Prophylactic treatment with CN-105 improves functional outcomes in a murine model of closed head injury

Introduction: Traumatic brain injury (TBI) has become the signature wound of recent military operations and training over the past two decades. A treatment that would protect against secondary brain injury in TBI and improve the long-term functional outcomes of traumatic brain injury on service personnel is therefore of great interest. One agent that has shown promise in modifying the post-traumatic neuroinflammatory response in murine closed-head injury models of TBI is a novel, small 5 amino acid apolipoprotein E (ApoE) mimetic peptide, CN-105. The goal of this study was to determine whether CN-105 would maintain its neuroprotective effects if administered prior to closed-head injury in a clinically relevant murine model.Materials and Methods: CN-105 was synthesized by Polypeptide Inc. (San Diego, CA) to > 99% purity. We examined the efficacy of prophylactic administration of CN-105 in a well-established closed head injury model associated with reproducible vestibulomotor deficits. CN-105 was dissolved in sterile 0.9% saline and administered to male 12 week old C57-BL/6 mice intravenously (IV) through a tail vein and/or by intraperitoneal (IP) injection in a volume of 100 microliters at various time points prior to injury. Vehicle treated animals received IV and/or IP injection of 100 microliters of normal saline at the same time points. Animals were randomly assigned to treatment groups immediately following injury and all behavioral observations were conducted by investigators blinded to treatment. Vestibulomotor function was assessed using an automated Rotarod (Ugo Basile, Comerio, Italy). An in vivo assessment of the pharmacokinetics of CN-105 following IV or IP administration in healthy fed adult male CD-1 mice was conducted by Charles River (Worcester, MA). Mouse brains were harvested and sectioned 7 days post TBI with 25 ml phosphate buffered saline (PBS) and fixed overnight in 4% paraformaldehyde in PBS. Brains were left in sucrose 30% prior to being sectioned in 40 µm sections by microtome. Microglial activation in rat hippocampi was assessed in 5 vehicle and 5 CN-105 treated rats who had been received CN-105 three hours prior to injury using F4/80 immunohistochemical staining.Results: Intravenous (IV) administration of CN-105 up to thirty minutes prior to closed head injury significantly improved durable vestibulomotor function compared to vehicle control-treated animals. In pharmacokinetic studies in uninjured CD-1 mice, IP administration of CN-105 resulted in an ~ 3-fold increase in the time to reach maximal plasma concentration (Tmax) and an ~1.5-fold increase in mean plasma residence time (MRT) compared to IV administration although the terminal elimination half-lives (T1/2) were similar. When CN-105 was co-administered by IP and IV dosing 6 hours prior to injury, a durable improvement in vestibulomotor function was observed up to 28 days following injury. Microglial counted in CN-105 treated specimens were significantly less (p = 0.0327) than those counted in vehicle specimens. Conclusion: CN-105 improves functional outcomes and reduces hippocampal microglial activation when administered prior to injury. This may be adaptable in the future as a means of preventing or reducing TBI-related secondary brain injury and improving clinical outcomes in service members who are identified as high-risk for TBI, are pre-treated, and subsequently suffer a TBI in the line of duty.