Robert Peach

Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection

Summary Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P1 receptor, we elucidate cellular and signaling mechanisms that are important in initiating cytokine storm. Whereas S1P1 receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P1 agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events that are both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P1 signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases in which amplification of cytokine storm is a significant pathological component could be chemically tractable.

Sphingosine-1-phosphate receptor-1 (S1P1) is expressed by lymphocytes, dendritic cells, and endothelium and modulated during inflammatory bowel disease

The sphingosine-1-phosphate receptor-1 (S1P1) agonist ozanimod ameliorates ulcerative colitis, yet its mechanism of action is unknown. Here, we examine the cell subsets that express S1P1 in intestine using S1P1-eGFP mice, the regulation of S1P1 expression in lymphocytes after administration of dextran sulfate sodium (DSS), after colitis induced by transfer of CD4+CD45RBhi cells, and by crossing a mouse with TNF-driven ileitis with S1P1-eGFP mice. We then assayed the expression of enzymes that regulate intestinal S1P levels, and the effect of FTY720 on lymphocyte behavior and S1P1 expression. We found that not only T and B cells express S1P1, but also dendritic (DC) and endothelial cells. Furthermore, chronic but not acute inflammatory signals increased S1P1 expression, while the enzymes that control tissue S1P levels in mice and humans with inflammatory bowel disease (IBD) were uniformly dysregulated, favoring synthesis over degradation. Finally, we observed that FTY720 reduced T-cell velocity and induced S1P1 degradation and retention of Naïve but not effector T cells. Our data demonstrate that chronic inflammation modulates S1P1 expression and tissue S1P levels and suggests that the anti-inflammatory properties of S1PR agonists might not be solely due to their lymphopenic effects, but also due to potential effects on DC migration and vascular barrier function.

XIAP is not required for human tumor cell survival in the absence of an exogenous death signal

BackgroundThe X-linked Inhibitor of Apoptosis (XIAP) has attracted much attention as a cancer drug target. It is the only member of the IAP family that can directly inhibit caspase activity in vitro, and it can regulate apoptosis and other biological processes through its C-terminal E3 ubiquitin ligase RING domain. However, there is controversy regarding XIAPs role in regulating tumor cell proliferation and survival under normal growth conditions in vitro.MethodsWe utilized siRNA to systematically knock down XIAP in ten human tumor cell lines and then monitored both XIAP protein levels and cell viability over time. To examine the role of XIAP in the intrinsic versus extrinsic cell death pathways, we compared the viability of XIAP depleted cells treated either with a variety of mechanistically distinct, intrinsic pathway inducing agents, or the canonical inducer of the extrinsic pathway, TNF-related apoptosis-inducing ligand (TRAIL).ResultsXIAP knockdown had no effect on the viability of six cell lines, whereas the effect in the other four was modest and transient. XIAP knockdown only sensitized tumor cells to TRAIL and not the mitochondrial pathway inducing agents.ConclusionsThese data indicate that XIAP has a more central role in regulating death receptor mediated apoptosis than it does the intrinsic pathway mediated cell death.

Results From the First-in-Human Study With Ozanimod, a Novel, Selective Sphingosine-1-Phosphate Receptor Modulator

The sphingosine‐1‐phosphate 1 receptor (S1P1R) is expressed by lymphocytes, dendritic cells, and vascular endothelial cells and plays a role in the regulation of chronic inflammation and lymphocyte egress from peripheral lymphoid organs. Ozanimod is an oral selective modulator of S1P1R and S1P5R receptors in clinical development for the treatment of chronic immune‐mediated, inflammatory diseases. This first‐in‐human study characterized the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of ozanimod in 88 healthy volunteers using a range of single and multiple doses (7 and 28 days) and a dose‐escalation regimen. Ozanimod was generally well tolerated up to a maximum single dose of 3 mg and multiple doses of 2 mg/d, with no severe adverse events (AEs) and no dose‐limiting toxicities. The most common ozanimod‐related AEs included headache, somnolence, dizziness, nausea, and fatigue. Ozanimod exhibited linear PK, high steady‐state volume of distribution (73–101 L/kg), moderate oral clearance (204–227 L/h), and an elimination half‐life of approximately 17 to 21 hours. Ozanimod produced a robust dose‐dependent reduction in total peripheral lymphocytes, with a median decrease of 65% to 68% observed after 28 days of dosing at 1 and 1.5 mg/d, respectively. Ozanimod selectivity affected lymphocyte subtypes, causing marked decreases in cells expressing CCR7 and variable decreases in subsets lacking CCR7. A dose‐dependent negative chronotropic effect was observed following the first dose, with the dose‐escalation regimen attenuating the first‐dose negative chronotropic effect. Ozanimod safety, PK, and PD properties support the once‐daily regimens under clinical investigation.

Cardiac Safety of Ozanimod, a Novel Sphingosine‐1‐Phosphate Receptor Modulator: Results of a Thorough QT/QTc Study

Ozanimod is a novel, selective, oral sphingosine‐1‐phosphate (1 and 5) receptor modulator in development for multiple sclerosis and inflammatory bowel disease. This randomized, double‐blind, placebo‐controlled, positive‐controlled, parallel‐group thorough QT study characterized the effects of ozanimod on cardiac repolarization in healthy subjects. Eligible subjects were randomized to 1 of 2 groups: ozanimod (escalated from 0.25 to 2 mg over 14 days) or placebo (for 14 days). A single dose of moxifloxacin 400 mg or placebo was administered on days 2 and 17. The primary end point was the time‐matched, placebo‐corrected, baseline‐adjusted mean QTcF (ΔΔQTcF). A total of 113/124 (91.1%) subjects completed the study. The upper limits of the 2‐sided 90% confidence intervals for ΔΔQTcF for both ozanimod 1 and 2 mg were below the 10‐millisecond regulatory threshold. No QTcF >480 milliseconds or postdose change in QTcF of >60 milliseconds was observed. There was no evidence of a positive relationship between concentrations of ozanimod and its active metabolites and ΔΔQTcF. Although ozanimod blunted the observed diurnal increase in heart rate, excursions below predose heart rates were no greater than with placebo. Results demonstrate that ozanimod does not prolong the QTc interval or cause clinically significant bradycardia, supporting ozanimods evolving favorable cardiac safety profile.

Ozanimod (RPC1063), a selective S1PR1 and S1PR5 modulator, reduces chronic inflammation and alleviates kidney pathology in murine systemic lupus erythematosus

Ozanimod (RPC1063) is a specific and potent small molecule modulator of the sphingosine 1-phosphate receptor 1 (S1PR1) and receptor 5 (S1PR5), which has shown therapeutic benefit in clinical trials of relapsing multiple sclerosis and ulcerative colitis. Ozanimod and its active metabolite, RP-101075, exhibit a similar specificity profile at the S1P receptor family in vitro and pharmacodynamic profile in vivo. The NZBWF1 mouse model was used in therapeutic dosing mode to assess the potential benefit of ozanimod and RP-101075 in an established animal model of systemic lupus erythematosus. Compared with vehicle-treated animals, ozanimod and RP-101075 reduced proteinuria over the duration of the study and serum blood urea nitrogen at termination. Additionally, ozanimod and RP-101075 reduced kidney disease in a dose-dependent manner, as measured by histological assessment of mesangial expansion, endo- and exo-capillary proliferation, interstitial infiltrates and fibrosis, glomerular deposits, and tubular atrophy. Further exploration into gene expression changes in the kidney demonstrate that RP-101075 also significantly reduced expression of fibrotic and immune-related genes in the kidneys. Of note, RP-101075 lowered the number of plasmacytoid dendritic cells, a major source of interferon alpha in lupus patients, and reduced all B and T cell subsets in the spleen. Given the efficacy demonstrated by ozanimod and its metabolite RP-101075 in the NZBWF1 preclinical animal model, ozanimod may warrant clinical evaluation as a potential treatment for systemic lupus erythematosus.

Tu1616 Rpc1063 Is a Potent S1p1r Agonist With Efficacy in the Samp1yitfc Ileitis Model: New Evidence for Dysregulation of S1p Metabolism in Preclinical Models and Patients With Inflammatory Bowel Disease

Body: Synthetic triterpenoids are effective anti-cancer agents. They also inhibit IL17 and improve autoimmune disease in mice, but have not been tested in murine IBD models. One prototype triterpenoid, CDDO-Im, inhibits STAT3 pathway activation. This transcription factor plays a role in the pathogenesis of IBD. Aims: The goals of the study were two-fold: To determine if ex vivo treatment with CDDO-Im would be effective for attenuating colonic IL-17 isoform secretion; and to determine if oral treatment with CDDOIm would modulate colonic IL-17 and improve DSS-induced colitis in mice. Methods: Study 1: Colitis was induced in male C57BL/6 mice (n = 9) by adding DSS 2% to the drinking water for 6 days. A control group of mice (n = 4) received filtered water. On day 6, colons were collected and organ culture was performed. Colonic strips (3 mm) were exposed to CDDO-Im at 0.5, 1, and 2 μM concentrations in the presence of IL-23 + IL-1 β (10 ng/ml of each). After 24 hours, supernatant was collected. IL-17 andIL-17F were measured by ELISA. Study 2: Using the same colitis paradigm described above, 20 mice were randomized into 3 groups (table 1). Mice were dosed by orogastric gavage with vehicle (10% Cremophor, 10% DMSO, 80% PBS) or CDDO-Im (20mg/kg) on study days 0, 1, 3 and 5. Body weights, water consumption and disease activity index (DAI) scores (0-12 scale) were recorded daily. Colons were collected on day 6. Distal colon was retained for colonic histology. Using coded slides, histology scores were determined with a 40-point severity scale. The proximal colon samples were analyzed for IL-17, IL-17F, IL-6 (by ELISA), and colonic MPO. Colonic nuclear protein extracts were used to measure the binding of STAT3 to a consensus site (specific Trans-Am® kit). Results: In colonic strips from DSS-treated mice, ex vivo treatment with CDDO-Im dose-dependently decreased both IL-17 and IL-17F secretion. The calculated IC50 values were 0.51 μM and 0.62 μM respectively. A similar inhibitory profile was found for CDDO-Im when using colonic strips from non-DSS-treated mice. In vivo, vehicle-treated mice lost weight between study days 2 and 6, while CDDO-treated animals gained weight (table 1). On study day 6, mean DAI, MPO, histology score, IL-17 and IL-6 values were all lower in the CDDO-Im group. These results are consistent with decreased colonic inflammation, compared to Vehicle/DSS treatment. Nuclear binding of STAT3 was also significantly attenuated (p , 0.05) in the CDDO-Im group of mice, compared to the corresponding vehicle cohort of animals. Summary: CDDO-Im attenuated IL-17 isoform secretion in an ex vivo model of colonic inflammation. In vivo, CDDO-Im improved multiple parameters of murine DSS-induced colitis, including STAT3 and IL-17. Conclusion: CDDO-Im has a unique pharmacological profile, which supports further testing in animal models of IBD. Table 1. Treatment Groups S-807 AGA Abstracts Values are Mean ± SEM. + p , 0.05 vs. Vehicle/Water or * p , 0.05 vs. Vehicle/DSS