Sudhir P. Deosarkar

Polymeric particles conjugated with a ligand to VCAM‐1 exhibit selective, avid, and focal adhesion to sites of atherosclerosis

The increased expression of VCAM‐1 on endothelial segments within plaque regions could be used as a target to deliver polymeric drug carriers selectively to sites of atherosclerosis. We probed the hypothesis that polymeric particles conjugated with a ligand for VCAM‐1 exhibit selective and avid adhesion to sites of atherosclerosis. Particles made from polystyrene or the biodegradable polymer poly(sebacic acid)‐block‐polyethylene glycol (PSA‐PEG) were conjugated with an antibody to VCAM‐1 (α‐VCAM‐1) or IgG (negative control). The particles were injected into the jugular vein of ApoE−/− (a murine model of atherosclerosis) or wild type mice and their adhesion to the aorta determined. α‐VCAM‐1 particles exhibited significantly greater adhesion to ApoE−/− mouse aorta [32 ± 5 (mean ± SEM) particles/mm2 for polystyrene particles and 31 ± 7 particles/mm2 for PSA‐PEG particles] compared to the level of adhesion to wild type mouse aorta (18 ± 1 particles/mm2 for polystyrene particles and 6 ± 1 particles/mm2 for PSA‐PEG particles). Within ApoE−/− mice, the α‐VCAM‐1 particles exhibited significantly greater adhesion to the aorta (32 ± 5 particles/mm2 for polystyrene particles and 31 ± 7 particles/mm2 for PSA‐PEG particles) compared to the adhesion of IgG particles (1 ± 1 particles/mm2 for polystyrene particles and 2 ± 1 particles/mm2 for PSA‐PEG particles). Detailed analysis of the adhesion revealed that α‐VCAM‐1 particles exhibited focal adhesion to plaque regions, in particular the periphery of the plaques, within the ApoE−/− mouse aorta. Combined the data demonstrate that polymeric particles conjugated with a ligand to VCAM‐1 exhibit selective, avid and focal adhesion to sites of atherosclerosis providing strong evidence that VCAM‐1 ligand bearing polymeric particles could be used for targeting drugs selectively to atherosclerotic tissue. Biotechnol. Bioeng. 2008;101: 400–407.

Phenyl methimazole suppresses dextran sulfate sodium-induced murine colitis

Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions.

E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe(x)), present on the surface of leukocytes, play a key role in leukocyte-endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe(x) and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe(x) microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe(x) mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe(x) microspheres. Perfusion of the sLe(x) microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5 dyn/cm² revealed that the microspheres attach to 4h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLe(x) microspheres with HECA-452 did not influence sLe(x) microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLe(x) microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe(x) does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe(x) antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe(x)-mediated adhesion to endothelial expressed E-selectin.

Simple modifications to methimazole that enhance its inhibitory effect on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression by human endothelial cells

The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor-α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves׳ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds.

A Quantitative Real-Time PCR Approach for the Detection and Characterization of Endothelial Cells in Whole Blood

Pathological inflammation and endothelial dysfunction in atherosclerosis causes endothelial cell detachment from affected vasculature giving rise to circulating endothelial cells (CECs). A blood-based assay that can detect and characterize CECs in atherosclerosis could serve as a valuable diagnostic. Thus, we sought to develop a prototypic assay that detects and characterizes the inflammatory state of endothelial cells present in blood. For this purpose, we spiked resting and inflamed human umbilical vein endothelial cells (HUVEC) into separate samples of whole blood. RNA was harvested and analyzed via quantitative real-time PCR (qPCR) using melanoma cell adhesion molecule (MCAM), as an endothelial marker, and vascular cell adhesion molecule (VCAM-1), which is increased on inflamed endothelium. We found that MCAM mRNA levels correlated with the number of HUVEC spiked into the blood. VCAM-1 mRNA levels were elevated, and correlated with the number of HUVEC, in blood spiked with inflamed HUVEC but not in blood spiked with resting HUVEC. VCAM-1 and MCAM mRNA levels were converted into numerical indices that indicate the inflammatory state of the HUVEC. Combined, the blood spiking studies demonstrate that a VCAM-1/MCAM qPCR assay can successfully detect inflamed endothelial cells in whole blood thus providing proof-of-concept for a diagnostic based on a coupled-phenotypic qPCR assay.

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells.

Abstract Inhibition of interleukin–6 (IL‐6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL‐6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB‐141. In the present work, we tested the hypothesis that C10 and COB‐141 inhibit TNBC cell expressed IL‐6 and investigated the potential for classical IL‐6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA‐MB‐231, Hs578T, MDA‐MB‐468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB‐141 inhibit MDA‐MB‐231 cell IL‐6 secretion, with COB‐141 being ˜6.5 times more potent than C10. Therefore, the remainder of the study focused on COB‐141 which inhibited IL‐6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT‐PCR), to inhibit IL‐6 mRNA in the TNBC panel. COB‐141 had little, if any, effect on metabolic activity indicating that the IL‐6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT‐PCR revealed that the TNBC cell lines do not express the IL‐6 receptor (IL‐6R&agr;). Trans‐AM assays suggested that COB‐141 exerts its inhibitory effect, at least in part, by reducing NF‐&kgr;B (p65/p50) DNA binding. In summary, COB‐141 is a potent inhibitor of TNBC cell expressed IL‐6 and the inhibition does not appear to be due to non‐specific toxicity. The TNBC cell lines do not have an intact classical IL‐6 signaling pathway. COB‐141s inhibitory effect may be due, at least in part, to reducing NF‐&kgr;B (p65/p50) DNA binding.

Inhibition of LPS‐Induced TLR4 Signaling Products in Murine Macrophages by Phenylmethimazole: An Assay Methodology for Screening Potential Phenylmethimazole Analogs

Preclinical Research

Abstract 1932: Phenylmethimazole decreases constitutively high basal levels of Interleukin 6 (IL-6) in a triple negative breast cancer cell line

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Interleukin 6 (IL-6) is a pro-inflammatory cytokine that plays an important role in the progression of many types of cancers by regulating macrophage recruitment and differentiation, as well as modulating the activation of the JAK/Stat signal cascade, and inducing pro-angiogenic factors. Several studies have correlated a negative prognosis in cancer with a high level of IL-6 in patient serum. Triple (i.e. progesterone, human epidermal growth factor-2, and estrogen receptor) negative breast cancer has been reported to be highly aggressive and invasive. MDA-MB-231, a triple negative breast cancer cell line, expresses high basal levels of IL-6. Recently our laboratory demonstrated that phenylmethimazole (C10), a novel toll-like receptor inhibitor, diminishes basal expression of IL-6 in pancreatic cancer and melanoma cells as well as their growth and migration. Based on these observations, we sought to test the hypothesis that C10 inhibits IL-6 expression in MDA-MB-231 cells. Here we report that triple negative breast cancer cells express considerably higher levels of IL-6 than estrogen receptor positive breast cancer cells and that C10 significantly reduces IL-6 expression in the triple negative breast cancer cells. This inhibition was observed at both the RNA and protein level. Given the importance of IL-6 in cancer progression, and our finding that C10 attenuates IL-6 expression in the MDA-MB-231 cell line, this study suggests that C10 may have therapeutic potential for triple negative breast cancers. Additional studies are currently underway to further explore these hypotheses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1932. doi:1538-7445.AM2012-1932