Corrigendum to "Microglia are both a source and target of extracellular cyclophilin A" [Heliyon 5(9) August 2019 e02390].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e02390.].

Assessment of the safety of the cationic arginine-rich peptides (CARPs) poly-arginine-18 (R18 and R18D) in ex vivo models of mast cell degranulation and red blood cell hemolysis.

Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on ß-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 µM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1-16 µM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 µM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.

Impact of poly-arginine peptides R18D and R18 on alteplase and tenecteplase thrombolysis in vitro, and neuroprotective stability to proteolysis.

The poly-arginine peptides R18D and R18 represent novel potential neuroprotective treatments for acute ischaemic stroke. Here we examined whether R18D and R18 had any significant effects on the thrombolytic activity of alteplase (tPA) and tenecteplase (TNK) on clots formed from whole blood in an in vitro thrombolysis plate assay. R18D and R18 were examined at concentrations of 0.25, 0.5, 1, 2, 4, 8 and 16 µM during the 1-h thrombolytic assay. We also included the well-characterised neuroprotective NA-1 peptide as a control. R18D, R18 and NA-1 all reduced tPA or TNK percentage clot lysis by 0-9.35%, 0-3.44% and 0-4.8%, respectively. R18D, R18 and NA-1 had a modest and variable effect on the lag time, increasing the time to the commencement of thrombolysis by 0-9.9 min, 0-5.53 min and 0-7.16 min, respectively. Lastly, R18 and NA-1 appeared to increase the maximal activity of the thrombolysis reaction. In addition, the in vitro anti-excitotoxic neuroprotective efficacy of R18D and R18 was not affected by pre-incubation for 1-2 h or overnight with tPA or TNK, whereas only R18D retained high anti-excitotoxic neuroprotective efficacy when pre-incubated in a synthetic trypsin (TrypLE Express). The present in vitro findings suggest that neither R18D or R18 when co-administered with the thrombolytic inducing agents tPA or TNK are likely to have a significant impact when used clinically during clot thrombolysis and confirm the superior proteolytic stability of the R18D peptide.

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.

Poly-arginine-18 (R18) Confers Neuroprotection through Glutamate Receptor Modulation, Intracellular Calcium Reduction, and Preservation of Mitochondrial Function.

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.

Effect of Polyarginine Peptide R18D Following a Traumatic Brain Injury in Sprague-Dawley Rats.

BACKGROUND: Despite extensive studies, there are still no clinically available neuroprotective treatments for traumatic brain injury. OBJECTIVES: In previous studies we demonstrated beneficial treatment effects of polyarginine peptides R18 (18-mer of arginine; 300 nmol/kg) and R18D (18-mer of D-arginine; 1000 nmol/kg) in a rat model of impact-acceleration closed-head injury. METHODS: We examined the efficacy of R18D when intravenously administered at a low (100 nmol/kg) and high (1000 nmol/kg) dose, 30 minutes after a closed-head injury in male Sprague-Dawley rats. RESULTS: At postinjury day 3, treatment with R18D at the high dose significantly reduced axonal injury (P = 0.044), whereas the low-dose treatment of R18D showed a trend for reduced axonal injury. Following assessment in the Barnes maze, both doses of R18D treatment appeared to improve learning and memory recovery compared with vehicle treatment at postinjury days 1 and 3, albeit not to a statistically significant level. Rotarod assessment of vestibulomotor recovery did not differ between R18D and the vehicle treatment groups. CONCLUSIONS: R18D modestly decreased axonal injury only at the highest dose used but had no significant effect on functional recovery. These findings warrant further studies with additional doses to better understand peptide pharmacodynamics and provide information to guide optimal dosing.

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.

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

There are virtually no clinically available neuroprotective drugs for the treatment of acute and chronic neurological disorders, hence there is an urgent need for the development of new neuroprotective molecules. Cationic arginine-rich peptides (CARPs) are an expanding and relatively novel class of compounds, which possess intrinsic neuroprotective properties. Intriguingly, CARPs possess a combination of biological properties unprecedented for a neuroprotective agent including the ability to traverse cell membranes and enter the CNS, antagonize calcium influx, target mitochondria, stabilize proteins, inhibit proteolytic enzymes, induce pro-survival signaling, scavenge toxic molecules, and reduce oxidative stress as well as, having a range of anti-inflammatory, analgesic, anti-microbial, and anti-cancer actions. CARPs have also been used as carrier molecules for the delivery of other putative neuroprotective agents across the blood-brain barrier and blood-spinal cord barrier. However, there is increasing evidence that the neuroprotective efficacy of many, if not all these other agents delivered using a cationic arginine-rich cell-penetrating peptide (CCPPs) carrier (e.g., TAT) may actually be mediated largely by the properties of the carrier molecule, with overall efficacy further enhanced according to the amino acid composition of the cargo peptide, in particular its arginine content. Therefore, in reviewing the neuroprotective mechanisms of action of CARPs we also consider studies using CCPPs fused to a putative neuroprotective peptide. We review the history of CARPs in neuroprotection and discuss in detail the intrinsic biological properties that may contribute to their cytoprotective effects and their usefulness as a broad-acting class of neuroprotective drugs.

In vitro cellular uptake and neuroprotective efficacy of poly-arginine-18 (R18) and poly-ornithine-18 (O18) peptides: critical role of arginine guanidinium head groups for neuroprotection.

We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.

Poly-Arginine Peptide-18 (R18) Reduces Brain Injury and Improves Functional Outcomes in a Nonhuman Primate Stroke Model.

Poly-arginine peptide-18 (R18) is neuroprotective in different rodent middle cerebral artery occlusion (MCAO) stroke models. In this study, we examined whether R18 treatment could reduce ischemic brain injury and improve functional outcome in a nonhuman primate (NHP) stroke model. A stroke was induced in male cynomolgus macaques by MCAO distal to the orbitofrontal branch of the MCA through a right pterional craniotomy, using a 5-mm titanium aneurysm clip for 90 min. R18 (1000 nmol/kg) or saline vehicle was administered intravenously 60 min after the onset of MCAO. Magnetic resonance imaging (MRI; perfusion-weighted imaging, diffusion-weighted imaging, or T2-weighted imaging) of the brain was performed 15 min, 24 h, and 28 days post-MCAO, and neurological outcome was assessed using the NHP stroke scale (NHPSS). Experimental endpoint was 28 days post-MCAO, treatments were randomized, and all procedures were performed blinded to treatment status. R18 treatment reduced infarct lesion volume by up to 65.2% and 69.7% at 24 h and 28 days poststroke, respectively. Based on NHPSS scores, R18-treated animals displayed reduced functional deficits. This study confirms the effectiveness of R18 in reducing the severity of ischemic brain injury and improving functional outcomes after stroke in a NHP model, and provides further support for its clinical development as a stroke neuroprotective therapeutic.

Poly-arginine-18 peptides do not exacerbate bleeding, or improve functional outcomes following collagenase-induced intracerebral hemorrhage in the rat.

BACKGROUND: Cationic arginine-rich peptides (CARPs) have demonstrated neuroprotective and/or behavioural efficacy in ischemic and hemorrhagic stroke and traumatic brain injury models. Therefore, in this study we investigated the safety and neuroprotective efficacy of the CARPs poly-arginine-18 (R18; 18-mer of arginine) and its D-enantiomer R18D given in the acute bleeding phase in an intracerebral hemorrhage (ICH) model. METHODS: One hundred and fifty-eight male Sprague-Dawley rats received collagenase-induced ICH. Study 1 examined various doses of R18D (30, 100, 300, or 1000 nmol/kg) or R18 (100, 300, 1000 nmol/kg) administered intravenously 30 minutes post-collagenase injection on hemorrhage volume 24 hours after ICH. Study 2 examined R18D (single intravenous dose) or R18 (single intravenous dose, plus 6 daily intraperitoneal doses) at 300 or 1000 nmol/kg commencing 30 minutes post-collagenase injection on behavioural outcomes (Montoya staircase test, and horizontal ladder test) in the chronic post-ICH period. A histological assessment of tissue loss was assessed using a Nissl stain at 28 days after ICH. RESULTS: When administered during ongoing bleeding, neither R18 or R18D exacerbated hematoma volume or worsened functional deficits. Lesion volume assessment at 28 days post-ICH was not reduced by the peptides; however, animals treated with the lower R18D 300 nmol/kg dose, but not with the higher 1000 nmol/kg dose, demonstrated a statistically increased lesion size compared to saline treated animals. CONCLUSION: Overall, both R18 and R18D appeared to be safe when administered during a period of ongoing bleeding following ICH. Neither peptide appears to have any statistically significant effect in reducing lesion volume or improving functional recovery after ICH. Additional studies are required to further assess dose efficacy and safety in pre-clinical ICH studies.

Microglia are both a source and target of extracellular cyclophilin A.

Glioblastoma (GBM) are lethal primary brain tumours whose pathogenesis is aided, at least partly, via a pro-tumorigenic microenvironment. This study investigated whether microglia, a cell component of the GBM microenvironment, mediates pro-tumorigenic properties via the action of cyclophilin A (CypA), a potent secretable chemokine and cytoprotectant that signals via the cell surface receptor, CD147. To this end, intracellular and secreted CypA expression was assessed in human primary microglia and BV2 microglial cells treated with the endotoxin, lipopolysaccharide (LPS) and the oxidative stress inducer, LY83583. We report that human primary microglia and BV2 microglia both express CypA and CD147, and that BV2 microglial cells secrete CypA in response to pro-inflammatory and oxidative stimuli. We also demonstrate for the first time that recombinant CypA (rCypA; 1nM-1000nM) dose-dependently increased wound healing and reduced basal cell death in BV2 microglial cells. To determine the cell-signalling pathways involved, we probed microglial cell lysates for changes in ERK1/2 and AKT phosphorylation, IκB degradation, and IL-6 secretion using Western blot and ELISA analysis. In summary, BV2 microglial cells secrete CypA in response to inflammatory and oxidative stress, and that rCypA increases cell viability and chemotaxis. Our findings suggest that rCypA is a pro-survival chemokine for microglia that may influence the GBM tumour microenvironment.

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

Poly-arginine peptide-18 (R18) has recently emerged as a highly effective neuroprotective agent in experimental stroke models, and is particularly efficacious in protecting cortical neurons against glutamic acid excitotoxicity. While we have previously demonstrated that R18 can reduce excitotoxicity-induced neuronal calcium influx, other molecular events associated with R18 neuroprotection are yet to investigated. Therefore, in this study we were particularly interested in protein expression changes in R18 treated neurons subjected to excitotoxicity. Proteomic analysis was used to compare protein expression patterns in primary cortical neuronal cultures subjected to: (i) R18-treatment alone (R18); (ii) glutamic acid excitotoxic injury (Glut); (iii) R18-treatment and glutamic acid injury (R18 + Glut); (iv) no treatment (Cont). Whole cell lysates were harvested 24 h post-injury and subjected to quantitative proteomic analysis (iTRAQ), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and subsequent bioinformatic analysis of differentially expressed proteins (DEPs). Relative to control cultures, R18, Glut, and R18 + Glut treatment resulted in the detection of 5, 95 and 14 DEPs respectively. Compared to Glut alone, R18 + Glut revealed 98 DEPs, including 73 proteins whose expression was also altered by treatment with Glut and/or R18 alone, as well as 25 other uniquely regulated proteins. R18 treatment reversed the up- or down-regulation of all 73 Glut-associated DEPs, which included proteins involved in mitochondrial integrity, ATP generation, mRNA processing and protein translation. Analysis of protein-protein interactions of the 73 DEPs showed they were primarily associated with mitochondrial respiration, proteasome activity and protein synthesis, transmembrane trafficking, axonal growth and neuronal differentiation, and carbohydrate metabolism. Identified protein pathways associated with proteostasis and energy metabolism, and with pathways involved in neurodegeneration. Collectively, the findings indicate that R18 neuroprotection following excitotoxicity is associated with preservation of neuronal protein profiles, and differential protein expression that assists in maintaining mitochondrial function and energy production, protein homeostasis, and membrane trafficking.

Poly-Arginine Peptides R18 and R18D Improve Functional Outcomes After Endothelin-1-Induced Stroke in the Sprague Dawley Rat.

We have previously demonstrated that R18 and its d-enantiomer, R18D, are neuroprotective at 24 hours following intraluminal filament occlusion of the middle cerebral artery (MCAO) in the rat. This study examined R18 and R18D effectiveness in improving functional outcomes at up to 56 days poststroke following endothelin-1-induced MCAO. Peptides were administered intravenously at doses of 100, 300, or 1000 nmol/kg, 60 minutes after MCAO. Functional recovery poststroke was assessed using multiple forelimb placing tests and horizontal ladder test, and NA-1 (TAT-NR2B9c), a neuroprotective currently in phase 3 clinical stroke trials, was used as a benchmark. The study demonstrated that R18 (300 and 1000 nmol/kg) was the most effective peptide in improving functional outcomes, followed by R18D (300 and 1000 nmol/kg), and NA-1 (300 and 100 nmol/kg). Furthermore, R18 at doses of 300 and 1000 nmol/kg was the most effective agent in restoring pre-stroke body weight, while R18 and R18D at doses of 300 and 1000 nmol/kg, but not NA-1 also significantly reduced the number of animals requiring hand feeding 48 hours after stroke. This study confirms that R18 and R18D are effective in improving long-term functional outcomes after stroke, and suggests that R18 may be more effective than NA-1.

Mitochondria and neuroprotection in stroke: Cationic arginine-rich peptides (CARPs) as a novel class of mitochondria-targeted neuroprotective therapeutics.

Stroke is the second leading cause of death globally and represents a major cause of devastating long-term disability. Despite sustained efforts to develop clinically effective neuroprotective therapies, presently there is no clinically available neuroprotective agent for stroke. As a central mediator of neurodamaging events in stroke, mitochondria are recognised as a critical neuroprotective target, and as such, provide a focus for developing mitochondrial-targeted therapeutics. In recent years, cationic arginine-rich peptides (CARPs) have been identified as a novel class of neuroprotective agent with several demonstrated mechanisms of action, including their ability to target mitochondria and exert positive effects on the organelle. This review provides an overview on neuronal mitochondrial dysfunction in ischaemic stroke pathophysiology and highlights the potential beneficial effects of CARPs on mitochondria in the ischaemic brain following stroke.

Perinatal Hypoxic-Ischemic Encephalopathy and Neuroprotective Peptide Therapies: A Case for Cationic Arginine-Rich Peptides (CARPs).

Perinatal hypoxic-ischemic encephalopathy (HIE) is the leading cause of mortality and morbidity in neonates, with survivors suffering significant neurological sequelae including cerebral palsy, epilepsy, intellectual disability and autism spectrum disorders. While hypothermia is used clinically to reduce neurological injury following HIE, it is only used for term infants (>36 weeks gestation) in tertiary hospitals and improves outcomes in only 30% of patients. For these reasons, a more effective and easily administrable pharmacological therapeutic agent, that can be used in combination with hypothermia or alone when hypothermia cannot be applied, is urgently needed to treat pre-term (≤36 weeks gestation) and term infants suffering HIE. Several recent studies have demonstrated that cationic arginine-rich peptides (CARPs), which include many cell-penetrating peptides [CPPs; e.g., transactivator of transcription (TAT) and poly-arginine-9 (R9; 9-mer of arginine)], possess intrinsic neuroprotective properties. For example, we have demonstrated that poly-arginine-18 (R18; 18-mer of arginine) and its D-enantiomer (R18D) are neuroprotective in vitro following neuronal excitotoxicity, and in vivo following perinatal hypoxia-ischemia (HI). In this paper, we review studies that have used CARPs and other peptides, including putative neuroprotective peptides fused to TAT, in animal models of perinatal HIE. We critically evaluate the evidence that supports our hypothesis that CARP neuroprotection is mediated by peptide arginine content and positive charge and that CARPs represent a novel potential therapeutic for HIE.

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.