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.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets. MMPs2 are a family of structurally related zinc-binding proteolytic enzymes that digest extracellular matrix proteins and participate in tissue remodeling and signaling events (1). Currently, ϳ23 MMPs have been identified, comprising secreted and membrane-bound forms, and different family members share some common structural and functional domains and have varying degrees of substrate specificity. Abnormal expression and activation of MMPs has been implicated in the pathogenesis and pathological progression of several different human diseases that are centered in many different tissues in the periphery and central nervous system (2, 3). Initial clinical exploration of synthetic MMP inhibitors was focused on oncology indications, as preventing the breakdown of tissue matrices and barriers was viewed as a potential mechanism to limit tumor metastasis.Despite intensive efforts over many years to develop synthetic MMP inhibitors, only a single MMP inhibitor, Periostat, a tetracycline derivative used in periodontal disease, has progressed into regular clinical use (4). Of the ϳ50 other clinical trials conducted with active site MMP inhibitors, all have failed due to the onset of significant dose-limiting musculoskeletal toxicity ...

To investigate the role of phospholipase C (PLC) in inflammatory processes, we tested 1-(6-((17␤-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), a widely used PLC inhibitor, in several in vitro and in vivo assays. We first examined the effects of U73122 on human phospholipase C-␤ (PLC-␤) isozymes and found that U73122 significantly inhibited recombinant human PLC-␤2, with an IC 50 of ϳ6 M. U73122 had little effect on PLC-␤1, PLC-␤3, or PLC-␤4. Consistent with its ability to inhibit PLC-␤2 enzymatic activity, U73122 reduced interleukin-8 and leukotriene B 4 -induced Ca 2ϩ flux and chemotaxis in human neutrophils in a concentration-dependent manner. In vivo, U73122 blocked carrageenan-induced hind paw edema in rats, carrageenan-induced macrophage and lymphocyte accumulation into subcutaneous chambers in dogs, lipopolysaccharide-induced macrophage, lymphocyte infiltration and prostaglandin E 2 production in a mouse peritonitis model, and 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice. These results implicate PLC-dependent signaling pathways in the development of acute and chronic inflammatory responses in vivo.

Inflammatory diseases such as rheumatoid arthritis and psoriasis are characterized by increases in circulating cytokines, which play an important role in modulation of the disease state. Several marketed bio-therapeutics target cytokines and act as effective treatment strategies. Previous in-vitro and in-vivo studies have suggested that cytokines may have both direct and indirect effects on drug metabolizing enzyme levels in the liver. Few studies have characterized models to evaluate the risk of potential drug interactions that might be mediated by changes in cytokine levels. In the present studies the potential of three cytokines (IL-2, IL-6 and TNF-α) to modulate gene expression and activity of the major human cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4) in cryopreserved human hepatocytes (CHH) was investigated. Significant decreases in the activity of all 6 CYP isoforms occurred in hepatocytes incubated with TNF-α or IL-6 (17-85%; and 22-76% of untreated control values, respectively). TNF-α down-regulated the gene expression of CYP1A2, 2D6 and 3A4 only, whereas IL-6 down-regulated gene expression of all of the tested CYP isoforms except 2D6. IL-2 had only mild effects on CYP activity and mRNA levels of examined isoforms. In CHH exposed to TNF-α, changes in CYP activity were not always paralleled by gene expression alterations for three of the examined CYP isoforms. These studies highlight several potential pitfalls in using isolated human hepatocytes for determination of drug interactions by bio-therapeutics including lack of correlation of mRNA and activity measurements for some CYP isoforms when using single time point determinations, and appropriateness of the model for indirect acting cytokine and cytokine modulators.