Distinct activity of pralatrexate in human cancer models in vivo and in vitro

B15 Distinct activity profile of pralatrexate (PDX) in human cancer models in vivo and in vitro.
 Background: PDX (pralatrexate) is a small-molecule inhibitor of dihydrofolate reductase (DHFR), a validated target in oncology. PDX was designed to increase membrane transport and intracellular accumulation compared to methotrexate (MTX), the first DHFR inhibitor used in the clinic despite a narrow therapeutic index, and pemetrexed (PMX), a multitargeted antimetabolite that inhibits multiple folate-dependent enzymes. This pilot study evaluated the mechanism of action of PDX and its differences from other antifolates.
 Methods: Inhibition of DHFR by the 3 antifolates was quantified in a cell-free assay against human recombinant DHFR. The short-term uptake (assessment of RFC-1 transporter) and folylpolyglutamate synthase (FPGS) activity (polyglutamylation) using radiolabeled PDX, MTX (both at 5 µM) and PMX (20 µM) was evaluated in H460 cells in vitro. We compared in vivo activity of PDX and MTX (both at 1 mg/kg and 2 mg/kg), PMX (150 mg/kg), and docetaxel (7 mg/kg and 12.5 mg/kg) in MV522 and H460, non small cell lung cancer human tumor xenografts models using mean tumor growth inhibition or regression as endpoints. We further evaluated reduced folate carrier (RFC-1) expression by RT PCR in MV522 and H460 xenografts, and measured activity and/or expression of DHFR, TS, and FPGS using kinetic assays and immunostaining ex vivo.
 Results: As reported recently (Diaz et al, Proc AACR 2007), apparent Ki values for DHFR inhibition in a cell-free assay were, respectively, 6 nM, 12 nM, and >200 nM for PDX, MTX, and PMX. The total uptake of radiolabeled drugs measured at 15 and 60 minutes was similar at both times (MTX), decreased (PMX) or increased (PDX) over time. A significantly smaller fraction of radiolabeled MTX or PMX entered the cells in comparison with PDX. Radiolabeled species (conceivably polyglutamylated PDX) of different Rf values than the drug in a cell-free system appeared in a time-dependent manner in PDX-treated cell extracts. PMX was polyglutamylatated to a lesser extent, and there was no apparent polyglutamylation of MTX. Only PDX demonstrated a clear dose-dependence on distribution of polyglutamylated species. In vivo, PDX was superior to PMX and MTX in both models. A greater dose-dependent inhibition of tumor growth was observed in the more rapidly growing H460 xenografts. Unlike docetaxel, the activity of MTX and PMX was model-dependent. RFC-1 expression was below the assay detection limit in both models. PDX, PMX and MTX demonstrated differences in regulation of enzymatic activity (TS, DHFR) and protein expression (DHFR) in H460 and MV522 tumor xenografts; only PDX downregulated most of these endpoints.
 Conclusions: The results from this pilot study corroborate earlier notions that PDX has a different activity profile relative to MTX and PMX. Some of the observed differences include enhanced uptake of PDX into tumor cells and subsequent greater intracellular polyglutamylation, which translates into more efficient tumor growth inhibition by PDX in NSCLC xenograft models in vivo. Current findings warrant further studies in other human tumor models including hematologic malignancies.
 Supported by Allos Therapeutics, Inc.