Episode 64: Are Nitazenes Being Missed? Stability in Post-Mortem Toxicology

[Music] Hi everyone, I'm Macaw back from Tox Lab, I'm Rebecca. I'm Rob, this week we're back looking at knit-a-zines. We're going to be asking the question, are we sure we're picking up all the cases? So Rebecca, before we look at knit-a-zines specifically, I want to think in terms of drugs more generally, when we're looking at drugs post-mortem, how do we know we're picking up all the cases? It's tricky, I mean you want to make sure that like your extraction method can actually extract the drugs you want to look for, you've then got questions on how stable drugs are in samples or in that post-mortem interval. There's lots of different factors that come into play. With regards to stability, how do we know whether a drug is stable? I mean I guess looking at literature for a lot of the common things. So follow up question, what happens when that drug is pretty much brand new and not being seen before? Ooh, that is a good question. And that's what we're going to be looking at this week. We're actually going to be looking at a story that happened a couple of years back now, and the data that it led to has finally been published. And shameless plug, this is a publication that we've been involved in. Before we jump into today's episode, if you want to step to stay with our content, you can find us on Instagram @v_tox_lab. We also have Pedro LinkedIn and we're fairly active on there. And for those of you who don't use social media, but still want to get in touch with websites, or comments, websites, you can email us @[email protected]. So before we talk about today's actual publication, I'm going to actually open up with the story that led to the development of this paper. Ooh. Because it is quite a story. So as I said, we're going to be talking about nitrazines, and we've talked a lot about nitrazines. Haven't we? Ultra potent opioids originally developed in the 1950s. Shelved, never found medical use, emerged again in 2019 onwards, as drugs, contaminants, and drug supplies. So I want to take us back to 2023, about two and a half years ago. Rishi Sunak was prime minister, and I guess that means Joe Biden would have been in the White House at this point. Greedy by Tate McRae was on the radio. I'm told. Oh, no idea. No, I've got no idea either. And we were amidst intoxicology, the movement of nitrazine drugs from niche outlier occasional cases to a lot more mainstream. And this is the story really of two cases that came through our lab. And so the first case was from a known heroin user, and everything about this case at first glance looked relatively straightforward. She was found deceased with the syringe still in her arm, which is not a common finding, but we do see from time to time. But what was unusual about this case is, for whatever reason, they decided to take two sets of toxicology samples. A sample where the individual first arrived at the mortuary, and a second sample a week later when the post-mortem happened. Both sets of samples came to our lab. We analyzed the sample, looked like a classic heroin case, identification of morphine, noscopine, also identified metanitrazine and xylyzine. And anyway, long story short, I wanted to check a few more things I wanted to re-analyze the sample, because at this point, nitrazines were still fairly new to us. And that initial sample that was collected on day zero, it wasn't enough sample left. So without really even thinking too hard, I picked up the vial from seven days later, and analyzed that. You remember what happened, don't you, Rebecca? Yeah, this was when I'd like just started properly getting into toxicology after my training had finished. And yeah, it was a really weird set of circumstances. And so we analyzed that second sample, and the metanitrazine was no longer there. Now I'm going to be honest, I doubted myself. I thought I had done something wrong, I've re-checked everything. I couldn't go back to that original sample, because there was no sample left. I was convinced I had done something rogue. Anyway, maybe a week later, and I was still thinking about this case at this point. We had a second case come in, and it was very similar in terms of circumstance. Another heroin user had sadly passed away. And like the first time, two sets of samples were taken. We analyzed the first sample, and sure enough, the pattern was very similar. Consistent with heroin use, plus metanitrazine, xyrazine. And sure enough, we analyzed the later sample, the metanitrazine, has vanished. So I'm scratching my head at this point. I don't know what's going on. But there was some indication that nittazines were a little bit unstable. We had a little bit of stability data. I wasn't quite prepared for this. That's all I thought about it, and I thought about it. And eventually I spoke to a friend of ours, and between us, we hatched a plan to try and test what we had been seeing. And so our paper this week is that study that we conceived. So Rebecca, why don't you dive in, unpack this paper for us? So as we've hinted at this paper, it's looking at stability data scenes in a rap model. So 12 male with star rats were given either metanitrazine and concentration of 0.02 milligrams per kilogram, or 0.001 milligrams per kilogram. Some were given an end as ethyl-isotinatrazine at concentration of 0.006 milligrams per kilogram. And others were given an end perilidine etanitrazine, a concentration of 0.003 milligrams per kilogram. So really, really small doses were like, yeah. And each metanitrazine was given two four different rats. The original dose of metanitrazine in this, the 0.02 milligrams per kilogram was too high and instantly killed to the rats. So the lower dose was used for the other two. So when these rats had died, which in four of them was incident, the other eight animals were euthanized, and then Venus blob was collected and stored in both preserved and on preserved containers. And urine was also collected. The rats were then stored in the fridge for a week, to mimic real-world conditions of storage and time frame of bodies being stored before a post-multimus carried out. And seven days later, blood sampling happened again. And these again were collected in preserved and preserved tubes and stored at -80 before being analyzed using Iris Masspeck following a liquid liquid extraction. These samples were then also stored in the fridge for a month, so we could look at how stable the nittatines were in that sample once they'd been collected. So when we took some rats, they were given nittatines. We took samples, we simulated that post-multimum interval and took samples again. And when looking at the results, there was a huge variation in the concentration of each nittatine that was measured between animals who were given the same dose across both the unpreserved and the preserved samples. When looking at concentrations of nittatines between the sample collected on day zero and the sample collected at seven days after they'd been in the fridge in preserved containers, there was a reduction in the concentration of the nittatines with an average of 26.7% of the most. So it was 6.7% of the methanitine that was measured on day zero remaining on day seven, 36.1% of the endazethyl isotinitine from day zero remaining on day seven, and 54% of the end perlidina etinitine that was measured on day zero remaining on day seven. And there was also this similar trend detected in the unpreserved samples too. So considerable loss of nittatines in that post-multimum interval? And that the samples collected on day seven that had been kept in the fridge for a month is another marker of stability within the sample itself. And there was also a reduction in the concentration of nittatines that measured between that day seven and a month after they'd been in the fridge. It was a mean average of 20.9% of the methanitine measured on day seven present amount later, 9.4% of the endazethyl isotinitine measured on day seven was present amount later, and 12.8% of the end perlidina etinitine that was measured on day seven. So considerable degradation within sample as well. Yeah, we did also look at the urine samples. Nittatines were detectable in six of the preserved samples and eight of the unpreserved samples which were analysed immediately. They couldn't collect urine on day seven to look at the post-multimum stability of nittatines in urine. But when the samples were retested a month later, there were similar concentrations of nittatines being detected in those samples. So up until this point, there was data that was collected in the sample. So up until this point, there was data that showed nittatines were unstable within samples. We've talked about a paper here before, haven't we, about the instability of nitro nittatines and how they can be converted to their amino and acetamidome metabolites. This was the first data, as far as I'm aware, that showed significant loss in that post-multimum interval. Particularly, in a relatively short period of time, we were losing considerable amounts of nittatines. So in this paper as well, a retrospective cohort study was conducted using data from NPSOM, which is the national programme on substance use mortality, looking at relevant cases from those reported to NPSOM by the 1st of March 2025. Nittatine cases were identified by searching the post-multimum drug fields by the numerical crates corresponding to nittatine compounds and those from the Birmingham and Solohol coronal area. And this area was chosen because they turn around their inquest conclusions really, really quickly, so the data is most complete from that area. 624 cases were reported to NPSOM where deaths occur between 2019 and 2023 in this coronal area. When looking at the presence of nittatines, the number of non-nittatine deaths in 2023, which was 142 deaths, significantly exceeded the number of deaths that were expected when looking at the trends for 2019 to 2022. When looking at the presence of more than 13,000 deaths in 2020, there was significantly more deaths in 2023, with more than 23,000 deaths being detected than was forecasted from the 2019 to 2022 trend, and it looked to be about a 33% increase. While the number of deaths without nittatines or heroin or morphine in 2023 did not exceed the expected number, this was towards the upper limit of the model with the actual number being 71 deaths, but the projected number of deaths was expected to be 53, and the upper confidence interval was 73. This paper also covers the profile of deaths following nittatine use. 285 deaths were reported to NPSOM by the 1st of March 2025 with the involvement of nittatines. The 1st case was a case of ice tin etacine in 2019 where an individual had intentionally purchased this in the dark web, and since then small clusters of nittatine use had occurred in 2021 with 23 cases, and in 2022 with 15 cases, and those are marked increase in 2023 with 151 cases, and the projected number of cases in 2024 was 236. Only 25 of these cases had a purchased intent, and nittatines were the intended purchase item in 6 of these cases. 12 nittatines were detected in this data set with 7 for the first time being detected in 2023. Ice tin etacine was the most commonly detected nittatine in 2021, covering 78% of these cases, and pyrolydino etanetazine in 2022 was the most common with covering 60% of the cases, and as etherl ice tin etacine was the most popular nittatine in 2023 and 40% of these cases, and proton etacine was the most common in 2021, and the price of nittatine was 40% of these cases. And pyrolydino etacine was detected most in 2024, covering 77% of the cases in that year. When looking at causes of death, nittazines were deemed to be the cause of death in 89.8% of cases involving nittazines, and 97.5% of these cases were deemed unintentional with four being suicides and the remaining three being of undetermined intent. When looking at the number of nittazines were detected across these cases, a single nittazine was detected in 87% of cases. Two nittazines were detected in 33 cases, and the most common combination was methanetazine and proton etacine, and three nittazines were detected in four cases. Polydrug use was present in all but four cases, with cocaine being the most commonly coded detected drug in 78.2% of cases, followed by the presence of heroin slash morphine in 72.3% of cases, and xylazine was detected in 33 cases. The loxonese was also detected in 47% out of the 285 cases. The majority of deaths in this data set were from England, which covers 90.2% and these occurred across the country. 8% of cases came from Wales and 1.7% of cases came from Northern Ireland. Nittazine cases predominantly involved males and a significantly greater proportion of nittazine cases occurred with individuals experiencing homelessness or living in hostels compared to non-nittazine cases. A history of drug use was significantly higher in nittazine cases compared to non-nittazine cases. There was a higher prevalence of drug use for injection. So this paper really was a paper in three parts, wasn't it? The stability data, the mathematical modelling, and then the profiling of the cases in which data was known. So let's go back to the stability data, first of all. What are the implications here? Firstly, let's go back to our story, our observation that led to this whole slightly bonkers experiment in the first place. I mean, the implication for me was, okay, I wasn't going mad, I hadn't done anything wrong, which had definitely plagued me for some weeks. But then once you get over that initial feeling of, oh, I was right, you then start to reflect on the real reality, don't you? That actually we just don't know how many cases could involve nittazines. We've got data that shows they're really unstable, both within the post-mortem interval and within the sample. We've got albeit low case number anecdotal evidence of nittazines disappearing in real life samples. How many cases out there could have been nittazine cases whereby they simply wasn't enough or any nittazine to detect by the time the sample was analysed? I mean, it's so hard to put a number on. I've definitely had the odd case where I'm convinced there was something like nittazine present that we just couldn't pick up for whatever reason. And it's just one of those unknowns that's just going to bother me for a long time. In some ways, it's quite frustrating, doesn't it? Because probably isn't just a case of, we'll get better equipment. This is the nature of the compounds, how they behave. So I want to look at the mechanism a little bit because we don't really understand the mechanism, but there are some speculations and some thoughts. So we know that nittazines are unstable in blood samples and that's been shown. We think through potentially bacterial conversion to five amino and then acetamidometabolites. What's going on in that short interval between death and sample collection? We know nittazines are really lipophilic, so they're probably being redistributed into the fatty tissue. Like what happens with other drugs, but generally we see drugs that have been taken for a long time, like SSRIs, for example, coming out of the fatty tissue during that post-mortem interval. But I feel like it's happening the other way around. You stole my lines, that's my line. No, you're absolutely right. And that is the hypothesis I've been postulating that this is like in inverted commas reverse post-mortem redistribution. I can't prove that. We haven't supported that with science as yet. It really is a speculative hypothesis. But it would explain why, particularly in these cases where individuals have injected nittazine directly into their bloodstream that very quickly it seems to be leaving that bloodstream into tissues that aren't the blood. At least that's my hypothesis. So let's look at the mathematical modeling then. That did seem to show that there could have been some cases we were missing. Yeah, it seemed to post on the model there was an increase that wasn't predicted based on the previous years trends. So there may have been some more cases. Possibly, but what does that mean? No, for sure. And finally, let's drill a little bit into the nittazine profile papers. You talked about that first case. Yeah. And actually that was a case I was involved in. And that was a really unusual case. Somebody deliberately seeking opioids from the dark web. But since then we've seen that trend haven't we towards this being less of a drug that individuals seek and more of a drug that contaminates typically heroin, but sometimes other drugs too. And he's more often than not, not what was intended when individuals took the substance. Yeah, it's especially scary with nittazines appearing in counterfeit pharmaceuticals like counter-oxycodones or even counterfeit bensos. And that's the trend that does really worry me because those individuals, especially with the benzodiazepines don't don't they're getting an opioid and then do when nittazines occur in heroin, the individuals are at least intending to purchase heroin and probably have a degree of opioid tolerance. Yeah. That isn't the case usually if they're deliberately seeking benzodiazepines, they may not have an opioid tolerance. And that general pattern of nittazines that were described that really does fit what we saw didn't it the move from isotinitazine to endezethylisotinitazine to metinitazine and then to protonitazine later on. And as we've seen, as we've talked about before, that trend really has continued, hasn't it? We saw then newer nittazines and now possibly we're looking at a move away from nittazines towards orphine opioids. And so I'm going to ask you a question now, Rebecca. How stable are orphine opioids? No idea. Precisely. I'd love to have this experiment repeated with the orphines. It'd be really interesting to see whether they're one more stable or less stable in the postmodement of interval, but also how stable they are in samples. Yeah. I think we've now moved drug classes and suddenly we're back to square one. We're back not knowing again. Always on the back for the toxicology. One thing we didn't actually mention at the start was that alongside all of the chaos, the BBC did get involved, didn't they? And they did make a little documentary about this story. So we'll drop a link as well to that. That is available. It was quite well produced, actually, I thought and does feature the sound of my voice at points, but it's a different retelling of the same story. So really in summary then, I mean, this is it's going to sound like a really, really bold and arrogant thing to say, but this is the story of science, isn't it? Yeah. A chance observation that led to some awkward inward reflection about whether I had done something wrong, followed by some really solid collaboration with some colleagues, designing a hypothesis, testing that hypothesis, having some results, and kind of confirming that initial observation. But also I do, you know, when I reflect on this story a bit, you do have to remember that these were two authentic cases, these were two people who sadly lost their lives due to nitazines and they'll never know that legacy that they left behind. So thank you again for listening this week. Do give us a like, give us a follow, share this with your friends, and also check out the paper. We'll put a link down below as we do every week. Hope you all have an amazing week and we'll see you next time. Bye!