Superiority of enoxaparin over heparin in combination with a GPIIb/IIIa receptor antagonist during coronary thrombolysis in dogs.

It is known that a low-molecular-weight heparin (LMWH) is more effective than unfractionated heparin in unstable angina/non-Q-wave myocardial infarction (UA/NQMI) and the platelet GPIIb/IIIa receptors play an important role in acute myocardial infarction (AMI). Therefore, enoxaparin might have a similar advantage over heparin when used with a GPIIb/IIIa antagonist (RPR109891) in coronary thrombolysis. After induction of coronary thrombosis in anesthetized dogs, infusion of saline, enoxaparin, heparin, RPR109891, enoxaparin+RPR109891, or heparin+RPR109891 was initiated followed 15 min later by recombinant tissue plasminogen activator (rt-PA). The incidence of reperfusion in the enoxaparin+RPR109891- and the heparin+RPR109891-treated groups was similar, but time to reperfusion tended to be shorter for enoxaparin versus heparin. Only 43% of the vessels reoccluded in the enoxaparin+RPR109891 group, compared to 100% vessels in the heparin+RPR109891 group. Enoxaparin+RPR109891 maintained flow for a significantly longer time compared to saline, enoxaparin, heparin, and heparin+RPR109891. Enoxaparin+RPR109891 and heparin+RPR109891 increased the template bleeding time by 2- and 3-fold and activated partial thromboplastin time (APTT) by 1.3- and 3-fold, respectively. These data suggest that enoxaparin is more effective and potentially safer than heparin when combined with a GPIIb/IIIa receptor antagonist during rt-PA-induced coronary thrombolysis.

Pharmacodynamic activity and antithrombotic efficacy of RPR120844, a novel inhibitor of coagulation factor Xa.

These studies were designed to examine the pharmacodynamic profile and antithrombotic efficacy of RPR120844, a competitive inhibitor of coagulation factor Xa, with a K(i) of 7 nM against human factor Xa. In vitro, RPR120844 doubled activated partial thromboplastin time (APTT) at concentrations of 1.54, 1.48, and 0.74 microM in plasma obtained from humans, dogs, and rats, respectively. Intravenous bolus administration of RPR 120844 at 0.3, 1, and 3 mg/kg to rats resulted in maximal increases in APTT of 1.8-, 2.6-, and 8.4-fold over baseline, respectively. The effect on prothrombin time (PT) was less pronounced, resulting in a 4.4-fold increase at 3 mg/kg. These effects were rapidly reversible; APTT and PT returned to control values by 30 min after dosing. Intragastric administration to rats at 50, 100, and 200 mg/kg resulted in modest increases in APTT and PT of 1.5- and 1.3-fold over baseline at the highest dose. Plasma levels were estimated by anti-Xa activity by using an amidolytic, chromogenic assay. Plasma levels were 0.65, 1.29, and 2.45 microM at 30 min after dosing at 50, 100, and 200 mg/kg, respectively. Intravenous administration to dogs at 0.1 and 0.3 mg/kg produced maximal increases in APTT of 1.7- and 2.4-fold over baseline, respectively. Intragastric administration to dogs at 50 mg/kg resulted in maximal increases in APTT and PT of 1.7- and 1.1-fold over baseline, with peak plasma levels of 3.9 microM observed at 15 min after dosing. In a rat model of FeCl2-induced carotid artery thrombosis, RPR120844 (3 mg/kg, i.v. bolus + 300 microg/kg/min constant infusion; n = 4) significantly increased time-to-occlusion from 18+/-1 min (vehicle, n = 4) to 60 min (maximal observation time) and reduced thrombus mass from 5.5 +/- 0.2 mg (vehicle) to 1.4 +/- 0.2 mg. These results indicate that RPR120844 is a potent, selective inhibitor of Xa that exhibits oral activity and is efficacious in a standard model of arterial thrombosis.

RPR120844, a novel, specific inhibitor of coagulation factor Xa inhibits venous thrombosis in the rabbit.

The in vivo antithrombotic activity of RPR 20844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p<0.05 vs. saline-treated animals) by RPR120844 at 30 and 100 microg/kg/min. At doses of 10, 30 and 100 microg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8+/-0.3, 1.5+/-0.9 and 2.4+/-0.6 microM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR 120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

Design of a new class of orally active fibrinogen receptor antagonists.

The integrin receptor recognition sequence Arg-Gly-Asp was successfully used as a template from which to develop a series of potent, selective, orally active, peptide-based fibrinogen receptor antagonists with a long duration of action. Simple modifications centered on the Arg and Gly residues quickly led to a modified peptide (1) with significantly enhanced ability to inhibit in vitro platelet aggregation. Substitution of the guanidino group in 1 by piperidine provided 3, which showed not only a further increase in potency but also a modest degree of oral efficacy. Finally, exploration of the nature of the C-terminal amino acid, with respect to its side-chain functionality and the carboxy terminus, yielded a group of molecules that showed excellent in vitro potency for inhibiting platelet aggregation, excellent integrin selectivity, a high level of oral efficacy, and an extended duration of action.

Inhibition of repetitive thrombus formation in the stenosed canine coronary artery by enoxaparin, but not by unfractionated heparin.

Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg + 5 microg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg + 10 microg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (P<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg + 10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (P<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.

Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery.

A canine model of electrolytic injury-induced coronary artery thrombosis and rtPA-induced thrombolysis was used to evaluate the relative antithrombotic efficacy of enoxaparin (a low molecular weight heparin), conventional therapy (heparin or heparin plus aspirin), and hirulog (a direct thrombin inhibitor), when used as adjunctive therapy during thrombolysis. After 60 min of clot aging, adjunctive therapy was begun at doses which elevated APTT approximately 2-fold over baseline. Fifteen minutes after the start of adjunctive therapy, recombinant tissue plasminogen activator (rtPA) was administered (100 microg/kg i.v. bolus + 20 microg/kg/min for 60 min). Adjunctive therapy continued for 1 h after termination of rtPA and blood flow was monitored for two additional hours. Enoxaparin (1 mg/kg i.v. bolus + 30 microg/kg/min, n = 10 for each treatment group) was the only adjunctive treatment that significantly increased the total minutes of flow (143 +/- 25 min out of a possible 240 min, vs 54 +/- 25 min for vehicle, p <0.05) and decreased thrombus mass (6.0 +/- 1.3 mg vs 11.8 +/- 3.2 mg for vehicle). Although hirulog (2 mg/kg i.v. bolus + 40 microg/kg/min) did not significantly increase the minutes of flow (120 +/- 27 min, p <0.06) or decrease thrombus mass (8.7 +/- 1.7 mg) compared to vehicle, these values were not significantly different than those measured in the enoxaparin group. However, the results with hirulog were achieved at the expense of a significantly greater increase in template bleeding time than that measured during enoxaparin treatment. Minutes of flow for heparin (50 U/kg i.v. bolus + 0.6 U/kg/min) and heparin plus aspirin (5 mg/kg i.v. bolus) were 69 +/- 20 and 60 +/- 23 min, respectively; thrombus masses were 8.2 +/- 1.3 and 7.3 +/- 1.0 mg, respectively. In summary, enoxaparin was more effective than conventional therapy in this model in terms of vessel patency and thrombus mass, and was as effective as hirulog, at least at a dose of hirulog that only modestly impaired hemostasis. Therefore, enoxaparin may prove to be a safe and effective alternative agent for adjunctive therapy during thrombolysis with rtPA.

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist.

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC50 values of 3.7, 4.6, 6.3, 126, 136 and 1600 microM, respectively. RG13965 (3, 10, and 30 mg/kg, i.v.) decreased the incidence of collagen/epinephrine-induced thrombosis in mice from 90% in untreated animals to 63, 37, and 0%, respectively. In hamsters, RG13965 (10 and 30 mg/kg, i.v.) prolonged the time required for formation of a hemostatic plug in severed mesenteric arteries by 1.6- and 3.6-fold, respectively. In a canine model of repetitive platelet thrombus formation in the coronary artery, RG13965 (0.1, 0.3, and 1 mg/kg, i.v.) reversibly inhibited cyclic flow reductions (CFRs) and inhibited ADP-induced ex vivo platelet aggregation by 29, 57, and 77%, respectively. RG13965 (1 mg/kg) completely inhibited CFRs for at least 40 min. Platelet count was not altered at any dose and template bleeding time was prolonged modestly (1.8-fold) at only the highest dose. RG13965 dose-dependently and reversibly inhibited thrombus formation at doses which did not completely inhibit ex vivo platelet aggregation and only modestly prolonged template bleeding time.

RG 12915: a potent 5-hydroxytryptamine-3 antagonist that is an orally effective inhibitor of cytotoxic drug-induced emesis in the ferret and dog.

RG 12915 [4-[N-(1-azabicyclo[2.2.2.]octan-3-(S)-yl)]2-chloro-cis 5a-(S)-9a-(S)-5a,6,7,8,9,9a-hexahydrobenzofurancarboxamide hydrochloride] is a potent and effective agent against cisplatin-induced emesis in the ferret after i.v. or p.o. administration. This agent (p.o.) is also highly protective against cisplatin-induced emesis in the dog, as well as cyclophosphamide/doxorubicin-induced emesis in the ferret. When administered either p.o. or i.v., RG 12915 has a lower ED50 value (0.004 mg/kg) than GR 38032F, BRL 43694 and metoclopramide for attenuating cisplatin-induced emetic episodes in the ferret. It also has a long duration of action against cisplatin-induced emesis in the ferret. In contrast to metoclopramide, RG 12915 lacks significant antidopaminergic activity both in vitro [( 3H]spiroperidol displacement), as well as in vivo (apomorphine-induced emesis). In radioligand binding assays, RG 12915 is a potent and selective displacer of binding of 5-hydroxytryptamine (5-HT)3 binding sites (IC50 value = 0.16 nM), whereas failing to displace binding of ligands for the alpha-1, alpha-2 and beta adrenergic, 5-HT1 or 5-HT2 or cholinergic-muscarinic sites with IC50 values less than 1 microM. At a p.o. dose (1 mg/kg) in which RG 12915 is highly protective against cisplatin-induced emesis in the dog, RG 12915 has no significant gastroprokinetic activity in the same species. In summary, RG 12915 is a potent and p.o. effective agent against cytotoxic drug-induced emesis in animal models. The antiemetic potency of RG 12915 against cisplatin is unrelated to antidopaminergic or gastroprokinetic activity, but may be related to its affinity for 5-HT3 binding sites.

Antiinflammatory effects of various drugs on acetic acid induced colitis in the rat.

The efficacy of various drugs used to treat ulcerative colitis, (sulfasalazine, 5-aminosalicylate, hydrocortisone) was investigated in a model of acetic acid-induced colitis in the rat. Subsequently, we tested the ability of antioxidant/5-lipoxygenase inhibitors (gossypol and nordihydroguiaretic acid [NDGA]) and a cyclooxygenase inhibitor (indomethacin) to attenuate the macroscopic colonic damage and/or neutrophil influx (myeloperoxidase activity [MPO]) associated with this model of colitis. Oral pretreatment with either sulfasalazine, gossypol, or NDGA significantly decreased colonic MPO activity induced by acetic acid. Intrarectal administration of such drugs resulted in an even larger reduction of the colonic inflammation, with gossypol being the most potent compound. Oral or intrarectal administration of corticosteroids (dexamethasone, hydrocortisone) also attenuated the parameters of acetic acid induced colitis. In contrast, pretreatment with indomethacin was ineffective, or when administered daily after colitis induction, indomethacin actually increased colonic neutrophil influx significantly. Our data suggest that both the route of drug administration and dosing regimen employed affect the antiinflammatory potency and/or efficacy of compounds on colitis induced by acetic acid in the rat. Drugs which were effective against this colitis may act by scavenging of oxygen derived free radicals.