HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ISOLATION, SPECTROSCOPIC CHARACTERIZATION, AND IMMUNOSUPPRESSIVE ACTIVITIES OF 2 RAPAMYCIN DEGRADATION PRODUCTS

A high performance liquid chromatographic method has been developed fo r the isolation of two degradation products of rapamycin which is curr ently under development as an immunosuppressive agent. Prior to isolat ion, the drug was incubated at 37 degrees C in rat bile or ammonium ac etate (pH 8.0). The isolation was achieved by a Supelco, PLC-18 21.2 x 250 mm, 18 mu m column using methanol/ammonium acetate gradient mobil e phase. After evaporation of methanol, the remaining eluates were lyo philized. The isolated degradation products were characterized by nega tive ion fast atom bombardment mass spectrometry (FAB MS) and proton n uclear magnetic resonance spectroscopy (H-1 NMR). Degradation product A was found to be a macrolide ring-opened hydrolysis product of rapamy cin where the C25 ester bond had been hydrolyzed. Degradation product B was determined to be a ring-opened isomer of rapamycin. B had less t han 4% of the potency of rapamycin in a thymocyte proliferation assay, while A had minimal activity at concentrations tested.