Detection by high-performance liquid chromatography of methotrexate and its metabolites in tumor tissue from osteosarcoma patients treated with high-dose methotrexate/leucovorin rescue.

Methotrexate (MTX) polyglutamates were detected in osteogenic sarcoma tumor samples obtained from patients 24 or 48 h after receiving high-dose MTX/leucovorin rescue therapy. Tumor samples were assayed by high-performance liquid chromatography, and polyglutamyl metabolites, along with MTX, were quantitated using both direct u.v. absorption at 313 nm and an enzyme titration assay. Good agreement between these two methods was found although the more sensitive enzyme assay detected peaks in some samples not detected by u.v. absorbance. A wide variation in MTX:MTX polyglutamate levels (1:1 to 25:1) was found among the six clinical samples studied. Also, no correlation between the extent of polyglutamate formation and plasma levels (determined at the time of tumor sampling) was observed. High intracellular levels of a derivative which appears to be the 7-hydroxy metabolite of MTX were also detected in four of six samples. This material coeluted with authentic standard, showed spectral properties like standard 7-OH-MTX, and did not inhibit dihydrofolate reductase.

Improved therapeutic index with high-dose methotrexate: comparison of thymidine-purine rescue with citrovorum factor rescue in mice.

Two biochemically different rescue agents, citrovorum factor (CF) and thymidine-inosine-allopurinol (TIA), were compared in an attempt to identify the mechanism for the increased therapeutic index achieved with high-dose methotrexate (MTX) plus rescue. Both CF and TIA were capable of protecting mice from MTX dosages up to 2000 mg/kg. Treatment of L1210-bearing mice with 2000 mg/kg MTX plus CF or TIA produced a 70 and 100% increase in life span, respectively, compared with 29% increase in life span achieved with the maximally tolerated dose of MTX alone. Bioassay of surviving peritoneal L1210 cells showed that a 4.5-log tumor kill occurred 24 hr after 2000 mg/kg MTX, while 400 mg/kg MTX produced only a 2-log cell kill. This differential tumor kill in the 4-hr period after MTX and prior to the onset of rescue accounted for the observed increase in animal survival times. In addition, treatment with 2000 mg/kg MTX resulted in a one-log-greater tumor kill of cells metastasized to the brain than did treatment with 400 mg/kg MTX. Following 2000 mg/kg MTX, additional tumor kill, as measured by bioassay, occurred during the period of TIA rescue but not during CF rescue, which was consistent with the observed differences in survival times between CF- and TIA-rescued mice. DNA synthesis in tumor and host tissue, as measured by the rate of [3H]dCyd incorporation into DNA, was cyclic after TIA administration but not after CF administration. The cyclic nature of DNA recovery in TIA-treated mice paralleled plasma kinetics of thymidine. It is postulated that " thymineless " intervals created by the rapid disappearance of thymidine resulted in inhibition of DNA synthesis and additional tumor cell kill during TIA rescue. Normal tissue did not appear to be adversely affected by exposure to these " thymineless " intervals.