Thrombolytic effect of a modified plasminogen activator (SUN9216) on photochemically induced thrombosis (PIT) models in the rat femoral and middle cerebral artery

POSTER

178

THE EFFECT OF PRIMARY THROMBUS

FIBRINOLYTIC FORMATION

AGENTS

1l*Stassen JM, 1Nvstrom A, *Vanlinthout and *Hovlaerts M. ‘Department

of Hand

Sweden and *Center University of Leuven,

Surgery,

NUS,

for Molecular Belgium.

ON

I, *Moreau

University

and Vascular

H

of Umea, Biology,

The antithrombotic effect of a range of known plasminogen activators was studied in a platelet dependent thrombosis model in hamsters (Thromb. Haemostas. 65: 425, 1991). This model allows continuous in vivo monitoring of thrombus formation and embolization. The left femoral vein was exposed and mounted on a transilluminator, a standardised vessel wall trauma was applied and thrombus formation was continuously monitored over 40 min by image analysis. Thrombus accumulation was quantified from the change with time of the light transmission through the vessel at the trauma location. To test the effect of recombinant tissue-type plasminogen activator (rt-PA), recombinant single-chain urokinase-type plasmminogen

179 THROMBOLYTIC

MODIFIED EFFECT OF A ACTIVATOR (SUN9216) ON PLASMINOGEN PHOTOCHEMICALLY INDUCED THROMBOSIS (PIT) MODELS IN THE RAT FEMORAL AND MIDDLE CEREBRAL ARTERY Matsuno H. Uematsu T. Takiauchi Y. Nakashima M Department of Pharmacology, Hamamatsu University School of Medicine, Hamamatsu, Japan

The thrombolytic effect of SUN9216, which is a novel modified t-PA with deletion of the finger, the EFG domain and the mannose-rich carbohydrate chain on the kringle 1 of the wild-type t-PA, were investigated using photochemically induced thrombosis (PIT) models in the rat femoral artery (Matsuno et al., Br. J. Pharmacol., 1992) and middle cerebral artery (MCA). When SUN9216 was administered either as an i.v. infusion (1.0 mg/kg) for 30 min or as a single bolus injection (1.0 mg/kg), arterial patency after reperfusion was markedly improved compared to t-PA administered as an i.v. infusion for 30 min (3.0 mg/kg). In this case, a higher concentration of plasminogen activator activity in plasma was observed

180 PROLONGED PRESENCE OF DSPAal ANTIGEN IN PLASMA PARALLELS LONG-LASTING FIBRINOLYTIC ACTIVITY IN RATS AND MONKEYS. P Verhallen. AS. Bhargava. P Brinamann. M.Hildeb rand. Research Laboratories, Schering AG, Berlin, Germany. DSPAot (Desmodus Salivary Plasminogen Activator al) is a novel thrombolytic protein with extreme fibrinspecificity. During its pharmacokinetic evaluation in rats and monkeys, the relationship between antigen- and functional-levels of DSPAof was studied in detail. Antigen was determined by a sandwich ELISA technique, employing immunopurified anti-DSPAol-IgG rabbit antiserum. Enzymatic activity of DSPAot was measured spectra-photometrically as lysis-rate of preformed human fibrin clots containing human plasminogen and expressed in DSPAot concentration by reference to a standard curve. Plasma level time-profiles of DSPAol were obtained in rats by sampling a series of time-points up to 180 min after i.v. bolus administration of either 3, 10 or 30 mg/kg DSPAot. Individual antigen levels measured at various

PRESENTATIONS

P-6: Experimental

Animal

Studies

65

chain urokinase-type two (rscu-PA), activator plasminogen activator (tcu-PA), a t-PA mutant lacking amino acids 6 to 173 and mutation of Nl84E (K2-Pt) and a recombinant t-PA/u-PA chimera with amino acids 1 to 3 and 87 to 274 of rt-PA and amino acids 138 to 411 of rscu-PA (KlK2-Pu), 60 hamsters were assigned to 3 dose-groups with 4 animals each in comparison to a control group of 10 animals. A single IV bolus 30 s before infliction of the trauma resulted in 50% inhibition of formed thrombus for doses of 8; 8; 1; 3 and 0.01 mg/kg of rt-PA, rscu-PA, tcu-PA, K2-Pt and KlK2-Pu respectively. These results indicate that rt-PA, rscu-PA, tcu-PA and K2-Pt have a lower antithrombotic effect, in comparison with KlK2-Pu. This can not be explained by its prolonged halflife alone, since KlK2-PU and K2-Pt had comparable halflifes around 20 min. Although these results may overestimate the observed effect of KiK2-PU because of high sensitivity of the hamsters and therefore need to be studied in an additional animal species, it is confirmed that the U-PA proteolytic domain exerts a higher in vivo antithrombotic potency than that of t-PA.

after the administration of SUN9216 which persisted for longer than that of after t-PA. Furthermore we studied the inhibiton of thrombotic cerebral ischemia using this drug in a recently described model (Matsuno et al., J. Pharmacol. Methods, 1993). A single bolus injection of SUN9216, at 30 min after thrombus formation in MCA, also significantly reduced the ischemic area as compared to the untreated animals 24 hrs after induction of thrombus. It is concluded that SUN9216 is an effective thrombolytic agent when administered as a single i.v. bolus injection and its thrombolytic potency is also significantly higher than t-PA when given as an i.v. infusion. Additionally the treatment with this drug in thrombotic cerebral ischemia reduced the area of cerebral infarction. Author’s Vascular

present address: Biology, University

time-points,

Center for Molecular of Leuven, Belgium

and

ranged from 125 ng/ml to 515 pg/ml, while ranged from 55 ng/ml to 537 pg/ml. Individual DSPAot antigen and activity levels were strongly correlated with a ratio of 0.94kO.02 (r=0.977, n=l69). Cynomolgus monkeys were treated with either 3, 10 or 30 mg/kg by i.v. bolus administration, which was repeated after 90 min. For plasma level monitoring of DSPAot, blood samples were obtained at various time points up to 270 min. Altogether, individual antigen levels varied from 557 ng/ml to 525 pg/ml, while activity levels ranged from 80 ng/ml to 719 pg/ml. Individual DSPAof antigen and activity levels correlated well with a ratio of 0.77+0.02 (r=0.956, n=l20). In summary, in two different species antigen and activitylevels of DSPAot correlated very well with a ratio of one (rats) or close to one (monkeys), indicating that DSPAot remains functionally present for up to 3 hrs after i.v. bolus adminstration activity

levels