title: Amelioration of endotoxin-induced uveitis treated with an IκB kinase β inhibitor in rats.
abstract: OBJECTIVE
  Endotoxin-induced uveitis (EIU) is an animal model for acute ocular inflammation. Several substances play major roles in the development of inflammatory changes in EIU, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. These inflammatory cytokines trigger the degradation of IκB by activating IκB kinases (IKKs). Released nuclear factor kappaB (NFκB) subsequently translocates to the nucleus, where NFκB expresses its proinflammatory function. IMD-0354, N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide, selectively inhibits IKKβ, particularly when induced by proinflammatory cytokines, such as TNF-α and IL-1β. In the present study, we examined whether IKKβ inhibition has therapeutic effects on EIU by using IMD-0354 and its prodrug IMD-1041.
  
  
  METHODS
  Six-week-old male Lewis rats were used. EIU was induced with subcutaneous injections of 200 μg of lipopolysaccharide (LPS) from Escherichia coli that had been diluted in 0.1 ml of phosphate-buffered saline. IMD-0354 was administered intraperitoneally at 30, 10, 3, or 0 mg/kg, suspended in 1.0 ml of 0.5% carboxymethyl cellulose sodium. The prodrug IMD-1041 (100 mg/kg) was also administered orally. The rats were euthanized 24 h after LPS injection, and EIU severity was evaluated histologically. The number of infiltrating cells and the protein, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) concentrations in the aqueous humor were determined. TNF-α and MCP-1 concentrations were quantified with enzyme-linked immunosorbent assay. Eye sections were also stained with anti-NFκB and phosphorylated I-κBα antibodies.
  
  
  RESULTS
  The number of infiltrating cells in aqueous humor was 53.6±9.8×10(5), 72.5±17.0×10(5), 127.25±32.0×10(5), and 132.0±25.0×10(5) cells/ml in rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, respectively. The total protein concentrations of aqueous humor were 92.6±3.1 mg/ml, 101.5±6.8 mg/ml, 112.6±1.9 mg/ml, and 117.33±1.8 mg/ml in rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively. Infiltrating cells and protein concentrations were significantly decreased by treatment with IMD-0354 (p<0.01). IMD-0354 treatment significantly reduced the concentration of TNF-α (p<0.05) and MCP-1 (p<0.01) in aqueous humor. The number of NFκB positive nuclei was reduced when treated with IMD-0354. Furthermore, IMD-0354-treated EIU rats showed only background levels of phosphorylated I-κBα; however, it was strongly expressed in the iris-ciliary body cell cytoplasm of the IMD-0354 untreated EIU rats. Oral administration of IMD-1041 also decreased the cell number (p<0.01) and protein concentration (p<0.05) of aqueous humor in EIU.
  
  
  CONCLUSIONS
  Acute uveitis was ameliorated by inhibition of IKKβ in rats. IMD-0354 and its prodrug IMD-1041 seem to be promising candidates for treating intraocular inflammation/uveitis.
authors.affiliation: Laboratory of Ocular Cell Biology and Visual Science, Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors.forename: Anton
authors.initials: A
authors.lastname: Lennikov
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Kitaichi
authors.forename: Kousuke
authors.initials: K
authors.lastname: Noda
authors.forename: Ryo
authors.initials: R
authors.lastname: Ando
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Dong
authors.forename: Junichi
authors.initials: J
authors.lastname: Fukuhara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kinoshita
authors.forename: Kenichi
authors.initials: K
authors.lastname: Namba
authors.forename: Miho
authors.initials: M
authors.lastname: Mizutani
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Fujikawa
authors.forename: Akiko
authors.initials: A
authors.lastname: Itai
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Ohno
authors.forename: Susumu
authors.initials: S
authors.lastname: Ishida
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001082:Aqueous Humor / Q000187:drug effects* / Q000276:immunology; D001549:Benzamides / Q000494:pharmacology* / Q000627:therapeutic use; D018932:Chemokine CCL2 / Q000235:genetics / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins; D015870:Gene Expression / Q000187:drug effects; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007274:Injections, Intraperitoneal; D008297:Male; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology; D020556:Neutrophil Infiltration / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D051381:Rats; D011917:Rats, Inbred Lew; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology; D014605:Uveitis / Q000139:chemically induced / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology
chemical_list: D001549:Benzamides; C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D004731:Endotoxins; D016328:NF-kappa B; D011355:Prodrugs; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; C492919:N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide; D051550:I-kappa B Kinase