School: Shorewood High School
City: Shoreline, WA
Grades taught: 9-12

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

Contributing Investigator: Evan Kharasch, M.D., Ph.D.
Department: Anesthesiology
Institution: University of Washington

Project Description

The Biological Basis for Interindividual Variability in Drug Metabolism
Research in Dr. Thummel's lab focuses on cytochrome P450 enzymes and understanding the role these proteins play in drug metabolism. A large family of enzymes, cytochrome P450s, or CYPs, are responsible for metabolizing a broad range of endogenous and xenobiotic compounds, including vitamins, fatty acids, steroids, hormones, drugs, toxins, carcinogens, and dietary constituents.  CYPs are widely distributed throughout the body, but are primarily found in the liver and small intestine. As different CYPs show varied substrate specificity, inductive and inhibitory regulation, tissue distribution, and polymorphic genetic expression, the variability of metabolism among different pharmaceutical agents and different individuals is vast.  This variability plays a role in how extensively or poorly an individual may metabolize certain compounds. Understanding the basis for interindividual variability in drug metabolism may some day allow for individualized drug therapy based upon one’s genotype.

StarNet Summer Research Project on CYP2B6
My research focused on the variable expression of CYP2B6 in human liver microsomes and on the metabolism of Methadone by this enzyme. Methadone is a long-acting opioid analgesic used in post operative pain management and the treatment of heroin addiction.  Some patients who receive Methadone are also being treated with Ritonavir, a protease inhibitor used to treat HIV infection.  My research focused on understanding the role that CYP2B6 plays in Methadone metabolism and determining the extent of inhibition, if any, of this enzyme by Ritonavir.  To approach this experimental question, I phenotyped a 64 sample liver bank for CYP2B6 expression using SDS PAGE / Western Blot technology to separate the microsomal proteins and immunochemically detect CYP2B6 with a secondary antibody conjugated with phosphatase.  I used linear regression analysis to quantify the level of protein expression and found that I was able to detect and quantify CYP2B6 in all liver bank samples.  Significant variability among individuals existed, as CYP2B6 is polymorphically expressed.

I then prepared a matched set of 14 liver microsomal samples.  Seven pairs were identified, with each pair having similar CYP3A4 expression (the predominant hepatic CYP isoform), but one member of the pair having high CYP2B6 levels, and the other member expressing low levels of the enzyme.  I incubated these samples separately with R-Methadone and with S-Methadone at two different concentrations, physiological concentration and at a saturating condition.  I then collected the primary and secondary metabolites, EDDP and EMDP, using solid phase extraction and separated and quantified them using high pressure liquid chromatography and mass spectrometry.  The results suggested that those livers expressing high levels of CYP2B6 were metabolizing Methadone at a higher rate, and possibly showing a greater specificity for the S-Methadone substrate.

However, the two livers demonstrating the greatest Methadone metabolism in this assay also expressed the enzyme CYP3A5, a highly polymorphically expressed protein (10% in Caucasians, 30% in Asians, 50-60% in those of African descent). From these preliminary findings, a second assay was developed to distinguish between CYP3A4, 3A5, and 2B6 involvement in Methadone metabolism.  cDNA expressed supersomes were used to incubate 3A4, 3A5, and 2B6 with Ritonavir (a known inhibitor of 3A4).  R-Methadone and S-Methadone substrates were then introduced separately, using a regenerating system, and metabolite formation was measured.  The findings were surprising.  CYP2B6 demonstrated greater metabolism of Methadone (36% greater for the R enantiomer, 68% for the S enantiomer) than CYP3A4.  CYP3A5 did not demonstrate appreciable Methadone metabolism.  Interestingly, Ritonavir inhibited Methadone metabolism in both 3A4 and 2B6 at all three concentrations assayed.  The next step is to repeat the Ritonavir incubations and perform a different inhibition assay using inhibitory antibodies.  In addition, kinetic studies will be conducted on CYP3A4 and CYP2B6 with Methadone (R-, S-, and racemic) to characterize the Km and Vmax of these enzymes with these substrates.  Additionally, I will genotype the liver bank for CYP2B6 allelic variants using PCR technology over the course of the coming year.

Acknowledgements
I offer my sincerest gratitude to the members of the Thummel Lab for their kindness, mentorship, and support: Kenneth Thummel, PhD; Yang Xu, PhD; Rheem Totah, PhD; and Tina and Neena. I would also like to extend my thanks and acknowledge the significant contributions of the Kharasch Lab to my research: Evan Kharasch, MD, PhD; Pam Sheffels, MS; and Dale Wittington, BS.