Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

In vitro covalent binding studies in which xenobiotics are shown to undergo metabolism-dependent covalent binding to macromolecules have been commonly used to shed light on the biochemical mechanisms of xenobiotic-induced toxicity. In this paper, 18 drugs (nine hepatotoxins and nine nonhepatotoxins) were tested for their proclivity to demonstrate metabolism-dependent covalent binding to macromolecules in human liver S-9 fraction (9000 g supernatant) or human hepatocytes, as an extension to previous work that used human liver microsomes published in this journal [ Obach et al. ( 2008 ) Chem. Res. Toxicol. 21 , 1814 -1822 ]. In the S-9 fraction, seven out of the nine drugs in each category demonstrated some level of metabolism-dependent covalent binding. Inclusion of reduced glutathione, cofactors needed by conjugating enzymes, and other parameters (total daily dose and fraction of total intrinsic clearance comprised by covalent binding) improved the ability of the system to separate hepatotoxins from nonhepatotoxins to a limited extent. Covalent binding in human hepatocytes showed that six out of the nine hepatotoxins and four out of eight nonhepatotoxins demonstrated covalent binding. Taking into account estimates of total daily body burden of covalent binding from the hepatocyte data showed an improvement over other in vitro systems for distinguishing hepatotoxins from nonhepatotoxins; however, this metabolism system still displayed some false positives. Combined with the previous study using liver microsomes, these findings identify the limitations of in vitro covalent binding data for prospective prediction of hepatotoxicity for new drug candidates and highlight the need for a better understanding of the link between drug bioactivation, covalent adduct formation, and toxicity outcomes. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets, interindividual differences in susceptibility, etc.) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments.

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D 2 O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H 2 O or D 2 O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site uring the process of drug discovery, it is highly desirable to increase the number of successful drug candidates for preclinical, clinical and commercial development. Therefore, the drug discovery process is constantly scrutinized and improved [1]. Adding to this pressure is the generation of vast numbers of new chemical entities resulting from combinatorial chemistry technology [2]. Drug metabolism plays an important role in the drug discovery process [3]. Specifically, the identification of metabolites during the early stage of development can be helpful to medicinal chemists trying to block some of the metabolic hot spots and produce an appropriate drug that is less susceptible to metabolism and increase the half-life of the drug. Therefore, rapid identification of drug metabolites is imperative for drug development [4,5].Hydrogen/deuterium exchange is a well-established technique for studying structure, stability, folding dynamics, and intermolecular interactions in proteins in solution [6]. During solution phase H/D exchange, labile protons in the side chains and amide hydrogens, which are not protected from solution generally exchange rapidly. Exchanges of these unprotected hydrogens occur on the order of a few to a few tenths per second under the experimental conditions described in the aforementioned studies. If however, amide or side chain hydrogens are protected from solution (e.g., when they are hydrogen-bonded in structurally stable secondary-structure elements), the exchange rates can be considerably reduced. Methods in which H/D exchange experiments are combined with either nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry are also well-established [7,8]. NMR methods, when coupled with H/D exchange are the ideal choice for monitoring individual residues or each amide hydrogen; however, these methods are limited to highly purified proteins or metabolites that are soluble at high concentrations, thus eliminating the possibility of determining structural features of drugs and metabolites that are i...

TAK-875, a GPR40 agonist, was withdrawn from Phase III clinical trials due to drug-induced liver injury (DILI). Mechanistic studies were conducted to identify potential DILI hazards (covalent binding burden (CVB), hepatic transporter inhibition, mitochondrial toxicity, and liver toxicity in rats) associated with TAK-875. Treatment of hepatocytes with radiolabeled TAK-875 resulted in a CVB of 2.0 mg/day, which is above the threshold of 1 mg/day considered to be a risk for DILI. Covalent binding to hepatocytes was due to formation of a reactive acyl glucuronide (AG) and, possibly, an acyl-CoA thioester intermediate. Formation of TAK-875AG in hepatocytes and/or in vivo was in the order of non-rodents > human (in vitro only) > rat. These data suggest that non-rodents, and presumably humans, form TAK-875AG more efficiently than rats, and that AG-mediated toxicities in rats may only occur at high doses. TAK-875 (1000 mg/kg/day) formed significant amounts of AG metabolite (≤32.7 μM) in rat liver that was associated with increases in ALT (×4), bilirubin (×9), and bile acids (×3.4), and microscopic findings of hepatocellular hypertrophy and single cell necrosis. TAK-875 and TAK-875AG had similar potencies (within 3-fold) for human multi-drug resistant associated protein 2/4 (MRP2/4) and bile salt export pump, but TAK-875AG was exceptionally potent against MRP3 (0.21 μM). Inhibition of MRPs may contribute to liver accumulation of TAK-875AG. TAK-875 also inhibited mitochondrial respiration in HepG2 cells, and mitochondrial Complex 1 and 2 activities in isolated rat mitochondria. In summary, formation of TAK-875AG, and possibly TAK-875CoA in hepatocytes, coupled with inhibition of hepatic transporters and mitochondrial respiration may be key contributors to TAK-875-mediated DILI.

The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications.