University of Michigan College of Pharmacy Group

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SUBCELLULAR DRUG TRANSPORT

Cells on Pores. Left: Confocal micrograph of MDCK epithelial cell sheet differentiated on nanoengineered membranes with 400 nanometer pores. Red and Green crosshairs show horizontal (top) and vertical (right) cross-section through the cell sheet. Right: Transmitted light micrograph of nanoengineered membranes with randomly arranged pores of 1 (top), 3(middle), and 8 (bottom) micron diameters.

One of our objectives is to map out drug transport pathways that may be important for the efficacy of chemotherapeutic agents. For this purpose, we are developing biochemical fractionation and analysis techniques to measure the mass of drug in different intracellular compartments through time, after cells are exposed to a drug pulse. Currently, we are studying the transport pathways of small molecules in epithelial cells differentiated on scaffolds with nanoengineered pores of varying size, density and spatial patterns(see photo above). Do differentiated epithelial cells know when they are sitting on a pore? How does pore size, density, and distribution affect epithelial cell structure and function? These questions are fundamental to our understanding of the transport function of epithelial cells, and answering them will provide important information for building computational models of cellular function.

Relevant references:

Chen, VY, Posada, MM, Zhao, L, and Rosania GR (2007). Rapid doxorubicin efflux from the nucleus of drug resistant cancer cells following extracellular drug clearance. Pharmaceutical Research Nov;24(11):2156-67.[PubMed]

Chen VY, Posada MM,, Blazer LL, Zhao T, and Rosania GR (2006) The role of the Vps4a-exosome pathway in the intrinsic egress route of DNA-binding anticancer drug. Pharmaceutical Research. 23(8): 1687-1695. [PubMed]

Shedden K, Xie XT, Chandaroy P, Chang YT, Rosania GR. (2003) Expulsion of small molecules in vesicles shed by cancer cells: association with gene expression and chemosensitivity profiles. Cancer Research. 2003 Aug 1;63(15):4331-7 [PubMed]

CHEMICAL ADDRESS TAGS

Yellow Swollen Lysosomes. MDCK cells were labeled with three fluorescent dyes staining DNA (red), mitochondria (blue) and lysosomes (yellow), and then incubated with a lysosomotropic small molecule drug (chloroquine). The drug concentrates in lysosomes, which become more numerous and swollen, while mitochondria and nuclei remain the same number and size.

Supertargeted chemistry refers to the study of the relationship between the intracellular transport properties of small molecules and their chemical structures. We would like to study how CHEMICAL ADDRESS TAGS -localization signals encoded in the chemical structure of small molecules- can be used to control the biodistribution of drugs at a microscopic level. In order to understand subcellular transport behavior, we have pioneered the development of combinatorial libraries of fluorescent probes specifically for the purpose of studying subcellular transport phenomena. To analyze the cellular pharmacokinetics of large number of probes, we are at the forefront of using kinetics high content screening instruments to capture the system dynamics of subcellular transport. To relate the chemical structure of small molecules to the observed distribution and transport behaviors, we have pioneered quantitative, structure-localization relationship studies (QSLR) of intracellular small molecule distribution. In addition, for linking large amounts of image data and relating image data to chemical structure, we are pioneering the development of a new image visualization platform dubbed Cheminformatic Assisted Image Array (CAIA).

Relevant references:

Shedden K, Li Q, Liu F, Chang YT and Rosania GR (2009) Machine vision assisted analysis of structure-localization relationships in a combinatorial library of prospective bioimaging probes. Cytometry A (in press)

Maria M Posada, Kerby Shedden, Young Tae Chang, Qian Li and Gus R. Rosania (2007). Prospecting for live cell bioimaging probes with cheminformatic assisted image arrays (CAIA). Proceedings of the 4th IEEE International Symposium on Biomedical Imaging: from Nano to Macro. Pp. 1108-1111. [article]

Shedden K, Brumer J, Chang YT and Rosania GR (2003) Chemoinformatic analysis of a supertargeted library of styryl molecules. J. Chem. Info and Comp. Sci. 43(6):2068-80. [PubMed]

Rosania, GR, Ding, L, Yoon, H-S, and Chang, YT (2003) Combinatorial approach to organelle-targeted fluorescent library based on the styryl scaffold. J Am Chem Soc. Feb 5;125(5):1130-1 [PubMed]

CHEMINFORMATICS, MACHINE VISION, MATHEMATICAL MODELING.

The Virtual Intestine. With a cell based molecular transport simulator, the interaction of small molecules with the three dimensional structure of the intestine can be modeled on a computer, allowing analysis and prediction of the fraction of a drug dose that is absorbed. Because most medications are administered orally, intestinal epithelial cells are the most important gateway for drugs entering the body.

In order to be able to simulate the intracellular distribution and transport of small molecules, we developed a mathematical model of epithelial cells, that captures the changes in mass of drug in various organelles, when a cell monolayer is exposed to a transcellular apical-to-basolateral drug concentration gradient. This is the condition that is most physiologically relevant to systemic drug absorption, distribution and clearance (in the case of oral drugs, for example, the transcellular concentration gradient provides the driving force for intestinal drug absorption into the body). Also, we are at the forefront of visualization of cellular pharmacokinetic simulation results. In addition, cell based molecular transport simulators are being developed to facilitate cheminformatic analysis of the chemical diversity of large collections of molecules being assembled as probes for chemical biology, part of the large scale Molecular Libraries Screening Center Network established by the NIH.

Relevant references:

Trapp, S; Rosania GR, Horobin R. and Kornhuber J.. (2008) Quantitative modeling of selective lysosomal. targeting by passive diffusion of xenobiotic compounds: model development and simulations for single cells. European Biophysical Journal 37(8):1317-28.

Zhang X., Zheng N and Rosania GR (2008) Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines. Journal of Computer Aided Molecular Design Sep;22(9):629-45.

Xinyuan Zhang, Kerby Shedden, Gus Rosania. (2006). A cell-based molecular transport simulator for pharmacokinetic prediction and cheminformatic exploration. Molecular Pharmaceutics; 3(6) pp 704 - 716. [PubMed]

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