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Temozolomide Pharmacodynamics in Patients with Metastatic Melanoma: DNA Damage and Activity of Repair Enzymes O⁶-Alkylguanine Alkyltransferase and Poly(ADP-Ribose) Polymerase-1

Authors' Affiliations: ¹ Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; ² Cancer Research UK Medical Oncology Unit and ³ Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom; and ⁴ Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, United Kingdom

Requests for reprints: E. Ruth Plummer, Northern Institute for Cancer Research, Medical School, University of Newcastle upon Tyne, Paul O'Gorman Building, Framlington Place, Newcastle uponTyne NE2 4HH, United Kingdom. Phone: 44-191-246-4414; Fax: 44-191-246-4301; E-mail: E.R.Plummer{at}ncl.ac.uk.

Purpose: Temozolomide, a DNA methylating agent used to treat melanoma, induces DNA damage, which is repaired by O⁶-alkylguanine alkyltransferase (ATase) and poly(ADP-ribose) polymerase-1 (PARP-1)–dependent base excision repair. The current study was done to define the effect of temozolomide on DNA integrity and relevant repair enzymes as a prelude to a phase I trial of the combination of temozolomide with a PARP inhibitor.

Experimental Design: Temozolomide (200 mg/m² oral administration) was given to 12 patients with metastatic malignant melanoma. Peripheral blood lymphocytes (PBL) were analyzed for PARP activity, DNA single-strand breakage, ATase levels, and DNA methylation. PARP activity was also measured in tumor biopsies from 9 of 12 patients and in PBLs from healthy volunteers.

Results: Temozolomide pharmacokinetics were consistent with previous reports. Temozolomide therapy caused a substantial and sustained elevation of N⁷-methylguanine levels, a modest and sustained reduction in ATase activity, and a modest and transient increase in DNA strand breaks and PARP activity in PBLs. PARP-1 activity in tumor homogenates was variable (828 ± 599 pmol PAR monomer/mg protein) and was not consistently affected by temozolomide treatment.

Conclusions: The effect of temozolomide reported here are consistent with those documented in previous studies with temozolomide and similar drug, dacarbazine, demonstrating that a representative patient population was investigated. Furthermore, PARP activity was not inhibited by temozolomide treatment and this newly validated pharmacodynamic assay is therefore suitable for use in a proof-of-principle phase I trial a PARP-1 inhibitor in combination with temozolomide.

Key Words: Pharmacodynamic analysis • COMET assay • Immunoblot PARP assay

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