deoxycholic-acid and Venous-Thrombosis

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.

Topics: Administration, Oral; Animals; Anticoagulants; Cell Line; Deoxycholic Acid; Drug Carriers; Heparin, Low-Molecular-Weight; Macaca fascicularis; Male; Models, Molecular; Organic Anion Transporters, Sodium-Dependent; Rats; Rats, Sprague-Dawley; Symporters; Venous Thrombosis

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.

Topics: Administration, Oral; Animals; Anticoagulants; Biological Availability; Deoxycholic Acid; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Stability; Haplorhini; Heparin, Low-Molecular-Weight; Humans; Kidney; Male; Mice; Mice, Inbred ICR; Protein Engineering; Rats; Rats, Sprague-Dawley; Tablets; Venous Thrombosis

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.

Topics: Administration, Oral; Animals; Anticoagulants; Capsules; Chemistry, Pharmaceutical; Deoxycholic Acid; Dimethyl Sulfoxide; Disease Models, Animal; Drug Compounding; Enoxaparin; Factor Xa Inhibitors; Heparin, Low-Molecular-Weight; Intestinal Absorption; Ligation; Macaca fascicularis; Male; Mice; Mice, Inbred ICR; Powders; Rats; Rats, Sprague-Dawley; Solvents; Vena Cava, Inferior; Venous Thrombosis

The use of heparin as the most potent anticoagulant for the prevention of deep vein thrombosis and pulmonary embolism is nevertheless limited, because it is available to patients only by parenteral administration. Toward overcoming this limitation in the use of heparin, we have previously developed an orally active heparin-deoxycholic acid conjugate (LMWH-DOCA) in 10% DMSO formulation. The present study evaluates the anti-thrombogenic effect of this orally active LMWH-DOCA using a venous thrombosis animal model with Sprague-Dawley rats. When 5 mg/kg of LMWH-DOCA was orally administered in rats, the maximum anti-FXa activity in plasma was 0.35 +/- 0.02, and anti-FXa activity in plasma was maintained above 0.1 IU/ml [the minimum effective anti-FXa activity for the prevention of deep venous thrombosis (DVT) and pulmonary embolism (PE)] for five hours. LMWH-DOCA (5 mg/kg, 430 IU/kg) that was orally administered reduced the thrombus formation by 56.3 +/- 19.8%;on the other hand, subcutaneously administered enoxaparin (100 IU/kg) reduced the thrombus formation by 36.4 +/- 14.5%. Also, LMWH-DOCA was effectively neutralized by protamine that was used as an antidote. Therefore, orally active LMWH-DOCA could be proposed as a new drug that is effective for the longterm prevention of DVT and PE.

Topics: Administration, Oral; Animals; Anticoagulants; Deoxycholic Acid; Heparin; Heparin, Low-Molecular-Weight; Humans; Male; Models, Chemical; Rats; Rats, Sprague-Dawley; Time Factors; Venous Thrombosis