Synergistic activity of troxacitabine (Troxatyl) and gemcitabine in pancreatic cancer.
BACKGROUND
Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials.


METHODS
The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired t test using GraphPad prism software.


RESULTS
Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. In vivo testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine.


CONCLUSIONS
These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.
Damaraju
Vijaya L
VL
Department of Oncology, University of Alberta, and Cross Cancer Institute, Edmonton, Alberta, Canada. vijayada@cancerboard.ab.ca <vijayada@cancerboard.ab.ca>
Bouffard
David Y
DY
Wong
Clarence K W
CK
Clarke
Marilyn L
ML
Mackey
John R
JR
Leblond
Lorraine
L
Cass
Carol E
CE
Grey
Mike
M
Gourdeau
Henriette
H
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000473:pathology; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D003596:Cytosine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D004148:Dioxolanes / Q000008:administration & dosage* / Q000378:metabolism / Q000493:pharmacokinetics; D004357:Drug Synergism; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D016896:Treatment Outcome; D014316:Tritium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000493:pharmacokinetics; D023041:Xenograft Model Antitumor Assays; D000093542:Gemcitabine
D004148:Dioxolanes; D003841:Deoxycytidine; D014316:Tritium; C074908:troxacitabine; D003596:Cytosine; D014529:Uridine; D000093542:Gemcitabine