Metabolism of Thalidomide in Liver Microsomes of Mice, Rabbits, and Humans

Abstract

Thalidomide is increasingly important in clinical treatment, not only of various inflammatory conditions but also in multiple myeloma and other malignancies. Moreover, the metabolism of thalidomide varies considerably among different species, indicating a need to understand its mechanistic basis. Our previous in vivo studies showed the plasma half-life of thalidomide to be much shorter in mice than in humans, with rabbits showing intermediate values. We were unable to detect hydroxylated thalidomide metabolites in humans and suggested that interspecies differences in thalidomide hydroxylation might account for the differences in plasma half-life. We sought here to establish whether these species differences in the formation of hydroxylated thalidomide metabolites could be discerned from in vitro studies. Liver microsomes of mice, rabbit, and human donors were incubated with thalidomide and analyzed using liquid chromatography-mass spectrometry. Hydrolysis products were detected for all three species, and the rates of formation were similar to those for spontaneous hydrolysis, except in rabbits where phthaloylisoglutamine formation increased linearly with microsomal enzyme concentration. Multiple hydroxylation products were detected, including three dihydroxylated metabolites not observed in vivo. Thalidomide-5-O-glucuronide, detected in vivo, was absent in vitro. The amount of 5-hydroxythalidomide formed was high in mice, lower in rabbits, and barely detectable in humans. We conclude that major interspecies differences in hepatic metabolism of thalidomide relate closely to the rate of in vivo metabolite formation. The very low rate of in vitro and in vivo hydroxylation in humans strongly suggests that thalidomide hydroxylation is not a requirement for clinical anticancer activity.Thalidomide (␣-phthalimidoglutarimide, Thal), despite its teratogenicity (McBride, 1961;Lenz, 1962), is attracting increasing clinical interest, initially as an anti-inflammatory agent (Sheskin, 1965;Zwingenberger and Wnendt, 1996;Calabrese and Fleischer, 2000) and more recently for the treatment of malignancies, particularly of multiple myeloma (Singhal et al., 1999;Eisen, 2000). Differences in metabolism among different species represent an important feature of its pharmacology. It has been suggested that Thal exerts its teratogenicity through an active metabolite, which is produced by human and rabbit liver microsome preparations but not by mouse liver microsomes (Gordon et al., 1981). After in vivo administration, Thal is both hydrolyzed chemically to a series of acid derivatives and hydroxylated by liver enzymes. In a previous study , we showed that the plasma half-life of Thal in mice was much shorter than that in patients with multiple myeloma, with New Zealand White rabbits showing an intermediate half-life. We also showed that hydroxylated metabolites were not detected in multiple myeloma patients but were found in C57/Bl/6 mice and rabbits, suggesting that relative rates of Thal hydroxylation in different species could...

Influence of Mustard Group Structure on Pathways of in Vitro Metabolism of Anticancer N-(2-Hydroxyethyl)-3,5-dinitrobenzamide 2-Mustard Prodrugs

“…The two livers selected for this study were chosen because they are representative of cancer drug metabolism in a larger liver bank (Zhou et al, 2000;Lu et al, 2004;Tang et al, 2005). However, the potential for interindividual TABLE 1…”

“…In vitro hepatic metabolism of compounds 1 through 4 was studied using the 9000g postmitochondrial supernatant (S9) of human (HL5 and HL18), dog (beagle), rat (male and female, Sprague-Dawley) and mouse (pooled male and female CD-1 athymic nu/nu) livers, prepared as described previously (Tang et al, 2005). The human livers selected were chosen because we have shown previously that they are representative of drug metabolism across a diverse range of cancer chemotherapeutic drugs (Zhou et al, 2000;Lu et al, 2004;Tang et al, 2005). Hepatic S9 preparations (8 mg of protein/ml) and dinitrobenzamide prodrug substrate (250 M) were incubated for 30 min at 37°C in a final volume of 0.5 ml of sodium-potassium phosphate buffer (67 mM, pH 7.4) under air in the presence of cofactor (NADPH or NADH, 1 mM).…”

Substrates, Inducers, Inhibitors and Structure-Activity Relationships of Human Cytochrome P450 2C9 and Implications in Drug Development

“…The R and Senantiomers of thalidomide interconvert rapidly in plasma, with protein binding of 55% and 65%, respectively [322,323]. Thalidomide mainly underwent rapid and spontaneous hydrolysis in aqueous medium at physiological pH and biological matrixes such as plasma and blood [322,[324][325][326][327], whereas CYP2C-mediated metabolism plays a minor role in its elimination [328][329][330][331][332]. CYP1A1/2, 2E1, 2C9 and 2C19 are involved in the metabolism of thalidomide (Fig.…”

Utilização de animais em pesquisas: breve revisão da legislação no Brasil

“…The drug thalidomide, for example, prescribed as a hypnotic and sedative for humans, including pregnant women, resulted in many cases of congenital malformations in children 8 . This was partly due to misinterpretation of the actual effect of thalidomide, as rodents' metabolism of the drug is different from humans' metabolism, and, as a result, numerous reports of "thalidomide babies" 9 were observed.…”