Temozolomide is an oral methylating agent used in the treatment of gliomas and melanomas. The majority (>70%) of DNA damage caused by temozolomide is rapidly repaired by the base excision repair pathway (BER). Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme with a key role in BER. The aim of this study was to investigate DNA damage and PARP activity, in relation to plasma temozolomide concentration, in patients receiving temozolomide for the first time. The study involved the development of a PARP-1 activity assay for use in clinical samples. A phase II study of temozolomide (200 mg/m2/day for 5 days every 28 days, 2 to 9 cycles) was conducted in 12 patients with malignant melanoma who consented to have additional blood tests at 1, 4 and 24 hr for analysis of temozolomide-pharmacokinetics (PK), PARP-1 activity and DNA damage. Tumour biopsy samples were obtained from most patients for similar analysis. There was no significant toxicity, one patient required a dose reduction due to CTC grade 3 thrombocytopaenia. PARP-1 activity was determined in permeabilised peripheral blood lymphocytes (PBLs) stimulated with a blunt ended oligonucleotide in the presence of NAD+ (adapted from Pfieffer et al, Analytical Biochem. 275, 118, 1999). Poly(ADP-ribose) polymer formation was measured by immunoblot with a monoclonal antibody (10H) followed by chemiluminescence detection and quantification using a Fuji LAS 3000. Polymer formation was accurately estimated by reference to a purified polymer standard. The assay was also adapted to measure PARP-1 activity in tumour homogenates. DNA strand breaks were measured in PBLs by COMET assay. Plasma temozolomide was measured by HPLC and O6-alkyl guanine alkyltransferase (ATase) activity by an enzymatic assay. Limited temozolomide PK demonstrated the rapid absorption and elimination of the compound. The mean PARP activity in human PBLs was 190 pmol polymer/ 106 cells (range 10-695 pmol). There was a small but significant increase in the maximally stimulatable PARP activity 4 (mean 280 pmol/106 cells, range 75-695) and 24 hours (mean 155 pmol/106 cells, range 10-500) after dosing. PARP-1 activity in tumour homogenates was variable (mean 1364 pmol/mg prot, range 1.6-3609), generally much higher than in PBLs and did not change with treatment. Levels of DNA breaks were increased at 4 hours (mean tail moment increased to 177% baseline), but had returned to normal by 24 hr after treatment. Mean Atase activity was decreased at 4 (11.4 fmol/mcg DNA) and 24 hours (10.8) over baseline (18.0). This small clinical study allowed validation of a pharmacodynamic assay for PARP-1 inhibition and provides data to support the ongoing first clinical trial of a temozolomide-PARP inhibitor combination. It demonstrated that PARP-1 activity can be reproducibly measured in PBLs and the activity of the enzyme is not altered markedly by temozolomide.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]