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theses

Amphotericin B methyl ester (AME) similar to Amphotericin B (AMB), is a macrolide antibiotic highly effective against complex systemic fungal infections. Clinical data suggested AME was neurotoxic, but subsequent analysis of lots used in its only clinical-trial revealed contamination with AMB and multi-methylated AMB-derivatives. Accruing evidence suggests AMB not highly-purified AME causes neurotoxicity, thus, it is likely that AMB contributed to neurotoxicity observed during the AME trials. Preliminary assessment of adult Balb/c mice given alternate day intraperitoneal (i.p) AME to mimic the human dose used (5 mg/kg of body weight [b.w]) compared with similarly treated AMB mice was carried out after 1-month and 2-months of injections. Light microscopy of cerebral white matter stained with hematoxylin/eosin and luxol fast blue suggested AME caused less demyelination than AMB. AMB induced CNS demyelination persisted after 3-months of drug-cessation following 2-months of injections. The nuclear area factor (NAF) analysis of AMB-brains provides preliminary data that AMB may cause apoptotic change in cerebral white matter of mice. Myelin lesions were absent in AME-brains, and even after 5-months of day i.p treatment of adult mice with 7-mg/kg b.w of AME alone, ultrastructural myelin changes were minimal. Differentiated human oligodendrocyte cell line (MO3.13) exposed to 0.5-30µg/ml AMB or AME for 24 hours demonstrated a progressive increase in cytotoxicity as determined by dye exclusion. The lowest concentration of AMB to induce cell injury was 1µg/ml, whereas injury first appeared in AME-exposed cells at10 µg/ml, a concentration far exceeding those expected in clinical use. AMB at1µg/ml showed apoptotic nuclear change in cells colocalizing for propidium iodide and fluorescein diacetate. Confirmation with Hoechst and fluorescent inhibition of caspases labeling revealed a significant decrease in the NAF and an increase in caspase-3 and -9 activity, suggesting induction of the intrinsic mitochondrial apoptotic pathway. AMB also caused increased cytochrome c release with a significant loss of mitochondrial membrane potential (ΔΨΜ) that was refractory to rescue with Trolox as determined with JC-1 dye. Cytotoxicity was not observed in similarly treated AME-cells. Our data suggests that AMB, but not AME may induce oligodendrocytes cytotoxicity and mitochondrial damage, eventually diminishing ΔΨΜ with consequent apoptosis.

ETD_5992

Ph.D.

Includes bibliographical references

by Oluchukwu U. Nnodi

doi:10.7282/T3N015SR

ETD doctoral

The author owns the copyright to this work.