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Ecarin clotting time (ECT) is currently developed for the specific monitoring of antithrombin drugs, such as hirudin, argatroban, and hirulog. Aqueous reagent and dry chemistry technology have become available for ECT monitoring of antithrombin agents. Currently, many heparinoids and heparinomimetic drugs are being developed. These agents activate heparin cofactor 11 (HCII) and primarily mediate their effects by inhibiting thrombin. Atthough the test is specific for antithrombin agents, heparin cofactor II-mediated thrombin inhibitors are capable of prolonging the ECT. In order to study the relative effects of some of these agents, ECT was measured in human plasma supplemented with PI-88 (a sutfated pentomanose ; Progen Industries Limited. Sydney, Australia), aprosulate, pentosan polysulfate, dermatan Sulfate, unfractionated heparin (UFH), and recombinant hirudin (r-hirtadin). All agents were supplemented to the citrited-pooled plasma prepared from 10 healthy volunteers at a graded dosage of 0 to 100 These techniques gave comparable results for all of the agents used (PI-88. r2 = 0.99; r-hirudin, r2= 0.98, UFH. r2 =0.98; dermatan sutfate, r2 = 0.95; aprosulate, r2 = 0.95; pentosan polysulfate, r2 = 0.94). The relative anticoagulant effects of various agents used on ECT varied widely, exhibiting their potency in the following order: r-hirudin = pentosan polysulfate > dermatan sulfate > PI-88 > aprosulate > UFH. The sensitivity of ECT was adjusted by varying the concentration of the ecarin reagent. The results suggest that HCIImediated inhibition of thrombin can be detected by using ECT reagents.

Arterial thrombosis in the rabbit was established as a novel model to evaluate the effects of antithrombotic agents. Endothelial injury was produced by applying electrical stimulation to the femoral artery. The process of primary endothelial injury, and subsequent platelet activation and fibrin formation were confirmed by electron microscopy. In this model, vessel occlusion occurred within 30 min after stimulation without changes in heart rate and blood pressure. Using this model, several agents were evaluated for their antithrombotic activities: aspirin (30 mg/kg, p.o.), ticlopidine (10-100 mg/kg, p.o.), heparin (300 unit/kg, i.v.), PPACK (10-33 micrograms/kg/min, i.v.), WEB-2347 (1 mg/kg, p.o.) and nicardipine (10 micrograms/kg, i.v.). Fifty per cent decrease in vessel temperature (T1/2), assessed by a thermic probe, averaged 15.1 +/- 1.2 (n = 11, p.o.) and 15.6 +/- 1.9 min (n = 8, i.v.) in the vehicle groups, and this was significantly prolonged by aspirin (23.0 +/- 2.6 min), ticlopidine at a dose of 100 mg/kg (24.6 +/- 2.5 min), heparin (27.2 +/- 2.8 min) and PPACK at a dose of 33 micrograms/kg (30.0 min). However, WEB-2347 and nicardipine were without effect. The effect of aprosulate, a new class of polyanion with anticoagulant activity, was further examined. Aprosulate (1-30 mg/kg, i.v.) inhibited thrombus formation in a dose-dependent manner. These results show that acute occlusive thrombus can be readily and reproducibly formed in the rabbit femoral artery and suggest that this thrombus formation depends on the activation of both platelets and blood coagulation. The merit of this model lies in its simplicity for evaluating the antithrombotic effects of antiplatelet and anticoagulant agents and is therefore expected to be extensively used in the future.

Tissue factor pathway inhibitor (TFPI) is a naturally occurring, Kunitz-type serine protease inhibitor whose anticoagulant activity is due to an inhibition of the extrinsic coagulation pathway. Heparin injection has previously been shown to increase the plasma levels of TFPI. In this study, plasma samples were obtained from a multiple dose phase I tolerance study with a synthetic analogue of heparin, namely Aprosulate (PALLAS). Volunteers were randomized into four treatment groups: (A) 35 mg Aprosulate b.i.d. s.c.; (B) 70 mg Aprosulate b.i.d. s.c.; (C) 70 mg Aprosulate o.d. + placebo o.d. s.c.; (D) 40 mg of a low molecular weight heparin, Enoxaparin o.d. + placebo o.d. s.c. All treatments were for 7 days, with blood samples taken periodically over this time period. TFPI antigen levels were determined using Imubind TFPI ELISA kits (American Diagnostica, Greenwich, CT). TFPI antigen levels were observed to rapidly increase to levels two- to three-fold over baseline in all groups. Aprosulate caused a slightly larger increase in TFPI antigen levels than Enoxaparin, though this may be related to the doses chosen for this study. These data indicate that plasma concentrations of TFPI are increased following Aprosulate administration. TFPI may be important in mediating the antithrombotic activity of Aprosulate.

Aprosulate sodium was the first representative of a new class of synthetic polyanions showing antithrombotic efficacy in different animal models. In several clinical trails (Phase I) in human volunteers aprosulate demonstrated anticoagulant properties, too. Searching for other active substances of similar structure, a series of bis-aldonic acid amides were synthesized. These compounds exhibited interesting antithrombotic and anticoagulant activities. The pharmacodynamic activities of the compounds LW 10121, LW 10125, LW 10114, 10244, and LW 10168 are summarized in this article. These substances prolonged the APTT to 150% of the blank values at concentrations of 1.5 to 13.5 micrograms/mL. The thrombin time and anti-Xa test were only slightly influenced by concentrations up to 100 micrograms/mL. All compounds were investigated in a jugular vein hemostasis model in rats to examine their antithrombotic potential. They all had an antithrombotic activity lower than aprosulate, except compound LW 10121, which seemed to be a little more active. The subcutaneous injection of 10 mg/kg LW 10121 resulted in a longer duration of action than aprosulate and heparin. On the basis of the chemical structure and the profile of action, it is assumed that the new compounds may possess the same mode of action as aprosulate, but the mechanism of action may be different from heparin and low molecular weight heparins.

The in vivo effects of a new synthetic inhibitor of blood coagulation, aprosulate sodium, were investigated. Intravenous bolus injection of aprosulate or standard heparin in rats produced an immediate prolongation of the APTT which were characterized by a moderate dose-dependency and long-lasting duration when compared with those of standard heparin. Standard heparin inhibited plasma factor Xa activity, but aprosulate did not even at the highest dose used. Both agents inhibited thrombus formation in a dose-dependent manner in an arterio-venous shunt model. At antithrombotic doses, standard heparin prolonged the bleeding time measured by the tail transection method, but aprosulate did not. The present results suggest that aprosulate has promising in vivo profile as an anticoagulant and antithrombotic agent.

In a phase I clinical trial the effect of the highly sulfated polyanion "Aprosulate" was studied in healthy volun teers using different coagulation and platelet function param eters. Eighteen healthy volunteers aged 21 to 30 years received two single subcutaneous doses of aprosulate (0.5 mg/kg body weight; 1.0 mg/kg body weight), or unfractionated heparin (Calciparin® 7,500 IU). The washout period between the dif ferent drugs/doses was at least 7 days. Coagulation and platelet function parameters (activated partial thromboplastin time, Heptest, fibrinogen, von Willebrand factor, ristocetin cofactor, platelet adhesion to siliconized glass, and platelet-induced thrombin generation time [a new method for measuring throm bin generation in platelet-rich plasma in the presence of plate lets]) were assessed during 24 hours after each injection. Aprosulate led to a significant and dose-dependent prolonga tion of activated partial thromboplastin time and Heptest. This effect lasted for 4 hours (activated partial thromboplastin time) to 8 hours (Heptest). Activated partial thromboplastin time was not prolonged after the injection of unfractionated heparin (7,500 IU). Fibrinogen, von Willebrand factor, and ristocetin cofactor remained unchanged with both drugs. Platelet induced thrombin generation time was slightly prolonged and platelet adhesion was slightly diminished up to 2 hours using 0.5 mg/kg aprosulate, and up to 4 hours using 1.0 mg/kg aprosulate while the platelet induced thrombin generation time system was not influenced by the subcutaneous injection (7,500 IU) of unfrac tionated heparin. Both drugs and doses were well tolerated. Plasma transaminase concentrations alanin aminotransferase and aspartate aminotransferase serum values were slightly in creased in some volunteers but returned to normal during or after the study (

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

Aprosulate sodium, a new synthetic polyanion, was evaluated for anticoagulant/antiprotease activities in vitro. Aprosulate prolonged activated partial thromboplastin time (aPTT), Heptest and thrombin time (TT) with the sensitivity order of aPTT > Heptest > TT. Prothrombin time (PT) was hardly affected by the agent. Since these results indicated that inhibition of the intrinsic coagulation cascade was important for the anticoagulant activity of aprosulate, amidolytic assays were subsequently performed to characterize antiprotease activities of the agent in the common and intrinsic pathways. Aprosulate inhibited amidolytic activity of thrombin in the presence of heparin cofactor II, but was devoid of direct and antithrombin-mediated anti-factor Xa, and direct anti-factor XIIa activities. However, aprosulate was found to be a potent inhibitor of amidolytic activity of factor Xase (IXa/VIIIa). These data suggest that the direct anti-factor Xase activity of aprosulate primarily contributes to its anticoagulant activity in the intrinsic coagulation cascade.

Low molecular weight heparins, namely CY 216 and CY 222 (Sanofi/Choay); OP 622 and OP 386 (Opocrin); PK 10169 (Pharmuka); an oligosaccharide prepared from heparin by heparitinase II digestion; chemically sulfated glycosaminoglycans and polysaccharide namely Suleparoid (Syntex), Aprosulate (Luitpold-Werk); chemically modified glycosaminoglycans GAGPS and MPS (Luitpold-Werk) as well as unmodified heparin stimulate two to three fold the synthesis of a heparan sulfate with antithrombotic activity secreted by endothelial cells in culture. The stimulation is concentration dependent and specific for the endothelial cell. The [35S]-heparan sulfate synthesized in the presence of heparin and/ or the tested antithrombotic agents has shown a high degree of sulfation of the iduronic acid residues as revealed by the analyses of the disaccharide products formed from the heparan sulfate by the action of bacterial heparitinases. The features of the above compounds in common with heparin are their polymeric nature and a high charge density, as well as their pharmacological activities as potent antithrombotic agents “in vivo”. These combined observations reinforce the proposition that the antithrombotic activity of heparin, low molecular weight heparins and the chemically modified polysaccharides could be related to the increased production of this peculiar heparan sulfate by endothelial cells.

Aprosulate or lactobionic acid is a highly sulfated analogue of heparin which is currently undergoing clinical trials in Europe as a potential antithrombotic drug. Aprosulate exerts a strong anticoagulant effect in plasma as a result of its interaction with heparin cofactor II. In this study, the ability of protamine sulfate to neutralize the anticoagulant activity of Aprosulate was investigated. In vitro, ex vivo, and in vivo coagulation studies were performed using various clotting assays such as the APTT, Heptest, and thrombin time as a measure of the anticoagulant activity of Aprosulate. In the first study, protamine sulfate when administered in vitro to plasma samples containing various concentrations of Aprosulate was found to effectively neutralize the anticoagulant activity of the Aprosulate in both normal human and normal monkey plasma systems. However, the relative index of neutralization of Aprosulate was assay dependent. Protamine sulfate was also found to antagonize the anticoagulant effects of Aprosulate in an ex vivo study. The ex vivo supplementation of protamine sulfate to plasma samples collected at various time intervals following the subcutaneous administration of Aprosulate to a group of primates completely neutralized the anticoagulant activity of the Aprosulate. In a third in vivo study, protamine sulfate when injected intravenously into the bloodstream of a group of primate was found to completely neutralize the anticoagulant effects of a previously administered dosage of Aprosulate. The results of these three studies clearly suggest that protamine sulfate can be used to effectively neutralize the anticoagulant activity of Aprosulate.

This paper describes the first human study with aprosulate, a new chemically synthesized anticoagulant with a defined molecular structure and a molecular weight of 2388. Twelve healthy male volunteers received subcutaneous injections of placebo on the first day followed by ascending doses of aprosulate in the range of 0.25 mg/kg to 2.0 mg/kg body weight on alternate days. Anticoagulant, pharmacokinetic and safety parameters were assessed for 48 hours after each injection. The activated partial thromboplastin time and the Heptest showed a dose-dependent increase for up to ten hours after each application. A trend towards prolongation of the bleeding time was indicated with higher doses. In general, the tolerance was good. Plasma transaminase concentrations were raised in some volunteers but returned spontaneously to normal during or after the study.