Novel Therapy for Treating Human Glioblastomas

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

• Normal cells survive at a pH7.4
• Glioblastomas can survive at pH6.9
• 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.

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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.

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Why new therapy is required?

• Poor clinical prognosis
• High recurrence rate
• Patients typically live only 6-12 months
  following diagnosis regardless of therapeutic
  regimen

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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

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Testing efficacy of the drug

• Inhibition constant
• IC50concentration 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.

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Methodology

• Determination of NHE1 IC50
• Cells grown in Petri dishes
• 37 degrees Celsius
• pH7.4
• Cells are subjected to the drug in vitro
• changes in pH are measured spectrofluorometrically
  using a fluorescent dye, 2,7bis(carboxyethyl)-5
  ,6-carboxyfluorescin acetoxy-methyl ester (BCECF)
  
• Excitation wavelengths 507/440nm
• Emission wavelength 535 nm

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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.

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Graph produced by Data
Figure 1
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Graphs continued
Figure 2
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Graphs continued
Figure 3
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Graphs continued
Figure 4
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Data Analysis
Concentration (µM) NHE1i Slope Control Slope 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) 4.320662768
x (IC50) µM 75
NHE1i slope Determined from Figure 2 Control
Slope Determined from Figure 1 KiNHE1i
slope/ Control slope Y-intercept and
Coefficient Determined from Figure 4 IC50
Determined From equation Given by figure 4
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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.

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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

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Acknowledgements

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