AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies

Abstract

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress.

Potentiation of anti-cancer agent cytotoxicity by the potent poly(ADP-ribose) polymerase inhibitors NU1025 and NU1064

“…The elution profiles of cells allowed to recover for 2 h after inradiation British Joumal of Cancer (1998) [3H]Thymidine retained Figure 6 Effect of NU1025 on the repair of DNA damage after potentialty lethal y-irradiation damage. Cells were exposed to 8 Gy of y-irradiaton and eluted immediatety (V) or allowed to recover for up to 2 h in control medium (A) (Webber et al 1996). In dialysed serum there was a decrease in cell survival followinc a 16-h exposure to increasing concentrations of nolatrexed.…”

Resistance to antifolates

“…On the other hand, for antifolates that do not form polyglutamates and are cleared rapidly from cells, such as AG337, ZD9331 (TS inhibitors), or trimetrexate (DHFR inhibitor), optimum activity requires frequent administration (i.e. daily, infusions, etc) (Jackson et al, 1984;Webber et al, 1996;Jackman et al, 1997). The rate and extent of polyglutamation in cells is determined by a variety of factors.…”

A phase I study of nolatrexed dihydrochloride in children with advanced cancer. A United Kingdom Children’s Cancer Study Group Investigation

“…Nolatrexed is a non-competitive TS inhibitor with an inhibition constant of 11 nM, with activity against rodent and human tumour cell lines in vitro, and against selected tumour models in vivo (Webber et al, 1996). Phase I studies in adults of nolatrexed administered either as a 5-day continuous infusion (Rafi et al, 1995), or orally every 6 hours for 5 days (Rafi et al, 1996), demonstrated dose-limiting myelosuppression with oral mucositis and a recommended Phase II dose of 800 mg/m 2 24 h −1 .…”