A potential role for human UDP-glucuronosyltransferase 1A4 promoter single nucleotide polymorphisms in the pharmacogenomics of tamoxifen and its derivatives.

Tamoxifen (Tam) is a selective estrogen receptor modulator used to inhibit breast tumor growth. Tam can be directly N-glucuronidated via the tertiary amine group or O-glucuronidated after cytochrome P450-mediated hydroxylation. In this study, the glucuronidation of Tam and its hydroxylated and/or chlorinated derivatives [4-hydroxytamoxifen (4OHTam), toremifene (Tor), and 4-hydroxytoremifene (4OHTor)] was examined using recombinant human UDP-glucuronosyltransferases (UGTs) from the 1A subfamily and human hepatic microsomes. Recombinant UGT1A4 catalyzed the formation of N-glucuronides of Tam and its derivatives and was the most active UGT enzyme toward these compounds. Therefore, it was hypothesized that single nucleotide polymorphisms (SNPs) in the promoter region of UGT1A4 have the ability to significantly decrease the glucuronidation rates of Tam metabolites in the human liver. In vitro activity of 64 genotyped human liver microsomes was used to determine the association between the UGT1A4 promoter and coding region SNPs and the glucuronidation rates of Tam, 4OHTam, Tor, and 4OHTor. Significant decreases in enzymatic activity were observed in microsomes for individuals heterozygous for -163G/A and -217T/G. These alterations in glucuronidation may lead to prolonged circulating half-lives and may potentially modify the effectiveness of these drugs in the treatment of breast cancer.

Novel resveratrol-based substrates for human hepatic, renal, and intestinal UDP-glucuronosyltransferases.

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by ß-glucuronidase hydrolysis and LC-MS/MS analysis. NI-12a was glucuronidated at both the -COOH and -OH functions, NI-ST-05 formed a novel N-O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent Km values of 240 and 6.2 µM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen.

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 µM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.