Hydrolysis of methotrexate-immunoglobulin conjugates by liver homogenates and characterization of catabolites.
Methotrexate (MTX) linked to antitumor antibodies inhibits tumor growth better than free MTX, free antibody, or MTX linked to normal rabbit IgG (NRG), in spite of the less effective inhibition of the target enzyme dihydrofolate reductase (DHFR) by conjugated MTX. In addition to the demonstrated higher uptake of MTX linked to antitumor antibodies (compared with the uptake of free MTX or nonspecific IgG conjugates), a contributory factor to the superior tumor inhibitory action of MTX-IgG conjugates may be the prolonged release of active drug from the internalized conjugate. Therefore, we have investigated whether an MTX-IgG conjugate could be hydrolyzed to release free MTX or fully active MTX-containing fragments after incubation with liver homogenates and have characterized the catabolites according to the presence of free MTX and their capacity to inhibit DHFR. Catabolism was optimal at pH 4.6, activated by dithiothreitol, and inhibited by antipain and N-alpha-p-tosyl-L-lysine chloromethyl ketone, thus implicating lysosomal enzymes. Liver homogenates produced an MTX-containing, low-molecular-weight fraction that was isolated by gel filtration. Further purification of this fraction by DEAE-cellulose chromatography gave two MTX-containing peaks, neither of which migrated as free MTX on thin-layer chromatography or inhibited DHFR more effectively than the parent conjugate. However, the presence of amino acid residues in these catabolites could contribute to their observed prolonged intracellular retention and superior antitumor action.
Uadia
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PO
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Blair
A H
AH
Ghose
T
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D000976:Antipain / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007136:Immunoglobulins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
D007136:Immunoglobulins; D014107:Tosyllysine Chloromethyl Ketone; D000976:Antipain; D013762:Tetrahydrofolate Dehydrogenase; D004229:Dithiothreitol; D008727:Methotrexate