Imaging Pulmonary NF-kappaB Activation and Therapeutic Effects of MLN120B and TDZD-8

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions

Abstract 5288A: Multivalent fluorescent probes for in vivo tumor targeting

Abstract Conventional fluorescence probes are monovalent molecules that interact with cellular or extra-cellular target molecules through affinity binding. To improve the binding affinity and sensitivity of detection of the fluorescence probe, we describe a novel strategy for the synthesis designed to contain multiple targeting moieties and a near infrared fluorescent dye Iflour750 for optimal in vivo detection. In this study, we applied several multivalent probes to in vivo tumor targeting and monitored the targeting process using fluorescence imaging. Deoxyglucose based fluorescence probes have been applied to tumor labeling as the high metabolic rate of the tumor cells causes preferential labeling of the tumor mass. With a multivalent 2-deoxy-D- glucose Iflour750 (2-DG-IF750) probe, we demonstrated sensitive in vivo targeting as compared to a control mono-valent 2DG probe in several tumor models, including PC3M-luc2 prostate tumor cells, MDA-MB-231-luc2 mammary fat tumor cells as well as LL/2-luc and H460-luc2 lung cancer cells. Distinct tumor signal was visualized within a few hours after intravenous delivery and reached the peak of signal/background ratio between 6-24 hours. With a multivalent cyclooxygenase (COX) binding fluorescence probe Indomethacin-IF750, we observed an interesting targeting of COX2 negative colon tumor cells HCT116-luc2, but not the COX2 positive HT29-luc2, hypothetically due to selective binding to COX1. We also tested a multivalent RGD probe for targeting integrin avb3, the expression of which is associated with tumor angiogenesis and metastasis. With our iFlur750-(RGD)4 probe, we demonstrated in vivo targeting of U87-MG-luc2, HCT116-luc2 and HT29-luc2 tumor cells. Finally, we demonstrated that optical imaging was conveniently applied to evaluating the PK/PD profile of the probes. Our multivalent probes showed a fast clearance with a half life of less than 1 hour, which is a desirable feature for achieving maximal tumor/background contrast. In summary, we have developed a new class of multi-valent fluorescence probes with improved specificity and sensitivity of tumor targeting as compared to mono-valent analogs. Utility of this class of multivalent probes is likely to pass beyond pre-clinical models, as imaging guided tumor surgery is on the verge of clinical applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5288A. doi:10.1158/1538-7445.AM2011-5288A</jats:p

Pharmacokinetics and pharmacodynamics of the novel monobactam LYS228 in a neutropenic murine thigh model of infection

Objectives: The neutropenic murine thigh infection model and a dose-fractionation approach were used to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship of LYS228, a novel monobactamantibiotic with activity against Enterobacteriaceae including carbapenem-resistant strains. Methods: Mice (n"4 per group) were inoculated with Enterobacteriaceae strains via intramuscular injection. Two hours post-bacterial inoculation, treatment with LYS228 was initiated. Animals were euthanized with CO2 24 h after the start of therapy and bacterial counts (log10 cfu) per thigh were determined. PK parameters were calculated using free (f) plasma drug levels. Results: Following a dose-fractionation study, non-linear regression analysis determined that the predominant PK/PD parameter associated with antibacterial efficacy of LYS228 was the percentage of the dosing interval that free drug concentrations remained above the MIC (%fT.MIC). In a dose-dependent manner, LYS228 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing b-lactamase enzymes of all classes (e.g. ESBLs, NDM-1, KPC, CMY-2 and OXA-48). The range of the calculated static dose was 86-649 mg/kg/ day for the isolates tested, and the magnitude of the driver of efficacy was 37-83%fT.MIC. %fT.MIC was confirmed as the parameter predominantly driving efficacy as evidenced by a strong coefficient of determination (r2"0.68). Neutrophils had minimal impact on the effect of LYS228 in the murine thigh infection model. Conclusions: LYS228 is efficacious in murine thigh infection models using b-lactamase-producing strains of Enterobacteriaceae, including those expressing metallo-b-lactamases, ESBLs and serine carbapenemases, with the PK/PD driver of efficacy identified as%T.MIC

In vivo gene delivery of NF-κB reporters to the lung and response to LPS treatment.

<p>Albino C57BL/6 mice (n = 3 per group) were injected intravenously with NF-κB reporter DNA using JetPEI. Mice were imaged following i.p. injection of luciferin at 1, 5, 7, and 14 days following transfection. After the imaging on the day 14, mice were intratracheally challenged with LPS at 1 mg/kg and re-imaged after 3 hours. Bioluminescence images (A) and quantification of bioluminescence signal from the lungs (B). Quantification was performed by drawing a region of interest (ROI) over the chest area followed by quantification using the LivingImage software. Data presented as mean ± SEM.</p

Impact of ageing on presentation and outcome of mitral regurgitation due to flail leaflet: a multicentre international study

Define the impact of age at diagnosis on degenerative mitral regurgitation (MR) prognosis.Methods and resultsThe Mitral regurgitation International DAtabase (MIDA) is a multicentre registry of MR due to flail leaflets including 862 patients (65 \ub1 12 years) diagnosed by echocardiography. The 498 older patients ( 6565 years at diagnosis) were compared with the 364 younger (<65) with regard to presentation and the outcome was compared with that expected in the general population. Older vs. younger patients had MR of similar severity and ventricular overload but presented with more MR consequences and incurred higher mortality [risk ratio (rr) 95% confidence interval (95% CI) 4.7 (2.5-10.0), P < 0.001] independently of co-morbidity. Compared with expected survival [relative risk (95% confidence interval)], excess mortality, non-significant in younger patients [1.1 (0.6-2.0), P = 0.65], was prominent in older patients [1.4 (1.2-1.7), P < 0.001]. Compared with expected, excess heart failure (HF) occurred in younger [9.3 (6.5-13.3), P < 0.0001) and in older patients [6.7 (5.6-8.1), P < 0.0001]. Excess atrial fibrillation (AF) was even higher in younger [6.9 (4.5-10.6), P < 0.0001] than in older patients [3.5 (2.6-4.7), P < 0.0001; P < 0.001 for comparison between age groups]. Subsequent excess mortality [rr (95% CI)] was associated with occurrence of HF and/or AF in both age groups [13.5 (7.4-24.6), P < 0.001]. Mitral surgery was associated with reduced long-term mortality in older patients and lower rate of HF in both the age groups (all P < 0.01).ConclusionsBoth older and younger patients incurred excess risk of complications. Older patients suffered excess mortality, AF, and HF, whereas younger incurred excess morbidity linked to subsequent long-term excess mortality. The excess risks of uncorrected degenerative MR should be considered in deliberating surgical management, which significantly reduced mortality in older patients and HF in younger patient

Effect of IKK2 inhibition on NF-κB induction in vivo.

<p>At 2 weeks after in vivo gene delivery with the NF-κB2-luc2 reporter, mice (n = 7) were pre-treated with MLN120B at 300 mg/kg orally or vehicle control 16 and 1-hour prior to LPS delivery. Mice were imaged immediately before LPS injection (T = 0) and at 4 hours. Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p

Kinetics of NF-κB activation.

<p>TNFAIP3-luc2 reporter transfected mice were challenged with LPS (1 mg/kg, intratracheally, n = 11) or TNFα (1 µg/mouse, intravenously, n = 4) or saline (n = 4). Mice were imaged at 0 (prior to injection), 3, 6, and 24 hours after injection. Bioluminescent images from one representative mouse per group (A) and quantification of lung signals (B) are shown. Data presented as mean ± SEM.</p

In vitro comparison of NF-κB reporters.

<p>RAW264.7 cells were transiently transfected with NF-κB reporters. Cells were treated with LPS at 0–1 µg/ml concentrations overnight and imaged with IVIS after adding luciferin. Quantification of luciferase signal (A). Fold change of cells treated with 1 µg/ml LPS compared to vehicle treated cells (B). Data presented as mean ± SEM; n = 4 wells per reporter construct and LPS concentration of 1 µg/ml.</p

Effect of glycogen synthase kinase beta inhibition on NF-κB induction in vivo.

<p>NF-κB2-luc2 reporter transfected mice (n = 6 per group) were pre-dosed with either TDZD-8 (10 mg/kg, i.p.), or DMSO vehicle control 16 and 1-hour prior to LPS challenge. Mice were imaged immediately before LPS injection (T = 0) and at 3 hours post injection. Another group of mice was not treated with LPS and served as negative controls (n = 3). Bioluminescence images (A) and quantification of lung signals (B) are shown. Cytokines measured in bronchial lavage fluid as specified (C). Data presented as standard box and whisker plots, *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U test.</p