The development of preclinical strategies for facilitation of lead candidate selection

Chris Kulczar, Purdue University


Chapter 1 details a background of techniques used for modeling the blood-brain barrier (BBB). The BBB represents a diffusive barrier to both paracellular and transcellular movement of many compounds in and out of the brain. The main rate- limiting barriers of the BBB include exclusive tight junctions that prevent the movement of hydrophilic molecules through intercellular gaps, and efflux proteins in the membrane which pump many hydrophobic molecules back into the blood. In addition, the BBB contains metabolizing enzymes, including Cytochrome P450s. This barrier acts to protect the vulnerable tissues of the brain from harmful xenobiotics, but also can serve as a restrictive barrier for potential therapeutic compounds for the growing number of neurological diseases, including Alzheimer’s disease, Parkinson’s disease, stroke, depression, brain cancers, and many others. A number of in vitro cell screens have been established to mimic the BBB for permeation testing. Principally, the best model should include primary human brain microvessel endothelial cells (BMECs), however, due to the growing interest in BBB permeation and lack of tissues, the supply is limited. As an alternative method, many groups have investigated the use of primary animal BMECs, usually of murine, porcine, or bovine source. These models prove effective at restricting paracellular movement; however, one must question the effects of animal isoforms on modeling uptake, efflux, and metabolism of transcellular markers. This proves important as in vivo most, if not all, therapeutic compounds will cross the BBB transellularly. Consequently, much work has been done to establish immortalized human BMECs. These immortalized cell lines alleviate the high cost and lack of supply found with primary human cells, but potentially may serve as a better model for transcellular permeation over animal cell lines. Currently, the most widely characterized immortalized human BMEC cell line is the human cerebral microvessel endothelial cell line (hCMEC/D3). These cells express tight junction proteins, efflux proteins, cyp450 enzymes, and are conducive to in vitro testing. However, while these cells express tight junctions, their function is less than ideal and leads to a leaky monolayer which may allow faster permeation through the paracellular route or paracellular permeation of compounds that move transcellularly in vivo leading to poor prediction of BBB permeability. One method of investigating the reason behind these leaky tight junctions was to take a closer look at the BBB itself. Chapters 2 and 3 discuss the optimization and establishment of a direct contact coculture in vitro model in which both endothelial cells and astrocytes are plated on the apical side of the Transwell® allowing for more physiologically relevant signaling between the cells. Early results suggest decreased paracellular permeation in this configuration compared to endothelial monocultures and indirect cocultures. In addition, this setup should allow for easier transition to high-throughput equipment. Additional studies in Chapter 4 attempt to show the utility of a new mono-PEGylated Human Serum Albumin (HSA) as a potential enhancer of drug solubilization, permeation, and eventual cytotoxicty. Multi-gram batches of short (5 kDa) and long (20 kDa) PEG-HSA were synthesized with high efficiency (77%) and characterized in collaboration with Dr. Jonathan Mehtala and Dr. Alex Wei in the Department of Chemistry. Furthermore, effects of PEG-HSA on permeation of paclitaxel through peripheral and BBB in vitro cell models as well as changes in cytotoxicity against MCF-7 cells was investigated. Finally, Chapter 5 includes an investigation into the characterization, formulation, and in vitro/in vivo testing of a potent V-ATPase inhibitor known as Saliphenylhalamide. The V-ATPase is an endogenous protein that is responsible for acidifying intercellular compartments and has been targeted in the past with in vitro success for treatment of cancer and osteoporosis among other diseases. However, here we investigate its use as an anti-viral therapeutic as acidification of endosomes by the V-ATPase is thought to be a critical step in replication of alpha viruses. Initial characterization showed poor water solubility and acid liability. Therefore, two solubility enabling formulations were created and tested for in vitro permeability and in vivo murine pharmacokinetics. (Abstract shortened by ProQuest.)




Knipp, Purdue University.

Subject Area

Pharmacy sciences

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