FDA and drug companies alike want ADME-tox testing performed earlier and earlier in a drug’s life cycle.

If the ADME-tox status of your new, hoped-to-be blockbuster is not yet certain, the FDA does not want to hear "What doesn't kill you makes you stronger." Rather, some in vitro data would be nice. So would in vivo validation. And, whatever computer modeling—so-called in silico predictions—the lab can muster to support ongoing development, human testing, and eventual approval would be appropriate.

Fortunately, the tools that provide this information are evolving. At the same time the need-to-know emphasis has shifted from a compound's ADME (absorption, distribution, metabolism, and excretion) to its potential toxicity.

"A number of years ago, quite a few compounds failed in the clinic," says Han van de Waterbeemd, PhD, global project leader for C-Lab, AstraZeneca, Basingstoke,

click to enlarge  1 dpf zebrafish were treated with 0.1% DMSO (Control) or 25 µM Celebrex (Celebrex) for 48 hours. Abnormal pericardial edema and hemorrhage/thrombosis can be observed in Celebrex-treated live zebrafish.

UK. "And we started analyzing why that was, and ADME was one of the major reasons that came out." Subsequently, more in vitro assays were designed, and then implemented much earlier. The strategy was successful to the point that now clinical failures are more often traced to toxicity. "Today, people are measuring tox earlier and earlier, performing these assays in drug discovery instead of drug development where they were done in the past."

The repositioning in surveillance has sparked a business boom in early toxicity detection. A report published in mid-2007 by Kalorama Information, New York, estimates that the market for early tox will grow from the present $947 million to $1.52 billion by 2010. Part of this cost is driven by consultancies, but most is comprised of advances in, and earlier, and more extensive use of in vitro, in vivo, and in silico applications. The report cites innovation in all three areas, but senior analyst, Jack Gardner, comments via email that, "No one thing will answer all that early tox requires." Many techniques are promising, "but they are part of the whole process, and none is the magic bullet," Gardner says.

Yet, there is magic in the making. The "C" in van de Waterbeemd's C-lab stands for "computational." It's his alchemist's job to turn bins of base-data into health-affirming, in silico, predictive gold. "Tox is more complex [than ADME] because there are many reasons why something can be wrong with a compound," he says. "It often depends on the dose you give, the food you eat, your age—a lot of factors." So, whereas the software to build a model is largely available in the marketplace, the raw material—the often company-centric experience—the experimental data used for the training set is absolutely key.

click to enlarge  . The heart (purple), liver (red) and intestine (green). At high concentrations, Compound-3 (1 mM) caused malformation of the heart, liver and intestine (B); Compound-3-treated embryos showed heart edema, underdeveloped liver and intestine. Compound-4 (1 mM) caused enlarged intestine (C). Compound-5 (100 μM) caused defective heart and liver development (smaller liver) (D). Compound-6 (100 μM [E]), Compound-7 (0.01 μM [F]), Compound-8 (100 μM [G]), BMS-A (0.01 μM [H]), and BMS-B (0.01 μM [I]) caused abnormal heart, liver and intestine morphology. BMS-C (1 μM) caused abnormal heart and liver morphology (J). In summary, smaller heart and an enlarged pericardial compartment were observed for Compounds-3, 5, 6, 7, 8, BMS-A, B, and C. Small liver was observed for Compounds-3 and 5. No visible liver structure was observed for Compounds-6, 7, BMS-A and B. Intestine with defective lumen morphology was observed for Compound-3. Enlarged intestine was observed for Compound-4. No visible intestine structure was observed for Compounds-6, 7, BMS-A, BMS-B.

Depending on the nature of the query, one might be able to glean enough data from the literature, but even when combined with proprietary data, there are blind spots. "Particularly in the tox area there's an intense interest from regulatory bodies and others to get pharma to join up their databases on toxicity, van de Waterbeemd observes. "So, there's a strong argument to say ‘yeah, let's share,' but for economic reasons, that still hasn't happened yet." But it will, because the payoff is too sweet.

 Not as slippery when wet
As virtual reality comes of age, in vitro reality gains in sophistication. Driven by circumstance, cell-based assays are increasingly tailored to specific needs. "One of our specialties is in permeability, particularly using MDR1-MDCK cells," says Philip Burton, CEO and cofounder of ADMETRx, Kalamazoo, Mich. And it's a good thing—permeability assays have exploded in demand. "I expected this for some time," says Burton, "There was an FDA draft guidance in September 2006 in which they drew a line in the sand [stating that] there's now enough clinical evidence to show that drug transporters can make significant contributions to clinically relevant drug-drug interactions. So, an NDA [New Drug Application] now needs to contain an evaluation on this."

The guidance also delineates how this is to be done, proposing a series of tests to be performed, which, as it is now worded is to include in vitro permeability studies in the MDCK cell model. For the moment, Burton sees this directive as being primarily concerned with P-glycoprotein, an extensively expressed ATP-binding cassette (ABC) transporter, but he predicts the scope will broaden to include others from the ABC transporter family. "Since that guidance was issued, we've just been flooded with requests to do transporter characterization, and this is just the beginning."

The third prong of the advancing ADME/tox attack is the evaluation of drug candidates in vivo, and the wet science here is getting very wet, as in with zebrafish. Long used as a model for certain diseases, big pharma has adopted these little creatures to assay preclinical toxicity. Patricia McGrath, president and CEO of Phylonix, Cambridge, Mass., is not surprised. "The data are compelling—highly predictive of what effects will be seen in mammalian models." And zebrafish have certain advantages over their lab-mates. Because of their diminutive size, smaller amounts of compound are utilized as compared to work with mice. "If your drug is in short supply, as it usually is in preclinical stages, then you can still get good results with a small amount of compound. You can also use a lot of animals, so your statistical confidence increases."

Zebrafish are particularly suited for the subset issue of developmental toxicity; generation time is rapid, and the transparent embryos are easily observed, growing as they do, outside the mother. This ability was exploited recently by Bristol-Myers Squibb, which hired Phylonix to assay 12 compounds for developmental toxicity. "Only one out of the 12 didn't show up with the same profile as was in the mammalian database," says McGrath. "It was a very good correlation."

About the Author
Neil Canavan is a freelance journalist of science and medicine based in New York.

This article was published in Drug Discovery & Development magazine: Vol. 10, No. 10, October, 2007, pp. 34-36.