QUANTITATION OF 6-THIOGUANINE RESIDUES IN PERIPHERAL-BLOOD LEUKOCYTE DNA OBTAINED FROM PATIENTS RECEIVING 6-MERCAPTOPURINE-BASED MAINTENANCE THERAPY

The antimetabolite 6-mercaptopurine is widely utilized in maintenance therapy for childhood acute lymphoblastic leukemia, Following p.o. adm inistration, this prodrug undergoes extensive biotransformation, resul ting in the generation of a plethora of metabolites including 2'-deoxy -6-thioguanosine triphosphate, Incorporation of 6-thioguanine (6-TG) b ases into DNA is generally considered to be central to thiopurine-medi ated cytotoxicity. We have developed a novel precolumn derivatization HPLC technique for quantifying 6-TG base accumulation into leukocyte D NA obtained from acute lymphoblastic leukemia patients receiving 6-mer captopurine maintenance therapy. The method is based on enzymatic degr adation of DNA to 2'-deoxyribonucleosides and the derivatization of re leased 2'-deoxy-6-thioguanosine with a thiol-reactive reagent containi ng a 7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2'-deoxy- 6-thioguanosine-7 amino-4-methylcoumarin-3 acid adduct is resolved by reversed-phase HPLC and quantified fluorometrically. Assay response is linear from 15 pmol to 60 fmol 6-TG bases/mu g DNA with a limit of qu antitation 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, significan t interindividual variation in the accumulation of 6-TG bases into leu kocyte DNA was revealed. The determined levels of drug base incorporat ion ranged from 95 to 710 fmol 6-TG bases/mu g DNA (6-TG base:nucleoti de ratio 1:32,000 to 1:4,000). The assay may provide a novel methodolo gy for pharmacological monitoring of thiopurine therapy either in the routine clinical setting or during studies of alternative routes of dr ug delivery.