity (e.g., evaporation, condensation) or oxidation. A standard calibration plot was not included in this report.

Armenian et al. also observed naloxone hydrochloride exhibits temperature-dependent degradation [15]. Naloxone hydrochloride ampoules were subject to several conditions (sustained temperatures of − 20 °C, + 45 °C, or alternating weekly between the two) for 1 month. The most degradation occurred in the presence of heat with only 39% of the original concentration remaining after 4 weeks. Several factors may explain the degree of degradation observed. First, although ampoules were subject to moderate heat, the exposure was sustained for the duration of the study. Second, only one vial was used per thermal condition, which was sampled weekly. Therefore, the humidity and oxidation may have contributed to the extent of degradation. It is also important to note that an experimental control was not included in the study. Since HPLC was used to identify and quantify naloxone in this study, further characterization of degradation products by liquid chromatography-mass spectrometry (LC-MS) would have been useful in their overall analysis.

The limitations described above, in addition to other factors, were considered for our experimental design to reduce the impact of confounding factors. For example, no ampoules were tampered with until the end of the study where samples were run simultaneously via HPLC. This eliminated any impact that humidity or oxidation may have had on naloxone stability. Since all samples were run on HPLC at the same time, other external factors (e.g., column temperature) could also be controlled. Each sample was also measured in triplicate readings to ensure homogeneity of ampoule contents. Importantly, our study also included control ampoules, which were stored according to the manufacturer’s storage conditions.

However, this study is not without its limitations. First, we attempted to emulate conditions that would be generally encountered upon vehicle storage in winter or summer months. However, these conditions vary considerably. Ideally, assembled THN kits should have been stored in a car parked outside in the winter or summer months. Depending on the location within Canada, internal vehicle temperatures may differ. Temperature extremes may also be dampened by the THN kit housing and the location of placement within the vehicle. Second, although ampoules retain naloxone concentrations following thermal stress, we did not make any functional assessment of the naloxone samples (e.g., binding affinity to opioid receptors) in vitro or in vivo. Third, although ampoules were not tampered with until the end of the study, we still cannot eliminate secondary reactions or degradation that may occur from time of last exposure until the HPLC run. However, because ampoules were stored according to the product monograph during this time and all ampoules were exposed for the same duration of time, any effect would be negligible. Last, it would be useful to extend the duration of exposure beyond 1 month to assess how long naloxone hydrochloride can tolerate these conditions before the compound begins to break down.

This study is the first in Canada to assess the practical impact of heat and freeze-thaw cycles on naloxone hydrochloride stability. Our findings suggest that exposure of naloxone hydrochloride ampoules to temperatures up to 80 °C or daily freeze thaw cycles for 1 month appear to have no impact on its stability. Therefore, naloxone hydrochloride ampoules should remain efficacious should THN kits be temporarily stored outside of the recommended storage conditions. Despite naloxone’s stability, and because the clinical implications were not evaluated, pharmacists should continue to counsel patients on the importance of appropriate naloxone storage to ensure optimal efficacy. Furthermore, if any signs of precipitate become visible in the ampoule, the ampoules should be replaced. However, the naloxone itself remains stable in solution, and injection of such a product is warranted to reverse a life-threatening opioid overdose.