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Jun 15, 2008 (Vol. 28, No. 12)

Tackling Drug-Interaction Issues Early On

Researchers Explore a Number of Strategies to Better Predict Drug Responses in the Clinic

  • Troubleshooting

    One can’t have a discussion on drug-drug interactions without addressing cytochrome P450 (CYP). The metabolism and clearance of most drugs are primarily mediated by CYP. Adverse drug-drug interactions frequently occur because of altered P450 activities, explains Jim Cali, Ph.D., senior scientist at Promega (www.promega.com).

    “For example, nuclear receptor-mediated induction of a P450 gene by a component of St. John’s Wort reduces efficacy of oral contraceptives by increasing their rate of metabolism.”

    One of Promega’s areas of strength, he states, is in bioluminescent luciferase reporter gene assays, which are ideal for studying gene regulation by nuclear receptors. According to Dr. Cali, Promega has also developed luminogenic P450 enzyme assays that use luciferin derivatives as P450 substrates. By combining reporter gene assays with enzyme assays, one can predict P450 induction at both the transcriptional level and at the level of enzyme activity during the earliest stages of drug discovery.

    “We have developed three assays for the CYP3A4 enzyme, which accounts for about 30% of the P450 in human liver and oxidizes about half of all drugs that are eliminated after prior metabolism,” explains Dr. Cali. “Luciferin-PFBE is ideal for cell-based measurement of CYP3A gene inductions by drugs. The cell-based assay is rapid and simple and can be performed in a nonlytic mode that leaves cells intact for additional analysis.”

    In addition to its utility for measuring induction in cell-based assays each substrate can be used as a probe for in vitro biochemical assays of recombinant CYP3A4 to detect P450 inhibition by test compounds. Dr. Cali points out that running a reporter assay is only doing part of the job. “We get a more complete picture by pairing reporter assays with enzyme assays.”

    The value of this approach is illustrated by considering the effects of bergamottin, a component of grapefruit juice, on CYP3A4 expression. Although bergamottin activates CYP3A4 gene transcription, its net effect is to reduce activity, because it is also an inhibitor of the CYP3A4 enzyme. This explains how grapefruit juice consumption slows clearance of CYP3A4-metabolized drugs such as the cholesterol-lowering statins.

    “Reporter gene technology paired with P450 enzyme assays is a good way to get a full picture of how certain compounds impact drug disposition through nuclear receptor interactions,” notes Dr. Cali.

  • Regulatory Guidelines

    It’s a matter of public record that regulatory guidelines focus on the use of human hepatocytes as the gold standard of testing. Some comments appear on the FDA site saying that this needs to change and that the focus should not be solely on human hepatocytes, given the variability of reproducibility from donor to donor.

    Dr. Cheng agrees. “We use nuclear receptors as the first line—they definitely have a lot of merit in drug discovery. Those hepatocytes derived from human donors serve as our second-tier assays, only because there is so much variability in the cell lines from donor to donor.”

    The FDA acknowledges the evolution of reporter technology, explains Dr. Murray. “They will allow the use of data from nuclear receptors, certainly, but they will also want more information, which will require using human hepatocytes to follow up.” He notes that in dealing with some therapeutic areas, drug-drug interactions may not be quite as big an issue. However, in dealing in his field of antiviral discovery for HIV, “you could get into trouble pretty quickly unless you keep your eyes open.”

    Dr. Murray is working with human PXR, CAR AhR and also rat and monkey PXR and also “keeps an eye on what vendors have available. Dog PXR, for example, would be something we’d use, as well. When picking final candidates to nominate for development, we are often screening multiple compounds against every receptor that is available.”

    As for overall reproducibility, Dr. Tang emphasizes that the FDA guideline is the standard to follow. “So far, based on our results, we have not seen any significant outliers when comparing human hepatocytes to the Puracyp cell lines. But whoever implements the results of these finds has to understand the potential drawbacks.”

  • Looking into the Future

    Dr. Murray notes that some of the receptors already have well-characterized systems in place—human PXR and AhR in particular. Assays for CAR activation, he notes, are somewhat less mature, “but when assays for all three of these important receptors are in place, we’ll feel more comfortable. The more information we have, the lower the risk when moving forward. I do see receptor-based technology as being strongly predictive of the potential for clinical drug-drug interactions.”

Readers' Comments

Posted 07/25/2008 by The pre-competetive period

The collaboration of Eli Lilly, Pfizer, and Merck within Enlight Biosciences to solve common drug discovery problems early in the so-called "pre-competetive phase" is an important part of what Elizabeth Lipp is describing here.

More power to the participants, because people's health will ultimately benefit.

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