Treatment of solid L1210 murine tumors with local hyperthermia and temperature-sensitive liposomes containing methotrexate.

Abstract

Abstract Liposomes can be designed to break down and release their contents preferentially in an anatomical region subjected to local hyperthermia. Such “temperature-sensitive” liposomes, containing 3H-radiolabeled methotrexate ([3H]MTX), were made by sonication of dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine (7:3, by weight) and injected i.v. into mice with L1210 tumor implanted in each hind foot. The lipid was radiolabeled with [14C]dipalmitoyl phosphatidylcholine. Encapsulated [3H]MTX at a dose of 0.0058 mg/kg was cleared from the plasma more rapidly when one foot was heated to 42° in a water bath than when neither foot was heated. This finding indicated release of methotrexate (MTX) from liposomes in the small fraction of blood circulating through the heated foot. Four hr after injection of liposomes, the heated tumors contained an average of 14 times as much [3H]MTX as did the unheated ones. A large dose (300 mg/kg) of free, unlabeled MTX effectively blocked incorporation of [3H]MTX but not of [14C]dipalmitoyl phosphatidylcholine into the tumors, indicating that the differences observed with heating did not simply represent liposomes sequestered in the extravascular space of the tumors. Similar inhibition was seen with a dose of dl-l-calcium leucovorin (300 mg/kg), a compound which competes with MTX for membrane transport. A larger fraction of injected lipid than of [3H]MTX remained in the circulation after 4 hr, and there was less than a factor of 2 difference in [14C]dipalmitoyl phosphatidylcholine uptake between heated and unheated tumors. These findings combined with those from our previous studies in vitro suggest the following picture. The temperature-sensitive liposomes break down preferentially in the heated tumor. Some of the released drug is taken up into the tumor cells via the usual transport mechanisms for MTX, and the rest circulates in the bloodstream to be cleared into other tissues and finally from the body by liver and kidneys. The lipid continues to circulate longer partly in association with serum apolipoproteins. In chemotherapy studies with MTX (3 mg/kg), the major findings were these: (a) liposome-encapsulated MTX caused a greater delay in tumor growth than did an equivalent concentration of free MTX with or without heating; and (b) heating alone of the tumor to 42° for 1 hr delayed tumor growth for about 0.7 days, whereas heating increased by about 2.8 days the growth delay observed with heat-sensitive liposomes containing MTX (3 mg/kg). The nonadditive effect (about 2.1 days) corresponds to an extra cell kill of 4- to 16-fold. The magnitude of the nonadditive effect was probably limited by such factors as saturation of cellular transport mechanisms for MTX and saturation of the ability of the serum to release MTX from liposomes at the phase transition.