Patel SJ, Milwid JM, King KR, Bohr S, Iracheta-Velle A, Li M, et al. Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure. Nat Biotechnol 2012;30:179-183. Available at: www.nature.com (Reprinted with permission.)
Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen-induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety. (HEPATOLOGY 2012;)
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Drug-induced liver injury (DILI) limits the development and application of many therapeutic compounds and presents major challenges to the pharmaceutical industry and clinical medicine. Acetaminophen-containing compounds are among the most frequently prescribed drugs and are also the most common cause of DILI. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and acetaminophen- induced hepatotoxicity. We demonstrate that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation and death caused by liver-toxic drugs. We identify a small-molecule inhibitor of Cx32 that protects against liver failure and death in wild-type mice when co-administered with known hepatotoxic drugs. These findings indicate that gap junction inhibition could provide a pharmaceutical strategy to limit DILI and improve drug safety.
Drug-induced liver injury (DILI) is the most frequent reason for drug withdrawal either during early development or later from the market. It can also lead to a recommendation of limited dosage.1, 2 DILI is initiated by direct hepatotoxic effects of a drug or a reactive metabolite of a drug. Overdosing on N-acetyl-para-amino-phenol (APAP; acetaminophen), a widely used antipyretic and analgesic agent, is the most frequent cause of acute liver failure in young adults in the United States. CYP1A2, CYP2E1, and CYP3A4 drive conversion of APAP into N-acetyl-p-benzoquinone imine (NAPQI), which is then conjugated to glutathione and detoxified to mercapturic acid.3 When excessive doses of APAP are ingested, the glucuronidation pathway is overwhelmed, and the rapid generation of NAPQI can lead to the depletion of intrahepatic glutathione stores and reduced detoxification of the drug. NAPQI can also covalently bind to intracellular proteins, interfere with mitochondrial and nuclear function, generate reactive oxidative species, and ultimately lead to apoptosis and necrosis in the liver.3 Currently, the only available therapies are N-acetylcysteine (NAC), supportive care, and liver transplantation. Because of the zonation of the liver that confines most P450 enzymes to perivenular hepatocytes, APAP toxicity initially affects those, and then spreads into the parenchyma and the entire hepatic lobule. Published evidence suggests that liver gap junctions are involved in such spreading. Gap junctions are plasma membrane spatial microdomains constituted by assemblies of channel proteins called connexins (Cx). The channels provide direct intercellular communication pathways, allowing cell-to-cell rapid exchange of ions and metabolites up to approximately 1 Kd in size. Cx32 is the major gap junction protein in the liver, but it is also found in pancreas, kidney, and nervous tissue. Several studies have shown that interfering with liver gap junctions function greatly reduces liver damage due to several toxic agents including carbon tetrachloride, D-galactosamine,4 and APAP itself.5 In the latter study, the authors used a line of transgenic rats carrying a dominant negative mutant of Cx32 and found a reduced hepatotoxic effect of APAP compared to wild-type rats.5 In this new study, Patel et al.6 have confirmed and expanded these results, and most importantly, have been able to devise a pharmacological strategy to limit DILI spreading through the blockade of gap junctions.
Patel et al. first showed that mice deficient in Cx32 (Cx32−/−) are protected against liver damage, acute inflammation, and death caused by thioacetamide (TAA), a hepatotoxin model. They carefully checked that phase I and phase II drug metabolism efficiency was similar in Cx32+/+ and Cx32−/− mice, ruling out the possibility that Cx32−/− mice were protected through defective drug metabolism.
Because hepatotoxin exposure leads to oxidative stress and because free radicals propagate through gap junctions,7, 8 Patel et al. hypothesized that gap junctions might be responsible for the dissemination and amplification of oxidative stress in the liver after TAA treatment. They first verified that drug-induced hepatotoxicity was indeed dependent on oxidative stress by showing that antioxidants (dimethyl sulfoxide, N-acetylcysteine) reduced liver injury. They also validated that Cx32 gap junctions propagated oxidative stress from hepatotoxin-injured cells to neighboring hepatocytes by comparing the accumulation of intracellular free radicals within the livers of Cx32−/− and Cx32+/+ mice.
Patel et al. then sought to identify small molecule inhibitors of Cx32 and selected 2-aminoethoxydipenyl-borate (2APB). 2APB is a membrane-permeable reagent widely used to block inositol 1,4,5-trisphosphate–induced calcium release,9 and that also activates or inhibits various transient receptor potential channels.10 Tao et al. previously showed that 2APB directly and reversibly inhibited connexin channels composed of Cx26 and Cx32 in vitro.11 Here, Patel et al. first confirmed using liver slices that 2APB very efficiently blocked gap junction function. They then pretreated mice with 2APB before administration of TAA or APAP. This experiment showed a dramatic protection against the hepatotoxic effects of both drugs in the same range as that seen in Cx32−/− mice. Because pretreatment is not an option in clinical practice, they then tested the coformulation of 2APB with APAP or TAA. In that case, mice were still largely preserved from severe liver damage as judged by a variety of parameters. Most importantly, 2APB reduced APAP-induced mortality from 80% to 30% and completely abolished TAA-induced mortality.
Even though coformulation might in theory be applicable to drugs with a high incidence of DILI, the most common situation is that of patients seeking treatment after ingestion of compounds at hepatotoxic doses. Patel et al. thus treated mice with 2APB at various time points following APAP or TAA administration. At 1.5 hours after intoxication, protection by 2APB was still almost complete. Remarkably, even when administered 6 hours after hepatotoxin exposure, at a time where hepatic necrosis is already evident, the treatment could reduce serum alanine aminotransferase (ALT) levels and hepatocellular damage and necrosis, although data on mortality were not reported.
Altogether, these results strongly argue for a role of liver gap junctions in the propagation of DILI and suggest for the first time that this mechanism can be targeted with drugs (Fig. 1). A number of issues will likely have to be solved before human trials. For instance, 2APB has other targets besides Cx32, and its toxicity needs to be investigated further and/or new blockers need to be identified. Tao et al. have shown that 2APB also block Cx32 hemichannels that connect the cytosol to the extracellular space11; because there is now accumulated evidence that connexin hemichannels are involved in cell death signaling,12 2APB might protect from cell death at least partly independently of blocking gap junctions. Very interestingly, promising results were obtained by Patel et al. in a postabsorption setting, suggesting that their strategy could be used in patients seen after intoxication with the hope of limiting the requirement for liver transplantation. However, and intriguingly, previous studies have shown that administration of hepatotoxic chemicals such as dimethylnitrosamine or carbon tetrachloride decreased the protein levels of Cx32 in cultured hepatocytes13 and in rat liver.14 Remarkably, Cx32 levels became undetectable as soon as 24 hours following dimethylnitrosamine treatment, before the peak of ALT,14 which may be a cellular response to injury designed to protect healthy cells from the diffusion of toxic molecules. However, if such a decreased Cx32 expression was also to be found in the TAA or APAP setting, it could indicate that the protective effect of 2APB may be partly related to an effect on other targets, such as those mentioned above. In any case, it suggests that the therapeutic window for using Cx32 blockers in the course of an acute intoxication may be narrow. On the other hand, Cx32 blockers used in coformulation may help rescue a number of hepatotoxic drugs in the course of their clinical development.