The metabolism and pharmacokinetics of phospho-sulindac (OXT-328) and the effect of difluoromethylornithine

Abstract

Background and purpose: Phospho-sulindac (PS; OXT-328) prevents colon cancer in mice, especially when combined with difluoromethylornithine (DFMO). Here, we explored its metabolism and pharmacokinetics.

Experimental approach: PS metabolism was studied in cultured cells, liver microsomes and cytosol, intestinal microsomes and in mice. Pharmacokinetics and biodistribution of PS were studied in mice.

Key results: PS undergoes reduction and oxidation yielding PS sulphide and PS sulphone; is hydrolysed releasing sulindac, which generates sulindac sulphide (SSide) and sulindac sulphone (SSone), all of which are glucuronidated. Liver and intestinal microsomes metabolized PS extensively but cultured cells converted only 10% of it to PS sulphide and PS sulphone. In mice, oral PS is rapidly absorbed, metabolized and distributed to the blood and other tissues. PS survives only partially intact in blood; of its three major metabolites (sulindac, SSide and SSone), sulindac has the highest C(max) and SSone the highest t(1/2) ; their AUC(0-24h) are similar. Compared with conventional sulindac, PS generated more SSone but less SSide, which may contribute to the safety of PS. In the gastroduodenal wall of mice, 71% of PS was intact; sulindac, SSide and SSone together accounted for <30% of the total. This finding may explain the lack of gastrointestinal toxicity by PS. DFMO had no effect on PS metabolism but significantly reduced drug level in mouse plasma and other tissues.

Conclusions and implications: Our findings establish the metabolism of PS define its pharmacokinetics and biodistribution, describe its interactions with DFMO and largely explain its gastrointestinal safety.

Figure 1

Metabolism of PS by rat and HLMs. (A) HPLC profile of PS and its metabolites generated by RLM 10 min after PS was incubated with RLM. PS and its metabolites were extracted and fractionated by HPLC. (B) MS spectrum of PS sulphone fraction collected from HPLC. (C) MS/MS spectrum of the major fragment ions of the PS sulphone ion. (D) Metabolic transformations of PS by liver microsomes.

Figure 2

Kinetics of PS and its metabolites by rat and HLMs. PS (35 µM) was incubated with RLM (A) or HLM (B) at 37°C for up to 90 min. PS and its metabolites were extracted at the designated time points and assayed.

Figure 3

Metabolism of PS by RLC. Kinetics of PS and its metabolites by RLC. PS (100 µM) was incubated with RLC at 37°C for up to 7 h. PS and its metabolites were extracted at the designated time points and assayed.

Figure 4

Kinetics of PS and its metabolites in SW480 cancer cells. PS (100 µM) was incubated with SW480 cells at 37°C for up to 6 h. PS and its metabolites were extracted at the designated time points and assayed.

Figure 5

Comparison of the pharmacokinetics of PS and sulindac in mice. Equimolar doses of sulindac (100 mg·kg⁻¹) and PS (158 mg·kg⁻¹) in corn oil were administered to mice as a single dose by gastric gavage. Plasma levels of PS metabolites were determined at the times shown.

Figure 6

The pharmacokinetics of PS administered in carboxymethyl cellulose or PBS to mice. PS (150 mg·kg⁻¹) was administered to mice as a single dose orally in carboxymethyl cellulose (upper graph) or i.p. in PBS (lower graph). Plasma levels of PS metabolites were determined at the times shown.

Figure 7

Effect of DFMO on the metabolism, pharmacokinetics and tissue distribution of PS. (A) PS (35 µM) was incubated with HLM for up to 60 min in the absence or presence of 5 mM DFMO. PS and its metabolites were extracted at the indicated time points and assayed. (B) Study mice were given water with or without 2% DFMO for 2 days. Following that, a single dose of PS 200 mg·kg⁻¹ was administered by gastric gavage, and plasma levels of PS metabolites were measured. (C) The livers, kidneys and hearts of the mice studied in Panel B were harvested, and the PS metabolites were measured. Data were expressed as the sum of the levels of PS and its metabolites in each tissue.

Figure 8

Overall metabolic pathways of PS in vitro and in vivo. PS is subjected to a series of reduction/oxidation reactions of its sulphoxide group and/or hydrolytic cleavages of its carboxy ester bond. The end result is eight metabolic products through three distinct and ultimately converging pathways.