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.