FK1706, a novel non-immunosuppressant neurophilin ligand, ameliorates motor dysfunction following spinal cord injury through its neuroregenerative action

Abstract

Injured spinal cord axons fail to regenerate in part due to a lack of trophic support. While various methods for replacing neurotrophins have been pursued, clinical uses of these methods face significant barriers. FK1706, a non-immunosuppressant neurophilin ligand, potentiates nerve growth factor signaling, suggesting therapeutic potential for functional deficits following spinal cord injury. Here, we demonstrate that FK1706 significantly improves behavioral outcomes in animal models of spinal cord hemisection and contusion injuries in rats. Furthermore, we show that FK1706 is effective even if administration is delayed until 1 week after injury, suggesting that FK1706 has a reasonable therapeutic time-window. Morphological analysis of injured axons in the dorsal corticospinal tract showed an increase in the radius and perimeter of stained axons, which were reduced by FK1706 treatment, suggesting that axonal swelling and retraction balls observed in injured spinal cord were improved by the neurotrophic effect of FK1706. Taken together, FK1706 improves both behavioral motor function and the underlying morphological changes, suggesting that FK1706 may have therapeutic potential in meeting the significant unmet needs in spinal cord injury.

Introduction

The prevalence of spinal cord injury in the United States is approximately 200,000, with 13,000 additional spinal cord injuries annually, with the causes of these injuries ranging from traffic accidents and community violence to recreational activities and workplace-related injuries (Sekhon and Fehlings, 2001). The majority of spinal cord injury patients range from 20 to 40 years of age and approximately 55% of acute spinal cord injury occurs in the cervical region. The severity of spinal cord injury varies depending on the spinal level, its width, and the extent of the injury, and can lead to paraplesia or tetraplesia. Loss of sensorimotor function due to injury requires lengthy hospitalization after the initial incident as well as extensive rehabilitative care, creating physical, emotional, and economic burdens on both the individual and society. Furthermore, all patients with spinal cord injury face a lifelong series of acute and chronic non-neurological complications that can be life-threatening. There are no approved therapeutic agents for spinal cord injury, so the primary objective of current therapies is to prevent further injury by physically stabilizing the spine and by inhibiting the inflammatory response that results from the injury. Although palliative therapy using the corticosteroid, methylprednisolone, can reduce inflammation of the injured area, and standard medical practice can help control complications arising from chronic denervation, spinal cord injury still represents an area of significant unmet medical need.

Members of the neurotrophin family, including nerve growth factor (NGF) (Kim et al., 1996, Grill et al., 1997, Ramer et al., 2000, Tuszynski et al., 2002), brain-derived growth factor (BDNF) (Bregman et al., 1997, Kobayashi et al., 1997, Novikova et al., 2002), and neurotrophin-3 (NT-3) (Bregman et al., 1997, Blits et al., 2000, Ramer et al., 2000), might have therapeutic potential following spinal cord injury. These molecules, and their respective receptors, are upregulated after central nervous system (CNS) injury (Mocchetti and Wrathall, 1995) and are capable of supporting neuronal survival, stimulating the neurite outgrowth needed for reorganization of the injured CNS, and inducing the expression of key enzymes for neurotransmitter synthesis necessary to compensate for reduced innervation. However, despite the promising work on these compounds, clinical use has been limited by poor oral bioavailability and adverse effects resulting from non-specific neuronal stimulation (Apfel et al., 1998, McArthur et al., 2000). Thus, an orally available compound that enhances endogenous neurotrophin signaling, with no neurotrophic properties of its own, would be highly desirable.

FK1706 is a novel, non-immunosuppressant neurophilin ligand, a class of ligands characterized by binding with high affinity to FK506 binding proteins. We have previously shown that FK1706 potentiates NGF-induced neurite outgrowth in vitro, with no neurotrophic activity of its own (Price et al., 2005). Furthermore, motor dysfunction after spinal cord hemisection is improved in rats treated with FK506 or FK1706 subcutaneously (Voda et al., 2005). In the present study, we evaluated the effect of oral administration of FK1706 in two different rat models of spinal cord injury, a hemisection and a contusion model. In addition, we also expanded the study to determine the therapeutic time-window of FK1706 by delaying treatment initiation following contusion injury.

Hemisection models are frequently used for studying behavioral alterations related to neural regeneration (Huang et al., 1999, Bradbury et al., 2002, Dergham et al., 2002, Schucht et al., 2002, Voda et al., 2005). In addition, traumatic spinal cord injury produces morphological changes, including organelle accumulation, focal swelling, and axonal detachment (Yaghmai and Povlishock, 1992), as well as axonal swelling and disruption, and produces retraction balls (Lu et al., 2001). Therapeutics should produce functional improvement and provide anatomical evidence for that improvement. Therefore, we also measured changes in axon morphology in the spinal cord contusion model.