Emerging cytoprotective peptide therapies for stroke

KEYWORDS:

• Stroke
• neuroprotection
• NA-1
• CN-105
• R18
• cationic arginine-rich peptides (CARPs)

1. Introduction

Globally, stroke remains a major cause of death and disability. The development of a clinically effective neuroprotective drug to reduce the severity of brain injury and improve outcomes after stroke remains an urgent priority and unmet need. Furthermore, given the demonstrated effectiveness of thrombectomy and thrombolysis in patients with thrombo-embolic stroke with a favorable perfusion status and salvageable tissue in the ischemic penumbra, a neuroprotective drug administered at initial patient contact has the potential for extending the therapeutic time window for these interventions, and decreasing tissue damage and the risk of hemorrhagic and edema-related complications following reperfusion. Neuroprotective drugs could also have a valuable role in health care systems with limited access to stroke recanalization therapies.

While many previous small-molecule drugs targeting specific components in the pathophysiology of ischemic or hemorrhagic stroke (e.g. excitotoxicity and intracellular calcium influx, oxidative stress and inflammation) have been developed, they have invariably failed to demonstrate efficacy in clinical trials. The main reasons for this include inadequate preclinical evaluation, differences in study design between experimental and clinical studies [1], and limited potency because of a single mechanism of action or only modest neuroprotective effect of the agent [2]. Cationic arginine-rich peptides (CARPs) are a new emerging class of neuroprotective agents with multimodal cytoprotective actions. These include the ability to antagonize calcium influx, target and stabilize mitochondria, scavenge toxic molecules and reduce oxidative stress, inhibit proteolytic enzymes, induce pro-survival signaling, as well as having a range of anti-inflammatory properties (reviewed in depth in [3]). Moreover, CARPs have shown consistent efficacy in a variety of in vitro and animal models of ischemic and hemorrhagic stroke [3], and have demonstrated a favorable safety profile [4]. With respect to CARP neuroprotection, while cationic charge can be imparted by positively charged arginine, lysine and histidine residues, and while other amino acids can influence efficacy (e.g. tryptophan), arginine is the amino acid essential for neuroprotection [3,5–7].

The first CARP to be evaluated in clinical trials for ischemic stroke is the NA-1 peptide. The recent ESCAPE-NA1 study demonstrated efficacy of the peptide when administered as an adjunct therapy to thrombectomy [8]. Other CARPs that are currently undergoing evaluation and have shown encouraging results include polyarginine-18 (R18), which has been developed as a treatment for ischemic stroke [9–14] and the CN-105 peptide for hemorrhagic stroke [15,16]. This editorial provides a concise update on the current status of these agents as potential clinical stroke therapeutics.

2. NA-1 peptide

The NA-1 peptide (originally named TAT-NR2B9 c: sequence YGRKKRRQRRR-KLSSIESDV: net charge +7) is undergoing clinical assessment as an acute neuroprotective treatment for ischemic stroke [8,17]. The peptide consists of a sequence (KLSSIESDV) derived from the intracellular terminal carboxyl region of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit protein, fused to the cationic arginine-rich cell penetrating peptide TAT (YGRKKRRQRRR; net change +8), which facilitates passage across the blood brain barrier and entry into cells including neurons. The NR2B9 c 9-mer peptide was selected to inhibit the postsynaptic density protein-95 (PSD-95) adaptor protein binding to the NR2B subunit, and thereby block downstream cell signaling associated with overstimulation of the NMDA receptor, including nitric oxide synthase activation and the subsequent production of toxic levels of nitric oxide [18]. Given that NA-1 is cationic and arginine-rich, we and others [19,20] have proposed that the peptide’s mechanism of action is linked with the intrinsic neuroprotective properties associated with CARPs.

Aside mechanism of action, NA-1 has demonstrated neuroprotective efficacy in multiple rodent [18,21–23] and non-human primate [24] stroke models. The rodent studies have confirmed efficacy in stroke models with or without reperfusion, whereas the non-human primate stroke study utilized models incorporating reperfusion. In addition, NA-1 was found to be safe in a phase 2 study, when administered to patients after endovascular aneurysm repair surgery (the ENACT study) [25]. The recent large multi-center phase 3 ESCAPE-NA1 study demonstrated no overall improvement in functional outcomes in patients with large artery cerebral occlusion undergoing thrombectomy with or without prior alteplase administration, who were given a single intravenous infusion (1032 nmol/kg; over 10 min) of NA-1 within 12 hours of stroke onset [8]. However, post-hoc subgroup analysis revealed significant benefit in patients who did not undergo alteplase thrombolysis, who had improved functional outcomes, reduced mortality and reduced infarct volumes. While requiring further confirmation, the positive effect of NA-1 on stroke outcomes is important as it represents the first clinical study demonstrating neuroprotection for a therapeutic agent in stroke, albeit as an adjunct therapy to endovascular thrombectomy.

The unexpected nullifying effect of alteplase on NA-1 efficacy was shown to be attributable to reduced and subtherapeutic levels of the peptide in plasma as a result of proteolytic degradation of the peptide by plasmin, the enzyme activated by alteplase. On this basis, it is hypothesized that the administration of NA-1 prior to alteplase treatment (e.g. 30 to 60 min), will provide sufficient time for the peptide to exert its neuroprotective actions before being degraded by plasmin. Importantly, the FRONTIER NA-1 phase 3 trial [17], which is currently in progress will be able to provide the confirmatory data to support this hypothesis, as the peptide is being administered by field paramedics within three hours of symptom onset, prior to confirmation of stroke subtype and alteplase administration. Moreover, this trial will also provide information regarding any beneficial effects of NA-1 in people who suffer a hemorrhagic stroke. Interestingly, in experimental rat models of intracerebral hemorrhage, NA-1 has been shown to reduce cell death, MMP9 activation, inflammation and cerebral edema and to improve behavioral performance [26]. It is therefore feasible that NA-1 may represent an effective broad-acting neuroprotective therapeutic not only for ischemic stroke when administered before alteplase (or when alteplase cannot be administered or is contraindicated), but also in patients with hemorrhagic stroke.

3. R18 peptide

Poly-arginine peptide-18 (R18; sequence RRRRRRRRRRRRRRRRRR; net charge +18) is a neuroprotective agent under development that has demonstrated strong efficacy in vitro in the neuronal excitotoxicity model [5,27] and in rodent [9–13] and non-human primate [14] models of middle cerebral artery occlusion (MCAO), both with or without reperfusion. R18 was selected as a lead peptide on the basis of its high arginine content and peptide positive charge following comparative studies with other poly-arginine peptides, demonstrating that in the excitotoxicity model neuroprotective efficacy increases with increasing arginine residues and peptide charge, plateauing at R15 to R18 [5], and its superior efficacy in the permanent MCAO model when compared with the shorter R12 and R15 peptides [9].

Furthermore, in experimental studies R18 has consistently out-performed NA-1 in terms of neuroprotective efficacy. For example, in the in vitro excitotoxicity model, R18 displayed almost complete protection, whereas NA-1 was largely ineffective [5]. In a study comparing the effects of R18 and NA-1 at a 1000 nmol/kg dose in the rat model of transient MCAO, R18 reduced infarct volume by 35.1% compared to a 26.1% reduction for NA-1 [11]. Similarly, in an endothelin-1 induced transient vasoconstrictive MCAO stroke model, R18 was shown to be superior when compared with NA-1 in terms of improving functional outcomes, time taken for animals to regain their pre-stroke body weight, and a lower number of animals requiring additional post-stroke syringe feeding [13]. The effectiveness of R18 has also been confirmed in the macaque transient MCAO stroke model, in which infarct lesion volume was reduced by up to 69.7% and functional deficits were significantly reduced [14]. Notably also, there was no evidence that R18 or its D-enantiomer (R18D) exacerbated bleeding in a collagenase-induced intracerebral hemorrhage rat stroke model [28], suggesting the peptides would be safe if administered on initial contact in patients with a hemorrhagic stroke prior to actual confirmation of the stroke subtype.

Importantly, R18 and R18D have been shown to have a comparable level of neuroprotective efficacy in in vitro and in vivo models of ischemia [12,13,27]. However, given that peptides comprising D-amino acids are resistant to proteolytic degradation [29,30], R18D is less likely to be degraded by plasmin. Based on the rodent and non-human primate stroke studies undertaken with R18 and other CARPs previously administered to humans (e.g. R9D, NA-1, protamine) [4], it is predicted there are unlikely to be any serious safety concerns with either R18 or R18D. However, prior to progressing to full animal safety and toxicity assessment, further studies will be required to assess the stability of the peptides and their neuroprotective efficacy in animal stroke models when co-administered with alteplase. In addition, further preclinical studies will be required to assess their dose efficacy and safety in the intracerebral hemorrhage model, prior to consideration for use in patients with hemorrhagic stroke.

4. CN-105 peptide

The CN-105 peptide (sequence: Ac-VSRRR-NH₂; net charge: +3) has been developed based on amino acids, namely arginine residues, present in the parent neuroprotective peptide COG133 (LRVRLASHLRKLRKRLL) comprising the heparin-binding and LDL receptor binding domains within the apolipoprotein E protein [15,31] and the identification of the importance of charged amino acids (e.g. arginine and lysine) for bioactivity [32]. While CN-105 has been shown to have broad efficacy in improving histological and functional outcomes in rodent models of ischemic stroke [33], as well as subarachnoid hemorrhage [34], intracerebral hemorrhage [15], and traumatic brain injury [35], currently its main clinical application appears to be for the treatment of hemorrhagic stroke [16].

Following demonstration that CN-105 was safe when administered intravenously to healthy volunteers as a single dose (14, 42, 140, 420 or 1,400 nmol/kg) or in repeated doses (1,400 nmol/kg 6 hourly for 72 h) [36], a phase 2 open label first-in-disease trial in intracerebral hemorrhage has recently been completed [16] and a phase 2, randomized, double-blind, placebo-controlled study is underway [37]. The protocol involves administering CN-105 intravenously within 12 hours of symptom onset, and every 6 hours for up to a maximum of 3 days (total of 13 doses). While trial results are not yet available, the primary outcome measures include the number and severity of adverse events, and in-hospital 30-day mortality and neurological outcomes. In addition, a phase 2, randomized, double-blind, placebo-controlled study is examining CN-105 as a neuroprotectant to reduce post-operative cognitive decline in patients undergoing non-cardiac, non-neurological surgery [38], with the peptide being administered intravenously at 140, 420 or 1,400 nmol/kg, 1 hour before and every 6 hours after surgery for 3 days.

5. Conclusion and future perspectives

A large number of different CARPs have now demonstrated neuroprotective efficacy in animal models of stroke [3], providing confidence in the potential of this class of peptides with multimodal cytoprotective mechanisms of action to translate to an effective clinical stroke therapeutic. Indeed, the NA-1 and CN-105 peptides have paved the way in demonstrating the translational process from pre-clinical to clinical studies for ischemic and hemorrhagic stroke, respectively. Even more promising is the first clinical demonstration of a positive effect on clinical outcomes in a subgroup of patients with large artery ischemic stroke treated with the neuroprotective agent NA-1 [8], against which other promising CARPs such as the R18 peptide will now need to be compared [39]. As with any class of drug, the future challenge for CARPs will be to better characterize their neuroprotective mechanisms of action so that the peptide amino acid sequence can be tailored to improve their efficacy, affinity to specific targets and stability, as well as to provide broad spectrum neuroprotection for different stroke subtypes, and possibly other neurological disorders 22.

Declaration of interest

N Knuckey and B Meloni are named inventors on several patents for the use of CARPs as therapeutic agents. In addition, N Knuckey, B Meloni. and D Blacker. are shareholders in Argenica Therapeutics, which is a company developing R18 as a stroke therapeutic. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or conflict with the subject matter or materials discussed in this manuscript apart from those disclosed 10.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose 6.

Additional information

Funding

This paper was not funded.