Polymerization of the triphosphates of AraC, 2',2'-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase.

OSI-7836 (4'-thio-araC, T-araC) is a nucleoside analogue that shows efficacy against solid tumor xenograft models. We examined how the triphosphates of OSI-7836 (T-araCTP), cytarabine (araCTP), and gemcitabine (dFdCTP) affected the initiation of new DNA strands by the pol alpha primase complex. Whereas dFdCTP very weakly inhibited primase, both T-araCTP and araCTP potently inhibited this enzyme. Primase polymerized T-araCTP and araCTP more readily than its natural substrate, CTP, and incorporation resulted in strong chain termination. dFdCTP, araCTP, and T-araCTP inhibited pol alpha competitively with respect to dCTP. When exogenously added primentemplates were used, pol alpha incorporated all three analogues into DNA, and incorporation caused either weak chain termination (dFdCTP), strong termination (araCTP), or extremely strong termination (T-araC). Furthermore, pol alpha polymerized T-araCTP only nine-fold less well than dCTP, whereas it polymerized araCTP and dFdCTP 24- and 83-fold less well, respectively. The presence of these three analogues in the template strand resulted in significant pausing by pol alpha, although the site and severity of pausing varied between the analogues. During the elongation of primase-synthesized primers, a reaction that is thought to mimic the normal sequence of events during the initiation of new DNA strands, pol alpha polymerized all three compounds. However, incorporation of araCTP and dFdCTP resulted in minimal chain termination, while incorporation of T-araCTP still caused extremely strong termination. The implications of these results with respect to how these compounds affect cells are discussed.

Quantification of 2'-fluoro-2'-deoxyuridine and 2'-fluoro-2'-deoxycytidine in DNA and RNA isolated from rats and woodchucks using LC/MS/MS.

Apatmers are synthesized using 2'-fluoropyrimdines in place of normal pyrmidines to stabilize them against enzymatic degradation, and thereby improve their therapeutic efficacy. Despite this stabilizing effect, the apatmers can still be degraded by nucleases in the blood. Primer template extension studies have demonstrated that mammalian DNA polymerases can incorporate these 2'-fluoropyrimidines into growing strands of DNA. The toxicologic effects of these compounds have been examined in rats and woodchucks, animals known to be susceptible to the toxic effects of other modified pyrimidines. Whether these nucleosides can be incorporated into DNA in vivo has not been established. These studies report the development of methodologies and the results of studies designed to determine if and to what extent 2'-fluoropyrimidines are incorporated into tissue DNA following long-term treatment. Rats were dosed intravenously with either 2'-fluorouridine (2'-FU) or 2'-fluorocytidine (2'-FC) at doses of 5, 50, and 500 mg/kg/day for 90 days. Woodchucks were dosed intravenously with either 2'-FU or 2'-FC at doses of 0.75 or 7.5 mg/kg/day for 90 days. The amounts of 2'-FU or 2'-FC in DNA and RNA were quantified using newly developed LC/MS/MS methodologies. Administration of 2'-FU to rats and woodchucks resulted in incorporation of the compound into DNA from liver, spleen, testis, muscle, and kidney. Incorporation also occurred in RNA from rat liver (only tissue examined). Similarly, administration of 2'-FC to rats and woodchucks resulted in incorporation into liver DNA (only tissue examined). These data demonstrate that 2'-fluoropyrimidines are incorporated into DNA and RNA of various tissues of rats and woodchucks following long-term administration.