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Heparin-like sulfated polysaccharides are potential drug candidates owing to their ability to interact with angiogenic factors and inhibit angiogenesis, tumor growth, and metastasis. This study aimed to improve the delivery of heparin-like anticancer polysaccharides for accumulation at the tumor site. We designed a nanocarrier system using protamine attached to polyethylene glycol (PEG) and evaluated the stability, tumor targeting, and tumor growth inhibition of the nanocarrier loaded with heparin derivatives. When mixed with various polyanionic heparin derivatives, the polycationic PEG-protamine formed stable self-assembled nanocomplexes via ionic interactions, with flexible PEG chains located on the outside. Among the complexes, a nanocomplex loaded with a low-molecular-weight heparin-suramin conjugate (LHsura) had the most suitable average size (101.9nm) for the enhanced permeability and retention effect and allowed accumulation of LHsura at the tumor site for up to 48h. In a tumor-bearing mouse model, the PEG-protamine and LHsura nanocomplex (10mg/kg/3days, intravenously), which could be extravasated through the tumor vasculature, significantly inhibited tumor growth, more than LHsura alone did. Overall, the self-assembled nanocomplexation of PEG-protamine and LHsura helped control the release and extravasation of LHsura, which resulted in an antitumor effect on the target tumor cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Context: As a class of angiogenesis inhibitors, heparin conjugates have shown significant effectiveness in several studies., Objectives: The purpose of our current study is to evaluate the effectiveness and safety of infusing the conjugate of low molecular weight heparin and taurocholate (LHT7), which has been developed as a potent angiogenesis inhibitor., Methods: To evaluate its safety, the method of intravenous infusion was compared with its i.v. bolus administration. Intravenous infusion was administered at a rate of 400 μl/min/kg of body weight for 30 min. Pharmacokinetic (PK) analysis, organ accumulation, and plasma concentration profiles of LHT7 were measured. The anticancer effect of LHT7 was evaluated in murine and human xenograft models, and preclinical studies were performed in SD rats and beagle dogs., Results: The results of the PK studies showed reduced organ accumulation in mice and the AUC(0-96 h) (area under the curve) was increased up to 1485 ± 125 h × μg/ml. The efficacy, at dose 1 mg/kg/2 d was higher for i.v. infusion than for i.v. bolus administration in both murine and human cancer models. The preclinical studies showed the safety dose of LHT7 is less than 20 mg/kg in SD rats and in the next safety analysis in beagle dogs showed that there were no organ-specific adverse effects in higher doses, such as, 12 mg/kg. LHT7 showed sustained effects with minimized adverse events when administered through i.v. infusion., Conclusions: LHT7 (i.v. infusion) could be safely used for further clinical development as a multi-targeting anti-angiogenic agent.

Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-β1 (TGF-β1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-β1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-β1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-β1 and CXCL12 (TGF-β1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-β1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-β1 and CXCL12 with LHTD4 were 0.85 and 0.019 μM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-β1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-β1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-β1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-β1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-β1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The preclinical safety of a newly developed oral anticoagulant, the low molecular weight heparin-deoxycholate conjugate (OH09208), was evaluated by a comprehensive evaluating program in compliance with standard guidelines. The single dose oral toxicity study in rats receiving 2000 and 5000 mg kg(-1) of OH09208 did not reveal any mortality, unusual body weight changes or necropsy findings. The results of the 4-week oral toxicity study with a 4-week recovery program in rats receiving OH09208 in doses of 100, 300 and 1000 mg kg(-1) day(-1) did not reveal any mortality, or indicate any unusual clinical signs, or show any toxicokinetic relationships to the administration of OH09208. Although the increase in liver enzymes in one male dog treated with 300 mg kg(-1) day(-1) and one female dog treated with 1000 mg kg(-1) day(-1) could not be excluded from the effect of the test substance, no other toxicologically significant changes were observed in the 4-week oral toxicity study with a 4-week recovery in beagle dogs. Thus, while the no-observed-adverse-effect level value from the 4-week study in both male and female rats was 1000 mg kg(-1) day(-1), those from the 4-week study in male and female beagle dogs were 300 and 1000 mg kg(-1) day(-1), respectively. Furthermore, OH09208 did not induce anaphylactic reactions in guinea pigs, micronucleated bone marrow cells in male ICR mice, chromosomal aberration in Chinese hamster lung cell lines, bacterial reverse mutation, and any abnormalities in hERG current assay, mouse central nervous system and dog cardiovascular studies. Overall, there were no unexpected toxicities in this preclinical study that might have precluded the safe administration of OH09208 to humans., (Copyright © 2015 John Wiley & Sons, Ltd.)

Low molecular weight heparin (LMWH) and its derivatives have been reported to possess antiangiogenic effect via electrostatic interaction with various angiogenic growth factors such as VEGF165. However, clinical applications of LMWH for anticancer therapy have been restricted due to its anticoagulant effect and insufficient therapeutic efficacy. To overcome these limitations and enhance the antiangiogenic efficacy, LMWH was conjugated with suramin fragments that have a binding affinity to the heparin-binding domain (HBD) of proteins. The conjugation of suramin fragments to LMWH enhanced the antiangiogenic effect of LMWH by increasing the binding affinity to VEGF165, while decreasing its anticoagulant activity. The chemical conjugate of LMWH and suramin fragments (LHsura) showed a substantial inhibitory effect on VEGF165-mediated cell proliferation, migration, and tube formation of HUVECs without significant cytotoxicity in vitro. Finally, we confirmed the anticancer effect of LHsura (61.4% vs control) in a SCC7-bearing mouse model.

Purpose: To overcome definite limitations of angiogenesis inhibitors such as insufficient therapeutic efficacy as a single drug and resisting or conflicting effect under chronic treatment, it is required to develop a new regimen to improve the therapeutic effect., Methods: The combination effect of a multi-targeting angiogenesis inhibitor (LHT7) and a selective cyclooxygenase-2 inhibitor (celecoxib) on neovascularization in tumor growth was studied both in vitro and vivo experiments., Results: While hypoxia-mediated COX-2 overexpression and macrophage recruitment were observed at LHT7-treated tumor tissues, it was well-controlled by the combination of celecoxib and LHT7. On the other hand, the in vitro tube formation and the in vivo tumor vessel formation and structure were inhibited by either LHT7 or celecoxib, but the inhibition effect was further enhanced by using them together. However, the combination therapy did not further enhance the inhibitory effect on tumor growth in terms of volume compared to single drug uses, which attributed not to increased cellular apoptosis but to decreased cell proliferation., Conclusions: COX-2 inhibition could enhance the therapeutic effect of anti-angiogenic drugs both by inhibiting the inflammatory reactions induced by hypoxia and by altering the vascular stabilization that is mediated by an assembly with mural cells.

Angiogenesis is well recognized as a pivotal process in tumor progression from the very early premalignant stages, thus making it an important target in cancer chemoprevention as well as in cancer treatment. In the present study, we introduce a recently developed oral heparin conjugate (LHTD4) for use as an inhibitor of angiogenesis and evaluate its therapeutic and preventive effects in two different animal models of lung cancer. The antiangiogenic activities of LHTD4 were evaluated using tube formation and Matrigel plug assays. VEGF- and bFGF-induced tube formations were reduced up to 77.2 and 67.3%, respectively, by LHTD4. Hemoglobin content was also significantly decreased by LHTD4 in the Matrigel plugs that were transplanted into mice. We observed that the VEGF- and bFGF-mediated phosphorylations of the receptors, VEGFR-2 and FGFR-1, were also inhibited by LHTD4. The in vivo antiangiogenic and anticancer effects of LHTD4 that developed following oral administration were verified in a tumor xenograft model of human A549 lung cancer cells: tumor volume was found to have decreased by 60.2% and the expressions of CD34 and Ki-67 in the LHTD4-treated group were affected. Finally, in a chemically induced lung carcinogenesis model, the number and area of each nodule were significantly reduced in the LHTD4-treated groups by 49.2% and 30.1%, respectively. In addition, the degree of angiogenesis in the lung tissue itself was decreased in the drug treatment group by 52.9%. Taken together, these results suggest that LHTD4 could be a promising candidate for angiogenesis inhibitor for the treatment and prevention of cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Angiogenesis is a key feature of cancer development, thus it is a good target for cancer therapy. However, drugs that have been designed to block angiogenesis mainly capture growth factors in circulation, resulting not only in the transient inhibition of tumor progression but also in producing undesirable side effects. Nanoparticular drug delivery systems, on the other hand, may help overcome such drawbacks and improve the efficacy of anti-angiogenic therapies by altering the biodistribution and pharmacokinetics, improving tumor targeting ability, and reducing side effects. In this light, we propose a new approach of anti-angiogenic therapy that combines strategies of long circulating, passive tumor targeting, and anti-angiogenesis efficacy using a new polyelectrolyte complex system that combines LHT7, a previously developed heparin-based angiogenesis inhibitor, with a protamine to form a self-assembling nanocomplex with a mean diameter of 200nm, which is effective for anti-angiogenesis therapy. At first, LHT7 was modified with polyethylene glycol (PEG). We observed that PEG-LHT7/protamine nanocomplex was stable in buffer and slowly dissociated in plasma (9% dissociation for 24h). Compared to the free form of PEG-LHT7, the mean residence time of PEG-LHT7/protamine nanocomplex was found higher (15.9h) with its increased accumulation in tumor. Most importantly, PEG-LHT7/protamine nanocomplex was diffused and extravasated through the dense collagen matrix of the tumor. Thus, the study describes a successful application of functionalized PEG-LHT/protamine nanocomplex that can inhibit angiogenesis with long circulating, passive targeting, and tumor extravasating ability., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Despite the therapeutic benefits of the angiogenesis inhibitors shown in the clinics, they have encountered an unexpected limitation by the occurrence of acquired resistance. Although the mechanism of the resistance is not clear so far, the upregulation of alternative angiogenic pathways and stabilization of endothelium by mural cells were reported to be responsible. Therefore, blocking multiple angiogenic pathways that are crucial in tumor angiogenesis has been highlighted to overcome such limitations. To develop an angiogenesis inhibitor that could block multiple angiogenic factors, heparin is an excellent lead compound since wide array of angiogenic factors are heparin-binding proteins. In previous study, we reported a heparin-derived angiogenesis inhibitor, LHT7, as a potent angiogenesis inhibitor and showed that it blocked VEGF signaling pathway. Here we show that LHT7 could block the fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) in addition to VEGF. Simultaneous blockade of these angiogenic factors resulted in inhibition of multiple stages of the angiogenic process, including initial angiogenic response to maturation of the endothelium by pericyte coverage in vitro. In addition, the treatment of LHT7 in vivo did not show any sign of vascular normalization and directly led to decreased blood perfusion throughout the tumor. Our findings show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of the angiogenesis, and could potentially be used to overcome the resistance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material., (Copyright © 2014 John Wiley & Sons, Ltd.)

Thrombogenesis is a major cause of morbidity and mortality in cancer patients. Prophylaxis with low-molecular-weight heparin (LMWH) is recommended for cancer patients, but requires non-parenteral delivery methods for long-term treatments. In this study, we sought to generate a new oligomeric-bile acid conjugate of LMWH that can be used for oral delivery. We first synthesized a tetramer of deoxycholic acid (tetraDOCA), which was site-specifically conjugated at the end saccharide of LMWH. When LMWH-tetraDOCA conjugate (LHe-tetraD) was orally administered at a dose of 5 mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62±0.05 IU/mL without any evidence of liver toxicity, gastrointestinal damage, or thrombocytopenia. The cancer-associated thrombosis was induced in tumor-bearing mice by local heat application, and the fibrin deposition in tumors was evaluated. The oral administration of LHe-tetraD (either a single dose or multiple daily doses for up to 10 days) in mice substantially abolished the coagulation-dependent tropism of fibrinogen in the heated tumors and significantly decreased hemorrhage, compared to the mice treated with saline or subcutaneous injection of LMWH. Thus, the anticoagulation effect of oral LHe-tetraD invokes the benefits of oral delivery and promises to provide an effective and convenient treatment for cancer patients at risk of thrombosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To achieve a clinically rational regimen for cancer chemoprevention with improved efficacy and safety, the combination effect of celecoxib and newly developed oral angiogenesis inhibitor, LHD4, on chemoprevention was evaluated. The chemopreventive effects of celecoxib, LHD4, and the combination of celecoxib and LHD4 were evaluated in a murine colorectal carcinogenesis model. After 17 experimental weeks, mouse colon tissues were collected and examined in terms of polyp volume and degree of carcinogenesis, inflammation, and angiogenesis. Mice in the celecoxib-treated or LHD4-treated groups had total polyp volumes of 47.0±9.7 and 120.1±45.2 mm, respectively, which represented decreases of 65.6 and 22.3% from the control (154.5±33.5 mm). However, the polyp volume in the combination group was 22.8±9.3 mm, a decrease of 85.2% from the control. In the comparison of carcinogenesis, the percentage of normal tissue (i.e. excluding proliferative tissue) was found to be 40.6% in the control, 51.7% in the celecoxib, 56.9% in the LHD4, and 81.7% in the combination group. In accordance with attenuated carcinogenesis, both inflammation and angiogenesis were also well controlled. Together, these results suggest that the combinatory use of celecoxib and a newly developed oral heparin conjugate could be a promising regimen for chemoprevention by intervening in both inflammation and angiogenesis.

Here, we report reduced graphene oxide (rGO) nanosheets coated with an anti-angiogenic anticancer taurocholate derivative of low-molecular-weight heparin (LHT7) as a tumor-targeting nanodelivery platform for anticancer drugs. Surface coating of LHT7 onto rGO was confirmed using fluorescein isothiocyanate-labeled LHT7, monitored as fluorescence quenching due to associated rGO. Unlike plain rGO, LHT7-coated rGO (LHT-rGO) nanosheets maintained a stable dispersion under physiological conditions for at least 24h. Moreover, LHT-rGO provided greater loading capacity for doxorubicin (Dox) compared with uncoated rGO nanosheets. Following intravenous administration into KB tumor-bearing mice, in vivo tumor accumulation of LHT-rGO/Dox was 7-fold higher than that of rGO/Dox 24h post dosing. In tumor tissues, LHT-rGO/Dox was shown to localize not to the tumor vasculature, but rather to tumor cells. Intravenously administered LHT-rGO/Dox showed the greatest anti-tumor effect in KB-bearing mice, reducing tumor volume by 92.5%±3.1% compared to the untreated group 25days after tumor inoculation. TUNEL assays revealed that the population of apoptotic cells was highest in the group treated with LHT-rGO/Dox. Taken together, our results demonstrate that LHT-rGO nanosheets confer improved dispersion stability, tumor distribution and in vivo antitumor effects, and may be further developed as a potential active nanoplatform of various anticancer drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Intestinal transporters are limited to the transport of small molecular substrates. Here, we describe the development of apical sodium-dependent bile acid transporter (ASBT)-targeted high-affinity oligomeric bile acid substrates that mediate the transmembrane transport of low molecular weight heparin (LMWH). Several oligomers of deoxycholic acid (oligoDOCA) were synthesized to investigate the substrate specificity of ASBT. To see the binding of oligoDOCA on the substrate-binding pocket of ASBT, molecular docking was used and the dissociation rate constants (KD) were measured using surface plasmon resonance. The KD for tetrameric DOCA (tetraDOCA) was 50-fold lower than that for monomeric DOCA, because tetraDOCA interacted with several hydrophobic grooves in the substrate-binding pocket of ASBT. The synthesized oligoDOCA compounds were subsequently chemically conjugated to macromolecular LMWH. In vitro, tetraDOCA-conjugated LMWH (LHe-tetraD) had highest selectivity for ASBT during its transport. Orally administered LHe-tetraD showed remarkable systemic anticoagulation activity and high oral bioavailability of 33.5±3.2% and 19.9±2.5% in rats and monkeys, respectively. Notably, LHe-tetraD successfully prevented thrombosis in a rat model of deep vein thrombosis. These results represent a major advancement in ASBT-mediated LMWH delivery and may facilitate administration of many important therapeutic macromolecules through a non-invasive oral route., (Copyright © 2013. Published by Elsevier B.V.)

Various angiogenesis inhibitors and apoptosis-targeting agents have been therapeutically applied in preclinical cancer models, some of which have been tested in clinical trials. In a previous study, we demonstrated that LHT7, a low molecular weight heparin (LMWH)-taurocholate conjugate, has strong antiangiogenic and tumor-suppressive activity and diminished anticoagulant properties. In this study, we developed LHT7-ApoPep-1, an apoptosis-homing peptide-conjugated variant of LHT7. LHT7-ApoPep-1 exhibited antiangiogenic activity in endothelial cell tube-formation assays and apoptotic cell-targeting ability in tumor cell binding assays; it also showed little toxicity toward healthy cells. Administration of LHT7-ApoPep-1 in mouse xenograft models of breast carcinoma delayed tumor growth compared to LHT7-only, and histological evaluations revealed decreased vessel formation and increased apoptotic area in tumor tissues. Moreover, an examination of LHT7-ApoPep-1-Cy7.5 localization within the body using in vivo live imaging showed accumulation at the tumor site of tumor-bearing mice, with a more prolonged circulation time and enhanced intensity compared to LHT7-Cy7.5. Inspection of the tumor microenvironment revealed that Cy5.5-labeled LHT7-ApoPep-1 was located on and near CD31-positive vessels in tumor tissue. We conclude that LHT7-ApoPep-1 has antiangiogenic and apoptosis-targeting properties and exerts antitumor effects by suppressing tumor vessel growth and homing to apoptotic cells within the tumor., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10 mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study was designed to develop a solid oral dosage form of deoxycholic acid (DOCA)-conjugated low-molecular-weight heparin (LMWH) and to evaluate its oral absorption, distribution, and metabolic stability for the prospect of providing an orally bioavailable LMWH. The LMWH derivative (LHD) was synthesised and then formulated with solubilisers and other pharmaceutical excipients to form a solid tablet. Its absorption and distribution after oral administration were evaluated in mice, rats, and monkeys. The in vitro metabolic stability of LHD was examined by liver microsome assays. More than 80% of LHD was released from the tablet within 60 minutes, guaranteeing rapid tablet disintegration after oral administration. Oral bioavailability of LHD in mice, rats and monkeys were 16.1 ± 3.0, 15.6 ± 6.1, and 15.8 ± 2.5%, respectively. After the oral administration of 131I-tyramine-LHD, most of the absorbed drug remained in the blood circulation and was eliminated mainly through the kidneys. LHD was hardly metabolised by the liver microsomes and showed a stable metabolic pattern similar to that of LMWH. In a rat thrombosis model, 10 mg/kg of orally administered LHD reduced thrombus formation by 60.8%, which was comparable to the anti-thrombotic effect of the subcutaneously injected LMWH (100 IU/kg). Solid tablets of LHD exhibited high oral absorption and statistically significant therapeutic effects in preventing venous thromboembolism. Accordingly, LHD tablets are expected to satisfy the unmet medical need for an oral heparin-based anticoagulant as an alternative to injectable heparin and oral warfarin.

Heparin, an anticoagulant that is widely used clinically, is also known to bind to several kinds of proteins through electrostatic interactions because of its polyanionic character. These interactions are mediated by the physicochemical properties of heparin such as sequence composition, sulfation patterns, charge distribution, overall charge density, and molecular size. Although this electrostatic character mediates its binding to many proteins related with tumor progression, thereby providing its antiangiogenic property, the administration of heparin for treating cancer is limited in clinical applications due to several drawbacks, such as its low oral absorption, unsatisfactory therapeutic effects, and strong anticoagulant activity which induces hemorrhaging. Here, we evaluated novel, orally active, low molecular weight heparin (LMWH) derivatives (LHD) conjugated with deoxycholic acid (DOCA) that show reduced anticoagulant activity and enhanced antiangiogenic activity. The chemical conjugate of LMWH and DOCA was synthesized by conjugating the amine group of N-deoxycholylethylamine (EtDOCA) with the carboxylic groups of heparin at various DOCA conjugation ratios. The LMWH-DOCA conjugate series (LHD1, LHD1.5, LHD2, and LHD4) were further formulated with poloxamer 407 as a solubilizer for oral administration. An in vitro endothelial tubular formation and in vivo Matrigel plug assay were performed to verify the antiangiogenic potential of LHD. Finally, we evaluated tumor growth inhibition of oral LHD administration in a SCC7 model as well as in A549 human cancer cell lines in a mouse xenograft model. Increasing DOCA conjugation ratios showed decreased anticoagulant activity, eventually to zero. LHD could block angiogenesis in the tubular formation assay and the Matrigel plug assay. In particular, oral administration of LHD4, which has 4 molecules of DOCA per mole of LMWH, inhibited tumor growth in SCC7 mice model as well as A549 mice xenograft model. LHD4 was orally absorbable, showed minimal anticoagulant activity and inhibits tumor growth via antiangiogenesis. These findings demonstrate the therapeutic potential of LHD4 as a new oral anti-cancer drug., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Introduction: Thrombogenic occlusion, at the anastomotic microvessels, contributed impaired blood flow to flap failure. The effect of an orally active low molecular weight heparin (LMWH) derivative conjugated with deoxycholic acid (DOCA) on the patency of anastomosis of the crushed rat artery was investigated, expecting its antithrombogenic effect., Materials and Methods: 60 Femoral arteries of 30 rats, divided into three groups of 20 each, were reanastomosed. LMWH-DOCA conjugate was orally administered prior to and after operation for 5 consecutive days. On the sixth day of operation, the patency of the anastomosed artery was evaluated., Results: The patency of oral LMWH-DOCA (10 mg/kg) group was significantly enhanced from 15% to 45%, compared to non-treated control group. On the other hand, when the dosage of LMWH-DOCA was reduced to 1mg/kg, its efficacy on anastomosis was not as efficacious in terms of patency. The intima of crushed artery was impaired and thrombus formation was examined in the control group. In the drug treated group, the patency was only compromised by a thin layer of thrombus that covered the inner layer of the vessel without causing any damage to the internal elastic lamina., Conclusion: The medication of oral LMWH-DOCA conjugate has been vetted in microvascular anastomosis of the crushed artery. LMWH-DOCA was potentially useful for improving the patency in compromised vessels after microsurgery., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Purpose: Orally absorbable anticancer medications have great advantages for conventional cancer therapies to patients. Here we evaluated the potent anticancer effect of orally absorbable LHD, a chemical conjugate of low-molecular-weight heparin and deoxycholic acid, on tumor graft growth models., Methods: We characterized the angiogenic factors, such as VEGF, heparanase, and MMPs, of murine squamous cell carcinoma (SCC7), melanoma (B16F10) or lung carcinoma (LLC1). Two weeks after oral administration of LHD into these cancer-cell-bearing mice, we evaluated the antiangiogenic activity of LHD., Results: Although all cancer cells expressed the angiogenic factors, SCC7 cells had much higher angiogenic potential and grew rapidly after implantation into mice. When orally administered, LHD delayed tumor graft growth regardless of cancer types. Particularly, LHD powerfully diminished the SCC7-derived tumor growth. Also, the expression of angiogenic factors in all kinds of tumor tissues was decreased, thereby attenuating the neovascularization in tumor tissue., Conclusion: Our study shows that LHD has potent anticancer and antiangiogenic effect on at least three kinds of tumor cells. LHD can be specifically used for preventing neovascularization in tumor tissue because it has therapeutical potential as an antiangiogenic drug and can be orally absorbed.

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low-molecular heparin (LMWH), for treating cancer are limited because of heparin's anticoagulant activity and risk of hemorrhages. LMWH-taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF(165)) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF-mediated KDR (VEGF-receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF(165) dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF(165) inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA-MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline-type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF(165). Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA-MB231 human breast cancer cell lines., (Copyright 2008 UICC.)

Purpose: Orally active anticancer drugs have great advantages for the treatment of cancer. Compelling data suggest that heparin exhibits critical antimetastatic effects via interference with P-selectin-mediated cell-cell binding. However, heparin should be given parenterally because it is not orally absorbed. Here, we evaluated the inhibitory effect of orally absorbable heparin derivative (LHD) on experimentally induced metastasis., Experimental Design: We developed LHD, which is a chemical conjugate of low molecular weight heparin and deoxycholic acid, and measured the plasma concentration of LHD after oral administration. To evaluate the antimetastatic effect of LHD, we carried out experimental lung metastasis assays in vivo using murine melanoma or human lung carcinoma cells and interruption assay between murine melanoma cells and activated platelets and human umbilical vascular endothelial cells in vitro., Results: In mice, the plasma concentration was approximately 7 microg/mL at 20 minutes after oral administration of LHD (10 mg/kg), indicating that bleeding was not induced at this dose. Interestingly, we found that LHD dramatically attenuated metastasis experimentally induced by murine melanoma or human lung carcinoma cells and that its antimetastatic activity was attributed to the interruption of the interactions between melanoma cells and activated platelets and between melanoma cells and human umbilical vascular endothelial cells by blocking selectin-mediated interactions. Furthermore, it prevented tumor growth in secondary organs., Conclusions: On the basis of these findings, the present study shows the possibility of LHD as a suitable first-line anticancer drug that can be used for preventing metastasis and recurrence because it has therapeutic potential as an antimetastatic drug, has lower side effects, and can be orally absorbed.

The development of orally active heparin will have tremendous clinical importance since it can be used to effectively prevent deep vein thrombosis (DVT) in a long-term chronic treatment. We developed in this study a new orally active heparin derivative (Db-LHD), which has heparin chemically conjugated with deoxycholic acid and DMSO molecules by secondary interactions. Db-LHD was prepared in the powder form in soft capsules. When we administered Db-LHD capsules to monkeys, its oral physiological availability was increased up to 16.6%. The maximum anti-FXa activity at 5 mg/kg of Db-LHD was more than twice the minimum effective anti-FXa activity (MEC, 0.1 IU/mL) for preventing DVT, and the anti-FXa activity in plasma was maintained for 10 h above the MEC in monkeys. Also, we evaluated anti-thrombogenic effect of Db-LHD in a rat thrombosis model. A subcutaneous administration of enoxaparin (100 IU/kg), which was the highest recommended dose for the prevention of venous thromboembolism, reduced thrombus formation by 38.9+/-14.2%. On the other hand, 5 mg/kg (425 IU/kg) of orally administered Db-LHD reduced thrombus formation by 51.0+/-2.0. We propose a new orally active heparin, Db-LHD, in a solid dosage form to effectively prevent DVT and PE.

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.

Heparin, a potent inhibitor of blood coagulation, exhibits antitumoral action in tumor progression such as in angiogenesis and metastasis but is not orally absorbed in the body, making it an attractive candidate as an oral drug for antiangiogenic cancer therapy. We generated LHD or orally active heparin using low molecular weight heparin (LMWH) and deoxycholic acid that is effectively absorbed in the gastrointestinal tract. Using the in vitro endothelial tubular formation and chicken chorioallantoic membrane angiogenesis assay, we found that antiangiogenic activity of this LHD was similar to that of LMWH. From the in vivo Matrigel plugs assay, LHD treated orally could effectively inhibit angiogenesis into the plugs induced by basic fibroblast growth factor, whereas LMWH treated orally could not due to no oral absorption. In addition, when this LHD was orally administered into the tumor bearing mice, it significantly inhibited tumor growth by its antiangiogenic therapeutic mechanism, and when accompanied with doxorubicin, it appeared to have an additive effect. Collectively, LHD having antiangiogenic activity could be orally absorbable and inhibit tumor growth via inhibiting angiogenesis. These findings demonstrate the therapeutic potential of LHD in the clinical trials, which is suggested as a new oral therapeutic remedy for cancer therapy.

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.

Heparin is available to patients only by parenteral administrations due to its low oral bioavailability. For the oral delivery of low molecular weight heparin (LMWH), LMWH-DOCA was synthesized by chemical conjugation of LMWH and deoxycholic acid (DOCA) and this conjugate was formulated in a microemulsion system. The coupling ratios of DOCA to LMWH for LD1 and LD2 were 1.33 and 2.37, respectively. The microemulsion was composed of tricaprylin, surfactant mixture (Tween 80 and Span 20) and LMWH-DOCA in water, and their volume ratio was 5:3:1:1. Pharmacokinetic parameters of LMWH were not significantly changed by conjugation with DOCA; however, when LMWH-DOCA in tricaprylin microemulsion was orally administered in mice, its bioavailability was increased up to 1.5%. Furthermore, the enhancing effect of the conjugated DOCA in the tricaprylin microemulsion on the absorption of LMWH in the intestine was more significantly increased in monkey than in mice. Since the tricaprylin microemulsion could dissolve LMWH-DOCA, this formulation could maximize the enhancing effect of the conjugated DOCA on the absorption of LMWH in the intestine. Finally, it was expected that 20 mg/kg of LMWH-DOCA in the tricaprylin microemulsion was enough to prevent deep vein thrombosis (DVT) and pulmonary embolism (PE).

Background: Heparin administration is usually limited to intravenous or subcutaneous injection. Oral delivery of heparin is an alternative to this and has been in great demand for treating patients who are at a high risk of deep vein thrombosis or pulmonary embolism. In this study, new heparin derivatives were synthesized to enhance the oral absorption of heparin in the gastrointestinal tract. Methods and Results- By using heparin of 3000 Da [LMWH(3 kDa)], heparin of 6000 Da [LMWH(6 kDa)], and unfractionated heparin (UFH), we synthesized 3 kinds of conjugates of heparin and deoxycholic acid (DOCA): LMWH(3 kDa)-DOCA, LMWH(6 kDa)-DOCA, and UFH-DOCA. After oral administration of 100 mg/kg of heparin-DOCA, the maximum activated partial thromboplastin times of the LMWH(3 kDa)-DOCA, LMWH(6 kDa)-DOCA, and UFH-DOCA were 31.0+/-6.0, 87.8+/-11.1, and 51.0+/-8.7 seconds, respectively. The peak plasma concentrations of LMWH(3 kDa)-DOCA, LMWH(6 kDa)-DOCA, and UFH-DOCA were 0.06+/-0.02, 0.76+/-0.15, and 0.41+/-0.13 IU/mL, respectively. The bioavailability of LMWH(6 kDa)-DOCA at the 20-mg/kg dosage was calculated to be 7.8%., Conclusions: LMWH(6 kDa)-DOCA was found to have a high anticoagulant effect when administered orally and could be used as a new oral anticoagulant agent. Furthermore, the present work proposed a new method for oral delivery of macromolecules and polysaccharide drugs.

The immobilization of heparin onto polymeric surfaces using hydrophilic spacer groups has been effective in curtailing surface induced thrombus formation. In this study, the effect of hydrophilic spacers (PEO) on the binding kinetics of immobilized heparin with antithrombin III (ATIII) and thrombin was investigated. Monodispersed, low molecular weight heparin was fractionated on an ATIII affinity column to isolate high-ATIII affinity heparin. This high-ATIII affinity fraction was immobilized onto a styrene/p-amino styrene random copolymer surface using hydrophilic poly(ethylene oxide) (PEO) spacer groups. Styrene/p-amino styrene random copolymer was chosen as the model surface to provide quantitative and reproducible surface concentrations of available amine groups, grafted PEO spacers, and immobilized heparin. The polymer substrate was coated onto glass beads, tolylene diisocyanate modified PEO was covalently coupled to the surface, followed by heparin immobilization. The bioactivity of immobilized heparin was 16.2%, relative to free heparin, and a 1:1 binding ratio between heparin and PEO was achieved. The binding of ATIII and thrombin to control surfaces (no heparin), soluble heparin, heparin immobilized directly onto the surface, and heparin immobilized via spacer groups, were compared. Soluble heparin bound both thrombin and ATIII, while heparin immobilized directly onto the surface bound only thrombin. Spacer-immobilized heparin bound both ATIII and thrombin, although to a lesser extent than soluble heparin. Thus, the enhanced bioactivity of spacer-immobilized heparin, compared to direct-immobilization, may be attributed to the retention of ATIII binding.