The combination of brequinar and fluorouracil FU has been
The combination of brequinar and 5-fluorouracil (5-FU) has been evaluated to assess whether brequinar-induced uridine depletion would improve 5-FU activity. In cells, 5-FU is metabolized into FdUMP and inhibits thymidylate synthetase, which catalyzes the formation of thymidine monophosphate from deoxyuridine monophosphate (Longley, Harkin, & Johnston, 2003; Wilson, Danenberg, Johnston, Lenz, & Ladner, 2014). A combination of brequinar and 5-FU was proposed to increase generation of FdUMP by decreasing intracellular nucleotide concentrations. Initial evaluation of this combination utilized a low dose of brequinar to assess whether pretreatment would improve the activity of 5-FU or 5-FU with leucovorin. The results indicated that pretreatment with brequinar, 24 or 48 h in advance of 5-FU dosing, improves potency and that brequinar pretreated Murine had significantly higher concentrations of FdUMP compared to cells treated with 5-FU alone (Chen & Erlichman, 1992). However, the addition of leucovorin to this combination diminishes the effectiveness as brequinar pretreatment did not significantly improve the 5-FU and leucovorin combination (Chen & Erlichman, 1992). A larger study evaluating 5-FU and brequinar in vivo suggested the combination provided a synergistic effect in colon cancer cells at low uridine concentrations. However, at higher concentrations, this combination did not perform well. Studies were also conducted in colon 26 (estimated intracellular [uridine] of 10 μM) and colon 38 (estimated intracellular [uridine] of 50–100 μM) cell lines (Peters et al., 1987; Peters, Kraal, & Pinedo, 1992). When applied in vivo, the 5-FU + brequinar combination significantly decreased the colon 26 tumor weight, but did not significantly affect the colon 28 tumor compared to 5-FU alone (Peters et al., 1992). A third compound, dipyridamole (DPM), was then added to improve the combination in vitro. DPM is an equilibrative nucleoside transport inhibitor believed to hinder nucleobase/nucleoside transport across the cell membrane. This three inhibitor cocktail combination reduced cell growth at low concentrations of uridine, but was unable to inhibit cell growth in the presence of 50 μM uridine (Peters et al., 1992). Further in vivo studies on the combination demonstrated a significant decrease in tumor weight when comparing brequinar as a single agent to brequinar plus 5-FU (Pizzorno, Wiegand, Lentz, & Handschumacher, 1992). The combination was tested in a Phase I study, in which weekly doses as high as 600 mg/m2 brequinar and 600 mg/m2 of 5-FU were administered. However, no objective responses were observed in 25 patients with refractory solid tumors (Buzaid et al., 1995). Brequinar doses as high as 400 mg/m2 were observed to decrease baseline uridine levels by >50% (Buzaid et al., 1995). Beyond combinations with 5-FU, brequinar has also been evaluated in combination with cisplatin. Unfortunately, the results in a phase I study were similar with no objective responses (Burris III et al., 1998). Other combination therapy studies have been pursued with non-DHODH inhibitors of de novo biosynthesis. 5-FU and DPM have been evaluated in combinations with other de novo pyrimidine biosynthesis inhibitors. PALA and 5-FU were evaluated in multiple anticancer clinical trials, but the combination showed little advantage in comparison to 5-FU alone (Ardalan, Jamin, Jayaram, & Presant, 1984; Weiss, Ervin, Meshad, & Kufe, 1982). The combination of PALA, 5-FU, and leucovorin did not perform much better in a clinical trial for pancreatic ductal adenocarcinoma. From a total of 26 patients, only 3 showed partial responses to combination therapy (12%) (Whitehead et al., 2004). Beyond 5-FU, PALA was investigated with DPM. This combination progressed to a phase I clinical trial, but only 4 of 65 patients responded to the therapy (Markman, Chan, Cleary, & Howell, 1987). An additional phase II study evaluating PALA and DPM for soft tissue sarcoma concluded that the combination was not better than PALA alone, which had previously failed to induce objective responses in clinical trials (Casper et al., 1991). Aside from PALA, a combination of acivicin and DPM has been evaluated in patients diagnosed with diverse tumor types. However, this did not reach objective responses despite achieving plasma concentrations of DPM that were sufficient to inhibit nucleoside transport in vitro (Fischer, Pamukcu, Bittner, & Willson, 1984; Willson et al., 1988).