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Apr 1, 2013 (Vol. 33, No. 7)

Predicting Drug Toxicity in Humans

  • As he explained, the human hepatocyte imaging assay allows simultaneous evaluation of a panel of relevant hepatocyte biomarkers with relatively high sensitivity and specificity in detecting drugs known to cause severe DILI, with 1% to 5% false-positivity. “Using this imaging assay for evaluation of multiple toxicity biomarkers during pre-clinical lead drug candidate optimization offers us a relatively high (95–99%) assurance against DILI despite the low incidence of occurrence.”

    Biopharmaceutical companies are looking to avoid false positives with their drugs. For instance, troglitazone may be safe in rat models, but 1 in 2,000 humans suffers from fatal liver injury. “This lack of liver toxicity in animal safety studies provided a false sense of safety.” It was withdrawn from the U.S. market in 2000. Most of these DILIs cannot be detected during Phase I and II clinical trials.

    It is essential to screen all the drugs for DILIs even if the incidence may be low, according to Dr. Kwok, citing valproic acid, which is on the market today as a life-saving anti-epileptic drug with a low incidence of severe DILI (1:1,000) in young children less than two years of age, or 1:50,000 in older children and in adults. “We do all the assays involving a variety of endpoints including cytosolic biomarkers such as glutathione, ATP levels, and reactive oxygen species in hepatocytes,” said Dr. Kwok.

  • Exploring Mitochondrial Function

    Mitochondrial bioenergetic balance may be a pertinent marker for early preclinical safety testing, especially in predicting DILI, according to Nathalie Compagnone, Ph.D., founder and CEO of Innovative Concepts in Drug Development (ICDD). Mitochondria are central to drug development as targets in CNS diseases, cancer, and metabolic diseases and as one of the main off-target sites causing undesirable side effects and toxicity. Dr. Compagnone’s team has developed a cell-based high-throughput screening (HTS) methodology to investigate the mechanisms underlying DILI and cardiac toxicity.

    The Mitosafe® technology developed at ICDD engages multiple integrated mitochondrial targets and the interactions are modeled. The functional bioassays reflect bioenergetic balance and redox status, and reveal mitochondrial DNA depletion, among other parameters.

    The Mitostream® technology engages multiple integrated cellular targets, with the interactions modeled via measurement of mitochondrial behavior. The technology measures the quantitative and qualitative levels of cellular adaptation, drug toxicity and sensitivity profiles, and disease signatures. “Mitosafe is for early deselection of hits using a mechanistic approach, and Mitostream is for anticipation of lead/candidate clinical tolerance,” said Dr. Compagnone.

    Dr. Compagnone and her team have developed a bioenergetic balanced screen (BBS) for fast, affordable, and efficient early toxicity testing by measuring the oxygen consumption rate, ATP levels, lactose production, and cell viability in live, nonpermeabilized cells. It identifies modulators of mitochondrial oxidative phosphorylation and glycolysis leading to ATP synthesis. It is used in identifying and validating a functional readout in HTS format that can be used as a marker to screen early compounds with potential toxicity, at dosages relevant to the clinical setting, she claimed.

    “Our bioassays tend to reduce the bias commonly used in measuring cytotoxicity by assessing pharmacological effects at clinically relevant doses leading to mitochondrial dysfunction, an early step of hepato- and cardiotoxicity,” said Dr. Compagnone.

    “If a drug produces unwanted modulation of mitochondrial dysfunction at a clinically relevant dose it can adversely affect the function of organs such as the liver, the brain, the heart, and the skin, which are sensitive to mitochondrial functioning for energy production.” Using clinically relevant doses also allows comparison of toxicity and efficacy on the same scale leading to a better definition of the safety window, defined by the range of circulating and bioactive doses.

  • Compliance with FDA

    Click Image To Enlarge +
    TRAC gene expression assay kit in use: Fast hybridization is achieved by coupling the probes to magnetic beads after hybridization is completed. Sovicell recently signed a collaboration agreement with Plexpress in Finland to market its TRAC technology for gene expression screening.

    Gene expression profiling of large numbers of samples and intermediate numbers of target genes (about 30) may be accomplished using TRAC technology. “The TRAC method increases the sensitivity and specificity of drug-drug interaction screening,” said Heinnerk Boriss, Ph.D., CEO, Sovicell. The method can therefore be readily applied to biomarker discovery and validation as well as nonclinical diagnostics. In fact, gene panels for other applications, such as oncology, are readily available.

    Dr. Boriss said that TRAC methodology is an advance over the current standard in gene expression profiling in that it eliminates three of the four steps: cell lysis, mRNA extraction, cDNA conversion, and amplification.

    “Hybridization with target probes is conducted directly in the lysed cells,” thereby saving time and money, Dr. Boriss said. “It also increases the accuracy of the final result” by eliminating three steps, which introduce substantial variability in the quantification. Gene expression screening with TRAC technology eliminates costly, time-consuming, and error-prone steps in existing methods, said Dr. Boriss.

    “Measuring the induction of drug metabolizing enzymes with mRNA expression profiling has a 30% higher sensitivity than the traditional activity screens,” he explained. It is well known that some drugs inhibit one or more of the drug metabolizing enzymes such as cytochromes (CYP P450), which may result in decreased metabolism of other drugs, raising their levels in the body to toxic proportions. Other drugs may induce the expression of these CYP enzymes, which increases metabolic rate, effectively lowering the concentration of other drugs, thereby resulting in a loss of efficacy.

    It has been shown that mRNA expression profiling increases the sensitivity of CYP induction screening—measuring CYP3A4 mRNA as a marker for pregnane X receptor-activated induction of drug-metabolizing enzymes. This observation led FDA and EMA to adopt mRNA expression screening into their guidelines for drug-drug interaction studies, noted Dr. Boriss.

    “The sheer simplicity of the TRAC technology enables comprehensive screening for potential expression of drug-metabolizing enzymes, which in turn will contribute to safer medications,” argued Dr. Boriss.


Readers' Comments

Posted 11/06/2013 by Flossie

This article fundamentally misunderstands the process. The fact that animals do not predict effects 100% of the time is only an issue for stage 1 clinical trials. No drug goes to market without being tested on humans first. Even then, animal tests are suffiiently predictive that there hven't been any stage 1 deaths for over 30 years. Furthermore, most ADRs are under or overdoses of otherwise safe drugs. Very poorly researched article.

Posted 04/01/2013 by AHarrill

"Idiosyncratic drug toxicity is human-specific, occurring as a rare event (<1/5,000) and therefore, impossible to study in clinical trials or experimental animals."

I take issue with the blanket statement that idiosyncratic DILI is impossible to model. Roth/Ganey have demonstated that addition of inflammatory mediators can sensitize animals to drugs that are otherwise benign to animals, such as trovafloxacin.

In addition, there has been an accumulating body of literature that suggests that genetic variants underlie drug toxicities (although almost certainly genetics is not the sole contributing factor in all cases). This has been shown for augmentin, ximelagatran, flucloxacillin, abacavir, and others. Given the genetic basis of many of these reactions, it is unlikely that average human hepatocytes from only a few donors will encompass a sufficient frequency of rare variants to detect idiosyncratic DILI that has a genetic basis.

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