A drug’s efficacy and safety determine whether it will be approved and ultimately prescribed. To characterize a drug’s impact, researchers must study how it is distributed and circulates in the body; this makes up the basis of pharmacokinetics, often known simply as PK. A drug’s PK consists primarily of four mechanisms: absorption, distribution, metabolism and excretion (ADME). Overall, a drug’s ADME indicates how much someone will be exposed to the drug and where, which affect its pharmacological impact. A drug’s toxicity (Tox) is often explored along with ADME, hence the field of ADME/Tox.
When asked about the top technical challenges in ADME/Tox, Maureen Bunger, technical director at Triangle Research Labs (part of Lonza), says, “One of the key challenges for ADME/Tox is finding meaningful models and appropriate measures that capture human-relevant pharmacokinetics and pharmacodynamics,” which is what a drug does to the body. “In order to understand these parameters in humans before clinical trials, human models should ideally be used in preclinical and nonclinical testing.”
Changes in today’s medicines can make ADME/Tox even more complex.
“Many new drugs in development are designed to be more slowly metabolized and are being identified in more human-relevant models during screening processes,” Bunger explains. “Standard animal models that are used in preclinical work for demonstrating pharmacokinetics and toxicity potential of new drugs often fall short in predicting clinical outcomes for these new characteristic drugs.”
That opens the door for advances in ADME/Tox technology.
A new dimension
“There’s a growing desire across the global scientific community to work with cells cultured in three dimensions,” says Brad Larson, principal scientist at BioTek Instruments Applications Lab. “Toxicology and metabolism cell models can behave differently in 2D vs. 3D cultures.”
With spherical collections of cells—such as liver cells developed as microtissues for ADME/Tox studies—scientists face some tricky challenges. “In many spheroid models, the cells aren’t attached to a surface,” says Larson. “So when aspirating media, it’s very easy to inadvertently evacuate the spheroids along with the spent media.” Therefore, automating 3D cell cultures, especially those using nonscaffold methods, requires advanced liquid-handling instrumentation. “BioTek’s liquid-handling instrumentation is optimized with unique methods to remove media and wash cells and spheroids,” Larson explains. “These methods are less disruptive to the spheroid and enable automated liquid handling without risk of losing the spheroid.”
A second challenge when working with 3D-cultured cells is image capture and analysis. A 2D monolayer puts all the cells in the same focal plane, which simplifies imaging. “Conversely, 3D structures are typically thicker than the depth-of-field capability of the microscope objective used,” Larson says. Imaging software can incorporate Z-stacking of the spheroid images to reduce out-of-focus portions of 3D cell structures and improve image quality. BioTek’s Lionheart FX Automated Live Cell Imager and Cytation Cell Imaging Multi-Mode Reader are both well suited for 2D and 3D cell-culture applications, including advanced imaging and analysis. Additionally, the Cytation can simultaneously read and image cells, enabling unique and time-saving workflows.
Primary potential
To give researchers more tools, Lonza offers high-quality human primary hepatocytes. “The challenge of using primary hepatocytes is that once separated from the tissue, the cells undergo a rapid de-differentiation and lose much of their metabolic activity,” Bunger points out. “This presents a challenge when researchers need to model more slowly metabolizing compounds.”
Consequently, Lonza added two technologies to keep human hepatocytes metabolizing longer. “Quasi Vivo System is a mesoscale, fluidic cell-culture system that enables the culture of metabolically active primary hepatocytes for more than 28 days,” Bunger says. “Cells can be cultured in standard 24-well formats on coverslips then transferred into the Quasi Vivo System for extended periods of culture time.” The latter feature means that existing protocols for hepatocytes are easy to adapt to the Quasi Vivo System.
This technology will provide even more options ahead. “Lonza is also in the process of establishing protocols for combining the Quasi Vivo System with our existing RAFT 3D Cell Culture System,” Bunger explains. “The RAFT 3D Cell Culture System provides a way to make collagen-rich 3D tissue structures from single or multiple cell types.” She adds that the two technologies combined can “support cultures of multiple cell types in a tissue-like environment.”
In our bodies, drugs are foreign substances—xenobiotics—that can be altered, or biotransformed.
“One of the key challenges of ADME/Tox and drug development is to better predict xenobiotic biotransformation in the organism,” says Ramon Lavado, assistant professor of environmental science at Baylor University. “Biotransformation is a broad and growing field of toxicology.”
Lavado and his colleagues study ADME/Tox using cellular- and molecular-based technologies, plus some analytical chemistry techniques. These include chromatography, gene expression, and metabolic assays. “Progress has been made in research on the key drivers of biotransformations, including the isolation, screening and characterization of enzymes, their utilization, the manipulation of alteration and augmentation of metabolic pathways,” Lavado says, “but nowadays we only know about 30% of the total biotransformation pathways occurring in mammal tissues.”
In the future, Lavado would like to see some improvements in technology. In toxicology, he says, “we’re limited by the analytical measurements, so faster and more sensitive methods would be necessary to advance.” He adds, “Also, technology has to advance in the field of alternatives to animal uses.”
As in many other areas of R&D, computation could change the way scientists study ADME/Tox.
In fact, it already does in some cases. A team of Romanian scientists, for example, used an in silico study of ADME in treatments for fungal infections caused by Candida [1]. They used the computational approach to predict pharmacokinetic and pharmacotoxicological properties of newly synthesized compounds.
This is not a completely new approach. In 2000, scientists from Trega Biosciences reported that a “computer simulation model has been developed and validated to predict ADME outcomes, such as rate of absorption, extent of absorption, etc. using a limited number of in vitro data inputs [2].” Ahead, even more computational methods could be used in ADME/Tox studies.
Creating new tools and techniques that help scientists study ADME/Tox could make significant differences in the new medicines that can be developed, as well as reducing the costs. These advances can also reduce the odds of an unsafe medicine getting to market or farther along in development than it should. Ultimately, efficient and effective methods of assessing ADME/Tox will make tomorrow’s medicines safer and more powerful than ever.
References
[1] Stana, A, et al., “New Thiazolyl-triazole Schiff Bases: Synthesis and Evaluation of the Anti-Candida Potential,” Molecules, 21:1595-1613, 2016. [PMID: 27879678]
[2] Norris, DA, et al., “Development of predictive pharmacokinetic simulation models for drug discovery,” J Control Release, 65:55-62, 2000. [PMID: 10699270]
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