Evaluation of Acute and Convalescent Antibody Concentration Against Pneumococcal Capsular Polysaccharides for the Diagnosis of Pneumococcal Infection in Children with Community-Acquired Pneumonia.

We evaluated whether the quantification of IgG to pneumococcal capsular polysaccharides is an accurate diagnostic test for pneumococcal infection in children with pneumonia in Nepal. Children with pneumococcal pneumonia did not have higher convalescent, or higher fold change, IgG to pneumococcal polysaccharides than children with other causes of pneumonia. Caution is needed in interpreting antibody responses in pneumococcal infections.

Euonymus alatus Leaf Extract Attenuates Effects of Aging on Oxidative Stress, Neuroinflammation, and Cognitive Impairment.

Our study aimed to explore the impact and mechanism of Euonymus alatus leaf extract on age-dependent oxidative stress, neuroinflammation, and progressive memory impairments in aged mice. Twenty-four-month-old mice received EA-L3 (300 mg/kg/day) or the reference drug, donepezil (DPZ, 5 mg/kg/day), for 6 weeks, and learning and memory functions were detected using the Passive Avoidance Test (PAT). As expected, cognitive function deficits were detected in aged mice compared with young mice, and these deficits were significantly mitigated by dietary treatments with EA-L3. In parallel, it upregulated the brain-derived neurotrophic factor (BDNF) and subsequently activated the extracellular-signal-regulated kinase (ERK)/cAMP response element-binding (CREB) signaling in the mouse hippocampus and scopolamine-induced B35 and SH-SY5Y neuroblastoma cells. EA-L3 showed strong anti-inflammatory effects with decreased NF-κBp65, cyclooxygenase 2 (COX-2), and tumor necrosis factor alpha (TNF-α), increased interleukin (IL)-10, and doublecortin (DCX) protein expression in the hippocampus of aged mice. Similar results were also confirmed in LPS-induced BV-2 microglia and neuroblastoma cells upon treatment with EA-L3 extract. In addition, EA-L3 notably dose-dependently decreased ROS in BV2 cells after exposure to LPS. Taken together, EA-L3 might be used as a dietary supplement to alleviate oxidative stress, the deterioration of hippocampal-based memory tasks, and neuroinflammation in elderly people.

Efficient Assessment of Tumor Vascular Shutdown by Photodynamic Therapy on Orthotopic Pancreatic Cancer Using High-Speed Wide-Field Waterproof Galvanometer Scanner Photoacoustic Microscopy.

To identify the vascular alteration by photodynamic therapy (PDT), the utilization of high-resolution, high-speed, and wide-field photoacoustic microscopy (PAM) has gained enormous interest. The rapid changes in vasculature during PDT treatment and monitoring of tumor tissue activation in the orthotopic pancreatic cancer model have received limited attention in previous studies. Here, a fully two-axes waterproof galvanometer scanner-based photoacoustic microscopy (WGS-PAM) system was developed for in vivo monitoring of dynamic variations in micro blood vessels due to PDT in an orthotopic pancreatic cancer mouse model. The photosensitizer (PS), Chlorin e6 (Ce6), was utilized to activate antitumor reactions in response to the irradiation of a 660 nm light source. Microvasculatures of angiogenesis tissue were visualized on a 40 mm2 area using the WGS-PAM system at 30 min intervals for 3 h after the PDT treatment. The decline in vascular intensity was observed at 24.5% along with a 32.4% reduction of the vascular density at 3 h post-PDT by the analysis of PAM images. The anti-vascularization effect was also identified with fluorescent imaging. Moreover, Ce6-PDT increased apoptotic and necrotic markers while decreasing vascular endothelial growth factor (VEGF) expression in MIA PaCa-2 and BxPC-3 pancreatic cancer cell lines. The approach of the WGS-PAM system shows the potential to investigate PDT effects on the mechanism of angiographic dynamics with high-resolution wide-field imaging modalities.

Chlorin E6-Curcumin-Mediated Photodynamic Therapy Promotes an Anti-Photoaging Effect in UVB-Irradiated Fibroblasts.

Skin photoaging due to ultraviolet B (UVB) exposure generates reactive oxygen species (ROS) that increase matrix metalloproteinase (MMP). Chlorin e6-photodynamic therapy (Ce6-PDT), in addition to being the first-line treatment for malignancies, has been shown to lessen skin photoaging, while curcumin is well known for reducing the deleterious effects of ROS. In the current study, PDT with three novel Ce6-curcumin derivatives, a combination of Ce6 and curcumin with various linkers, including propane-1,3-diamine for Ce6-propane-curcumin; hexane-1,6-diamine for Ce6-hexane-curcumin; and 3,3'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(propan-1-amine) for Ce6-dipolyethylene glycol (diPEG)-curcumin, were studied for regulation of UVB-induced photoaging on human skin fibroblast (Hs68) and mouse embryonic fibroblast (BALB/c 3T3) cells. We assessed the antiphotoaging effects of Ce6-curcumin derivatives on cell viability, antioxidant activity, the mechanism of matrix metalloproteinase-1 and 2 (MMP-2) expression, and collagen synthesis in UVB-irradiated in vitro models. All three Ce6-curcumin derivatives were found to be non-phototoxic in the neutral red uptake phototoxicity test. We found that Ce6-hexane-curcumin-PDT and Ce6-propane-curcumin-associated PDT exhibited less cytotoxicity in Hs68 and BALB/c 3T3 fibroblast cell lines compared to Ce6-diPEG-curcumin-PDT. Ce6-diPEG-curcumin and Ce6-propane-curcumin-associated PDT showed superior antioxidant activity in Hs68 cell lines. Further, in UVB-irradiated in vitro models, the Ce6-diPEG-curcumin-PDT greatly attenuated the expression levels of MMP-1 and MMP-2 by blocking mitogen-activated protein kinases (MAPKs), activator protein 1 (AP-1), and tumor necrosis factor-α (NF-κB) signaling. Moreover, Ce6-diPEG-curcumin effectively inhibited inflammatory molecules, such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, while accelerating collagen synthesis. These results demonstrate that Ce6-diPEG-curcumin may be a potential therapy for treating skin photoaging.

Anti-Obesity Effect of Chlorin e6-Mediated Photodynamic Therapy on Mice with High-Fat-Diet-Induced Obesity.

This study aimed to evaluate the efficacy of Chlorin e6 (Ce6)-based photodynamic therapy (PDT) for anti-obesity activities in high-fat-diet (HFD)-induced obesity mouse models. We induced obesity in C57BL/6 mice by HFD and administered Ce6 (2.5 or 5 mg/kg) orally with 3 h of incubation. The mice were then exposed to light of high fluence rate (4.96 mW/cm2) or low fluence rate (2.56 mW/cm2) in the designed LED mouse chamber 2-3 days a week for up to 8 weeks. The study also analyzed the pharmacokinetics and optimization of the drug by evaluating the absorption, distribution, metabolism, and excretion (ADME) of Ce6 in the rat models. Both low doses (2.5 mg/kg) and high doses (5 mg/kg) of Ce6 with high irradiation dose showed better anti-obesity effects than other groups with decreased body weight. The lipid accumulation in the liver and adipocyte size in epididymal adipose tissues were found to be decreased by Ce6-PDT in comparison to vehicle-treated HFD groups. We also observed increased levels of the lipidomic biomarkers, such as leptin and LDL cholesterol, while observing decreasing levels of total cholesterol and adiponectin in the Ce6-PDT-treated mice. These findings may provide valuable insight into Ce6-PDT as an alternative and non-invasive therapeutic methodology for obesity and obesity-related diseases.

Novel Chlorin e6-Curcumin Derivatives as a Potential Photosensitizer: Synthesis, Characterization, and Anticancer Activity.

Novel series of chlorin e6-curcumin derivatives were designed and synthesized. All the synthesized compounds 16, 17, 18, and 19 were tested for their photodynamic treatment (PDT) efficacy against human pancreatic cancer cell lines: AsPC-1, MIA-PaCa-2, and PANC-1. The cellular uptake study was performed in the aforementioned cell lines using fluorescence-activated cell sorting (FACS). 17, among the synthesized compounds with IC50 values of 0.27, 0.42, and 0.21 µM against AsPC-1, MIA PaCa-2, and PANC-1 cell lines, respectively, demonstrated excellent cellular internalization capability and exhibited higher phototoxicity relative to the parent Ce6. The quantitative analyses using Annexin V-PI staining revealed that the 17-PDT-induced apoptosis was dose-dependent. In pancreatic cell lines, 17 reduced the expression of the anti-apoptotic protein, Bcl-2, and increased the pro-apoptotic protein, cytochrome C, which indicates the activation of intrinsic apoptosis, the primary cause of cancer cell death. Structure-activity relationship studies have shown that the incorporation of additional methyl ester moiety and conjugation to the enone moiety of curcumin enhances cellular uptake and PDT efficacy. Moreover, in vivo PDT testing in melanoma mouse models revealed that 17-PDT greatly reduced tumor growth. Therefore, 17 might be an effective photosensitizer for PDT anticancer therapy.

Chlorin e6-associated photodynamic therapy enhances abscopal antitumor effects via inhibition of PD-1/PD-L1 immune checkpoint.

We hypothesized that photodynamic therapy (PDT) with Chlorin e6 (Ce6) enhances antitumor abscopal effects via inhibition of the programmed cell death-1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint. By using syngeneic melanoma and pancreatic tumor mouse models, we studied the Ce6-PDT-induced immune responses in local and distant tumor microenvironments. In addition, the Ce6-PDT's target in the PD-1/PD-L1 interaction was analyzed in MC38-hPD-L1 colon cancer and PD-1 expressing Jurkat T cell coculture. The tumors in the irradiated and non-irradiated sites in the abscopal effective (Abseff) group of both mouse models were regressed, proving the abscopal effect. The immunogenic effect in the Abseff group was associated with an expansion of T cell and other immune cells infiltration without changes in the CD39+ population in either the right or left tumors compared to control group. Furthermore, the abscopal ineffective (Absineff) group demonstrated lesser increase of T cells, decreased immune cell infiltration, and increased CD39-expressing Treg cells without suppression of tumor growth. In the coculture with PD-1-expressing Jurkat T cell, Ce6-PDT efficiently suppressed the PD-1/PD-L1 interactions by increasing the proliferation and cytotoxic activity of CD8+ T cells while decreasing CD39-expressing Treg cells in a dose-dependent manner. Likewise, the inhibition of PD-1/PD-L1 interactions was also correlated with the increased production of IL-2 and Granzyme B. Our findings imply that Ce6-PDT is a promising immunotherapy with the potential to improve the abscopal effect.

Efficient Synthesis of Chlorin e6 and Its Potential Photodynamic Immunotherapy in Mouse Melanoma by the Abscopal Effect.

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an antitumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of Chlorin e6 (Ce6) from Spirulina platensis and address the phototoxic effect of Ce6 in vitro along with antitumor activity in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. The C57BL/6 mice were subcutaneously inoculated on the left and right flank with B16F10 cells. The mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors 3 h after the injection. The immune response was studied by analyzing Interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The upregulated gene and protein expression of IFN-γ, TNF-α, and IL-2 revealed antitumor immunity due to Ce6-PDT. The findings of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising antitumor immune response.

Effect of Photodynamic Therapy with Chlorin e6 on Canine Tumors.

This work aims to prepare pure Chlorin e6 (Ce6) and establish Ce6-mediated photodynamic therapy (Ce6-PDT) as a better therapy option for canine tumors as well as mouse tumor models. Five dogs suffering from various cancers were treated with Ce6-PDT from one to several times. After receiving the Ce6 (2.5 mg/kg) for 3 h, tumors were illuminated superficially or interstitially with 660 nm light. Two dogs underwent Ce6-guided fluorescence imaging by photodynamic diagnosis (PDD). Cell proliferation and apoptosis were detected by the 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and western blot assay, respectively. Ce6-PDT efficacy was also determined using melanoma and pancreatic cancer mouse models. Two veterinary patients with mammary carcinoma and histiocytic sarcoma had their tumors significantly diminished and showed improved health after receiving Ce6-PDT. Moreover, in the cases of canine tumors, the adjunctive use of Ce6-PDD revealed cancers that were not visible with white light viewing and provided a visual contrast from surrounding tissues. Also, in vivo, Ce6-PDT remarkably reduced melanoma and pancreatic tumors in the mouse model. These findings could pave the way for a better understanding of the underlying processes of Ce6-PDT, making it an effective and safe candidate for use in human and veterinary applications to abolish various cancers.

Euonymus alatus Twig Extract Protects against Scopolamine-Induced Changes in Brain and Brain-Derived Cells via Cholinergic and BDNF Pathways.

In the current study, the therapeutic and preventive effects of Euonymus alatus (EA) twig extract were investigated in a mouse model of cognitive deficit and B35 cells. Twig extract 1 was extracted with 70% ethanol and later twig extract 2 was extracted through liquid-liquid extraction with 70% ethanol and hexane. EA twig 2 (300 mg/kg) along with the standard drug donepezil (5 mg/kg) were orally administered to the mice for 34 days. Scopolamine was given intraperitoneally for 7 days. Administration of EA twig extract 2 significantly improved the passive avoidance test (PAT) in mice. EA twigs extract also restored the scopolamine-reduced brain-derived neurotrophic factor (BDNF)/extracellular regulated kinase (ERK)/cyclic AMP responsive element binding protein (CREB) signaling in B35 cells and the mouse hippocampus. In addition, EA twig extract significantly inhibited the acetylcholine esterase (AChE) activity in B35 cells in a dose-dependent manner. Chromatography and ESI MS analysis of EA twig extract revealed the presence of flavonoids; epicatechin, taxifolin, aromadendrin, and naringenin with catechin being the most abundant. These flavonoids exerted protective effects alone and had the possibility of synergistic effects in combination. Our work unmasks the ameliorating effect of EA twig extract 2 on scopolamine-associated cognitive impairments through the restoration of cholinergic systems and the BDNF/ERK/CREB pathway.

Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK.

BACKGROUND: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation. METHODS: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKß1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot. RESULTS: Genetic deletion of AMPKß1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the ß1 subunit. CONCLUSIONS: Together, these data suggest that the development of therapeutic agents targeting AMPKß1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease.

AMPK is essential for IL-10 expression and for maintaining balance between inflammatory and cytoprotective signaling.

BACKGROUND: AMP-activated protein kinase (AMPK) exerts its anti-inflammatory effects by suppressing redox-sensitive nuclear factor kappa B (NF-κB) and pro-inflammatory cytokines including TNF-α. However, it is unclear whether AMPK regulates anti-inflammatory cytokine expressions in the presence of oxidative stress-induced inflammation. We sought to elucidate the mechanisms whereby AMPK regulates inflammatory cytokine expressions under NADPH oxidase (NOX)-induced oxidative stress. METHODS: HT-29 human colonic epithelial cells transfected with AMPKα shRNA and mouse models with AMPKα knocked out in epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) were used to examine the effects of AMPK and NOX on signaling pathways and cytokine expressions. RESULTS: In HT-29 cells, 5-hydroxytryptamine (5-HT)-induced NOX activity was enhanced by AMPKα silencing, and resulted in inflammatory cell death. AMPKα deletion specific for colon epithelial cells (AMPKαfl/fl-Vil-Cre) or macrophages (AMPKαfl/fl-Lyz2-Cre) intensified 5-HT- or dextran sulfate sodium (DSS)-induced upregulations of NOX2, TNF-α, and IL-6, but completely abolished basal and 5-HT- or DSS-induced upregulation of IL-10 in colon epithelium. Furthermore, 5-HT- and DSS-induced changes were accompanied by marked upregulations of increased inflammatory signaling pathways linked to NF-κB, AP-1, and STAT3 transcription factors, and to GATA, a cell fate-directing signaling. In addition, AMPKα deletion significantly fortified 5-HT- or DSS-induced downregulations of cytoprotective signaling pathways (Nrf2, HIF-1α, and KLF4). CONCLUSION: Basal AMPKα maintains an anti-inflammatory state by inhibiting NOX, balancing pro-/anti-inflammatory signaling pathways, and directing IL-10 production. When these regulatory roles of AMPK are diminished by oxidative stress, colon epithelium undergoes inflammation despite IL-10 production.

Potent Inhibitory Effect of BJ-3105, a 6-Alkoxypyridin-3-ol Derivative, on Murine Colitis Is Mediated by Activating AMPK and Inhibiting NOX.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation in the gastrointestinal tract. Biological therapeutics and orally available small molecules like tofacitinib (a JAK inhibitor) have been developed to treat IBD, but half of the patients treated with these drugs fail to achieve sustained remission. In the present study, we compared the therapeutic effects of BJ-3105 (a 6-alkoxypyridin-3-ol derivative) and tofacitinib in IBD. BJ-3105 induced activation of AMP-activated protein kinase (AMPK) in the kinase activity measurement and recovery from cytokine-induced AMPK deactivation in HT-29 human colonic epithelial cells. Similar to tofacitinib and D942 (an AMPK activator), BJ-3105 inhibited IL-6-induced JAK2/STAT3 phosphorylation and TNF-α-stimulated activation of IKK/NF-κB, and consequently, stimulus-induced upregulations of inflammatory cytokines and inflammasome components. In addition, unlike tofacitinib or D942, BJ-3105 inhibited NADPH oxidase (NOX) activation and consequent superoxide production induced by activators (mevalonate and geranylgeranyl pyrophosphate) of the NOX cytosolic component Rac. In mice, oral administration with BJ-3105 ameliorated dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS-induced colitis-associated tumor formation (CAT) much more potently than that with tofacitinib. Moreover, BJ-3105 suppressed the more severe form of colitis and CAT formation in mice with AMPK knocked-out in macrophages (AMPKαfl/fl-Lyz2-Cre mice) with much greater efficacy than tofacitinib. Taken together, our findings suggest BJ-3105, which exerted a much better anti-colitis effect than tofacitinib through AMPK activation and NOX inhibition, is a promising candidate for the treatment of IBD.

Synthesis, activity and mechanism of alkoxy-, carbamato-, sulfonamido-, thioureido-, and ureido-derivatives of 2,4,5-trimethylpyridin-3-ol against inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic immuno-inflammation in gastrointestinal tract. We have evaluated the activity of the compounds to inhibit the adhesion of monocytes to colon epithelial cells is triggered by a pro-inflammatory cytokine, tumour necrosis factor (TNF)-α. The in vitro activity of the compounds, 13b (an ureido-derivative), 14c, 14j, 14k, 14n (thioureido-), 18c and 18d (sulfonamido-), was in correlation with in vivo anti-colitis activity revealed as significant recovery in body- and colon-weights and colon myeloperoxidase level, a biochemical marker of inflammation reflecting neutrophil infiltration. In vivo, TNBS-induced changes in the expression of inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-10, and TGF-ß), NLRP3 inflammasome components (NLRP-3, Caspase-1, and IL-18), and epithelial junction molecules (E-cadherin, claudin2/3, and ZO-1) were blocked and recovered by oral administration of the compounds (1 mg/kg). Compound 14n which showed the best efficacy can be a promising lead for orally available therapeutics for pathology of IBD.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73-1.0), specificity of 0.66 (95% CI 0.52-0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75-0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47-0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Synthesis and evaluation of 6-heteroarylamino-2,4,5-trimethylpyridin-3-ols as inhibitors of TNF-α-induced cell adhesion and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an inflammatory disease of the gastrointestinal tract with complex pathogenesis. Here, we synthesized 6-heteroarylamino analogues to inhibit TNF-α-induced adhesion of monocytes to colon epithelial cells which are implicated in the initial inflammation process of IBD. The best analogue, 16a, showed IC50 = 0.29 µM, which is about five orders of magnitude better than that of 5-aminosalicylic acid (5-ASA), a positive control. Oral administration of 6f and 16a dramatically ameliorated 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colon inflammation in rat. The ameliorating effects were accompanied by a high level of recovery in colon and body weights and in the myeloperoxidase (MPO) level. Consistently, the compounds suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1). Moreover, they significantly suppressed the expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 while increasing the level of IL-10, an anti-inflammatory cytokine.

Synthesis and biological evaluation of pyridine-linked indanone derivatives: Potential agents for inflammatory bowel disease.

A series of pyridine-linked indanone derivatives were designed and synthesized to discover new small molecules for the treatment of inflammatory bowel disease (IBD). Compounds 5b and 5d exhibited strongest inhibitory activity against TNF-α-induced monocyte adhesion to colon epithelial cells (an in vitro model of colitis). In TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced rat colitis model, oral administration of the compounds 5b and 5d ameliorated colitis with significant recovery in altered expressions of E-cadherin, TNF-α and IL-1ß expressions, indicating 5b and 5d as potential agents for therapeutics development against IBD.

Ameliorating effect of TI-1-162, a hydroxyindenone derivative, against TNBS-induced rat colitis is mediated through suppression of RIP/ASK-1/MAPK signaling.

The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro-inflammatory cytokines including TNF-α. Once generated, TNF-α stimulates production of various pro-inflammatory cytokines and disrupts mucosal barrier by inducing inflamed mucosal epithelial cell death. In the present study, we investigated inhibitory effects of TI-1-162, a hydroxyindenone derivative, against TNF-α-induced and TNBS-induced colon inflammation. TI-1-162 showed inhibitory effect on the TNF-α-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells (IC50 = 0.83 ±â€¯0.12 µM), which is an in vitro model representing the initial step of colitis. In addition, TI-1-162 suppressed TNF-α-stimulated caspase-3 activation and HT-29 cell apoptosis. These in vitro inhibitory activities of TI-1-162 correlated to recovery changes in in vivo colon tissues, such as downregulation of adhesion molecules (ICAM-1, VCAM-1) and chemokines (CCL11, CXCL1, CXCL2, CXCL3, CX3CL1) revealed by gene expression array and Western blot analyses. Such molecular recovery of colon epithelium from TNBS-treated rats corresponded to the recovery in body weight, colon weight/length, and myeloperoxidase level by TI-1-162 (10 and 30 mg/kg/day, orally). In relation to action mechanism, TI-1-162 did not disturb TNF-α binding to its receptor, but suppressed phosphorylation of RIP-1, ASK-1, JNK and p38, and nuclear translocation of NF-kB and AP-1, which corresponded to down regulation of inflammatory cytokines in TNF-α-treated cells (HT-29 and U937) and TNBS-treated rat colon tissues. Taken together, the results indicate that the protective effects of TI-1-162 against colon inflammation and epithelial cell death are associated with its inhibitory action in RIP/ASK-1/MAPK signaling pathway downstream to TNF receptor 1.

Discovery and structure-activity relationship studies of 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives as a novel class of potential therapeutics for inflammatory bowel disease.

To develop effective therapeutics for inflammatory bowel disease (IBD), 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives, were designed and synthesized and their structure-activity relationships (SAR) were investigated. Compounds 7, 25, 26, 32, 39, 41, 52, 54, and 55 showed potent inhibitory effect (>70%) on the TNF-α-induced adhesion of monocytes to colon epithelial cells, which is one of the hallmark events leading to IBD. Such inhibitory activity of the compounds correlated with their suppressive activities against the TNF-α-induced production of ROS; ICAM-1 and MCP-1 expression, critical molecules involved in monocyte-epithelial adhesion; and NF-κB transcriptional activity. In addition, compounds 41 and 55 significantly suppressed the lipopolysaccharide (LPS)-induced expression of the TNF-α gene, with compound 55 showing better efficacy. This inhibition of TNF-α expression by compounds 41 and 55 corresponded to their additional inhibitory activity against AP-1 transcriptional activity, which is another transcription factor required for high level TNF-α expression. The strong inhibitory activity of compound 55 against an in vivo colitis model was confirmed by its dose-dependent inhibitory activity in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, demonstrating compound 55 as a new potential candidate for the development of therapeutics against IBD.