Cell imaging is an increasingly useful tool in predictive ADME/Tox strategies, according to Karen Tilmant, Ph.D., research scientist, investigative nonclinical safety, at UCB Pharma, who spoke at Mondial Research Group’s “Conference on Predictive Human Toxicity and ADME/Tox Studies” held recently in Brussels.
“One test that is gaining importance is the automated micronucleus test, based on cell imaging of fluorescent-labeled nuclei and micronuclei,” said Dr. Tilmant. The formation of micronuclei is a result of chromosome loss (aneugens) or detachment of a chromosome fragment (clastogens).
“The manually performed micronucleus test is validated and widely used, but it is low throughput. Also, counting can vary with the person who evaluates the microscope slides. The automated micronucleus test has advantages of being medium throughput, providing objective reading, and it can easily be applied in screening.”
The goal of predictive ADME/Tox, of course, is to reduce late-stage attrition. Identifying genotoxicity early is especially critical, noted Dr. Tilmant, “because it is often an insurmountable hurdle in drug development. Even a small mutation can lead to cancer.”
UCB’s in vitro toxicology group screens compounds with various automated imaging tests “to rank compounds from the same family and to allow the selection of the best compounds for further testing.” Improved cell imaging and high-content screening techniques received a fair amount of discussion at the conference, no doubt because of expanding applications. “Besides genotoxicity tests, cell imaging allows work on different other toxicity markers, through different immunomarkers and fluorescent dyes,” Dr. Tilmant said.
Brain Absorption Assay Advance
Estimating the unbound fraction of a compound in the brain is a key parameter when developing drugs for the central nervous system. Currently, dialysis systems and brain slice assays are the methods of choice. Hinnerk Boriss, Ph.D., CEO of Sovicell, described the company’s two-year-old Transil technology, which, he said, is faster and cheaper than other methods and has been shown to be as accurate as dialysis in work recently published by GlaxoSmithKline.
“The old way of looking at how difficult it is for a drug to pass the blood brain barrier (BBB) is actually the wrong question. Every drug gets into the brain, just to different extents. The right question is what happens when the drug actually gets into brain,” said Dr. Boriss. He noted that many drugs have drastically differing BBB penetration profiles—e.g., Sumatriptan (poor BBB penetrator) and Sertraline (high penetrator)—but are also effective in the brain.
Transil uses porous silica beads as a carrier for biological molecules such as membranes and proteins. Sovicell’s Transil Brain Absorption kit is made with reconstituted porcine brain lipid membranes immobilized on the beads.
After mixing and incubation for two minutes the beads are separated by low-speed centrifugation.
“If you do LC/MS, which is the standard method in this field, then you need longer gradients to interpolate the biological matrix from the compound of interest to quantify them. With our method there is no need to separate the biological matrix because that’s the beads. We just remove the beads and you can straight away analyze and quantify the samples. It’s three to four times faster for analyzing and quantifying the results.”
Dr. Boriss emphasized that lipid binding is a more important indicator than protein binding “because there are very few proteins that contribute to binding. It’s about 28 micromolar albumin that contributes to binding versus about 125 micromolar lipid and the affinity of drugs to lipids is three to four orders of magnitude higher on average—higher than binding to albumin.”
Sovicell offers assay chips and assay services on a small scale. Dr. Boriss believes the technology has the potential to enable companies to ramp up screening activities, reduce investments in instruments, or redirect other activities to instruments previously tied up with processing samples from dialysis.