The antimetabolite 6-mercaptopurine is widely utilized in maintenance therapy for childhood acute lymphoblastic leukemia. Following p.o. administration, this prodrug undergoes extensive biotransformation, resulting in the generation of a plethora of metabolites including 2′-deoxy-6-thioguanosine triphosphate. Incorporation of 6-thioguanine (6-TG) bases into DNA is generally considered to be central to thiopurine-mediated cytotoxicity. We have developed a novel precolumn derivatization HPLC technique for quantifying 6-TG base accumulation into leukocyte DNA obtained from acute lymphoblastic leukemia patients receiving 6-mercaptopurine maintenance therapy. The method is based on enzymatic degradation of DNA to 2′-deoxyribonucleosides and the derivatization of released 2′-deoxy-6-thioguanosine with a thiol-reactive reagent containing a 7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2′-deoxy-6-thioguanosine-7-amino-4-methylcoumarin-3-acetic acid adduct is resolved by reversed-phase HPLC and quantified fluorometrically. Assay response is linear from 15 pmol to 60 fmol 6-TG bases/µg DNA with a limit of quantitation corresponding to the incorporation of 1 6-TG residue per 50,000 bases. In a small cohort of acute lymphoblastic leukemia patients receiving p.o. 6-mercaptopurine-based maintenance therapy, significant interindividual variation in the accumulation of 6-TG bases into leukocyte DNA was revealed. The determined levels of drug base incorporation ranged from 95 to 710 fmol 6-TG bases/µg DNA (6-TG base:nucleotide ratio 1:32,000 to 1:4,000). The assay may provide a novel methodology for pharmacological monitoring of thiopurine therapy either in the routine clinical setting or during studies of alternative routes of drug delivery.
↵1 This work was supported by grants from the Norwegian Cancer Society and the Käte Forsberg Legacy. D. J. W. is a Norwegian Cancer Society Senior Researcher.
↵2 To whom requests for reprints should be addressed.
- Received December 21, 1994.
- Accepted March 6, 1995.
- ©1995 American Association for Cancer Research.