Cardioprotection by systemic dosing of thymosin beta four following ischemic myocardial injury

Thymosin beta 4 (Tβ4) was previously shown to reduce infarct size and improve contractile performance in chronic myocardial ischemic injury via two phases of action: an acute phase, just after injury, when Tβ4 preserves ischemic myocardium via antiapoptotic or anti-inflammatory mechanisms; and a chronic phase, when Tβ4 activates the growth of vascular or cardiac progenitor cells. In order to differentiate between the effects of Tβ4 during the acute and during the chronic phases, and also in order to obtain detailed hemodynamic and biomarker data on the effects of Tβ4 treatment suitable for use in clinical studies, we tested Tβ4 in a rat model of chronic myocardial ischemia using two dosing regimens: short term dosing (Tβ4 administered only during the first 3 days following injury), and long term dosing (Tβ4 administered during the first 3 days following injury and also every third day until the end of the study). Tβ4 administered throughout the study reduced infarct size and resulted in significant improvements in hemodynamic performance; however, chamber volumes and ejection fractions were not significantly improved. Tβ4 administered only during the first 3 days following injury tended to reduce infarct size, chamber volumes and improve hemodynamic performance. Plasma biomarkers of myocyte injury were significantly reduced by Tβ4 treatment during the acute injury period, and plasma ANP levels were significantly reduced in both dosing groups. Surprisingly, neither acute nor chronic Tβ4 treatment significantly increased blood vessel density in peri-infarct regions. These results suggest the following: repeated dosing may be required to achieve clinically measureable improvements in cardiac function post-myocardial infarction (MI); improvement in cardiac function may be observed in the absence of a high degree of angiogenesis; and that plasma biomarkers of cardiac function and myocardial injury are sensitive pharmacodynamic biomarkers of the effects of Tβ4

Confocal microscopy images of CHL-1 tumor samples collected 30 min post IV injection of either GSK2849330 (A, C, D, E) or IgG control (B, F, G, H) conjugated with VivoTag 680 (Red).

<p>The low magnification (20x) images show accumulation of the GSK2849330 (A) and the IgG control (B) labeled antibodies in the connective stromal tissue (S), but uptake of GSK2849330 into CHL-1 HER3 expressing tumor cells (T) was clearly distinguished. The high magnification images (63x) show GSK2849330 binding and internalization (arrows) in the individual tumor cell. Signal overlap between the Alexa 488 anti-human IgG secondary (green, C) and the directly labeled antibody (red, D) indicates specificity of the antibody localization on tumor cells (E). The CHL-1 tumor samples with IgG control showed little or no binding to tumor cells (F, G, H).</p

Non invasive imaging assessment of the biodistribution of GSK2849330, an ADCC and CDC optimized anti HER3 mAb, and its role in tumor macrophage recruitment in human tumor-bearing mice

<div><p>The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced ‘AccretaMab’ monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled ⁸⁹Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with ⁸⁹Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of ⁸⁹Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both ⁸⁹Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.</p></div

Immunohistochemistry analysis of tumors from the USPIO MRI study.

<p>Panels A and B show Prussian blue and F4/80 IHC staining for vehicle and GSK2849330 groups respectively. A region of tumor reflecting cytoplasmic iron staining coregistered with a sub population of plasma membrane F4/80 positive (F4/80+ve) cells is observed in C. The quantitative data (cells/mm²) reflects an increase in number of F4/80+ve macrophages (D) and a trend toward increased coregistered F4/80+ve macrophages with USPIO (E) in the GSK2849330 treated group compared to the vehicle group. Data is presented as mean±SEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176075#pone.0176075.s005" target="_blank">S5 Table</a>). ***P<0.01: unpaired t-test (two-tailed).</p

Tumor intensity at 48, 72, and 96 hours post injection of VivoTag 680-GSK2849330 in BxPC3 xenograft tumor bearing mice as measured in-vivo using optical imaging.

<p>Group 1 (blocking) received 5 mg/kg of VivoTag 680-GSK28493330 24 hours post injection of GSK2849330 (50 mg/kg). Groups 2, 3, and 4 received a single dose of VivoTag 680-GSK28493330 (5, 1, and 0.5 mg/kg, respectively). Groups 1 (blocking) and 2 (5 mg/kg) were significantly different compared to groups 3 (1 mg/kg) and 4 (0.5 mg/kg) at all time points. Group 1 (blocking) intensity decreased significantly over time: *p<0.05: 48 vs 96 hours, ***p<0.001: 48 vs 72 and 96 hours using 2-way ANOVA. Group 2 (5 mg/kg) showed significantly higher intensity compared to group 1 (blocking) at 96 hours: #p<0.05 using 1-way ANOVA. Data is presented as mean±SEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176075#pone.0176075.s003" target="_blank">S3 Table</a>).</p

Tumor uptake (A), blood uptake (B) and the normalized tumor to blood ratio (C) at 24, 48, 72, and 144 hours post injection of ⁸⁹Zr-GSK2849330 in MIA-PaCa-2 and CHL-1 xenograft tumor bearing mice as measured in-vivo using Immuno-PET imaging.

<p>All groups (1,2, and 3) received 0.5 mg/kg of ⁸⁹Zr-GSK2849330, but group 3 received 50 mg/kg of GSK2849330 16 hours prior to the injection of ⁸⁹Zr-GSK2849330. (*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001: group 2 vs group 3; ^#p<0.05, ^##p<0.01, ^###p<0.001: group 1 vs group 3 using 2-way ANOVA. ^ɸp<0.05: group 1 vs group 2 using unpaired t-test). Data is presented as mean±SEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176075#pone.0176075.s001" target="_blank">S1 Table</a>).</p

Dose escalation study of ⁸⁹Zr-GSK2849330 in CHL-1 xenograft tumor bearing mice as measured ex-vivo using a Gamma Counter at 72 hours.

<p>Each group received a tracer dose (~0.14 mg/kg) of ⁸⁹Zr-GSK2849330 plus a dose of non-labeled GSK2849330 (0, 0.3, 1, 3, or 10 mg/kg) (panels A and B). The normalized liver uptake to blood uptake ratio (Panel C) showed a significant decrease with the increased non labeled GSK2849330 (***p<0.01 vs 0 mg/kg; ^##p<0.01, ^###p<0.001 vs 0.3 mg/kg using One-way ANOVA). The normalized tumor uptake to blood uptake ratio (Panel D) showed a significant decrease with the increased non labeled GSK2849330 from 0.3 and 1 mg/kg compared to the 10 mg/kg group (^#p<0.05 vs 1 mg/kg; ** p<0.01, *** p<0.001 vs 10 mg/kg using One-way ANOVA). Data is presented as mean±SEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176075#pone.0176075.s002" target="_blank">S2 Table</a>).</p

Experimental results from the USPIO MRI study.

<p>No difference in body weight (A) was observed between groups. Tumor growth (B) was significantly inhibited in the GSK2849330 treated group (red) compared to the vehicle group (black). **p<0.01, ***p<0.001 using 2 way ANOVA. Axial MRI slices (C) showing T₂*-w images pre-USPIO and 24 hours post injection of ferumoxytol (Post-USPIO). Red arrows indicate xenograft tumor location. Tumor signal/noise ratio (S/N (T₂*-w)) decreased significantly post-USPIO injection in the GSK2849330 treated group (red bars). However, no difference was found in the vehicle group (D). Data is presented as mean±SEM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176075#pone.0176075.s004" target="_blank">S4 Table</a>). **p<0.01: Unpaired t-test (two-tailed).</p

Reduced Acute Vascular Injury and Atherosclerosis in Hyperlipidemic Mice Transgenic for Lysozyme

Hyperlipidemia promotes oxidant stress, inflammation, and atherogenesis in apolipoprotein E-deficient (ApoE((−/−))) mice. Mice transgenic for lysozyme (LZ-Tg) are resistant to acute and chronic oxidative stress and have decreased circulating levels of pro-oxidant advanced glycation end-products (AGEs). Herein we report that TIB-186 macrophages transduced with adenovirus-expressing human LZ (AdV-LZ) containing the AGE-binding domain facilitated AGE uptake and degradation and that AdV-LZ-transduced macrophages and peritoneal macrophages from LZ-Tg mice suppressed the AGE-triggered tumor necrosis factor-α response. We assessed atherosclerosis in LZ-Tg mice crossed with ApoE((−/−)) mice (LZ/ApoE((−/−))) and found increased serum LZ levels and decreased AGE and 8-isoprostanes levels, although hyperlipidemia remained similar to ApoE((−/−)) controls. Atherosclerotic plaques and neointimal lesions at the aortic root and descending aorta were markedly decreased (by 40% and 80%, respectively) in LZ/ApoE((−/−)) versus ApoE((−/−)) mice, as were inflammatory infiltrates. The arterial lesions following femoral artery injury in LZ/ApoE((−/−)) mice were suppressed (intimal to media ratio decreased by 50%), as were AGE deposits and vascular smooth muscle cell activation, compared to ApoE((−/−)) mice. Despite hyperlipidemia, development of atheroma and occlusive, inflammatory arterial neointimal lesions in response to injury was suppressed in LZ/ApoE((−/−)) mice. This effect may be due to the antioxidant properties of LZ, which is possibly linked to the AGE-binding domain region of the molecule