title: Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA obtained from patients receiving 6-mercaptopurine-based maintenance therapy.
abstract: 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/microgram 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/microgram 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.
authors.affiliation: Department of Clinical Pharmacology, Norwegian Radium Hospital, Montebello, Oslo.
authors.forename: D J
authors.initials: DJ
authors.lastname: Warren
authors.forename: A
authors.initials: A
authors.lastname: Andersen
authors.forename: L
authors.initials: L
authors.lastname: Slørdal
mesh_terms: D002460:Cell Line; D002648:Child; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000097:blood / Q000737:chemistry*; D004273:DNA, Neoplasm / Q000097:blood / Q000737:chemistry*; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D015122:Mercaptopurine / Q000627:therapeutic use*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood* / Q000188:drug therapy / Q000378:metabolism; D013866:Thioguanine / Q000032:analysis*; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D004247:DNA; D015122:Mercaptopurine; D013866:Thioguanine