Ok Cheol Jeon

High antiangiogenic and low anticoagulant efficacy of orally active low molecular weight heparin derivatives

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.

Anticoagulant efficacy of solid oral formulations containing a new heparin derivative.

The need for an efficacious and safe oral anticoagulant that does not require monitoring has been largely unmet. Many efforts have centered on preparing orally available heparin to improve patient compliance. In this study, novel orally active heparin derivatives (LHD), i.e. low molecular weight heparin (LMWH) conjugated with deoxycholic acid (DOCA), were evaluated in vitro and in vivo for their enhancement effect of oral heparin absorption. After oral administration of 10 mg/kg of water-soluble LHD, Ws-LHD1.5 showed optimum oral efficacy and its bioavailability was about 24% in rats. The oral absorption of LHD1.5 was also enhanced by several solubilizers, among which Poloxamer 407 provided the best results. When 5 mg/kg of LHD1.5 with Poloxamer 407 was orally administered to monkeys, the maximum anti-FXa activity in plasma was 0.26 +/- 0.04 IU/mL and its bioavailability was 17.4%. In a rat thrombosis model, 5 mg/kg of orally administered LHD1.5 formulated with Poloxamer reduced thrombus formation by 63.9 +/- 16.6%, which was higher than the efficacy of clinically used enoxaparin (49.4 +/- 17.8% at 100 IU/kg, sc). Considering the oral absorption efficacy and therapeutic effect, the conjugation ratio was optimized as about 1.5 molecules of DOCA per mole of heparin. Therefore, LHD1.5 with Poloxamer 407 can be further formulated as a solid oral anticoagulant drug.

Diabetes Correction in Pancreatectomized Canines by Orally Absorbable Insulin-Deoxycholate Complex

Oral insulin therapy has great potential benefits over conventional therapy for diabetic patients as well as mimicking the physiological fate of insulin. Here we evaluated the characteristics of insulin and deoxycholate-based synthetic N(alpha)-deoxycholyl-L-lysyl-methylester (DCK) complex, and diabetes correction in pancreatectomized canines after oral administration. After the insulin/DCK complexation was made, the insulins folding structure, stability against digestive enzymes, lipophilicity and permeability to Caco-2 monolayer were evaluated in vitro. Diabetic canines were kept under fasting conditions, and Eudragit-coated gelatin capsules containing insulin or insulin/DCK powder were singly or triply administered. Evaluation of glucodynamics, pharmacokinetics, oral glucose tolerance test (OGTT) and reproducibility were carried out. After complexation with DCK, the folding structure of insulin did not become denatured and the resistance against digestive enzymes was powerfully improved. The lipophilicity and permeability of insulin/DCK (coupling ratio up to 1:10) were also highly increased. The insulin/DCK complex, administered orally into diabetic canines at the doses of 21, 42, and 81 IU/kg, reduced the plasma glucose levels by about 28%, 44% and 67%, respectively, while the plasma insulin concentrations increased. During OGTT, insulin/DCK nearly maintained the normoglycemic state in the diabetic canines, whereas the hyperglycemic state of placebo-treated controls was not corrected. During oral administration of insulin/DCK, it repetitively showed similar therapeutic efficacy in diabetic canines for 3 days. The therapeutic efficacy of insulin/DCK was exhibited in its digestive enzyme resistance, deoxycholate-based lipophilicity for enhancing permeability and intact insulin delivery without chemical modification, providing potential oral therapeutic remedy as an alternative to injectable insulin medication.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells

Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration–approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL)‐induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half‐life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG) that has a much longer half‐life in rodents than native‐type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half‐life over native‐type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride‐induced fibrosis/cirrhosis in rats by simultaneously down‐regulating multiple key fibrotic markers that are associated with aHSCs. Conclusion: TRAIL‐based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209–223)

Potentiation of anti-angiogenic activity of heparin by blocking the ATIII-interacting pentasaccharide unit and increasing net anionic charge.

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.

Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis.

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.

Pharmacokinetic evaluation of an oral tablet form of low-molecular-weight heparin and deoxycholic acid conjugate as a novel oral anticoagulant

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.

Antithrombotic efficacy of an oral low molecular weight heparin conjugated with deoxycholic asset on microsurgical anastomosis in rats

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.

Combinational chemoprevention effect of celecoxib and an oral antiangiogenic LHD4 on colorectal carcinogenesis in mice

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 mm3, respectively, which represented decreases of 65.6 and 22.3% from the control (154.5±33.5 mm3). However, the polyp volume in the combination group was 22.8±9.3 mm3, 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.