School: Honaker High School
Location: Honaker, VA
Grades taught: 11-12 (Human Anatomy and Physiology,  Biology II Topics, Chemistry)

Principle Investigator: Kenneth E. Thummel, Ph.D.
Department: Pharmaceutics
Institution: University of Washington

Project Description
Research in Dr. Thummel’s lab is focused on analyzing the factors that determine interindividual variability in drug metabolism. The two primary enzyme systems studied in Dr. Thummel’s lab are the cytochrome P450 and UGT enzyme families. Both are large families of enzymes responsible for the metabolism of a variety of endogenous and xenobiotic compounds such as steroids, hormones, environmental pollutants, dietary chemicals and drugs from almost all therapeutic classes. Studies have shown a significant amount of interindividual variability in the metabolism of many cytochrome P450 and UGT substrates. Understanding the genetic basis of this observed variability may lead to more individualized drug therapy and increased efficacy.

My research focused on the role of UGTs in the metabolism of hydromorphone, an opioid analgesic. Although expressed in many tissues, the UGTs are primarily active in the liver and small intestine. The UGTs conjugate their substrates with glucuronic acid (glucuronidation), which serves as a detoxification and elimination mechanism. The goals of this research were to (1) determine which of the UGT enzymes are responsible for the glucuronidation of hydromorphone, (2) determine the genotype of the human liver bank for the UGT2B7*1 and UGT2B7*2 polymorphisms, (3) quantify the level of UGT2B7 expression in microsome samples from the liver bank, and (4) determine the activity of the UGT2B7 protein in the microsome samples.

A panel of human UGT cDNA supersomes (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) was screened for production of the hydromorphone-3-glucuronide metabolite. Only 2B4 and 2B7 produced the metabolite, with 2B7 activity significantly higher than 2B4. The UGT2B7 gene has one single nucleotide polymorphism (SNP) resulting in a protein sequence change from histidine to tyrosine that may be of significance in hydromorphone metabolism. PCR-RFLP techniques were used to genotype the 63 sample liver bank for the UGT2B7 polymorphism. Genotype assignments of selected samples were confirmed by direct sequencing. Quantification of the UGT2B7 protein levels in the microsomes was not completed but my preliminary experiments indicate high variability among the liver microsome samples. The human liver microsomal UGT2B7 activity was measured by incubation with hydromorphone and then quantification of the metabolite by high pressure liquid chromatography. My preliminary experiments indicate little or no difference in activity among the UGT2B7 genotypes. The next steps for this research are to complete the quantification of 2B7 in the human liver microsomes, complete the activity assays on the human liver microsomes, and determine the Km and Vmax of each 2B7 genotype per unit of protein.