Simultaneous, But Not Consecutive, Combination With Folinate Salts Potentiates 5-Fluorouracil Antitumor Activity In Vitro and In Vivo

Abstract

The combination of folinate salts to 5-fluoruracil (5-FU)-based schedules is an established clinical routine in the landscape of colorectal cancer treatment. The aim of this study was to investigate the pharmacological differences between the sequential administration of folinate salts (1 h before, as in clinical routine) followed by 5-FU and the simultaneous administration of both drugs. Proliferation and apoptotic assays were performed on human colon cancer cells exposed to 5-FU, calcium (CaLV), or disodium (NaLV) levofolinate or their simultaneous and sequential combination for 24 and 72 h. TYMS and SLC19A1 gene expression was performed with real-time PCR. In vivo experiments were performed in xenografted nude mice, which were treated with 5-FU escalating doses and CaLV or NaLV alone or in simultaneous and sequential combination. The simultaneous combination of folinate salts and 5-FU was synergistic (NaLV) or additive (CaLV) in a 24-h treatment in both cell lines. In contrast, the sequential combination of both folinate salts and 5-FU was antagonistic at 24 and 72 h. The simultaneous combination of 5-FU and NaLV or CaLV inhibited TYMS gene expression at 24 h, whereas the sequential combination reduced SLC19A1 gene expression. In vivo experiments confirmed the enhanced antitumor activity of the 5-FU + NaLV simultaneous combination with a good toxicity profile, whereas the sequential combination with CaLV failed to potentiate 5-FU activity. In conclusion, only the simultaneous, but not the consecutive, in vitro and in vivo combination of 5-FU and both folinate salt formulations potentiated the antiproliferative effects of the drugs.

Major innovations and clinical applications of disodium-levofolinate: a review of available preclinical and clinical data

“…However, Na-Lv can be simultaneous administrated with 5-FU, owing to the highest solubility. 3,11,15 The differences between sequential and simultaneous administration of 5-FU with either Ca-FA or Na-Lv have been investigated in human colon cancer cells and in xenograft nude mice. 3 In vitro data indicated that the simultaneous administration of both Ca-FA and Na-Lv with 5-FU enhanced the antiproliferative effect of 5-FU and induced apoptosis in cancer cells.…”

“…3 This opposite effect was mainly due to different regulation on gene expression: simultaneous combination of Na-Lv and 5-FU inhibited the expression of TS gene, whereas sequential administration resulted in a reduced expression of the soluble carrier 19A member 1 gene, the major folate transporter in mammalian cells. 3,36 Low levels of folate transporter reduced intracellular folate concentration and impaired the stability of ternary complex that blocks TS. 3,33,34 In vivo experiments confirmed the impact of simultaneous administration of Na-Lv and 5-FU to strengthen antitumor activity 3 and, consistently, a low expression of the soluble carrier 19A member 1 gene correlated to poor response to Na-Lv and 5-FU.…”

“…33–35 This results in a persistent blockade of TS with enhanced inhibition of DNA biosynthesis. 3,32,34…”

“…3 However, a remarkable difference between the two folinate salts was described: Na-Lv and 5-FU showed a synergistic action in both 24-hour and 72-hour simultaneous administration, whereas Ca-FA had just an additive effect to 5-FU activity. 3 Surprisingly, the sequential infusion of Na-Lv or Ca-FA and 5-FU had an antagonistic action. 3 This opposite effect was mainly due to different regulation on gene expression: simultaneous combination of Na-Lv and 5-FU inhibited the expression of TS gene, whereas sequential administration resulted in a reduced expression of the soluble carrier 19A member 1 gene, the major folate transporter in mammalian cells.…”

Letter to the editor: Re: Ratti M, Hahne JC, Toppo L, et al. Major innovations and clinical applications of disodium-levofolinate: a review of available preclinical and clinical data. Ther Adv Med Oncol. 2019

“…3 Furthermore, in vitro and in vivo experiments conducted by Di Paolo et al confirmed the enhanced antitumor activity and good toxicity profile of the simultaneous combination of sodium-based levoleucovorin and 5-FU, while the sequential combination with calcium-based leucovorin failed to potentiate 5-FU activity. 4…”