(T1430-10-55) Development of a Novel Tocotrienol Analogue, Tocoflexol, as a Powerful Radioprotective Agent

Purpose: Vitamin E is a well-known antioxidant and effectively scavenges free radicals generated by radiation exposure. Tocols collectively are promising agents for radiation protection with minimal toxicity. Although α-tocopherol is the most studied form of vitamin E, recent studies have shown that tocotrienols are more potent radioprotector than α-tocopherol (AT). Despite their superior biological activity, tocotrienols have shorter plasma half-life and require a higher dose to achieve therapeutic levels in the body. This is due to the fact that their transporter, the α- tocopherol transfer protein (ATTP), shows preferential binding for AT, putting the tocotrienols at higher risk for metabolism and biliary excretion due to longer residence time in the liver. The reason for the low affinity of the tocotrienols for ATTP is the rigid tail structure which makes them rotationally restricted and does not allow them to fit in the binding pocket of the protein. We have designed and synthesized a novel class of compounds, named tocoflexols, which are aimed to provide radioprotective effects by overcoming the pharmacokinetic limitations of tocotrienols.

Methods: In tocoflexols, a mono- or dienyl chain substitutes the tridienyl chain of the farnesyl tail of tocotrienol. To optimize resources and increase the chances of success, we subjected the possible candidates to a rigorous computer-based screening. Molecular dynamic simulations were performed using SYBYL 8.1 to screen potential tocol derivates for enhanced binding to alpha tocopherol transfer protein. There is consistent evidence that the hypercholesterolemic, cytoprotective and anticancer properties activities of vitamin E are directly correlated to their cellular uptake. We compared the cellular uptake of tocoflexol with that of the tocols by incubating mouse NSC-34 cells with 5 uM alpha tocopherol, delta, and gamma tocotrienol for various time points. Cellular uptake levels were determined using GC/MS. We also studied the radioprotective activity of delta tocoflexol using male CDF21 mice subjected to lethal doses of radiation. For this study mice were pre-treated, 24 before radiation with a subcutaneous injection of 0.1 ml of tocoflexol (300 mg/kg body weight). The vehicle group received 5% tween 80 in PEG. Mice were subjected to 9.5 Gy total body irradiation using Mark I Model 68A cesium-137 animal irradiator.

Results: Our computational analysis has shown that tocoflexol has superior binding capacity to ATTP, the key transporter that reduces elimination rate of tocols. We demonstrated an accelerated cellular uptake of tocoflexol and tocotrienols compared to alpha tocopherol. Delta tocoflexol has 6.7 times higher cellular uptake rate compared to alpha tocopherol. We also observed that subcutaneous administration of tocoflexol in mice prior to lethal total-body irradiation (9.5 Gy) is powerfully radioprotective, providing 100% survival compared to 40% survival in vehicle-treated irradiated mice.

Conclusion: Collectively, our studies show that tocoflexol has potential for the therapeutic use to protect against radiation due to its combination of tocopherol-like pharmacokinetic properties (ATTP binding) and tocotrienol-like pharmacological properties (cell uptake).

Acknowledgments: The University of Arkansas has received patent protection for parts of this research. A potential royalty stream may occur consistent with the University of Arkansas policy. This research was supported in part by grants from the National Center for Research Resources award 1ULl RR029884, the IDeA Networks of Biomedical Research Excellence (INBRE).

Tocol uptake measured in vitro (NSC-34 cells)



Molecular dynamics predicts that removing the center double bond from tocotrienols makes binding with ATTP more feasible



Survival curve for tocoflexol radioprotection study in mice. Tocoflexol provides 100% protection against lethal doses of radiation, n=10