High grade malignant gliomas are the most frequent type of lethal adult brain tumor, and there is no current effective treatment. Amiloride is an FDA-approved diuretic that inhibits the proliferation of malignant glioma cells and, at high concentrations, selectively kills human glioma cells. Intracranial infusion of amiloride significantly decreased the rate of tumor growth of intracerebral human glioma xenografts in athymic rats, and killed glioma cells in poorly vascularized tumor regions. A primary objective of this application is to identify the cellular mechanisms by which amiloride selectively kills malignant glioma cells. Recently published data indicate that glioma cytotoxicity could arise from amiloride's dual inhibition of the sodium calcium exchanger (NCX) and of the type 1 sodium proton exchanger (NHE1). Nonspecific cellular toxicities of the more potent, lipophilic amiloride derivatives correspond with their intracellular permeation. We hypothesize that conjugating amino acids or peptides to the C (5) position and to the C(2) guanidine moiety of amiloride can generate novel hydrophilic amiloride derivatives, and limit drug activities to cell surface transporters. C (2) amiloride glycine conjugate inhibits NCX>NHE1, and is at least 50-fold more potent than amiloride in selectively killing glioma cells. C (5) amiloride glycine conjugate inhibits NHE1""""""""NCX, and when coupled to an opioid-like pentapeptide created an inactive prodrug. This prodrug liberates bioactive C (5)-Am-Gly when incubated with enkephalinase. In a similar fashion, we envision that glioma-specific peptidases, such as metalloproteinases, could regionally activate amiloride-peptide prodrugs. The investigator proposes to further analyze the cellular mechanisms by which inhibitors of NCX and NHE1 selectively kill glioma cells. Syntheses of novel amiloride amino acid and peptide conjugates will be guided by these mechanistic studies, by their inhibitory activities on NCX and NHE1, and by screening their anti-cancer properties in a panel of human glioma cell lines and primary astrocytes. The most selective and efficacious of these novel amiloride derivatives will be infused intracranial into human glioma xenografts implanted intracerebrally into athymic rats. The pharmacokinetics, neurotoxicities, and neuropathology of these compounds also will be evaluated.
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