Transcript Slide 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 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 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. 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. Why new therapy is required? Poor clinical prognosis High recurrence rate Patients typically live only 6-12 months following diagnosis regardless of therapeutic regimen 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 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. 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 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. Graph produced by Data Figure 1: Control pHi Recovery 7.700 7.600 7.500 7.400 pHi 7.300 7.200 7.100 7.000 6.900 HR 6.800 0 200 400 600 800 time (seconds) 1000 1200 1400 Graphs continued… Figure 2: 500µM C2-Am-Gly 7.250 pHi 7.150 7.050 6.950 HR NHE1i 6.850 0 500 1000 time (seconds) 1500 Graphs continued… Figure 3: 500µM C2-Am-Gly NHE Inhibition 7.000 6.990 y = 0.0002x + 6.6939 R2 = 0.9856 6.980 6.970 pHi 6.960 6.950 6.940 6.930 6.920 6.910 6.900 900 1000 1100 time (seconds) 1200 1300 Graphs continued… Figure 4: IC50 Determination from Ki vs Concentration 1 0.9 0.8 0.7 Ki 0.6 y = -0.2052Ln(x) + 1.3866 R2 = 0.9899 Ki 0.5 Log. (Ki) 0.4 0.3 0.2 0.1 0 0 50 100 150 200 250 concentrations (uM) 300 350 400 450 500 Data Analysis Concentration (µM) NHE1i Slope Control Slope NHE1i slope: Determined from Figure 2 Ki 500 0.0005 0.0036 0.08 100 0.0015 0.0038 0.39 10 0.0028 0.003 0.93 y-intercept 1.3866 coefficient -0.2052 ln x (when y = 0.5) x (IC50) µM 4.320662768 75 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 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. 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 Acknowledgements Fredric A. Gorin, Dept. of Neurology. Michael Nantz, Dept. of Chemistry. Hasan Palandoken, Dept. of Chemistry. Bill Harley, Dept. of Neurology.