b-1287 and Inflammation

The systemic administration of immunosuppressive and anti-inflammatory drugs is routinely employed in organ transplantation to minimize graft rejection and improve graft survival. Localized drug delivery has the potential to improve transplant outcomes by providing sustained exposure to efficacious drug concentrations while avoiding systemic immunosuppression and off-target effects. Here, we describe the synthesis of a novel prodrug and its direct covalent conjugation to pancreatic islets via a cleavable linker. Post-transplant, linker hydrolysis results in the release of a potent anti-inflammatory antagonist of TLR4, localized to the site of implantation. This covalent islet modification significantly reduces the time and the minimal effective dose of islets necessary to achieve normoglycemia in a murine transplantation model. In streptozotocin-induced diabetic C57BL/6 mice a syngeneic transplant of ∼100 modified islets achieved a 100% cure rate by the end of a 4-week monitoring period, compared to a 0% cure rate for untreated control islets. Overall, this direct prodrug conjugation to islets is well tolerated and preserves their functionality while affording significantly superior transplant outcomes. The development of drug-eluting tissues that deliver sustained and localized doses of small-molecule therapeutics represents a novel pathway for enhancing success in transplantation.

Topics: Animals; Cell Line, Tumor; Cell Survival; Diabetes Mellitus; Glucosamine; Inflammation; Islets of Langerhans; Islets of Langerhans Transplantation; Lipid A; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Sulfonamides; Toll-Like Receptor 4

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.

Topics: Acetylglucosamine; Analysis of Variance; Animals; Binding Sites; DNA Primers; Drug Discovery; Enterocolitis, Necrotizing; Enterocytes; Humans; Inflammation; Lipid A; Macrophages; Mice; Protein Binding; Real-Time Polymerase Chain Reaction; Small Molecule Libraries; Toll-Like Receptor 4; Tritium

Bacteria can naturally secrete outer membrane vesicles (OMVs) as pathogenic factors, while these vesicles may also serve as immunologic regulators if appropriately prepared. However, it is largely unknown whether Pseudomonas aeruginosa OMVs can activate inflammatory responses and whether immunization with OMVs can provide immune protection against subsequent infection. We purified and identified OMVs, which were then used to infect lung epithelial cells in vitro as well as C57BL/6J mice to investigate the immune response and the underlying signaling pathway. The results showed that OMVs generated from P. aeruginosa wild-type strain PAO1 were more cytotoxic to alveolar epithelial cells than those from quorum-sensing (QS)-deficient strain PAO1-ΔlasR. The levels of Toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-6, increased following OMV infection. Compared with lipopolysaccharide (LPS), lysed OMVs in which the membrane structures were broken induced a weak immune response. Furthermore, expression levels of TLR4-mediated responders (i.e., cytokines) were markedly downregulated by the TLR4 inhibitor E5564. Active immunization with OMVs or passive transfer of sera with a high cytokine quantity acquired from OMV-immunized mice could protect healthy mice against subsequent lethal PAO1 challenges (1.5 × 10(11) CFU). Collectively, these findings indicate that naturally secreted P. aeruginosa OMVs may trigger significant inflammatory responses via the TLR4 signaling pathway and protect mice against pseudomonal lung infection.

Topics: Animals; Bacterial Outer Membrane Proteins; Cell Line; Cytoplasmic Vesicles; Epithelial Cells; Humans; Immunization; Inflammation; Interleukin-1beta; Interleukin-6; Lipid A; Lipopolysaccharides; Lung; Mice; Mice, Inbred C57BL; Pseudomonas aeruginosa; Pseudomonas Infections; Pseudomonas Vaccines; Pulmonary Alveoli; Respiratory Mucosa; Signal Transduction; Toll-Like Receptor 4; Vaccination

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

Topics: Animals; Anti-Bacterial Agents; Antigens, Surface; Blotting, Western; Cell Line; Dose-Response Relationship, Drug; Drosophila Proteins; Enzyme Activation; Exons; Fibronectins; Hot Temperature; Humans; Inflammation; Interleukin-10; Lipid A; Lipopolysaccharides; Lymphocyte Antigen 96; Matrix Metalloproteinase 9; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Plasmids; Polymyxin B; Protein Structure, Tertiary; Receptors, Cell Surface; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spleen; Time Factors; Toll-Like Receptor 4; Toll-Like Receptors; Transfection