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May 1, 2009 (Vol. 29, No. 9)

ADMET Increasingly Integral to Discovery

Evolving Technology and Regulations Rapidly Advance a Formerly Extraneous Tool

  • Human-Based In Vitro Systems

    Click Image To Enlarge +
    IVAL’s IdMOC (integrated discrete multiple organ co-culture) system allows researchers to co-culture multiple cell types on one plate.

    A major concern of the pharmaceutical industry is that even after time-consuming and expensive preclinical trials in multiple species of animals, drugs still fail in clinical trials, mainly due to adverse drug toxicity, noted Albert P. Li, Ph.D., president and CEO, In Vitro ADMET Laboratories (IVAL), part of Advanced Pharmaceutical Sciences. “The take-home message is that human beings may respond to drug toxicity differently from nonhuman animals. For the accurate prediction of human drug toxicity, one needs human-specific information.

    “During preclinical trials, the human-specific information can only be obtained from human-based in vitro systems—experimental systems with human cells or tissue fractions. This approach has proven to be effective for the prediction of human drug metabolism.

    “Before 2000, a major cause of clinical trial failure was human pharmacokinetics (PK)—up to 30 percent. Now, due to the use of both human in vitro systems and in vivo animal models, human PK contributes to less than five percent of clinical trial failure. Prediction of human drug toxicity can benefit from the successful experience with drug metabolism and pharmacokinetics.”

    Dr. Li further added that one major adverse drug effect, drug-drug interactions, is now evaluated mainly with human-based in vitro assays. “FDA now requires results with human in vitro hepatic systems for the definition of pharmacokinetic drug-drug interactions. In vivo animal studies are not required for this purpose, mainly due to our clear understanding of the vast differences between animals and humans in drug-metabolizing enzymes,” said Dr. Li.

    Dr. Li and his team are working with human hepatocytes for the prediction of human hepatotoxicity. Human hepatocytes as an experimental system seems like an intuitive choice, because the species (human), metabolism (human), and cell type (human parenchymal cells are known targets for hepatotoxicants)—are relevant.

    “The main goal is to accurately predict human drug properties,” he continued. “Human cells, if used correctly, will provide the right information.”

    Dr. Li argued that, if the experimental system incorporates human-specific metabolism, human target cells, and mechanistically relevant endpoints, it should provide information useful for the accurate prediction of adverse drug effects in human.

    “These MTE (metabolism, target, endpoint) requirements are essential to the accurate prediction of human drug toxicity,” he added.

    To achieve the requirements of MTE, Dr. Li and his team work with primary cells derived from human tissues. “We’ve successfully cultured primary cells from other systems in the body, as well as hepatocytes,” Dr. Li noted. “With these cells, we can now evaluate toxicity in all major human organs, including heart, kidney, and bone marrow. While primary cultures from nonhepatic target organs retain organ-specific functions, they may not be used universally for the evaluation of drug toxicity because of the absence of hepatic drug metabolism.

    “And this touches on a major deficiency of in vitro experimental systems,” concluded Dr. Li. “Multiple organ interactions can be a key to drug toxicity, because a drug may be biotransformed by multiple organs, a drug and its metabolites may have multiple organ effects, or metabolites from one organ may have effects on other organs. Consequently, an ideal in vitro system for human toxicity evaluation will have human hepatic metabolism, human target organs, and multiple organ interactions.”

    IVAL’s IdMOC (integrated discrete multiple organ co-culture) system allows researchers to co-culture multiple cell types on one plate.

    “The IdMOC plate consists of multiple, inner wells within a larger interconnecting chamber,” explained Dr. Li. “Multiple cell types are individually cultured in the inner well, and the chamber is filled with a single, universal medium, allowing well-to-well communication. The overlying medium can be analyzed for test material metabolism, and individual cell types can be evaluated for possible organ-specific bioaccumulation, cytotoxicity, and efficacy.” 

  • Multiparameter Screening Assay

    Toxicity has become an increased area of interest and guidance by regulating bodies, particularly the EMEA and FDA. To that end, says David Hayes, Ph.D., director R&D, Millipore, we’ve been increasing our focus on creating platforms and panels with this in mind, focusing on toxicity-related endpoints for renal, hepato, cardio, or neuro systems by creating a mix of serum and cellular assays to be developed.

    “Virtually all toxic responses are preceded by the transcriptional activation of stress-response pathways. The promoters of genes activated in this manner, therefore, have the potential to be used to provide markers of toxicity. So the program was designed to make cell lines where a series of promoters were used to drive a common soluble reporter protein each tagged with unique epitope. The ELISA assay can then read the common reporter protein, independent of the specific tag.”

    Dr. Hayes also mentioned that Millipore, in collaboration with CXR BioSciences, had just released ToxReporter™, a new panel of engineered cell lines and assays that can screen compounds for toxicity early in the drug discovery process. The ToxReporter panel detects a variety of cellular responses linked to toxic pathways such as oxidative stress, inflammatory response, cell-cycle control, DNA damage, and apoptosis.

    “Toxicity induces certain pathways that produce an adaptive response. Once it’s switched on we get a reporter analysis. We don’t have to destroy a cell to get the report,” added Dr. Hayes. “Five lines have already been launched, and we have four lines that will launch in May.”

    The ability of high-content analysis to harness the image of cells under the microscope with powerful analytical algorithms is being exploited to develop kits that provide information on hepatotoxicity and neurotoxicity. A dual culture system of neurons and astrocytes cells allowed kits to be developed that demonstrated the neuro-protective properties of glial (astrocytes) cells, reflecting a more in vivo like situation.

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