Pharmacokinetics in mice and growth-inhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4'-tetramethoxystilbene

Abstract

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a naturally occurring polyphenol with cancer chemopreventive properties in preclinical models of carcinogenesis, including those of colorectal cancer. Recently, a variety of analogues of resveratrol have been synthesised and investigated in in vitro assays. One analogue, 3,4,5,4'-tetramethoxystilbene (DMU 212), showed preferential growth-inhibitory and proapoptotic properties in transformed cells, when compared with their untransformed counterparts. As part of a chemoprevention drug development programme, the pharmacokinetic properties of DMU 212 were compared with those of resveratrol in the plasma, liver, kidney, lung, heart, brain and small intestinal and colonic mucosa of mice. DMU 212 or resveratrol (240 mg kg(-1)) were administered intragastrically, and drug concentrations were measured by HPLC. Metabolites were characterised by cochromatography with authentic reference compounds and were identified by mass spectrometry. The ratios of area of plasma or tissue concentration vs time curves of resveratrol over DMU 212 (AUC(res)/AUC(DMU212)) for the plasma, liver, small intestinal and colonic mucosa were 3.5, 5, 0.1 and 0.15, respectively. Thus, resveratrol afforded significantly higher levels than DMU 212 in the plasma and liver, while DMU 212 exhibited superior availability compared to resveratrol in the small intestine and colon. Resveratrol was metabolised to its sulphate or glucuronate conjugates, while DMU 212 underwent metabolic hydroxylation or single and double O-demethylation. DMU 212 and resveratrol inhibited the growth of human-derived colon cancer cells HCA-7 and HT-29 in vitro with IC(50) values of between 6 and 26 microM. In the light of the superior levels achieved in the gastrointestinal tract after the administration of DMU 212, when compared to resveratrol, the results provide a good rationale to evaluate DMU 212 as a colorectal cancer chemopreventive agent.

Figure 1

Chemical structures of (A) resveratrol and (B) DMU 212.

Figure 2

Structures of five putative metabolites of DMU 212.

Figure 3

Concentrations of resveratrol (squares, dotted line) and DMU 212 (rhombi, solid line) in the plasma and tissues of mice that received a single dose of drug (240 mg kg⁻¹) i.g. Values are the mean±s.d. (n=3). Star indicates that the values differ significantly (P<0.05, one-way ANOVA). For details of dosing, extraction and HPLC analyses, see Materials and Methods.

Figure 4

(A) HPLC analysis of liver extracts of mice that received resveratrol (240 mg kg⁻¹) p.o. (i) and of a mixture (ii) of (i) and biosynthesised resveratrol glucuronide. Liver tissue was obtained 60 min postadministration. Peak allocation is (1) resveratrol glucuronide, (2) resveratrol sulphate and (3) resveratrol. The chromatogram is representative of three. For details of dosing, extraction and HPLC analyses, see Materials and Methods. (B) HPLC analysis of liver extracts of mice that received DMU 212 (240 mg kg⁻¹) p.o. (i) a mixture (ii) of (i) with authentic standards DMU 212, 4,4′-di-desmethyl-DMU 212 (DMU 295), 4′-desmethyl-DMU 212 (DMU 281), 3′-hydroxy-DMU 212 (DMU 214), 4-desmethyl-DMU 212 (DMU 291) and 3-desmethyl-DMU 212 (DMU 807). Liver tissue was obtained 60 min postadministration. Peak allocation is (4) 4,4′-di -desmethyl-DMU 212 (DMU 295) (5) 4′-desmethyl-DMU 212 (DMU 281), (6) 3′-hydroxy-DMU 212 (DMU 214), (7) 4-desmethyl-DMU 212 (DMU 291), (8) 3-desmethyl-DMU 212 (DMU 807) (9) DMU 212 and (10) internal standard. The chromatogram is representative of three. For details of dosing, extraction and HPLC analyses, see Materials and Methods. (C) HPLC analysis of extracts of an incubate of mouse liver microsomes with DMU 212 (1 mM) (i) and of a mixture (ii) of (i) with authentic DMU 212, 4,4′-di-desmethyl-DMU 212 (DMU 295), 4′-desmethyl-DMU 212 (DMU 281), 3′-hydroxy-DMU 212 (DMU 214), 4-desmethyl-DMU 212 (DMU 291) and 3-desmethyl-DMU 212 (DMU 807). Incubations were terminated after 20 min. Peak allocation is (4) 4,4′-di-desmethyl-DMU 212 (DMU 295) (5) 4′-desmethyl-DMU 212 (DMU 281), (6) 3′-hydroxy-DMU 212 (DMU 214), (7) 4-desmethyl-DMU 212 (DMU 291), (8) 3-desmethyl-DMU 212 (DMU 807) (9) DMU 212. The chromatogram is representative of three. For details of incubation, extraction and HPLC analysis, see Materials and Methods.

Figure 5

Effect of resveratrol (A, C) and DMU 212 (B, D) on the growth of HT-29 (A, B) and HCA-7 colon cancer cells (C, D). Symbols indicate the following agent concentrations: closed squares control cells, open rhombi 1 μM, crosses 5 μM, closed rhombi 10 μM, closed triangles 25 μM, open circles 50 μM and open squares 100 μM. IC₅₀ values computed for the 168 h time point are inserted. Values are the mean±s.d. of four independent experiments.