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

Elizabeth Lipp

Evolving Technology and Regulations Rapidly Advance a Formerly Extraneous Tool

Preclinical in vitro testing for ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties has come a long way in the decade since it became an important consideration in the discovery process. But it still has a long way to go.

“Before the late 1980s to early 1990s, ADME testing was not a significant component of the drug discovery effort,” noted Robert J. Guttendorf, Ph.D., vp, pharmacology and experimental therapeutics, Sequoia Pharmaceuticals.

“As a result, poor ADME was a significant contributor to attrition of drug candidates. As higher-throughput methods allowed us to be more involved in the drug discovery process, by the 2000s we were able to optimize ADME properties while simultaneously optimizing intrinsic pharmacologic activity. We became an integral part of the discovery effort, enabling teams to solve two key problems in parallel.”

Dr. Guttendorf noted that, while eventually trimming attrition due to poor ADME from 40% to 10% is a great thing, “drug discovery is not working by the old blockbuster standard anymore. Overall attrition of drug candidates is still 90 percent, and the burden has merely been shifted from ADME to toxicology and pharmacology. But since pharmacokinetics is crucial to optimizing pharmacology and toxicology in vivo, we still have significant work to do.”

There is no doubt that there are still challenges. Hinnerk Boriss, Ph.D., CEO at Sovicell, observed that “the bottleneck is in the tox part of the field. It’s difficult to come up with meaningful tox assays, because they only run a short time, and PK typically acts over the long term. So it’s hard to tell long-term toxicity and whether it has any relativity to long-term, low-level toxicity.”

At the “Predictive Human Toxicity and ADME/Tox Studies” meeting, researchers had the opportunity to assess ADMET’s impact on the quality of leads that are nominated for clinical development, as well as see what new tools and methodologies are becoming available.

“ADMET is no longer a nice-to-do tool. It has become an integral part of drug discovery and development,” noted Katya Tsaioun, Ph.D., president, Apredica. “New ADMET tools are being developed and validated. These tools are more predictive of human mechanisms of toxicities and metabolism.  A lot of interesting work is being done in ADMET right now to allow organ-specific (particularly hepato- and cardio-) toxicity, and the mechanistic modes of toxicity to be predicted early in the lead-optimization process.”

A co-culture of primary rat hippocampal neurons and astrocytes created using a GE InCell 1000 High Content Imager.

ADME and Drug Discovery

While many bottlenecks have been dealt with in ADME over the years, Dr. Guttendorf said, there is still much work to be done. “Escalating research costs have been outpacing our ability to produce enough  new chemical entities (NCEs) to offset them, and both the quantity and quality of data required by regulatory agencies have been on the rise. When you look at the new drug application process, total drug approval rate has been declining. Think about what good compounds were rejected in the quest for the excellent or perfect compounds needed to offset expenditures and patent expiries. The blockbuster model is not working.

“It has been estimated that by 2012, big pharma companies will need to generate two to nine high-quality NCEs per year to sustain growth, and to do this under the current paradigm is impossible,” Dr. Guttendorf continued. “In specialty pharma, success rates look more like 18 percent of all candidates, versus 8 to 10 percent in big pharma. Big contributions at this point are being made by small- to mid-size pharma, because they are more efficient and more focused.”

Dr. Guttendorf added that the flexibility of small to mid-sized biotech, in terms of what targets they can pursue, has been a key to their success in advancing compounds.

But attrition persists. To address attrition owing to lack of efficacy, Dr. Guttendorf said, better predictability of human outcomes from preclinical models is needed, which will require an improved ability to extrapolate pharmacokinetics and pharmacodynamics, including an ability to account for human variability.

“For pharmacokinetics, in silico modeling and mathematical models have improved greatly over the last few years, but the bigger deficiency is the definitiveness of current in vitro models—you can only go so far with these models, so we’re left with in vivo preclinical models. For now there’s no substitute for the whole organism, but we cannot predict as well across species,” Dr. Guttendorf noted.

“Those modified mouse models with human metabolic characteristics are good for now, but we ultimately need to create in vitro models to supplant animal models altogether. We need to continue advancing in silico models. Overall, we have made tremendous strides.”

We are not quite there though, he cautioned. “For pharmacodynamics we still rely on preclinical in vivo models, which is fine for something like antibiotics; the target is the same whether it is in an animal or a human. But when you’re looking at oncology or neurology, there is a much lower success rate because of the models we have now. When we get to a point where we can routinely and accurately link preclinical and clinical in vivo performance—biomarkers will play an increasingly critical role in this—we can fail early and fast or, more optimistically, succeed early and fast.”

Human-Based In Vitro Systems

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.” 

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

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.

Blood-to-Brain Absorption

“Sovicell has e a chemical model based on natural lipids that gives reliable estimates of the freely available drug in brain,” noted Dr. Boriss. “In fact, you get both an estimate of the brain-to-plasma distribution and the fraction of drug that is not bound to brain tissue. Due to its low price point, it can be applied in early stages of research before committing to particular structure.”

Dr. Boriss presented one of Sovicell’s plasma protein binding models, the TRANSIL High Protein Binding assay, “which addresses difficult to handle compounds. This relates to compounds which bind to 99 percent or more, or which are excessively sticky,” Dr. Boriss explained. “With standard assays, you run into analytical issues since there is so little compound left to quantify. TRANSIL is an assay that not only works in high-throughput format, but is also convenient, as it works well with highly lipophilic compounds and minimizes any bias due to unspecific binding.”

Recent FDA Guidelines

“At Apredica, we often find that drug-discovery teams are confused about how and when to address preclinical ADME-Tox issues and about what the FDA guidance call for,” Dr. Tsaioun said. “I find that, in working with them, it’s important to review the clinical implications, starting with target product profile, to do what-if scenarios, to explain why the FDA would care, and to bring up the importance of following protocols that are developed following FDA guidelines.

“One of the most important things to understand about ADMET,” Dr. Tsaiou added, “is that one needs to start evaluating the drug-like properties of drug candidates early enough so that the discovery team has choices and is not committed to one molecule. Don’t get attached to a molecule. Fail fast and focus on the candidates most likely to succeed. This knowledge can save you time and resources.

“In the end, the more you know about your compound’s ADMET properties, the less complex or expensive your IND package is going to be.

“ADMET assays don’t kill compounds, decision-making groups do,” concluded Dr. Tsaioun.

“For those groups to make good decisions about compounds they need to closely collaborate and communicate. Biology must talk to chemistry and  discovery scientists must communicate with development people, otherwise, good compounds get terminated and bad compounds are advanced for reasons that are not always scientific.”

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