G.M.T. Vogel

Experimental Study of Thrombogenicity and Foreign Body Reaction Induced by Heparin-Coated Coronary Stents

BACKGROUND Results of recent randomized clinical trials have revealed a significant reduction in angiographic restenosis rate when adjunctive stenting was performed after conventional coronary balloon angioplasty. The thrombogenicity of metal stents, however, remains a concern. In the present study, we compare the thrombogenicity of heparin-coated coronary stents with that of bare metallic coronary stents. METHODS AND RESULTS Thrombogenicity of metallic coronary stents (four heparin-coated and eight bare stents) was studied in a rat arteriovenous shunt model with the use of 125I-labeled fibrinogen and 51Cr-labeled platelets. Total clot weight after 30-minute follow-up was significantly lower in the heparin-coated stents compared with the bare stents (8.1 +/- 3.7 versus 25.8 +/- 4.6 mg; P < .001). Relative 125I and 51Cr activities in the stents were significantly higher in the bare stents than in the heparin-coated stents (125I, 1.03 +/- 0.43 versus 0.18 +/- 0.04, P = .003; 51Cr, 17.5 +/- 6.8 versus 4.4 +/- 1.0, P = .004). Subsequently, heparin-coated and bare stents were randomly implanted in the right coronary artery of 20 domestic pigs. Angiographic parameters were similar between both groups at baseline and after 6-week follow-up. Morphometry also did not show a significant difference in lumen area (bare, 1.03 +/- 0.83 mm2; heparin-coated, 1.12 +/- 0.73 mm2; P = NS) or neointimal hyperplasia (bare, 1.01 +/- 0.81 mm2; heparin-coated, 1.21 +/- 0.57 mm2; P = NS). CONCLUSIONS Heparin coating of metallic coronary stents decreases their thrombogenicity but does not improve late vessel patency and neointimal hyperplasia at follow-up in a porcine coronary model.

Caco-2 permeability, P-glycoprotein transport ratios and brain penetration of heterocyclic drugs

In this study the gastrointestinal absorption and P-glycoprotein (Pgp) efflux transport of heterocyclic drugs was investigated with the Caco-2 cell model. Based on the calculation of the physico-chemical properties a good oral absorption was predicted for all the drugs tested in this study which corresponded well with the measured Caco-2 permeabilities (Papp). Generally a high permeability of the tested heterocyclic drugs was measured being in agreement with earlier published human in vivo absorption data. Based on the transport data of domperidone and verapamil it was found that the Pgp efflux transporter was expressed in the Caco-2 cells. Many of the drugs tested were indicated to be potential Pgp efflux substrates. Since Pgp is expressed at the Blood Brain Barrier (BBB) as well, it was expected that CNS penetration will be impaired if a drug is a Pgp substrate. However, no correlation could be found between brain penetration in rats and the Pgp efflux ratio as measured with the Caco-2 cells. From the data it is concluded that Pgp efflux ratios as determined in in vitro High Throughput Screening (HTS) tests, where the transport conditions are fixed (pH gradient, concentration, etc.), cannot routinely be used to predict a possible limited brain penetration.

Comparison of two experimental thrombosis models in rats effects of four glycosaminoglycans

Two experimental thrombosis models in rats have been compared with regard to the composition of the formed thrombi and the effects of various treatments on thrombus formation. In the first model thrombosis is induced in the vena cava by a combination of venous stasis and hypercoagulability; these thrombi consist merely of red cells and fibrin with only a few platelets. In the second model thrombosis is induced in an arterio-venous shunt in which the formed thrombi consist of red cells, fibrin and a large amount of platelet aggregates adhering to the foreign material. Antiplatelet serum and acetylsalicylic acid, which reduce blood platelet activity, inhibited thrombus formation only in the arteriovenous shunt model. Dicumoxane, an oral anticoagulant, was active in both models. The glycosaminoglycans heparin, Org 10172, Fragmin and the pentasaccharide, representing the AT-III binding sequence of heparin, were active in both models. However, there were qualitative and quantitative differences between the effects of the glycosaminoglycans suggesting differences in their modes of action.

From heparin to EP217609: The long way to a new pentasaccharide-based neutralisable anticoagulant with an unprecedented pharmacological profile

The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.

Antithrombotic properties of a direct thrombin inhibitor with a prolonged half‐life and AT‐mediated factor Xa inhibitory activity

Summary.  Rebound thrombin generation after successful thrombolysis might be related to (i) too short‐term anticoagulant therapy and to (ii) the inability of heparin derivatives to inhibit clot‐bound thrombin. To meet these shortcomings, a compound was synthesized, which consists of a pentasaccharide conjugated to a direct thrombin inhibitor. This compound (Org 42675) has a 10 times longer half‐life compared with the original half‐life of the direct thrombin inhibitor, while the thrombin inhibitory activity is maintained. An extra advantage of this product is the inhibitory activity on thrombin generation via antithrombin III (AT)‐mediated factor (F)Xa inhibition. Org 42675 inhibited in vitro clot‐bound thrombin with similar activity to the direct thrombin inhibitor argatroban. In experimental models in rats, Org 42675 showed on a molar base similar antithrombotic activity to unfractionated heparin, was more active than argatroban and was more active than fondaparinux sodium (AT‐mediated FXa inhibitor) in arterial thrombosis. Finally, Org 42675 was far more active than the three reference compounds in an experimental thrombolysis model in rabbits. These properties of Org 42675, with its FXa and (clot‐bound) thrombin inhibitory activity in combination with its long half‐life, make this compound a powerful drug that is likely to be effective in the prevention of re‐occlusion after successful thrombolysis in man.

Time courses of the antithrombotic effects, bleeding enhancing effects and interactions with factors Xa and thrombin after administration of iow molecular weight heparinoid Org 10172 or heparin to rats

Time response curves of the anti-thrombotic effects, bleeding enhancing effects, effects on APTT, anti-Xa activities, anti-thrombin activities and thrombin generation inhibitory activities of the low molecular weight heparinoid Org 10172 and heparin have been compared in rats. The time courses of these effects were similar for heparin but quite different for Org 10172. Org 10172 induced anti-thrombotic and anti-Xa effects which lasted approximately 3 times longer than those at the same anti-Xa doses of heparin, whereas the bleeding enhancing effects and effects on APTT of Org 10172 were of shorter duration than those of heparin. The half-life of the anti-thrombin effect after Org 10172 seemed somewhat longer than after heparin administration. Thrombin generation inhibition by Org 10172 showed a slightly longer duration than by heparin. The similarities between the time courses of the anti-thrombotic effect and the anti-Xa activity after Org 10172 administration suggest that the most appropriate parameter to monitor Org 10172 treatment is the plasma anti-Xa level.

Effects of intra-arterial cannulation on blood platelet consumption in rats

Experimental arterial thrombosis has been induced by an indwelling cannula in the abdominal aorta of rats. The involvement of blood platelets was studied by the determination of the blood platelet count and the survival of 51Cr labeled platelets. Cannulation transformed the platelet disappearance patterns from linear to curvi linear and exponential curves. The blood platelet consumption rate increased 2–3 fold immediately after cannulation and remained at that level for at least 8 days. The consumption rate was higher when longer cannulae were used. Initially the increased blood platelet consumption was not compensated by an increase in platelet production, this resulted in a decreasing platelet count. After 3 to 7 days a long lasting steady state thrombocytopenia was achieved, under these circumstances the platelet turnover in cannulated and normal rats was the same. Withdrawal of the cannula normalized the platelet consumption within a few hours, leading to a linear disappearance pattern of the residual labeled platelets. The platelet count was readily restored to normal values via a rebound overshoot.

PLACENTAL TRANSFER OF Org 10172, A LOW-MOLECULAR WEIGHT HEPARINOID, IN THE AWAKE LATE-PREGNANT GUINEA PIG

The placental transfer of Org 10172, a low-molecular weight heparinoid, was determined in 12 awake late-pregnant guinea pigs. Nine animals receiving placebo served as controls. After 5 days i.v. treatment with Org 10172 (2 x 300 anti-Xa U/kg/day), anti-Xa activity in fetal plasma amounted to 2.4% of the maternal concentration. The Org 10172 transfer across the placenta was also evaluated with 3H-labelled Org 10172. One hour after the administration of the latter compound, fetal Org 10172-bound 3H-activity had reached 1.5% of the maternal value. The associated extremely low placental Org 10172 transfer indicates that the Org 10172 transport across the placenta of the guinea pig is membrane-limited and that the placental permeability is negligibly low. Since the hemochorial placenta of the guinea pig closely resembles that of the human, it is likely that similar transplacental transfer properties of Org 10172 apply to man.

Analysis of platelet survival curves in an arterial thrombosis model in rats.

Abstract A mathematical formula, which can objectively describe blood platelet disappearance patterns under normal, mild and severe thrombotic conditions, has been derived from experimental data. Platelet kinetics were studied in rats with an experimentally induced arterial thrombosis, in rats during the termination of the thrombotic stimulus and in normal rats. Normal populations and a cohort of young, radiolabeled platelets were used in the experiments. Our investigations strongly suggested that under normal and thrombotic conditions and under conditions in which the thrombotic stimulus is terminated, blood platelets ultimately disappeared from the circulation after the same finite life span. The obtained data could be best explained by assuming that two mechanisms can operate simultaneously: disappearance by ageing occurs only under non-thrombotic conditions, whilst both mechanisms operate together under thrombotic conditions. The derived mathematical formula, which describes both processes, offers a useful method to analyse data obtained from platelet survival studies.