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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

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]

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.

Fluorofenidone (AKF-PD) is an analog of pirfenidone and shows stronger antifibrotic effect and lower toxicity compared to pirfenidone in preclinical studies. However, the inhibitory and inducible effects of AKF-PD on human CYP450s are unclear. The aim of this study was to evaluate the ability of AKF-PD to inhibit and induce CYP450s in vitro. In inhibition study, the inhibitory effects of CYP1A2, CYP3A4, CYP2C9, CYP2E1, CYP2C19 and CYP2D6 by AKF-PD were evaluated with the metabolic rate of probe drug of each enzyme in pooled human liver microsomes. The enzyme inducible potential of AKF-PD was evaluated by the mRNA expression and enzyme activity of CYP1A2, CYP2B6 and CYP3A4 in human hepatocytes. The results suggested that AKF-PD produced weak inhibition on CYP1A2 and CYP2C19, while no inhibitory effects were found on the other enzymes. Since the plasma concentration of AKF-PD is much lower than the IC50 values of both CYP1A2 and CYP2C19, the inhibitory effects can be reasonably ignored. On the other hand, AKF-PD showed no inducible effects on CYP1A2 while showed potential inducible ability on CYP2B6 and CYP3A4 in some test groups. Further study of this novel anti-fibrotic drug should take into account in clinical therapies. [ABSTRACT FROM AUTHOR]

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Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

Abstract Background and aims Glutathione S-transferase A3 (GSTA3) is known as an antioxidative protease, however, the crucial role of GSTA3 in liver fibrosis remains unclear. As a recently we developed water-soluble pyridone agent with antifibrotic features, fluorofenidone (AKF-PD) can attenuate liver fibrosis, present studies were designed to explore the role of GSTA3 in liver fibrosis and its modulation by AKF-PD in vivo and in vitro. Methods Rats liver fibrosis models were induced by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4). The two activated hepatic stellate cells (HSCs) lines, rat CFSC-2G and human LX2 were treated with AKF-PD respectively. The lipid peroxidation byproduct malondialdehyde (MDA) in rat serum was determined by ELISA. The accumulation of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescein fluorescence analysis. The expression of α-smooth muscle actin (α-SMA), fibronectin (FN), and phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 beta (GSK-3β) were detected by western blotting (WB). Results GSTA3 was substantially reduced in the experimental fibrotic livers and transdifferentiated HSCs. AKF-PD alleviated rat hepatic fibrosis and potently inhibited HSCs activation correlated with restoring GSTA3. Moreover, GSTA3 overexpression prevented HSCs activation and fibrogenesis, while GSTA3 knockdown enhanced HSCs activation and fibrogenesis resulted from increasing accumulation of ROS and subsequent amplified MAPK signaling and GSK-3β phosphorylation. Conclusions We demonstrated firstly that GSTA3 inhibited HSCs activation and liver fibrosis through suppression of the MAPK and GSK-3β signaling pathways. GSTA3 may represent a promising target for potential therapeutic intervention in liver fibrotic diseases.

Cell death and sterile inflammation are major mechanisms of renal fibrosis, which eventually develop into end-stage renal disease. “Necroptosis” is a type of caspase-independent regulated cell death, and sterile inflammatory response caused by tissue injury is strongly related to necrosis. Fluorofenidone (AKF-PD) is a novel compound shown to ameliorate renal fibrosis and associated inflammation. We investigated whether AKF-PD could alleviate renal fibrosis by inhibiting necroptosis. Unilateral ureteral obstruction (UUO) was used to induce renal tubulointerstitial fibrosis in C57BL/6J mice. AKF-PD (500 mg/kg) or necrostatin-1 (Nec-1; 1.65 mg/kg) was administered simultaneously for 3 and 7 days. Obstructed kidneys and serum were harvested after euthanasia. AKF-PD and Nec-1 ameliorated renal tubular damage, inflammatory-cell infiltration, and collagen deposition, and the expression of proinflammatory factors (interlukin-1β, tumor necrosis factor [TNF]-α) and chemokines (monocyte chemoattractant protein-1) decreased. AKF-PD or Nec-1 treatment protected renal tubular epithelial cells from necrosis and reduced the release of lactate dehydrogenase in serum. Simultaneously, production of receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL) was also reduced 3 and 7 days after UUO. AKF-PD and Nec-1 significantly decreased the percentage of cell necrosis, inhibiting the phosphorylation of MLKL and RIPK3 in TNF-α- and Z-VAD–stimulated human proximal tubular epithelial (HK-2) cells. In conclusion, AKF-PD and Nec-1 have effective anti-inflammatory and antifibrotic activity in UUO-induced renal tubulointerstitial fibrosis, potentially mediated by the RIPK3/MLKL pathway.

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

Jing Tang,1 Jianming Li,2 Guo Li,2 Haitao Zhang,2 Ling Wang,3 Dai Li,4 Jinsong Ding2 1School of Pharmaceutical Sciences, Changsha Medical University, 2Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 3Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, 4Xiangya Hospital, Central South University, Changsha, China Abstract: Idiopathic pulmonary fibrosis is a progressive, fatal lung disease with poor survival. The advances made in deciphering this disease have led to the approval of different antifibrotic molecules, such as pirfenidone and nintedanib. An increasing number of studies with particles (liposomes, nanoparticles [NPs], microspheres, nanopolymersomes, and nanoliposomes) modified with different functional groups have demonstrated improvement in lung-targeted drug delivery. In the present study, we prepared, characterized, and evaluated spermidine (Spd)-modified poly(lactic-co-glycolic acid) (PLGA) NPs as carriers for fluorofenidone (AKF) to improve the antifibrotic efficacy of this drug in the lung. Spd-AKF-PLGA NPs were prepared and functionalized by modified solvent evaporation with Spd and polyethylene glycol (PEG)-PLGA groups. The size of Spd-AKF-PLGA NPs was 172.5±4.3 nm. AKF release from NPs was shown to fit the Higuchi model. A549 cellular uptake of an Spd–coumarin (Cou)-6-PLGA NP group was found to be almost twice as high as that of the Cou-6-PLGA NP group. Free Spd and difluoromethylornithine (DFMO) were preincubated in A549 cells to prove uptake of Spd-Cou-6-PLGA NPs via a polyamine-transport system. As a result, the uptake of Spd-Cou-6-PLGA NPs significantly decreased with increased Spd concentrations in incubation. At higher Spd concentrations of 50 and 500 µM, uptake of Spd-Cou-6-PLGA NPs reduced 0.34- and 0.49-fold from that without Spd pretreatment. After pretreatment with DFMO for 36 hours, cellular uptake of Spd-Cou-6-PLGA NPs reached 1.26-fold compared to the untreated DFMO group. In a biodistribution study, the drug-targeting index of Spd-AKF-PLGA NPs in the lung was 3.62- and 4.66-fold that of AKF-PLGA NPs and AKF solution, respectively. This suggested that Spd-AKF-PLGA NPs accumulated effectively in the lung. Lung-histopathology changes and collagen deposition were observed by H&E staining and Masson staining in an efficacy study. In the Spd-AKF-PLGA NP group, damage was further improved compared to the AKF-PLGA NP group and AKF-solution group. The results indicated that Spd-AKF-PLGA NPs are able to be effective nanocarriers for anti–pulmonary fibrosis therapy. Keywords: idiopathic pulmonary fibrosis, fluorofenidone, spermidine, polyamine transport system, nanoparticles

Background/Aims: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. Methods: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-β1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 μmol/L), AKF-PD (400 μg/mL), PFD (400 μg/mL), and GW788388 (5 μmol/L). Results: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-β1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 μg/mL) significantly decreased TGF-β1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. Conclusion: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury. [ABSTRACT FROM AUTHOR]

The aim of the present research was to study the therapeutic impacts of fluorofenidone (AKF-PD) on pig serum (PS)-induced liver fibrosis in rats and the complex molecular mechanisms of its effects on hepatic stellate cells (HSCs). Wistar rats were randomly divided into normal control, PS and PS/AKF-PD treatment groups. The activated human HSC LX-2 cell line was also treated with AKF-PD. The expression of collagen I and III, and α-smooth muscle actin (α-SMA) was determined by immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blotting and/or RT-qPCR analyses were used to determine the expression of transforming growth factor (TGF)-β1, α-SMA, collagen I, mothers against decapentaplegic homolog (Smad)-3, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). AKF-PD attenuated the degree of hepatic fibrosis and liver injury in vivo, which was associated with the downregulation of collagen I and III, and α-SMA at the mRNA and protein levels. In vitro, AKF-PD treatment significantly reduced the TGF-β1-induced activation of HSCs, as determined by the reduction in collagen I and α-SMA protein expression. The TGF-β1-induced upregulation of the phosphorylation of Smad 3, ERK1/2, p38 and JNK was attenuated by AKF-PD treatment. These findings suggested that AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs activation via the TGF-β1/Smad and MAPK signaling pathways, and therefore that AKF-PD may be suitable for use as a novel therapeutic agent against liver fibrosis. [ABSTRACT FROM AUTHOR]

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]

Background/Aims: We evaluated the therapeutic effects of fluorofenidone (AKF-PD), a novel pyridone agent, targeting oxidative stress and fibrosis in obstructive nephropathy. Methods: AKF-PD was used to treat renal interstitial fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of NOX2 (gp91phox), fibronectin and extracellular signal regulated kinase (ERK) were detected by western blot. A level of Malondialdehyde (MDA), an oxidative stress marker, was measured by ELISA. In addition, ROS and the expressions of NOX2, collagen I (a1), fibronectin and p-ERK were measured in angiotensin (Ang) II-stimulated rat proximal tubular epithelial cells (NRK-52E) in culture. Results: In NRK-52E cells, AKF-PD reduced AngII induced expressions of ROS, NOX2, fibronectin, collagen I (a1) and p-ERK. In UUO kidney cortex, AKF-PD attenuated the degree of renal interstitial fibrosis, which was associated with reduced the expressions of collagen I (a1) and fibronectin. Furthermore, AKF-PD downregulated the expressions of NOX2, MDA and p-ERK. Conclusion: AKF-PD treatment inhibits the progression of renal interstitial fibrosis by suppressing oxidative stress and ERK/MAPK signaling pathway.

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.

Abstract: The aim of this study was to investigate the protective effect of fluorofenidone (5‐methyl‐1‐[3‐fluorophenyl]‐2‐[1H]‐pyridone, AKF‐PD) on ultraviolet (UV)‐A‐induced senescence in human dermal fibroblasts (HDF) and examine the mechanisms involved. HDF were treated with AKF‐PD. Senescence‐associated (SA)‐β‐galactosidase level, cell viability and expression of p16 were evaluated. In addition, UV‐A‐irradiated HDF were treated with AKF‐PD, rapamycin and MHY1485; SA‐β‐galactosidase staining, 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assay and western blot for SIRT1 were performed; and phosphorylated mammalian target of rapamycin (p‐mTOR) expression and reactive oxygen species (ROS) levels were measured. Intracellular ROS was detected by the 2′,7′‐dichlorofluroescein diacetate probe. Our results showed that AKF‐PD substantially attenuated the changes of p16 expression, SA‐β‐galactosidase staining and cellular proliferation induced by UV‐A irradiation in HDF. AKF‐PD rescued the increased mTOR phosphorylation and reduced SIRT1 expression induced by UV‐A irradiation in HDF. AKF‐PD and rapamycin together had a synergistic effect on p‐mTOR reduction and SIRT1 increase. mTOR activator MHY1485 partly blocked the above effects. Moreover, intracellular ROS level induced by UV‐A irradiation could partly decrease by AKF‐PD, and MHY1485 could reduce this effect. Our results indicated that AKF‐PD could alleviate HDF senescence induced by UV‐A‐irradiation by inhibiting the p‐mTOR and increasing SIRT1. Moreover, AKF‐PD may be a potential treatment material for skin. [ABSTRACT FROM AUTHOR]

Interleukin ( IL)-1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL-1β is dependent upon caspase-1-containing multiprotein complexes called inflammasomes and IL-1R1/MyD88/ NF-κB pathway. In this study, we explored whether a potential anti-fibrotic agent fluorofenidone ( FD) exerts its anti-inflammatory and anti-fibrotic effects through suppressing activation of NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and the IL-1β/ IL-1R1/MyD88/ NF-κB pathway in vivo and in vitro. Male C57 BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α-smooth muscle actin (α- SMA), fibronectin, collagen I, caspase-1, IL-1R1, MyD88 were measured by Western blot and/or RT- PCR. The human actue monocytic leukaemia cell line (THP-1) were incubated with monosodium urate ( MSU), with or without FD pre-treatment. The expression of caspase-1, IL-1β, NALP3, apoptosis-associated speck-like protein containing (ASC) and pro-caspase-1 were measured by Western blot, the reactive oxygen species ( ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome-associated molecules were measured by Co-immunoprecipitation. RLE-6TN (rat lung epithelial-T-antigen negative) cells were incubated with IL-1β, with or without FD pre-treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL-1β, IL-6, monocyte chemotactic protein-1 ( MCP-1), myeloperoxidase ( MPO), α- SMA, fibronectin, collagen I, caspase-1, IL-1R1 and MyD88 in mice lung tissues. And FD inhibited MSU-induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome-associated molecules, decreased the level of caspase-1 and IL-1β in THP-1 cells. Besides, FD inhibited IL-1β-induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM-induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL-1β/ IL-1R1/MyD88/ NF-κB pathway. [ABSTRACT FROM AUTHOR]

Pirfenidone, a pyridone compound, is an effective and novel antifibrotic agent. In this article, we describe the design, synthesis and activity evaluation of novel antifibrotic agents, 1-(substituted aryl)-5-trifluoromethyl-2(1H) pyridones modified with carbohydrate. Most of the title compounds exhibited comparable or better inhibitory activity than fluorofenidone. Notably, compound 19a demonstrated the highest cell-based inhibitory activity against NIH 3T3 (IC50 = 0.17 mM).

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]

Fluorofenidone is a novel derivative of l -mimosine. It has remarkable anti-fibrotic properties. In this study, we established that fluorofenidone ameliorates pulmonary fibrosis (PF) both in vivo and in vitro by specifically inhibiting the expression of eukaryotic translation initiation factor 3a (eIF3a). eIF3a plays an important role in the development and progression of PF. An animal model of PF was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Rats were orally administered with fluorofenidone (250, 500 mg/kg/d·[i.g.]) and pirfenidone (500 mg/kg/d·[i.g.]) for 28 days. Primary pulmonary fibroblasts were cultured to determine the effect of fluorofenidone on TGF-β 1 -induced (5 ng/ml) proliferation and differentiation of fibroblasts. The expression/level of eIF3a, TGF-β 1 , α-SMA, collagen I, and collagen III were analyzed by ELISA, real-time PCR, and western blot. The cell proliferation rate was determined by MTS assay. The results indicate that fluorofenidone significantly improves the pathological changes in lung tissues and reduces the deposition of collagen by inhibiting eIF3a in rats with bleomycin-induced PF. Moreover, in a culture of pulmonary fibroblasts, fluorofenidone decreased the up-regulation of TGF-β 1 -induced eIF3a by inhibiting the proliferation of cells and reducing the expression of α-SMA, collagen I, and collagen III. These findings suggest that eIF3a is a new and special target of fluorofenidone, which could be potentially used in the development of a drug that treats PF. [ABSTRACT FROM AUTHOR]

Signaling through the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, especially JAK2/STAT3, is involved in renal fibrosis. Fluorofenidone (FD), a novel pyridone agent, exerts anti-fibrotic effects in vitro and in vivo. Herein, we sought to investigate whether FD demonstrates its inhibitory function through preventing JAK2/STAT3 pathway. In this study, we examined the effect of FD on activation of rat renal interstitial fibroblasts, glomerular mesangial cells (GMC), and expression of JAK2/STAT3. Moreover, we explored the histological protection effects of FD in UUO rats, db/db mice, and phosphorylation of JAK2/STAT3 cascade. Our studies found that pretreatment with FD resulted in blockade of activation of fibroblast and GMC manifested by fibronectin (FN) and α-smooth muscle actin (α-SMA) protein expression and decline of STAT3 tyrosine phosphorylation induced by IL-6 or high glucose. In unilateral ureteral obstruction rats and a murine model of spontaneous type 2 diabetes (db/db mice), treatment with FD blocked the expression of FN and α-SMA, prevented renal fibrosis progression, and attenuated STAT3 activation. However, FD administration did not interfere with JAK2 activation both in vivo and in vitro. In summary, the molecular mechanism by which FD exhibits renoprotective effects appears to involve the inhibition of STAT3 phosphorylation. [ABSTRACT FROM AUTHOR]

Interleukin-1 beta (IL-1β) is a potent pro-inflammatory and pro-fibrotic cytokine that plays an important role in renal fibrosis. Fluorofenidone (AKF-PD) is a novel pyridone agent that exerts a strong renal anti-fibrotic effect. We previously found that administration of AKF-PD could significantly attenuate IL-1β production in vitro and in vivo. However, the underlying mechanism is not fully understood. Here we show that AKF-PD has no effect on the expression of pro-IL-1β in activated mouse macrophages in vitro. Instead, AKF-PD inhibits the inflammasome, lowering caspase-1 levels and thereby decreasing cleavage of pro-IL-1β into IL-1β. AKF-PD was found to block inflammasome activity induced by various signals, including ATP, alum crystals, and Salmonella typhimurium . These results provide a novel mechanistic insight into how AKF-PD exerts its anti-inflammatory and anti-fibrotic activities, and suggest that AKF-PD might block IL-1β production via suppression of inflammasomes in renal fibrosis. In addition, the results suggest that AKF-PD may be of therapeutic potential in other inflammasome-related diseases. [ABSTRACT FROM AUTHOR]

Background/Aims: We evaluated the therapeutic effects of fluorofenidone (AKF-PD), a novel pyridone agent, targeting oxidative stress and fibrosis in obstructive nephropathy. Methods: AKF-PD was used to treat renal interstitial fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of NOX2 (gp91phox), fibronectin and extracellular signal regulated kinase (ERK) were detected by western blot. A level of Malondialdehyde (MDA), an oxidative stress marker, was measured by ELISA. In addition, ROS and the expressions of NOX2, collagen I (a1), fibronectin and p-ERK were measured in angiotensin (Ang) II-stimulated rat proximal tubular epithelial cells (NRK-52E) in culture. Results: In NRK-52E cells, AKF-PD reduced AngII induced expressions of ROS, NOX2, fibronectin, collagen I (a1) and p-ERK. In UUO kidney cortex, AKF-PD attenuated the degree of renal interstitial fibrosis, which was associated with reduced the expressions of collagen I (a1) and fibronectin. Furthermore, AKF-PD downregulated the expressions of NOX2, MDA and p-ERK. Conclusion: AKF-PD treatment inhibits the progression of renal interstitial fibrosis by suppressing oxidative stress and ERK/MAPK signaling pathway. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Fluorofenidone (AKF-PD) is a novel pyridone agent. The purpose of this study is to investigate the inhibitory effects of AKF-PD on liver fibrosis in rats and the involved molecular mechanism related to hepatic stellate cells (HSCs). Rats treated with dimethylnitrosamine or CCl4 were randomly divided into normal, model, AKF-PD treatment, and pirfenidone (PFD) treatment groups. The isolated primary rat HSCs were treated with AKF-PD and PFD respectively. Cell proliferation and cell cycle distribution were analyzed by bromodeoxyuridine and flow cytometry, respectively. The expression of collagen I and a-smooth muscle actin (a-SMA) were determined by Western blot, immunohistochemical staining, and real-time RT-PCR. The expression of cyclin D1, cyclin E, and p27kip1 and phosphorylation of MEK, ERK, Akt, and 70-kDa ribosomal S6 kinase (p70S6K) were detected by Western blot. AKF-PD significantly inhibited PDGF-BB-induced HSC proliferation and activation by attenuating the expression of collagen I and a-SMA, causing G0/G1 phase cell cycle arrest, reducing expression of cyclin D1 and cyclin E, and promoting expression of p27kip1. AKF-PD also downregulated PDGF-BB-induced MEK, ERK, Akt, and p70S6K phosphorylation in HSCs. In rat liver fibrosis, AKF-PD alleviated hepatic fibrosis by decreasing necroinflammatory score and semiquantitative score, and reducing expression of collagen I and a-SMA. AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs proliferation and activation via the ERK/MAPK and PI3K/Akt signaling pathways. AKF-PD may be used as a potential novel therapeutic agent against liver fibrosis. [ABSTRACT FROM AUTHOR]

Fluorofenidone (AKF-PD) is a novel pyridone derivate that targets transforming growth factor-β1 (TGF-β1) signaling. Previous studies have proven that AKF-PD functions as an antifibrotic agent in pulmonary fibrosis and renal fibrosis models. Activated TGF-β1 signaling is thought to be a major feature of pulmonary hypertension (PH). TGF-β1 exerts powerful pro-proliferation effects on pulmonary arterial smooth muscle cells (PASMCs), and hence, prompts vascular remodeling. This study is designed to investigate the effect of AKF-PD on vascular remodeling in a rat model of hypoxia-induced PH. PH was induced in rats by 4 weeks of hypoxia. The expression of TGF-β1, collagen I, and collagen III was analyzed by ELISA, immunohistochemistry, real-time PCR, or Western blot. Proliferation of cultured PASMCs was determined by the BrdU incorporation method and flow cytometry. The results showed that AKF-PD treatment (0.5 or 1.0 g·(kg body mass)·d−1) for 4 weeks attenuated pulmonary vascular remodeling and improved homodynamic parameters. TGF-β1 level was significantly down-regulated by AKF-PD both in vivo and in vitro. Furthermore, hypoxia- and TGF-β1-induced PASMC proliferation and collagen expression were both significantly suppressed by AKF-PD. These results suggest that AKF-PD ameliorates the progression of PH induced by hypoxia in rats through its regulation of TGF-β1 expression, PASMC proliferation, and the extracellular matrix. [ABSTRACT FROM AUTHOR]

Aim Oxidative stress plays an important role in the progression of renal interstitial fibrosis. The nicotinamide adeninedinucleotide phosphate ( NADPH) oxidase ( Nox) family is considered one of the major sources of reactive oxygen species ( ROS). In the present study, we investigated the inhibitory effects of a novel anti-fibrotic agent, Fluorofenidone ( AKF-PD), upon Nox-mediated oxidative stress and deposition of extracellular matrix ( ECM) in the development of renalinterstitial fibrosis. Methods AKF-PD was used to treat renal fibrosis in unilateral ureteral obstruction ( UUO) obstructive nephropathy in rats. The expression of Nox homologues, p- Akt, collagen I and III were detected by immunoblotting or immunohistochemistry. Levels of 8-iso prostaglandin F2alpha (8- Iso PGF2a) was measured by enzyme linked immunosorbent assay. In addition, ROS and the expression of collagen I (1a), Nox subunits and p- Akt was measured in angiotensin ( Ang) II-stimulated rat proximal tubular epithelial ( NRK-52E) cells in culture. Results AKF-PD treatment significantly attenuated tubulo-interstitial injury, ECM deposition and oxidative stress in fibrotic rat kidneys. In addition, AKF-PD inhibited the expression of ROS, Collagen I (1a), Nox2, p- Akt in Ang II-stimulated NRK-52 E cells. Conclusion AKF-PD attenuates the progression of renal interstitial fibrosis partly by suppressing NADPH oxidase and ECM deposition via the PI3K/Akt signalling pathway, suggesting AKF-PD is a potential novel therapeutic agent against renal fibrosis. [ABSTRACT FROM AUTHOR]

Background Fluorofenidone ( AKF- PD) is a novel pyridone agent. The purpose of this study is to investigate the inhibitory effects of AKF- PD on dimethylnitrosamine ( DMN)-induced liver fibrosis in rats and the involved molecular mechanism related to hepatic stellate cells ( HSCs). Materials and methods Wistar rats were randomly divided into normal control, DMN, DMN/ AKF- PD treatment and DMN/pirfenidone ( PFD) treatment groups. AKF- PD and PFD treatments were, respectively, performed for two activated HSCs lines, rat CFSC-2G and human LX2. The cell proliferation was analysed by MTT. The expression of collagen I was determined by immunohistochemical staining and real-time RT- PCR. The expression of α-smooth muscle actin (α- SMA), tissue inhibitor of metalloproteinases-1 ( TIMP-1), extracellular signal regulated kinase ( ERK1/2), p38 MAPK (p38), and c-Jun N-terminal kinase/stress-activated protein kinase ( JNK) were also detected by real-time RT- PCR and/or Western blot. Results AKF-PD significantly reduced PDGF-BB-induced proliferation and activation of HSCs, as determined by reducing protein expression of α-SMA and TIMP-1. AKF-PD treatment attenuated PDGF-BB-induced upregulation of phosphorylation of ERK1/2, p38 and JNK. In fibrotic rat liver, AKF-PD reduced the degree of liver injury and hepatic fibrosis, which was associated with reduced the expression of collagen I, α-SMA, TIMP-1 at both mRNA and protein levels. Conclusion AKF- PD treatment inhibits the progression of hepatic fibrosis by suppressing HSCs proliferation and activation via MAPK signalling pathway. [ABSTRACT FROM AUTHOR]

Pirfenidone, a pyridone compound, is an effective and novel antifibrotic agent. In this article, we describe the design, synthesis and activity evaluation of novel antifibrotic agents, 1-(substituted aryl)-5-trifluoromethyl-2(1H) pyridones modified with carbohydrate. Most of the title compounds exhibited comparable or better inhibitory activity than fluorofenidone. Notably, compound 19a demonstrated the highest cell-based inhibitory activity against NIH 3T3 (IC50 = 0.17 mM). [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The present study was designed to investigate the inhibitory effects of fluorofenidone on Ang II-induced apoptosis in renal tubular cells and the related signaling pathway. Methods: Rat proximal tubular epithelial cells (NRK-52E) were used to examine the anti-apoptosis effects of fluorofenidone. Cell proliferation was assessed by methyl thiazolyl tetrazolium assay. Apoptosis was examined by AO/EB staining and TUNEL assay. The expression of Fas/FasL pathway members, including Fas, FasL, Bax, Bcl-2, Caspase-8, and Caspase-3 was detected by real-time RT-PCR and/or Western blot, respectively. The activity of Caspase-8 and Caspase-3 was detected by spectrophotometry. Results: Fluorofenidone didn''t affect the proliferation of NRK-52E cells, but significantly inhibited the apoptosis of NRK-52E cells induced by Ang II. Fluorofenidone significantly reduced Ang II-induced increases in Fas, FasL, Bax, Caspase-8 and Caspase-3 at the mRNA level. Consistent with these observations, fluorofenidone also prevented Ang II-mediated up-regulation of FasL and Bax at the protein level. Additionally, Ang II-induced activation of Caspase-8 and Caspase-3 as well as Ang II-initiated downregulation of Bcl-2 at both mRNA and protein levels was all prevented by fluorofenidone. Conclusions: Fluorofenidone can inhibit Ang II-induced apoptosis of renal tubular cells through blockage of the Fas/FasL pathway. [Copyright &y& Elsevier]

Background/Aims: Fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD], a novel pyridone agent, showed potent antifibrotic properties. The aim of the present study was to investigate the effects of AKF-PD on diabetic nephropathy and kidney fibrosis, and to obtain an insight into its mechanisms of action. Methods: We administered AKF-PD to diabetic db/db mice for 12 weeks. Moreover, we performed in vitro cultures using murine mesangial cells exposed to high ambient glucose concentrations. Results: AKF-PD reduced renal hypertrophy, mesangial matrix expansion and albuminuria in the db/db mice. The upregulated expression of α1(I)- and α1(IV)-collagen and fibronectin mRNAs, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and tissue inhibitors of metalloproteinase 1 (TIMP-1) mRNAs and proteins was inhibited by AKF-PD treatment in the renal cortex of db/db mice. The maximal effective dose of AKF-PD was about 500 mg/kg body weight. AKF-PD inhibited the upregulated expression of α1(I)- and α1(IV)-collagens, TGF-β1, TIMP-1 and α-SMA induced by high glucose concentrations in cultured mesangial cells. Conclusions: Our data indicate that AKF-PD diminishes the abnormal accumulation of mesangial matrix through the inhibition of upregulated expression of TGF-β target genes in kidneys of db/db mice, resulting in attenuation of renal fibrosis and amelioration of renal dysfunction despite persistent hyperglycemia. Therefore, AKF-PD, a potent antifibrotic agent, holds great promise in the treatment of diabetic nephropathy. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Fluorofenidone (FD) is a novel pyridone agent with significant antifibrotic effects in vitro. The purpose of this study is to investigate the effects of FD on renal interstitial fibrosis in rats with obstructive nephropathy caused by unilateral ureteral obstruction (UUO). With pirfenidone (PD, 500 mg/kg/day) and enalapril (10 mg/kg/day) as the positive treatment controls, the rats in different experimental groups were administered with FD (500 mg/kg/day) from day 4 to day 14 after UUO. The tubulointerstitial injury, interstitial collagen deposition, and expression of type I and type III collagen, transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), α-smooth muscle actin (α-SMA), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed. FD treatment significantly attenuated the prominently increased scores of tubulointerstitial injury, interstitial collagen deposition, and protein expression of type I and type III collagen in ureter-obstructed kidneys, respectively. As compared with untreated rats, FD also significantly reduced the expression of α-SMA, TGF-β, CTGF, PDGF, and inhibitor of TIMP-1 in the obstructed kidneys. Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy through its regulation on fibrogenic growth factors, tubular cell transdifferentiation, and extracellular matrix. [ABSTRACT FROM AUTHOR]

Aim: Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone) is a novel pyridone agent. The aim of the present study is to investigate the effects of fluorofenidone on angiotensin (Ang)II-induced fibrosis and the involved molecular mechanism in rat proximal tubular epithelial cells. Methods: NRK-52E cells, a rat proximal tubular epithelial cell line, were incubated with medium containing AngII, with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), losartan, fluorofenidone (2, 4 and 8 mmol/L) and pirfenidone (8 mmol/L) for 24 h. Cells in the serum-free medium were controls. The expression of three subunits of NADPH oxidase, including p47phox, Nox-4 and p22phox, were determined by real-time reverse transcription polymerase chain reaction (RT–PCR) and western blot. NADPH oxidase activity was measured directly by superoxide dismutase (SOD) inhibitable cytochrome C reduction assay. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein fluorescence analysis. The mRNA and protein expression of collagen I and transforming growth factor (TGF)-β1 were determined by real-time RT–PCR and enzyme-linked immunosorbent assay. Results: Fluorofenidone significantly inhibited TGF-β1 and collagen I expression upregulation induced by AngII or TGF-β1 respectively. Moreover, fluorofenidone greatly reduced the elevation of expression and activity of NADPH oxidase and inhibited ROS generation induced by AngII in rat proximal tubular epithelial cells. These responses were also attenuated by DPI, losartan, and pirfenidone. Conclusion: Fluorofenidone acted as an anti-oxidative and anti-fibrotic agent through the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-β1 expression in rat proximal tubular epithelial cells. [ABSTRACT FROM AUTHOR]

Background: Fluorofenidone is a novel antifibrotic drug and its aqueous solubility is low. Aim: This study was to prepare and characterize inclusion complexes of fluorofenidone (AKF-PD) with β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD). Method: The AKF-PD/cyclodextrins (CDs) inclusion complexes were prepared by coprecipitation and freeze-drying, respectively. The solubility enhancement of AKF-PD was evaluated by phase solubility method. Inclusion complexation in solid phase was studied by X-ray diffraction (XRD) and differential thermal analysis (DTA). The dissolution profiles of AKF-PD/CDs inclusion complexes were investigated and compared with those of their physical mixtures and AKF-PD alone. Results: The phase solubility diagrams of AKF-PD with β-CD and HP-β-CD were of AL-types, and the solubility of AKF-PD could be increased by 51.5% for β-CD at 0.014 M and 794.0% for HP-β-CD at 0.254 M. The results from XRD and DTA suggested that AKF-PD could form inclusion complex with β-CD or HP-β-CD. The dissolution rate of AKF-PD from the inclusion complexes was much more rapid than AKF-PD alone. Conclusions: The formulation of AKF-PD/CDs inclusion complexes showed superior performance in improving dissolution properties of AKF-PD. [ABSTRACT FROM AUTHOR]

Paraquat (PQ) is a widely used herbicide that is severely toxic to humans and animals. Pulmonary fibrosis is a disorder that can result from PQ poisoning. Fluorofenidone (AKF-PD) is a novel small molecule pyridone drug with a widespread and clear anti-organ fibrosis effect; however, its mechanism of action on PQ poisoning-induced pulmonary fibrosis is not clear. The purpose of the present study was to investigate the protective effect and underlying mechanism of AKF-PD on PQ poisoning-induced pulmonary fibrosis. Human alveolar epithelial cells (HPAEpiC) and Sprague-Dawley rats were treated with AKF-PD in the presence or absence of PQ. Hematoxylin-eosin and Masson staining were used to observe the morphological changes in lung tissue. Cell Counting Kit-8 and lactate dehydrogenase assays were used to evaluate the viability of HPAEpiC cells. ELISA was used to detect inflammatory factors and the collagen content. Finally, the effects of AKF-PD on pulmonary fibrosis, as well as the underlying mechanisms, were evaluated via western blotting, reverse transcription-quantitative PCR and immunofluorescence analysis. AKF-PD effectively alleviated PQ-induced pulmonary fibrosis and reduced the expression of oxidative stress and inflammatory factors. Moreover, AKF-PD treatment effectively inhibited the PI3K/Akt/mTOR signaling pathway and upregulated autophagy. Overall, these findings suggested that AKF-PD can alleviate PQ-induced inflammation and pulmonary fibrosis by inhibiting the PI3K/Akt/mTOR signaling pathway and by upregulating autophagy. [ABSTRACT FROM AUTHOR]