Incorporation of OSI-7836 into DNA of Calu-6 and H460 xenograft tumors.
OSI-7836 (4'-thio-beta-D-arabinofuranosylcytosine) is a novel nucleoside analog in phase I clinical development for the treatment of cancer. As with other nucleoside analogs, the proposed mechanism of action involves phosphorylation to the triphosphate form followed by incorporation into cellular DNA, leading to cell death. This hypothesis has been examined by measuring and comparing the incorporation of ara-C, OSI-7836, and gemcitabine (dFdC) into DNA of cultured cells and by investigating the role of deoxycytidine kinase in OSI-7836 toxicity. We report here additional studies in which the role of cell cycling on OSI-7836 toxicity was investigated and incorporation of OSI-7836 into DNA of xenograft tumors measured. The role of the cell cycle was examined by comparing the toxicity of OSI-7836 in A549 NSCLC cells that were either in log phase growth or had reached confluence. A novel validated LC-MS/MS assay was developed to quantify the concentrations of OSI-7836 in DNA from Calu-6 and H460 human tumor xenografts in mice. Results showed that apoptosis induced by OSI-7836 was markedly greater in cycling cells than in confluent non-cycling cells despite only a modest increase in intracellular OSI-7836 triphosphate concentration. The LC-MS/MS assay developed to measure OSI-7836 incorporation into DNA had an on-column detection limit of 0.25 fmol, a quantification limit of 0.5 fmol, and a sensitivity of approximately 0.1 pmol OSI-7836/micromol dThy. Concentrations of OSI-7836 in splenic DNA (0.4 pmol OSI-7836/micromol dThy) averaged fivefold less than the average concentration in Calu-6 and H460 xenograft DNA (3.0 pmol OSI-7836/micromol dThy) following a 400 mg/kg dose of OSI-7836. Concentrations of OSI-7836 in Calu-6 tumor DNA isolated 24 h following a dose of 400, 1000, or 1600 mg OSI-7836/kg were approximately 1.3, 1 and 1.3 pmol OSI-7836/micromol dThy, respectively. Concentrations of OSI-7836 in DNA from H460 and Calu-6 xenografts did not appear to increase during repeated administration of 400 mg OSI-7836/kg on days 1, 4, 7, and 10. The majority of OSI-7836 in DNA from Calu-6 and H460 tumors of mice dosed with 1600 mg/kg was located at internal nucleotide linkages, similar to dFdC and ara-C. In conclusion, cell cycling studies supported the hypothesis that OSI-7836 cytotoxicity is dependent upon DNA synthesis. A validated LC-MS/MS assay was developed that could quantify OSI-7836 in DNA from tissues. The assay was used to show that OSI-7836 was incorporated in internal linkages in tumor DNA in a manner that was dose-independent at the doses tested and did not appear to accumulate during repeated dosing. The results suggest that if DNA incorporation is a toxic event, the relationships between administered dose, DNA incorporation, and toxicity are complex.
Richardson
Frank
F
OSI Pharmaceuticals, Inc., 2860 Wilderness Place, Boulder, CO 80301, USA. frichardson@osip.com
Black
Chris
C
Richardson
Katherine
K
Franks
April
A
Wells
Edward
E
Karimi
Susan
S
Sennello
Gina
G
Hart
Karen
K
Meyer
Denny
D
Emerson
David
D
Brown
Eric
E
LeRay
Jeremy
J
Nilsson
Christy
C
Tomkinson
Blake
B
Bendele
Raymond
R
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism*; D001087:Arabinonucleosides / Q000378:metabolism*; D002453:Cell Cycle; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D051379:Mice; D009368:Neoplasm Transplantation; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D004273:DNA, Neoplasm; C411255:4'-thio-arabinofuranosylcytosine