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Small intestinal mucosal injury is a frequent adverse effect caused by nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms are not completely understood, but topical (luminal) effects have been implicated. Many carboxylic acid-containing NSAIDs, including diclofenac (DCF), are metabolized to acyl glucuronides (AGs), and/or ether glucuronides after ring hydroxylation, and exported into the biliary tree. In the gut, these conjugates are cleaved by bacterial ␤-glucuronidase, releasing the potentially harmful aglycone. We first confirmed that DCF-AG was an excellent substrate for purified Escherichia coli ␤-D-glucuronidase. Using a previously characterized novel bacteria-specific ␤-glucuronidase inhibitor (Inhibitor-1), we then found that the enzymatic hydrolysis of DCF-AG in vitro was inhibited concentration dependently (IC 50 ϳ164 nM). We next hypothesized that pharmacologic inhibition of bacterial ␤-glucuronidase would reduce exposure of enterocytes to the aglycone and, as a result, alleviate enteropathy. C57BL/6J mice were administered an ulcerogenic dose of DCF (60 mg/kg i.p.) with or without oral pretreatment with Inhibitor-1 (10 g per mouse, b.i.d.). Whereas DCF alone caused the formation of numerous large ulcers in the distal parts of the small intestine and increased (2-fold) the intestinal permeability to fluorescein isothiocyanate-dextran, Inhibitor-1 cotreatment significantly alleviated mucosal injury and reduced all parameters of enteropathy. Pharmacokinetic profiling of DCF plasma levels in mice revealed that Inhibitor-1 coadministration did not significantly alter the C max , half-life, or area under the plasma concentration versus time curve of DCF. Thus, highly selective pharmacologic targeting of luminal bacterial ␤-D-glucuronidase by a novel class of small-molecule inhibitors protects against DCF-induced enteropathy without altering systemic drug exposure.

are often taken chronically, such drugs should feature a good benefit-risk profile and be devoid of long-term safety concerns. However, studies like the one published by Yoriki et al. in this issue of Journal of Gastroenterology and Hepatology are important because they create new conceptual approaches to developing novel therapies that are based on insights into the mechanisms underlying NSAID-induced enteropathy. References 1 Bjarnason I. Gastrointestinal safety of NSAIDs and over-the-counter analgesics. Int. J. Clin. Pract. 2013; 67 (Suppl. 178): 37-42. 2 Wallace JL. NSAID gastropathy and enteropathy: distinct pathogenesis likely necessitates distinct prevention strategies. Br. J. Clin. Pharmacol. 2012; 165: 67-74. 3 Boelsterli UA, Redinbo MR, Saitta K. Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies. Toxicol. Sci. 2012. doi: 10.1093/toxsci/kfs310. 4 LoGuidice A, Ramirez-Alcantara V, Proli A, Gavillet B, Boelsterli UA. Pharmacologic targeting or genetic deletion of mitochondrial cyclophilin D protects from NSAID-induced small intestinal ulceration in mice. Toxicol. Sci. 2010; 118: 276-85. 5 Ramirez-Alcantara V, LoGuidice A, Boelsterli UA. Protection from diclofenac-induced small intestinal injury by the JNK inhibitor SP600125 in a mouse model of NSAID-associated enteropathy. Am. J. Physiol. (Gastrointest. Liver Physiol.) 2009; 297: G990-8. 6 Zhu Y, Zhang QY. Role of intestinal cytochrome P450 enzymes in diclofenac-induced toxicity in the small intestine. J. Pharmacol. Exp. Ther. 2012; 343: 362-70. 7 Yamada S, Naito Y, Takagi T et al. Rebamipide ameliorates indomethacin-induced small intestinal injury in rats via the inhibition of matrix metalloproteases activity. J. Gastroenterol. Hepatol. 2012; 27: 1816-24. 8 Somasundaram S, Rafi S, Hayllar J et al. Mitochondrial damage: a possible mechanism of the "topical" phase of NSAID induced injury to the rat intestine. Gut 1997; 41: 344-53. 9 Yoriki H, Naito Y, Takagi T et al. Hemin ameliorates indomethacin-induced small intestinal injury in mice through the induction of heme oxygenase-1. J. Gastroenterol. Hepatol. 2013; 28: 632-8. 10 Vile GF, Basu-Modak S, Waltner C, Tyrrell RM. Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts. 278: L312-9. 12 Cantoni L, Valaperta R, Ponsoda X et al. Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity. J. Hepatol. 2003; 38: 776-83. 13 Siegert SWK, Holt RJ. Physicochemical properties, pharmacokinetics, and pharmacodynamics of intravenous hematin: a literature review. Adv. Ther. 2008; 25: 842-57. 14 Wallace BD, Wang H, Lane KT et al. Alleviating cancer drug toxicity by inhibiting a bacterial enzyme. Science 2010; 330: 831-5. 15 LoGuidice A, Wallace BD, Bendel L, Redinbo MR, Boelsterli UA. Pharmacologic targeting of bacterial b-glucuronidase alleviates nonsteroidal anti-inflammatory drug-induced enteropathy in mice.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Lefluno-mide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH 2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-X L inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepato-toxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabi-lization. (HEPATOLOGY 2007;45:412-421.) A cetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, when taken at high doses or, rarely in particularly susceptible people at therapeutic doses, APAP can precipitate severe liver injury that can develop into fulminant liver failure. 1 The clinical significance of this adverse effect is underscored by APAP being, among all drugs, the single major cause of drug-induced hepatotoxicity in the United States and the United Kingdom. 2 The mechanisms underlying APAP-induced liver injury have been studied for several decades, and excellent recent reviews have summarized the cellular and molecular pathways of this toxic response. 3-5 Although the initial steps in the sequence of events leading to hepatocyte ne-crosis (bioactivation of APAP and glutathione depletion) have been well known for many years, the more distal events (signaling pathways that lead to the precipitation of cell death) are less clear. However, recently, the mito-chondrial permeability transition (mPT) has been identified as a pivotal mechanism mediating APAP-induced cell death. 6,7 According to this concept, a combination of mi-tochondrial oxidant stress, increased Ca 2 levels, and other factors may favor ...