1. Active Inactive Monitoring Anti-Cancer Drug Metabolism Amanda Jones, Varuni Subramaniam and Amanda J. Haes www.chem.uiowa.edu/faculty/haes/group/AJH_home.html University of Iowa; Department of Chemistry; Iowa City, Iowa 52242 GAANN Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer among children. The treatment of ALL utilizes anti-cancer drugs which are metabolized by intracellular enzymes; however, the products have poor selectivity and non-specific toxicity. For instance, the anti-cancer drug 6-mercaptopurine (6-MP) forms three specific metabolites from three different enzymes, resulting in an active, inactive and toxic form of the drug. In this research, capillary electrophoresis (CE) will be utilized to directly monitor 6-MP metabolism. Varying enzyme activity and drug concentration will yield information relating to the rate of formation of the metabolite. We expect that a better understanding of the enzyme kinetics associated with the anti-cancer drug will provide important insights into the effective enzyme threshold levels for proper dosages, and ultimately, the improvement of current cancer treatments. Acknowledgements Haes Research Group Conclusions and Future Work “Leukemia, Lymphoma, Myeloma, Facts 2009-2010,” The Leukemia and Lymphoma Society, Inc., 2009. Seidman, E. G.; Theoret, Y. Optimized use of 6-mercaptopurine drug in the treatment of immune-mediated gastrointestinal disorders; (Hopital Sainte-Justine, Can.). Application: WO, 2000; pp 77 pp. Kalra, S.; Paul, M. K.; Balaram, H.; Mukhopadhyay, A. K. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2007, 850, 7. References The Basics: Capillary Electrophoresis Acute Lymphoblastic Leukemia (ALL) Detector Outlet reservoir Light source - Capillary Power supply + Inlet reservoir  Leukemia is a fast-growing cancer of the white blood cells and bone marrow which forms abnormal cells that do not develop and cannot fight infections.  The number of leukemia cells grows quickly crowding out the normal red blood cells, white blood cells and platelets the body needs.  ALL is the most common type of leukemia in children 1 to 7 years old, accounting for about 5,760 new cases this year.  There are about 4,000 new cases of ALL in the United States each year  Survival rate for children 85%, adults 50% Advantages:  Small sample volumes  Fast analysis  Improved separation efficiency Normal Bone Marrow Bone Marrow with Leukemia Cells Results Enzymatic Reaction with 6-Mercaptopurine Study of 6-MP consumption (t = 6.5 min) and 6-TUA formation (t = 10 – 11 min) as a function of time Comparison of the peak area of 6-MP and 6-TUA as a function of time.  Therapeutically-active metabolite: Hypoxanthine- guanine phophoribosyltransferase (HCPRT) forms thioinonsinic monophosphate (TIMP)  Inactive metabolite: Xanthine oxidase (XO) forms 6-thiouric acid (6TUA)  Study the activity of these enzymes for improved dosage determination and personalized medication Time + Neutrals Detector response + 1. Peak Identification 2. Instrumental Parameters 3. Concentration Optimization After Optimization Combination of electrophoresis and electroosmosis yields high separation efficiencies The diffuse double layer moves all of the molecules towards the cathode 1:1 and 1:3 dilution of 50 μM 6-MP for peak identification 0.1 U of XO for peak identification Variation of injection parameters, 10 kV for 20 sec versus 10 kV for 5 sec, to improve peak shape Variation of internal standard concentration to improve signal to noise ratio Optimized Conditions Run Buffer: 25mM potassium phosphate buffer pH 7.5, Wavelength of detection: 280 nm, Concentration: 50 μM 6-MP, 50 μM 4-AAP and 0.1 XO, Injection: 10kV 5sec In the future, the kinetics of 6-MP metabolism will be monitored by varying XO activity, a real sample mimic will be studied with in-capillary mixing and the inhibition capacity of allopurinol will be evaluated. +