Comparison of neuroprotective efficacy of poly-arginine R18 and R18D (D-enantiomer) peptides following permanent middle cerebral artery occlusion in the Wistar rat and in vitro toxicity studies

Abstract

We have previously demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties, with poly-arginine peptide R18 identified as being highly effective at reducing infarct volume following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. Since peptides synthesised using D-isoform amino acids have greater stability than L-isoform peptides due to increased resistance to proteolytic degradation, they represent potentially more effective peptide therapeutics. Therefore we compared the neuroprotective efficacy of R18 and its D-enantiomer R18D following permanent MCAO in the Wistar rat. Furthermore, as increased peptide stability may also increase peptide toxicity, we examined the effects of R18 and R18D on cultured cortical neurons, astrocytes, brain endothelial cells (bEND.3), and embryonic kidney cells (HEK293) following a 10-minute or 24-hour peptide exposure duration. The in vivo studies demonstrated that R18D resulted in a greater reduction in mean infarct volume compared to R18 (33%, p = 0.004 vs 12%, p = 0.27) after intravenous administration at 300 nmol/kg 30 minutes after MCAO. Both R18D and R18 reduced cerebral hemisphere swelling to a comparable degree (27%, p = 0.03 and 30%, p = 0.02), and improved neurological assessment scores (1.5, p = 0.02 and 2, p = 0.058 vs 3 for vehicle). No abnormal histological findings specific to peptide treatments were observed in hematoxylin and eosin stained sections of kidney, liver, spleen, lung and heart. In vitro studies demonstrated that R18 and R18D were most toxic to neurons, followed by astrocytes, HEK293 and bEND.3 cells, but only at high concentrations and/or following 24-hour exposure. These findings further highlight the neuroprotective properties of poly-arginine peptides, and indicate that R18D at the dose examined is more potent than R18 in Wistar rats, and justify continued investigation of the R18 peptide as a novel neuroprotective agent for stroke.

Assessment of therapeutic window for poly‐arginine‐18D (R18D) in a P7 rat model of perinatal hypoxic‐ischaemic encephalopathy

“…The present study adds to our previous findings which demonstrate the neuroprotective effectiveness of R18 and R18D in a P7 rat model of perinatal HIE (Edwards et al, ), and other acute neuronal injury models (Chiu et al, ; Edwards et al, ; Meloni et al, ; Meloni, Brookes, et al, ; Meloni, Milani, et al, ; Milani et al, ; Milani, Clark, et al, ; Milani, Cross, et al, ; Milani, Knuckey, et al, ). Importantly, the present study examined the neuroprotective efficacy of R18D when administered 30, 60 or 120 min after HI.…”

Proteomic analysis of cortical neuronal cultures treated with poly-arginine peptide-18 (R18) and exposed to glutamic acid excitotoxicity

“…Therefore, the ability of R18 treatment to attenuate excitotoxicityinduced protein changes underlying loss of mitochondrial integrity, provides evidence that the peptide helps preserve the function of the organelle in times of cellular stress. In line with the ability of CARPs to maintain mitochondrial function and energy generation, in this and previous studies R18 was demonstrated to increase MTS metabolism in uninjured neurons and in neurons after exposure to glutamic acid [14].…”

“…Recent studies in our laboratory have identified cationic arginine-rich peptides (CARPs), which include poly-arginine peptides, as a novel class of neuroprotective agents. In particular, we have demonstrated that poly-arginine-18 (R18, 18-mer of arginine) is neuroprotective in in vitro neuronal excitotoxicity models and in vivo in rodent models of stroke [11][12][13][14][15][16][17][18], hypoxic-ischaemic encephalopathy (HIE) [19], and traumatic brain injury (TBI) [20,21].…”

Cationic Arginine-Rich Peptides (CARPs): A Novel Class of Neuroprotective Agents With a Multimodal Mechanism of Action

“…Consistent with in vitro findings, CARPs (e.g., R9D, R12, R18, R18D, protamine; Tables 1, 3) were also demonstrated to provide significant neuroprotection and improve functional outcomes in rat models of permanent and/or transient middle cerebral artery occlusion (MCAO), perinatal hypoxia-ischemia and traumatic brain injury (15,16,(18)(19)(20)(21)(22)(23)(24)212) and a non-human primate MCAO stroke model (26). Positive neuroprotective effects with R9D and R18D, which are the D-enantiomers of R9 and R18, also confirmed the lack of stereo-specificity for CARP efficacy in vivo.…”