Comparison of the thrombolytic activity of the novel plasminogen activator, LY210825, to anisoylated plasminogen-streptokinase activator complex in a canine model of coronary artery thrombolysis.

We have compared the thrombolytic efficacy of a novel single-chain, recombinant tissue-type plasminogen activator variant, LY210825, containing the second kringle and serine protease domains of native tissue-type plasminogen activator, with anisoylated plasminogen-streptokinase activator complex (APSAC). Male hounds (16-22 kg) were anesthetized, the left circumflex coronary artery was isolated and an electromagnetic flow probe was placed around the artery proximal to the first main branch for the measurement of coronary blood flow. An occlusive thrombus was formed after electrolytic injury of the intima of the coronary artery. After an occlusion period of 1 hr, either LY210825 (n = 8) or APSAC (n = 6) was administered as a single i.v. injection of 0.45 mg/kg. Blood was drawn (3.8% citrate) for determination of plasma fibrinogen, plasminogen and alpha-2 antiplasmin. Time to reperfusion was significantly faster with LY210825 than with APSAC, 20 +/- 2 vs. 54 +/- 8 min, respectively. The incidence of reocclusion was similar for both agents. APSAC produced significant depletion of alpha-2 antiplasmin, plasminogen and circulating fibrinogen, whereas LY210825 caused only slight consumption of plasminogen and only small decreases in fibrinogen. After a single injection of LY210825, thrombolytic concentrations of plasminogen activator were available immediately, whereas there was a significant delay in lytic concentrations of active streptokinase-plasmin complex. Consequently, LY210825 reperfused the coronary artery faster than did APSAC. In addition, LY210825 spared plasma fibrinogen, plasminogen and alpha-2 antiplasmin and therefore, could potentially minimize the risk of bleeding complications.

Thrombolytic activity of a novel plasminogen activator, LY210825, compared with recombinant tissue-type plasminogen activator in a canine model of coronary artery thrombosis.

LY210825, a recombinant tissue-type plasminogen activator (rt-PA), which contains the kringle-2 and serine protease functional domains of native tissue-type plasminogen activator, was previously produced by site-directed mutagenesis in a Syrian hamster cell line. We studied the thrombolytic potential of this molecule in a canine thrombosis model. Male hounds (16-22 kg) were anesthetized; a 2.0-cm segment of the left circumflex coronary artery (LCX) was isolated proximal to the first main branch, and the dogs were instrumented with an electromagnetic flow probe to measure coronary blood flow. An occlusive thrombus was formed after injury of the intimal surface of the LCX with an electrical current applied by a needle-tipped anode placed distal to the electromagnetic flow probe. After 1 hour of occlusion, either LY210825 or rt-PA was administered intravenously according to the following protocols: 1) a 1-hour infusion of either 0.25 mg/kg LY210825 or 0.4 mg/kg rt-PA, 2) single injections of 0.15-0.6 mg/kg LY210825, and 3) a single injection of 0.45 mg/kg LY210825 and a 3-hour infusion of 1.0 or 1.7 mg/kg rt-PA. Plasma half-lives of LY210825 and rt-PA were 58 +/- 7 and 3.3 +/- 0.3 minutes, respectively. LY210825 produced more rapid reperfusion of the LCX than did rt-PA. In the third study, 90% of the rt-PA-treated vessels reoccluded within 1 hour after cessation of drug, whereas only 25% of the LY210825-treated vessels reoccluded during a 4-hour washout period. There were significant, but relatively small, reductions produced by both plasminogen activators on plasma fibrinogen and plasminogen (25-35% decreases). Because of its longer plasma half-life, LY210825 could be administered intravenously as a single injection. In a canine model of coronary artery thrombosis, LY210825 was a more effective thrombolytic agent than was rt-PA.

Correlation of the in vivo anticoagulant, antithrombotic, and antimetastatic efficacy of warfarin in the rat.

Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an in vivo study of warfarin in two independent rat disease models--a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIc (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and failed to produce any antithrombotic or antimetastatic effects. The substantial correlation (very similar dose-response effects) among the anticoagulant, antithrombotic and antimetastatic efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antithrombotic and the antimetastatic effects. The poor therapeutic index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

The effects of 1-methyl-5-thiotetrazole in a rat liver vitamin K-dependent carboxylase assay.

1-Methyl-5-thiotetrazole (NMTT), a metabolite of moxalactam (MoxamR), was studied for its potential inhibition of vitamin K-dependent carboxylation. The assay system utilized a detergent solubilized rat liver microsomal preparation. Vitamin K1H2 was artificially produced in situ by the NADH-dependent reduction of exogenous phylloquinone and the resultant carboxylation monitored by 14CO2 incorporation into a soluble peptide substrate. Warfarin, used as a reference inhibitor, gave results expected from the literature - 50% inhibition at a pharmacologically excessive level of 1.0 mM. Carboxylation was unaffected by 1.0 mM NMTT and was marginally (0-14%) diminished by 5.0 mM NMTT. Carboxylation was 25% diminished at 10.0 mM NMTT, a concentration far above that achieved in human testing of moxalactam. When NMTT was pre-incubated with the liver microsomal carboxylase enzyme preparation, 10.0 mM NMTT again caused merely a 25% diminution of carboxylation in the assay. These results do not support a role for NMTT as an inhibition of Vitamin K-dependent carboxylation which would produce pharmacological side effects during moxalactam therapy. During these studies it was found that dramatic consumption of NADH occurs in the presence of liver microsomal preparations (independent of vitamin K and of NMTT) and that NMTT effects on these processes may explain the small carboxylation diminution observed at 10.0 nM NMTT in the carboxylase assay.