Thrombolysis with Intracoronary Administration of YM866, a Novel Modified Tissue-Type Plasminogen Activator, in a Canine Model of Coronary Artery Thrombosis

Thrombolysis a Novel

with

Intracoronary

Modified

in a Canine

Administration

Tissue-Type

Model

of YM866,

Plasminogen

of Coronary

Artery

Activator, Thrombosis

Tomihisa Kawasaki', Masao Katoh2, Seiji Kaku', Hiroshi Gushima3, Toichi Takenaka', Yoshiki Yui4 and Chuichi Kawai4 'Cardiovascular 'Research

and Atherosclerosis

Strategy and Evaluation 4Third Division

Research Laboratories Division

, Department

, Institute for Drug Discovery Research, 2Molecular Medicine Research,

, Yamanouchi Pharmaceutical

Co., Ltd., 21 Miyukigaoka,

Tsukuba, Ibaraki

of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606-01, Received

May

18, 1993

Accepted

305, Japan

Japan

July 30, 1993

ABSTRACT-We compared the thrombolytic activity of YM866, a novel modified tissue-type plasminogen activator, with that of t-PA by intracoronary administration in a canine thrombosis model of copper coil induced 6-hr-old thrombi. Either drug was administered by a single injection (10 min) or multiple injection (4 x 10 min) under heparinization (300 IU/kg, i.v.). The reperfusion rate of YM866 was 4 times higher than that of t-PA when administered by single injection. Time to reperfusion of YM866 by single injection was shorter than that of either agent by multiple injection. No group showed any decrease in plasma fibrinogen levels. No acute reocclusion was seen in animals with YM866 by single injection. The improved throm bolytic activity of YM866 by single injection correlated with the relatively higher antigen levels of this agent due to its prolonged biological half-life. These results suggest that single intracoronary administration of YM866 is a safe and effective thrombolytic method for rapid recanalization and lowered acute reocclusion without activation of systemic fibrinolysis. Keywords: Tissue-type plasminogen Thrombi (aged)

activator (t-PA) (modified), Thrombolysis

Owing to its strict fibrin specificity, tissue-type plas minogen activator (t-PA) is a potent thrombolytic agent. However, due to the extremely short half-life of this agent (T1/2 < 5 min), administration must be by high-dosage infusion. This increases the risk of systemic bleeding, and results in a high incidence of acute reocclusion (1). To eliminate these shortcomings, attempts have been made to modify the t-PA molecule, and several mutants of t-PA that have prolonged plasma half-life and that can be ad ministered by intravenous (i.v.) bolus injection have been reported (2 4). YM866 is a novel modified t-PA with deletion of the K1 domain of the molecule and with a point mutation at the site of the K2 domain linkage to the L-chain (del 92-173, 275Arg -> Glu) (5) . It has been shown in vitro to possess a pronounced affinity for fibrin, and to retain the same specific activity as t-PA (6). The plasma clearance of YM866 calculated from pharmacokinetics in rats has been 7-fold slower than that of t-PA (6). We previously showed that YM866 administered by i.v. bolus injection

(intracoronary),

produced a superior thrombolytic effect to t-PA by i.v. bolus injection or infusion in a canine model of copper coil-induced coronary artery thrombosis (7). The benefit of thrombolysis is enhanced by early treat ment and the re-establishment of coronary patency (8). This requirement makes widespread intracoronary (i.c.) thrombolysis impractical, and most clinical trials of t-PA have examined thrombolysis by the i.v. route. Against this trend, Gemmil et al. reported a regime in which addi tional i.c. t-PA was given to patients after i.v. bolus injec tion of the agent failed to clear the occluded coronary artery (9), while Pitney et al. used an i.c. infusion of t-PA to allow angioplasty in patients who had previously been unsuitable for the procedure (10). In the present study, we compared the thrombolytic activity of YM866 with that of t-PA by intracoronary ad ministration in a canine model of coronary artery throm bosis. As i.c. thrombolysis is generally undertaken later in the course of thrombus formation than thrombolysis by i.v. injection, we first prepared a thrombosis model of 6

hr-old thrombi, and used this model to evaluate thrombolytic activity of YM866 by i.c. administration comparison

the in

with that of t-PA.

MATERIALS

AND METHODS

Thrombolytic agents YM866 is a recombinant tissue-type plasminogen activa tor analogue which contains a finger domain, growth fac tor domain, kringle-2 domain, and a serine protease domain, as well as a point mutation at the kringle-2-serine protease linkage site (del 92-173, 275Arg Glu) (5). Prepa rations of YM866 used in this study contained more than 98% of the single-chain form. Lyophilized preparations of YM866 and t-PA (ACTIVASE®, 50 mg/vial; Genen tech, Inc., South San Francisco, CA, USA) were dis solved in distilled water for injection and diluted with sa line. The specific activities of YM866 and t-PA as deter mined by fibrin clot lysis assay calibrated with the inter national standard (83/157) were 570,000 and 600,000 IU/mg, respectively (6). Preparation of aged thrombi and determination of throm bolytic activity Sixty adult mongrel dogs of both sexes weighing ap

respectively. Continuous monitoring of precordial leads was performed for the detection of arrhythmias. Coro nary thrombosis was induced by placing a copper coil (length, 8 mm; diameter, 2 mm) over an intracoronary wire into the left anterior descending coronary artery (LAD) distal to the first diagonal branch under fluoroscopy, as described previously (11). The presence of an occlusive thrombus was confirmed angiographically. The thrombus was allowed to age for 6 hr before the administration of test drug. Heparin (Novo Heparin®; Novo BioLabs, Copenhagen, Denmark) was given to all animals by i.v. bolus at 300 IU/kg before administration of a thrombolytic agent. The experimental protocol is shown in Fig. 1. Test drug (YM866 or t-PA) was ad ministered by a single injection (10 min) or multiple injec tions (4 x 10 min) into the LAD ostium through an an giography catheter using a constant-rate infusion pump (STC-523; Terumo Co., Ltd., Tokyo). Coronary angio grams were obtained to check the patency of the LAD. Animals showing no evidence of coronary reperfusion at 60 min were considered to have failed to attain reperfu sion. Three further angiograms were obtained at 20-min intervals after the confirmation of reperfusion in the sin

proximately 15 kg were used. Animals were anesthetized with 20 mg/kg sodium pentobarbital and then intubated and artificially ventilated with room air. Catheters were

gle injection group or after the 4th injection (at 60 min) in the multiple injection group. Reperfusion was defined as TIMI grade 2 or 3 and reocclusion as 0 or 1 (12). The reperfusion rate, time to reperfusion, and reocclusion rate calculated from coronary angiography served as

placed in the cephalic vein, femoral vein, and femoral artery for maintenance of anesthesia, collection of blood samples, and monitoring of blood pressure/heart rate,

parameters for the thrombolytic activity of the test drug. Lidocaine (Xylocaine®; Fujisawa-Astra Co., Ltd., Osaka) was used to prevent the development of arrhyth

Fig. 1. Experimental protocol. Six hours after the induction of an occlusive thrombus, test drug was administered by a single (10 min) or multiple injection (4 x 10 min). Coronary angiography (CAG) was performed periodically for up to 1 hr in the single injection groups or 2 hr in the multiple injection groups after the start of drug injection. Three further angiograms were obtain ed at 20-min intervals after the confirmation of reperfusion in the single injection groups or after the end of the 4th injection in the multiple injection groups.

mias during the occlusion and reperfusion

processes.

Measurement of fibrinolytic parameters Citrated blood samples were collected periodically in 1 pM PPACK (D-Phe-Pro-Arg-chloromethylketone; Cal biochem Co., Ltd., San Diego, CA, USA) (13) for meas urement of fibrinogen, plasminogen, and a2-plasmin inhi bitor. Plasma samples were preserved frozen at 701C un til assay. Fibrinogen was measured by the thrombin time method (14) (Fibrinogen B Test®; Wako Pure Chemical Co., Ltd., Osaka), and plasminogen and a2-plasmin inhi bitor were determined by the use of synthetic substrates (15) (Testzym PLG Kite and Testzym APL Kit®; Daiichi Pure Chemicals Co., Ltd., Tokyo). Measurement

of YM866 and

t-PA concentrations

in

plasma Plasma antigen levels of YM866 and t-PA were deter mined in PPACK-added plasma samples by enzyme linked immunosorbent assay (16) standardized against YM866 and t-PA, respectively. RESULTS Thrombolytic activity of YM866 and t-PA An occlusive thrombus usually developed within 10 to

Fig. 2. Thrombolytic activity with intracoronary administration of YM866 and t-PA by a single injection on 6-hr-old thrombi. Times to reperfusion are indicated as the mean±S.E.M. of animals that achieved successful reperfusion, while reperfusion and reocclusion rates are expressed as percentages in groups of 5 animals each. Open circles represent the time to reperfusion in each animal.

20 min after the insertion of the copper coil; occlusion of the artery was signaled by an elevation of the ST segment in the left precordial ECG and confirmed as complete oc clusion by coronary angiography. Figures 2 and 3 illus trate the reperfusion rate, time to reperfusion, and reoc clusion rate following i.c. administration of YM866 and t-PA in the 6-hr-old thrombi. By single injection (Fig. 2), the reperfusion rate of YM866 was 4 times higher than that of t-PA. Time to reperfusion of t-PA was shorter than that of YM866: all animals of the t-PA group attained reperfusion within 10 min after the cessation of injection, whereas many animals given YM866 did not attain reperfusion until more than 10 min after injection. The incidence of reoc clusion was zero with YM866 and low with t-PA. By multiple injection (Fig. 3), the reperfusion rate and time to reperfusion of two agents were comparable. All animals in both groups attained reperfusion, after the 2nd injection at the earliest. There was no clear difference in re occlusion rate between the agents. Changes in fibrinolytic parameters Changes in plasma levels of fibrinogen, plasminogen, and a2-plasmin inhibitor following the administration of test drugs are shown in Figs. 4 and 5. Data are plotted as percentages of the baseline. No change in plasma fibrino

Fig. 3. Thrombolytic activity with intracoronary administration of YM866 and t-PA by multiple injection on 6-hr-old thrombi. Times to reperfusion are indicated as the mean±S.E.M. of animals that achieved successful reperfusion, while reperfusion and reocclusion rates are expressed as percentages in groups of 5 animals each. Open circles represent the time to reperfusion in each animal.

Fig. 4. Changes in plasma fibrinogen, plasminogen, and a2-plasmin inhibitor levels after a single injection of YM866 (A) and t PA (B). Values are the mean±S.E.M. of 5 animals. Data are plotted as percentages of the baseline. (A), 0, 0.025; A, 0.05; /, 0.1 mg/kg; (B), •, 0.1; A, 0.2; / 0.4 mg/kg.

Fig. 5. Changes in plasma fibrinogen, plasminogen, and a2-plasmin inhibitor levels after multiple injection of YM866 (A) and t-PA (B). Values are the mean±S.E.M. of 5 animals. Data are plotted as percentages of the baseline. 0, 0.05; A, 0.1; /, 0.2 mg/kg.

Fig. 6. Plasma antigen concentrations after a single injection (A) at a dose of 0.1 mg/kg or multiple injection (B) at a dose of 0.2 mg/kg of YM866 and t-PA. Values are the mean±S.E.M. of 5 animals. •, YM866; 0, t-PA.

gen or plasminogen levels was seen in any group. a2-Plas min inhibitor levels in the plasma after single injection of YM866 at 0.1 mg/kg or t-PA at 0.4 mg/kg or multiple in jections of YM866 at 0.2 mg/kg decreased to 67%, 69% and 60% of the baseline, respectively. With the single in jection of t-PA (Fig. 4), plasma a2-plasmin inhibitor dropped rapidly and tended to reach an inverse plateau, whereas with both regimens of YM866, it declined progressively over time. Changes in plasma antigen levels of YM866 and t-PA Figure 6 illustrates the time-course of plasma YM866 and t-PA antigen levels over the 60 (single injection) and 120 (multiple injection)-min periods after the start of in jection of both agents. By single injection (Fig. 6A), plas ma YM866 antigen levels were markedly sustained com pared with that of t-PA. By multiple injection with t-PA at 0.2 mg/kg (Fig. 6B), plasma antigen increased to be tween 92±15 to 122--±:18ng/ml at the end of each injec tion, but rapidly decreased to between 30±6.1 to 36± 5.5 ng/ml by the start of the next injection, and fell rapidly at the end of the 4th injection. With YM866 at 0.2 mg/kg, however, the absence of any decrease during the discon tinuation period meant that plasma antigen increased gradually and remained at higher levels than with t-PA. DISCUSSION The benefit of thrombolysis is enhanced by early treat ment and the reestablishment of coronary patency. The ideal thrombolytic agent should therefore allow simple and rapid administration by the intravenous route, and provide effective and rapid restoration of coronary paten cy without adverse side effects (17). Theoretically, t-PA is

only activated at the site of a fibrin-bound thrombus, and should thus be as effective when given intravenously as when given intraarterially. In practice, however, a num ber of clinical trials (9, 10, 18) have shown intraarterial use to be superior, in that lysis could be attained substan tially faster and with smaller doses of thrombolytic agent. The aim of the present study was to explore the potential of i.c. thrombolysis with YM866 as a supplement to con ventional thrombolytic methods. In the previous study, the residual plasma YM866 and t-PA antigen at 10 min after i.v. bolus injection was 61 % and 20% of the peak levels, respectively (7). Therefore in this study, we ad ministered both agents by a single injection (10 min) or multiple injection (4 x 10 min). By single i.c. injection, the reperfusion rate with YM866 was 4 times higher than that with t-PA. By multi ple injection, reperfusion with YM866 was comparable to that with t-PA. We previously reported that the intra venous doses of the agents producing a 100% reperfu sion rate in the same thrombus model were 0.2 mg/kg of YM866, 0.8 mg/kg of t-PA by bolus injection, and 0.3 mg/kg of t-PA by infusion (7). Both agents were twice as potent when given by single i.c. injection than by i.v. bolus injection. By multiple i.c. injection, however, t-PA was two to three times as potent as by i.v. infusion, whereas YM866 showed only comparable potency to ad ministration by i.v. bolus injection. For both agents, time to reperfusion was faster when administered by single injection than when given by multi ple injection. By single injection, reperfusion with t-PA was seen in all animals within 10 min after the end of injec tion, whereas reperfusion with YM866 was as late as 20 min or more after injection. This finding seems to reflect the respective plasma half-lives of the agents, since both agents had the same affinity for fibrin (6) and the same resistance to plasminogen activator inhibitor-1 (PAI-1) (M. Katoh et al., unpublished data). By multiple injec tion, successful reperfusion with both agents occurred later than at the end of the 2nd injection. This indicates that the thrombolytic effect of both agents was exerted only after its plasma concentration had reached a certain level. With both drugs, the rate of reocclusion was lower af ter a single injection than after multiple injections. The oc currence of acute reocclusion in this thrombosis model is probably closely related to the extent of residual thrombi at the site of thrombus; the higher concentrations of both agents by single injection maybe credited with decreasing residual thrombi. A relationship between coronary reoc clusion and residual thrombus after thrombolytic therapy has been described clinically (19). Additional doses of a thrombolytic agent (1), or adjunctive therapy with an an tiplatelet agent such as anti-GPIIb/IIIa antibody (20) or

von Willebrand Factor inhibitor (21), or with an anti coagulant such as synthetic thrombin inhibitor (22) help to minimize residual thrombus, as well as to prevent the growth of the thrombus. a2-Plasmin inhibitor levels in the plasma decreased to within 60% to 70% of the baseline after the single injec tion of YM866 at 0.1 mg/kg or t-PA at 0.4 mg/kg or multiple injection of YM866 at 0.2 mg/kg. However, no change in plasma fibrinogen or plasminogen was detected in any group. At doses that produced a 100% reperfusion rate when YM866 was given intravenously, plasma fibrino gen and a2-plasmin inhibitor levels were decreased to ap proximately 30% and 60%, respectively (7). Local ad ministration of smaller amounts of thrombolytic agents offers higher reperfusion rates and the likelihood of a reduced incidence of systemic side effects. Concentrations of plasma antigen were sustained mark edly longer with YM866 than with t-PA. These findings are essentially consistent with the mean residence time of YM866 as calculated from its pharmacokinetics in rats (6) and dogs (I. Miyamoto et al., unpublished data), and which can probably be extrapolated to humans. By multiple injec tion, antigen levels of YM866 were maintained at 2 to 3 times those of t-PA, but the reperfusion rate with YM866 was comparable to that of t-PA. This may be a reflection of the finding that the clot lysis activity of YM866 in the plasma of dogs was about one-third that of t-PA (6). In the assay of fibrin clot lysis using purified human fibrino

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gen, in contrast, the specific activities of the agents were shown to be comparable. This discrepancy may be ex plained by a difference between the agents in their resistance to putative inhibitors other than PAI-1 of the thrombolytic system in plasma. The results of this study suggest that YM866 by single intracoronary injection produces excellent thrombolysis and prevents acute reocclusion without systemic fibrinolyt ic activation. Furthermore, this regime may provide safe and effective thrombolysis in patients in whom early i.v. thrombolysis is unsuccessful and may allow angioplasty in patients who were not previously suitable for this proce dure.

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