TNK-Tissue Plasminogen Activator in Acute Myocardial Infarction
Results of the Thrombolysis in Myocardial Infarction (TIMI) 10A Dose-Ranging Trial
Background TNK-tissue plasminogen activator (TNK-TPA) is a genetically engineered variant of TPA, which in experimental models has a slower plasma clearance and greater fibrin specificity and is 80-fold more resistant to plasminogen activator inhibitor-1 than alteplase TPA.
Methods and Results The Thrombolysis in Myocardial Infarction (TIMI) 10A trial was a Phase 1, dose-ranging pilot trial designed to evaluate the pharmacokinetics, safety, and efficacy of TNK-TPA in patients with acute myocardial infarction. One hundred thirteen patients with acute ST-segment elevation myocardial infarction presenting within 12 hours and without contraindications to thrombolysis were enrolled and treated with a single bolus of TNK-TPA over 5 to 10 seconds with doses ranging from 5 to 50 mg. TNK-TPA demonstrated a plasma clearance of 151±55 mL/min and a half-life of 17±7 minutes. Comparable values for wild-type TPA are 572±132 mL/min and 3.5±1.4 minutes, respectively. Systemic fibrinogen and plasminogen levels fell by only 3% and 13%, respectively, at 1 hour after TNK-TPA administration. TIMI grade 3 flow at 90 minutes was achieved in 57% to 64% of patients at the 30- to 50-mg doses. Seven patients (6.2%) experienced a major hemorrhage, which occurred at a vascular access site in six patients.
Conclusions TNK-TPA has a prolonged half-life so it can be administered as a single bolus. TNK-TPA appears to be very fibrin specific, and the initial patency and safety profiles are encouraging. Further study of this new thrombolytic agent is ongoing.
Thrombolytic trials in acute MI over the past decade have shown that early achievement of TIMI grade 3 flow in the infarct-related artery is associated with reduced mortality.1 2 3 4 5 6 7 8 In addition, rapid time to treatment2 9 10 and prehospital treatment may also be beneficial,11 12 and these can be facilitated by simpler thrombolytic dosing regimens, such as a bolus regimen. Thus, research has focused on developing thrombolytic agents that are more potent, safer, and easier to administer.
Using TPA as the model, Keyt and colleagues13 developed >1000 variants of TPA and discovered one with particularly promising attributes (Table 1⇓).The novel plasminogen activator, designated TNK-TPA, is similar to wild-type TPA but has amino acid substitutions at three sites: a threonine (T) is replaced by an asparagine, which adds a glycosylation site to position 103; an asparagine (N) is replaced by a glutamine, thereby removing a glycosylation site from site 117; and four amino acids, lysine (K), histidine, arginine, and arginine, are replaced by four alanines at the third site. Together, these substitutions led to a prolonged half-life of the molecule, increased fibrin specificity, and increased resistance to inhibition by PAI-1.13 In an animal model of acute arterial occlusion, a bolus of TNK-TPA was found to produce more rapid recanalization and a greater degree of clot lysis compared with a front-loaded TPA regimen.14 15
The TIMI 10A trial was a phase I, dose-ranging, pilot trial with several goals: to evaluate the initial safety profile of TNK-TPA in humans, to establish the pharmacokinetic profile of TNK-TPA in patients with acute MI, to assess the effects of increasing doses of TNK-TPA on coagulation parameters, and to evaluate angiographic efficacy using both TIMI grade 3 flow and the TIMI frame count.16
Patients between the ages of 19 and 69 were screened for enrollment at 18 hospitals between January and November 1995 (see “Appendix”). Inclusion criteria for the trial were the presence of ischemic pain lasting ≥30 minutes, associated with ST-segment elevation of ≥0.1 mV in two or more contiguous leads or new left bundle-branch block and ability to treat within 12 hours of symptom onset. Exclusion criteria in this phase I trial were prior stroke or transient ischemic attack, a reliably obtained blood pressure of >170/100 mm Hg, significant bleeding within 6 months, major surgery, trauma (including head trauma associated with the presenting MI) within 2 months, a history of dementia or major cognitive deficit, prolonged cardiopulmonary resuscitation within 2 weeks, treatment of acute MI with thrombolytic therapy within the prior 4 days, cardiogenic shock, inability to undergo cardiac catheterization, body weight of <60 kg, pregnancy or current lactation, prothrombin time of ≥14 seconds or international normalized ratio of ≥1.4, allergy to heparin or history of multiple allergies, current cocaine abuse, other serious illness, inability to follow the protocol, current participation in another experimental drug protocol, and previous participation in TIMI 10A.
The TIMI 10A protocol was as follows: Eligible patients were treated with a single bolus of TNK-TPA administered over 5 to 10 seconds. Eight doses of TNK-TPA (Genentech) were studied sequentially in an ascending, open-label fashion: 5, 7.5, 10, 15, 20, 30, 40, and 50 mg. By design, no control arm of TPA was included in this Phase I, dose-escalation study, but it was planned for the Phase II study. All patients received 150 to 325 mg of aspirin daily. Intravenous heparin was administered as a 5000 U bolus and an initial 1000 U/hr infusion for 48 to 72 hours, which was titrated to an activated partial thromboplastin time of 55 to 85 seconds.17 β-Blockers were recommended,18 and other medications were administered at the discretion of the treating physicians. Patients underwent coronary angiography at 90 minutes and at 60 and 75 minutes when feasible. To ensure that patients with failed thrombolysis achieve early reperfusion, PTCA was performed at the discretion of the treating physician after the 90-minute angiogram was obtained. Furthermore, the protocol stated that if a patient developed hemodynamic compromise or decompensation before the 90-minute angiogram, rescue PTCA or other interventions before the 90-minute angiogram were permitted. Patients were monitored in-hospital and returned at 30 days for follow-up and a blood sample to evaluate for the formation of TNK-TPA antibodies. The study protocol was reviewed and approved by each hospital's institutional review board, and written informed consent was obtained from each patient before enrollment.
Study End Points
End points in the trial included pharmacokinetics, coagulation parameters, the rate of TIMI grade 3 flow at 90 minutes,3 the TIMI frame count,16 serious bleeding, and anaphylaxis. Clinical end points were defined as in previous TIMI trials.4 18 19 20 Pharmacokinetics of TNK-TPA were characterized using standard methods.21 Coagulation assays were collected in SCAT-1 tubes containing 50 μmol/L d-Phe-Pro-Arg-chloromethyl ketone, 150 KIU/mL aprotinin, and 4.5 mmol/L EDTA and assayed as previously reported.22 Plasmin/α2-antiplasmin complexes were assayed using an ELISA assay.23 All angiograms were analyzed at the Angiographic Core Laboratory by personnel who were blinded to dose assignment. TIMI flow grade3 and TIMI frame count16 were analyzed according to the prespecified definitions.
The number of patients were originally specified to be 5 patients per dose. However, to gain a greater initial experience with the safety of each ascending dose, after the first three doses a protocol amendment increased this number to 21 patients per dose. An interim analysis of efficacy (the rate of TIMI grade 3 flow) after every 7 patients was planned. If the rate of TIMI grade 3 flow was less than a prespecified boundary24 25 and if no safety problems were identified, the dose was increased to the next prespecified dose. To gain more safety and patency information, after completion of the eight doses, an additional 20 patients were treated with the 30-mg dose. Thus, 5 patients were treated with the 5-, 7.5-, and 10-mg doses, 7 patients with the 15- and 20-mg doses, 41 patients with the 30-mg dose, and 21 and 22 patients with the 40- and 50-mg doses, respectively. Continuous variables were compared with the Student's t test, and categorical variables were compared with χ2 analysis.
One hundred thirteen patients were enrolled in the trial (Table 2⇓). The mean age of the patient population was 54 years, 84% were male, 38% presented with anterior MI, 49% were “not-low-risk” as defined in the TIMI II trial,18 and the mean time to treatment was 3.0 hours. There were no major differences in baseline characteristics among the doses tested.
The plasma clearance of TNK-TPA ranged from 125±25 to 216±98 mL/min across the 5- to 50-mg doses, approximately one third of that previously observed with recombinant wild-type TPA (572±132 mL/min).26 The corresponding plasma half-life of elimination of TNK-TPA ranged from 11±5 to 20±6 minutes compared with 3.5 minutes as previously reported for TPA.26 Fig 1⇓ shows the TNK-TPA plasma levels over time for several doses of TNK-TPA and the TPA levels from 10 patients in the TAPS trial treated with the front-loaded regimen of TPA.26 There was a dose-dependent increase in TNK-TPA levels. As shown, after a single 5- to 10-second bolus of TNK, the plasma concentration was very similar to that of other patients treated with TPA given in the front-loaded, 90-minute regimen.26
The effect on systemic coagulation factors at 1 hour across the range of doses of TNK-TPA is shown in Fig 2⇓. There was a dose-dependent increase in the consumption of α2-antiplasmin, the fluid-phase inhibitor of plasmin, and a consequent increase in plasmin/α2-antiplasmin complexes, indicating a dose-dependent increase in the level of systemic plasmin generation. However, there was on average only a 3% reduction in fibrinogen and a 13% reduction in plasminogen even at the 50-mg dose. Similar results were observed at 3 hours. Previous studies have shown that TPA, either the 90-minute or the 3-hour regimen, leads to a 50% to 60% decrease in fibrinogen and plasminogen and a 70% to 80% decrease in α2-antiplasmin.22 27 28
TIMI grade 3 flow at 90 minutes in the infarct-related artery appeared to be higher in patients treated with the 30- to 50-mg doses as compared with the lower doses (P=.032) (Fig 3⇓). The rate of TIMI grade 3 flow was 59% at the 30-mg dose and 64% at the 50-mg dose. Patency, defined as TIMI grade 2 or 3 flow, of the infarct-related artery was 85% overall and did not appear to differ across the full range of doses tested. No differences in the rate of TIMI grade 3 flow or TIMI grade 2 or 3 flow combined were noted between the three different infarct related arteries or when stratifying patients by time to treatment (≤3 hours versus >3 hours). Rescue PTCA was performed immediately after the 90-minute angiogram in 16 of 17 patients with TIMI grade 0 or 1 flow in the infarct-related artery and was successful in all patients. Thus, patency was achieved after the 90-minute angiogram in 99% of patients.
The TIMI frame count was also used to objectively assess coronary flow.16 The percentages of patients with a TIMI frame count of <40 frames at 90 minutes, which corresponds closely to TIMI grade 3 flow,16 were 62% and 68% at the 30- and 50-mg doses, respectively. The percentage of patients with a TIMI frame count of ≤27 frames, which corresponds to truly normal coronary flow in the absence of acute MI,16 was 45% at the 30- to 50-mg doses.
Fig 4⇓ shows the angiographic results at 60, 75, and 90 minutes for the 30- and 50-mg doses. After only 60 minutes, TIMI grade 2 or 3 flow was achieved in 79% to 82% of patients, indicating that among patients who achieved reperfusion by 90 minutes, most already had patent infarct-related arteries by 60 minutes.
Mortality at 30 days was 3.5% (Table 3⇓). Reinfarction was observed in 4.4% of patients, and new-onset pulmonary edema occurred in 2.7%. Serious bleeding occurred in 7 patients (6.2%): at a vascular access site in 6 patients and after CABG in 1 patient. These hemorrhagic events were distributed across the doses, occurring at the 15-mg (1 patient), 20-mg (2), 30-mg (1), 40-mg (2), and 50-mg (1) doses. There were no strokes or intracranial hemorrhages. Antibodies to TNK-TPA were not detected in any patient at 30 days, although 1 patient had a positive titer before treatment with TNK-TPA.
In this pilot trial of TNK-TPA, we observed that TNK-TPA has a prolonged half-life, so it can be administered as a single bolus. In addition, TNK-TPA appears to be more fibrin specific than wild-type TPA. The initial infarct-related artery patency profile shows encouraging rates of TIMI grade 3 flow at the 30- to 50-mg doses. Although not sized to fully evaluate safety events, the initial safety profile appears to be acceptable and TNK-TPA is not antigenic.
TNK-TPA: A Single-Bolus Agent
TNK-TPA was developed with the goal of producing a thrombolytic agent that could be administered as a single bolus. The mutation at the 103 site with the addition of a glycosylation site leads to a decrease in the clearance of the molecule. This study documented that the half-life of TNK-TPA is approximately threefold longer than that of wild-type TPA in patients with acute MI. Consequently, a single 5- to 10-second bolus of TNK-TPA achieves blood levels similar to those of the 90-minute infusion of TPA (Fig 1⇑). Furthermore, its ease of administration could facilitate more rapid treatment, which has been shown to improve survival in acute MI,2 9 29 and make more feasible the promising strategy of prehospital treatment with thrombolysis.10 11 12
TNK-TPA Structure and Function
TNK-TPA has several other features that make it a promising new advance in thrombolytic therapy.15 The potency of TNK-TPA was maintained and perhaps enhanced by the mutation at site 117, which deleted a glycosylation site. In addition, the substitution in the catalytic region (296–299) leads to an increase in the fibrin specificity of TNK-TPA and renders it 80-fold more resistant to inhibition from PAI-1.30 31 In in vitro models of thrombolysis, TNK-TPA was noted to be 8- to 13-fold more potent at clot lysis on a per-milligram-weight basis.13 In a rabbit carotid occlusion model, the average time to reperfusion was 11±2 minutes for TNK-TPA compared with 23±7 minutes for TPA (P=.02).13 The total duration of reperfusion was improved, and the average total residual thrombus weight was less with TNK-TPA than with TPA.13
Fibrin Specificity of TNK-TPA
TNK-TPA had very little effect on measures of systemic coagulation. Fibrinogen levels fell by only 3% and plasminogen fell by only 13% on average. The size of the decrease was constant across all dose levels. In comparison, front-loaded TPA leads to a 50% decrease in fibrinogen and a 60% reduction in plasminogen at 3 hours.28 Furthermore, the very sensitive measure of plasmin generation, α2-antiplasmin, fell by only 15% to 25% by 3 hours at the higher doses compared with a 70% to 80% reduction in patients treated with front-loaded TPA.28 Thus, TNK-TPA appears to be more fibrin specific than TPA. Whether greater fibrin specificity will translate into improved clot lysis or a reduction in bleeding and intracerebral hemorrhage remains to be determined. It is nevertheless encouraging to note that a lower rate of surgical site bleeding was observed in animal studies compared with TPA14 and that a relatively low rate of major hemorrhage was observed in this angiographic trial−6.2% compared with 11% to 23% in other recent studies with front-loaded TPA,4 19 suggesting that TNK-TPA may be associated with a lower rate of hemorrhage. A comparison is being carried out in a larger, prospective comparative study of TNK-TPA and TPA.
It has been noted that marked depletion of systemic plasminogen may adversely affect clot lysis, the so-called “plasminogen steal” phenomenon.32 It is hypothesized that very low systemic levels of plasminogen lead to diffusion of plasminogen out of the coronary thrombus, thereby depleting the substrate and decreasing the amount of plasmin generated on the clot surface, ultimately decreasing clot lysis.32 In the recent trial of double-bolus TPA, a lower rate of TIMI grade 3 flow and infarct-related artery patency was observed in the double-bolus regimen.33 Because the double-bolus regimen was associated with a >80% depletion of systemic plasminogen, it was hypothesized that plasminogen steal may have reduced the efficacy of the more aggressive regimen. As such, the fibrin specificity of TNK-TPA should permit rapid plasminogen activation in the clot to proceed even when given as a single bolus.
We observed that TIMI grade 3 flow was achieved by 90 minutes in 57% to 64% of patients treated with the 30- to 50-mg doses. This compares favorably with front-loaded TPA4 6 or the newer agent reteplase (recombinant PA) in other trials.34 A randomized trial under way to compare directly the rates of TIMI grade 3 flow between the 30- to 50-mg doses of TNK-TPA and front-loaded TPA. Overall, infarct-related artery patency with TIMI grade 2 or 3 flow was ≈81% to 89% across the higher doses of TNK-TPA, which is similar to that of TPA given in either the front-loaded regimen4 6 or the double-bolus regimen.33 This suggests that there may be a ceiling in overall patency that can be achieved with the combination of a thrombolytic agent, aspirin, and heparin.
TIMI Frame Count
This trial was the first angiographic trial to prospectively use the TIMI Frame Count to evaluate early patency. This method, developed by Gibson and colleagues,16 is an objective and continuous measure of coronary flow. Using this measure, we observed that 60% to 68% of patients achieved a corrected TIMI Frame Count of <40, corresponding closely to TIMI grade 3 flow, which compares favorably with patients treated with front-loaded TPA in the TIMI 4 trial, in which 53% of patients achieved this degree of reperfusion.
Interestingly, use of the TIMI Frame Count has shown that even TIMI grade 3 flow after the use of first-generation thrombolytic agents for acute MI is not necessarily normal flow.16 Using the more strict criteria for truly normal flow (TIMI frame count ≤27), 45% of patients treated with the higher doses of TNK-TPA achieved full reperfusion, whereas only 27% of patients treated with TPA in the TIMI 4 trial achieved truly normal flow by 90 minutes (P<.01). If these results can be confirmed in subsequent studies, they will suggest that TNK-TPA achieves more rapid and complete thrombolysis than the current standard, front-loaded TPA.
In this initial pilot trial in patients with acute MI, TNK-TPA was shown to have a prolonged half-life so that it can be administered as a single bolus. TNK-TPA is very fibrin specific, and the initial patency and safety profiles are encouraging. Further study of this promising new thrombolytic agent is ongoing.
TIMI 10A Appendix
Study Chairman's Office
Harvard Medical School, Brigham and Women's Hospital, Boston, Mass: Study chairman: Eugene Braunwald, MD, principal investigator: Christopher P. Cannon, MD; project director: Carolyn H. McCabe, BS; coinvestigators: Elliott M. Antman, MD; Susan J. Marble, RN, MS.
Angiographic Core Laboratory
VA Medical Center, West Roxbury, Mass: Principal investigator: C. Michael Gibson, MS, MD; coinvestigators: Rahul Chaturvedi, MD; William L. Daley, MD; Clyde Meckel, MD; Thomas Rocco, MD; Lori Raymond; Christine McLean; Michael J. Rizzo; Nilda E. Martin.
Coagulation Core Laboratory
University of Vermont (Colchester): Coprincipal investigators: Russell P. Tracy, PhD, Edwin G. Bovill, MD; research coordinator: Elaine Cornell, BS.
Genentech, Inc, South San Francisco, Calif: Ted W. Love, MD; Norma Lynn Fox, PhD, MPH; Judy Breed, RN; Nishit B. Modi, PhD; Dorene Jansen, MPH; James Reimann, PhD; Fong Wang Clow, PhD; and Boehringer Ingelheim GmBH, Rhein, Germany.
Clinical Centers in Order of Enrollment
Iowa Heart Center/Mercy Medical Center (Des Moines)−principal investigator: Magdi Ghali, MD; research coordinator: Theresa Coulson, RN; University of Miami/Jackson Memorial Hospital, Miami, Fla−principal investigator: Rafael F. Sequeira, MD; coinvestigator, Manuel R. Mayor, MD; research coordinators: Gayatri Girwarr, MD, Pura Teixeiro, RN; Rhode Island Hospital (Providence)−principal investigator: George R. McKendall, MD, coprincipal investigator: David O. Williams, MD; research coordinator: MaryJane McDonald, RN; Brigham and Women's Hospital, Boston, Mass−principal investigator: James M. Kirshenbaum, MD; coinvestigator: Christopher P. Cannon, MD; research coordinator: Jill Cloutier; Hennepin County Medical Center, Minneapolis, Minn−principal investigator: Timothy D. Henry, MD; research coordinators: Lorri Knox, RN, Charlene Boisjolie, RN; Baystate Medical Center, Springfield, Mass−principal investigator: Marc J. Schweiger, MD; research coordinators: Barbara Burkott, RN, Deborah Warwick, RN; Trinity Mother Frances Hospital, Tyler, Tex−principal investigator: Robert Carney, MD; research coordinator: Gregory Murphy, RPH; University of Texas/Medical Branch at Galveston−principal investigator: David Cutler, MD; research coordinator: Gaylene Malmberg, RN; Alta Bates Medical Center, Berkeley, Calif−principal investigator: Robert M. Greene, MD; research coordinators: Eileen Healy, RN; Vickie Perry, RN; University of Vermont/Fletcher Allen Health Care (Burlington)−principal investigator: Matthew Watkins, MD; coinvestigator: Burton E. Sobel, MD, research coordinator: Michaelanne Rowen, RN; Parkview Memorial Hospital/Stucky Research Center, Fort Wayne, Ind−principal investigator: William Wilson, MD; research coordinator: Jane Cuttitta, RN. Broward General Medical Center, Fort Lauderdale, Fla−principal investigator: Alan Niederman, MD; research coordinator: Terri Kellerman, RN; Sarasota Memorial Hospital, Sarasota, Fla−principal investigator: Martin Frey, MD; research coordinator: Holly Taylor; Montefiore Medical Center, Bronx, NY−principal investigator: Hiltrud S. Mueller, MD; research coordinators: Linda Kunkel, RN, Joseph Cosico, RN; Lancaster General Hospital, Lancaster, Pa−principal investigator: Paul Casale, MD; research coordinator: Joann Tuzi, RN; University of Alabama (Birmingham)−principal investigator: William J. Rogers, MD; research coordinator: Terri Morgan, RN; LDS Hospital, Salt Lake City, Utah−principal investigator: Jeffrey L. Anderson, MD; coprincipal investigator: Labros Karagounis, MD; research coordinator: Ann Allen, RN; St Luke's Hospital, New York, NY−principal investigator: Judith S. Hochman, MD; research coordinator: Mary McAnulty, RN.
Selected Abbreviations and Acronyms
|PAI-1||=||plasminogen activator inhibitor-1|
|PTCA||=||percutaneous transluminal coronary angioplasty|
|TIMI||=||Thrombolysis in Myocardial Infarction|
|TPA||=||tissue plasminogen activator|
The study was sponsored by Genentech, Inc (South San Francisco, Calif) and Boehringer Ingelheim GmBH (Rhein, Germany).
- Received August 12, 1996.
- Revision received September 4, 1996.
- Accepted September 9, 1996.
- Copyright © 1997 by American Heart Association
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