Enhancement of 5-Fluorouracil Cytotoxicity by Pyridoxal 5′-Phosphate and Folinic Acid in Tandem

Abstract

The current study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (HPteGlu) into methylene tetra hydro pteroylglutamate (CH-HPteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyzes formation of CH-HPteGlu by transfer of the C of serine to HPteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The median-effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29 and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the K for binding to SHMT, and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.

Pharmacologic modulation of 5-fluorouracil by folinic acid and high-dose pyridoxine for treatment of patients with digestive tract carcinomas

“…Patients with colorectal carcinoma and with pancreas adenocarcinoma had induction treatment using the regimen said Folfirinox consisting in two-day courses repeated every 14 days of combined oxaliplatin (L-OHP; 85 mg/m 2 , Day 1), irinotecan (CPT11; 180 mg/m 2 , Day1), folinic acid (FA; [6R,S]-5-formyl tetrahydropteroylglutamate; [6R,S]-5-HCO-H 4 PteGlu; 200 mg/m 2 /day, Days 1 and 2), and 5-fluorouracil (FUra; 1000 mg/m 2 day, Days 1 and 2) distributed in one rapid iv injection (400 mg/m 2 /day), and one iv infusion during 22 h (600 mg/m 2 /day). We used the Folfirinox regimen described above instead of slightly different simplified versions 30 , in order to insure two consecutive days of exposure to the modulators FA and PN, owing to the rapid plasma and cellular clearance of their metabolites whose intracellular pool expansion forms the rationale of the present study 11 , 24 , 25 , 27 , 31 . Patients with Ras-WT colorectal carcinoma received the anti EGF-R chimeric monoclonal antibody cetuximab in addition to chemotherapy every 14 days.…”

“…From these data we assumed that, in tumors, naturally occurring PLP levels are too small to allow intracellular SHMT-dependent conversion of H 4 PteGlu into CH 2 -H 4 PteGlu in amounts required to improve inhibition of TS by FdUMP by stabilizing the ternary complex 27 . To test for variations of SHMT activity resulting from PLP level changes in tumor cells, we conducted experiments in the human colon carcinoma HT29, and HCT116 cell lines, and in the murine leukemia L1210 cell line in vitro to investigate for interactions between FUra, FA, and PLP on cell growth 27 . Supplementation of cancer cells exposed to FUra with high concentrations of PLP and FA strongly potentiated the cytotoxic activity of FUra in the three cell lines and resulted in synergistic interaction in HT29 and in L1210 cells, while summation was found in HCT116 cells.…”

“…Supplementation of cancer cells exposed to FUra with high concentrations of PLP and FA strongly potentiated the cytotoxic activity of FUra in the three cell lines and resulted in synergistic interaction in HT29 and in L1210 cells, while summation was found in HCT116 cells. Experiments in mice have shown that erythrocyte levels of PLP after parenteral administration of high dose B6 vitamer in the form of pyridoxamine (PM), or pyridoxine (PN), rise to concentrations within the range of K d values of SHMT binding to cofactor, suggesting that modulation of FUra by vitamin B6 could be achieved in vivo 27 . These studies also demonstrated that newly synthesized PLP was rapidly cleared from cells; levels reached baseline concentrations by 12 h after injection, with no cumulative effect when administrations were repeated at 12-h interval.…”

Effects in Cancer Cells of the Recombinant L-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained L-Methionine Elimination

“…The variable rapidity of L-methionine elimination according to concentration of PLP in medium is probably the result of differences in amounts of cofactor uptake by MGL-loaded erythrocytes (Maeda et al, 1976). Furthermore, on the basis of the naturally occurring rapid decline of PLP concentration in red blood cells (Zempleni and Kübler, 1994;Machover et al, 2018), evidence of elimination of L-methionine by the bioreactor from medium containing a low concentration of PLP, as well as PN or PM instead of PLP, suggest that sustained MGL function within loaded erythrocytes in the longer term is probably relative to the newly synthesized intracellular cofactor (Anderson et al, 1971;di Salvo et al, 2011). These findings together with data from previously reported erythrocyte pharmacokinetics of vitamin B6 (Zempleni and Kübler, 1994;Machover et al, 2018) suggest the possibility of modulating the bioreactor's MGL activity in vivo by supplying cells with B6 vitamers.…”

“…With the aim at preserving MGL function for long periods of time in vivo, we constructed a heterologous bioreactor by encapsulation of the MGL-BL929 in human erythrocytes. These cells internalize L-methionine and have the capacity to synthesize PLP, the cofactor required to preserve the intracellular activity of the PLP-dependent L-methionine g-lyase (Zempleni and Kübler, 1994;Machover et al, 2018). Carrier erythrocyte lifespan does not differ from or is only slightly shorter than that of intact cells (Bax et al, 1999), a characteristic that allows maintenance of therapeutic blood levels of loaded cells for prolonged periods of time.…”