Synthesis and evaluation of an injectable everolimus prodrug.

A novel water-soluble everolimus prodrug, glutathione-everolimus, was designed and synthesized by introducing an endogenous tripeptide with an acetyl as the linker. The improvement in water solubility allowed the conjugate to be developed into an injectable drug. The results of biological evaluation in vitro and in vivo suggested that the prodrug was more effective and long acting than everolimus. Meanwhile, the pharmacokinetics study in vivo confirmed that the delivery of everolimus through the injection of the prodrug can overcome the low bioavailability of oral everolimus.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

Folate receptor (FR)-ß has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted.