We kicked off this week in an exciting fashion: mixing our custom formulation of naloxone and a reference solution of noroxymorphone for an accelerated aging stability test. Brad and Jonathan had the pleasure of spending Sunday (mother’s day) assembling our “pop-up-lab” space at the AlphaLab Health Office inside of Allegheny General Hospital suburban campus. On Monday, our small molecule chemist Al Ritter arrived (after a long drive from Kentucky), and we got to work mixing up drugs!
Our popup lab at AlphaLab health
First, (and most obviously) because the FDA requires stability tests of drugs before approval. The test we initiated this week won’t be the only test we submit to the FDA, but it will serve as a proof of concept.
Second, The Resilient System has unique characteristics that make the stability of naloxone more interesting. To quickly detect and treat an opioid overdose, the Resilient System is meant to be worn by users for extended periods of time. This means that the naloxone inside of the device will be near body temperature (roughly 40° C) for extended periods of time. Since heat increases the speed at which a substance degrades, we need to prove that Naloxone can be kept at body temperature for extended periods.
Some existing naloxone products claim a shelf life of up to 6 months when occasionally exposed to high heat. Our goal for the Resilient Monitor is 3 years. So, we have a lot to prove to the FDA, but there’s some great data from past research to support our optimism here.
One study by Pruyn et. al looked at naloxone products that were up to 27 years past expiration and found nearly all samples to be within quality standards for a drug. 1
Another study by Lai et al heat cycled naloxone ampoules between 80 degrees C and room temperature for 30 days and found minimal degradation. 2
So, there’s some convincing data that naloxone will last a long time if kept in the right conditions. In fact, the only data we could find showing significant degradation of naloxone with time and heat was done by the world health organization (WHO) in 1983, where the drug was exposed to temperatures between 50°C and 70°C for 30 days. 3 In this study, the naloxone was left in the open, exposed to oxygen, so we hypothesize that oxygen is the primary culprit for naloxone’s degradation.
Accelerated aging tests rely primarily on the Arrhenius equation, which states that with every 10°C increase in temperature, the speed of a reaction approximately doubles.4 We use this to find an accelerated aging factor:
Accelerated Aging Factor (AAF) =2(X/10)
where X is the difference between the aging experiment temperature and the expected storage temperature in °C. Since our expected “storage temperature” in this case is body temperature (40°C), we used 60°C for the aging experiment temperature.
You can then divide your desired shelf life by the AAF to find the required duration of the accelerated aging:
Accelerated Aging Time (AAT) = required life expectancy / (AAF)
Using these equations, we realized we needed to age naloxone for 33 weeks, which we rounded up to 9 months. So now we need to keep a drug at 60°C for 9 months. This is the easy part. You buy an oven and stick the sample inside.
Over the course of the next 9 months, we’ll periodically send samples to our partners at Exemplify Biopharma so they can run High-Precision liquid chromatography on the samples and tell us how well our naloxone is aging. They’ll be looking for two things:
This is straightforward. We need at least 90% of the original naloxone concentration to remain to be within standard.
This is more interesting. Turns out that naloxone degrades into noroxymorphone, which is an opioid agonist like fentanyl. For some pretty obvious reasons, we don’t want to have an additional opioid in the drug that we’re administering to reverse an opioid overdose and save a life. Fortunately, past research has shown presence of noroxymorphone to be incredibly limited in even the most expired naloxone solutions, but we decided to test for this out of an abundance of caution.
This meant we had to prepare a noroxymorphone solution as a reference so the lab would know how to detect its presence in an LCMS test. Since Noroxymorphone is a controlled substance, we had the fun experience of registering with the DEA and building a mini fort-Knox in our lab to securely store controlled drugs.
The “ingredient list” for injectable naloxone is quite simple:
To make a 25ml solution of naloxone, we first dissolved the desired amount of salt and naloxone in a smaller volume of water. We then added water to the solution until we had nearly 25ml of water before adjusting the pH.
To adjust the pH, we added a single drop of hydrochloric acid, which in our case was way too much. We balanced this out by adding a solution of sodium hydroxide (a base) until reaching a pH between 3 and 4.5. One cool observation was that when adding the sodium hydroxide, each drop would cause some naloxone to briefly precipitate out of the solution, with some cool visuals.
Naloxone precipitating out of solution when a base is added
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Once we had the pH corrected, we then passed the naloxone through a sterile syringe filter into our cartridges, which resemble the cartridges system we intend to use in the final Resilient System.
We followed a very similar procedure for the noroxymorphone solution.
We of course took many steps to make the solution as pure and sterile as possible for a non-GMP manufacturing environment:
It had been about 20 years since I last performed any kind of chemistry lab work, and it was impressive to work besides the consummate professionals Jonathan Birabaharan and Al Ritter in getting these samples prepared. Time will tell how successful we were in preparing stable solutions, but it was exciting and encouraging to take our first giant step in the drug development world.
If we meet our goals in this study, we’ll be excited to publish these results and add to the growing body of work that supports an extended shelf life of naloxone products. Opioid harm reduction organizations already instruct consumers to use expired naloxone in cases where it is needed 5, and no alternatives are available. Hopefully, we’ll keep seeing naloxone shelf lives extend, increasing availability.
1. Pruyn S, Frey J, Baker B, Brodeur M, Graichen C, Long H, et al. Quality Assessment of Expired Naloxone Products from First-Responders’ Supplies. Prehospital Emergency Care [Internet]. 2019 [cited 2024 Mar 20];23:647–53. Available from: https://www.tandfonline.com/doi/abs/10.1080/10903127.2018.1563257
2. Lai D, Pham AT, Nekkar Rao PP, Beazely MA. The effects of heat and freeze-thaw cycling on naloxone stability. Harm Reduct J [Internet]. 2019 [cited 2024 May 14];16. Available from: /pmc/articles/PMC6391798/
3. Accelerated stability studies of widely used pharmaceutical substances under simulated tropical conditions [Internet]. [cited 2024 May 14]. Available from: https://iris.who.int/handle/10665/61480
4. F1980 Standard Guide for Accelerated Aging of Sterile Barrier Systems and Medical Devices [Internet]. [cited 2023 Jul 4]. Available from: https://www.astm.org/f1980-21.html?gclid=CjwKCAjwqZSlBhBwEiwAfoZUIC8XM16JffHPca_ZJ92LE9Z7cpqlcnwmRMTZkQSM2NtRQcvnpTOhHxoC4HEQAvD_BwE
5. About Expired Naloxone — NEXT Distro [Internet]. [cited 2024 May 14]. Available from: https://nextdistro.org/mightynaloxone
