In Vitro Metabolism Study of Combretastatin A-4 in Rat and Human Liver Microsomes

Abstract

ABSTRACT:The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6 of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding paraquinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.

Identification of the Human UDP-Glucuronosyltransferases Involved in the Glucuronidation of Combretastatin A-4

“…In a recent study, we elucidated the in vitro/vivo metabolic fate of CA-4 through identifying the structures of a series of phase I metabolites and the glucuronide conjugate. Formation of the sulfate metabolite has also been demonstrated both in vitro and in vivo (Aprile et al, 2007(Aprile et al, , 2009). UDPglucuronosyltransferases (UGTs) are the major phase II drug-metabolizing enzymes in humans, catalyzing the conjugation with UDP-glucuronic acid of numerous endogenous and exogenous compounds.…”

“…Indeed, because it has been demonstrated (Brown et al, 1995) that CA-4G is characterized by very low pharmacodynamic activity, a different rate of glucuronidation could affect the CA-4 therapeutic effect. Moreover, glucuronidation could also modulate the formation of quinone species whose role in CA-4 pharmacodynamics is at the present time unknown (Aprile et al, 2007). This article describes the full in vitro kinetic characterization of the hepatic UGT isoforms involved in the CA-4 glucuronidation pathway.…”

Synthesis, biological evaluation, and structure–activity relationships of tri- and tetrasubstituted olefins related to isocombretastatin A-4 as new tubulin inhibitors

“…10 It is important to note that the Z olefinic bridge is able to undergo rapid Z-E isomerization under the influence of heat, light, and protic media 10a and that this olefinic bridge represents a weak point for metabolic stability, 11 resulting in a dramatic loss in antitumor activity. To circumvent the problem of Z-E isomerization, a substantial range of CA-4 analogues have been designed and synthesized with the objective to replace the olefinic bridge and improve the intrinsic stability as well as the therapeutic index of CA-4.…”

Combretastatins: More Than Just Vascular Targeting Agents?

“…The combretastatin metabolite profile has been described as complex. O-demethylation and aromatic hydroxylation were identified as the two principal phase I biotransformation pathways of CA-4 (Aprile et al, 2007). Z-E isomerization of the olefin bond was primarily observed during metabolic O-demethylation and aromatic hydroxylation of CA-4.…”

“…Z-E isomerization of the olefin bond was primarily observed during metabolic O-demethylation and aromatic hydroxylation of CA-4. Metabolites arising from aromatic hydroxylation of ring B were readily oxidized into para-quinone metabolites (Aprile et al, 2007). In this study, the resulting metabolites were not analyzed for activity or toxicity.…”