CHARACTERIZATION OF UGTS ACTIVE AGAINST SUBEROYLANILIDE HYDROXAMIC ACID (SAHA): VARIATIONS IN SAHA GLUCURONIDATION ASSOCIATED WITH UGT GENETIC VARIANTS
Open Access
- Author:
- Balliet, Renee Marie
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 18, 2009
- Committee Members:
- Philip Lazarus, Dissertation Advisor/Co-Advisor
Philip Lazarus, Committee Chair/Co-Chair
Laura Carrel, Committee Member
Lisa M Shantz, Committee Member
Christopher Herzog, Committee Member - Keywords:
- SAHA
pharmacogenetic
UGT - 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. Variations in patient response and toxicities have been observed; however, no underlying cause has been identified for these differences. A mechanism that could be responsible for the observed differences is altered drug metabolism. A major mode of SAHA metabolism is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. The UGT superfamily of enzymes catalyzes the glucuronidation of a variety of endogenous compounds such as bilirubin and steroid hormones, as well as xenobiotics such as drugs and environmental carcinogens. These enzymes are located in the endoplasmic reticulum of cells and make xenobiotics and endogenous compounds more water soluble through their conjugation to glucuronic acid in a reaction with the hydrophilic co-substrate, UDPGA. This conjugation alters the biological properties of the compound to enhance its excretion in the urine or bile and typically converts substrates into products that are less pharmacologically active. These enzymes have been associated with altered drug metabolism and correlated with patient response and toxicity. The primary goals of this thesis include: identification of the UGTs responsible for SAHA glucuronidation, characterization of the UGTs that are most active against SAHA, in vitro studies of UGT genotype/SAHA glucuronidation phenotype association, and characterization of the promoter region of the UGT1A10 gene. To identify and characterize the UGTs active against SAHA, homogenates from UGT-overexpressing HEK293 cell lines were used. The hepatic UGTs 2B17 and 1A9 and the extra-hepatic UGTs 1A8 and 1A10 exhibited the highest overall activity against SAHA as determined by Vmax/KM (16±6.5, 7.1±2.2, 33±6.3, and 24±2.4 nL.min-1.mg UGT protein-1, respectively), with UGT2B17 exhibiting the lowest KM (300 μM) for SAHA of any UGT in vitro. While the UGT1A8p.Ala173Gly variant exhibited a 3-fold (P<0.005) decrease in glucuronidation activity for SAHA as compared to 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 as well as for UGT2B17 genotype. HLM from subjects homozygous for UGT2B17*2 exhibited a 45% (P<0.01) decrease in SAHA glucuronidation activity and a 75% (P<0.002) increase in KM for SAHA as compared to the HLMs from subjects homozygous for the wild-type UGT2B17*1 allele. Further stratification of the HLM glucuronidation data by gender, identified a significant difference in SAHA glucuronidation between males and females, with females having significantly less glucuronidation. Characterization of the promoter of one of the most active glucuronidators of SAHA, UGT1A10, led to the identification of a novel deletion which was able to cause differential induction of luciferase reporter activity in colon derived cell lines. These data suggested that tissue specific regulation of UGTs may ultimately determine glucuronidation levels in specific tissues. Overall, these results suggest that several UGTs play important roles in the metabolism of SAHA and that females and UGT2B17-null males could potentially exhibit altered SAHA clearance rates and differences in overall response to the drug.