How it started
During my PhD at Lund University, I evaluated succinate prodrugs as potential treatment for drug-induced mitochondrial dysfunction. The succinate prodrugs were originally developed as therapeutic for genetic mitochondrial disease, with mitochondrial complex I being most commonly affected. Because succinate is used for energy production by mitochondrial complex II it can bypass complex I and serve as an alternative energy substrate. Mitochondria are a common target for a wide range of chemicals, such as organophosphates, and highly metabolic tissues are often a target of drug-related toxicities. Therefore, acute intoxication due to intentional or accidental exposure presents an additional target indication which can benefit from this pharmaceutical concept.
At the Children’s Hospital of Philadelphia (CHOP) and under the lead of Todd Kilbaugh, Associate Professor of Anaesthesiology, Critical Care, and Paediatrics, we have started to develop a multi-species pre-clinical toxicology and drug development platform to evaluate mitochondrial-targeted therapeutics as treatment for metabolic dysfunction due to chemical exposure. This toxicology program started in 2017 and is part of the NIH funded initiative CounterACT with the mission to support research and development of therapeutics that can mitigate the health effects of toxic chemicals, including traditional chemical warfare agents, toxic industrial chemicals, and pesticides. The succinate prodrugs is the first pharmacological strategy that is evaluated using this platform. We started off with cellular models of organophosphate, carbamate and Sodium Fluoroacetate poisoning, where we were able to improve mitochondrial function in vitro with the succinate prodrugs.1 Currently, we are leading this new drug class further down the pipeline and are testing the succinate prodrugs in experimental rodent models acute chemical exposure. The most recent of our work on ‘Succinate prodrugs in combination with atropine and pralidoxime protect cerebral mitochondrial function in a rodent model of acute organophosphate poisoning’ is available in the related content to this blog post.
Acute pesticide poisoning has become a worldwide health concern, with hundreds of millions of cases of acute poisoning worldwide each year.2-5 Anticholinergic compounds, such as organophosphates (OPs), are responsible for the majority of all pesticide-related deaths.6 Nerve agents of the organophosphate (OP) compound class pose an additional threat to our society and this threat has become even more evident in recent years. Sarin gas has been used in the Syrian civil war, A-234, a so called Novichok agent, was used in the Salisbury attack in 2018 and North Korean agents used VX gas in the murder of Kim Jong-Nam at the Kuala Lumpur airport in 2017. Death from acute OP poisoning is primarily due to respiratory failure, with survivors often demonstrating significant neurologic sequelae.7-9 Current standard of care (SOC) treatment for acute OP poisoning consists of supportive critical care and anticholinergic drugs, such as atropine and pralidoxime.6,10 While SOC treatment may alleviate acute symptoms and reduce short term mortality, survivors are at risk for development of significant cognitive and motor function deficits.6,11,12
The immediate lethal potential of OPs is due to their acetylcholinesterase inhibitory effects. However, an increasing body of evidence indicates that there are other important pathways that may contribute to the overall toxicity and development of neurodegeneration of these agents. Mitochondrial dysfunction has been reported as one of the primary non-cholinergic toxic effects of OPs.13 Targeting these effects with mitochondrially directed therapeutics, such as succinate prodrugs, to support mitochondrial function presents a novel treatment approach. Succinate prodrugs do not act on OPs themselves but act as a countermeasure to compensate for the mitochondrial toxicity.14 Prodrugs of succinate more easily pass through the cell membrane than regular succinate and can be directly metabolized through mitochondrial complex II of the oxidative phosphorylation system, thereby increasing mitochondrial ATP production.1,14-17 Additional studies have demonstrated that the succinate prodrugs are also able to support impaired mitochondrial respiration due to partial mitochondrial CIV inhibition, despite the site of toxicity being downstream of the target of the succinate prodrugs.1,14-18
We hypothesized that co-treatment of SOC treatment with atropine and pralidoxime with a succinate prodrug would support mitochondrial function and reduce brain injury during acute poisoning with the OP diisopropylfluorophosphate (DFP). To this end, we developed a rat model of acute DFP intoxication and evaluated the efficacy of the succinate prodrug candidate NV354 as adjunctive therapy to SOC treatment with atropine and pralidoxime. We demonstrate that mitochondrial CIV function was impaired in the brain during the acute phase of OP poisoning with DFP. This effect was not prevented by SOC treatment with atropine and pralidoxime or treatment with succinate prodrug NV354 alone. Interestingly, SOC in combination with the succinate prodrug NV354 prevented the OP-induced inhibition of mitochondrial CIV function. Our data indicate that the mitochondrial toxicity of DFP can be mitigated by NV354 as adjunctive therapy to SOC. Moreover, we detected signals of brain injury in response to DFP in our model, which were considerably reduced by SOC treatment, treatment with the succinate prodrug NV354, and the combinational therapy of SOC with the succinate prodrug NV354. Succinate prodrugs present a novel approach for therapeutic development in this area and show promise as adjunctive therapy to SOC treatment with atropine and pralidoxime.
What is next?
Based on these promising results we believe that further studies are warranted to investigate the potential of this novel drug class as preventive measure for the development of long-term cognitive- and motor function deficits following OP exposure. Furthermore, we will continue to build our multi-species pre-clinical toxicology and drug development platform to evaluate this and other novel mitochondrial-targeted therapeutics as treatment for metabolic dysfunction due to chemical exposure. This will not only include the development of more high-throughput models for large drug screens to bridge the gap between our cellular and rodent models, but also the addition of porcine models, which allows a close simulation of the clinical care setting.
1 Janowska, J. I. et al. Mitochondrial respiratory chain complex I dysfunction induced by N-methyl carbamate ex vivo can be alleviated with a cell-permeable succinate prodrug. Toxicology in vitro : an international journal published in association with BIBRA 65, 104794, doi:10.1016/j.tiv.2020.104794 (2020).
2 Boedeker, W., Watts, M., Clausing, P. & Marquez, E. The global distribution of acute unintentional pesticide poisoning: estimations based on a systematic review. BMC Public Health 20, 1875, doi:10.1186/s12889-020-09939-0 (2020).
3 Eddleston, M. Patterns and problems of deliberate self‐poisoning in the developing world. QJM: An International Journal of Medicine 93, 715-731, doi:10.1093/qjmed/93.11.715 (2000).
4 Mukherjee, S. & Gupta, R. D. Organophosphorus Nerve Agents: Types, Toxicity, and Treatments. Journal of Toxicology 2020, 3007984, doi:10.1155/2020/3007984 (2020).
5 Brenet, A. et al. Organophosphorus diisopropylfluorophosphate (DFP) intoxication in zebrafish larvae causes behavioral defects, neuronal hyperexcitation and neuronal death. Scientific reports 10, 19228, doi:10.1038/s41598-020-76056-8 (2020).
6 Eddleston, M., Buckley, N. A., Eyer, P. & Dawson, A. H. Management of acute organophosphorus pesticide poisoning. Lancet (London, England) 371, 597-607, doi:10.1016/s0140-6736(07)61202-1 (2008).
7 Misik, J., Pavlikova, R., Cabal, J. & Kuca, K. Acute toxicity of some nerve agents and pesticides in rats. Drug and chemical toxicology 38, 32-36, doi:10.3109/01480545.2014.900070 (2015).
8 John, J. J., Nagar, D. P., Gujar, N. L. & Bhattacharya, R. Oxidative and histopathological alterations after sub-acute exposure of diisopropyl phosphorofluoridate in mice: Beneficial effect of N‑acetylcysteine. Life sciences 228, 98-111, doi:10.1016/j.lfs.2019.04.067 (2019).
9 Terry, A. Functional Consequences of Repeated Organophosphate Exposure: Potential Non-Cholinergic Mechanisms. Pharmacology & therapeutics 134, 355-365, doi:10.1016/j.pharmthera.2012.03.001 (2012).
10 Sungur, M. & Güven, M. Intensive care management of organophosphate insecticide poisoning. Critical Care 5, 211, doi:10.1186/cc1025 (2001).
11 Masoud, A., Kiran, R. & Sandhir, R. Impaired mitochondrial functions in organophosphate induced delayed neuropathy in rats. Cellular and molecular neurobiology 29, 1245-1255, doi:10.1007/s10571-009-9420-4 (2009).
12 Farkhondeh, T., Mehrpour, O., Forouzanfar, F., Roshanravan, B. & Samarghandian, S. Oxidative stress and mitochondrial dysfunction in organophosphate pesticide-induced neurotoxicity and its amelioration: a review. Environmental science and pollution research international 27, 24799-24814, doi:10.1007/s11356-020-09045-z (2020).
13 Gao, J. et al. Diisopropylfluorophosphate Impairs the Transport of Membrane-Bound Organelles in Rat Cortical Axons. The Journal of pharmacology and experimental therapeutics 356, 645-655, doi:10.1124/jpet.115.230839 (2016).
14 Ehinger, J. K. et al. Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency. Nature communications 7, 12317, doi:10.1038/ncomms12317 (2016).
15 Avram, V. F. et al. Cell-Permeable Succinate Rescues Mitochondrial Respiration in Cellular Models of Statin Toxicity. International journal of molecular sciences 22, doi:10.3390/ijms22010424 (2021).
16 Piel, S. et al. Cell-permeable succinate prodrugs rescue mitochondrial respiration in cellular models of acute acetaminophen overdose. PloS one 15, e0231173, doi:10.1371/journal.pone.0231173 (2020).
17 Piel, S. et al. Bioenergetic bypass using cell-permeable succinate, but not methylene blue, attenuates metformin-induced lactate production. Intensive care medicine experimental 6, 22, doi:10.1186/s40635-018-0186-1 (2018).
18 Owiredu, S. et al. Ex vivo use of cell-permeable succinate prodrug attenuates mitochondrial dysfunction in blood cells obtained from carbon monoxide-poisoned individuals. American journal of physiology. Cell physiology 319, C129-c135, doi:10.1152/ajpcell.00539.2019 (2020).