Development of new promising antimetabolite, DFP-11207 with self-controlled toxicity in rodents

Figures & data

Figure 1 Molecular structure of DFP-11207 and 5-fluorouracil.

Note: DFP-11207 consists of three biologically important ingredients, 1-ethoxymethyl-5-fluorouracil, 5-chloro-2,4-dihydroxypyridine, and citrazinic acid.
Abbreviation: DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy) isoni cotinate.

Figure 2 Enzymatic conversion of DFP-11207 to three metabolites, EM-FU, CDHP, and CTA in the serum, liver, and small intestine of rats in vitro.

Notes: DFP-11207 (1 mM/assay mixture) was incubated with rat serum or with crude extracts of the liver and small intestine of rats. After 10, 20, 30, and 60 min, the reaction was terminated and the products, EM-FU (●), CDHP (○), and CTA (▲) were measured using HPLC system. Data represent mean values for duplicate assays.
Abbreviations: CDHP, 5-chloro-2,4-dihydroxypyridine; CTA, citrazinic acid; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; EM-FU, 1-ethoxymethyl-5-fluorouracil; HPLC, high-performance liquid chromatography.

Table 1 Inhibitory effect of DFP-11207 on the activities of DPD and OPRT

Inhibition of DPD				Inhibition of OPRT
Drug	Conc (μM)	Activity (nmol/mL/min)	Inhibition (%)	Drug	Conc (μM)	Activity (nmol/mL/min)	Inhibition (%)
Control	0	0.667	0	Control	0	0.335	0
DFP-11207	20	0.131	80.4	DFP-11207	20	0.003	99.1
	2	0.152	77.2		2	0.079	76.4
	0.2	0.374	43.9		0.2	0.242	27.8
	0.02	0.518	22.3

Notes: Crude extracts of rat liver and human tumor xenografts (SC-2) were used as enzyme souses of DPD and OPRT, respectively. Data represent mean values for duplicate enzyme assays.

Abbreviations: Conc, concentration; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; DPD, dihydropyrimidine dehydrogenase; OPRT, orotate phosphoribosyltransferase.

Figure 3 Inhibitory effects of DFP-11207 and citrazinic acid on intracellular phosphorylation of 5-FU and its subsequent incorporation into RNA in human colorectal tumor cells.

Notes: One to 100 µM DFP-11207 and CTA were added to colorectal tumor cells (1×10⁷ cells) and incubated with 1 µM 5-FU containing [6-³H] 5-FU at 37°C for 45 min. After the reaction, treated cells were isolated by centrifugation at 4°C, immediately treated with 10% PCA to separate the acid-soluble fraction containing the nucleotide form of 5-FU and acid-insoluble fraction containing 5-FU-incorporated RNA. Data represent mean values for duplicate cellular assays.
Abbreviations: 5-FU, 5-fluorouracil; CTA, citrazinic acid; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl) benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; PCA, percloric acid.

Figure 4 Conversion of EM-FU, a major metabolite of DFP-11207, to 5-FU by various liver microsomes.

Notes: EM-FU (1 mM) derived from DFP-11207 and FT as a positive control were incubated for 60 min with liver microsomes (0.85 mg proteins) from different origins, and 5-FU produced in the reaction mixture was detected using HPLC system (A). The reaction was assayed triplicate. Also EM-FU (1 mM) was incubated with human liver microsomes in the presence of 100 µM coumarin, α-naphthoflavone, sulfaphenazole, quinidine, p-nitrophenol, and toleandomycin, respectively, which specifically inhibit corresponding CYP activity (B). Data represent mean values ± SD for triplicate assays (A) and mean values for duplicate assays (B), respectively.
Abbreviations: CTA, citrazinic acid; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; EM-FU, 1-ethoxymethyl-5-fluorouracil; 5-FU, 5-fluorouracil; FT, tegafur; HPLC, high-performance liquid chromatography.

Figure 5 CTA and 5-FU levels in blood, small intestine, and tumor tissues of rats after oral administration of DFP-11207.

Notes: DFP-11207 (53.4 mg/kg, 75 µmol/kg) was administered to human tumor (AZ521)-bearing xenograft in nude rats. After 1, 2, 4, 8, and 12 h, rats were sacrificed and their blood (as serum), small intestines, and tumors were isolated to prepare the crude extracts containing CTA (A) and 5-FU (B). For this, tissues were homogenized with 10 mM potassium phosphate-buffered saline (pH 7.2) and then centrifuged at 9,000× g for 30 min. The resultant supernatants (one volume) were treated with four volumes of ethylacetate for 10 min, and obtained organic layer was dried at 40°C. The dried product was dissolved into a small volume of 10 mM phosphate-buffered saline and aliquot of the preparation was applied to HPLC system to measure the concentration of 5-FU and CTA. Data represent mean values ± SD for three rats.
Abbreviations: CTA, citrazinic acid; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; 5-FU, 5-fluorouracil; HPLC, high-performance liquid chromatography.

Figure 6 Comparative 5-FU levels in rat plasma following oral administration of DFP-11207 and S-1.

Notes: A total of 50 µmol/kg of DFP-11207 and S-1 were orally administered to rats (N=3) weighing ~200 g. At indicated time, the blood was isolated and 5-FU levels in the blood were comparatively determined by HPLC. Values are mean ± SD for three rats.
Abbreviations: DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; 5-FU, 5-fluorouracil; HPLC, high-performance liquid chromatography; S-1, tegafur-gimeracil-oteracil.

Figure 7 Antitumor activity and toxicity of DFP-11207 and S-1 in human colorectal cancer KM12C xenografts in nude rats.

Notes: Seventy-five to 112.5 µmol/kg of DFP-11207 and S-1 were orally administered to KM12C-bearing nude rats (six rats/group) once daily for 14 consecutive days, and antitumor effect (A), body weight changes (B) and the change of hematological parameters (WBC (C) and platelet count (D)) were evaluated. Values are mean ± SD for six nude rats.
Abbreviations: DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isoni cotinate; S-1, tegafur-gimeracil-oteracil; WBC, white blood cell.

Figure 8 Histochemical evaluation of GI tissues (jejunum) in rats treated with each 112.5 µmol/kg of DFP-11207 and S-1.

Notes: Portions of the jejunum in rats treated with DFP-11207 and S-1, respectively, were isolated and evaluated pathologically by H&E stain. As control group showing 5-FU-induced GI damage, tegafur plus gimeracil alone was administered to rats as same way. Magnification ×10.
Abbreviations: DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy) isonicotinate; 5-FU, 5-fluorouracil; GI, gastrointestinal; S-1, tegafur-gimeracil-oteracil.

Table 2 Antitumor activity of DFP-1207 compared with 5-FU or gemcitabine on human gastrointestinal tumor xenografts in nude rats

Xenografts	Drug	Dose (mg/kg)	Tumor volume (mm^3±SD)	TGI (%)	P-value
HT-29 (CRC)	Vehicle (0.5% HPMC)		1,710±85		P<0.01 vs 5-FU
	DFP-11207	300	819±54	52.2
	5-FU, ip	15	1,211±76	29.2
MKN-45 (GC)	Vehicle (0.5% HPMC)		2,403±166		P<0.001 vs 5-FU
	DFP-11207	300	868±70	63.9
	5-FU, ip	20	1,740±70	27.6
BxPC3 (PC)	Vehicle (0.5% HPMC)		807±51		P=0.001 vs gem
	DFP-11207	300	323±29	60.0
	Gemcitabine	50	539±37	33.2
PANC-1 (PC)	Vehicle (0.5% HPMC)		1,708±92		NS vs gem
	DFP-11207	300	1,243±28	27.2
	Gemcitabine	50	1,320±27	22.7

Notes: Human tumor cells (5×10⁶ cells) were inoculated into the right axilla of nude rats. When each tumor volume reached ~100–200 mm³, DFP-11207 was orally administered once daily for 14–21 days, 5-FU and gemcitabine were intraperitoneally injected daily for 5 days and weekly for 3 weeks, respectively. Antitumor activities of the drugs were evaluated at day 29 for HT-29, day 21 for MKN-45, day 45 for BxPC3, and day 31 for PANC-1 after tumor implantation.

Abbreviations: DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy) isonicotinate; 5-FU, 5-fluorouracil; TGI, tumor growth inhibition; CRC, colorectal cancer; HPMC, hydroxypropyl methylcellulose; PC, pancreatic cancer; GC, gastric cancer; ip, intraperitoneal; NS, not significant.

Figure 9 Possible biological metabolism and mechanism of action of DFP-11207 in rats.

Notes: After oral administration, DFP-11207 is quickly hydrolyzed to three major metabolites, EM-FU, CDHP, and CTA in GI cells, and resultant EM-FU is further metabolized to 5-FU by liver microsomes. CDHP inhibits the degradation of 5-FU into inactive catabolite in the liver, which results in a higher 5-FU levels in the body. CTA mainly distributes and inhibits the phosphorylation of 5-FU in GI cells leading to decrease in GI toxicity.
Abbreviations: CDHP, 5-chloro-2,4-dihydroxypyridine; CTA, citrazinic acid; DFP-11207, 5-chloro-2-(3-(3-(ethoxymethyl)-5-fluoro-2,6-dioxo-1,2,3,6-tetrahydopyrimidine-1-carbonyl)benzoyloxy)pyridine-4-yl-2,6-bis(propionyloxy)isonicotinate; EM-FU, 1-ethoxymethyl-5-fluorouracil; 5-FU, 5-fluorouracil; GI, gastrointestinal; FUMP, 5-fluorouridine-5′-monophosphate.