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.

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.

Neuroprotective Cationic Arginine-Rich Peptides (CARPs): An Assessment of Their Clinical Safety.

Cationic arginine-rich peptides represent a novel class of peptides being developed as neuroprotective agents for stroke and other acute and chronic neurological disorders. As a group, cationic arginine-rich peptides have a diverse range of other biological properties including the ability to traverse cell membranes, modulate immune responses, antagonise ion channel receptor function, as well as possessing cardioprotective, anti-nociceptive, anti-microbial and anti-cancer properties. A sound understanding of their safety profile is essential for the design of future clinical trials and for ensuring translational success with these compounds. At present, while many neuroprotective cationic arginine-rich peptides have been examined in preclinical animal neuroprotection studies, few have been assessed in human safety studies. Despite this, the safety of the prototypical cationic arginine-rich peptide, protamine, which has been in clinical use for over 70 years to reverse the anticoagulant effects of heparin and as an excipient in certain insulin preparations, is well established. In addition, the poly-arginine peptide R9 (ALX40-4C) was developed as an anti-human inmmunodeficiency virus therapeutic in the mid-1990s, and more recently, the neuroprotective cationic arginine-rich peptides TAT-NR2B9c (NA-1), CN-105 and RD2 are being evaluated for the treatment of ischaemic stroke, haemorrhagic stroke and Alzheimer's disease, respectively. Based on the available clinical data, cationic arginine-rich peptides as a group appear to be safe when administered at therapeutic doses by a slow intravenous infusion. While protamine, owing to its isolation from salmon milt and homology with human sperm protamine, can trigger anaphylactic and anaphylactoid reactions in a small proportion of patients previously exposed to the peptide (e.g. diabetic patients), who are allergic to fish or have undergone a vasectomy, such reactions are unlikely to be triggered in individuals exposed to non-protamine cationic arginine-rich peptides.

Assessment of recombinant tissue plasminogen activator (rtPA) toxicity in cultured neural cells and subsequent treatment with poly-arginine peptide R18D.

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) in ischaemic stroke has been associated with neurotoxicity, blood brain barrier (BBB) disruption and intra-cerebral hemorrhage. To examine rtPA cellular toxicity we investigated the effects of rtPA on cell viability in neuronal, astrocyte and brain endothelial cell (bEnd.3) cultures with and without prior exposure to oxygen-glucose deprivation (OGD). In addition, the neuroprotective peptide poly-arginine-18 (R18D; 18-mer of D-arginine) was examined for its ability to reduce rtPA toxicity. Studies demonstrated that a 4- or 24-h exposure of rtPA was toxic, affecting neuronal cell viability at ≥ 2 µM, and astrocyte and bEnd.3 cells viability at ≥ 5 µM. In addition, a 4-h exposure to rtPA after a period of OGD (OGD/rtPA) exacerbated toxicity, affecting neuronal, astrocyte and bEnd.3 cell viability at rtPA concentrations as low as 0.1 µM. Treatment of cells with low concentrations of R18D (0.5 and 1 µM) reduced the toxic effects of rtPA and OGD/rtPA, while on some occasions a higher 2 µM R18D concentrations exacerbated neuronal and bEnd.3 cell toxicity in OGD/rtPA exposed cultures. In exploratory studies we also demonstrated that OGD activates matrix metalloproteinase-9 (MMP-9) release into the supernatant of astrocyte and bEnd.3 cell cultures, but not neuronal cultures, and that OGD/rtPA increases MMP-9 activation. Furthermore, R18D decreased MMP-9 activation in OGD/rtPA treated astrocyte and bEnd.3 cell cultures. In summary, the findings show that rtPA can be toxic to neural cells and that OGD exacerbates toxicity, while R18D has the capacity to reduce rtPA neural cellular toxicity and reduce MMP-9 activation in astrocytes and bEnd.3. Poly-arginine-18 peptides, which are being developed as neuroprotective therapeutics for ischaemic stroke, therefore have the additional potential of reducing cytotoxic effects associated with rtPA thrombolysis in the treatment of ischaemic stroke.

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

Hypoxic-ischaemic encephalopathy (HIE) remains the leading cause of mortality and morbidity in neonates, with no available neuroprotective therapeutic agent. In the development of a therapeutic for HIE, we examined the neuroprotective efficacy of the poly-arginine peptide R18D (arginine 18 mer synthesised with D-arginine) in a perinatal model of hypoxia-ischaemia (HI; common carotid and external carotid occlusion + 8%O2 /92%N2 for 2.5 hr) in the P7 Sprague-Dawley rat. R18D was administered intraperitoneally 30 min (doses 10, 30, 100, 300 and 1,000 nmol/kg), 60 min (doses 30 and 300 nmol/kg) or 120 min (doses 30 and 300 nmol/kg) after HI. Infarct volumes and behavioural outcomes were measured 48 hr after HI. When administered 30 min after HI, R18D at varying doses reduced infarct volume by 23.7% to 35.6% (p = 0.009 to < 0.0001) and resulted in improvements in the negative geotactic response and wire-hang times, at a dose of 30 nmol/kg. When administered 60 min after HI, R18D at the 30 nmol/kg dose reduced total infarct volume by 34.2% (p = 0.002), whilst the 300 nmol/kg dose improved wire-hang time. When administered 120 min after HI, R18D at the 30 and 300 nmol/kg doses had no significant impact on infarct volume, but the 300 nmol/kg dose improved the negative geotactic response. This study further confirms the neuroprotective properties of poly-arginine peptides, demonstrating that R18D can reduce infarct volume and improve behavioural outcomes after HI if administered up to 60 min after HI and improve behavioural outcomes up to 2 hr after HI.

Assessment of R18, COG1410, and APP96-110 in Excitotoxicity and Traumatic Brain Injury.

Cationic arginine-rich and poly-arginine peptides (referred to as CARPs) have potent neuroprotective properties in in vitro excitotoxicity and in vivo models of stroke. Traumatic brain injury (TBI) shares many pathophysiological processes as stroke, including excitotoxicity. Therefore, we evaluated our lead peptide, poly-arginine R18, with the COG1410 and APP96-110 peptides, which have neuroprotective actions following TBI. In an in vitro cortical neuronal glutamic acid excitotoxicity injury model, R18 was highly neuroprotective and reduced neuronal calcium influx, while COG1410 and APP96-110 displayed modest neuroprotection and were less effective at reducing calcium influx. In an impact-acceleration closed-head injury model (Marmarou model), R18, COG1410, and APP96-110 were administered intravenously (300 nmol/kg) at 30 minutes after injury in male Sprague-Dawley rats. When compared to vehicle, no peptide significantly improved functional outcomes, however the R18 and COG1410 treatment groups displayed positive trends in the adhesive tape test and rotarod assessments. Similarly, no peptide had a significant effect on hippocampal neuronal loss, however a significant reduction in axonal injury was observed for R18 and COG1410. In conclusion, this study has demonstrated that R18 is significantly more effective than COG1410 and APP96-110 at reducing neuronal injury and calcium influx following excitotoxicity, and that both R18 and COG1410 reduce axonal injury following TBI. Additional dose response and treatment time course studies are required to further assess the efficacy of R18 in TBI.

Assessment of the Neuroprotective Effects of Arginine-Rich Protamine Peptides, Poly-Arginine Peptides (R12-Cyclic, R22) and Arginine-Tryptophan-Containing Peptides Following In Vitro Excitotoxicity and/or Permanent Middle Cerebral Artery Occlusion in Rats.

We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000 nmol/kg 30 min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100 nmol/kg and ineffective at reducing infarct volume when administered at 30 nmol/kg 30 min after MCAO, unlike R18 (30 nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.

Assessment of CMV, RSV and SYN1 promoters and the woodchuck post-transcriptional regulatory element in adenovirus vectors for transgene expression in cortical neuronal cultures.

In order to investigate protein function in rat primary cortical neuronal cultures, we modified an adenoviral vector expression system and assessed the strength and specificity of the cytomegalovirus (CMV), rous sarcoma virus (RSV), and rat and human synapsin 1 (SYN1) promoters to drive DsRed-X expression. We also incorporated the woodchuck post-transcriptional regulatory element (WPRE) and a CMV promoter-enhanced green fluorescent protein (EGFP) reporter cassette. We observed that the RSV promoter activity was strong in neurons and moderate in astrocytes, while the CMV promoter activity was weak-to-moderate in neurons and very strong in astrocytes. The rat and human SYN1 promoters exhibited similar but weak activity in neurons, despite inclusion of the WPRE. We confirmed that the WPRE enhanced RSV promoter-mediated DsRed-X expression in a time-dependent fashion. Interestingly, we observed very weak SYN1-mediated DsRed-X expression in astrocytes and HEK293 cells suggesting incomplete neuronal-restrictive behavior for this promoter. Finally, using our adenoviral expression system, we demonstrated that RSV promoter-mediated Bcl-X(L) overexpression attenuated neuronal death caused by in vitro ischemia and oxidative stress.