Interactions of carboxypeptidase G₂ with 6S-leucovorin and 6R-leucovorin in vitro: implications for the application in case of methotrexate intoxications

Carboxypeptidase G₂ (CPG₂) is used when unexpected toxicity or renal failure occurs during high-dose methotrexate therapy. Leucovorin is also administered to antagonise the effects of methotrexate on purine anabolism. To investigate the effects of CPG₂ on leucovorin rescue, we incubated the enzyme with both stereoisomers and analysed the degradation. A method for separating the stereoisomers of leucovorin, the internal standard aminopterin and the degradation products by capillary electrophoresis with 2.6-dimethyl-β-cyclodextrin as a chiral selector has been developed. The active 6S-leucovorin is degraded much faster than the inactive 6R-isomer. The maximum observed degradation velocity was 31 μM/min for 6S-leucovorin and 20 μM/min for 6R-leucovorin, respectively, with an initial concentration of each stereoisomer of 250 μM. Similar results were obtained at lower concentrations of leucovorin isomers. Thus, the selectivity of CPG₂ for methotrexate in comparison to leucovorin is not as high as anticipated in the literature as only the active 6S-leucovorin and not the mixture of the diastereomers should be taken into account. We conclude that the protective effects of leucovorin are antagonized by CPG₂. Therefore, CPG₂ should be administered to patients with caution.