Although general anaesthetics (GA) have been in use since 1846 and hundreds of millions of people every year worldwide undergo surgery, we still lack a full explanation of the mechanism of action of most agents used in general anaesthesia. This knowledge gap has hindered our capacity to address some aspects of general anaesthesia that remain problematic, in particular recovery from deep anaesthesia, especially in elderly patients. Considering the shift to older demographics and the increased reliance on general anaesthesia for most surgeries, there is a clear need to better understand why recovery can sometimes be difficult or unduly prolonged, and whether designer drugs can help with this process. This lack of a capacity to counteract the anaesthesia process has serious secondary consequences, such as increased length of hospital stay, and more post-operative complications, including post-operative cognitive decline.
The aim of this study is to investigate whether two propofol analogues are effective in reversing the effects of propofol on syntaxin1A mobility at the synapse when provisioned in combination with clinically relevant concentrations of propofol. This work will be done in fly brains and mammalian cell cultures in the laboratory of Professor Bruno van Swinderen at the Queensland Brain Institute.
The study will provide a platform for screening and testing candidate reversal agents for general anaesthesia, based on our growing understanding of a relevant presynaptic process that is targeted alongside the better-understood post-synaptic receptor targets. This platform will help establish novel approaches to reversing GA, based on a better understanding of the underlying molecular processes.
Professor Andre van Zundert, Royal Brisbane and Women’s Hospital, Queensland.
Associate Investigator: Professor Bruno van Swinderen, Queensland Brain Institute.
The project was awarded $A69,687 through the ANZCA research grants program for 2023.
Recovery from general anaesthesia is an entirely passive process, with no currently available reversal agents available for expediting recovery time in patients. In part, this is due to lack of knowledge about anaesthetic mechanisms of action, especially potential presynaptic mechanisms. We found that common general anaesthetics such as propofol impair neurotransmission, and that this likely happens by way of impairing the mobility of presynaptic proteins such as syntaxin1A and munc-18. We therefore sought to investigate whether non-anaesthetic analogues of propofol (e.g., 2,4diisopropylphenol) could counteract the presynaptic effects of propofol, and also whether they exerted any effects on their own.
More broadly, our main goal was to develop a platform to test candidate reversal agents in diverse biological preparations, such as cultured cells and potentially live animals. Irrespective of the outcome of 2,4-diisopropylphenol, this would serve as a paradigm for effectively screening a wide range of potential reversal agents in the future. Discovery of reversal agents that could overturn the presynaptic effects of common drugs such as propofol in these reductionistic and small animal preparations would then present as candidates for further trials in live mammals and eventually clinical trials. The goal would be to not only provide better control of general anaesthesia procedures, but also to improve recovery outcomes in vulnerable patient populations that are more likely to be affected by exposure to general anaesthetic drugs.
Our outcomes were successful insofar as we did develop a platform for testing the effect of candidate reversal agents for sophisticated presynaptic readouts such as single-molecule imaging and neurotransmission. However, much of our efforts were dedicated to optimising these novel paradigms, and as a consequence, our final datasets remain of low sample size and thus underpowered. Additionally, in the process of optimizing our assays we settled on an alternate approach to stimulating neurosecretory cells, using BaCl2 as opposed to high K+.
Although our data remain preliminary and underpowered, we could conclude that 2,4-diisopropylphenol is unlikely to present as a promising propofol reversal agent. While neurotransmission readouts were tentatively promising, our single-molecule imaging (super-resolution) experiments suggest that the analogue does not counteract the effect of propofol. Finalizing this dataset should provide a more definitive conclusion, at the level of significance testing. We aim to still achieve a sample size of ~10 cells for each experiment, so that this conclusion might be solidified.
In the course of achieving our outcomes, we added an additional layer to our experiments: a new paradigm to test the effect of potential reversal agents in live nematode worms (which can be soaked in the drug, to assess possible effects on behaviour and survival more generally). This proved a useful assay, as we found that 2,4-diisopropylphenol rapidly killed the animals. We have since adopted this live animal assay as a crucial test to assess the potential toxicity of these agents.
In more recent experiments using the same cell platform combined with live animal screening, we have found other propofol analogues that do not kill the nematodes, while also seeming to rescue the presynaptic readouts. These will form the basis for our next ANZCA project report.
We have established a screening platform to discover potential reversal agents for general anaesthesia, focussing on two presynaptic endpoints: neurotransmission and single molecule imaging of presynaptic protein dynamics. In this first approach we focussed on a propofol to examine effects in a live animal model, Caenorhabditis elegans. This is an important addition to this study. While it is likely that some non-anaesthetic analogues might reverse presynaptic phenotypes in a reductionistic preparation such as cell cultures, it is important to also verify that these drugs are not toxic in a live animal preparation. We have accomplished this goal in this study, showing that out first candidate agent, 2,4-diisopropylphenol is lethal to nematode worms.
By establishing this approach linking presynaptic effects to whole-animal behaviour, we have designed a working platform for screening other potential reversal agents. This platform is aimed specifically at uncovering presynaptic effects, which present a newly discovered conserved target for many general anaesthetics. Any uncovered effective presynaptic reversal agents could then be trialled in combination with existing post-synaptic drugs, to better control emergence from general anaesthesia.
Conclusions
In this first trial for testing potential reversal agents for common general anaesthetics such as propofol, results were mixed. We have successfully set up a platform to test these drugs at a level never tested before, namely investigating presynaptic functions such as neurotransmission and presynaptic protein dynamics. Additionally, we have developed an efficient and cost-effective live animal assay (nematodes) to test whether any drugs might be toxic, irrespective of their potential presynaptic effects. In our first trial we found that 2,4-diisopropylphenol (a non-anaesthetic analogue of propofol) kills nematode worms. While this is a disappointing result, it does pave the way for effectively screening these drugs, before proceeding to more clinically-oriented trials. We also exerted considerable effort optimising both of our presynaptic assays, settling on a BaCl2 approach to activate neurosecretory cells. Consequently, sample sizes remain quite low some of our results for this first investigation, although the data taken together indicate that 2,4diisopropylphenol is unlikely to overturn the effect of propofol on presynaptic function – the key question central to this project.
Trials are ongoing for uncovering alternative presynaptic drugs, focussing on fluorinated analogues of propofol. These appear to be much more promising, with excellent outcomes and form the basis of our second funded ANZCA project. Additionally, the fluorinated propofol analogues we have tested so far do not kill the nematode worms. The same approaches as optimised in the project reported here therefore being taken to screen these new candidate reversal agents.
Published
Cylinder DM, van Zundert AAJ, Solt K, van Swinderen B. Time to Wake Up! The Ongoing Search for General Anesthetic Reversal Agents. Anesthesiology. 2024 Mar 1;140(3):610-627. doi: 10.1097/ALN.0000000000004846. PMID: 38349760; PMCID: PMC10868874.
Conference Presentation
Cylinder DM, Hines A, Kerwin A, Dunn J, Liu ZY, van Zundert AAJ, van Swinderen B. Investigating Reversal Mechanisms of General Anaesthesia. Australasian Neuroscience Society (2023) Brisbane, QLD, Australia.