Experimental Neurology 185 (2004) 154 – 159

[Link]/locate/yexnr

Comparison of Tenecteplase with Alteplase on clinical rating scores

following small clot embolic strokes in rabbits
Paul A. Lapchak, a,b,c,* Dalia M. Araujo, b and Justin A. Zivin a,b,c
a
Department of Neuroscience, University of California San Diego, La Jolla, CA 92093-0624, USA
b
VASDHS, San Diego, CA 92161, USA
c
Veterans Medical Research Foundation, San Diego, CA 92161, USA

Received 5 August 2003; revised 9 September 2003; accepted 16 September 2003

Abstract

Tenecteplase (TNK) was engineered to have increased fibrin specificity and an increased half-life compared to Alteplase. Although
Tenecteplase is currently being tested in a Phase II clinical trial in acute ischemic stroke patients, little is known about the pharmacology and
dose – response or therapeutic window for Tenecteplase in embolic stroke models. In the present study, we compared Tenecteplase with
Alteplase on behavioral outcome in rabbits with embolic strokes. Male New Zealand white rabbits were embolized by injecting a suspension
of small blood clots into the middle cerebral artery (MCA) via a catheter. The rabbit small clot embolic stroke model (RSCEM) was used for
a dose – response profile analysis of Tenecteplase (0.1 mg/kg – 3.3 mg/kg) and Alteplase (0.9 mg/kg – 3.3 mg/kg) given intravenously 1 h
following embolization. In additional studies, Tenecteplase (0.9 mg/kg) or Alteplase (3.3 mg/kg) was administered 3 (or 6) h following
embolization to determine the therapeutic window for the thrombolytics. For both studies, behavioral analysis was conducted 24 h following
embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50%
of the rabbits. Using the RSCEM, a drug is considered beneficial if it significantly increases the P50 compared with the control group. The P50
of controls 24 h after embolization was 1.13 F 0.15 mg. Rabbits treated 1 h post-embolization with Tenecteplase (0.1, 0.25, 0.9, 1.5 or 3.3
mg/kg) had P50 values of 1.48 F 0.33, 2.20 F 0.44, 2.76 F 0.37, 2.15 F 0.29 and 2.78 F 0.31 mg, respectively. In Alteplase-treated rabbits,
only the 3.3 mg/kg dose significantly increased the group P50 by 189% compared to control. Tenecteplase was also effective at increasing the
P50 value to 2.21 F 0.43 mg if there was a 3-h delay following embolization, but not if there was a 6-h delay before administration. Alteplase
was only effective if administered 1 h following embolization where it significantly increased the P50 value to 3.27 F 0.40 mg. This study
indicates that Tenecteplase has a wide therapeutic range, a therapeutic window of at least 3 h and a durable effect. Moreover, the safety profile
for Tenecteplase is similar to that of Alteplase. Tenecteplase does not increase the rate of intracerebral hemorrhage (ICH) above that produced
by Alteplase. However, the therapeutic range and window for Alteplase is more limited than that for Tenecteplase. Our preclinical studies
suggest that Tenecteplase has a better pharmacological profile than Alteplase and supports further investigation of Tenecteplase in
randomized double-blinded clinical trials in stroke patients.
D 2003 Elsevier Inc. All rights reserved.

Keywords: TNK; tPA; Reperfusion; Embolism; Neuroprotection

Introduction (Smalling, 1996). By virtue of mutations at amino acids 103

and 117, Tenecteplase has a reduced clearance rate and is
Tenecteplase (TNK) is a second-generation genetically 80-fold more resistant to PA inhibitor-1 (PA-I-1) because of
modified form of wild-type Alteplase (Davydov and Cheng, the substitution of alanine residues at amino acid positions
2001; Lapchak, 2002a) produced by multiple point muta- 296 through 299, compared to Alteplase. Tenecteplase has
tions designed to prolong the biological half-life from 4 min been studied extensively in clinical trials for the treatment of
(Alteplase) to 18 min and to increase its specificity for fibrin acute myocardial infarction (AMI) (Angeja et al., 2001;
Cannon et al., 1998; Davydov and Cheng, 2001; Keyt et al.,
1994). A recent systematic review of AMI thrombolytic
* Corresponding author. Department of Neuroscience, University of
California San Diego, MTF 316, 9500 Gilman Drive, La Jolla, CA 92093-
trials and meta-analysis of AMI clinical trial results con-
0624. Fax: +1-858-822-4106. cluded that the efficacy of Tenecteplase was similar to all
E-mail address: plapchak@[Link] (P.A. Lapchak). other thrombolytics that have been tested, which included

0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/[Link].2003.09.009
 P.A. Lapchak et al. / Experimental Neurology 185 (2004) 154–159 155

Reteplase, Alteplase and Streptokinase (Dundar et al., particles with tracer quantities of 15-Am radiolabeled micro-
2003). Moreover, the safety profile of Tenecteplase in a spheres. The specific activity of the particles was deter-
preclinical ischemia model is similar to that of Alteplase mined by removing an aliquot, after which appropriate
(Chapman et al., 2001). Although Tenecteplase has been volumes of PBS solution were added so that a predeter-
studied in detail in AMI, little is known about the potential mined weight of injection, clot particles were rapidly
usefulness of Tenecteplase for the treatment of stroke. injected through the catheter and both the syringe and
The main objective of the present study was to assess the catheter were flushed with 5 ml of normal saline.
pharmacological profile of Tenecteplase in a rabbit model of
embolic stroke that in many ways reproduces human stro- Drug administration
ke(Zivin and Waud, 1992; Lapchak et al., 2002a,b). We
directly compared the pharmacological profile of Tenecte- Tenecteplase was given 1, 3 or 6 h post-embolization as a
plase with that of Alteplase, the only FDA-approved treat- bolus injection over 1 min, whereas Alteplase was given 1
ment for acute ischemic stroke (Lapchak, 2002a; The or 3 h post-embolization as a bolus injection (20% of the
National Institute of Neurological Disorders and Stroke rt- dose over 1 min) followed by an infusion (80% of the dose)
PA Stroke Study Group, 1995). The rabbit small clot over 30 min. Genentech, Inc. (South San Francisco, CA)
embolism model (RSCEM) uses administration of small- supplied Tenecteplase and Alteplase in the same formulation
sized blood microclots resulting in random infarcts through- used clinically that was then reconstituted with sterile water.
out the brain (Lapchak and Zivin, 2003; Lapchak et al., Rabbits were observed continuously for a minimum of 2
2002a,b). The infarcts cause behavioral deficits that can be h after embolization and treatment. Neurologic function was
quantitated using a dichotomous rating scale (Lapchak and scored 24 h post-embolization by an observer who was
Zivin, 2003; Lapchak et al., 2002a,b). The present study naive to the treatments. For durability of response experi-
included a dose – response analysis, therapeutic window ments, rabbits were rated 4 days following embolization.
analysis and assessment of the durability of response. For all behavioral analyses, observers who were naive to the
treatments completed the analysis.

Methods RSCEM analyses

The procedures used in this study were approved by the For the RSCEM, a quantal dose – response data analysis
Department of Veterans Affairs and the subcommittee on technique was used, as described previously(Lapchak and
animal studies at the VA San Diego Healthcare System Zivin, 2003; Lapchak et al., 2002a,b), and reviewed in detail
(VASDHS). Throughout the study, the veterinarian and staff by Zivin and Waud (1992). A wide range of clot doses
on duty at the VASDHS closely monitored the health status resulting in normal and abnormal animals, with small
of the rabbits. numbers of microclots causing no grossly apparent neuro-
logic dysfunction and many microclots invariably caused
Animals and model encephalopathy or death. Using a simple dichotomous rating
system, with a reproducible composite result and low inter-
Male New Zealand white rabbits were anesthetized using rater variability ( < 5%), each animal was rated by a naive
halothane (5% induction, 2% maintenance by facemask), observer as either behaviorally normal or abnormal. Abnor-
the bifurcation of the right carotid artery was exposed and mal rabbits include those with one or more of the following
the external carotid was ligated just distal to the bifurcation, symptoms: ataxia, leaning, circling, lethargy, nystagmus,
where a catheter was inserted anteriorly into the common loss of balance, loss of sensation and occasionally, paraple-
carotid and secured with ligatures. The incision was closed gia. The effective stroke dose or P50 value was then
around the catheter with the distal ends left accessible calculated as the amount of microclots that produce neuro-
outside the neck; the catheter was filled with heparinized logic dysfunction (impairment) in 50% of the rabbits within
saline and plugged with an injection cap. Rabbits were a specific treatment group. For all experiments in this study,
allowed to recover from anesthesia for a minimum of 3 rabbits were randomly allocated into treatment groups
h until they awoke and behaved normally. before the embolization procedure. The randomization se-
For the RSCEM, microclots were prepared from blood quence was not revealed until all post-mortem analyses were
drawn from a donor rabbit and allowed to clot at 37jC, as complete.
described in detail previously(Lapchak and Zivin, 2003;
Lapchak et al., 2002a,b). For the RSCEM, small clots were Statistics
prepared by allowing blood drawn from a donor rabbit to
clot at 37jC, as described previously by Lapchak and Zivin For the RSCEM, a separate curve was generated for each
(2003) and Lapchak et al. (2002a,b). The ‘‘microclots’’ were treatment tested. The unpaired Student’s t test was used for
resuspended in PBS, then washed and allowed to settle, all comparisons, with the Bonferroni correction for multiple
followed by aspiration of the supernatant and spiking of the comparisons, where appropriate.
156 P.A. Lapchak et al. / Experimental Neurology 185 (2004) 154–159

Results

Dose– response relationship for Tenecteplase and Alteplase

Quantal analysis was used to determine whether Tenec-

teplase improved behavioral ratings after an embolic stroke.
The quantal response curves were constructed from the raw
data presented in Table 1. Fig. 1 shows the quantal curve for
the 0.9 mg/kg dose of Tenecteplase and for the vehicle-
treated group. When administered starting 1 h post-emboli-
zation, Tenecteplase (0.9– 3.3 mg/kg) significantly ( P <
0.05) increased the P50 by 96 – 144% compared to the
vehicle-treated controls (1.13 F 0.15 mg; n = 16), indicating
a significant effect of the drug over the dose range (Fig. 2).
No significant differences between vehicle and either the
Fig. 1. Percentage of rabbits behaviorally abnormal or dead as a function of
0.10 or 0.25 mg/kg doses of Tenecteplase were observed microclot weight (mg) injected into the carotid artery system. The dotted
(Fig. 2). Fig. 1 also shows that Alteplase significantly curve (left) shows that 50% of vehicle-treated control rabbits will be
increased behavioral ratings when given at a dose of 3.3 abnormal or dead 24 h after injection of 1.13 F 0.15 mg (P50) of clots. The
mg/kg. However, Alteplase was not effective at either the solid curve (middle) indicates that when Tenecteplase (0.9 mg/kg) is given
0.9 or 1.5 mg/kg dose (Fig. 2). 1 h post-embolization, the P50 is significantly increased to 2.76 F 0.37 mg
( P < 0.05). The dashed curve (right) shows that Alteplase (3.3 mg/kg)
significantly increased the P50 to 3.27 F 0.40 mg ( P < 0.05).
Delayed administration of Tenecteplase and Alteplase

These experiments assessed the effects of treating embol- delayed by 1 or 3 h, there were significant increases in
ized rabbits with Tenecteplase (0.9 mg/kg) starting 1, 3 or 6 the P50 value [2.76 F 0.37 mg (n = 15) and 2.21 F 0.43 mg
h post-embolization or Alteplase (3.3 mg/kg) starting 1 or 3 (n = 23), respectively] compared to control; however, the
h post-embolization. When Tenecteplase treatment was mean at 3 h was reduced compared to the 1-h time point
(Fig. 3). Tenecteplase was not effective if given 6 h after
embolization: the P50 value for the group was 1.35 F 0.38
Table 1 mg (n = 20). Fig. 3 also shows that Alteplase (3.3 mg/kg)
Effect of Tenecteplase (0.9 mg/kg) on behavioral outcome of embolized was effective at increasing the P50 value to 3.27 F 0.40 mg
rabbits (n = 16) if administered 1 h following embolization, but not
Vehicle Tenecteplase (1 h) Tenecteplase (3 h) if there was a 3-h delay following embolization.
Clot NORM AB Clot NORM AB Clot NORM AB
dose dose dose
(mg) (mg) (mg)
0.08 1 0 0.119 1 0 0.049 1 0
0.09 1 0 0.96 1 0 0.13 2 0
0.98 1 0 1.05 1 0 0.42 1 0
1.05 1 0 1.07 1 0 0.56 1 0
1.21 0 1 1.85 1 0 0.58 1 0
1.27 0 1 2.08 1 0 0.74 2 0
1.30 0 1 2.10 0 1 1.01 1 0
1.31 0 1 2.44 1 0 1.21 1 0
1.44 0 1 2.45 1 0 1.25 1 0
1.58 1 0 2.48 1 0 1.32 1 0
1.97 0 1 2.53 0 1 1.40 1 0
2.02 0 1 3.09 1 0 1.44 0 1
2.21 0 1 3.14 0 1 1.60 0 1
2.22 0 1 4.06 0 1 1.85 0 1
2.40 0 1 4.12 0 1 2.10 1 0
4.29 0 1 2.13 0 1
2.27 0 1
2.80 1 0 Fig. 2. Dose – response analysis for the effect of Tenecteplase (0.10 – 3.3 mg/
3.00 1 0 kg) or Alteplase (0.9 – 3.3 mg/kg) administered 1 h following embolization
3.12 0 1 on behavioral outcome (P50) measured 24 h following embolism.
3.50 0 1 Tenecteplase at doses from 0.9 – 3.3 mg/kg significantly increased the P50
NORM—rabbits with normal rating scores; AB—rabbits with abnormal ( P < 0.05), but the lower doses did not ( P > 0.05). Alteplase significantly
rating scores. increased the P50 rating when administered at 3.3 mg/kg.
 P.A. Lapchak et al. / Experimental Neurology 185 (2004) 154–159 157

showing that Tenecteplase can improve clinical rating scores

or decrease behavioral deficits in an embolic stroke model
that reproduces many aspects of human stroke is lacking.
Thus, in the present study, we assessed the pharmacolog-
ical effects of Tenecteplase, a thrombolytic with a pharma-
cological profile that may be considered to be superior to that
of tPA (Davydov and Cheng, 2001; Smalling, 1996) in the
embolic stroke model that was used in the preclinical
development of tPA (Zivin et al., 1988). The results in the
RSCEM showed that Tenecteplase significantly improved
behavioral ratings scores following an embolic stroke, even
when administered within 3 h of embolization. The obser-
vation that Tenecteplase improves behavior in the RSCEM is
consistent with an earlier finding using a rat stroke model
and intrarterial injections of TNK (Zhang et al., 2000). We
also found that Tenecteplase was effective at improving
Fig. 3. Therapeutic window. The solid curve shows that Tenecteplase (0.9 behavioral deficits over a wide range of doses, whereas
mg/kg) administered 1 or 3 h post-embolization results in a significant
increase ( P < 0.05) in P50 measured in embolized rabbits. The P50
Alteplase was only effective at the highest dose tested in
measured in rabbits treated 6 h following embolization is not significantly this study. In the RSCEM, the magnitude of the improvement
different from control values ( P > 0.05). The dotted curve shows that by Tenecteplase was comparable to that of Alteplase (Fig. 1,
Alteplase was only effective ( P < 0.05) when administered 1 h following see also Lapchak and Zivin, 2003; Lapchak et al., 2002b). In
embolization. the present study, Alteplase increased the P50 value by 189%
compared to a 144% increase measured following Tenecte-
Durability of the Tenecteplase response plase treatment. Although the magnitude of the response is
similar, it should be emphasized there are some important
This experiment determined whether the effects of Ten- differences between the two thrombolytic therapies. In the
ecteplase (0.9 mg/kg) measured as behavioral improve- RSCEM, Alteplase was only effective when administered
ments, which we observed 1 day following embolization within 1 h of embolization. When Alteplase was adminis-
and treatment, would be measurable 4 days after adminis- tered at long delays (e.g. 3 h) following embolization, we
tration of Tenecteplase. With Tenecteplase administered at 1 found that there was no significant behavioral improvement
h, a significant increase in the P50 (2.10 F 0.07 mg; n = 13) (Lapchak and Zivin, 2003; Lapchak et al., 2002b), whereas
was noted compared to the 4-day vehicle-treated group Tenecteplase was effective out to 3 h following emboliza-
(P50 = 0.92 F 0.22 mg; n = 12). tion. Although Alteplase is only effective out to 1 h following
embolization in the RSCEM, it is useful up to 3 h or more
following a stroke in patients (Angeja et al., 2001; The
Discussion National Institute of Neurological Disorders and Stroke rt-
PA Stroke Study Group, 1995; Zivin, 1999), indicating a
Alteplase (tissue plasminogen activator, or tPA; Acti- difference between rabbits and humans. Therefore, since we
vase) is the only FDA-approved treatment for acute ische- have found that Tenecteplase is effective out to 3 h following
mic stroke (see Lapchak, 2002a, for review). Thrombolytics embolization in rabbits, it is likely that the time window for
are considered to be valuable agents because recanalization Tenecteplase in humans may be significantly longer than 3 h.
allows not only for restoration of blood flow to oxygen- Last, based upon results from the rabbit large clot embolism
deprived tissues, but also can allow for access of small model from our laboratory, both Tenecteplase and Alteplase
molecule neuroprotective or neuroregenerative drugs to the produced similar rates of intracerebral hemorrhage (ICH)
penumbra of the infarcted tissue (Lapchak, 2002a,b; Zivin, following an embolic stroke (Chapman et al., 2001; Lap-
1999). Currently, there are at least 10 thrombolytics in chak, 2002b; Lapchak and Araujo, 2001; Lapchak et al.,
preclinical and clinical development for acute ischemic 2002a,b). This is a very important aspect requiring consid-
stroke including Tenecteplase, recombinant desmodus eration when novel tPA-like thrombolytics are to be devel-
rotundus salivary plasminogen activator (rDSPA; vampire oped. For instance, a recent early phase clinical trial with
bat tPA) and microplasmin (reviewed in Lapchak, 2002a). rDSPA showed that there was a intracerebral hemorrhage
Based upon the recent literature, each thrombolytic appears rate in the range of 23.5– 30.7%, approximately 3.6 – 4.8
to have different pharmacological and side effect profiles times that observed with Alteplase (Hacke, 2003; The
(Lapchak, 2002a). One of the most studied thrombolytics in National Institute of Neurological Disorders and Stroke rt-
AMI, Tenecteplase, recently entered into a dose-finding PA Stroke Study Group, 1995). This suggests that the side-
clinical trial in stroke patients (E. Clark Haley Jr., P. Lyden, effect profile of rDSPA will outweigh the therapeutic benefits
personal communication). However, preclinical evidence of the drug.
158 P.A. Lapchak et al. / Experimental Neurology 185 (2004) 154–159

The present preclinical data together with the results the Christopher Reeve Paralysis Foundation (PAL). We
from Tenecteplase AMI clinical trials indicate that Tenecte- especially thank Dr. Semba and Dr. McKeever (Genentech
plase may be superior to Alteplase as a thrombolytic to Inc.) for supplying Tenecteplase and Alteplase. Jiandong
improve clinical rating scores. In the ASSENT-2 AMI trial, Wei and Donghuan Song are recognized for their expert
while both Alteplase and Tenecteplase were equally neuro- technical expertise.
protective, Alteplase was reported to produce significantly
more noncerebral bleeding compared to Tenecteplase (Van
de Werf et al., 2001). However, the ICH rate (< 1%) was References
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