The Effect of the Calpain Inhibitor Gabadur on Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a leading cause of mortality and chronic disability. It afflicts people of all ages with 2.2 million traumatic brain injuries occurring per year. A successful treatment to stop the progression of traumatic brain injury and prevent the consequent neurological and functional deficits has not yet been determined. Calpain over-activation is a major contributor to cell dysfunction and neurodegeneration following TBI. Therefore, calpain inhibition is a viable therapeutic strategy to stop neurodegeneration and improve neurological function post-TBI. Gabadur is a novel calpain-inhibitor therapeutic derived from the calpain protease inhibitor leupeptin linked to the FDA-approved anti-epileptic drug, pregabalin, as a carrier molecule. This formulation permits the entire compound to cross the blood-brain barrier and target the site of neural injury after peripheral administration. We hypothesize that the administration of Gabadur can halt calpain over-activation post-injury and reduce neurological and functional deficits that occur following severe traumatic brain injury. A single 80 mg/kg dose of Gabadur was administered intraperitoneally (i.p) immediately following severe injury induced by the controlled cortical impact (CCI) rodent model of TBI. Control rats were subjected to the same injury, but received saline vehicle i.p. At 48 hours post-TBI, rats were euthanized and tissue from the injured hemisphere was collected. In comparison to control rats, rats given Gabadur immediately post-TBI displayed a significant reduction in the number of degenerating cortical neurons, as shown by Fluoro-Jade B staining. Smaller lesion sites were observed. Cytoskeletal protection was observed in Gabadur-treated rats via western blot for calpain-cleaved αII-spectrin breakdown product and microtubule-associated protein 2 (MAP-2). These results suggest that a single i.p. administration of Gabadur after CCI injury is able to reach the injury site and decrease TBI-induced neurodegeneration. Spatial memory and motor function were evaluated in sham, TBI-1XPBS treated rats, and TBI-Gabadur treated rats to test Gabadur’s ability to improve function post-TBI. TBI-Gabadur rats received two 80mg/kg doses, once immediately post-injury and again at 16 hours post-injury. Short- and long- term functional deficits induced by TBI were identified in open field activity, the active place avoidance task, and Morris water maze (MWM); somatosensory and motor deficits were detected up to 5 days post-injury. Gabadur administration did not significantly improve motor nor somatosensory recovery nor did it ameliorate functional deficits in active place avoidance. However, improvements in short-term and long-term memory were seen in TBI-Gabadur rats in the MWM task. TBI-Gabadur rats also appeared to acquire the MWM task faster than TBI-1XPBS rats as indicated by faster mean latencies to the hidden platform on particular training days. Western blot analysis at 3 weeks post-injury assessed changes in hippocampal and cortical protein levels for membrane associated scaffolding protein, PSD-95, the NMDA receptor subunits, NR1 and NR2B, the GluA2 subunit, interneuron marker, glutamic acid decarboxylase (GAD), and the inflammatory marker, glial fibrillary acidic protein (GFAP). Gabadur-treatment prevented TBI-induced increases in the excitotoxic NR2B containing NMDA receptor (GluN2B), as well as GluA2 subunit levels of the AMPA receptor. Gabadur treatment did not affect TBI-induced decreases in GAD nor TBI-induced increases in GFAP. Additional western blot studies at 48 hours post-TBI addressed whether a single 80mg/kg dose of Gabadur’s diastereomer, Gabadur A, has similar actions to Gabadur; like Gabadur, Gabadur A also inhibited calpain and prevented calpain substrates from cleavage.