Evaluation of thrombolytic and systemic effects of the novel recombinant plasminogen activator BM 06.022 compared with alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis.

The thrombolytic and systemic effects of BM 06.022 were evaluated and compared with those of alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis. BM 06.022 consists of the kringle-2 and protease domains of human tissue plasminogen activator (t-PA) and is unglycosylated because of its expression in Escherichia coli cells. Thrombus formation in anesthetized open chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery at a high level site of obstruction. In heparinized dogs, none of six vehicle-treated animals exhibited reperfusion. Reperfusion was achieved in four of six dogs at 18.3 +/- 6 min after intravenous bolus injection of 140 kU/kg (0.24 mg/kg) of BM 06.022, whereas four of six dogs exhibited reperfusion later (p less than 0.05) at 76.5 +/- 16.1 min during infusion of 1.33 mg/kg of alteplase (0.13 mg/kg as initial bolus injection, followed by 0.66 mg/kg over 1 h and 0.53 mg/kg over 2 h). Significantly later (p less than 0.05) reperfusion than that achieved with BM 06.022 was achieved in five of six dogs at 57.8 +/- 12.1 min after intravenous injection of 0.4 U/kg of anistreplase. Streptokinase (21,000 IU/kg over 60 min) and urokinase (20,000 IU/kg as an intravenous bolus injection, followed by 20,000 IU/kg over 89 min) each induced reperfusion in three of six dogs but at 67 +/- 12 and 84.3 +/- 17.1 min (p less than 0.05 vs. BM 06.022), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of heparin and systemic lysis on coronary blood flow after reperfusion induced by the novel recombinant plasminogen activator BM 06.022 in a canine model of coronary thrombosis.

OBJECTIVES: We sought to evaluate whether anticoagulation by an intravenous heparin infusion prevents deterioration of coronary blood flow restored by the novel recombinant plasminogen activator BM 06.022, and to compare the effects of profound fibrinogenolysis with those of an intravenous bolus injection of heparin. BACKGROUND: Recent clinical studies indicate that heparin appears to be effective in reducing reocclusion when combined with recombinant tissue-type plasminogen activator (rt-PA), but that heparin is associated with an increased bleeding incidence. Therefore, the need for heparin has to be critically evaluated in the development of BM 06.022. METHODS: BM 06.022 is an unglycosylated variant of human tissue-type plasminogen activator. Thrombus formation in anesthetized open chest dogs was induced by electrical injury. Left circumflex coronary artery blood flow was monitored for 4 h using an electromagnetic flow probe. Twenty dogs were randomized to receive intravenous heparin (100 IU/kg bolus plus 100 IU/kg per h) in group B or saline solution in group A before an intravenous bolus injection of 200 kU/kg (= 0.34 mg/kg) BM 06.022 1 h after thrombus formation. Another 14 dogs were randomized to receive a single intravenous bolus injection of 200 IU/kg heparin plus 200 kU/kg BM 06.022 in group D or saline solution plus 1,000 kU/kg BM 06.022 in group C. RESULTS: In the absence of a systemic lytic state, heparin infusion prolonged (p < 0.05) the cumulative patency time (sum of time intervals during which the coronary artery was patent) to 204.3 +/- 7.4 min (group B) compared with 34.6 +/- 10.8 min with saline solution (group A), and increased (p < 0.05) the area under the curve for coronary blood flow versus time (AUCFlow) to 34.0 +/- 3.4 ml.h.min-1 compared with 7.7 +/- 4.6 ml.h.min-1. Profound fibrinogenolysis after administration of 1,000 kU/kg BM 06.022 (group C) and a single intravenous heparin injection (group D) did not differ in their effects on the cumulative patency time (182 +/- 30.3 vs. 177.5 +/- 25.4 min) and AUCFlow (36.0 +/- 10.3 vs. 30.5 +/- 4.8 ml.h.min-1), but these values were improved (p < 0.05) compared with those obtained after administration of saline solution plus 200 kU/kg BM 06.022 (group A). CONCLUSIONS: In the absence of a systemic lytic state, intravenous heparin is required as an adjunct to BM 06.022 to maintain coronary blood flow in dogs.

Thrombolysis with an Escherichia coli-produced recombinant plasminogen activator (BM 06.022) in the rabbit model of jugular vein thrombosis.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of the expression in Escherichia coli. The thrombolytic and pharmacokinetic properties as well as the hemostasis effects of BM 06.022 were investigated in the rabbit model of jugular vein thrombosis. The thrombi were 125I-fibrin labeled. Intravenous bolus injection of 50, 100, 200, and 400 kU/kg BM 06.022 or 400, 800, and 1600 kU/kg alteplase over 15 s to six rabbits/dose produced a dose-dependent increase of thrombolysis determined 2 h post injection. The dose-response curve of BM 06.022 was located left compared with that of alteplase. The effective dose of 50% thrombolysis (ED50) obtained by half-logarithmic regression analysis was 163 kU/kg (= 0.28 mg/kg) for BM 06.022 and 871 kU/kg (= 1.09 mg/kg) for alteplase. At equipotent doses (50% thrombolysis), the residual concentration of fibrinogen was 74.2% and 76.5%, that of plasminogen 66.7% and 69.4%, and that of alpha 2-antiplasmin 47.3% and 46% for BM 06.022 and alteplase, respectively. Pharmacokinetic analysis for plasma activity at a dose of 400 kU/kg revealed a half-life of 18.9 +/- 1.5 min for BM 06.022, whereas alteplase was distributed with a half-life of 2.1 +/- 0.1 min, accounting for 86.7 +/- 1.9% of the total AUC, followed by a beta-phase with a half-life of 13.8 +/- 0.9 min. Plasma clearance of BM 06.022 was 4.7 +/- 0.7 ml min-1 kg-1 compared with 20 +/- 1.2 ml min-1 kg-1 for alteplase.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemodynamic profile of an inhibitor of phosphodiesterase III, adibendan (BM 14.478): comparison with nitroprusside and dobutamine in conscious dogs.

1. This study was performed to investigate whether cardiac positive inotropic as well as peripheral vasodilator properties of adibendan contribute to its overall haemodynamic profile in conscious dogs. 2. Haemodynamic measurements were carried out in conscious chronically instrumented dogs after administration of adibendan, sodium nitroprusside or dobutamine. 3. The cardiovascular changes induced by adibendan (0.01 and 0.03 mg kg-1) resembled those of dobutamine (1.0-4.0 micrograms kg-1 min-1): left ventricular dP/dt60 (LV dP/dt60), stroke volume (SV) and cardiac output (CO) increased to a similar extent, but mean arterial pressure (MAP) and heart rate (HR) remained unchanged. 4. In contrast to dobutamine, higher doses of adibendan (0.1-1.0 mg kg-1) decreased MAP and LVEDP. These effects were of a similar magnitude to those observed following nitroprusside administration (0.5-12.5 micrograms kg-1 min-1). In contrast to nitroprusside, adibendan still showed additional effects on LV dP/dt60 and CO. 5. From these results, it is concluded that both the peripheral vasodilator and the cardiac positive inotropic action of adibendan contribute to its overall haemodynamic profile.

Hirudin and sulotroban improve coronary blood flow after reperfusion induced by the novel recombinant plasminogen activator BM 06.022 in a canine model of coronary artery thrombosis.

Thrombus formation in anesthetized, open-chest dogs was induced by electrical injury to the intimal surface of the left circumflex coronary artery. One-hour postocclusion, administration of vehicle to heparinized dogs (n = 12) did not induce reperfusion despite concomitant treatment with acetylsalicylic acid (ASA), sulotroban, or saline. Intravenous bolus injection of 140 kU/kg (= 0.24 mg/kg) of the unglycosylated t-PA variant BM 06.022 induced reperfusion in 4 out of 6 dogs, followed by flow deterioration. Pretreatment with i.v. ASA did not improve coronary blood flow (CBF). Conjunctive treatment with the thromboxane A2-receptor antagonist, sulotroban, (10 mg/kg i.v. bolus, followed by 10 mg/kg/h) or with recombinant hirudin, a specific thrombin inhibitor, (1 mg/kg/h) 30 min prior to i.v. injection of BM 06.022, prolonged (p < 0.01) the cumulative patency time (sum of time-intervals in which the coronary artery was patent) to 147.4 +/- 9.2 min in 4 out of 6 reperfused dogs and 129.9 +/- 12.3 min in 7 out of 8 dogs, respectively, compared to the saline plus BM 06.022 treatment (47.5 +/- 13.1 min) in 4 out of 6 dogs. The terminal CBF was higher (p < 0.01) after sulotroban plus BM 06.022 (7.0 +/- 1.7 ml/min) and hirudin plus BM 06.022 (6.3 +/- 1.5 ml/min) than after saline plus BM 06.022 (0.8 ml/min). These findings demonstrate that drugs with antithromboxane or antithrombin activity may improve CBF after reperfusion.

Pharmacokinetic properties of an Escherichia-coli-produced recombinant plasminogen activator (BM 06.022) in rabbits.

The recombinant plasminogen activator BM 06.022 consists of the kringle 2 and the protease domains of human t-PA and is unglycosylated because of its expression in Escherichia coli. The pharmacokinetic properties of BM 06.022 following intravenous injection over 1 min were characterized in anesthetized male New Zealand white rabbits. BM 06.022 was injected at doses of 50, 100, 200, and 400 kU/kg bw (n = 5-6/dose). Activity concentrations in plasma were determined using an indirect spectrophotometric assay. The maximum plasma concentration and the area under the plasma concentration vs. time curve (AUC0-00) of BM 06.022 increased linearly with dose. The systemic clearance ranged from 2.5 to 3.0 ml.min-1.kg-1 and did not show dose-dependency, in contrast to alteplase which was studied at doses of 200, 400, 800, and 1600 kU/kg. A direct comparison of clearance rates of BM 06.022 and alteplase at doses of 200 and 400 kU/kg each revealed a 8.5-fold slower clearance rate of BM 06.022. The majority (18/23) of rabbits with BM 06.022 injection showed a pharmacokinetic profile which was best characterized by a one-compartment model in contrast to alteplase (10/23). The dose-groups of BM 06.022 showed an average dominant half-life ranging from 11.6 to 15.4 min, which was about five-times longer than the dominant half-life values of alteplase (2.3 to 4.5 min). Assuming a two-compartment model in the remaining animals, the initial alpha-phase of BM 06.022 accounted for 40.1 +/- 13.2% (n = 5) of the total AUC, whereas the alpha-phase of alteplase accounted for 82.7 +/- 3% (n = 13) of the total AUC.

Pharmacokinetic and thrombolytic properties of unglycosylated recombinant tissue-type plasminogen activator (BM 06.021) produced in Escherichia coli.

Recombinant tissue-type plasminogen activator (rt-PA) was produced in Escherichia coli cells in order to obtain an unglycosylated rt-PA (BM 06.021) with increased thrombolytic potency due to altered pharmacokinetic properties. The pharmacokinetics were studied in rabbits upon intravenous infusion of 200 kU/kg over 30 min. The thrombolytic dose-response effects were evaluated in a rabbit model with 125I-labeled venous thrombi upon intravenous infusion over 4 h. The thrombolytic effects after intravenous bolus injection of 200 kU/kg BM 06.021 were investigated in a canine model of coronary artery thrombosis. All studies were performed comparing BM 06.021 with glycosylated rt-PA (alteplase). BM 06.021 demonstrated a longer (p less than 0.05) half-life (5.6 +/- 2.6 vs. 2.1 +/- 0.3 min) and a lower (p less than 0.05) clearance rate (7.5 +/- 0.8 vs. 22.2 +/- 3.1 ml.min-1.kg-1) than alteplase in rabbits upon intravenous infusion. The dose-response curve of BM 06.021 for thrombolysis in a rabbit model of jugular vein thrombosis was located to the left of that for alteplase with a 2.1-fold lower effective dose of 50% thrombolysis (ED50) of BM 06.021 (207 vs. 436 kU/kg). Intravenous bolus injection of 200 kU/kg BM 06.021 induced the same reperfusion rate (4/6) as intravenous infusion of 800 kU/kg alteplase over 90 min in a canine model of coronary artery thrombosis. The residual thrombus wet weight did not significantly differ between BM 06.021 and alteplase (5.7 +/- 1.8 vs. 6.3 +/- 1.1 mg). The results indicate that unglycosylated rt-PA (BM 06.021) has a higher in vivo thrombolytic potency than glycosylated rt-PA (alteplase).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential fibrinolytic properties of the recombinant plasminogen activator BM 06.022 in human plasma and blood clot systems in vitro.

The effects of the unglycosylated recombinant plasminogen activator BM 06.022, consisting of the kringle 2 and protease domains of tissue-type plasminogen activator (t-PA), on clot lysis were evaluated in an in vitro system. Fresh and aged 125I-labelled human platelet-poor (PPP) and platelet-rich plasma (PRP) and whole blood clots were immersed in human plasma. Clot lysis was quantitated by measurement of released 125I. Fresh PPP clots were time- and concentration-dependently lysed by BM 06.022, alteplase, melanoma t-PA (mt-PA), and urokinase. Fifty per cent clot lysis at 4 h required 3.2-, 6.4- and 15.2-fold higher nM concentrations of mt-PA, BM 06.022, and urokinase respectively compared with alteplase. Maximal lysis (Emax) at 4 h was similar (84.1-87.6%) for BM 06.022, alteplase, and mt-PA, but lower (65.3 +/- 0.6%) for urokinase. Emax for BM 06.022 was lower (P < 0.05) than for alteplase for fresh and aged PRP and whole blood clot lysis. These data suggest that in vitro BM 06.022 achieved, compared with alteplase, the same maximal efficacy in fresh PPP-clot lysis despite a lower potency, but was less effective in lysing aged and fresh PRP and whole blood clots.

Double bolus administration of the novel recombinant plasminogen activator BM 06.022 improves coronary blood flow after reperfusion in a canine model of coronary thrombosis.

Reocclusion of coronary arteries is a major problem after successful thrombolysis in patients with acute myocardial infarction. We evaluated in a canine model of coronary thrombosis which is known to elicit reocclusion, whether an increased single i.v. bolus dose or two boli of the novel t-PA variant BM 06.022 improved coronary blood flow after reperfusion compared to i.v. injection of a standard single dose of BM 06.022. Double bolus administration, but not an increased single bolus dose of BM 06.022 significantly increased the maximum achieved coronary blood flow, prolonged the cumulative patency time, maintained blood flow at the end of the experiments, and reduced residual thrombus wet weight. Thus, double bolus administration improves coronary blood flow after reperfusion in the dog.

Does intention-to-treat analysis answer all questions in long-term mortality trials? Considerations on the basis of the ANZ trial.

AIMS: Intention-to-treat (ITT) analyses are the gold standard in endpoint analyses of randomized clinical trials. Valuable information, however, may be lost in this approach as the actual time on trial medication is not accounted for in patients who withdraw early. Since compliance per se can be a prognostic factor and the actual treatment time is a variable likely to influence clinical outcome, this information should be added to an ITT analysis concept. Thus, the aim is to elucidate the influence of actual treatment time on the results of ITT analysis using available data from a well-conducted, large-scale clinical trial. METHODS AND RESULTS: The ANZ trial, a carvedilol study in heart failure, is characterized by a considerable number of early withdrawals in the carvedilol group. Using the ANZ trial database, the percentage of withdrawals was calculated as well as the number of days on treatment. Fourteen out of 21 deaths (67%) in the carvedilol group occurred after discontinuation of carvedilol. At the time of death, 14 out of 29 (48%) patients in the placebo group had withdrawn from treatment. Mean time of patients on medication who withdrew and died later were 301 days for placebo, but only 204 days for carvedilol. We performed a conventional ITT analysis, and an ITT-based Cox-proportional hazards model (modified ITT) in which the actual time on trial medication was entered into the model as an explanatory variable. The risk ratio in conventional ITT analysis was 0.71 (95% confidence interval (CI) 0.41 to 1.24, p = 0.228) and 0.50 (95% CI 0.28-0.90, p < 0.001 ) in modified ITT analysis. CONCLUSIONS: Incorporation of the actual treatment time substantially influences the result of this exemplary ITT analysis. The resulting risk ratio is clearly in accordance with clinical implications and in close agreement with those of other beta-blocker trials in heart failure. Entering the actual treatment time into a Cox-proportional hazards model as an explanatory variable is reasonable if not necessary from a clinical point of view and thus may strengthen the validity of ITT analysis.

Local drug delivery via transvascular injection.

BACKGROUND: In this experimental series we tested drug distribution and systemic leakage using local drug delivery with a new transvascular injection system. METHODS: Porcine femoral and carotid arteries (n = 56) underwent local drug application with a new 5 French (Fr) over-the-wire needle-injection catheter system (NIC) using three needles. A radioactive indicator [C14-Carvedilol, 2.0 milliliter (ml); 0.03 milligram (mg)] was injected in two carotid and two femoral vessels in parallel. Serial blood withdrawal was performed thereafter. After randomization to different explantation times, the vessels, perivascular tissue, liver and spleen were removed [0.5, 1, 1.5, 3 and 4 hours after injection, respectively]. Radioactivity was determined in a scintillation counter or with autoradiography. The indicator amount was calculated in relation to total drug amount (100%). RESULTS: Use of the NIC caused vessel texture alteration in non-diseased porcine vessels, seen as vessel wall penetration and perivascular edema. After single injection the maximum of the indicator was found in perivascular tissue 0.5 hours at the application site (carotid perivascular tissue: 7.48%; femoral perivascular tissue: 2.56%). Thereafter, radioactivity in the artery increased and perivascular content declined. The maximum in femoral arteries (1 hour; 1.96%) occurred earlier and was significantly lower compared to carotid arteries (2 hours; 7.75%). Four hours post-injection, 1.4% of total drug amount was detectable in the carotid arteries and 0.6% was detected in the femoral arteries. Systemic content was measured after C14-Carvedilol application with a maximum in serum of 28% (10 minutes), liver 30% (0.5 hour) and spleen 0.6% (0.5 hour). After 3 hours, still 5% of the indicator was still measureable in the serum and liver and less than 0.1% was measurable in the spleen. LDD with the NIC system is dependent on the vascular anatomy. The data indicate redistribution from perivascular to vascular space thus allowing a prolonged vascular and perivascular drug delivery. The amount deliverable is lower than expected due to substantial systemic drug contamination with this catheter.

Comparison of the haemodynamic profiles of the hypotensive action of verapamil and carvedilol.

Comparative haemodynamic investigations with carvedilol and verapamil were carried out on conscious, instrumented dogs. Arterial blood pressure, right atrial pressure (RAP), cardiac output (CO), heart rate, stroke volume and total peripheral resistance (TPR) were determined after intravenous (i.v.) injection of incremental doses (0.01-3 mg/kg) of the drugs. Carvedilol reduced the blood pressure in a dose-dependent manner, concomitant with a reduction in TPR. The RAP and the CO were not affected, indicating that arterial vasodilatation was induced by carvedilol. Verapamil showed a decrease in the blood pressure with a reduction of CO and SV. Moreover, at high doses the RAP was increased, indicating a reduction of the cardiac performance. Thus, in our experimental model remarkable differences between the haemodynamic effects of i.v. injections of carvedilol and verapamil have been observed, whereas after oral administration blood pressure also decreases after verapamil due to a reduction of TPR.

Experimental and clinical pharmacology of carvedilol and other drugs combining vasodilation and beta-adrenoceptor antagonism in a single molecule.

Combinations of beta-adrenoreceptor blocking agents and vasodilators have been widely used because of their favorable hemodynamic actions and their high efficacy. In comparison with the free combination of the two active principles, substances that combine vasodilation and beta-blockade in a single molecule may lead to a simplification of therapy and thus to an improvement in compliance. Substances that show clear beta-blocking and vasodilating actions at therapeutic doses have already been introduced to therapy or are in an advanced stage of clinical development. The vasodilating action of amosulalol, carvedilol and labetalol is achieved by blockade of the alpha 1-receptors. In contrast, partial agonistic action on beta 2-receptors is responsible for the vasodilatation with dilevalol. This mechanism probably also plays an important role in the vasodilatation induced by celiprolol. While classical beta-blocker lead to a rise in peripheral resistance and to a marked fall in cardiac output, peripheral resistance falls during treatment with vasodilating beta-blockers. The cardiac output is either only slightly reduced or virtually unchanged. Surprisingly, three months' treatment with the vasodilating beta-blocker bucindolol in patients with severe heart failure led to a rise in cardiac output and in ejection fraction and to a reduction of the heart rate and pulmonary wedge pressure. An improvement of left-ventricular function was also obtained on administration of carvedilol in patients with coronary heart disease. Theoretically, it is conceivable that substances with additional alpha 1-blocking actions, such as labetalol, carvediolol or amosulalol, or with partial agonistic activity such as celiprolol or dilevalol, would have a clearly more favourable effect on the blood lipid profile than the classical beta-blocking agents. Initial results appear to confirm this, but final conclusions will only be possible when the results of prospective comparative studies are available.

Increase in the cardiotonic action and in the therapeutic margin of digoxin by the adrenergic beta-stimulant doxaminol in cats.

The interaction between digoxin and the beta-sympathomimetic drug doxaminol was investigated in cats with acute heart failure induced by pentobarbital sodium. Doxaminol, 50 micrograms/kg X min, infused for 60 min caused a dose-dependent rise in dp/dtmax with little increase in heart rate. The maximum increase of 4.3 mHg/s was obtained after about 37 min. Digoxin, 10 micrograms/kg X min, and the combination of both drugs were infused until cardiac arrest. The maximum increase of dp/dtmax was observed after 29 min in both experiments; it was 5.7 mHg/s with digoxin alone and 7.3 mHg/s with the combination (p = 0.025). The combined infusion of epinephrine (0.3 micrograms/kg X min) plus digoxin (10 micrograms/kg X min) caused a maximal increase of dp/dtmax by 7.9 mHg/s. The cardiotoxic dose of digoxin was markedly reduced by epinephrine, not by doxaminol. The relevance of this difference to man cannot be assessed definitely because the ECG changes produced by digoxin in cats are different from those seen in man.