Dose-dependent Antithrombotic Effect Research Articles

Discovery of Potent and Orally Bioavailable Pyridine N-Oxide-Based Factor XIa Inhibitors through Exploiting Nonclassical Interactions.

Activated factor XI (FXIa) inhibitors are promising novel anticoagulants with low bleeding risk compared with current anticoagulants. The discovery of potent FXIa inhibitors with good oral bioavailability has been challenging. Herein, we describe our discovery effort, utilizing nonclassical interactions to improve potency, cellular permeability, and oral bioavailability by enhancing the binding while reducing polar atoms. Beginning with literature-inspired pyridine N-oxide-based FXIa inhibitor 1, the imidazole linker was first replaced with a pyrazole moiety to establish a polar C-H···water hydrogen-bonding interaction. Then, structure-based drug design was employed to modify lead molecule 2d in the P1' and P2' regions, with substituents interacting with key residues through various nonclassical interactions. As a result, a potent FXIa inhibitor 3f (Ki = 0.17 nM) was discovered. This compound demonstrated oral bioavailability in preclinical species (rat 36.4%, dog 80.5%, and monkey 43.0%) and displayed a dose-dependent antithrombotic effect in a rabbit arteriovenous shunt model of thrombosis.

Sulfated polysaccharide from the red algae Gelidiella acerosa: Anticoagulant, antiplatelet and antithrombotic effects

A sulfated polysaccharide from the red algae Gelidiella acerosa (GaSP) was obtained through enzymatic extraction and subjected to chemical characterization by HPSEC, elemental microanalysis, FT-IR and NMR spectroscopies. The GaSP anticoagulant activity was investigated through APTT and PT tests and platelet aggregation assessed by turbidimetry. The antithrombotic and hemorrhagic activities were evaluated by venous thrombosis and hemorrhagic tendency models, respectively. FT-IR and NMR demonstrated that GaSP is a sulfated agaran. HPSEC and elemental microanalysis revealed a peak molar mass of 284.8 kDa and a degree of sulfation of 0.63, respectively. This molecule prolonged the coagulation time in 2.1 times and inhibited the platelet aggregation by 45%. Furthermore, it showed significant dose-dependent antithrombotic effect of 40%, 64% and 80% at 0.1, 0.5 and 1 mg/kg, respectively, without hemorrhage. These results suggest that GaSP has promising antithrombotic.

Prevention of Stent Thrombosis in Rats by a Direct Oral Factor Xa Inhibitor Edoxaban

Background/Aims: Stent thrombosis is a serious complication after percutaneous coronary intervention and femoropopliteal endovascular intervention. The aim of this study was to determine the antithrombotic effects of a direct factor Xa inhibitor edoxaban alone or in combination with antiplatelet agents in a rat model of stent thrombosis. Methods: Stainless steel stents (4 stents per rat) were placed in an extracorporeal arteriovenous shunt. The shunt was inserted into the carotid artery and the jugular vein in each rat to circulate blood. Stent thrombosis was induced by exposing the stents to arterial blood for 30 min. Protein content of the thrombus was measured. Edoxaban and antiplatelet agents (aspirin, clopidogrel, and ticagrelor) were orally administered before the thrombus induction. Results: Edoxaban (0.3–3 mg/kg), clopidogrel (1–10 mg/kg), aspirin (10–100 mg/kg), and ticagrelor (0.3–3 mg/kg) exerted significant and dose-dependent antithrombotic effects in a rat stent thrombosis model. The effect of edoxaban was comparable to that of antiplatelet agents. The combination of submaximal doses of edoxaban and clopidogrel or aspirin significantly potentiated the antithrombotic effects compared with antiplatelet agents alone. Conclusion: These results suggest that edoxaban alone and in combination with clopidogrel or aspirin are promising antithrombotic therapies for the prevention of stent thrombosis.

Antithrombotic Activity of DAB-15, a Novel Diazepinobenzimidazole Compound.

Antithrombotic activity of a new orally administered antiplatelet compound DAB-15 was compared to that of acetylsalicylic acid, ticlopidine, and clopidogrel in the experimental model of arterial thrombosis in rats caused by surface application of 50% ferric chloride (III) on the carotid artery. Compound DAB-15 exerted a dose-dependent antithrombotic effect and was superior to acetylsalicylic acid, ticlopidine and clopidogrel by 5, 7, and 4.9 times, respectively (by ED50). This necessitates studying of the action mechanism of this antiplatelet compound with consideration of its influence on different stages of the pathogenesis of platelet aggregation.

Evaluation of a multifunctional staphylokinase variant with thrombin inhibition and antiplatelet aggregation activities produced from salt-inducibleE.coliGJ1158

Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models.

Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats

BackgroundEdoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants. ObjectivesTo compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage. MethodsRats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60minutes. Tail template bleeding time was measured after making an incision on the tail. ResultsIn rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose–response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin. ConclusionsThese results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.

Evaluation of the Antithrombotic and Hemorrhagic Effects of a Novel Ultra Low Molecular Weight Heparin (ULMWH)

Semuloparin (S) (Sanofi‐Aventis, Paris, France) is an ULMWH exhibiting high anti‐Xa (aXa) activity due to preservation of antithrombin‐binding sites. Antithrombotic (jugular vein clamping model) and hemorrhagic (tail transection model) effects of S, the LWMH enoxaparin (E) and heparin (H) were compared in Sprague‐Dawley rats treated subcutaneously (SC) with doses of 1.0 and 2.5 mg/kg (n=5). Blood samples collected prior to euthanasia were analyzed using aPTT, aXa and Heptest (HT) assays. 1.0 mg/kg SC H significantly prolonged bleeding time compared to control (C). All agents had equivalent antithrombotic effects. S and E significantly increased HT prolongation and aXa activity compared to C and H (HT: p&lt;0.001 A vs H; p&lt;0.05 E vs H; Xa: p&lt;0.001 A vs H; p&lt;0.01 E vs H). At 2.5 mg/kg SC, only S did not significantly prolong bleeding time compared to C. Only H significantly increased aPTT compared to C. Prolongation of HT and aXa activity was observed with all agents. S exhibited a more potent aXa activity than H (p&lt;0.001 S vs H). All agents demonstrated a dose‐dependent antithrombotic effect. Bleeding time was lower with S at both dosages, suggesting a better safety profile. AXa activity with S was closely related to its antithrombotic activity. These studies suggest that S represents a new alternative for the prevention of thrombotic disorders with an improved bene&amp;#64257;t/risk pro&amp;#64257;le compared with standard H and E.

A small-molecule factor XIa inhibitor produces antithrombotic efficacy with minimal bleeding time prolongation in rabbits

BMS-262084 is a 4-carboxy-2-azetidinone-containing irreversible inhibitor of FXIa, which is selective over other coagulation proteases. We evaluated the in vitro and in vivo properties of BMS-262084 in rabbits. Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrolytic-mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. BMS-262084 was infused IV from 1h before thrombus induction or cuticle transection to the end of the experiment. In vitro, BMS-262084 prolonged activated partial thromboplastin time (aPTT) with EC(2x) (concentration required to double aPTT) of 10.6μM in rabbit plasma, and did not prolong prothrombin time (PT), thrombin time (TT) and HepTest. In vivo, BMS-262084 produced dose-dependent antithrombotic effects in rabbits with antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT of 0.4, 0.7 and 1.5mg/kg/h IV, respectively. BMS-262084 increased ex vivo aPTT dose-dependently without changes in PT and TT. The antithrombotic effect of BMS-262084 was significantly correlated with its ex vivo aPTT, supporting the use of ex vivo aPTT as a pharmacodynamic biomarker. BMS-262084 did not alter ex vivo rabbit platelet aggregation to ADP and collagen. BT (fold-increase) determined at 3 and 10mg/kg/h of BMS-262084 were 1.17±0.04 and 1.52±0.07*, respectively (*P<0.05 vs. control). This study demonstrated that BMS-262084 prevented experimental thrombosis at doses with low BT effects in rabbits, and suggests that a small molecule FXIa inhibitor may represent a promising antithrombotic therapy.

Evaluation of AR-H067637, the active metabolite of the new direct thrombin inhibitor AZD0837, in models of venous and arterial thrombosis and bleeding in anaesthetised rats

AZD0837, currently in clinical development, is a once-daily oral anticoagulant that is bioconverted to AR-H067637, a selective, reversible direct thrombin inhibitor (DTI). When developing a new DTI, the antithrombotic effects are commonly investigated in in vivo animal models; this report shows the effect of AR-H067637 in venous and arterial thrombosis and bleeding models in anaesthetised rats. Thrombus formation was induced by topical application of ferric chloride to the carotid artery or to the caval vein with partial stasis. Cutaneous incision bleeding time and muscle transection blood loss were assessed, with or without acetylsalicylic acid (ASA). Activated partial thromboplastin time (APTT), ecarin coagulation time (ECT) and thrombin coagulation time (TCT) were used as plasma biomarkers of anticoagulant effect. Dalteparin was used as a reference compound. AR-H067637, given by continuous infusion, displayed a dose-dependent antithrombotic effect, with 50% inhibition (IC50) of thrombus size in venous and arterial thrombosis models obtained at plasma concentrations of 0.13 μM and 0.55 μM, respectively, without increased bleeding. Dose-dependent increased bleeding and blood loss were seen at plasma concentrations ≥1 μM AR-H067637. At the highest AR-H067637 plasma concentration tested, bleeding time and blood loss increased two and four times the vehicle group. Addition of ASA moderately potentiated bleeding time and blood loss. APTT, ECT and TCT were dose-dependently prolonged. These studies demonstrate that the DTI AR-H067637 inhibits thrombus formation in rat venous and arterial thrombosis models with no or minor increases in bleeding.

AVE5026: A Novel, Extractive Heparinoid with Enriched Anti-Xa Activity and Enhanced Antithrombotic Activity.

AVE5026 represents a new generation of antithrombotic with high anti-Xa and low anti-IIa activities (anti Xa/IIa ratio > 30) when compared to heparin and low molecular weight heparins. This new heparin is a polydisperse mixture of oligomeric heparin fragments with an average molecular weight of 2000–3000 daltons prepared by partial and controlled depolymerization of porcine mucosal heparin. Unlike other depolymerized heparins where the antithrombin (AT) affinity components and anti-Xa activity is decreased with the decrease in the molecular weight, AVE5026 exhibits a relatively higher proportion of AT components which translates into a higher anti-Xa activity. A great majority of the oligosaccharide components in AVE5026 are composed of tetra-, hexa-, octa-and deca-saccharides. Heparinase is capable of digesting AVE5026 into mainly tetrasaccharide components. Despite its lower antithrombin actions, AVE5026 produces its anticoagulant effect in such whole blood assays as ACT and TEG. In the whole blood based global anticoagulant assays such as PT, APTT, AVE5026 produces modest anticoagulant effects in comparison to enoxaparin and heparin. Similarly in the plasma base system its anticoagulant effects are relatively weaker than enoxaparin and heparin. In the amidolytic anti-Xa assay, AVE5026 produced stronger inhibitory effects in comparison to enoxaparin (IC50 = 2.5 and 4.1 μg/ml, respectively). In the amidolytic anti-IIa assays AVE5026 produced modest inhibitory effects (IC50 = 38 μg/ml). Interestingly, in the AT deficient plasma in the APTT, AVE5026 produced comparable anticoagulant effects as in the NHP, indicating a stronger thrombin generation effect. Such an effect was not observed in the heptest and anti-Xa assays. In the purified AT system, AVE5026 produced a relatively stronger anti-Xa effect than enoxaparin (IC50 = 2.0 and 3.8 μg/ml, respectively). Interestingly in the thrombin generation assays using both intrinsic and extrinsic activators, AVE5026 produced concentration dependent inhibition of Xa (IC50=7–8 μg/ml) and IIa generation (IC50=>10 μg/ml), whereas fondaparinux did not produce any effect. In the agonist induced platelet aggregation studies with agonists such as ADP, collagen, epinephrine and arachidonic acid, AVE5026 did not produce any inhibitory effects. However, it strongly blunted TF mediated activation of microparticles and p-selectin expression (IC50=8 μg/ml). In comparison to enoxaparin, AVE5026 showed no cross reactivity with the HIT antibodies in the platelet aggregation system. AVE5026 produced a dose dependent antithrombotic response after IV and SC administration in the rat laser model. In the rabbit stasis thrombosis model AVE5026 produced a dose dependent antithrombotic effect by both IV and SC routes. The relative bleeding effects of AVE5026 in a rat tail bleeding and rabbit blood loss model were negligible in comparison to enoxaparin and heparin by both IV and SC routes. Repeated administration of AVE5026 in dose ranges of 1–5 mg/kg od, sc for 14 days did not result in accumulation effect of the Xa actions. Furthermore, in contrast to enoxaparin, there was no enhancement of the hemorrhagic profile. This superior safety efficacy index in animal models in comparison to other LMWH may translate into improved antithrombotic efficacy with decreased bleeding risk.

Dose-Dependent Antithrombotic Effects of Apixaban, an Oral Direct Factor Xa Inhibitor, in Prevention and Treatment of Thrombosis in Rabbit Models of Arteriovenous-Shunt and Venous Thrombosis at Doses That Preserve Hemostasis.

Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor, which is currently in late stage clinical development for the prevention and treatment of thromboembolic diseases. The dose-dependent antithrombotic and antihemostatic profile of apixaban was determined in the rabbit models of arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), and cuticle bleeding time (BT), and compared to those of the direct thrombin inhibitor lepirudin, the indirect FXa inhibitor fondaparinux and the oral anticoagulant warfarin. We induced the formation of thrombus in the AVST and VT models by placing threads in the extracorporeal shunt and vena cava, respectively, and bleeding by the incision of cuticles in anesthetized rabbits. In the AVST and VT prevention models, apixaban (0.03 to 3 mg/kg/h), lepirudin (0.006 to 0.75 mg/kg/h) and fondaparinux (0.01 to 1 mg/kg/h) were infused IV 30–60 min before the initiation of thrombosis. Warfarin (0.1 to 3 mg/kg/day) was administered orally for 4 days before the study. Control thrombus weight averaged 290±11 mg and 64±2 mg in AVST and VT, respectively, and control BT averaged 179±5 s (n=6 per group). Apixaban exhibited similar dose-related efficacy as lepirudin, fondaparinux, and warfarin in preventing AVST and VT. At doses that prevented 80 to 90% of thrombus formation in AVST and VT, apixaban, fondaparinux, lepirudin and warfarin increased BT by 20±2, 30±5, 500±10, 502±20%, respectively (n=6 per group). Doses for 50% reduction of control thrombus weight in AVST, VT were 0.27±0.03, 0.11±0.02 mg/kg/h IV for apixaban, 0.04±0.01, 0.05±0.01 mg/kg/h IV for lepirudin, 0.05±0.01, 0.05±0.005 mg/kg/h IV for fondaparinux and 0.53±0.04, 0.27±0.02 mg/kg PO for warfarin, respectively. To increase BT by 3-fold required higher doses of apixaban and fondaparinux (>3 mg/kg/h IV), lepirudin (0.24±0.05 mg/kg/h IV) and warfarin (0.70±0.07 mg/kg PO). In a VT treatment model, apixaban, lepirudin and fondaparinux, administered IV as a bolus injection supplemented with a continuous infusion after thrombus formation, were all able to arrest thrombus growth. However clot regression was only observed following administration of apixaban (0.6 mg/kg+0.87 mg/kg/h IV) where the preformed thrombus decreased from an initial weight of 38±2 mg (n=6) to 26±4 mg (n=6; P<0.05). In summary, apixaban and fondaparinux were effective in the prevention and treatment of experimental thrombosis at doses that preserve hemostasis in rabbits. Warfarin and lepirudin also prevented thrombus formation but with greater increases in BT. Furthermore, these standard anticoagulant agents have well-known limitations including narrow therapeutic index, frequent laboratory monitoring, or the requirement of parenteral administration. The favorable preclinical antithrombotic and antihemostatic profile of apixaban demonstrated here is consistent with clinical efficacy and safety results in recent Phase II trials, and indicates that direct inhibition of FXa with apixaban is a promising approach for the prevention and treatment of venous thromboembolism.

Dose-dependent antithrombotic activity of an orally active tissue factor/factor VIIa inhibitor without concomitant enhancement of bleeding propensity

The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial thrombosis without prolonging bleeding time. The pharmacodynamic properties of this selective inhibitor are compared to the behaviour of a mixed factor VIIa/factor Xa inhibitor.

Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

3-( S)-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a series of novel dipeptide analogues by incorporating tetrahydro-β-carboline-3-carboxylic acid skeleton as an amino acid surrogate (∗Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers permeability assay. As a result, the overall biological properties of these molecules were significantly improved depending on the nature of the amino acid residues introduced onto the 3-position of the tetrahydro-β-carboline moiety. It was very interesting to notice that these dipeptide analogues ( 5b, c, h, i, n, o, p, q) displayed a remarkable dual antiaggregatory activity in both of ADP- and PAF-induced platelet aggregation assay, and their aggregation response was significantly higher than that of aspirin ( p < 0.01). In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic effect using in vivo rat arterial thrombosis model. The potency of antithrombotic activity of 5h, i, n, p was significantly higher than that of aspirin ( n = 12, p < 0.01) at equal dose (5 μmol/kg).

Synthesis and Structure−Activity Relationships of Novel Selective Factor Xa Inhibitors with a Tetrahydroisoquinoline Ring

A series of novel 2,7-disubstituted tetrahydroisoquinoline derivatives were designed and synthesized. Among these derivatives, compounds 1 and 2 exhibited potent inhibitory activity against factor Xa (FXa) and good selectivity with respect to other serine proteases (thrombin, plasmin, and trypsin). In addition, compound 2 exhibited potent anti-FXa activity after intravenous and oral administration to cynomolgus monkeys, showed a dose-dependent antithrombotic effect at 0.1, 0.3, and 1 mg kg(-1) h(-1) in a rat model of venous thrombosis, and significantly reduced the size of brain infarction in a middle cerebral artery occlusion model at a dose of 0.1 mg kg(-1) h(-1). These results suggest that compound 2 (JTV-803) is likely to be useful as both a venous and arterial antithrombotic agent.

Discovery of Novel Tetrahydroisoquinoline Derivatives as Potent and Selective Factor Xa Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Varying the ratio of dietary n−6/ n−3 polyunsaturated fatty acid alters the tendency to thrombosis and progress of atherosclerosis in apoE −/− LDLR −/− double knockout mouse

We have investigated the influence of dietary n−6/ n−3 (ù-6/ù-3) polyunsaturated fatty acid-balance on the tendency to arterial thrombosis and the progress of atherosclerosis in apoE −/− LDLR −/− double knockout mouse. Homozygous apoE −/− LDLR −/− double knockout mouse (DKO mice, 129XC57BL/6J background) and male C57BL/6 mice aged 6 weeks were divided into four groups. Each group was fed a diet containing a different n−6/ n−3 ratio (Group l: 0.29; Group 2: 1.43; Group 3: 5.00; Group 4: 8), prepared with high linolenic (LNA) flaxseed oil ( n−3 rich) and high linoleic (LA) safflower oil ( n−6 rich). There were no statistical differences in the gain in body weight between the four groups. After 16 weeks, plasma triglyceride and LDL levels in Group 1 were significantly lower than in the other groups. Conversely, HDL was the highest. After 8 and 16 weeks, the tendency to arterial thrombosis was assessed using a He–Ne laser-induced thrombosis model. The degree of atherosclerosis was measured using the entire aorta method employing image analysis software. The n−6/ n−3 ratio had a dose-dependent antithrombotic effect (thrombus volume decreased 23%, Group 1 vs. Group 4), In addition, the extent of atherosclerosis was less in the animals fed a low n−6/ n−3 ratio compared with the high n−6/ n−3 ratio group (atherosclerotic area decreased 40%, Group 1 vs. Group 4). The lowest n−6/ n−3 ratio tested (0.29) was the most effective in suppressing the thrombotic and atherosclerotic parameters in these DKO mice.

Discovery of novel tetrahydroisoquinoline derivatives as potent and selective factor Xa inhibitors

A series of novel 2,7-disubstituted tetrahydroisoquinoline derivatives were designed and synthesized. Among these derivatives, compounds 1 and 2 (JTV-803) exhibited potent inhibitory activity against FXa and good selectivity with respect to other serine proteases (thrombin, plasmin, and trypsin). In addition, compound 2 exhibited potent anti-FXa activity after intravenous and oral administration to cynomolgus monkey, and showed a dose-dependent antithrombotic effect in a rat model of venous thrombosis.

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 ({4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine–glycine–aspartic acid–serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A (( S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 μg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

Antithrombotic Efficacy in a Rat Model of the Low Molecular Weight Heparin, Reviparin Sodium, Administered by the Oral Route

Previous studies in rats show that unfractionated heparin and the low molecular weight heparin logiparin have a dose-dependent antithrombotic effect and are found in endothelium and plasma when administered orally. Objectives of the present study were to determine if similar evidence of absorption could be observed with oral reviparin sodium. Thrombosis incidence was determined 4 h after application of 10% formalin in methanol to the exposed jugular vein. A dose-dependent antithrombotic effect was observed when 0.01 to 7.5 mg/kg (20 rats/group) was administered by stomach tube immediately following thrombus initiation. Thrombotic incidence was also significantly reduced when 0.025 mg/kg was given 4 and 2 h prior to, immediately after, and 2 and 3 h following thrombus initiation. Reviparin was recovered from endothelium and plasma in trace amounts at all doses. At 0.025 mg/kg, peak aortic endothelial reviparin concentrations were found at 1 and 2 h and peak plasma anti-Xa activity was detected at 2 h. Trace amounts of plasma TFPI were found only at 8 h after administration. Dose-dependent antithrombotic activity and recovery from endothelium and plasma support the hypothesis that orally administered reviparin sodium is absorbed.

Antithrombotic Activity of Orally Administered Low Molecular Weight Heparin (Logiparin) in a Rat Model

Previous studies in rats demonstrated that orally administered, unfractionated bovine lung heparin is absorbed and has a dose-dependent antithrombotic effect. The objective of this study was to determine if an oral low molecular weight heparin had a similar antithrombotic effect in the same model. Thrombosis was induced in rats by application of 10% formalin in 65% methanol to the exposed jugular vein. Immediately following, saline, unfractionated heparin (3.3–60 mg/kg) or the low molecular weight heparin, Logiparin (0.025–15 mg/kg; 20–30 rats per group) was placed in the stomach and 4 h later the jugular vein was inspected for a thrombus. Compared to saline, oral Logiparin reduced the incidence of thrombosis at all doses with a dose-dependent effect suggested. A significant increase was observed in the activated partial thromboplastin time and in plasma heparin concentrations, determined by Accuclot^TM Heptest^® and anti-factor Xa chromogenic assay for rats given oral Logiparin versus saline. A dose-dependent increase in plasma heparin concentration was observed when estimated by the anti-Xa chromogenic assay. Heparin was recovered in 9% of aortic endothelial samples when ≧0.8 mg/kg Logiparin was administered. A 50% reduction in thrombosis was observed at 0.1 mg/kg for oral Logiparin versus 7.5 mg/kg for unfractionated bovine lung heparin indicating that oral Logiparin is an effective antithrombotic agent at doses lower than unfractionated heparin. Orally administered low molecular weight heparin may be useful for the prevention and treatment of thrombosis.