Following an acute myocardial infarction (AMI), there is replacement of myocytes by fibrotic tissue and ongoing apoptotic loss of viable cardiac myocytes in the infarct border zone as a result of reperfusion injury and ongoing ischemia from microvascular obstruction. If the infarction is large, left ventricular (LV) dysfunction may develop due to scar expansion and LV dilation, ultimately leading to the development of congestive heart failure. Congestive heart failure is the leading admission diagnosis for hospitalization in the United States and carries a 5-year mortality rate of 50%.1
Although medical therapy can improve symptoms and extend survival to a limited degree, cardiac transplantation remains the only curative procedure available.
Recent studies in animals with experimental AMI have observed significant recovery of LV function after the delivery of bone marrow–derived stem cells.2–4
Although the mechanisms of benefit are subject to ongoing investigation,5,6
the potential has led to the initiation of multiple clinical trials to test the concept that delivery of autologous bone marrow mononuclear cells (BMMNCs) into the infarct region can improve cardiac function after AMI.7–11
have demonstrated that BMMNC administration results in a small, but significant, improvement in LV function and attenuation of adverse LV remodeling.
The timing of cell administration after AMI may be of fundamental importance in determining the success of cell therapy in this patient population, given the temporal changes that occur in the myocardium in the days to weeks after AMI that can affect stem cell survival and efficacy. Initial up-regulation of chemokine production, such as stromal derived factor-1 (SDF-1), can direct cell homing and differentiation,15
while stem cell-secreted growth factors such as vascular endothelial growth factor (VEGF) and insulin growth factor-1 can improve perfusion (through angiogenesis) and reduce apoptotic cell death in the infarct border zone.16
However, the development of a vigorous inflammatory response, coupled with the release of reactive oxygen species and cytokines such as tumor necrosis factor-a in the infarct region after an AMI, can adversely affect cell survival17
and limit the efficacy of this therapeutic approach.
No clinical trial published to date has been sufficiently powered to adequately answer questions about the efficacy of late cell delivery after AMI. Recently, the National Heart, Lung and Blood Institute (NHLBI) established the Cardiovascular Cell Therapy Research Network (CCTRN) to answer important questions in cell therapy, such as the optimal timing of cell delivery after AMI. The 1st TIME trial developed by the CCTRN is currently investigating outcome differences in LV function in patients randomized to cell delivery at 3 versus 7 days after AMI.18
However, many patients eligible for cell therapy may not be suitable for randomization at these time-points, due to clinical instability or to hospitalization at facilities without access to cell therapy. To study the usefulness of cell therapy in this important subset of AMI patients, the CCTRN developed a 2nd randomized, placebo-controlled trial called LateTIME, in which autologous BMMNCs are delivered 2 to 3 weeks after AMI.
Organizational Structure and Oversight
The CCTRN was established by the NHLBI to devel-op, coordinate, and conduct multiple collaborative protocols testing the effects of stem cell therapy on cardiovascular disease. The Network builds on contemporary findings of the cell-therapy basic science community, translating this newly acquired information to the cardiac clinical setting in the Phase I/II study paradigm. The Network consists of 5 clinical research centers (Cleveland Clinic Foundation, University of Florida, Minneapolis Heart Institute/University of Minnesota, Texas Heart Institute, and Vanderbilt University), a data coordinating center (DCC) (University of Texas School of Public Health) that provides trial management and data analysis, a cell-processing quality-control center, and 5 core laboratories. Together, these Network components provide standardization of cell-therapy preparation and endpoint measurements. All clinical centers participate in the selection and design of Network protocols, which are also reviewed by an independent Protocol Review Committee and a Gene Therapy/Cell Therapy Data Safety and Monitoring Board (DSMB), under the aegis of the NHLBI. Each clinical center and the DCC has independent institutional review board (IRB) approval and oversight. By recruiting from multiple centers, the Network accelerates the speed with which its studies can be completed, increases the broad applicability of study findings, and amplifies the dissemination of its public health findings.
Objectives and Design
The primary objective of LateTIME, a Phase-I/II, randomized, double-blinded, placebo-controlled clinical trial, is to determine whether delayed (2–3 wk) administration of intracoronary autologous BMMNCs to patients after a moderate-to-large MI can safely produce a measurable improvement in LV global and regional function as determined by cardiovascular magnetic resonance (CMR) at 6 months, compared with baseline. Secondary objectives are to determine whether the administration of cell therapy in comparison with placebo will produce smaller end-diastolic and end-systolic volumes at 6 months, compared with baseline. Secondary objectives are to determine whether the administration of cell therapy in comparison with placebo will produce smaller end-diastolic and end-systolic volumes at 6 months. Patients will be monitored for 2 years to evaluate the effects of cell therapy on the clinical endpoints of death, reinfarction, repeat target-vessel revascularization, hospitalization for congestive heart failure, and placement of implantable cardiac defibrillators (ICDs). The effects of cell phenotype on therapeutic efficacy will be investigated through ancillary studies at 2 biorepository core facilities (the University of Minnesota and the University of Florida, Gainesville).
Patients will be randomized in a 2:1 ratio, BMMNC to placebo. The overall anticipated sample size required for LateTIME is 87 patients (58 cell therapy and 29 placebo). Hypothesis testing for the primary outcome will be carried out at the 0.05 level.
Enrollment and Study Population
The 87 patients required for LateTIME will be recruited from all sites participating in the NHLBI CCTRN and their developed satellite referral centers. Each patient will have undergone successful percutaneous revascularization of a major coronary artery after a moderate-to-large infarction with a left ventricular ejection fraction (LVEF) ≤0.45 as evaluated by echocardiography. All prospective patients will be screened by investigators or study coordinators and will be enrolled in the trial after meeting inclusion criteria () and signing the informed consent and Health Insurance Portability & Accountability Act forms.
TABLE I. Major Inclusion and Exclusion Criteria
Eligibility and Randomization
Once inclusion and exclusion criteria have been evaluated and informed consent has been obtained, eligible patients will be randomly assigned to one of the selected treatment strategies in an interactive Web-based randomization session. Patients will be randomized in a 2:1 (BMMNC-to-placebo) ratio using randomly selected block sizes of 6 or 9 and stratified by the center. The patients and research staff, including the CCTRN physicians and interventional cardiologists, will be blinded to treatment assignment. Patients will follow a prescribed schedule of procedures () and be evaluated at 6 months for efficacy of treatment. All patients will be monitored for 2 years to obtain an extensive safety profile.
TABLE II. Schedule of Procedures in LateTIME
Bone Marrow Aspiration
All patients will receive a physical examination, including evaluation of vital signs, height, weight, and New York Heart Association (NYHA) functional class. On the morning of study product infusion, patients will undergo bone marrow aspiration in accordance with standard operating procedures developed by the CCTRN. Patients will be lightly sedated or receive conscious sedation as determined by the attending physician. Approximately 80 to 90 mL of bone marrow will be aspirated from the iliac crest using standard techniques. Only one bone marrow aspiration will be attempted. As determined from preclinical experience, 150 to 200 million total nucleated cells (TNCs) can be routinely harvested with this volume of bone marrow aspirate, containing small fractions of CD34+ and CD133+ cells. The specific population of cells administered in LateTIME will be determined at the biorepository core facilities and correlated with outcomes.
Cell Preparation and Cell Product
Once harvested, the cells will be transported to the institution's cell-therapy laboratory. Each site will use the Sepax® system19
(Biosafe America, Inc.; Houston, Tex) for BMMNC isolation. The Sepax system is a closed system that enables faster isolation of the cellular product and, potentially, increased safety for the patient. Each site's cell-therapy laboratory director and participating personnel have been trained with the Sepax system, thereby ensuring a more uniform cellular product. The cells will be harvested and washed 3 times in human serum albumin/saline buffer before re-suspension in 5% human serum albumin/saline. The composition of CD34+ and CD133+ cells will be determined by fluorescent-activated cell-sorting analysis. Viability of the cells will be determined by trypan blue exclusion; ≥70% viability will be required before transplantation. A 14-day sterility culture will be performed on the final product. Because this testing will not be available before product infusion, a negative Gram stain will be required before the product is released. Although cells are autologous in this protocol, the standard tests for infectious diseases will be performed. Additional testing deemed necessary by regulations or institutional policy (or both) will be performed. Final product (lot) release criteria testing results () will be available before transport of BMMNCs to the hospital for administration. The product will be labeled and tracked with adhesive labels containing the patient's study identification.
TABLE III. Final Product Release Criteria Testing
All patients randomized to the active group will receive 150 × 106
TNCs (70%–80% BMMNCs) as determined by a hematology analyzer. If the bone marrow aspiration yield is less than the target amount and the patient has already been randomized to therapy, then he or she will receive all available cells. Patients randomized to the placebo group will receive 5% albumin in normal saline, to which 100 mL of autologous blood will be added to ensure that the color and consistency of the placebo infusion matches that of the active group. If the patient consents, all cells not required for study product delivery will be provided to the CCTRN biorepository core facilities for cell characterization and functional analysis.
Infusion of Cellular Product or Placebo
The final cellular product or placebo (approximate total volume, 30 mL) will be infused less than 12 hours after bone marrow aspiration. The patients will be heparinized to an activated clotting time of >200 sec, and the infusate will be delivered via an over-the-wire percutaneous transluminal coronary angioplasty catheter (stop-flow) in 6 aliquots of approximately 5 mL each, delivered over 2 minutes of balloon inflation within the previously placed stent at the lowest possible pressure necessary to obstruct coronary blood flow (3–6 atm). Two minutes of reperfusion will occur after each cycle of cell infusion.
All patients will be monitored overnight on a telemetry unit in the hospital and seen by a physician the following morning, before discharge. Patients are advised to take aspirin for life and 75 mg of clopidogrel for 24 months, as well as the usual medications for post-AMI care. Patients with LVEFs of <0.40 will be advised to take an aldosterone antagonist unless contraindicated by a creatinine level ≥2.5 mg/dL or a serum potassium level ≥5 mEq/L.
Determination of Outcomes
A 1.5T CMR scanner will be used, with precise magnetic resonance imaging (MRI) protocols developed by the MRI core laboratory (University of Florida). Cardiovascular Angiography Analysis System/Magnetic Resonance Ventricular (CAAS/MRV) cardiac function and perfusion analysis software (PIE Medical Imaging B.V.; Maastricht, The Netherlands) will be used to measure global LV myocardial mass, volumes, and LVEF, as well as regional parameters. Regional systolic wall motion, ejection fraction, thickening, and radial displacement in the infarct and border zones will be determined. Regional ejection fraction will be calculated for the basal, midwall, and apical portions of the LV cavity. Areas of microvascular obstruction, infarct size, and degree of transmurality will be quantified by delayed gadolinium-enhanced MRI.
Wall Motion Imaging.
Data on both global and segmental LV function will be obtained using a steady-state free-precession or fast-gradient echo technique. Long-axis cine images in the 2-chamber and 4-chamber projections will be acquired. In addition, a set of contiguous short-axis slices (8–10 mm thick) will be obtained from the mitral valve annulus through the apex of the LV throughout the cardiac cycle. Data will be analyzed using the CAAS/MRV software. Global measurements evaluated will include end-diastolic volume, end-systolic volume, stroke volume, ejection fraction, and LV mass. Volumetric measurements will be performed by direct planimetry on the contiguous short-axis images at both end-systole and end-diastole. Regional measurements will include wall thickening and wall motion, and will be calculated using 100 chords spaced every 3.6°, originating from the centroid of the LV. Regional data will be reported using the American Heart Association 17-segment model.20
The minimum spatial and temporal resolution requirements of the steady-state free-precession sequence are 2.5 × 2.5-mm voxels and 40 ms, respectively.
Baseline Perfusion Imaging. A 2-chamber long-axis cine image will be obtained on the basis of axial scout images, with the imaging plane spanning the center of the mitral valve coaptation point and through the apex of the LV. Based on this, a 4-chamber long-axis cine image will be obtained. Subsequently, a T1-weighted gradient-echo baseline perfusion sequence will be performed using an intravenous gadolinium chelate (for example, gadolinium-DTPA). Three short-axis slices will be obtained (positioned from the 2-chamber and 4-chamber cine images) to encompass the basal, middle, and apical thirds of the LV during a bolus administration of gadolinium chelate (0.15–0.2 mmol/kg). Imaging will be acquired for a total of 60 dynamics per slice, ensuring that the passage of contrast material through the myocardium is captured for semiquantitative analysis.
Viability Imaging. Fifteen to 20 minutes after the administration of gadolinium-chelate contrast agent, delayed-enhancement imaging (DE-MRI) will be performed with a T1-weighted inversion-recovery prepared gradient-echo sequence. The inversion delay time will be iteratively adjusted for optimal nulling of normal myocardium. Contrast-enhanced viability imaging will be performed with 2 techniques: the standard 2-dimensional technique, which acquires a single slice during each breath hold, will be performed in the short-axis projections, using the same plane prescription as the functional short-axis cine series; and a high-resolution 3-dimensional technique will be used to acquire 10 short-axis slices during a single breath hold. Regions of irreversible myocardial damage are manifested by “hyperenhancement” (bright white areas) on the images, while normal or viable tissue is “nulled” (black) on the acquired images. The presence, location, and extent of irreversibly damaged tissue will be qualitatively and quantitatively evaluated on a segmental basis. Pre- and post-therapy imaging, both cine wall motion and DE-MRI, will be carefully matched for consistency and accuracy, using internal landmarks that include the insertion sites of the right ventricular free wall and the papillary muscles.
All CCTRN participants will be closely monitored for adverse events, and this information will be transmitted to the IRBs of all centers, the U.S. Food and Drug Administration, and the DSMB through the DCC. The DSMB will meet at least twice yearly to review the performance of the participating sites, to evaluate the accruing safety data, and to ascertain the feasibility of continuing the study. A set of stopping rules has been developed in consultation with the DSMB.
In addition, the DCC will oversee and coordinate the collection, standardization, integration, and analysis of study data from the various study components (enrolling sites and core facilities) and the preparation and distribution of the required reports to each of the safety-oversight entities. The DCC will facilitate and monitor regulatory and safety compliance at each site and core laboratory and will conduct site visits to each site and core laboratory to ensure protocol adherence and regulatory compliance, both on a regular basis and for cause.
Assuming the independence and normalcy of the observations, we calculated the sample size using a 2-sample t
-test statistic. The literature21
suggests that the absolute change (effect size, δ) achievable in global LVEF is δ=4% and that the common group SD of the difference of LVEF over time is σD
= 6%. These produced a study sample size of 86, administratively rounded up to 87, with 58 participants in the active group and 29 in the control group. For a regional assessment of left ventricular function, we are assuming an absolute change in regional ejection fraction of δ=6.7% and a common group SD of σD = 9.5%, from the 2004 BOOST clinical trial report7
; these assumptions produced a sample size of 77 patients. On the basis of the assumptions described above for global and regional LV function, the overall anticipated sample size required for LateTIME is 87 patients.
Exact testing for categorical variables and Student's t-testing for continuous variables will be used to evaluate the compatibility of baseline variables between treatment groups. All hypothesis testing, and all effect sizes and their 95% confidence intervals, will be evaluated using the general linear mixed model. Both unadjusted and adjusted treatment effects will be computed for primary and secondary endpoints (); adjustments will be for clinical centers, as well as for baseline covariates whose association with the dependent variable is generally accepted. In keeping with standard methodology for clinical trials, the primary and secondary comparisons will compare the randomized study groups using an intention-to-treat analysis. Logistic regression will be used to evaluate the effect of cell administration on the combined endpoint of death, reinfarction, repeat target-vessel revascularization, hospitalization for congestive heart failure, and ICD placement. No adjustments for multiple comparisons will be made.
TABLE IV. Outcomes in LateTIME; Active versus Placebo Comparisons