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Comparative Assessment of the Proteolytic Stability and Impact of Poly-Arginine Peptides R18 and R18D on Infarct Growth and Penumbral Tissue Preservation Following Middle Cerebral Artery Occlusion in the Sprague Dawley Rat

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Abstract

Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm3 reduction; p < 0.001) and preserving penumbral tissue (21.6% increase; p < 0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm3 reduction; p < 0.001, penumbra: 12.5% increase; p < 0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm3 reduction; p = 0.026), but only a modest impact on penumbral preservation (6.9% increase; p = 0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1–3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.

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Data Availability Statement

The data that support the findings of the present study will be made available upon request from the corresponding author.

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Acknowledgements

We thank Prof Frank Mastaglia for editorial changes and suggestions to the manuscript.

Funding

The present study was supported by the Perron Institute for Neurological and Translational Science, and the Department of Neurosurgery at Sir Charles Gairdner Hospital.

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Correspondence to Bruno P. Meloni.

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Conflicts of interest

Bruno P. Meloni and Neville W. Knuckey are the holders of several patents regarding the use of arginine-rich peptides as neuroprotective treatments. Diego Milani, Vince W. Clark, Ryan S. Anderton, David J. Blacker, Adam Edwards, Neville W. Knuckey, and Bruno P. Meloni are shareholders in Argenica Therapeutics, which is a company developing R18 as a stroke therapeutic. The other authors declare no conflict of interest.

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All work was performed in compliance with the Animal Ethics Committee of the University of Western Australia and following the guidelines outlined by the Australian Code for the Care and Use of Animals for Scientific Purposes.

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Milani, D., Clark, V.W., Feindel, K.W. et al. Comparative Assessment of the Proteolytic Stability and Impact of Poly-Arginine Peptides R18 and R18D on Infarct Growth and Penumbral Tissue Preservation Following Middle Cerebral Artery Occlusion in the Sprague Dawley Rat. Neurochem Res 46, 1166–1176 (2021). https://doi.org/10.1007/s11064-021-03251-y

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