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3 Plasmin-Forming Activity and Its Enhancement by Fibrin, Fibrinogen and FDPs

3 Plasmin-Forming Activity and Its Enhancement by Fibrin, Fibrinogen and FDPs

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Reteplme, a recombinant plmminogen activator



195



100



80



g



.g

6D

.2



e

-'g 40

d



20



0



0



20



10



PAI-1 Wlml]



Figure 22. Inhibition of reteplase and rt-PA by PAI- 1 (12). 5 ng of the respective single- and

two-chain forms of reteplase and rt-PA were incubated with increasing amounts of PAI- 1.

The relative activity was calculated as (activity in the presence of PAI-1 divided by activity

in the absence of PAI-1) x 100. (The closed squares = reteplase [single-chain form]; closed

triangles = rt-PA; open squares = reteplase [two-chain form]).



5.5



Affinity for fibrin and lysine



The comparison of the fibrin binding of reteplase and alteplase reveals

significant differences. Alteplase binds completely to a fibrin clot, whereas

65-70% of reteplase is found in the supernatant of the clot. This was

confrmed in a clot-binding assay in whch 200 ng each of reteplase and

alteplase were mixed in increasing concentrations of fibrinogen, to whch

thrombin was added to induce clot formation. As shown in Figure 23,

alteplase almost completely binds to a fibrin clot formed from approximately

100 pg of fibrinogen, whereas less than 40% of reteplase is bound at levels of

100 and 150 pg (25).



196



Dr Michael Waller and Dr Ulrich Kohnert



Figure 23. Comparison of the fibrin binding of 200 ng of alteplase and reteplase. The

plasminogen activators were mixed with increasing concentrations of fibrinogen. The

formation of the clot was induced by the addition of thrombin. The amount of bound

plasminogen activators was calculated as the difference between the plasminogen activator

added and the activator in the supernatant of the clot after centrifugation. Reteplase and

alteplase were determined by an ELISA using polyclonal anti-t-PA-antibodies and a

standard curve for each plasminogen activator (25).



The non-specific bindmg of reteplase to fibrin in thls in vitro model is

almost completely suppressed by 0.3 mM E-aminocaproic acid (EACA) (a

lysine analogue) whereas rt-PA has a residual affinity of 55% (12). These

results arise from the different structures of t-PA and reteplase. Besides the

binding of t-PA via the htgh-affinity fibrin binding site on the finger domain,

the in vitro model also allows binding via the non-specific lysine bindmg site

on kringle-2. The lysine binding site is maintained in reteplase. Reteplase and

rt-PA possess the same affinity for lysine and lysine analogues (12).



5.6



Expression of reteplase in E. coli



A complementary DNA library from a Bowes melanoma cell line was

screened with a mixture of three oligodeoxynucleotides which were designed

on the basis of the published t-PA sequence (26). A full-length t-PA

complementary DNA clone was reconstituted from several overlapping

clones. The coding sequence for the finger, EGF and knngle domains of the tPA (nucleotides 199-714) was removed according to the intron-exon

organization of the t-PA gene (31). The coding sequence of reteplase was

introduced into the vector plasmid pKK223-3 as previously described (32).

The resulting plasmid pA27 fd was introduced into E. coli K12 C600+ by

transformation (33). The production level was improved by cotransformation

with the puBS520 plasmid containing the DNA Y gene (34).



Reteplase, a recombinant plasminogen activator



5.7



197



Preparation of active reteplase from inclusion bodies



The production of reteplase in E. colz leads to the formation of inclusion

bodies (32,35). Lysozyme was added to the E. coli, and the cells were lysed

by high-pressure dispersion (LAB 60, APV-Gaulin, Lubeck, Germany). The

inclusion bodies were isolated by centrifugation (20000 g, 1 hour, 4°C) and

solubilized with dithioerythritol and guanidium hydrochloride (36). The tho1

groups were derivatized with glutathone by incubating the solubilized protein

in 0.05 M Tris-HC1, pH 9.3, 6 M guanidine, 0.1 M GSSG (3.5 hours, 25OC).

Refolding of the reteplase molecules was acheved by adding 300 ml of

mixed disulphde solution to 10 litres of refolding buffer [0.7 M LargninelHC1, pH 8.6, 2 mM GSH, 1 mh4 EDTA] in three portions at 24-hour

intervals (3 5,37).



5.8



Purification of reteplase



After refolding, the reteplase preparation was cleared by filtration and

purified by affinity chromatography on Etythrina trypsin inhlbitor @TI) Sepharose and by cation-exchange chromatography (38,39).



5.9

-



-



Checking methods during manufacture



The identity of reteplase was determined by SDS-PAGE and Western blot

analyses.

The activity of the finished product was determined by the clot lysis test.

The activity of reteplase in biologcal fluids was determined by the

plasmin-forming assay (40).



6.



PRECLINICAL PHARMACOLOGICAL PROFILE

OF RETEPLASE



6.1



Clot lysis in vitro: static model



In a static model of in vitro clot lysis, reteplase exhibited a lower

thrombolytic potency than rt-PA and melanoma t-PA (13). In an assay in

whch human platelet-poor plasma (PPP), platelet-rich plasma (PRP) and

whole blood clots were incubated in human citrate plasma, reteplase required

a 6.4-fold hgher molar concentration than rt-PA to acheve 50% lysis of PPP

clot at 4 hours (13). Despite the lower potency, reteplase showed the same

maximal efficacy as rt-PA in lysing PPP clots, but was less effective in the



Dr Michael Waller and Dr Ulrich Kohnert



198



case of PRP and whole blood clots (13). Paradoxically, t h s effect may confer

clinical benefits since old thrombi tend to contain a higher concentration of

platelets than new ones. If reteplase has reduced thrombolytic efficacy

towards old thrombi, it may selectively spare older clots that seal small-vessel

wall injuries (30), reducing the risk of intracranial bleeding.



6.2



Dynamic plasma clot lysis model



The activities of reteplase and alteplase were compared in the dynamic

plasma clot lysis model (Figure 24 (41)).



Hose

Clip



Figure 24. Schematic description of the dynamic plasma model. In order to avoid the shear

stress-induced coagulation of the plasma, the pressure was provided by a butfer

compartment filled with 0.01 M Tris/HCl, pH 7.4,0.01% Tween 80.The mixture of buffer

and the plasma above the clot was avoided by the installation of a bubble trap.



Reteplase, a recombinant plasminogen activator



199



In t h s assay, the plasminogen activator is pressed into the clot by a

peristaltic pump, mimicking the in vivo situation more closely than the static

model. At low concentrations, both reteplase and alteplase have a similar

activity. At htgh concentrations, as achieved during the treatment of AMI,

reteplase is more potent than alteplase (Figure 25 (25)).



Figure 25. Comparison of the plasma clot lysis activity of alteplase and reteplase in the

dynamic plasma model. Increasing concentrations of both plasminogen activators were

added to 1 ml plasma on top of a preformed clot. All values are the mean f S.D. of five

experiments.



6.3



Clot penetration studies in vitro



The ability of reteplase and alteplase to penetrate into plasma clots was

analysed by adding the i h b i t e d plasminogen activators to the surface of the

clot and determining their location by immunostaining before and after

washmg the clot. These experiments demonstrated that alteplase was tightly

bound to the fibrin matrix and accumulated at the surface of the clot. As a

consequence, activation of plasminogen and the subsequent degradation of the

fibrin matrix is supposed to occur from the surface to the interior of the clot,

depending on the permanent supply of plasminogen from the plasma. In

contrast, reteplase penetrated into the clot due to its lack of fibrin binding,

whch in turn allowed the activation of plasminogen inside the clot (25). The

ability of plasma clot penetration might be a prerequisite for the achevement

of a htgh in vivo efficacy of fibrin-specific thrombolytic agents, especially

when applied by bolus injection.



Dr Michael WalIer and Dr Ulrich Kohnert



200



6.4



Clot lysis in vivo



In contrast to the static in vitro clot lysis model, in a rabbit jugular vein

model, reteplase was 5.3 times more effective than rt-PA in lysing venous

thrombi following intravenous bolus injection (13). This efficacy benefit may

have been due to slower plasma clearance of reteplase: pharmacokinetic

analysis revealed a much longer half-life for reteplase (18.9 k 1.5 minutes)

compared with rt-PA (2.1 k 0.1 minutes), and a 4.3-fold lower clearance rate,

following the same bolus dose (13). The improved clot lysis in vivo may also

reflect the lower fibrin binding of reteplase compared with rt-PA, discussed

above, whch may allow better clot penetration, particularly following a bolus

dose whch produces high peak plasma levels (25).



6.5



Canine model of coronary artery thrombosis



Reteplase provided faster reperfusion than rt-PA and all other

thrombolytics in a canine model of coronary artery thrombosis (42). Of six

dogs treated with an intravenous bolus injection of reteplase (140 kU/kg (0.24

mg/kg)) four achieved reperfusion at 18.3 6 minutes, compared with a

mean reperfusion time of 76.5 16.1 minutes in four out of six dogs treated

with rt-PA; 1.33 mgkg as an initial bolus, then by infusion (0.66 mgkg over

1 hour and 0.53 mg/kg over 2 hours) (p < 0.05 vs reteplase). Residual

fibrinogen was comparable in dogs treated with reteplase and rt-PA, as was

bleeding time measured at 90 minutes. These results demonstrate that, in the

animal model, reteplase fulfils its developmental goals by providing rapid

reperfusion in a hgh proportion of subjects without inducing a systemic lytic

state (42).

A double bolus of reteplase (140 kUkg + 140 kUkg, 45 minutes apart) in

another canine study (13) sigtllficantly prolonged the duration of arterial

patency, and this effect was further enhanced by co-administration of platelet

and thrombin irhbitors (43). These results suggest that double bolus dosing

optimizes the clinical performance of reteplase.



*



6.6



*



Animal pharmacokinetics



The goal of prolonging the half-life of reteplase relative to rt-PA has been

achieved. Following a single intravenous bolus dose of 200 kU/kg in different

mammals, reteplase exhlbits a half-life of between 7.2 k 0.5 minutes (nonhuman primate) and 15.4 k 2.6 minutes (rabbit), which is up to 10-fold longer

than rt-PA. Plasma clearance of reteplase is significantly lower than that of rt-



Reteplase, a recombinant plasminogen activator



20 1



PA in all species (44,45), presumably as a result of the lack of carbohydrate

side chains and deletion of the krtngle- 1 and EGF domains.



6.7



Toxicology



Reteplase exhibits low potential for acute and long-term toxicity,

demonstrated by studies in rabbits, rats and dogs (13). In rats, the minimum

lethal dose exceeds 8400 kU/kg. Following dosing in dogs for 14 days, there

was a dose-dependent reduction of fibrinogen, plasminogen and Qantiplasmin 2 hours post-injection. Mutagenicity, assessed in Salmonella

typhimurium and rat bone marrow erythrocytes, revealed no mutagenic

activity (13).



7.



CLINICAL PHARMACOLOGY



Reteplase efibits a highly favourable pharmacologtcal profile in humans,

consistent with its developmental goals of a prolonged half-life compared with

rt-PA, low b l d n g risk, and low potential for antigenicity.



7.1



Pharmacokinetics



7.1.1



Healthy subjects



In healthy volunteers, the area under the activity concentration-time curve

(AUC) for reteplase increased in a linear and dose-dependent fashion.

Following a single bolus dose of 5.5 U reteplase, the total plasma clearance

was 306 k 40 ml/minute and plasma half-life was 14.4 f 1.1 minutes (46). In

a second study, following a single bolus dose of 6 U of reteplase injected over

2 minutes, the half-life of reteplase activity (determined with a

plasminogenolytic assay) was 11.2

0.4 minutes and that of antigen

(assessed using an enzyme-linked immunosorbent assay [ELISA]) was 13.9 f

0.7 minutes, followed by a terminal half-life for antigen of 173 f 33 minutes.

Plasma clearance was 371 f 13 ml/minute for activity and 183 5 15

ml/minute for antigen. Compared with previously published values for rt-PA

(47), the activity half-life for reteplase was 3.3-fold higher and the clearance

3.3-fold lower.



+



Dr M, -hael Waller and Dr Ulrich Kohnert



202



7.1.2



Following AMI



Reteplase exhlbits similar pharmacokinetics in patients with AMI.

Following ahnistration of a single bolus dose of 10 U or 15 U reteplase, the

plasma half-life, assessed using ELISA, was approximately 19 minutes (48).

Thls is almost four times as long as the half-life reported for rt-PA in AMI

patients, measured after a 50 mg single bolus dose (49).



7.2



Haemostatic effects



7.2.1



Healthy subjects



Reteplase has modest effects on haemostatic variables, indicating a low

potential for causing bleeding (46,50). Intravenous bolus doses of reteplase

ranging from 0.11 U to 5.5 U in 18 subjects had no effect on plasma

fibrinogen, and reduced plasminogen levels only at higher doses. Fibrin Ddimers and az-antiplasmin were reduced in a dosedependent fashion (46). In

a separate randomized, singleblind, placebo-controlled, crossover study,

seven healthy volunteers received placebo or 6 U reteplase as a bolus injection

over 2 minutes (50). Fibrinogen levels remained unchanged and plasminogen

and az-antiplasmin levels fell to 83 f 1 % and 64 f 3%, respectively, of their

baseline levels, Reteplase was well-tolerated in both studes and no antibodes

were detected up to 1 year after dosing.



7.2.2



Following AMI



Transient and dosedependent reductions in fibrinogen, plasminogen and

%-antiplasmin have been observed in patients following AMI.

Two hours after a single bolus dose of reteplase, fibrinogen, plasminogen

and %-antiplasmin levels fell to 60%, 55% and 30% of their respective

baseline values following a 10 U dose, and to 44%, 41% and 26%,

respectively, following a 15 U dose (51). After a double bolus of 10 + 5 U

reteplase, given 30 minutes apart, levels of the same three variables fell to

45%, 42% and 19%, respectively, of their baseline values (52).



Reteplase, a recombinant plasminogen activator



8.



203



CLINICAL DATA



Clinical trials with reteplase, summarized in Tables 21 and 22, have

confrmed the profile of reteplase suggested by biochemical, pharmacologcal

and preclinical studies.

Table 21. Dose-fmding clinical trials using reteplase in patients with AMI

GRECO (51)

GRECO-DB (52)

MF4292 (53)

Location

Germany

Germany

Germany

Open-label

Study design

Open-label

Randomized, openlabel

AMI

AMI

Population

AMI

Dose strength and

10 U RP ( ~ 4 2 )

10 + 5 U RP (n=52)

15 U RP (n=9)

15 U RP (n=100)

form

10 + 5 U RP (n=S)

10 + 10 U RP (n=S)

Double bolus

Single bolus

Single bolus

Frequency

duration

Double bolus

Primary

90-minute patency

90-minute patency

90-minute patency

evaluation

and TIMI 3 rates

and TIMl3 rates

and TIMI 3 rates,

pharmacokinetics,

haemostasis

parameters

Key entry criteria

ST-segment

ST-segment

ST-segment

elevation

elevation

elevation

Onset of ischaemic

Onset of ischaemic

Onset of ischaemic

pain within 6 hours

pain within 6 hours

pain within 6 hours

IS-75 years old

18-75 years old

IS-75 years old

GRECO = German Recombinant Plasminogen Activator Study; GRECO-DB = German

Recombinant Plasminogen Activator Double Bolus Study; RP = Reteplase



The trials demonstrate that reteplase can achieve rapid reperfusion of

occluded coronary arteries in a hgh proportion of patients, whtle maintaining

a favourable safety profile. Through dose-finding studies, the optimal dosing

r q m e n has been identified as a double-bolus dose of 10 + 10 U reteplase,

administered 30 minutes apart. The TIMI flow grades, used in most trials of

thrombolytics, are summarized (Table 23).



204



Dr Michael WalIer and Dr Ulrich Kohnert



Table 22. Comparative studies using reteplase in patients with AMI

RAPID 1

RAPID 2 (55)

INJECT (56)

GUSTO III (57)

(phase II) (54)

USA, Canada,

USA, Germany, USA, Germany UK, Germany,

Location

Poland,

UK, Austria

Europe,

Sweden,

Australia, New

Zealand, South

Hungary,

Finland, Spain, Africa,

Lithuania,

Argentina

Austria

Randomized,

Randomized,

Randomized,

Study design

Randomized,

double-blind

Open-label

Open-label

Open-label

(angiograms

(angiograms

read blinded)

read blinded)

Population

AMI

AMI

AM7

AMI

Dose strength

15URP

l O + l0URP

l o + lOURP*

lo+ 1ouRP

and form

(~146)

(n=169)

(n=3004)

(n= 1505 9)

10+5URP

100 mg AP

1.5 MU SK*

(n= 152 )

(accelerated

(n=3006)

dose) (n=155)

10+ l 0 U R P

(n= 154)

100 mg AP

(standard dose)

(n=l54)

Double bolus

Double bolus

Double bolus

Frequency

Single bolus

Bolus + 1.5I-hour infusion

duration

Double bolus

hour infusion

Double bolus

Bolus + 3-hOI.U

infusion

35-day

30-day

90-minute

90-minute

Primary

mortality

mortality

patency and

evaluation

patency and

TIMI3

TIMI3

Within 6 hours

ST-segment

ST-segment

ST-segment

Key entry

elevation or

of symptom

elevation or

criteria

elevation

onset, STbundle branch

bundle branch

Onset of

block

block

segment

ischaemic pain

Onset of

elevation or

Onset of

within 6 hours

bundle branch

ischaemic pain

18-75 years old ischaemic pain

within 12 hours within 12 hours block

2 18 years old

2 18 years old

2 18 years old

*Seventy-four patients were randomized but were not treated. Overall, 2965 reteplase

patients and 2971 streptokinase patients received treatment. AP = alteplase; DB = doubleblind; GUSTO III = Global Use of Strategies to Open Occluded Coronary Arteries; INJECT

= International Joint Efficacy Comparison of Thrombolytics; RAPID I = Reteplase

Angiographic Phase II International Dose-fmding Study; RAPID 2 = Reteplase versus

Alteplase Patency Investigation During Acute Myocardial Infarction Study; RP = reteplase;

SK = streptokinase.



Reteplme, a recombinant plasminogen activator



205



Table 23. TIMI flow grades ( 5 8 )

Angiographic features of coronary artery flow

No penetration of contrast beyond the point of obstruction

0

Contrast penetrates the point of obstruction but does not completely opaclfy

1

the entire distal vessel

Complete contrast opacification of the infarct-related artery but neither

2

contrast opacifkation nor washout is delayed

3

Brisk, ‘normal’ flow



TIMI grade



8.1



GRECO study



The GRECO study (5 1) demonstrated that a single bolus dose of reteplase

acheves rapid reperfusion and hgh rates of early patency.

Forty-two patients with AMI received 5000 KJ heparin and a single bolus

of reteplase, either 10 U or 15 MU withn 6 hours of symptom onset. The

group receiving the 10 U dose reached the lower preset efficacy limit (90minute patency of 70%) and in accordance with the study protocol, the hgher

dose of 15 U was given to a further 100 patients. All patients also received

oral aspirin.

At 30, 60 and 90 minutes after injection of 10 U reteplase, angography

revealed TIMI grade 2 or 3 patency in 65%, 73% and 66%, respectively. In

the 15 MU group, these values were 66%, 74% and 75%, respectively. Very

early reocclusion (prior to 90 minutes) occurred in 5 of 30 (17%) patients in

the 10 U group, and 10 of 78 (13%) of those gven 15 MU reteplase.

Frequency of bleeding complications was as would be expected for standard

thrombolytic therapy.



8.2



GRECO-DB



A second dose-finding study, GRECO-DB (52), demonstrated that a

double bolus dose of reteplase is well tolerated and may offer efficacy

advantages over a single bolus r g m e n by extending the period of high

plasma levels of reteplase.

Fifty-one patients with AMI received 10 U of reteplase as a bolus dose

w i h n 6 hours of symptom onset, and a further 5 U bolus 30 minutes later.

Angography after 30, 60 and 90 minutes demonstrated TIMI grade 2 or 3

flow in 50%, 72% and 78% of patients, respectively. Early reocclusion (prior

to 90 minutes) occurred in 10% of patients, and reocclusion between 90

minutes and 24 hours occurred in 2% (1 patient). Bleeding complications

were no hgher than for standard thrombolysis. Thls study showed that the

double-bolus regimen reduces but does not completely eliminate incomplete

initial lysis or reocclusion. A subsequent study of 24 patients (53) showed



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