POTENTIAL THERAPUTIC ROLE OF FTY720 IN SPINAL CORD INJURY: BEHAVIORAL RESPONSE IN THE RAT MODEL

1. POTENTIAL THERAPUTIC ROLE OF FTY720 IN SPINAL CORD INJURY: BEHAVIORAL RESPONSE IN THE RAT MODEL SHANE ABDUNNUR – University of Texas – Houston DANNY LEE, BRUCE MATHERN, HAROLD YOUNG - Medical College of Virginia

2. Introduction • Spinal cord injury (SCI) affects nearly 11,000 people per year • Prevalence: 250,000 people • SCI disproportionately affects young healthy adults between the ages of 16-40 • Long-term morbidity and mortality implications • Inflammation after SCI has been postulated to impair short and long term functional recovery. • A novel immunosuppressant, FTY720 • Sphingosine-1-phosphate (S1P) receptor modulator • Sequesters T lymphocytes in peripheral/secondary lymphoid organs • Modulates systemic lymphocytic inflammatory reactions.

3. Hypothesis FTY720-treated rats will show expedited and greater overall functional recovery as compared with vehicle control-treated animals.

4. Spinal Cord Injury • Primary physical trauma • Secondary damage → Local Factors • Micro-circulatory and biochemical changes • Electrolyte shifts • Edema and loss of ATP/GTP production • Complex milieu of inflammatory cells and mediators are postulated to be an important determinant in the long-term functional status of SCI patients • Proinflammatory molecules include IL-1, TNF, and arachadonic acid metabolites • Secondary necrosis and apoptosis → further damage

5. T Lymphocytes • Because T lymphocytes play a critical role in the recruitment and activation of various immune cells, T cells are thought to be an integral participant in the progression of secondary cord injury after SCI. • Numerous studies have shown that T lymphocyte infiltration after SCI in rodents is correlated with increased cord necrosis as compared with T cell deficient controls. • T lymphocytes may present a target for medical intervention to prevent further damage after the initial insult in SCI patients.

6. Sphingosine-1-Phosphate • S1P is a bioactive phospholipid derived from numerous cells including RBCs, platelets, and endothelial cells. It is believed to mediate its cellular actions via 3 mechanisms: • S1P alters the microlipid environment in glia and neuronal cells, thus impairing the ability of oligodendroglia to produce myelin. • S1P is thought to act as an intracellular messenger. S1P can effect the release of calcium, independent of IP3. • S1P mediates its effects via activation of cell-surface S1P receptors, which are ubiquitously expressed in the CNS and on T cells.

7. FTY720 (2-amino-2-[2-(4-octylphenyl) ethyl] propane-1,3-diol) • FTY720 induces T cell S1P receptor internalization, thus attenuating T-cell responses to S1P gradients. Because S1P is a T cell chemoattractant, peripheral sequestration of T lymphocytes ensues. • FTY720 inhibits phospholipase A2, which reduces production of arachidonic acid-mediated S1P receptors on T lymphocytes. • S1P may also tighten the endothelial barrier, thus inhibiting CNS T-cell influx. • FTY720 readily crosses the BBB

8. Behavioral Studies • The Basso, Beattie, and Brensnahan locomotor rating scale (BBB) has been shown to be a reliable, reproducible, and valid measure of functional activity in rodents that corresponds to the pathologically defined size of cord lesion. • Rodents are rated on varying degrees of joint mobility, weight support, and forelimb/hindlimb coordination • The BBB scale was used to confirm satisfactory SCI and evaluate progressive locomotor function over a period of 5 weeks.

9. Materials and Methods • Animals: Long–Evans rats (~220 g). They were given food and water and were housed on a 12/12-h light–dark cycle. All protocols were approved by IACUC. • Spinal cord injury: • NYU impactor device • Dorsal laminectomy to expose the dura overlying T8 • The vertebral column stabilized by clamping • NYU impactor probe was dropped from a distance of 50 mm • Complete functional injury was confirmed using BBB immediately after surgery (BBB score of 0)

10. Materials and Methods • Post-SCI Care: Animals were monitored after the injury, and the bladders were expressed TID for the first week and BID thereafter for a total of 13 days. Vehicle control-treated animals received DMSO daily while the FTY720-treated rats received 0.25 mg/kg daily. All animals received gentamycin daily. • BBB Open Field Scoring: Open field locomotion score was evaluated by using the modified Basso-Beattie-Bresnahan (BBB) scoring system of 20 points. The mice were placed in an open field on a non-slippery surface. In each testing session, the mice were observed individually for 3 minutes by the observers. The mice were tested once per week, every week, for 5 weeks, including post-operative day (POD)#0 and #1.

11. Exclusionary Criteria • Of 20 rats, several were ultimately excluded from the study: • 1 due to intubation trauma • 2 due to immediate post operative BBB scores of >0 • 1 due to deterioration (repeated UTIs) at week 5 • 2 rats were treated as sham (surgical incision without subsequent SCI or treatment) • 1 due to unpaired caging • NOTE: N=30 in all experiments combined

12. FTY720-Treated Rats Demonstrate Improved Bladder Function • FTY720-treated rats were noted to have consistently decreased urine expression throughout the 5 week observation period as compared to DMSO vehicle-treated control rats • This difference, 5.23 ml, was most pronounced at POD #7 and decreased until the completion of bladder expression at POD #13 • No difference was noted by POD #13

13. FTY720-Treated Rats Demonstrate Improved Locomotor Coordination • After confirmation of adequate SCI on POD #0, the BBB locomotor function scoring system was used to assess hind leg functional recovery for 5 weeks • Improved locomotor function was first noted at post-op week 3 • Mean BBB scores were 13.5 for FTY720-treated rats and 11.5 for DMSO vehicle-treated control rats by week 5 • DMSO vehicle-treated control rats totally lacked HL-FL coordination whereas FTY720 treated rats were noted to have almost complete HL-FL coordination after 5 weeks

14. Conclusions and Discussion • Purpose: • Assess the functional recovery in SCI rats treated with FTY720 • FTY720 may positively impact the overall level of functional recovery after 3 weeks, which may be maintained as long as 5 weeks post SCI. • Additional beneficial effects of FTY720 may be increased bladder functionality as reflected in the bladder expression data.

15. Conclusions and Discussion • Results are consistent with the mechanism of action of FTY720 and what is currently known about S1P actions in the CNS. • Results are also consistent with the theory that T lymphocyte influx into the CNS after SCI is detrimental to functional recovery. Because this theory is not universally accepted, further studies in this area remain important in classifying pathological versus beneficial immunological response in SCI.

16. Conclusions and Discussion • Inferences from these data are guarded and provide a preliminary indication of possible effects of FTY720. • Other studies have included: • Histopathological assessment of spinal cord lesion in vehicle- control vs. FTY720-treated rats • FACS to assess T cell influx into the spinal cord

17. Future Studies • Apoptosis assay to determine protective effect of FTY720 in SCI • Does FTY720's modulation of intracellular calcium release exert protective effects in vivo? • Protection against necrosis? • Long-term studies and combination therapy • Does FTY720's effects last longer than 5 weeks? • Long-term implications of immunosuppression • Synergistic effects with other pharmacotherapy • Other potential effects: bladder and GI function, autonomic regulation, and infection