Stealth Liposomes: An Improved Sustained Release System for 1-/?-D-ArabinofuranosyIcytosine '

Newly developed liposomes with prolonged circulation half-lives and dose-independent pharmacokinetics (Stealth" liposomes) have been tested for their efficacy as a slow release system for the rapidly degraded, schedule-dependent, antineoplastic drug 1-0-D-arabinofuranosylcytosine (ara-C) in the treatment of murine L1210/C2 leukemia. Mice were given injections of either 10* cells or 10' cells by either the i.v. or the i.p. routes. Leukemia-bearing mice were treated with either i.v. or i.p. injec tions of free drug, i.v. or i.p. injections of liposome-entrapped drug, or 24-h i.v. infusions of free drug. Long-circulating liposomes contained, as the stealth component, either monosialoganglioside or polyethylene gly-col-distearoylphosphatidylethanolamine. Liposomes lacking the stealth components (non-stealth liposomes) were also injected for comparison. At lower dose ranges, stealth liposomes were superior to non-stealth liposomes in prolonging mean survival times of the mice, and all liposome preparations were superior to injections of the free drug. Drug entrapped in stealth liposomes, when administered at or near the maximum tolerated dose of 100 mg/kg ara-C were considerably superior to 24-h free drug infusions given at the same total drug dose. Therapeutic effect was related to the half-life of leakage of ara-C from the liposome formulations, as well as to circulation half-life, with maximum therapeutic effect achieved with long circulation half-lives and more rapid leakage rates. The thera peutic efficacy of non-stealth liposomes increased with increasing lipo some (and drug) dose as a result of saturation of liposome uptake by the mononuclear phagocyte system, which resulted in longer circulation half-lives for these liposomes at higher doses (Michaelis-Menten pharmaco kinetics). Liposome entrapment can protect rapidly degraded drugs from breakdown in n'TO,with release of the drugs in a therapeutically active form over periods of up to several days. The dose-independent pharma cokinetics and reduced mononuclear phagocyte system uptake of stealth liposomes gives them distinct advantages over non-stealth liposomes. experiments involving low doses of liposomal ara-C, liposomes were chromatographed over Sephadex G-50 in sterile, pyrogen-free NaCI (0.9%, USP) containing 10 mM /V-tris(hydroxymethyl)methyl-2-ami-noethanesulfonic acid, pH 7.4 (buffer). For higher doses of liposomal ara-C, where dilution of the liposomes was undesirable, liposomes were dialyzed through Spectrapore dialysis tubing (cutoff, M, 12) against several changes of buffer at 4Â°C.For the highest doses of liposomal ara-C, liposomes were concentrated by centrifugation at 100,000 x g for 1 h, and free ara-C was removed by washing the liposomes 3 times in buffer. Following the final washing step, the liposome pellet was made up to the desired concentration by the addition of buffer. In some liposome preparations, liposomes were pelleted by centrifugation, the supernatant containing free ara-C was removed, but the pellet was not washed. The pellet was resuspended in buffer to the desired drug concentration. In those preparations, the total amount of free ara-C was equivalent to the total amount of liposomal ara-C, as determined by chromatography of the samples over Sephadex G-50. Samples of the liposome preparations were taken before and after separation of the free drug, and the concentration of ara-C in the preparations was calculated from the specific activity of [5-'H]ara-C. Extruded vesicles were sized by quasielastic light scattering using a BI-90 particle sizer (Brookhaven Instruments, Holtsville, NJ). Phospholipid concentration was determined using the method of Bartlett (32), and trapped volumes of the liposomes were determined from the specific activity of [3H]ara-C. The average mean diameter of the lipo somes was approximately 190 to 220 nm and the trapped volume was in the range of 4 to 6 liters/mol phospholipid. These results could be reliably reproduced from sample to sample. Leakage of ara-C from Liposomes.