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Background: Venous thromboemblism is associated with a systemic hypercoagulable state that is secondary to tumor or proinflammatory activation of procoagulant mechanisms, as well as down-regulation of anticoagulant mechanisms. In the present investigation, the potential antithrombotic efficacy and mechanism for OT-304 analogs, novel small molecules targeting NFkB and oxidative stress pathways, was examined in various cellular systems. Methods: Human monocytic cells were incubated with OT-304 analogs for 3 hours and then stimulated with LPS (25 ng/ml) for another 3 hours. Real-time PCR for NF-κB (P50/P65 complex) nuclear translocation dynamics, TNF-alpha, EGR1, and tissue factor (TF) was then performed. Additionally, the effect of OT-304 analogs on oxidative stress, inflammation, and procoagulant pathways in human monocytes and endothelial cells were performed using Affymatrix Microarray. Furthermore, the anticoagulant efficacy of OT-304 analogs as compared to classical anticoagulants such as Low Molecular weight Heparins (LMWH) was determined in proinflammatory-mediated hypercoagulable state, cancer, and/or chemotherapy-associated platelet/fibrin clot formation assays using thrombelastography (TEG). TEG was performed using blood from healthy volunteers. Clot dynamics were initiated using different activators of coagulation that included different cancer types, different chemotherapeutic agents, and standard anti-angiogenesis agents such as bevacizumab, ranibizumab and others in human blood. Additionally, effects of OT-304 analogs as compared to unfractionated heparin and the LMWH enoxaparin on activated partial thromboplastin time (aPTT) in human plasma was carried out using a fibrometric method. Results: OT-304 analogs effectively inhibited NF-κB and oxidative stress pathways leading to down-regulation of cytokines and chemokines, including TNF-alpha. OT-304 analogs effectively inhibited EGR1, which was associated with down-regulation of the key procoagulant mediator tissue factor (TF). In human blood, OT-304 effectively inhibited hypercoagulation and prothrombotic states mediated by proinflammatory stimuli including LPS, NF-κB activators such as ceramide, different cancer types such as pancreatic, glioma and lung cancer, different chemotherapeutic agents such as doxorubicin and etopocide, and angiogenesis inhibitors such as bevacizumab (Avastin) or ranibizumab (Lucentis) as evident from the inhibition of platelet-fibrin clot dynamics. The IC50 required for blocking cancer or drug-associated thrombosis ranged from 1–3 uM. OT-304 analogs did not have any effect on aPTT up to concentrations ranging from 1– 100 uM in human plasma. In contrast, heparin or LMWH resulted in a dose-dependent (0.001 – 1 ug/ml) increase in aPTT. These data suggest OT-304 effectively inhibits prothrombotic events, regardless of the stimulus, with potentially no effect on hemostasis. Conclusions: OT304 analogs, by virtue of their ability to target and modulate more than one pathway, may represent a promising therapeutic strategy for inhibiting thrombosis associated with inflammation, cancer, and chemotherapy plus other adjunct therapies without compromising hemostasis.

Thyroid hormone has been recently shown to induce tumor growth and angiogenesis. These angiogenesis modulating activities are initiated at endothelial cell plasma membrane receptor via the integrin αVβ3, at or near the Arg-Gly-Asp (RGD) recognition site on the integrin. In the present study, we have investigated the effect of tetraiodothyroacetic acid (tetrac), a deaminated thyroid hormone analog that inhibits thyroid hormone-binding to the cell surface integrin, on angiogenesis and cancer cell resistance to doxorubicin both in vitro and in vivo. Two angiogenesis models were studied in which vascular endothelial growth factor, VEGF165 or basic fibroblast growth factor, FGF2 (1–2 μg/ml) or thyroid hormone, thyroxin (L-T4 or T3) were used either to induce tube formation in the human dermal micro-vascular endothelial cells (HDMEC), or to stimulate new blood vessel branch formation in the chick chorioallantoic membrane (CAM) models. In both models, Tetrac (0.1–10 μM) inhibited the pro-angiogenesis activity of VEGF, FGF2, L-T4 or T3 by more than 50% at 1.0 uM RT-PCR revealed that tetrac (1–3 μM) decreased abundance of angiopoietin-2 mRNA but did not affect the mRNA levels of angiopoietin-1, in VEGF-exposed endothelial cells, suggesting that specific angiogenic pathways are targeted by this compound. Additionally, microarray was used to examine changes in expression of Matrix Metalloproteinases (MMP) and Tissue Inhibitor of Metalloproteinases (TIMP) following VEGF treatment with and without tetrac. HDMEC cells treated with VEGF exhibited 3–5-fold increase in MMP-15 and MMP-19 expression and tetrac (3μM), inhibited expression of MMP-15 and MMP-19 by 3–9-fold, respectively. Expression of TIMP-3 was increased 5.4-fold following VEGF and tetrac treatment when compared to treatment with VEGF alone. This finding suggests that part of the mechanism by which tetrac inhibits VEGF-stimulated angiogenesis involves inhibition of certain MMPs and increase in TIMP expression. Investigation of the anti-proliferative function of tetrac was carried out using the αVβ3 expressing breast cancer cells MC7 and their drug resistant counterparts. Interestingly, proliferation of both cell lines was inhibited similarly by tetrac suggesting that this analog may circumvent drug resistance. In fact, tetrac was able to reverse resistance to doxorubicin in vitro and to suppress growth of doxorubicin resistant tumors in nude mice. Inhibition of the drug transporter p-glycoprotein was found to play a key role in mediating the action of tetrac. Taken together, findings presented in this study provide evidence that the anticancer function of tetrac can be attributed to its anti-angiogenic and drug resistance reversal activities.

Heparin is not orally absorbed, presumably because of its size and polyanionic charge and hence is administered parenterally, either by continuous or intermittent infusion or by subcutaneous (SC) injection. However, a formulation that would result in absorption of heparin after oral administration would provide an attractive alternative to parenteral heparin. In that regard several attempts to develop effective non-parenteral heparin formulations have been reported, but they have met with limited success. The present investigation determined the molecular structure, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of oral unfractionated heparin (UFH) containing oral absorption enhancer Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate, salcaprozate sodium (SNAC) and assessed the safety and tolerability of orally dosed heparin solid dosage form versus other routes. Sixteen healthy men were included in this single-dose, three-way crossover, randomized, open-label study. The different arms of the study were as follow: Treatment A- Subjects randomized to Treatment A received two capsules of UFH/SNAC soft gelatin capsules for a total dose of 75,000 U heparin sodium with 150 mL of water; Treatment B: Subjects randomized to Treatment B received 1 mL of heparin sodium USP parenteral 10,000 U/mL via SC injection; Treatment C: Subjects randomized to receive 75,000 U heparin sodium milled powder for oral solution with 150 mL of water; and Treatment D: Subjects randomized to receive 0.5 mL of heparin sodium USP parenteral 5,000 U/mL via IV bolus. Each subject was randomly assigned to receive three of the four treatments so that a total of 12 subjects received each treatment. To characterize the PK of SNAC after oral heparin/SNAC administration and the PD of heparin following the administration of heparin IV, SC, PO alone and PO as heparin/SNAC, 21 blood samples were drawn from all of the subjects receiving treatment A, B, C, and D at the following time points: within 30 minutes pre-dose, at 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 minutes, and at 1,1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 hours post-dose. Blood was drawn into sodium citrate tubes for assessment of anti-factor Xa, anti-factor IIa, aPTT, and SNAC. Disaccharide compositional analysis was performed using capillary high-performance liquid chromatography with electrospray ionization mass spectrometry detection. The PD of heparin was obtained from analysis of plasma anti-factor Xa, anti-factor IIa, aPTT, and total tissue factor pathway inhibitor (TFPI) data. The molecular weight properties and the disaccharide composition of orally administered UFH/SNAC and parenterally administered UFH are identical and consistent with the starting standard UFH administered and achieved anti-factor Xa: anti-factor IIa of 1:1. This is the first true pharmacokinetic study to measure the chemical compositions of heparin administered by different routes.