Development of an assay for the estimation of N10-propargyl-5,8-dideazafolic acid polyglutamates in tumor cells.
A method is described herein for the isolation and quantitation of polyglutamates of the thymidylate synthase (TS) inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) in tumor cells exposed to the drug in vitro. Cells were incubated with 50 microM 3H-CB3717 for 12 h and then disrupted by sonication. CB3717 and its polyglutamates were extracted by boiling in 0.01 M Tris-HCl pH 10. The extract was concentrated by lyophilization and analyzed by reverse phase HPLC (10 x 0.46-cm Polygosil 5-micron C18 column) using linear gradient elution (5-16% acetonitrile in 0.1 M sodium acetate, pH 5, over 15 min, 2 ml/min). Recovery of radioactivity at each stage of the method was greater than 70%. CB3717 and its polyglutamates were identified by co-chromatography with synthetic standards and by inhibition of partially purified TS. Quantitation was by means of radiochemical analysis. The 3H-CB3717 used in these studies was prepared by catalytic tritiation of diethyl-(2-chloro-4-nitrobenzoyl)-L-glutamate followed by consecutive alkylation with propargyl bromide and 2-amino-6-bromomethyl-3,4-dihydro-4-oxoquinazoline hydrobromide. The free diacid was prepared as required by hydrolysis in sodium hydroxide and purified by HPLC. Tritiation in only one position was confirmed by 3H NMR. Following the exposure of L1210 leukemia cells to 50 microM 3H-CB3717 for 12 h the total cellular radioactivity level was approximately 7 microM, of which 27% was present as polyglutamated metabolites with four and five glutamate residues.
Sikora
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Drug Development Section, Institute of Cancer Research, Sutton, Surrey, United Kingdom.
Newell
D R
DR
Jackman
A L
AL
Simmonds
A J
AJ
Jones
T R
TR
Calvert
A H
AH
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000032:analysis; D007939:Leukemia L1210; D051379:Mice; D009369:Neoplasms / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000032:analysis*; D011799:Quinazolines / Q000032:analysis*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors
D010455:Peptides; D011799:Quinazolines; D011099:Polyglutamic Acid; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase