Pharmaceutical development of a parenteral lyophilized formulation of the novel antitumor agent aplidine

Aplidine is a naturally occurring cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. Aplidine displays promising in vitro and in vivo antitumor activities against various solid human tumor xenografts and is therefore developed now for clinical testing. The aim of this study was to develop a stable parenteral pharmaceutical dosage form for clinical Phase I testing. Aplidine raw material was characterized by using several chromatographic and spectrometric techniques. These experiments showed that aplidine exists as two isomers. A stability-indicating HPLC assay was developed. Solubility testing showed that aplidine exhibits very poor aqueous solubility. Because solubilized aplidine showed substantial degradation under heat and light stress testing conditions, it was decided to develop a lyophilized dosage form. Freeze-drying was carried out with a 500 micrograms/mL solution of aplidine in 40% (v/v) tert-butanol in Water for Injection (WfI) containing 25 mg/mL D-mannitol as a bulking agent. Differential scanning calorimetry was applied to determine the optimal freeze-drying cycle parameters. The prototype, containing 500 micrograms aplidine and 25 mg D-mannitol per vial, was found to be the optimal formulation in terms of solubility, length of lyophilization cycle, and dosage requirements in the forthcoming Phase I clinical studies. Quality control of the freeze-dried formulation demonstrates that the manufacturing process does not affect the integrity of aplidine. The optimal reconstitution solution was found to be 15/15/70% (v/v/v) Cremophor EL/ethanol/WfI (CEW). Both reconstituted product and dilutions of the reconstituted product with normal saline (up to 1:100 v/v) appeared to be stable for at least 24 hours after preparation. Shelf-life data, available thus far, show that the lyophilized formulation is stable for at least 1 year when stored at +2-8 degrees C in the dark.