title: Molecular imaging of intracellular drug-membrane aggregate formation.
abstract: Clofazimine is a lipophilic antibiotic with an extremely long pharmacokinetic half-life associated with the appearance of crystal-like drug inclusions, in vivo. Here, we studied how clofazimine accumulates inside cells in the presence of supersaturating, extracellular concentrations of the drug (in the range of physiological drug concentrations). Based on a combination of molecular imaging, biochemical analysis and electron microscopy techniques, clofazimine mass increased inside cells in vitro, over a period of several days, with discrete clofazimine inclusions forming in the cytoplasm. These inclusions grew in size, number and density, as long as the drug-containing medium was replenished. With Raman confocal microscopy, clofazimine's spectral signature in these inclusions resembled that of amorphous clofazimine precipitates and was unlike that of clofazimine crystals. Additional experiments revealed that clofazimine first accumulated in mitochondria, with ensuing changes in mitochondrial structure and function. In turn, the degenerating organelles coalesced, fused with each other and condensed to form prominent drug-membrane aggregates (dubbed autophagosome-like drug inclusions or "aldis"). Like clofazimine, it is possible that intracellular drug-membrane aggregate formation is a common phenomenon underlying the reported phenotypic effects of many other small molecule drugs.
authors.affiliation: Department of Pharmaceutical Sciences, University of Michigan College of Pharmacy, 428 Church Street, Ann Arbor, Michigan 48109, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Baik
authors.forename: Gus R
authors.initials: GR
authors.lastname: Rosania
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D019069:Cell Respiration / Q000187:drug effects; D055598:Chemical Phenomena; D002991:Clofazimine / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects*; D007700:Kinetics; D050356:Lipid Metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D057054:Molecular Imaging; D010588:Phagosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013059:Spectrum Analysis, Raman
chemical_list: D000900:Anti-Bacterial Agents; D002991:Clofazimine