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Aims: Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. Methods: ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. Results: AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing. Conclusions: In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.

Wei Wu,1 Ze-neng Cheng2 1Department of Pharmacy, The First Hospital of Changsha, Changsha, Hunan, People’s Republic of China; 2Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, People’s Republic of ChinaCorrespondence: Ze-neng Cheng, Xiangya School of Pharmaceutical Sciences, Central South University, No. 172 Tongzipo Road, Yuelu District, Changsha, 410013, Hunan, People’s Republic of China, Tel +86-731-82650001, Email cheng15279817797@163.comObjective: Fluorofenidone (AKF-PD) is a novel antifibrotic small-molecule compound. The purpose of this study was to investigate the metabolic and excretory pathways of AKF-PD in rats.Methods: High-performance liquid chromatography with mass spectrometric (HPLC-MS) detection was used to analyze the metabolites in rat urine. The metabolites were separated by chromatography and their structure was confirmed. HPLC was used to determine the contents of the parent compound and its metabolites in feces and urine after quantitative administration to study the excretion pathway.Results: AKF-PD was mainly oxidized to the carboxyl group after methyl hydroxylation. After oral administration, the total amount of the prototype drug and its hydroxylated metabolites and carboxylated metabolites excreted from the urine and feces of rats was 87%. However, most of them are excreted in urine and feces in the form of carboxylated metabolites, and rarely excreted in the form of prototype drugs and hydroxylated metabolites. Which is that the urinary discharge of hydroxylated metabolites, fluorine ketones, and carboxylated metabolites were 0.2%, 1.1%, and 75.2%, respectively, while the fecal discharge were 0.2%, 0.3%, and 10.1%, respectively.Conclusion: AKF-PD is mainly oxidized into 2-hydroxymethyl and 5-carboxyl AKF-PD through the Phase I metabolic reaction in rats. AKF-PD is a highly permeable compound classified by biopharmaceutics and is mainly excreted from the urine in the form of metabolites.Keywords: fluorofenidone, metabolites, methyl hydroxylation, HPLC-MS

Acute lung injury (ALI) is a severe disease that presents serious damage and excessive inflammation in lungs with high mortality without effective pharmacological therapy. Fluorofenidone (AKFPD) is a novel pyridone agent that has anti-fibrosis, anti-inflammation, and other pharmacological activities, while the effect of fluorofenidone on ALI is unclarified. Here, we elucidated the protective effects and underlying mechanism of fluorofenidone on lipopolysaccharide (LPS)-induced ALI. In this study, fluorofenidone alleviated lung tissue structure injury and reduced mortality, decreased the pulmonary inflammatory cell accumulation and level of inflammatory cytokines IL-1β, IL-6, and TNF-α in the bronchoalveolar lavage fluid, and attenuated pulmonary apoptosis in LPS-induced ALI mice. Moreover, fluorofenidone could block LPS-activated phosphorylation of ERK, JNK, and P38 and further inhibited the phosphorylation of IκB and P65. These results suggested that fluorofenidone can significantly contrast LPS-induced ALI through suppressing the activation of the MAPK/NF-κB signaling pathway, which indicates that fluorofenidone could be considered as a novel therapeutic candidate for ALI. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is a severe disease that presents serious damage and excessive inflammation in lungs with high mortality without effective pharmacological therapy. Fluorofenidone (AKFPD) is a novel pyridone agent that has anti-fibrosis, anti-inflammation, and other pharmacological activities, while the effect of fluorofenidone on ALI is unclarified. Here, we elucidated the protective effects and underlying mechanism of fluorofenidone on lipopolysaccharide (LPS)-induced ALI. In this study, fluorofenidone alleviated lung tissue structure injury and reduced mortality, decreased the pulmonary inflammatory cell accumulation and level of inflammatory cytokines IL-1β, IL-6, and TNF-α in the bronchoalveolar lavage fluid, and attenuated pulmonary apoptosis in LPS-induced ALI mice. Moreover, fluorofenidone could block LPS-activated phosphorylation of ERK, JNK, and P38 and further inhibited the phosphorylation of IκB and P65. These results suggested that fluorofenidone can significantly contrast LPS-induced ALI through suppressing the activation of the MAPK/NF-κB signaling pathway, which indicates that fluorofenidone could be considered as a novel therapeutic candidate for ALI.

Currently, no antifibrotic drug in clinical use can effectively treat renal fibrosis. Fluorofenidone (AKFPD), a novel pyridone agent, significantly reduces renal fibrosis by inhibiting the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome; however, the underlying mechanism of this inhibition is not fully understood. The present study aimed to reveal the molecular mechanism underlying the suppression of NLRP3 inflammasome activation by AKFPD. It investigated the effect of AKFPD on NLRP3 activation and lysosomal cathepsins in a unilateral ureteral obstruction (UUO) rat model, and hypoxia/reoxygenation (H/R)-treated HK-2 cells and murine peritoneal-derived macrophages (PDMs) stimulated with lipopolysaccharide (LPS) and ATP. The results confirmed that AKFPD suppressed renal interstitial fibrosis and inflammation by inhibiting NLRP3 inflammasome activation in UUO rat kidney tissues. In addition, AKFPD reduced the production of activated caspase-1 and maturation of IL-1β by suppressing NLRP3 inflammasome activation in H/R-treated HK-2 cells and murine PDMs stimulated with LPS and ATP. AKFPD also decreased the activities of cathepsins B, L and S both in vivo and in vitro. Notably, AKFPD downregulated cathepsin B expression and NLRP3 colocalization in the cytoplasm after lysosomal disruptions. Overall, the results suggested that AKFPD attenuates renal fibrosis by inhibiting lysosomal cathepsin-mediated activation of the NLRP3 inflammasome. [ABSTRACT FROM AUTHOR]

This study is designed to compare drug encapsulation by cucurbit[7]uril and β-cyclodextrin, using fluorofenidone as a model drug. Single-crystal X-ray diffraction analysis was employed to successfully determine the crystal structures of fluorofenidone·H + @cucurbit[7]uril Form, fluorofenidone@cucurbit[7]uril Form, and fluorofenidone@β-cyclodextrin Form. Keto-enol tautomerization of fluorofenidone mediated by cucurbit[7]uril in acid solution is confirmed by crystal structures, pH titration, and nuclear magnetic resonance experiments. However, β-cyclodextrin cannot cause the keto-enol tautomerization of fluorofenidone under similar conditions. The phase solubility study demonstrates that cucurbit[7]uril has a much higher solubilization capacity for fluorofenidone than β-cyclodextrin in 0.1 M HCl since the K values of fluorofenidone with cucurbit[7]uril and β-cyclodextrin were 1223.97 ± 452.68 and 78.49 ± 10.56 M c , respectively. Excellent solubility can be attributed to the keto-enol tautomerization of fluorofenidone under the conditions of cucurbit[7]uril in acid solution. The enol form of fluorofenidone is encapsulated by cucurbit[7]uril by hydrogen bonding interaction and hydrophobic interaction to increase binding affinity. Rat pharmacokinetic studies demonstrate that the area under the plasma concentration-time curve from time 0 to 7 h value of fluorofenidone@cucurbit[7]uril complex is 1.70-fold greater than that of free fluorofenidone, and the mean residence time from time 0 to 7 h is slightly prolonged from 1.29 to 1.76 h ( -1 < 0.01) after oral administration. However, no significant difference is found between fluorofenidone and fluorofenidone@β-cyclodextrin complex. This work indicates that the induction of keto-enol tautomerization of drugs using macrocyclic molecules has the potential to be an effective method to improve their solubility and bioavailability, providing valuable insights for the application of macrocyclic molecules in the biomedical field.P < 0.01) after oral administration. However, no significant difference is found between fluorofenidone and fluorofenidone@β-cyclodextrin complex. This work indicates that the induction of keto-enol tautomerization of drugs using macrocyclic molecules has the potential to be an effective method to improve their solubility and bioavailability, providing valuable insights for the application of macrocyclic molecules in the biomedical field.

Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. ALF mouse models were established using APAP or lipopolysaccharide/ D -galactosamine (LPS/ D -Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/ D -Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/ D -Gal-induced mortality with delayed dosing. In summary, AKF-PD can protect against ALF caused by APAP or LPS/ D -Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF. [Display omitted] • Fluorofenidone (AKF-PD), a novel antifibrotic pyridone agent, mitigates acetaminophen (APAP)-induced acute liver failure (ALF) in mice by modulating the MKK4/JNK pathway. • In ALF induced by lipopolysaccharide/ D -galactosamine (LPS/ D -Gal) in mice, AKF-PD pretreatment effectively reduces liver injury by decreasing ROS levels and inflammation. • AKF-PD inhibits phosphorylation of MKK4 and JNK upon pretreatment, enhancing survival in LPS/ D -Gal-induced mortality cases with delayed dosing. [ABSTRACT FROM AUTHOR]

Cholestasis is characterized by intrahepatic accumulation of bile acids (BAs), resulting in liver injury, fibrosis, and liver failure. To date, only ursodeoxycholic acid and obeticholic acid have been approved for the treatment of cholestasis. As fluorofenidone (AKF-PD) was previously reported to play significant anti-fibrotic and anti-inflammatory roles in various diseases, we investigated whether AKF-PD ameliorates cholestasis. A mouse model of cholestasis was constructed by administering a 0.1 % 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) diet for 14 days. Male C57BL/6 J mice were treated with either AKF-PD or pirfenidone (PD) orally in addition to the DDC diet. Serum and liver tissues were subsequently collected and analyzed. We found that AKF-PD significantly reduced the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile salts (TBA), as well as hepatic bile acids (BAs) levels. Hepatic histological analyses demonstrated that AKF-PD markedly attenuated hepatic inflammation and fibrosis. Further mechanistic analyses revealed that AKF-PD markedly inhibited expression of Cyp7a1, an enzyme key to BAs synthesis, by increasing Fxr nuclear translocation, and decreased hepatic inflammation by attenuating Erk/-Egr-1-mediated expression of inflammatory cytokines and chemokines Tnfα, Il-1β, Il-6, Ccl2, Ccl5 and Cxcl10. Moreover, AKF-PD was found to substantially reduce liver fibrosis via inhibition of Tgfβ1/Smad pathway in our mouse model. Here, we found that AKF-PD effectively attenuates cholestasis and hepatic fibrosis in the mouse model of DDC-induced cholestasis. As such, AKF-PD warrants further investigation as a candidate drug for treatment of cholestasis. • Fluorofenidone can significantly improve both cholestasis and liver fibrosis. • Fluorofenidone markedly inhibits the expression of Cyp7a1, an enzyme key to bile acids synthesis, by increasing Fxr nuclear translocation. • Fluorofenidone decreases hepatic inflammation by attenuating Erk/-Egr-1-mediated cytokine and chemokine expression. • Fluorofenidone substantially reduces liver fibrosis via inhibition of Tgfβ1/Smad signaling. [ABSTRACT FROM AUTHOR]