Novel Therapy for Treating Human Glioblastomas

1. Novel Therapy for Treating Human Glioblastomas http://www.cancerhelp.org.uk/help/default.asp?page=96#cancer_cells_dont_stop By: Rajwant Singh Bedi Chemical Engineering

2. Glioblastoma Mutiforme • Most common type of malignant primary brain tumor • Typically contain more than one type of cells. • Characterized by rapid growth, these tumors can grow quite large before clinically relevant symptoms appear. • Symptoms include: • Motor weakness. • Severe headaches, nausea, vomiting. • Sometimes seizures. http://www.emedicine.com/med/topic2692.htm http://www.irsa.org/glioblastoma.html

3. Glioblastoma Mutiforme • Normal cells survive at a pH=7.4 • Glioblastomas can survive at pH=6.9 • Low pH is produced by lack of blood supply. No oxygen is received by cells for oxidative phosphorylation, so cells use glycolysis, which builds up protons inside the cells. • Glioblastomas use NHE1 to exchange H+ for Na+ ions to increase pH inside the cell and maintain homeostasis.

4. Previous attempts for a cure • Radiation Therapy • Slows down tumor proliferation, but does not cure the tumor. • Can also damage normal tissue. • Chemotherapy • Highly toxic drugs are needed to cross the blood-brain barrier. • These drugs may be toxic to other organs in the body. So, combinations of drugs are used to find the best drugs for the treatment. • Surgery • Impossible to identify and remove all of tumor tissue which extends into normal tissue.

5. Why new therapy is required? • Poor clinical prognosis • High recurrence rate • Patients typically live only 6-12 months following diagnosis regardless of therapeutic regimen

6. Novel Therapy • Amiloride derivatives • Carefully designed to exploit metabolic differences between normal and tumor cells • To produce cell death, drug must inhibit NHE1 and NCX to cause intracellular acidosis and loss of calcium regulation • Preferably only active in CNS or tumor

7. Testing efficacy of the drug • Inhibition constant • IC50—concentration of drug where 50% Inhibition of NHE1 occurs • Lower Inhibition constant is better as less amount of drug is required to successfully inhibit 50% of sodium/hydrogen exchange in the tumor cell.

8. Methodology Determination of NHE1 IC50 • Cells grown in Petri dishes • 37 degrees Celsius • pH=7.4 • Cells are subjected to the drug in vitro • changes in pH are measured spectrofluorometrically using a fluorescent dye, 2’,7’bis(carboxyethyl)-5,6-carboxyfluorescin acetoxy-methyl ester (BCECF) • Excitation wavelengths: 507/440nm • Emission wavelength: 535 nm

9. Ammonium Prepulse Method • Hepes Ringer • Baseline measurement of pH • NH4Cl • Acidification of cell • NMDG • Sodium free solution which stops sodium/hydrogen exchange • Add sodium to observe recovery +/-Drug • Drug inhibits NHE1, and thus recovery is slower than just adding Na-containing Hepes ringer.

10. Graph produced by Data Figure 1:

11. Graphs continued… Figure 2:

12. Graphs continued… Figure 3:

13. Graphs continued… Figure 4:

14. Data Analysis NHE1i slope: Determined from Figure 2 Control Slope: Determined from Figure 1 Ki=NHE1i slope/ Control slope Y-intercept and Coefficient: Determined from Figure 4 IC50: Determined From equation Given by figure 4

15. Conclusion • IC50 C2-Amiloride Glycine • 75 uM • Is lower than IC50 of Amiloride (124 uM) • Lower IC50 means that a lower concentration of drug is required to inhibit 50% of sodium/hydrogen exchange in the tumor cell.

16. Further Experimentation • In vivo experiments • Track tumor progression in animal mode using Proton Magnetic Resonance Spectroscopy (MRS) imaging in the presence or absence of new drugs

17. Acknowledgements • Fredric A. Gorin, Dept. of Neurology. • Michael Nantz, Dept. of Chemistry. • Hasan Palandoken, Dept. of Chemistry. • Bill Harley, Dept. of Neurology.