The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro

Abstract

Purpose To investigate the impact of 5-formytetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro. Methods Cells were exposed to (6S)5-formyltetrahydrofolate (5-formylTHF) for 24h, before or after a 6h exposure to antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells. Results A 24h delay between a 6h exposure to antifolates and a subsequent 24h exposure to 4 μM 5-formylTHF sustained the full activities of both antifolates. A 72h interval was required between a single exposure of up to 4 μM 5-formylTHF and subsequent exposure to drugs to sustain activities of the antifolates. When cells were incubated with 4 μM 5-formylTHF for 24h weekly, for 4 weeks, there was no significant increase in the IC50 for pralatrexate, but the MTX IC50 increased 2.5-fold as compared to cells growing continuously in 25nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16%, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15%. An extracellular concentration of methotrexate 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate. Conclusions Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical “rescue” regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of antifolate.

Schedule-Dependent Synergy Between the Histone Deacetylase Inhibitor Belinostat and the Dihydrofolate Reductase Inhibitor Pralatrexate in T-and B-cell Lymphoma Cells in vitro

“…The efficacy of PLX was shown to be related to the expression of RFC and DHFR ( Kinahan et al, 2020 ). This is in line with the findings that PLX is an excellent substrate for the RFC ( Figure 1 ; Wang et al, 2003 ; Visentin et al, 2014 ), but a poor substrate for PCFT and had a very low affinity for the folate receptors ( Table 1 ). In addition, it was shown that PLX was one log more potent than methotrexate in suppressing cell proliferation ( Tonner et al, 2006 ).…”

“…Although PLX has been tested in solid tumors ( Azzoli et al, 2007 ), it was ineffective. Compared with conventional antifolates, such as the classical methotrexate, PLX may be more tumor specific due to greater affinity for RFC ( Wang et al, 2003 ) and polyglutamylation efficiency by folylpolyglutamate synthetase ( O’Connor et al, 2011 ; Visentin et al, 2014 ). These two properties improve (tumor specific) cellular uptake and inhibit potential efflux following uptake.…”

Spontaneously-forming spheroids as anin vitrocancer cell model for anticancer drug screening

“…To determine how standard of care drugs would perform in a spheroids MARY-X screen, several FDA-approved drugs known to be effective in solid tumor types as well as blood-borne malignancies, were evaluated. Spheroids were added to a multi-well plate (~30–50 spheroids/well) and then treated with vehicle only (DMSO) and increasing therapeutically-relevant drug doses as follows: bortezomib, lapatinib and doxorubicin (0–2.5 μM) [ 28 – 32 ], cisplatin (0–10 μM) [ 32 ] and methotrexate (0–20 μM) [ 33 ]. Therapeutically-relevant is defined as a drug dose that is either recorded in literature [ 32 , 34 – 37 ] or previous experiments as being the effective in vitro dose as determined in 2D cancer models.…”

A phase 1 clinical trial of sequential pralatrexate followed by a 48‐hour infusion of 5‐fluorouracil given every other week in adult patients with solid tumors

“…41 In vitro studies suggested that a 24-hr delay between PDX exposure and subsequent exposure to leucovorin did not interfere with PDX cytotoxicity. 42 However, a 72-hr interval was required between a single exposure to leucovorin and subsequent exposure to PDX to sustain activity without requiring an increase in PDX concentration. Future studies that incorporate delayed administration of leucovorin may improve the side effect profile of this regimen.…”