ARCA Biopharma (United States)

BACKGROUNDUnconjugated bilirubin (UCB) is generally considered toxic but has gained recent prominence for its anti-inflammatory properties. However, the effects of it on the interaction between intestinal flora and organisms and how it influences immune responses remain unresolved.AIMTo investigate the role of UCB in intestinal barrier function and immune inflammation in mice with dextran-sulfate-sodium-induced colitis.METHODSAcute colitis was induced by 3% (w/v) dextran sulfate sodium salt in drinking water for 6 d followed by untreated water for 2 d. Concurrently, mice with colitis were administered 0.2 mL UCB (400 μmol/L) by intra-gastric gavage for 7 d. Disease activity index (DAI) was monitored daily. Mice were sacrificed at the end of the experiment. The length of the colon and weight of the spleen were recorded. Serum level of D-lactate, intestinal digestive proteases activity, and changes to the gut flora were analyzed. In addition, colonic specimens were analyzed by histology and for expression of inflammatory markers and proteins.RESULTSMice treated with UCB had significantly relieved severity of colitis, including lower DAI, longer colon length, and lower spleen weight (colon length: 4.92 ± 0.09 cm vs 3.9 ± 0.15 cm; spleen weight: 0.33 ± 0.04 vs 0.74 ± 0.04, P < 0.001). UCB administration inactivated digestive proteases (chymotrypsin: 18.70 ± 0.69 U/g vs 44.81 ± 8.60 U/g; trypsin: 1.52 ± 0.23 U/g vs 9.05 ± 1.77 U/g, P < 0.01), increased expression of tight junction (0.99 ± 0.05 vs 0.57 ± 0.03, P < 0.001), decreased serum level of D-lactate (31.76 ± 3.37 μmol/L vs 54.25 ± 1.45 μmol/L, P < 0.001), and lowered histopathological score (4 ± 0.57 vs 7 ± 0.57, P < 0.001) and activity of myeloperoxidase (46.79 ± 2.57 U/g vs 110.32 ± 19.19 U/g, P < 0.001). UCB also regulated the intestinal microbiota, inhibited expression of tumor necrosis factor (TNF) α and interleukin 1β (TNF-α: 52.61 ± 7.81 pg/mg vs 105.04 ± 11.92 pg/mg, interleukin 1β: 13.43 ± 1.68 vs 32.41 ± 4.62 pg/mg, P < 0.001), decreased expression of Toll-like receptor 4 (0.61 ± 0.09 vs 1.07 ± 0.03, P < 0.001) and myeloid differentiation primary response gene 88 (0.73 ± 0.08 vs 1.01 ± 0.07, P < 0.05), and increased expression of TNF-receptor-associated factor 6 (0.79 ± 0.02 vs 0.43 ± 0.09 P < 0.05) and inhibitor of kappa B α (0.93 ± 0.07 vs 0.72 ± 0.07, P < 0.05) in the colon.CONCLUSIONUCB can protect intestinal barrier function, regulate normal intestinal homeostasis, and suppress inflammation via the Toll-like receptor 4/ nuclear factor-κB signaling pathway.

Renal fibrosis is a characteristic of progressive renal diseases leading to end‐stage renal failure. Induction of renal fibrosis via the mouse unilateral ureteral occlusion (UUO) model occurs within 5 days making it a high throughput model to evaluate novel therapeutic targets. Transforming growth factor‐beta (TGF‐β) is a key signaling molecule in the pathogenesis of renal fibrosis mediated through its actions on its receptor, Activin A Receptor‐Like Kinase (ALK5). Previously, we have shown the anti‐TGF‐β antibody (1D11; clone 1D11.16.8) attenuates UUO‐induced renal fibrosis. Here, we evaluated and compared the dose‐dependent ability of an orally‐available ALK5 inhibitor (SB‐525334) to attenuate UUO‐induced tissue biomarkers. To minimize the number of necessary control groups and ensure all mice were handled similarly, all mice received intraperitoneal injections of PBS or 3 mg/kg 1D11 QD d‐1 through d4 and oral gavage administration of vehicle or SB‐525334 (10, 30 or 100 mg/kg) QD d‐1 through d5. On d0, mice (male, CD‐1, 8–9 weeks age) underwent sham (n=8) or permanent total ligation of the right ureter (UUO, n=12–13/group) via aseptic laparotomy. Five days post‐UUO or sham surgery, renal tissue from sham or obstructed kidney was collected for analysis of profibrotic mRNA biomarker expression, TGF‐β content, hydroxyproline content and renal fibrosis as expressed as collagen volume fraction (CVF; quantitated via histological image analysis of Picrosirius Red stained mid‐transverse kidney sections). Consistent with a pathophysiological response to ureteral obstruction, UUO reliably (100%) elicited hydronephrosis. UUO increased renal cortical mRNA expression of several fibrotic markers—fibronectin‐1 (FN‐1; 3.4±0.5 fold induction from sham), alpha‐smooth muscle actin (α‐SMA; 0.9±0.2 fold), collagen 1A1 (Col1A1; 12.7±4.2 fold) and collagen 3A1 (Col3A1; 5.8±0.8 fold) as well as increased renal cortical TGF‐β content (2.8±0.2 fold), hydroxyproline content (1.6±0.1 fold) and CVF (8.8±0.9 fold). 1D11 attenuated UUO‐induced increases in FN‐1 (45.5±5.6%), Col1A1 (61.3±9.6%) and Col3A1 (48.6±8.2%) mRNA expression, TGF‐β content (38.8±5.9% inhibition), hydroxyproline content (23.4±6.5%), and CVF (42.6±5.7%). High dose (100 mg/kg) SB‐525334 attenuated UUO‐induced increases in FN‐1 (77.8 ±6.4%), α‐SMA (111.0±23.6%), Col1A1 (77.1±4.1%) and Col3A1 (56.0±7.8%) mRNA expression, as well as TGF‐β content (35.2±6.3%), hydroxyproline content (46.5±4.4%), and CVF (73.7±4.8% inhibition). The current data demonstrate the importance of TGF‐β signaling in fibrotic consequences of ureteral obstruction. Additionally, UUO‐induced renal injury can be modulated by a parenterally administered biological agent (1D11) as well as by an orally administered small molecule (SB‐525334) thus eliminating the need for multiple dosing routes in future UUO studies where a reference agent is utilized.

Background & AimsInteractions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC’s anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis.MethodsMonocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC’s antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses.ResultsCVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight.ConclusionsCVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC’s antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).

Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.