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LMWH-taurocholate derivative (LHT7) has been reported as a novel angiogenesis inhibitor, due to its ability to bind to several kinds of growth factors, which play critical roles in tumor angiogenesis. In this study, we have highlighted the enhanced antiangiogenic activity of LHT7, by using cyclic RGDyk (cRGD), a targeting moiety that was chemically conjugated to LHT7 via amide bond. The SPR study revealed that cRGD-LHT7 bound to α(v)β(3) integrin as strongly as cRGD, and it bound to VEGF as strongly as LHT7. Importantly, in vitro anti-angiogenesis studies revealed that cRGD-LHT7 had a significant inhibition effect on HUVEC tubular formation. Finally, cRGD-LHT7 showed a greater inhibitory efficiency on the tumor growth in the U87MG xenograft model than the original LHT7, which was owed to its ability to target the tumor cells. All of these findings demonstrated that cRGD-LHT7 targeted α(v)β(3) integrin-positive cancer cells and endothelial cells, resulting in a greater anti-angiogenesis effect on the solid tumors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The oral delivery of macromolecules is a topic of much interest as this would undoubtedly improve patient acceptance and compliance with chronic regimens. Heparin and insulin are perhaps among the first candidates that should be considered for oral macromolecule delivery systems. Heparin is the most potent anti-coagulant known for the prevention of deep vein thrombosis and pulmonary embolism, and an orally active heparin would undoubtedly effectively reduce chronic thrombotic events. Here, we report on the development of an orally administrable chemical conjugate of heparin and hydrophobic deoxycholic acid (DOCA), which we refer to as LHD. LHD was pre-formulated with dimethyl sulfoxide (DMSO) as solubilizer to further improve its oral bioavailability (9.1% in monkey). LHD was found to be absorbed mainly in the jejunum and ileum of the small intestine, although it is in the ileum that the absorption is most notable. From the mechanism studies of LHD absorption using Caco-2 cell monolayers for mimicking the intestine, we found that LHD highly permeated by passive diffusion through the transcellular route and its permeation was partially affected by bile acid transporters. This study demonstrates the feasibility of chemically modified heparin for long-term oral administration as an effective therapy for venous thromboembolism in clinical trials.

Deoxycholic acid (DOCA) is a bile acid that facilitates the gastrointestinal (GI) absorption of low molecular weight heparin (LMWH) by bonding chemically to it. The calculated coupling ratio and bioactivity of LMWH-DOCA was 3.6 and 126.8 IU/mg, respectively. This study examined the efficacy of orally administered LMWH-DOCA in rat, mouse and monkey models. When 100 mg/kg (12,680 IU/kg) of LMWH-DOCA was administered to rats or mice, its maximum anti-factor Xa activity was 0.76+/-0.15 and 0.15+/-0.03 IU/ml, respectively. On the other hand, when a single dose of 100 mg/kg LMWH-DOCA mixed with either a bile solution or 200 mg/kg free DOCA was administered to mice, the anti-factor Xa activity of LMWH-DOCA was 0.42-0.45 IU/ml. Fluorescence studies confirmed that the free bile acid induced the morphological change in LMWH-DOCA in the buffer solution. In the monkey experiments, the bioavailability of LMWH-DOCA after administering 200 mg/kg free DOCA orally was 6.8%, which was 8 times higher than that of LMWH (0.8%) and 4 times higher than that of LMWH-DOCA in the absence of free DOCA (1.7%). The absorption of orally administered LMWH-DOCA occurred in all parts of the small intestine, particularly in the ileum without causing any damage such as fusion of the microvilli and dissolution or disorientation of the cell layers. The optimum oral formulation for LMWH-DOCA delivery in each animal model was determined according to the different absorption behavior of LMWH-DOCA.