We demonstrated that the APAP-induced increases in serum ALT and plasma 2,3-dinor TXB2 levels, as well as the rise in mortality rate, were drastically attenuated by ozagrel, a selective TXA2 synthase inhibitor, administered 30 min after the APAP injection. In addition, the histopathological changes produced by APAP were also suppressed by ozagrel. These results indicate that ozagrel protects against hepatotoxicity induced by APAP. The protective effect of ozagrel was comparable to that of NAC, the sole antidote for APAP hepatotoxicity.
A number of animal studies have demonstrated that ozagrel is protective against various forms of trauma and disease, including lung injury [23, 33], bronchial asthma [22, 40] and ischemia/reperfusion-induced organ injury [41, 42]. Ozagrel promptly inhibits TXA2 synthase in vitro and in vivo. In our previous study, we demonstrated that ozagrel protects against acute lung injury induced by fat embolism in guinea pigs . It is notable that ozagrel was protective despite being administered 60 min after APAP injection (Additional file 1: Figure 1S). From a clinical perspective, a drug that has efficacy when administered after the initiating insult has tremendous therapeutic potential.
Various inflammatory mediators are thought to be involved in the development of liver injury induced by APAP , and TXA2 appears to be one of these [20, 29]. In this study, we observed a significant increase in plasma 2,3-dinor TXB2 levels following APAP injection, which is in agreement with previous reports. Reilly et al. (2001)  observed significantly elevated APAP-induced hepatotoxicity in COX-2 deficient mice and in mice treated with a COX-2 inhibitor. These authors suggested that eicosanoids, such as PGE and PGI2, have an important hepatoprotective function, and that COX inhibition may exacerbate APAP-induced liver injury. However, the excessive production of 2,3-dinor TXB2 induced by APAP and the protective effects of ozagrel observed in this study suggest that TXA2 is an aggravating factor in APAP-mediated hepatotoxicity.
Jun and Fos have been reported to be associated with the degree of APAP-induced liver injury [43, 44]. In this study, ozagrel significantly suppressed the APAP-induced elevation in hepatic Jun and Fos mRNA expression. This result provides further support for a hepatoprotective function for ozagrel. In addition, APAP significantly induced expression of Chop and Bim mRNA, both of which play important roles in cell death during endoplasmic reticulum stress in various diseases. Nagy et al. (2007)  observed DNA fragmentation and CHOP induction in the livers of APAP-treated mice. We demonstrated that ozagrel attenuates the increase in the number of TUNEL-positive cells and suppresses the elevation in Chop mRNA expression induced by APAP in the liver. However, ozagrel did not repress the APAP-mediated increase in Bim mRNA expression. Badmann et al. (2011)  reported that Bim-deficient mice were substantially protected from APAP-induced liver damage, and suggested that Bim plays an important role in the development of liver injury induced by APAP. In cell death-related processes, the Bim pathway seems to be regulated not only by the transcriptional activation of Bim but also by other mechanisms, such as phosphorylation or proteasomal degradation of Bim protein  and binding to anti-apoptotic molecules, including Bcl-2 and Bcl-XL . Therefore, the effects of ozagrel on the Bim pathway remain unclear, and further study is needed to fully elucidate the effects of the drug. In this context, our finding that ozagrel does not affect APAP-induced Bim mRNA expression (in contrast to Chop, Jun and Fos mRNAs) is interesting and provides insight into the mechanisms underlying the protective effect of ozagrel against APAP hepatotoxicity.
The hepatotoxicity of APAP is triggered by a reactive metabolite, NAPQI, which is generated mainly by CYP2E1 [9–12]. Jaeschke et al. (2012)  and Bantel and Schulze-Osthoff (2012)  found that excessive NAPQI production depletes the hepatic GSH, and that this process is critical for the initiation of APAP hepatoxicity. In this study, ozagrel did not significantly attenuate the reduction in hepatic GSH content induced by APAP. In addition, ozagrel did not inhibit CYP2E1 activity in liver microsomes. These results suggest that the protective effect of ozagrel against APAP-induced hepatic injury is not due to inhibition of NAPQI production. This notion is supported by the in vitro results showing that ozagrel attenuates cellular injury induced by NAPQI in the RLC-16 hepatocyte cell line. These results suggest that the target of ozagrel, TXA2 synthase, may be situated downstream of NAPQI production and may play important roles in the development of APAP-induced liver injury. However, further detailed study (e.g., using TXA2 receptor knockout mice and TXA2 synthase knockdown) is required to fully uncover the roles of TXA2 in APAP hepatotoxicity.
OKY-1581 ((E)-2-methyl-3-[4-(pyridin-3-ylmethyl)phenyl]prop-2-enoic acid) was discovered as a selective inhibitor of TXA2 synthase, along with ozagrel, and shows protective effects against APAP hepatotoxicity in mice . However, the development of clinical OKY-1581 formulations has been abandoned because of adverse reactions observed in clinical trials. In comparison, ozagrel was found to be an ideal compound for use as a highly selective TXA2 synthase inhibitor , and it is in clinical usein Japan. Although further studies to evaluate the usefulness and safety of ozagrel in patients with APAP hepatotoxicity are needed, the results of this study suggest that the inhibition of TXA2 synthase by the drug is effective for the treatment of APAP-induced liver injury.
NAC is clinically used as an antidote for APAP intoxication, and it is thought that NAC provides cysteine, which is a precursor of GSH (which traps NAPQI), leading to a decrease in toxicity [16, 50]. In this study, NAC increased hepatic GSH content 2 h after the APAP injection and significantly prevented cell injury induced by NAPQI in RLC-16 cells. These results indicate that NAC provides GSH and detoxifies NAPQI. In comparison, although ozagrel exerted a remarkable hepatoprotective action against APAP-induced liver injury in mice, GSH content 2 h after APAP injection was not increased by the drug. These results indicate that ozagrel has a mode of action different from that of NAC in protection against APAP hepatotoxicity. For the development of new therapeutic strategies, it is interesting that ozagrel has a mechanism of action distinct from that of an existing agent, NAC.
Although the protective effect of ozagrel against cellular injury induced by NAPQI in RLC-16 cells was less robust than that of NAC, it may not indicate the inferiority of ozagrel as a therapeutic agent for APAP hepatotoxicity. The in vitro model of APAP hepatotoxicity using the cell culture system does not seem to fully agree with the in vivo model. For example, although the c-Jun N-terminal kinase inhibitor SP600125 drastically attenuates APAP-induced liver injury in the in vivo model, it has little effect in an in vitro model [44, 51]. Therefore, the in vitro model of APAP hepatotoxicity may not be adequate for comparison of the efficacy of drugs. Nonetheless, it may provide insight into the mechanisms underlying the protective effect of the agents against APAP liver injury. If ozagrel protects against APAP-induced liver injury only through the modulation of inflammatory cell activity, such as inhibition of neutrophils or Kupffer cells, it would not be able to exert a protective action in an in vitro model. Therefore, the results of the in vitro model demonstrate that ozagrel, at least in part, protects against APAP hepatotoxicity by inhibiting the oncotic necrosis of hepatocytes.