Thrombolytic efficacy of a modified tissue-type plasminogen activator, SUN9216, in the rat middle cerebral artery thrombosis model

ejp ELSEVIER

European Journal of Pharmacology262 (1994) 27-31

Thrombolytic efficacy of a modified tissue-type plasminogen activator, SUN9216, in the rat middle cerebral artery thrombosis model Kazuo Umemura *, Yoshinori Toshima, Mitsuyoshi Nakashima Department of Pharmacology, Hamamatu Umverslty School of Me&cme, 3600 Handa-cho, Hamamatsu 431-31, Japan

Recewed 16 December 1993, revised MS received 31 May 1994, accepted 3 June 1994

Abstract

We have developed a model whereby the middle cerebral artery m an experimental ammal can be occluded by a photochemical reaction between rose bengal and green hght This causes endothelial inJUry followed by platelet adhesion, aggregation and formation of a platelet-nch thrombus at the site of the photochemical reaction SUN9216, a mo&fled tissue-type plasmlnogen actwator, Is a new thrombolytlc agent which consists of the fibrin krlngle 1 domain of plasmmogen and the two krlngles, the serlne protease domains of the natwe t~ssue-type plasmmogen actwator The mannose glycosylatlon s~te on the krmgle 1 of tissue-type plasmmogen actwator Is modified to yield a compound with a longer half-hfe m the blood than natwe tissue-type plasmmogen actwator We evaluated the thrombolytlc effects of recombinant tissue-type plasmlnogen actwator and SUN9216 in the thrombotlcally occluded rat middle cerebral artery SUN9216 was administered by contmuous infusion or as a single bolus rejection 30 mm after the middle cerebral artery had been occluded by a thrombus Both SUN9216 and recombinant tissue-type plasmmogen actwator caused reopening of the middle cerebral artery by thrombolysls The efficacy of SUN9216 was higher than that of recombinant tissue-type plasmlnogen actwator Further, the area of lschaemlc cerebral damage caused by the middle cerebral artery occlusion was significantly (P < 0 05) reduced by SUN9216, but in this respect, recombinant tissue-type plasmmogen actwator was lneffectwe Key words Photochemlcally reduced thrombosis; T~ssue-type plasmmogen activator, Middle cerebral artery thrombosis, SUN9216, (Rat)

1. Introduction

Anglographic studies of lschaemic cerebral infarction suggest that acute thrombus formation and subsequent arterial occlusion are the critical pathological events in more than 70% of strokes (Fieschi et al., 1989). The potential for the treatment of acute cerebral artery thrombosis with thrombolytic agents has generated considerable interest (Levine and Brott, 1992; Del Z o p p o et al., 1986; Sloan, 1987). However, the use of thrombolytic agents m acute thrombotic stroke may predispose the patient to haemorrhage (Fletcher et al., 1976). It has been reported that intracerebral haemorrhage is a severe but infrequent comphcation assocmted wlth the use of recombinant tissue-

* Corresponding author Tel 053-435-2271,fax 053-435-2270 0014-2999/94/$07 00 © 1994 Elsevter Science B V All rights reserved SSD! 0014-2999(94)00345-8

type plasminogen activator in acute myocardial lnfarctlon (Gore et al., 1991; O ' C o n n o r et al., 1990). Recently climcal pilot stu&es on recombinant tissue-type plasmmogen activator in patients with acute lschaemic stroke have been performed (Brott et al., 1992, Harely et al., 1992). These stu&es have reported that treatment with high doses of recombinant tissue-type plasmmogen actwator in acute ischaemic infarction carries the risk of lntracerebral haemorrhage, especially 90 mm after the onset. Further, the rate of early neurological improvement is slow in patients treated with recombinant tissue-type plasminogen actwator (Harely et al., 1992). In our previous study (Umemura et al., 1993), in the rat middle cerebral artery thrombosis model, SUN9216, a mo&fied tissue-type plasmlnogen actwator which was Injected 30 mln after the middle cerebral artery occlusion, induced reopening of the vessel and produced a significant reduction in the size of the cerebral infarction

28

K Umemuraet al / EuropeanJournal of Pharmacology 262 (1994) 27-31

In the light of the above reports, we became interested to evaluate the effect of SUN9216 at various doses in the rat middle cerebral artery thrombosis model and to compare its efficacy wtth that of recombinant tissue-type plasminogen activator. We also wanted to verify whether or not the reduction in infarct size after thrombolytic reopening of the occluded middle cerebral artery depends on the dosage of thrombolytic agent. SUN9216 consists of the fibrin kringle 1 domain of plasminogen and the two kringles, the serine protease domains of the native tissue-type plasminogen activator. The mannose glycosylatlon site on the krlngle 1 of tissue-type plasmlnogen activator is modified to yield a compound with a longer half-life in blood than native tissue-type plasminogen activator. It has been reported that SUN9216 is cleared about 20 times slower than native tissue-type plasminogen activator and is 8.6-fold more potent as a thrombolytic agent than native tissue-type plasmlnogen activator, as determined in the rabbit after bolus injections (Larsen et al., 1991)

Xenon Lamp

, ~

/

V

Letlculostrmte branch

2. Materials and m e t h o d s Olfactory Tract

2.1. A m m a l preparanon

Wistar male rats weighing 240-260 g were used. Animal body t e m p e r a t u r e was maintained at 37.5°C with a heating-pad (K-module Model K-20, American Pharmaseal Company, USA). The middle cerebral artery thrombosis model in the rat has been described previously ( U m e m u r a et al., 1993). In brief, under pentobarbital anaesthesia and spontaneous respiration, a catheter for the administration of rose bengal or thrombolytic agents was inserted into the femoral vein. The scalp and temporalis muscle were folded over and a subtemporal craniotomy was performed by using a dental drill under an operating microscope. A 3-mmdiameter circular area of the window was illuminated with green light and the entire illuminated segment, including the proximal end of the lenticulostriate branch, became thrombotically occluded. Photo-illumination by green light (wave length, 540 nm) was achieved by using a xenon lamp (IA887: H a m a m a t s u Photonics, Hamamatsu, Japan) with a heat-absorbing filter and a green filter. The irradiation was directed by a 3-mm-diameter optic fibre mounted on a micromanlpulator. The head of the optic fibre was placed on the window In the skull base at a distance of 2 m m above the vessel, delivering an irradiation dose of 0.62 W / c m z (Fig. 1). The incisions were closed after the 90-mln observation period and a local anaesthetic was applied to the surgical wound every 3 h until the animals were killed. Twenty-four hours after the completion of the irradiation, the cerebrum was removed from anaesthetized rats by another blind investigator

Middle cerebral artery Fig 1 A schcmat=c illustration of the ammal preparation The head of the optic fibre was placed on a window m the skull base at a

&stance of 2 mm above the vessel and dehvered an lrra&atlon dose of 0 62 W/cm 2 A 3-mm-&ameter circular area of the window was dlummated and the enhre illuminated segment, including the proxareal end of the lentlculostnate branch, became thrombotlcally occluded for subsequent analysis. The cerebrum was coronally sectioned into 1-mm-thick slices from the frontal lobe with a mlcroslicer and 6 consecutive slices were stained with trlphenyltetrazohum chloride (Katayama, Japan) and photographed. For each animal, the ratio of infarction area to the whole area of the corresponding cerebrum was calculated by using a computerized image analysis system. PO 2 and PCO 2 were determined before the injection of rose bengal and the m e a n arterial blood pressure was monitored with a pressure transducer during the experiments. 2.2 Admmtstratton o f SUN9216 and recombinant nssue-type plasmmogen actwator

Thirty minutes after the thrombotic occlusion of the middle cerebral artery, various doses of SUN9216 or recombinant tissue-type plasmlnogen activator were administered as a single bolus dose in a volume of 0.5 ml or as a 30-mln continuous intravenous infusion in a volume of 1.0 ml via the femoral vein. The middle cerebral artery was examined for the presence of thrombus (or its lysxs), by using an operating micro-

K. Umemura et al / European Journal of Pharmacology 262 (1994) 27-31

scope, for 60 min after the injection of SUN9216 or recombinant tissue-type plasminogen activator. The time for reopening of the occluded middle cerebral artery by thrombolysis was noted when blood flow through the vessel could be observed through the operating microscope. Twenty-four hours after the administration of SUN9216 or recombinant tissue-type plasminogen activator, the area of ischaemlc damage was assessed by using a trlphenyltetrazohum chloride staining technique. An equal volume of saline was administered to a group of animals in the same manner as SUN9216 to serve as controls

2.3. Stattsucal analysts

Data are expressed as means _ S.E. Statistical analysis was done by using an analysis of variance or Fisher's exact test. P < 0.05 was considered significant.

29

3. Results Physiological variables after the operation were within the normal range (PO E = 88.4 + 3.5 mm Hg; PCO 2 = 40.2 ___0 6 mm Hg; p H = 7.42 + 0.02; mean arterial pressure = 112 + 4 mm Hg). The middle cerebral artery was completely occluded by thrombus about 6 min after the administration of rose bengal, as viewed with an operating microscope Neither photo-tllumlnatton nor rose bengal alone could induce thrombotic occlusion of the middle cerebral artery. In Ftg. 2, a representative photograph of a cerebral infarction stained with trlphenyltetrazolium chloride 24 h after the occlusion of the middle cerebral artery in untreated animals (controls) is shown. The left dorsolateral frontoparietal cortex and the left dorsolateral portion of the strlatum were infarcted. SUN9216, at doses of 0.3 and 1.0 m g / k g administered by continuous infusion, reopened the occluded middle cerebral artery in 7 out of 13 animals (53.8%)

Fig 2 A representatwe photograph of cerebral infarction stained with trlphenyltetrazohum chloride 24 h after the occlusion of the middle cerebral artery m control animals. Each section was cut coronally into i-ram-thick shces from the frontal lobe The left dorsolateral frontopanetal cortex and the left dorsolateral portion of the stnatum were consistently infarcted One bar represents 10 mm The order of numbers indicates the position from the frontal lobe

K Umemura et al / European Journal of Pharmacology 262 (1994) 27-31

30

Table 1 Effect of SUN9216 and recombinant tissue-type plasmlnogen actwator on the frequency of reopening of the occluded middle cerebral artery and duration of occlusion m the rat middle cerebral artery thrombosis model Dose ( m g / k g )

Number

Frequency of reopening b

10

0 / 1 0 (0%)

13 11 11 10

7/13 8/11 4/11 7/10

Duration of occluston (mm)

Sahne SUN9216 03 10 10 20

Infusion Infusion Bolus Bolus

(53 (72 (36 (70

8%) 7%) 4%) 0%)

a a a a

57 9 +- 3 3 631_+34 675_+48 517+_48

4. Discussion

Recombmant ttssue-type plasmmogen acttvator 1 0 Infusion 3 0 Infusion

10 11

1/10 (10 0%) 5/11 (45 5%) a

85 74 0 + 6 6

Data are expressed as m e a n s + S E a p < 0 05 vs animals treated with sahne Bolus SUN9216 was injected 30 mm after the middle cerebral artery occlusion Infusion SUN9216 or recombinant tissuetype plasmlnogen actwator was administered by continuous infusion over a 30-mln period, starting 30 mm after the m~ddle cerebral artery occlusion b The proporhon of animals m a group m which thrombolysls was achieved

( P < 0.05) and 8 out of 11 (72.7%) (P < 0.05), respectively, as compared with 0 out of 10 untreated animals (Table 1). At 1.0 and 2.0 mg/kg, given as bolus doses, SUN9216 could induce reopening of the occluded middle cerebral artery m 4 out of 11 animals (36.4%, P < 0 05) and 7 out of 10 ammals (70.0%, P < 0.05), respectively. There was no difference in the time re-

2O

t9 0

'"

Control

03 0 Infus,on

10 20 Bolus

quired to achieve thrombolysis between the bolus dose and the continuous infusion of SUN9216 At doses of 1 0 and 3 0 mg/kg gwen by continuous infusion, recombinant tissue-type plasminogen actwator induced reopening of the middle cerebral artery m 1 out of 10 ammals (10.0%) and 5 out of 11 animals (45.5%, P < 0 05) respectively. Further, each dose of SUN9216 significantly ( P < 005) reduced the area of ischaemic cerebral damage as compared with that of the controls but in this respect recombinant tissue-type plasminogen actwator was meffectwe (Fig. 3).

10 30 Infus,on

SUN9216 rt-PA Fig 3 Effect of SUN9216 or recombinant tissue-type plasmmogen activator on the cerebral infarction Open columns are ammals m which SUN9216 or recombinant Ussue-type plasmlnogen activator did not induce thrombolysls in the middle cerebral artery within 90 mm of the thrombotic occlusion Closed columns represent animals m which thrombolysls m the middle cerebral artery was achieved within 90 mln Units of SUN9216 and recombinant tissue-type plasmmogen actwator are m g / k g Bolus SUN9216 was injected as a single dose 30 mm after the middle cerebral artery occlusion, Infusion SUN9216 or recombinant tissue-type plasmmogen actwator was admlmstered as a continuous infusion over a 30 mln period, starting 30 mm after the middle cerebral artery occlusion *P < 005 vs ammals treated with sahne

In th~s study, we used a photochemical technique to cause thrombotic occlusion of the middle cerebral artery. After photo-illumination, the excited rose bengal molecules damage the vascular endothelium, perhaps by a photodynamlc action via generation of singlet molecular oxygen, 10 2 (Umemura et al, 1993). SUN9216 administered either as a single bolus injection or by continuous infusion induced thrombolysis in the occluded middle cerebral artery. Thrombolysls of the occluded middle cerebral artery could also be achieved with recombinant tissue-type plasmlnogen actwator. However, the efficacy of SUN9216 in the frequency (the proportion of animals in a group in which thrombolysis was achieved) of reopening of the occluded middle cerebral artery appeared to be greater than that of recombinant tissue-type plasminogen activator. There was no significant difference in the time to achieve thrombolysls between SUN9216 and recombinant tissue-type plasminogen activator when recombinant tissue-type plasminogen activator did reopen the occluded middle cerebral artery. The size of lschaemic cerebral lesions 24 h after the operation in animals in which SUN9216 induced thrombolytic reopening of the middle cerebral artery within 90 mm after occlusion was significantly reduced, an effect which was not observed w~th recombinant tissue-type plasmlnogen activator m this model. That a single bolus mjectlon of SUN9216 could reopen the occluded middle cerebral artery is attributable to its prolonged half-hfe in the blood and to its high plasminogen activating effect (Larsen et al., 1991). If these properties can be extrapolated to humans, patients with acute thrombotic stroke may greatly benefit from SUN9216 treatment. Surprisingly, the size of the ischaemic lesions was not reduced by recombinant tissue-type plasminogen activator in animals in which it did induce thrombolysls in the middle cerebral artery. At present the reason Is not clear to us. It may be that recombinant t~ssue-type plasmlnogen actwator induces only a partial reopening of the middle cerebral artery. Furthermore, recombl-

K Umemura et al/European Journal of Pharmacology 262 (1994) 27-31

nant tissue-type plasmlnogen activator could not induce reopening of small calibre vessels which are branch-arteries of middle cerebral artery or perforating arteries (personal observation). For SUN9216, there was no marked difference between its low and high doses. Thus, at high dose, only the frequency of reopening was increased; the time to induce thrombolysis after it was administered and the reduction in cerebral infarction size were identical with both low and high doses. Cheherazl et al. (1989) reported that, In a rabbit cerebral Ischaemic model, infarct size could be reduced by recombinant tissue-type plasminogen activator only when it was infused within 30 min after the cerebral artery occlusion. Brott et al. (1992) reported that for thrombolytic therapy in patients with cerebral infarction, major neurological improvement was not related to increasing doses of tissue-type plasmlnogen activator or to stroke type. These observations suggest that Infarct size depends on the duration of the middle cerebral artery occlusion and not on the dosage of the thrombolytlc agent, as long as a gwen dose does induce thrombolyt~c reopening of the occluded vessel. In our previous study (Umemura et al., 1993), a combination of SUN9216 and a thromboxane A 2 receptor antagonist, vapiprost, reduced the duration of mtddle cerebral artery occlusion, producing a decrease m the cerebral infarction size, to a greater extent than SUN9216 alone. In hne with this notion, some chnlcal studies (Gore et al., 1991; O'Connor et al., 1990; Brott et al., 1992; Harely et al., 1992) have demonstrated that a h~gh dose of recombinant t~ssuetype plasmlnogen actwator may predispose patients to haemorrhage. These findings suggest that in thrombolytic therapy for patients with acute ischaem~c stroke, a high dose of thrombolytic agent carries a high risk of lntracerebral haemorrhage but a combination of lowdose thrombolytic agent and a platelet aggregation inhibitor/vasodilator may be a more effectwe and logical approach to thrombolytlc therapy than a thrombolyhc agent alone In conclusion, the photochemically induced thrombosis model is a simple and useful approach for evaluating thrombolyt~c agents and may be extended to other mammahan species. Using this model, we found that SUN9216, the structure of which is a mo&fication

31

of native tissue-type plasminogen activator, was more efficacious in reducing the size of cerebral infarction than recombinant tissue-type plasmlnogen activator at equipotent doses.

References Brott, T , E C Haley, J r , D E Levy, W Barsan, J Brodenck, G L Sheppard, J Spdker, G L Kongable, S Massey, R Reed and J R Marler, 1992, Urgent therapy for stroke Part I Pdot study of tissue plasmmogen activator administered within 90 minutes, Stroke 23, 632 Chehrazl, B B , J A Selbert, P Klssel, L Hem and J M Brock, 1989, Evaluation of recombmant tissue plasmmogen actwator m embohc stroke, Neurosurgery 24, 355 Del Zoppo, G J , H Zeumer and L A Harker, 1986, Thrombolytlc therapy m stroke posslbdltles and hazards, Stroke 17, 595 Fleschx, C, C Argentmo, G L Lenzl, M L Sacchettl, D Toni and L Bozzao, 1989, Chmcal and instrumental evaluation of paUents with lschemlc stroke within the first SLXhours, J Neurol So 91, 311 Fletcher, A P , N Alkjaerslg and M Lewis, 1976, A pdot study of urokmase therapy m cerebral infarction, Stroke 7, 135 Gore, J M , M Sloan, T R Price, A M Y Randall, E Bovdl, D Collen, S Forman, G L Knatterud, G Sopko, M L Terrm and TIMI Investigators, 1991, Intracerebral hemorrhage, cerebral infarction, and subdural hematoma after acute myocardial infarction and thrombolytlc therapy in the thrombolysls in myocardial infarction study thrombolysls m myocardial lnfarctton phase II, pdot and cllmcal trial, Orculatlon 83, 448 Harely, Jr, E C , D E Levy, T Brott, G L Sheppard, M C W Wong, G L Kongable, J C Torner and J R Marler, 1992, Urgent therapy for stroke Part II Pdot of t~ssue plasmmogen activator administered 91-180 minutes from onset, Stroke 23, 641 Larsen, G R , G A Tlmony, P G Horgan, H K Barone, K S Henson, L B Angus and J B Stoudemlre, 1991, Protein engineering of novel plasmlnogen actwators with increased thrombolytlc potency m rabbits relatwe to actwase, J Blol Chem 266, 8156 Levme, S R and T G Brott, 1992, Thrombolytlc therapy m cerebrovascular disorders, Prog Cardlovasc Dis 34, 235 O'Connor, C M , R M Cahff, E W Massey, D B Mark, D J Kerelakes, R J Candela, C Abbott-Smith, B George, R S Stack, L Aronson, S Mantell and E J Topoi, 1990, Stroke and acute myocardial infarction in the thrombolytlc era chmcal correlates and long-term prognosis, J Am Coil Cardlol 16, 533 Sloan, M A , 1987, Thrombolysls and stroke past and future, Arch Neurol 44, 748 Umemura, K, K Wada, T Uematsu and M Nakashlma, 1993, Evaluation of the combination of a tissue-type plasmlnogen activator, SUN9216, and a thromboxane A 2 receptor antagomlst, vaplprost, m a rat middle cerebral artery thrombosis model, Stroke 24, 1077