Transcript Slide 1

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
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.
Acute Lymphoblastic Leukemia (ALL)
Enzymatic Reaction with 6-Mercaptopurine
The Basics: Capillary Electrophoresis
2.5 mM 6-MP
Detector response
+
+
Neutrals
Time
8
10
12
14
16
6
18
9
12
15
18
t=70 min
0.5 mA
6
21
Time (min)
2. Instrumental Parameters
0.1 U of XO for peak
identification
Absorbance (AU)
10 kV
5 sec
0.5 mA
10 kV
20 sec
4.5
5.0
5.5
6.0
Time (min)
6.5
7.0
3. Concentration
Optimization of Internal Standard Concentration
Variation of injection parameters,
10 kV for 20 sec versus 10 kV for 5
sec, to improve peak shape
10 M
4 M
5
6
Time (min)
7
11
12
2
0.01 mAU
0
50
100 150 200 250 300 350
Injection Time (min)
Comparison of the peak
area of 6-MP and 6-TUA
as a function of time.
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
15 M
2 mA
8
9
10
Time (min)
Conclusions and Future Work
30 M
4
7
Study of 6-MP consumption
(t = 6.5 min) and 6-TUA
formation (t = 10 – 11 min)
as a function of time
50 M
Acknowledgements
•Haes Research Group
0.5 mA
Time (min)
Combination of
electrophoresis and
electroosmosis yields high
separation efficiencies
6-MP Area
6-TUA Area
t=3 min
6
1:1 and 1:3 dilution of 50 μM 6-MP
for peak identification
The diffuse double
layer moves all of the
molecules towards
the cathode
Absorbance (AU)
-
Outlet
reservoir
XO
Absorbance (AU)
Detector
+
Inlet
reservoir
5 mM 6-MP
0.25 mA
Inactive
t=256 min
Absorbance (AU)
Capillary
Active
After Optimization
Optimization
1. Peak Identification
Advantages:
 Small sample volumes
 Fast analysis
 Improved separation
efficiency
Absorbance (AU)
Light
source
Power
supply
Results
 Therapeutically-active metabolite: Hypoxanthineguanine 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
Area (AU2)
 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.
Normal Bone
 The number of leukemia cells grows quickly crowding out the
Marrow
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 Bone Marrow with
 Survival rate for children 85%, adults 50%
Leukemia Cells
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Variation of internal standard
concentration to improve
signal to noise ratio
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.
References
GAANN
“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.