Characterization of UGTs Active against SAHA and Association between SAHA Glucuronidation Activity Phenotype with UGT Genotype

Abstract

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor used in the treatment of cutaneous T-cell lymphoma and in clinical trials for treatment of multiple other cancers. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To characterize the UGTs active against SAHA, homogenates from HEK293 cell lines overexpressing UGT wild-type or variant UGT were used. The hepatic UGTs 2B17 and 1A9 and the extrahepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by V max /K M (16 F 6.5, 7.1 F 2.2, 33 F 6.3, and 24 F 2.4 nLÁmin À1. Mg UGT protein À1 , respectively), with UGT2B17 exhibiting the lowest K M (300 Mmol/L) against SAHA of any UGT in vitro. Whereas the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P < 0.005) decrease in glucuronidation activity against SAHA compared with wild-type UGT1A8, the UGT1A8p.Cys277Tyr variant exhibited no detectable glucuronidation activity; a similar lack of detectable glucuronidation activity was observed for the UGT1A10p.Gly139Lys variant. To analyze the effects of the UGT2B17 gene deletion variant (UGT2B17*2) on SAHA glucuronidation phenotype, human liver microsomes (HLM) were analyzed for glucuronidation activity against SAHA and compared with UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P < 0.01) decrease in glucuronidation activity and a 75% (P < 0.002) increase in K M compared with HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Overall, these results suggest that several UGTs play an important role in the metabolism of SAHA and that UGT2B17-null individuals could potentially exhibit altered SAHA clearance rates with differences in overall response.

UGT2B Gene Expression Analysis in Multiple Tobacco Carcinogen-Targeted Tissues

“…The UDP-glucuronosyltransferase (UGT) family of phase II metabolic enzymes play a central role in the excretion of numerous endogenous compounds, including bilirubin (Bosma et al, 1994) and steroid hormones (Belanger et al, 1998(Belanger et al, , 2003, as well as exogenous compounds, including various drugs (Nagar and Remmel, 2006) and chemotherapeutic agents (Kemp et al, 2002;Sun et al, 2006Sun et al, , 2007Sun et al, , 2010Balliet et al, 2009). The UGTs also play an important role in the metabolism and detoxification of multiple tobacco carcinogens, including tobacco-specific nitrosamines like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Ren et al, 2000;Wiener et al, 2004a;Chen et al, 2008b), and polycyclic aromatic hydrocarbons like benzo(a)pyrene [B(a)P]-7,8-diol and dibenzo(a,l)pyrene-11,12-diol [DB(a,l)P] (Fang et al, 2002;Olson et al, 2011).…”

Identification and Functional Characterization of a Novel UDP-Glucuronosyltransferase 2A1 Splice Variant: Potential Importance in Tobacco-Related Cancer Susceptibility

“…Reaction mixtures were centrifuged for 10 min at 16,100g before the collection of supernatant. Glucuronide formation was determined by using an Acquity ultra-pressure liquid chromatography (UPLC) System (Waters, Milford, MA) as described previously (Fang et al, 2002;Wiener et al, 2004b;Dellinger et al, 2007;Balliet et al, 2009;Olson et al, 2009;Bushey et al, 2011). The flow rate was maintained at 0.5 ml/min, and a reverse-phase Acquity UPLC BEH C18 1.7-m 2.1 ϫ 100-mm column (Waters) was used to separate free substrate and the conjugated glucuronide.…”

Histone Deacetylase Inhibitors as Therapeutic Agents for Cancer Therapy: Drug Metabolism and Pharmacokinetic Properties

“…In the same study, up to 18% and 36% of the O-glucoronide and 4-AOB, respectively, were recovered in urine, with the parent accounting for < 1 % of the total dose, clearly indicating that Vorinostat was cleared primarily by metabolism in humans, and that the O-glucoronide and 4-AOB were the major metabolites. The main enzymes responsible for the formation of the Oglucoronide were identified as the UDP-glucoronosyltransferases (UGTs), such as the UGTs 2B17 and 1A9, which are expressed in the liver, and the extrahepatic UGTs 1A8 and 1A10 (Balliet et al,2009). UGT2B17 was one of the major enzymes contributing to the formation of the O-glucoronide of Vorinostat in humans (Balliet et al, 2009).…”

“…The main enzymes responsible for the formation of the Oglucoronide were identified as the UDP-glucoronosyltransferases (UGTs), such as the UGTs 2B17 and 1A9, which are expressed in the liver, and the extrahepatic UGTs 1A8 and 1A10 (Balliet et al,2009). UGT2B17 was one of the major enzymes contributing to the formation of the O-glucoronide of Vorinostat in humans (Balliet et al, 2009). Since UGTs are known to show extensive polymorphism, including UGT2B17, they have been associated with the variable PK and response of Vorinostat in patients (Balliet et al, 2009).…”

“…UGT2B17 was one of the major enzymes contributing to the formation of the O-glucoronide of Vorinostat in humans (Balliet et al, 2009). Since UGTs are known to show extensive polymorphism, including UGT2B17, they have been associated with the variable PK and response of Vorinostat in patients (Balliet et al, 2009). There have been no reports on allometric scaling or the predictions of human PK based on preclinical ADME data so far.…”