Abstract
Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.
Highlights
Gaukroger Received 15 November 1982; accepted 21 February 1983 responsible for the acquired resistance in some melanoma mutants. We have investigated these variables by a study in which three patients with advanced malignant melanoma resistant to other cytotoxic drugs were treated with high dose MTX
Plasma MTX concentrations were monitored by an EMIT assay during the infusion, to give immediate levels to assist in patient management, and the rate of infusion adjusted to maintain the concentration above 10-6 M
high pressure liquid chromatography (HPLC) analysis of these samples showed the major MTX metabolite, 70H-MTX, to be present in plasma at concentrations similar to those reported in patients with osteogenic sarcoma (Breithaupt et al, 1982) and that samples obtained after 24 h contained 70H-MTX at concentrations exceeding that of MTX (Figure 1)
Summary
Clinical studiesThree patients, 2 males and 1 female, with histologically proven stage III malignant melanoma, were treated with high dose methotrexate given by 24 h infusion. Plasma MTX concentrations were monitored by an EMIT assay during the infusion, to give immediate levels to assist in patient management, and the rate of infusion adjusted to maintain the concentration above 10-6 M. This was achieved in all patients, with peak concentrations > 10- M being observed. At the end of each infusion, calcium leucovorin was given i.v. at a dose of 30 mg, thereafter 19 mg was given at 6 hourly intervals until plasma MTX concentrations fell below 10-6 M. In all cases the patients urine was kept alkaline using sodium bicarbonate and a fluid load was given at the end of the infusion.
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