II study of intravenous allogeneic mesenchymal stromal cells in acute myocardial infarction

Cytotherapy, 2014; 0: 1e12

Randomized, double-blind, phase I/II study of intravenous allogeneic mesenchymal stromal cells in acute myocardial infarction

ANOOP CHULLIKANA1, ANISH SEN MAJUMDAR1, SANJAY GOTTIPAMULA1, SAGAR KRISHNAMURTHY1, A. SREENIVAS KUMAR2, V.S. PRAKASH3 & PAWAN KUMAR GUPTA3 1

Stempeutics Research Pvt Ltd, Bangalore, India, and Stempeutics Research Manipal, India, 2CARE Hospital Hyderabad, India, and 3M.S. Ramaiah Hospital Bangalore, India

Abstract Background aims. Cell therapy is promising as an exploratory cardiovascular therapy. We have recently developed an investigational new drug named Stempeucel (bone marrowederived allogeneic mesenchymal stromal cells) for patients with acute myocardial infarction (AMI) with ST-segment elevation. A phase I/II randomized, double-blind, single-dose study was conducted to assess the safety and efficacy of intravenous administration of Stempeucel versus placebo (multiple electrolytes injection). Methods. Twenty patients who had undergone percutaneous coronary intervention for AMI were randomly assigned (1:1) to receive intravenous Stempeucel or placebo and were followed for 2 years. Results. The number of treatmentemergent adverse events observed were 18 and 21 in the Stempeucel and placebo groups, respectively. None of the adverse events were related to Stempeucel according to the investigators and independent data safety monitoring board. There was no serious adverse event in the Stempeucel group and there were three serious adverse events in the placebo group, of which one had a fatal outcome. Ejection fraction determined by use of echocardiography showed improvement in both Stempeucel (43.06% to 47.80%) and placebo (43.44% to 45.33%) groups at 6 months (P ¼ 0.26). Perfusion scores measured by use of single-photon emission tomography and infarct volume measured by use of magnetic resonance imaging showed no significant differences between the two groups at 6 months. Conclusions. This study showed that Stempeucel was safe and well tolerated when administered intravenously in AMI patients 2 days after percutaneous coronary intervention. The optimal dose and route of administration needs further evaluation in larger clinical trials (http://clinicaltrials.gov/show/ NCT00883727). Key Words: acute myocardial infarction, bone marrow, cell therapy, mesenchymal stromal cells

Introduction Myocardial infarction is the single largest cause of death worldwide. The Global Burden of Diseases study reported that the incidence of death caused by coronary heart disease will double in developing countries by 2020 [1]. Despite significant advances in treatment, ventricular dysfunction remains the major cause of morbidity and mortality in these patients. Cellular therapy for myocardial infarction (MI) is gaining importance. Intracoronary infusion of different cell populations (circulating progenitor cells, bone marrowederived progenitor cells, bone marrow cells, peripheral blood stem cells, hematopoietic stem cells and allogeneic bone marrow mesenchymal stromal cells) have been used in acute MI (AMI) and in some instances with promising

results [2e6]. There are several publications discussing the role of stem cell therapy in ischemic heart disease [7e10]. Stempeucel is ex vivoecultured allogeneic bone marrowederived mesenchymal stromal cells (BMMSC) that have both myogenic and angiogenic potential and is being explored for its therapeutic potential for regeneration/repairing injured myocardial tissue. Furthermore, these cells are nonimmunogeneic [11e13] and have anti-inflammatory properties and facilitate vasculogenesis by increasing vascular endothelial growth factor (VEGF) level [14,15]. BMMSCs have been shown to home, accumulate and in some cases differentiate around the diseased micro-environment [16,17]. The increased vascular

Correspondence: Pawan Kumar Gupta, MD, Stempeutics Research Pvt Ltd, Akshay Tech Park, No. 72 & 73, 2nd Floor, EPIP Zone, Phase I-Area, Whitefield, Bangalore 560066, India. E-mail: [email protected] (Received 19 March 2014; accepted 14 October 2014) http://dx.doi.org/10.1016/j.jcyt.2014.10.009 ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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Table I. Eligibility criteria. Inclusion criteria Patients with STEMI ages between 20 and 70 years, either men or women with nonechild-bearing potential, after 2 days of successful PCI. Patient has global left ventricular systolic dysfunction with an ejection fraction of <50% and >30%. Electrocardiogram with sign of acute anterior MI with STelevation 2 mm in at least two of the following: leads I, AVL or V1-V6 or electrocardiogram with sign of acute inferoposterior MI with ST-elevation 1 mm on all of the following leads: II, III and V5-V6 or ST-elevation 2 mm in at least two of the leads. Target lesion is located in the proximal section of the left anterior descending, left circumflex or right coronary artery. Patient with AMI within 10 days before intraperitoneal administration. Normal liver and renal function. Able to understand study information provided. Able to give voluntary written consent.

permeability and expression of adhesion molecules such as integrin assist in MSC homing [18e20]. The migratory capacity of these cells is dependent on natural growth factors such as VEGF, stromal cellederived factor-1 (SDF1) and stem cell factor. The expression of VEGF, SDF1 and stem cell factor is highly unregulated in the hypoxic damaged tissue such as cardiac tissue and is responsible for the recruitment of the stem cells to assist in the repair mechanism and subsequent improvement in cardiac function [21e23]. On the basis of the homing properties of these cells, this study was designed to deliver the cells by intravenous route and to evaluate the safety and possible efficacy of Stempeucel in patients with AMI.

Exclusion criteria History of acute/chronic inflammatory condition or severe aortic stenosis or insufficiency; severe mitral stenosis or severe mitral insufficiency. Severe co-morbidity associated with a reduction in life expectancy of <1 year. Advanced renal dysfunction and creatinine 2 mg%. Advanced hepatic dysfunction. Clinically serious and/or unstable intercurrent infection, medical illnesses or conditions that are uncontrolled or whose control, in the opinion of the investigator, may be jeopardized by participation in this study or by the complications of this therapy. Previous MI. Patients already enrolled in another investigational drug trial. History of severe alcohol or drug abuse within 3 months of screening. Women with child-bearing potential or who are pregnant or lactating. Positive test for human immunodeficiency virus 1, hepatitis C virus, hepatitis B virus, syphilis and cytomegalovirus (immunoglobulin M). Patients contraindicated for MRI.

the National Institutes of Health website (http:// clinicaltrials.gov/show/NCT00883727) and was conducted from July 2009 to November 2011. Thirty patients were screened (as per criteria in Table I) after written informed consent was obtained. Computergenerated block randomization was performed centrally to randomly assign 20 patients to Stempeucel or to the placebo group in a 1:1 ratio (Figure 1). An independent data safety monitoring board (DSMB), which consisted of safety physicians and biostatisticians, was established to assess the progress of this study.

Preparation and composition of Stempeucel and placebo Methods Study design and criteria for enrollment This study was a phase I/II randomized, double-blind, placebo-controlled, multicentric trial to evaluate the safety and efficacy of Stempeucel administered to patients with ST-elevated myocardial infarction (STEMI) 2 days after percutaneous coronary intervention (PCI). The study conformed to the Declaration of Helsinki and followed International Conference on HarmonisationeGood Clinical Practice guidelines and was conducted in accordance with the Guidelines for Stem Cell Research and Therapy developed by the Department of Biotechnology and Indian Council of Medical Research jointly in 2007. The protocol was approved by the Drug Controller General of India (Indian Food and Drug Administration) and by the Institutional Ethical Committees of the four participating hospitals in India. The study was registered in

The investigational medicinal product (IMP) Stempeucel was constituted by allogeneic BMMSC obtained from bone marrow aspirates from consenting donors who were not human leukocyte antigen (HLA)matched to the recipients. Healthy bone marrow donors were tested according to 21 Code of Federal Regulations 640, Food and Drug Administration donor suitability and Indian Council of Medical Research guidance for healthy bone marrow donor screening. BMMSCs were obtained from bone marrow samples of healthy donors between the ages of 20 and 35 years after informed consent was obtained. The protocol was approved by the institutional ethics committee. Briefly, 60 mL of bone marrow aspirate was diluted (1:1) with knockout Dulbecco’s modified Eagle’s medium (KO-DMEM; Gibco-Invitrogen, Grand island, New York, USA), and centrifuged at 1800g for 10 min to remove the anti-coagulant. Bone marrow MNCs were isolated by means of the density

Study of mesenchymal stromal cells in myocardial infarction gradient centrifugation (1.077 g/mL) method as described earlier [24]. BMMSCs were isolated by means of plastic adherence from the donor’s bone marrow MNCs and cultured until passage 1. A donor master cell bank constituted of MSCs from individual BM samples was created and maintained under cryopreserved conditions. Subsequently, a working cell bank was prepared by combining MSCs from two or more donors and cryopreserved for manufacturing Stempeucel or by expanding the BMMSCs for additional passages (patent application No. PCT/IB2010/ 055424). For the work described in this report, pooled BMMSCs from three different individual donors were cultured, harvested and characterized at passage 4 and cryopreserved as the final product. The placebo preparation contained multiple electrolytes injection (PLASMA-LYTE A; Baxter, Deerfield, IL, USA). The IMP specification is given in Table II. Expanded BMMSCs (180e220 million cells) were cryopreserved and stored in 15 mL of PLASMALYTE A (multiple electrolytes injection, type 1, United States Pharmacopeia) containing 5% human serum albumin (Baxter Healthcare, California, USA) and 10% dimethyl sulfoxide (Sigma-Aldrich, Irvine, United Kingdom) in a cryobag (MacoPharma, Mouvaux, France). Stempeucel formulation consisted of 4.0  106 BMMSCs/mL, 1.6% human serum albumin with 3.33% dimethyl sulfoxide in multiple electrolytes injection (PLASMA-LYTE A) solution. Placebo consisted of PLASMA-LYTE A filled in 15-mL cryobags. Both Stempeucel and placebo were supplied in similar blinded cryobags. Release criteria Release criteria for Stempeucel included (i) Negative results of the microbiological testing, (ii) endotoxin content of <0.06 endotoxin units/mL, (iii) cell viability (trypan blue exclusion) of 85%, (iv) pH between 7.2 to 7.4, (v) karyotyping to be normal, 46 XY, (vi) measurement of cell surface markers by means of flow cytometry (Table II) and (vii) confirmation to differentiation to osteocytes, chondrocytes and adipocytes. Release criteria for placebo included (i) clear, colorless liquid on visual observation, (ii) pH between 6.5 to 8.0, (iii) endotoxin content of <0.06 EU/mL, (iv) no detection of mycoplasma and (v) pass the sterility test. Safety testing of the cells infused Stempeucel was subjected to karyotyping and soft agar assay. Karyotyping was performed by visualization of chromosomes with the use of the standard G-banding procedure and reported according to the International

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System for Human Cytogenetic Nomenclature (20 metaphases were scored) and were found to be within normal limits. The soft agar assay did not produce any colonies in any of the cell concentrations (2000, 4000 and 8000 cells per well) tested. Immunological profile The immunological profile of all patients was tested before infusion and at 1, 3 and 6 months after infusion of Stempeucel or placebo. Levels were measured to evaluate the immunological response in the recipients to Stempeucel or placebo; these included pro-inflammatory cytokines (interferon-g, interleukin (IL)-1, tumor necrosis factor-a) with the use of enzyme-linked immunosorbent assay kits (Abcam, San Francisco, CA, USA) and lymphocyte profile (cluster of differentiation [CD]3, CD4 and CD8) by means of flow cytometry. Differentiation Differentiation to osteocytes and adipocytes were performed with the use of published protocols [25e27] and to chondrocytes with the use of a kitbased assay. For osteogenic differentiation, Stempeucel was cultured in KO-DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Tauranga, New Zealand), 200 mmol/L glutamax (Invitrogen, Grand island, New York, USA), 108 mol/L dexamethasone (Sigma-Aldrich, St. Louis, Missouri, USA), 30 mg/mL ascorbic acid (Sigma-Aldrich) and 10 mmol/L b-glycerophosphate (Sigma-Aldrich) for 3 weeks. To assess the mineralization, osteogenic mediumetreated cells were then applied for Von Kossa staining to observe calcium deposits (Figure 2). For adipogenic differentiation, Stempeucel was cultured for up to 3 weeks in KO-DMEM supplemented with 10% FBS, 200 mmol/L glutamax (Invitrogen), 1 mmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine, 1 mg/mL insulin and 100 mmol/L indomethacin (all Sigma-Aldrich). Build-up of lipid droplets in the generated adipocytes was visualized by use of oil red O staining (SigmaAldrich) (Figure 2). Chondrogenic differentiation was performed with the use of a kit-based assay (STEMPRO Chondrogenesis Differentiation kit, Invitrogen) (Figure 2). Stempeucel efficiently differentiated in vitro into osteogenic, chondrogenic and adipogenic cells. Flow cytometry For analysis of the surface molecule expression of BMMSCs, the following monoclonal antibodies

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Figure 1. Flow chart depicts recruitment, randomization, treatment and follow-up. HbsAg, hepatitis B surface antigen; CMV, cytomegalovirus).

directly conjugated with fluorochromes were used: cluster of differentiation (CD) 73-phycoerythrin (PE) (clone AD2; Becton Dickinson, San Diego, CA,USA), CD 90-PE (clone 5E10; Becton Dickinson), CD 166-PE (clone 3A6; Becton Dickinson) and CD 105-PE (clone 166707; R&D Systems, Minneapolis, MN, USA) (positive markers >85% positive) and for CD 34-PE (clone 563, Becton Dickinson), CD45efluorescein isothiocyanate (FITC) (clone HI30, Becton Dickinson), CD14-FITC (clone M5E2, Becton Dickinson), CD19-FITC (clone HIB19, Becton Dickinson), HLA-DR-FITC (clone L243, (Becton Dickinson) and CD133-PE (clone AC133, Bergisch-Gladbach, Germany) (negative markers <5% positive) (Table II). Cells were stained with conjugated monoclonal antibodies at room temperature for 30 minutes, washed and fixed with 1% paraformaldehyde. Cells were analyzed with the use of a Guava Bench top flow cytometer (Millipore, Billerica, MA, USA), with Guava Express Pro Software (Version 5.2).

Testing of culture medium for endotoxins, mycoplasma and sterility The endotoxin level was performed by use of the limulus amebocyte lysate test (Gel clot method, Endosafe, Charles River), and mycoplasma testing was performed by use of polymerase chain reactioneenzyme-linked immunosorbent assay (MycoTOOL, Roche, Penzberg, Germany). This method is a kit-based method, validated internally as per International Conference on Harmonisation Q2 R1 [28]. Sterility was tested by means of direct inoculation method according to Indian Pharmacopeia [29].

Infusion parameters Stempeucel or placebo in the cryobag were thawed and re-suspended in 35 mL of PLASMA-LYTE A, resulting in 50 mL of suspension (viability of cells: 95.6%  0.36%; range, 95.32e96.02%; n ¼ 10). Premedication (100 mg of hydrocortisone and 45.5 mg of

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Table II. Investigational medicinal product release criteria.

Data collection

Serial No.

Paper case record forms were used to collect data. Source document verification was performed by monitors independent of the investigators.

1

2 3 4

Description Morphology

Cell count Viability Cell phenotype

Specifications Cells are fibroblastic and spindleshaped in active growing condition (Gibco-Invitrogen, Grand island, New York, USA) Cells are intact and round in shape after the trypsin action 180 to 220 million cells per bag 85% CD73 >85% CD34 <5% CD105 >85% CD45 <5% CD90 >85% CD133 <5% CD166 >85% CD14 <5% CD19 <5% HLA-DR <5%

pheniramine maleate) was given by intravenous route 15e30 minutes before administration of Stempeucel or placebo. Stempeucel was administered at a dose of 2 million cells/kg body weight. The volume of Stempeucel or placebo administered was 0.5 mL/kg body weight. The reconstituted volume of the Stempeucel or placebo was infused into the antecubital vein of the patients forearm through an indwelling cannula over a period of 30e45 minutes with the use of an infusion pump. Oxygen saturation was monitored 30 minutes before and 6 hours after infusion. Criteria for stopping the infusion included development of tachypnea, cyanosis or breathlessness or fall in oxygen saturation <85%. Patients were observed in the clinical facility for at least 24 hours after infusion. All patients received standard medications for AMI according to the treating investigator.

Statistical methods The SAS package (SAS Institute Inc, USA, version 9.2) was used for statistical evaluation. All data are presented as mean  standard deviation. AEs are summarized descriptively by total number of AE for each treatment group by system organ class. The number of patients with AEs was analyzed by means of the c2 test with Yates correction. The efficacy analyses were performed and summarized for improvement in LVEF, end-systolic volume and end-diastolic volume assessed by use of echocardiography, assessment of regional myocardial perfusion by use of SPECT and assessment of total volume of infarct by use of MRI. Change from baseline to 6 months was analyzed with the use of an analysis of variance model with factors for baseline, treatment and compared between two groups. Statistical significance was defined as a 2-sided P value of <0.05. Results Characterization of BMMSCs Stempeucel showed more than 90% expression of CD73 (99.04%), CD90 (94.00%) and CD166 (98.38%) and less than 5% expression of CD34 (0.14%), CD45 (0.06%) and HLA-DR (0.18%).

Clinical assessment Both safety and efficacy parameters were evaluated at baseline, 1 week and 1, 3 and 6 months after infusion of Stempeucel or placebo. Data were unblinded after 6 months and patients were further followed up until 2 years. Safety assessments included monitoring of all adverse events (AEs), assessment of electrocardiogram parameters, hematological (complete blood count) and biochemical parameters (liver function test, renal function test and lipid profile), proinflammatory cytokines, lymphocyte profile, physical examination and vital sign measurements. Efficacy end points included improvement in left ventricular ejection fraction (LVEF) assessed by 2-dimensional echocardiography (ECHO) performed at baseline and at 1, 3, 6, 12 and 24 months, assessment of regional myocardial perfusion by single photon emission computerized tomography (SPECT) and infarct size by magnetic resonance imaging (MRI) performed at baseline and 6 months.

Patients Demographic and baseline data were comparable between Stempeucel and placebo groups (Table III). Safety analysis was performed for all 20 patients who received Stempeucel or placebo. Efficacy analysis was performed in 19 patients, who had at least one post baseline efficacy assessment. Safety of Stempeucel There was no acute infusionerelated toxicity observed during or immediately after Stempeucel injection. There were 39 treatment-emergent adverse events (TEAEs) (AE starting during or after the infusion of the IMP) in the study (Table IV). Eighteen TEAEs were reported in seven patients in the Stempeucel group, and 21 TEAEs were reported in six patients in the placebo group (P ¼ 0.6392). None of the TEAEs in the Stempeucel group was related to

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Figure 2. Stempeucel differentiation to osteocytes, adipocytes and chondrocytes.

the IMP according to the opinion of the investigators and the independent DSMB report. Three serious adverse events (SAEs) were reported during the course of the study (ventricular tachycardia, pericardial effusion and AMI), all of which occurred in the placebo group. Two of these SAEs were assessed as unlikely and the third one (ventricular tachycardia) as probably related to the treatment according to the investigator. Two patients with SAEs recovered completely, whereas the one who had AMI (re-infarction secondary to subacute thrombosis) died because of the SAE on the next day of placebo administration. The hematological and biochemical parameters were comparable between the Stempeucel and placebo arms at various visits (data not reported). The immunological profile (IL-1, tumor necrosis factor-a and interferon-g) and lymphocyte profile (CD3, CD4 and CD8) were also comparable in both the

Stempeucel and placebo arms at various visits (data not reported). These results suggest that allogeneic BMMSC administration did not appear to elicit an immunological response. Efficacy of ECHO There was improvement in LVEF in both the Stempeucel and the placebo groups. At baseline, the mean LVEF was 43.06%  3.63% (range, 38e48%) (n ¼ 10) for the Stempeucel group and 43.44%  4.391% (range, 35e49%) (n ¼ 9) for the placebo group. The mean LVEF increased to 47.80%  8.12% (range, 32e59%) (change, 4.74 percentage points) and 45.33%  8.56% (range, 30e59%) (change, 1.89 percentage points) in the Stempeucel and placebo groups, respectively (P ¼ 0.26), at 6 months. Furthermore, at 2 years, the mean LVEF was 46.96%  7.56% (range, 35e58%) (change, 3.9

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Table III. Demographic and baseline characteristics. Categories Male, n (%) Female, n (%) Asian, n (%) Age (years), mean (SD) Height (cm), mean (SD) Weight (kg), mean (SD) PCI details Primary PCI, n (%) Secondary PCI, n (%) Treated artery LAD, n (%) LCX, n (%) RCA, n (%) Percent stenosisa Pre-PCI 100% stenosis, n (%) Pre-PCI 70e99% stenosis, n (%) Post-PCI 0% stenosis, n (%) Post-PCI 1e30% stenosis, n (1%) PCI procedure Angioplasty with stenting, n (1%) Clot extraction, n (1%) Thrombosuction n (%) Stent type Bare metal, n (%) Drug-eluting, n (%) Titan stent n, (%)

Stempeucel

Placebo

10 (100) 0 10 (100) 47.31 (12.10) 166.8 (8.74) 64.90 (16.99)

8 2 10 47.79 163.1 66.10

(80) (20) (100) (6.48) (4.44) (8.57)

All 18 2 20 47.55 164.9 65.50

(90) (10) (100) (9.45) (7.02) (13.11)

10 (100) 0

8 (80) 2 (20)

18 (90) 2 (20)

8 (80) 0 2 (20)

8 (80) 2 (20) 1 (10)

16 (80) 2 (10) 3 (15)

4 6 9 1

(40) (60) (90) (10)

7 (70) 4 (40) 11 (110) 0

11 10 20 1

(55) (50) (100) (5)

8 (80) 1 (10) 1 (10)

10 (100) 0 0

18 (90) 1 (5) 1 (5)

5 (50) 2 (20) 1 (10)

9 (90) 1 (10) 0

14 (70) 3 (15) 1 (5)

SD, standard deviation; LAD, left anterior descending artery; LCX, left circumflex artery; RCA, right coronary artery. Numbers and percentages are expressed as number of vessels divided by number of patients.

a

percentage points) and 45.00%  10.21% (range, 30e60%) (change, 1.56 percentage points) in the Stempeucel and placebo groups, respectively (P ¼ 0.41) (Figure 3 and Table V). Efficacy of SPECT Total perfusion score improved in both the Stempeucel and the placebo groups from baseline to 6 months. At baseline, the mean total perfusion score was 35.60  5.98 (range, 30e47) for the Stempeucel group and 33.44  8.60 (range, 21e51) for the placebo group, which increased to 36.78  7.34 (range, 30e51) (change, 1.18) and 35.67  8.94 (range, 22e51) (change, 2.23) respectively (P ¼ 0.21) (Table V). Efficacy of MRI At baseline, the mean total infarct volume was 77.09  53.38 mm (range, 14.82e165 mm) in the Stempeucel group and 82.18  58.85 (range, 0e179.63 mm) in the placebo group, which increased to 143.7  117.59 (range, 13.84e327 mm) (change, 66.61 mm) in the Stempeucel group and decreased to 66.12  47.99 (range, 0e141.6 mm) (change, e16.06) in the placebo group at 6 months (P ¼ 0.0562) (Table V).

Discussion The study met its primary end point of safety of Stempeucel in STEMI. This has been demonstrated by physical examination findings, laboratory evaluations including cytokine analysis, lymphocyte profile, electrocardiogram and AE analysis. The incidence of adverse events in the Stempeucel and placebo groups was comparable. Cardiac arrhythmia was not seen in the Stempeucel group, and none of the AEs were related to Stempeucel according to the investigators and the independent DSMB report. The hematological and biochemical parameters and immunological profile were comparable between the Stempeucel and placebo groups at various time points. Hence, it is concluded that Stempeucel is safe when administered by intravenous route in patients with AMI. The biggest fear for the use of allogeneic MSC therapy is transmission of infectious disease related to the use of animal compounds such as FBS (prions, bovine spongiform encephalopathy or other transmissible spongiform encephalopathies) and genetic transformation of cells leading to tumor formation. It has been reported in a few studies that MSC treatment was not associated with tumor formation, infection and transmission of prion diseases [6,30]. Lee et al. [31] followed up 16 patients with ischemic

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Table IV. Summary of treatment-emergent adverse events. Stempeucel (n ¼ 10) System organ class At least one symptom Blood and lymphatic system disorders Cardiac disorders Gastrointestinal disorders General disorders and administration site conditions Infections and infestations Investigations Metabolism and nutrition disorders Musculoskeletal and connective tissue disorders Nervous system disorders Respiratory, thoracic and mediastinal disorders Skin and subcutaneous tissue disorders Vascular disorders

Placebo (n ¼ 10)

Event

n (%)

Event

n (%)

18 1

7 (70) 1 (10)

21 0

6 (60) 0

4 2 1

3 (30) 1 (10) 1 (10)

8 4 2

5 (50) 4 (40) 1 (10)

2 1 1

1 (10) 1 (10) 1 (10)

0 1 1

0 1 (10) 1 (10)

7

4 (40)

2

1 (10)

3 1

1 (10) 1 (10)

0 2

0 2 (20)

0

0

1

1 (10)

0

0

1

1 (10)

stroke who received BMMSCs for a duration up to 5 years. The authors observed that none of the patients in the MSC group showed features suggestive of a prion disease such as myoclonus, rapidly progressing dementia or ataxia. Genetic changes in MSCs during long-term cultures were studied by Bernardo et al. [32], and they found no evidence suggestive of spontaneous transformation of these cells. In addition, Hare et al. [6] also concluded that there is no ectopic tissue formation after 2 years of BMMSC administration. Stempeucel has been tested for its tumorogenic potential in severe combined immunodeficient mice both by intramuscular and subcutaneous routes of injection in two different dose

Figure 3. Effect of Stempeucel on ejection fraction measured by echocardiogram at various time points. P ¼ 0.26, Stempeucel versus placebo at 6 months; P ¼ 0.41, Stempeucel versus placebo at 2 years).

levels: 0.5 million cells and 10 million cells. All the animals were injected once and were observed for a period of 26 weeks at regular intervals for any changes/reaction at the site of injection. Progressively growing tumors were produced in all the animals injected with the positive reference cells (DLD-1 colon cancer cells). Animals in the vehicle control group and the test group showed no significant reaction at the site of injection and no visible signs of nodule/tumor at the site of injection, which was proven histologically. Hence, it was concluded that these cells are nontumorogenic in severe combined immunodeficient mice of either sex until 6 months of follow-up. This is one of the few cardiovascular clinical trials published with the use of allogeneic BMMSCs by intravenous route after primary PCI for AMI. There was no overall effect of Stempeucel treatment on improvement of cardiac function at 6 months or 2 years versus placebo. This result is similar to that reported by Hare et al. [6] at 6-month follow-up. The authors had conducted a dose escalation study (0.5, 1.6 and 5  106 cells/kg) of allogeneic BMMSC in patients with AMI. In addition to proving the safety of MSCs in AMI, this study demonstrated a decrease in ventricular arrhythmias, better pulmonary function and increased LVEF in the BMMSC group compared with the placebo group, though not significant (P ¼ 0.737). Our study is similar to this study in several aspects: type of cells (allogeneic BMMSC), dose (2  106 cells/kg) and follow-up evaluations (ECHO, SPECT and MRI). Stempeucel therapy did not demonstrate an effect on the recovery of left ventricular function or on left ventricular volumes or infarct size. It has also been debated whether measurement of ejection fraction is an ideal tool to evaluate improvement in stem cell trials in AMI [33]. The increase in LVEF measured by ECHO in the Stempeucel group as compared with the placebo group did not correlate with MRI and SPECT findings. Similar findings were seen in a study by Mistry et al. [34], who compared four different imaging modalities (MRI versus ECHO versus contrast ECHO versus SPECT) in patients with recent STEMI. The limits of agreement between the ejection fractions measured by different imaging modalities varied widely, which may be caused by different tracing methods and imaging principles. The authors have recommended that ECHO is preferable from a cost-benefit point of view [34]. In addition, the CHRISTMAS study compared LVEF in 52 heart failure patients by means of ECHO, radionuclide ventriculography and MRI and suggested that ejection fraction measured with these different techniques were not interchangeable [35]. Hence, ejection fraction measured by ECHO may

.41 NA NA NA NA .26 .21 .45 .24 .0562 8.56 45.00  10.21 8.94 NA 10.682 NA 11.35 NA 47.99 NA      45.33 35.67 53.11 45.44 66.12 4.391 8.60 10.031 8.93 58.85      43.44 33.44 49.11 42.33 82.18 NA: These measurements were not performed in 2-year follow-up.

     LVEF (ECHO) Total perfusion score (SPECT) Total perfusion score by gated study (SPECT) LVEF by SPECT Total volume of infarct (mm) (MRI)

43.06 35.60 51.80 45.40 77.09

3.63 47.80 5.98 36.78 7.729 53.22 13.17 45.33 53.38 143.76     

8.12 46.96  7.56 7.34 NA 9.484 NA 14.33 NA 117.59 NA

6 months 6 months Baseline

Table V. Effect of Stempeucel on various cardiac parameters.

Stempeucel

2 years

Baseline

Placebo

2 years

P value (6 months) P value (2 years)

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still be the practical method for evaluation in these patients. Additionally, few clinical trials that used the intravenous route for delivering MSCs have been published. Martin et al. [36] randomly assigned 244 patients with steroid refractory graft-versus-host disease in a ratio of 2:1 to Prochymal (MSCs derived from unrelated volunteer adult donors) or placebo. Patients received eight infusions of 2  106 MSCs/kg over a period of 4 weeks (or volume equivalent for placebo). Those who had a partial response further received four infusions weekly after day 28. The study showed that the addition of Prochymal in patients with graft-versus-host disease with liver or gut involvement showed significant improvement without additive toxicity [36]. Weiss et al. [37] randomly assigned 62 patients with moderate to severe chronic obstructive pulmonary disease in a 1:1 ratio to either non-HLAematched allogeneic MSCs (Prochymal; Osiris Therapeutics Inc) or placebo (vehicle). Patients received four monthly infusions (100  106 cells/infusion) and were followed for 2 years. There were no infusional toxicities and no deaths or SAEs related to allogeneic MSC administration. There were no significant differences in the overall number of adverse events, frequency of chronic obstructive pulmonary disease exacerbations or worsening of disease in patients treated with allogeneic MSCs. Hence, it was concluded that systemic allogeneic administration of MSCs through the intravenous route is safe [37]. We included patients who had AMI within 10 days of Stempeucel or placebo injection and injected Stempeucel after 2 days of PCI. There have been studies focused on determining the appropriate timing of stem cell therapy albeit with the use of different types of cells and different routes of administration. The TIME study has shown that timing of administration of stem cells (either 3 days or 7 days) after MI is unlikely to have any major impact on the outcome [38]. Furthermore, the LateTIME randomized trial found that bone marrow cells injected through the intracoronary route 2e3 weeks after PCI did not have beneficial effects on cardiac function at 6 months [39]. Recently, the SWISS-AMI trial has reported that in patients with STEMI, either early (5e7 days) or late (3e4 weeks) administration of BM-MNC did not improve global LV function at 4-month follow-up [40]. Although these studies administered BMMNC through the intracoronary route, it appears that timing of stem cell therapy after AMI does not have much impact on clinical outcomes. The beneficial effect of MSCs may be due to the angiogenic potential and anti-inflammatory and antiapoptotic properties effected by the release of

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numerous cytokines. These cells play a vital supportive role within the marrow microenvironment, and their effects are mediated through cell-to-cell contact and through paracrine signaling [41e43]. MSCs expressed genes for a wide spectrum of arteriogenic cytokines such as VEGF, angiopoetin-1, fibroblast growth factor, matrix metalloproteinases, placental growth factor, IL-1 and IL-6, insulin-like growth factor, SDF, plasminogen activator [41], hepatocyte growth factor [42] and insulin growth factors [43]. Cytokines such as VEGF and fibroblast growth factors 1 and 2 can cause angiogenesis through either an autocrine or paracrine manner on the capillary endothelium [44]. Various types of stem cells have been used in AMI trials and include BMMNCs [45e47], granulocyte colony-stimulating factoremobilized bone marrow cells [48], autologous MSCs [49] and allogeneic MSCs [6]. Yet, consensus has not been reached on the ideal cell type, route and dose required to be administered for AMI. There are several studies that have shown improvement in cardiac function after administration of BMMNC in AMI [46,50e52]. A Cochrane meta-analysis has shown that there was an increase in ejection fraction in stem celletreated patients compared with placebo in the short term as well as the long term [53]. Another meta-analysis concluded that intracoronary BMMNC administration was associated with improvement in cardiac function and remodeling in STEMI [54]. Chen et al. [49] evaluated the effect of autologous BMMSCs delivered into the coronary arteries in patients with AMI. Improvements in myocardial perfusion and LVEF were observed in the cell group in comparison to placebo. This study also established that MSC therapy was safe in patients with AMI patients, with reduction in incidence of ventricular arrhythmia. The increase in the LVEF values observed with patients given Stempeucel must be examined in clinical trials with a large number of subjects. Furthermore, allogeneic MSCs may be uniquely suited for the early treatment of AMI. The ease of isolation, ex vivo expansion potential and allogeneic use of these cells would allow for a readily available off-the-shelf therapy that would be challenging with autologous cell strategies. In a recent publication, Hare et al. [55] have shown that the use of allogeneic and autologous MSCs was safe when administered intravenously; furthermore, they demonstrated that the use of allogeneic and autologous MSCs resulted in improvement of patient functional capacity, quality of life and ventricular remodeling in patients with ischemic cardiomyopathy [55]. Our study has a few limitations. We included patients with a wide range of ejection fractions (>30%

to <50%). Although we observed no differences in outcome of cell therapy in patients in lower ranges of ejection fractions versus higher ranges, the sample size becomes too small for this type of interpretation. Second, because this is a clinical trial, cells were not labeled to track the homing to the heart. Third, the sample size of the study was too small to detect any significant benefit in the two groups. Last, we did not follow up the patients for sufficiently long periods (eg, 5 years) for evaluating survival benefits. To summarize, this study has shown that intravenous administration of Stempeucel is safe in AMI. Further studies in a large number of patients, probably with the use of different subsets of AMI and different routes of administration with more robust end points, may be required to prove the efficacy of these cells. Acknowledgments This study was funded by Stempeutics Research Private Limited. Anoop Chullikana, Anish Sen Majumdar, Sanjay Gottipamula, Sagar Krishnamurthy and Pawan Kumar Gupta are employees of Stempeutics Research Private Limited. References [1] Murray C, Lopez A. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Disease, Injuries and Risk Factors in 1990 and Projected to 2020. Boston, MA: Harvard University Press; 1996. [2] Leistner DM, Fischer-Rasokat U, Honold J, Seeger FH, Schächinger V, Lehmann R, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol 2011 Oct;100:925e34. [3] Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S, et al. Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months’ follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation 2006;113:1287e94. [4] Kang H-J, Lee H-Y, Na S-H, Chang S-A, Park K-W, Kim H-K, et al. Differential effect of intracoronary infusion of mobilized peripheral blood stem cells by granulocyte colony-stimulating factor on left ventricular function and remodeling in patients with acute myocardial infarction versus old myocardial infarction: the MAGIC Cell-3-DES randomized, controlled trial. Circulation 2006;114(1 Suppl):I145e51. [5] Mansour S, Roy D-C, Bouchard V, Stevens LM, Gobeil F, Rivard A, et al. One-Year Safety Analysis of the COMPAREAMI Trial: Comparison of Intracoronary Injection of CD133þ Bone Marrow Stem Cells to Placebo in Patients after Acute Myocardial Infarction and Left Ventricular Dysfunction. Bone Marrow Res [Internet]. 2011 [cited 2014 Jun 18];2011. Available from: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC3200002/

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