RESUMEN
The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII. The qualitative analysis of GAGs showed the presence of the deltaDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.
Asunto(s)
Antitrombinas/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Cofactor II de Heparina/metabolismo , Tioglicósidos/administración & dosificación , Trombosis de la Vena/tratamiento farmacológico , Administración Oral , Animales , Interacciones Farmacológicas , Cofactor II de Heparina/administración & dosificación , Masculino , Conejos , Ratas , Tioglicósidos/sangre , Trombosis de la Vena/sangreRESUMEN
Fucoidans (high-molecular-weight sulfated polysaccharides extracted from brown seaweeds) have anticoagulant and antithrombotic effects. They inhibit thrombin by catalyzing both serpins (antithrombin and heparin cofactor II) according to their chemical structures and origins. In this study, a low-molecular-weight (LMW) fucoidan of 8 kDa was obtained by chemical degradation of a high-molecular-weight fraction. The antithrombotic and anticoagulant activities of this new compound were compared to those of a low-molecular-weight heparin (LMWH), dalteparin, following subcutaneous administration to rabbits. This LMW fucoidan exhibited dose-related venous antithrombotic activity, with an ED80 of about 20 mg/kg, 2 h after a single subcutaneous injection. Its activity was comparable to that of dalteparin (close to 200 anti-Xa IU/kg) and was maximal 30 min after a single subcutaneous injection. The activity remained stable (about 70%) from 1 to 4 h after injection, but disappeared by 8 h. The antithrombotic activity was not associated with either a prolongation of the thrombin clotting time (TCT) or an increase in anti-Xa activity, contrary to dalteparin. A slight prolongation of APTT occurred with both compounds. This venous antithrombotic activity was associated with a decrease in ex vivo thrombin generation and with a significant increase in the lag phase in a thrombin generation test. LMW fucoidan thus has potent antithrombotic activity and a potentially weaker haemorrhagic effect (i.e. a smaller effect on coagulation tests and a smaller prolongation of the bleeding time) than dalteparin.
Asunto(s)
Anticoagulantes/administración & dosificación , Polisacáridos/administración & dosificación , Animales , Anticoagulantes/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Dalteparina/administración & dosificación , Dalteparina/efectos adversos , Heparina de Bajo-Peso-Molecular/administración & dosificación , Heparina de Bajo-Peso-Molecular/efectos adversos , Inyecciones Subcutáneas , Masculino , Polisacáridos/efectos adversos , ConejosRESUMEN
A (-)-conduritol F derivative was condensed with 4-ethyl-7-hydroxy-2H-1-benzopyran-2-one and converted into (+)-4-ethyl-7-[(1'R,2'S,3'S,4'R)-2',3',4'- trihydroxycyclohexyloxy]-2H-1-benzopyran-2-one ((+)-2). Enantiomer (-)-2 was obtained from a (+)-conduritol F derivative. The carbaxyloside (-)-2 with the L-xylose configuration was more active than (+)-2 in the Wessler's model.
Asunto(s)
Anticoagulantes/farmacología , Benzopiranos/farmacología , Trombosis/tratamiento farmacológico , Administración Oral , Anticoagulantes/química , Anticoagulantes/uso terapéutico , Benzopiranos/química , Benzopiranos/uso terapéutico , Hidrólisis , Venas/patologíaRESUMEN
The aim of this study was to demonstrate the effectiveness of a new experimental approach for assessing the antithrombotic potential of low molecular weight heparins (LMWH) such as dalteparin. In this approach, sera obtained from healthy volunteers treated with various i.v. doses of dalteparin (30, 60 or 120 anti-Xa IU/kg) or placebo (physiological saline) were used as the thrombogenic challenge in a Wessler's stasis model of experimental venous thrombosis in the rat. Sera from placebo-treated volunteers showed a stable thrombogenic activity (0.25 ml/kg of serum producing thrombi of about 50 mg wet weight). Sera from healthy volunteers having previously received dalteparin however demonstrated dose- and time-related reductions in their thrombogenic activity. Half-lives of these effects were 300. 444 and > 480 min for 30, 60 and 120 anti-Xa IU/kg dalteparin respectively. These values were significantly higher than the corresponding anti-Xa and anti-IIa half-lives.
Asunto(s)
Anticoagulantes/administración & dosificación , Dalteparina/administración & dosificación , Tromboflebitis/tratamiento farmacológico , Animales , Coagulación Sanguínea/efectos de los fármacos , Humanos , Ratas , Tromboflebitis/sangreRESUMEN
To investigate the effects of new drugs on the prevention of venous thrombosis, the Wessler stasis model is extensively employed usually in the rabbit. In this model, a hypercoagulable state is achieved by the administration of either heterologous serum (often human serum) or an activated coagulation factor followed by total stasis of the vein in which the thrombus is to be formed. Although classically the stasis model is performed in rabbits, it is possible to induce experimental venous thrombosis in a number of animal species, including rats. However, the use of homologous serum as the activating or thrombogenic agent has been previously described only in the dog and in the rabbit. The purpose of the present study was to compare the activities of heterologous and homologous serum as hypercoagulating agents in a rat stasis model of venous thrombosis using Wessler's technique. Heparin was employed as a reference antithrombotic compound in order to compare the two thrombogenic challenges.
Asunto(s)
Sangre , Tromboflebitis/etiología , Animales , Fibrinolíticos/farmacología , Heparina/farmacología , Masculino , Ratas , Ratas Wistar , Tromboflebitis/sangreRESUMEN
beta-D xylosides have been shown to have venous antithrombotic properties after simple oral administration. Therefore, the arterial antithrombotic effect of these compounds was investigated in vivo, using the experimental thrombosis model induced by laser injury. The products tested were administered orally, 4 h before the thrombosis induction. Two beta-D xylosides were tested (LF 09-0055 and LP 05-0030), either after a simple oral administration at 50, 100, 200, and 400 mg/kg, or after repetitive oral administration at 200 mg/kg twice daily during 5 days. These compounds increased significantly the number of laser shots required to induce arterial thrombosis and decreased the number of emboli and the duration of embolization. At single-dose or repeated administrations, these xylosides did not affect diluted thrombin time in platelet-poor plasma collected after thrombosis inductions. They induced a dermatan sulfate-like activity in the plasma of treated rats, as measured by heparin cofactor II-mediated thrombin inhibition assay. These data suggest that these xylosides are potent arterial antithrombotic agents after single or repetitive oral administrations. beta-D xylosides constitute a very promising therapeutic class of orally active antithrombotic drugs.
Asunto(s)
Fibrinolíticos/administración & dosificación , Glicósidos/administración & dosificación , Trombosis/tratamiento farmacológico , Administración Oral , Animales , Modelos Animales de Enfermedad , Rayos Láser , Masculino , Ratas , Ratas WistarRESUMEN
Following a screening program for orally active antithrombotic drugs, it was found that a series of thioxyloside compounds presented with good venous antithrombotic properties. Of more than 500 compounds, LF 09-0055, LF 04-0212, and LF 05-0030 were the most active at inhibiting venous thrombus formation in the rat and rabbit Wessler model after intravenous and oral dosing. LF 05-0030 showed the greatest activity with an ED80 value of 6 mg/kg on oral administration in rats. This activity was maintained in several different models of venous thrombosis and shown to be devoid of anticoagulant effects or hemorrhage. Kinetic studies have demonstrated that maximal levels of activity, following either intravenous or oral dosing, occurred between 2 and 4 hours after administration. This may reflect the type of mechanism involved, since it has been well documented in the literature that xylosides are capable of initiating glycosaminoglycan (GAG) synthesis. Moreover, in vitro galactosyltransferase 1 (the second enzyme involved in GAG polymerization) enzymic assays showed that these thioxyloside derivatives were good acceptors for galactose transfer and therefore at initiating GAG formation. Further in vivo experimentation demonstrated that after treatment by these molecules an important elevation in circulating GAG occurred, with LF 05-0030 presenting the greatest activity, being five times higher than control levels. In addition it was found that dermatan sulfate levels, expressed as antithrombin activity by heparin cofactor II, were significantly increased over control values. As such, this dermatan sulfate moiety is believed to support the antithrombotic activity observed. Studies are underway to investigate the activity of these interesting molecules in atherosclerosis and other vessel wall diseases.
Asunto(s)
Fibrinolíticos/administración & dosificación , Glicósidos/administración & dosificación , Trombosis/tratamiento farmacológico , Administración Oral , Animales , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/metabolismo , Galactosiltransferasas/metabolismo , Glicósidos/metabolismo , Inyecciones Intravenosas , Conejos , RatasRESUMEN
Fucoïdans catalyse thrombin inhibition by antithrombin (AT) and heparin cofactor II (HCII); their affinity for each serpin varies according to the seaweed species from which they are extracted, as well as their chemical composition and molecular weight. We extracted a homogeneous fucoïdan fraction from Ascophyllum nodosum, a brown seaweed, and tested its anticoagulant and antithrombotic activities. At a fucoïdan concentration of 3.75 micrograms/ml, thrombin inhibition mediated by AT showed an apparent second-order rate constant (kapp) of 2 x 10(8) M-1 min-1, compared to 1.5 x 10(6) M-1 min-1 for the uncatalyzed reaction. The kapp value of thrombin inhibition via HCII was 1.17 x 10(9) M-1 min-1 at a fucoïdan concentration of 50 micrograms/ml, compared to 1.72 x 10(5) M-1 min-1 for the uncatalyzed reaction. In a Wessler model of venous thrombosis, the fucoïdan fraction, injected intravenously to rabbits 10 min before thrombosis induction, exhibited antithrombotic activity: 1.8 mg/kg was the dose which inhibited F Xa-induced thrombus formation by 80% (ED80), compared to a heparin ED80 of 0.1 mg/kg. At this ED80 the antithrombotic effect of the fucoïdan persisted longer than that of heparin (30 min versus 15 min). The thrombin clotting time (TCT) was significantly prolonged (73 s versus control 29 s, compared to 53 s with heparin) 10 min after a fucoïdan bolus infusion giving a plasma fucoïdan concentration of 14.6 +/- 2.7 micrograms/ml. The bleeding time was slightly increased after fucoïdan infusion at the ED80. Fucoïdan extracted from marine flora thus shows promise as an antithrombotic drug.
Asunto(s)
Anticoagulantes/farmacología , Fibrinolíticos/farmacología , Fucosa/farmacología , Animales , Anticoagulantes/aislamiento & purificación , Fibrinolíticos/aislamiento & purificación , Fucosa/aislamiento & purificación , Masculino , Conejos , Algas Marinas/enzimologíaRESUMEN
The venous antithrombotic profile of naroparcil or (4-[4-cyanobenzoyl]-phenyl)-1.5-dithio-beta-D-xylopyranoside was investigated in the rabbit following single i.v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i.v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i.v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same dose of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human alpha-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antitrombinas/farmacología , Venas Yugulares , Tioglicósidos/farmacología , Trombosis/prevención & control , Administración Oral , Animales , Coagulación Sanguínea/efectos de los fármacos , Hemorragia/inducido químicamente , Masculino , Tiempo de Tromboplastina Parcial , Conejos , Tiempo de TrombinaRESUMEN
The venous antithrombotic effects of a novel chemical entity, LF 1351, were investigated in rats following single oral administration, in comparison with i.v. administered heparin. LF 1351 demonstrated a dose-related antithrombotic effect in three models of venous thrombosis. The compound was approximately equipotent in two models involving complete stasis of the vena cava and administration of factor Xa or porcine serum, giving respective ED50 values of 48.7 mg/kg and 36.7 mg/kg. LF 1351 was less effective in a model involving partial stasis in the presence of an endothelial lesion. In this case, the antithrombotic effect did not exceed 60-65%, the ED50 being 150 mg/kg. Heparin (50-300 micrograms/kg; 7.5-45.0 U/kg) was effective in all three models. At the approximate ED80 value against factor Xa-induced thrombosis (75 mg/kg) the antithrombotic effect of LF 1351 persisted for 6 h. The antithrombotic effect of LF 1351 (300 mg/kg) occurred without significant changes in APTT or thrombin time.
Asunto(s)
Fibrinolíticos/farmacología , Glicósidos/farmacología , Nitrobencenos/farmacología , Tromboflebitis/prevención & control , Administración Oral , Animales , Modelos Animales de Enfermedad , Fibrinolíticos/administración & dosificación , Glicósidos/administración & dosificación , Heparina/administración & dosificación , Heparina/farmacología , Inyecciones Intravenosas , Masculino , Nitrobencenos/administración & dosificación , Ratas , Ratas Endogámicas , Tromboflebitis/etiología , Factores de TiempoRESUMEN
Venous thrombus formation is induced in rats using saline flushing in a venous bag created in the vena cava and subsequent stasis by stricture of the vein. Maximum thrombus weight is obtained in 15 minutes. Neither flushing nor partial stasis alone could trigger thrombus formation during this time. Transmission electron microscopy demonstrated that flushing induces discrete endothelial lesions with no evident desendothelialisation areas. Anticoagulant agents are found to be active in preventing thrombus formation. However, heparin or related compounds such as pentosan polysulphate are active at dose levels not demonstrating anticoagulant activity. Thrombosis could be induced in thrombocytopenic rats indicating the non-involving of platelets. Moreover, aspirin did not prevent thrombus formation. Ticlopidine, another antiplatelet agent, did show an activity which might not involved platelets. This model thus possesses the features necessary for reliable investigation of potential antithrombotic agents for human use.