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For peer review only

Rationale and design of a multicenter, prospective, randomized, controlled clinical trial to evaluate the efficacy

of the Adipose Graft Transposition Procedure (AGTP) in patients with a myocardial scar: the AGTP II trial.

Journal: BMJ Open

Manuscript ID bmjopen-2017-017187

Article Type: Protocol

Date Submitted by the Author: 11-Apr-2017

Complete List of Authors: Gastelurrutia, Paloma; Fundacio Institut d'Investigacio en Ciencies de la Salut Germans Trias i Pujol, Gálvez-Montón, Carolina; Fundacio Institut d'Investigacio en Ciencies de la Salut Germans Trias i Pujol Cámara, Maria Luisa; Hospital Universitari Germans Trias i Pujol Bustamante-Munguira, Juan; Hospital Universitari de Girona Doctor Josep Trueta García-Pavía, Pablo; Hospital Universitario Puerta del Hierro Majadahonda Avanzas, Pablo; Hospital Universitario Central de Asturias San Roman, J. Alberto; ICICOR- Hospital Clinico Universitario, Cardiology Pascual-Figal, Domingo; Hospital Clinico Universitario Virgen de la Arrixaca de Teresa, Eduardo ; Hospital Universitario Virgen de la Victoria Crespo-Leiro, Maria G.; Complexo Hospitalario Universitario A Coruna Manito, Nicolás ; Hospital Universitari de Bellvitge N��ez, Julio; Hospital Cl�nico Universitario, Cardiology Fernandez-Aviles, Francisco ; Hospital General Universitario Gregorio Maranon Caballero, Ángel; Hospital Universitari Germans Trias i Pujol Teis, Albert; Hospital Universitari Germans Trias i Pujol Lupón, Josep; Hospital Universitari Germans Trias i Pujol Brugada, Ramón ; Hospital Universitari de Girona Doctor Josep Trueta Martín, Carlos; Hospital Universitario Puerta del Hierro Majadahonda Silva, Jacobo; Hospital Universitario Central de Asturias Revilla, Ana; Hospital Clínico Universitario, Cánovas, Sergio J.; Hospital Clinico Universitario Virgen de la Arrixaca Melero, Jose María; Hospital Universitario Virgen de la Victoria Cuenca-Castillo, Jose; Complexo Hospitalario Universitario A Coruna, Cardiac Surgery González-Pinto, Ángel; Hospital General Universitario Gregorio Maranon Bayes-Genis, Antoni; Hospital Universitari Germans Trias i Pujol

<b>Primary Subject Heading</b>: Cardiovascular medicine

Secondary Subject Heading: Cardiovascular medicine

Keywords: Cardiac regeneration, adipose progenitor cells, chronic myocardial infarction, tissue engineering, pericardial adipose graft, Clinical trials < THERAPEUTICS

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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Rationale and design of a multicenter, prospective, randomized, controlled clinical

trial to evaluate the efficacy of the Adipose Graft Transposition Procedure (AGTP)

in patients with a myocardial scar: the AGTP II trial.

Paloma Gastelurrutia1,2, Carolina Gálvez-Montón1,2, Maria Luisa Cámara3, Juan

Bustamante-Munguira2,3,4, Pablo García-Pavia2,5, Pablo Avanzas6, J. Alberto San

Román2,7, Domingo Pascual-Figal2,8, Eduardo de Teresa2,9, Maria G. Crespo-Leiro2,10,

Nicolás Manito11, Julio Núñez2,12, Francisco Fernández-Avilés2,13, Ángel Caballero3,

Albert Teis3, Josep Lupón2,3,14, Ramón Brugada2,4, Carlos Martín5, Jacobo Silvia6, Ana

Revilla-Orodea2,7 Sergio J. Cánovas8, Jose M Melero2,9, Jose J Cuenca-Castillo2,10,

Angel Gonzalez-Pinto13, Antoni Bayes-Genis1,2,3,14

From: 1* ICREC Research Program, Fundació Institut d´Investigació en Ciències de la

Salut Germans Trias i Pujol (IGTP), Badalona, Spain; 2 CIBERCV, Instituto de Salud

Carlos III, Madrid, Spain, 3 Hospital Universitari Germans Trias i Pujol, Badalona,

Spain; 4 Hospital Universitari de Girona Josep Trueta, Girona, Spain; 5 Hospital

Universitario Puerta del Hierro, Madrid, Spain; 6 Hospital Universitario Central de

Asturias, Oviedo, Spain; 7 Hospital Clínico Universitario, Valladolid; ICICORELAB,

Valladolid, Spain; 8 Hospital Clínico Universitario Virgen de la Arrixaca, Universidad

de Murcia, IMIB-Arrixaca, Murcia, Spain; 9 Hospital Clínico Universitario Virgen de la

Victoria de Málaga, IDIMA, Málaga, Spain; 10 Complejo Hospitalario Universitario A

Coruña (CHUAC), A Coruña, Spain; 11 Hospital Universitari de Bellvitge, L`Hospitalet

de Llobregat, Spain; 12 Hospital Clinic de Valencia, Valencia, Spain; 13 Hospital General

Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio

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Marañón, Madrid, Spain; 14Department of Medicine, Universitat Autònoma de

Barcelona, Barcelona, Spain.

* Trial sponsor

Running head: AGTP II trial: rationale and design.

Word count: 2879

Corresponding Author:

Paloma Gastelurrutia, PhD. Germans Trias i Pujol Health Research Institute.

Can Ruti Campus, Camí de les Escoles s/n 08916. Badalona (Barcelona).

E-mail: [email protected]

Telephone +34 93 497 86 70; Fax +34 93 497 89 39

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ABSTRACT

Rationale: Cardiac adipose tissue is a source of progenitor cells with regenerative

capacity. Studies in rodent demonstrated that the intramyocardial delivery of cells

derived from this tissue improves cardiac function after MI. We developed a new

reparative approach for damaged myocardium that integrates the regenerative properties

of cardiac adipose tissue with tissue engineering. In the Adipose Graft Transposition

Procedure (AGTP), we dissect a vascularized flap of autologous pericardial adipose

tissue and position it over the myocardial scarred area. Following encouraging results in

acute and chronic MI porcine models, we performed the AGTP-I clinical trial

(NCT01473433, AdiFLAP Trial) to evaluate safety in patients with chronic MI

undergoing CABG. The good safety profile and trends in efficacy warranted a larger

trial.

Objective: The AGTP will be able to reduce the necrotic mass ratio of a non-

revascularizable MI, defined as a reduction of 10% in the estimated infarct size.

Setting and participants: The AGTP II Trial (NCT02798276) is an investigator-

initiated, prospective, randomized study to assess the efficacy of the AGTP in 108

patients with non-revascularizable MI who are admitted to 10 tertiary hospitals in Spain.

Intervention and primary and secondary outcome measures: Patients will be

assigned to standard clinical practice or AGTP. The primary endpoint is change in

necrotic mass ratio by gadolinium enhancement at 91, 365 days. Secondary endpoints

include improvement in regional contractibility by MRI at 91, 365 days; changes in

functional MRI parameters (LVEF, left and right ventricular geometric remodeling) at

91, 365 days; levels of NT-proBNP at 7, 91, 365 days; appearance of arrhythmias from

24h-Holter monitoring at 24h, 91, 365 days; all-cause death or re-hospitalization at 365

days; and cardiovascular death or re-hospitalization at 365 days.

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Ethics and dissemination: The Institutional Review Board approved the trial and will

comply with the Declaration of Helsinki, with all patients providing informed consent.

It may offer a novel, effective, and technically simple technique for patients with no

other therapeutic options. The results will be submitted to indexed medical journals and

national and international meetings.

Trial registration number: ClinicalTrials.gov: NCT02798276.

Strengths and limitations of this study:

- The AGTP is the first tissue engineering technique to use local adipose tissue

without laboratory manipulation.

- It is technically simple, requiring no learning curve for cardiac surgeons who

will simply dissect structures that are ordinarily modified in CABG surgery.

- There is no requirement for additional or expensive materials, nor do any ethical

or social constraints exist as the autologous tissue is sourced from a site in close

proximity to the injury.

- It minimizes the complications that can arise from the introduction of exogenous

stem cells or stem cell-derived cardiomyocytes, as well as those associated with

gene therapy.

- Access to local adipose tissue is also a critical aspect of the AGTP because its

factibility may be limited in patients with very little pericardial fat.

Keywords: Cardiac regeneration, adipose progenitor cells, chronic myocardial

infarction, tissue engineering, pericardial adipose graft, clinical trials.

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RATIONALE

Cardiac adipose tissue for heart regeneration

The regenerative capacity of the heart is limited. After a myocardial infarction (MI)

there is a massive loss of cardiac muscle that overloads the surviving myocardium and

ultimately leads to heart failure.1 Our group has focused on cardiac adipose tissue as a

source of progenitor cells with regenerative capacity, and as a biological matrix for

salvaging injured myocardium following MI. Under physiologic conditions, cardiac

adipose tissue displays biochemical and thermogenic cardio-protective properties.

Further, during pathological scenarios, cardiac adipose tissue can locally affect the heart

via the vasocrine or paracrine secretion of proinflammatory cytokines.2,3 We identified

and characterized a population of human adult mesenchymal-like cells derived from

cardiac adipose tissue (adipose tissue mesenchymal stem cells [cATMSCs]). Despite

residing in an adipocytic environment, cATMSCs have an inherent cardiac-like

phenotype and may play a role in heart homeostasis as well as act as a cellular reservoir

for myocardial tissue renewal. In vivo studies in murine models showed that the

intramyocardial delivery of cATMSCs improves cardiac function, reduces infarct size,

and avoids infarct-related reduction of wall thickness following MI.4 Moreover,

following the application of cATMSCs, cellular niches within the host myocardium

secrete pro-angiogenic factors and differentiate into an endothelial lineage that can

support local angiogenesis. Collectively, these preliminary results encouraged us to

expand the therapeutic uses of cATMSCs.

A new approach: preclinical trials

Our next aim was avoiding any damage that could be caused by intramyocardial

injection or the use of cellular material from allogeneic sources. Consequently, we

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developed a new approach that integrates cATMSC therapy with tissue engineering to

repair damaged myocardium (Figure 1). This innovative procedure involves the

dissection of a vascularized flap of autologous pericardial adipose tissue to cover the

infarcted myocardium.

Initially we tested this strategy, named Adipose Graft Transposition Procedure

(AGTP), in a porcine acute MI model. After a lateral thoracotomy to expose the

pericardium, adipose tissue from the fibrous pericardium was carefully dissected, while

maintaining its vascularization, to obtain a natural scaffold containing native

cATMSCs. The flap was reserved and a pericardiotomy was performed to access the

heart. After MI induction by permanent coronary artery ligation, the adipose pericardial

graft was adhered to healthy myocardium fully covering the ischemic area. At the 30-

day follow-up, we observed that AGTP-treated animals manifested a five-fold

improvement in the left ventricular ejection fraction (LVEF) vs. control animals, and a

smaller infarct size and myocardial remodeling.5 The AGTP was performed 30 min

after MI induction (an acute scenario not used in the clinical setting), thus we also re-

tested the AGTP in a chronic MI swine model, positioning the flap 2 weeks after

coronary coil deployment. Once again, the AGTP proved valuable in reducing the left

ventricular infarct area by as much as 34% vs. control animals, and in promoting

neovascularization in the infarcted myocardium.6 Microscopic evaluation revealed that

both models promoted vascular bridging between the ischemic myocardium and the

adipose graft. In terms of safety, the AGTP did not triggered arrhythmic events or the

sudden death of any of the included animals; therefore, we proceeded with further

bench-to-bedside studies.

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Safety clinical trial

The AGTP-I clinical trial (NCT01473433, AdiFLAP Trial) was designed to evaluate the

safety of the AGTP in patients with chronic MI undergoing a coronary artery bypass

graft (CABG) for other myocardial regions.7 Patients were enrolled if they were CABG

candidates and had an established, chronic, MI lesion that was not in the area supplied

by the CABG target vessel(s). Patients were assessed with late gadolinium enhancement

(LGE) MRI to identify a chronic non-revascularizable area to guide the AGTP. The

main safety endpoints were arrhythmia, hospital admission, and death during the 12-

month follow-up. Secondary endpoints included change in cardiac geometry and

function as determined by cardiac MRI, biomarkers of cardiac injury or strain (e.g.

troponin T and NT-proBNP), NYHA class, and Framingham-derived clinical score.7

Ten male patients were recruited from January 2012 to November 2013 in a

single center. All were candidates for myocardial revascularization with an established

chronic transmural myocardial scar in a distally non-revascularizable area. They did not

differ in disease severity or surgical risk variables, with a necrotic non-revascularizable

mass of 34±13 g vs. 25±4 g and a LVEF of 41±18% vs. 42±15% in the AGTP-treated

vs. the control arm, respectively. Patients were randomly allocated to either the control

or AGTP-treated arm; for both groups, the area that could be revascularized was treated

with CABG. The non-revascularizable scar was left untouched in the control arm and

was covered by an autologous pericardial adipose graft in the AGTP arm. The AGTP

location was inferior in four cases and anterior in one. No differences in safety were

observed in terms of clinical or arrhythmic events as assessed by clinical visits,

electrocardiogram (ECG), and 24-hour Holter monitoring during the 12-month study

duration. On follow-up, the AGTP-treated arm showed a borderline smaller left

ventricular end-systolic volume (LVESV; p=0.09) and necrosis ratio, assessed with

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LGE MRI (p=0.06). Interestingly, the AGTP patient with the largest necrotic area and

most dilated chambers experienced the most remarkable reduction in necrotic mass size

(-10.8%) and ventricular volumes (left ventricular end-diastolic volume (LVEDV): -

55.2 mL and LVESV: -37.8 mL) at the 1-year follow-up. Furthermore, this patient’s Q

waves vanished from leads I, II, and III in follow-up ECGs. Based on these results and

the good safety profile of the AGTP, we are undertaking a larger trial to assess the

efficacy of the AGTP.

DESIGN AND METHODS

Study aim

To compare the efficacy of the AGTP vs. standard clinical practice in reducing a non-

revascularizable MI at 3 months and/or 1 year in patients undergoing CABG of other

distal myocardial areas.

Primary hypothesis

The AGTP will be able to reduce the necrotic mass ratio of a non-revascularizable MI,

defined as a reduction of 10% in the estimated infarct size as determined by LGE MRI

at 91 and/or at 365 days in patients undergoing CABG of other myocardial areas.

Secondary hypothesis

1. Compared with those receiving the standard clinical practice, patients

undergoing the AGTP will show:

• Better regional contractibility

• Increased LVEF

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• Less remodeling (assessed by MRI)

• Lower levels of biomarkers (NT-proBNP and high-sensitive troponin T)

2. The AGTP will be safe with no increase in arrhythmias (24-h Holter monitoring)

or outcomes (all cause death or readmission, cardiovascular death or

readmission).

Trial population

Male or female patients aged ≥18 years, with a Q wave in the ECG and a history of a

transmural MI (≥50% late gadolinium enhancement) that is anatomically non-amenable

for surgical revascularization, who are candidates for CABG of other myocardial areas

will be included and randomized after they agree to participate and sign the informed

consent. Exclusion criteria include severe valvular disease that could be repaired

surgically, candidacy for surgical ventricular remodeling, contraindication for MRI,

extra-cardiac disease with a vital prognosis of <1 year, severe renal or hepatic failure,

previous cardiac surgery, pregnancy, or breastfeeding.

Trial design

The AGTP II Trial (NCT02798276) is an investigator initiated, prospective,

randomized, controlled, multicenter (10 tertiary hospitals in Spain) study comparing

two arms of patients with chronic MI undergoing CABG with a distally non-

revascularizable area. The protocol (V.2.2, 04th April 2017) has been approved by the

corresponding Institutional Review Board. One-hundred and eight patients with a

myocardial scar who are candidates for revascularization of other myocardial areas will

be included at the participating sites in Spain by the cardiology and cardiac surgery

team. These selected hospitals have a strong clinical cardiology and cardiac surgery

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team prepared to work together, along with adequate facilities for cardiac MRI. The

AGTP II trial is currently enrolling patients since the 1st of May 2017.

Randomization and blinding

After providing informed consent to the clinical professional, patients will be centrally

randomized at a 1:1 ratio into two arms by allocation order. The randomization scheme

was computer-generated by statisticians from the IMIM (Barcelona, Spain) and once a

patient is enrolled in the study, the local surgeon or study coordinator will contact the

coordinator center to be provided with the allocation group. Treatment allocation will be

blind to the patient and to the clinical team that performs the follow-up.

Study procedure and follow-up

The AGTP will be taught to other cardiac surgeons at study centers by a cardiac surgeon

(ML.C.3) skilled in the procedure who performed all surgeries in the AGTP-I trial.

Patients will be included in two arms, stratified according to the age of the scar (Figure

2):

A) Treated/AGTP patients (n=54): The surgeon will perform a sternotomy for the

CABG of revascularizable vessel(s). The pericardial tissue will be gently dissected

while maintaining its functional vascularization. The pericardium will be excised,

and the adipose flap fixed over the non-revascularizable scar using surgical glue.

Patients randomized to this AGTP group without sufficient fat tissue within the flap

will be excluded from the study, and analyzed in a subgroup.

B) Control patients (n=54): The surgeon will perform a sternotomy for the CABG of

the revascularizable vessel(s). The distally non-revascularizable area will be left

untouched (the current standard-of-care).

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Both groups of patients will be hospitalized for the usual time after a CABG. In

the AGTP-1 trial we found that the AGTP does not prolong the in-hospital stay. To

ensure adequate assessment of efficacy, all patients will undergo several tests (see Table

1) including MRI, ECG, 24-h Holter monitoring, and blood tests. A clinical cardiologist

blinded to the randomization group will conduct follow-up visits (Table 1). Additional

patient visits can also be made, if required, at the discretion of the clinical team. Cardiac

MRI will be performed in a standardized manner at each participant center with 1.5 T

equipment and assessed by a centralized core lab (ICICORELAB, Valladolid, Spain).

All images will be captured under ECG monitoring and in apnea. Images will be

obtained with three plane locators. For the study of ventricular function, we will obtain

steady-state free precession (SSFP) sequences (TrueFISP, bTFE, FIESTA) at short axis

(8 mm slices from the apex to the mitral ring), four-chamber and two-chamber views.

For the study of myocardial fibrosis/necrosis, a gadolinium-based contrast agent will be

intravenously administered and the necrotic myocardium will be assessed using delayed

enhancement. The necrotic size and the salvaged myocardium will be expressed as a

percentage of the total LV mass. In addition, the necrotic area will be expressed in

grams. All the results will be given in absolute numbers and normalized by body surface

area. In addition, LVESV and LVEDV will be assessed. Interobserver and intraobserver

variability will be assessed using randomly chosen (15%) MRI studies.

Risks and benefits

This clinical study is designed and will be implemented in accordance with the

Declaration of Helsinki, with all patients providing informed consent and patients’ data

being anonymized. The study has been assessed and approved by the hospital Ethics

Committee (reference number AC-16-025). Based on the preclinical and clinical AGTP

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phase I study, no safety issues are anticipated. Potential perioperative risks include

pericardium dissection and pericardial fat manipulation, both of which are normal

processes in conventional CABG. Surgical glue (Glubran2®, GEM srl, Viareggio, Italy)

is routinely used in surgeries, including conventional cardiac interventions in clinical

practice. Moreover, a high sensitivity troponin T curve will be obtained during the

surgical procedure to assess the patient’s wellbeing. Perioperative infarct will be

assessed according to the published definition of CABG-related MI.8 Finally, despite no

arrhythmic events detected in the AGTP-I trial, patients will be closely monitored

during their hospital admission. When discharged, the first visit will be 1 week post-

discharge, with follow-up extending to 1 year. Patients will be advised to contact the

clinical team in case of any discomfort. Patients are covered by an insurance policy.

Based on preclinical results, the expected benefits of the AGTP II trial include infarct

size reduction and an improvement in cardiac function. These benefits may be detected

by MRI, changes in surrogate biomarker concentration, and by clinical outcomes.

Criteria for study withdrawal

Patients may voluntarily leave the study at any time without giving any reason and with

no consequences for their medical care. They will also be withdrawn if any exclusion

criterion is detected after the informed consent signing, if they are lost to follow-up, or

fail to collaborate. However, irrespective of the reason of withdrawal, the investigators

will make all efforts to perform the end-of-study visit and collect all available

information from the patient.

Trial outcomes

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The main outcome is the proportion of patients who have a smaller necrotic mass ratio

of a non-revascularizable MI, defined as a reduction of 10% in the estimated infarct size

determined by LGE MRI at 91 and/or 365 days compared with the baseline assessment

(% and/or grams).

Secondary outcomes:

• Improvement in regional contractibility by MRI (time frame: 0-3-12 months)

• Percent changes in functional parameters by MRI: LVEF, right ventricular

ejection fraction (time frame: 0-3-12 months)

• Changes in mL in ventricular volumes by MRI: LVESV, LVEDV, right

ventricular end-systolic volume, right ventricular end-diastolic volume (time

frame: 0-3-12 months)

• Changes in L/min in cardiac output by MRI (time frame: 0-3-12 months)

• Changes in levels of natriuretic peptides (time frame: 0-1 week and 3-12

months)

• Adverse event rate and description: arrhythmia by 24-h Holter (time frame:

0-3-12 months), all-cause death or re-admission (time frame: 12 months),

and cardiovascular death or re-admission (time frame: 12 months).

Data analyses and sample size justification

Statistical analyses will be performed for all patients randomized and operated on. Any

randomized patient who abandons the study (for any cause) before the surgical

procedure will not be followed, but will be registered to investigate possible non-

candidacy for the AGTP. A subgroup analysis will be performed for patients with a

non-revascularizable MI ≤2 months prior to surgery. The aim of this analysis is to

assess the potentially increased benefit of the AGTP to this subgroup of patients in

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terms of remodeling and scar size/regional contractility vs. patients with older

chronified scars.

Descriptive analyses will be performed at the first step. Categorical variables

will be described by frequencies and percentages. Continuous variables will be

described by means and standard deviations or medians and interquartile ranges in case

of skewed distribution. Differences between groups will be compared using Student’s t-

test or one-way ANOVA for multiple comparisons, with Tukey’s test for post hoc

analyses. The comparison of continuous variables between groups will be performed

using analysis of variance for unpaired data once normality is demonstrated

(Kolmogorov-Smirnov test); otherwise, a nonparametric test (Mann-Whitney or

Kruskal-Wallis test) will be used. The MRI data will be analyzed as repeated measures

using ANOVA with Greenhouse–Geisser correction. A 2-sided p<0.05 will be

considered significant. Statistical analyses will be performed using SPSS statistical

package 19.0.1 version (SPSS Inc, Chicago, IL, USA). Monitoring will be performed by

the sponsor center, IGTP.

Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 54

subjects are necessary in the standard of care group and 54 in the AGTP group to

recognize as statistically significant a difference ≥10% reduction in baseline-to-12

month LGE MRI ratio change. Baseline LGE MRI ratio is estimated in 20% ± 7

according to AGTP-I trial.7 The correlation coefficient between the initial and final

measurement was assumed as 0.875. It has been anticipated a drop-out rate of 10%.

DISCUSSION

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The sequelae of MI have been decreasing since the establishment of early

revascularization procedures worldwide. However, a large number of patients still suffer

from myocardial scarring, including those treated outside of the optimal reperfusion

time window, or not having viable vessels available for CABG. Currently, experimental

cardiac tissue engineering approaches fall into two main categories: scaffolds and cells.

Scaffolds are sometimes supplemented with cytokines, growth factors, and peptides.

They may be of natural or synthetic origin and should recreate the myocardial

environment, support vascularization, ensure electrical and mechanical properties for

optimal host tissue coupling, and ultimately be degradable. Cells from several sources

have been tested (i.e. embryonic stem cells, adipose tissue-derived stem cells, cardiac

progenitor cells, skeletal myoblasts, induced pluripotent stem cells); although, to date,

the optimal cell population is not identified. From the clinical perspective, the main

limiting factors are the requirement for previously banked cells of autologous or

allogeneic origin, and, more importantly, the need for a proximal in-vitro set-up outside

of the confines of the operating theatre.

The AGTP is the first tissue engineering technique to use local adipose tissue

without laboratory manipulation. This approach minimizes the complications that can

arise from the introduction of exogenous stem cells or stem cell-derived

cardiomyocytes, as well as those associated with gene therapy (i.e. risks of

contamination due to laboratory manipulation, infection, immunoreactivity, viral

recombination, etc.). Access to local adipose tissue is a critical and advantageous aspect

of the AGTP. Adipose tissue is a natural niche of progenitor cells, with proven efficacy

in tissue regeneration and disease treatment.9 Based on previous studies, the AGTP may

stimulate neovascularization and improve local microcirculation and metabolism.4-6 The

paracrine effects derived from adipose-derived MSCs might also contribute to

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improving the microenvironment at the scar site, thereby enhancing myocardial

contractility. Further enrichment with exosomes, growth factors, and/or progenitor cells

could form the basis of future means to potentiate the regenerative capacity. Thus, with

this trial, we could establish a new concept in cardiac tissue engineering.

To summarize, the AGTP is technically simple, requiring no learning curve for

cardiac surgeons who will simply dissect structures that are ordinarily modified in

CABG surgery. Furthermore, there is no requirement for additional or expensive

materials, nor do any ethical or social constraints exist as the autologous tissue is

sourced from a site in close proximity to the injury.7

Funding Sources: The AGTP II trial is an investigator initiated study, sponsored by the

Germans Trias i Pujol Research Institute. It is supported by grants from Ministerio de

Educación y Ciencia (SAF2014-59892-R), Ministerio de Economía y Competitividad

(Juan de la Cierva, JCI-2012-14025), Fundació La MARATÓ de TV3 (201502,

201516), and Generalitat de Catalunya (SGR 2014 , CERCA Programme and PERIS

Programme, Departament de Salut SLT002/16/00234, SLT002_16_00209). Additional

funds will be provided by Red de Terapia Celular - TerCel (RD16/0011/0006) and

CIBER Cardiovascular (CB16/11/00403), as part of the Plan Nacional de I+D+I, and

cofunded by ISCIII-Sudirección General de Evaluación and Fondo Europeo de

Desarrollo Regional (FEDER), and “La Caixa” Banking Foundation. This work is

developed within the AdvanceCat project with the support of ACCIÓ (Catalonia Trade

& Investment; Generalitat de Catalunya) under the Catalonian European Regional

Development Fund (ERDF) operational program 2014-2020.

Contributorship statement: PG, CGM and ABG conceived the study. All authors

participated in the development of the protocol. PG, CGM, JBM, DPF, AT, ARO and

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ABG raised the manuscript. MLC, PGP, PA, ASR, ET, MCL, NM, JN, FFA, AC, JL,

RB, CM, JS, SJC, JMM, JCC and AGP revised the manuscript for relevant scientific

contents; AT, ASR, MCL and ARO with specific comments on imaging techniques and

JL and JL on statistical requirements. All authors approved the final version of the

manuscript.

Competing interests: None.

Data sharing statement: In addition to the paper and the protocol, the informed

consent documents and case report forms are available on request.

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REFERENCES

1. Olivetti G, Capasso JM, Meggs LG, et al. Cellular basis of chronic ventricular

remodeling after myocardial infarction in rats. Circ Res 1991;68:856-69.

2. Iacobellis G, Ribaudo MC, Assael F, et al. Echocardiographic epicardial adipose

tissue is related to anthropometric and clinical parameters of metabolic syndrome: a new

indicator of cardiovascular risk. J Clin Endocrinol Metab. 2003;88:5163-5168.

3. Mazurek T, Zhang L, Zalewski A, et al. Human epicardial adipose tissue is a source

of inflammatory mediators. Circulation 2003;108:2460-2466.

4. Bayes-Genis A, Soler-Botija C, Farré J, et al. Human progenitor cells derived from

cardiac adipose tissue ameliorate myocardial infarction in rodents. J Mol Cell Cardiol.

2010;49(5):771-80.

5. Gálvez-Montón C, Prat-Vidal C, Roura S, et al. Transposition of a pericardial-

derived vascular adipose flap for myocardial salvage after infarct. Cardiovasc Res.

2011;91(4):659-67.

6. Gálvez-Montón C, Prat-Vidal C, Roura S, et al. Post-infarction scar coverage using a

pericardial-derived vascular adipose flap. Pre-clinical results. Int J Cardiol.

2013;166(2):469-74.

7. Bayes-Genis A, Gastelurrutia P, Cámara ML, et al. First-in-man safety and efficacy

of the Adipose Graft Transposition Procedure (AGTP) in patients with a myocardial

scar. EBioMedicine 2106;7:248-54.

8. Thygesen K, Alpert JS, Jaffe AS, et al. Joint ESC/ACCF/AHA/WHF Task Force for

Universal Definition of Myocardial Infarction. Third universal definition of myocardial

infarction. J Am Coll Cardiol. 2012 Oct 16;60(16):1581-98.

9. Ma H, Liu J, Qian L. Fat for Fostering: Regenerating Injured Heart Using Local

Adipose Tissue. EBioMedicine 2106;7:25-6.

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FIGURE LEGENDS

Figure 1. Schematic illustration of the use of cATMSCs and the development of the

AGTP in preclinical and clinical studies. The figure was designed and hand-drawn by

CG-M.

Figure 2. Flowchart of the clinical trial design of AGTP II showing the procedures

performed in the two arms of the study. Arrows indicate the infarcted area covered by

the AGTP in the treatment group.

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Table 1. Study visits.

Selection and

randomization Surgery Follow-up

Visit 1 2 3 4 5

Time 0 0

1 week

post-

discharge (±7 days)

3 months post-


1 year

post-


Informed consent x

Inclusion/exclusion criteria

x

Vital signs x x x x x

Physical exam x

x x x

Concomitant medication

x

x x x

Pregnancy test x

Blood test x

x x x

ECG x

x x x

Holter x

x x

TTE x

x x

Cardiac MRI with gadolinium

x

x x

HF signs and symptoms

x

x x x

Adverse events x x x x x

Surgical procedure

x

ECG: Electrocardiogram; TTE: Trans-Thoracic Echocardiography; MRI: Magnetic Resonance Imaging; HF: Heart Failure. The surgical procedure will consist of a coronary artery bypass graft (CABG) on the amenable area for both groups. In addition, the treatment group will undergo the adipose graft transposition procedure (AGTP).

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Figure 1. Schematic illustration of the use of cATMSCs and the development of the AGTP in preclinical and clinical studies. The figure was designed and hand-drawn by CG-M.

297x209mm (300 x 300 DPI)

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Figure 2. Flowchart of the clinical trial design of AGTP II showing the procedures performed in the two arms of the study. Arrows indicate the infarcted area covered by the AGTP in the treatment group.

297x209mm (300 x 300 DPI)

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Código protocolo: ICOR-2016-01 Versión protocolo: 2.2

EudraCT Number: N/A Fecha protocolo: Abril 04, 2017

Eficacia de la transposición de un

pedículo adiposo pericárdico sobre el infarto de miocardio en pacientes

(AGTP II trial).

Efficacy of the Adipose Graft Transposition Procedure (AGTP) in

patients with a myocardial scar: The AGTP II trial.

Confidencial

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Título del estudio:

Eficacia y seguridad de la transposición de un pedículo adiposo pericárdico sobre el infarto de miocardio en pacientes subagudos (AGTP II trial).


EudraCT Number: N/A Fecha protoco: Abril 04, 2017

AGTP II Page 2 of 19

Resumen Antecedentes: Estudios pre-clínicos en el modelo porcino de infarto de miocardio han demostrado que la transposición de un pedículo adiposo pericárdico (AGTP) reduce el área de infarto. Un primer estudio fase I-II ha sido positivo en pacientes con infarto de miocardio crónico. Objetivo: Evaluar la eficacia del AGTP para la mejora de la función cardiaca en el infarto de miocardio en humanos. Métodos: Estudio multicéntrico, aleatorizado y controlado de eficacia. Se incluirán pacientes candidatos a revascularización quirúrgica de una o más zonas del miocardio con onda Q negativa en el ECG y una zona con retención de gadolinio por resonancia de ≥50% no revascularizable. Los pacientes se randomizarán en 2 ramas: pacientes control en los que se revascularizarán las zonas candidatas; y pacientes tratamiento o AGTP en los que se revascularizarán las zonas candidatas y la zona no revascularizable se cubrirá con el pedículo. La variable principal de estudio es la reducción de la zona necrótica en la zona del AGTP por RMN. Se analizarán otras variables de seguridad y eficacia incluyendo parámetros de RMN y biomarcadores. El seguimiento será de un año y los pacientes serán monitorizados de forma estrecha, vigilando la aparición de cualquier evento adverso a la semana y al año de la fecha de la intervención quirúrgica. Importancia potencial: Se trata de una novedosa aproximación a la regeneración cardiaca que podría suponer una opción terapéutica para pacientes que no tienen otra alternativa.

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Índice 1. Introducción 2. Objetivos del estudio

2.1 Endpoint primario 2.2 Endpoints secundarios

3. Diseño del estudio 4. Sujetos de estudio

4.1 Selección de pacientes y criterios de inclusión 4.2 Criterios de exclusión

5. Procedimientos 6. Riesgos y Beneficios 7. Abandono del estudio 8. Manejo y análisis de los datos

8.1 Gestión de datos y monitorización 8.2 Análisis estadístico 8.3 Potencia y cálculo de la muestra

9. Fechas previstas 10. Consideraciones regulatorias y éticas

10.1 Consideraciones regulatorias 10.2 Revisión ética 10.3 Confidencialidad 10.4 Consentimiento informado 10.5 Responsabilidad 10.6 Definición y presentación de informes sobre eventos adversos

10.6.1 Definición de eventos adversos de acuerdo a la Conferencia de Armonización Internacional (ICH) 10.6.2 Presentación de informes sobre eventos adversos

11. Referencias

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1. Introducción El infarto de miocardio causa necrosis de las células miocárdicas y reduce la función cardiaca. Hoy en día existen tratamientos como la angioplastia primaria y la trombolisis que son eficaces para limitar la muerte celular tras el infarto agudo de miocardio (IAM) (1-3). Sin embargo, la cicatriz post-infarto a menudo condiciona un remodelado ventricular global que puede evolucionar clínicamente hacia la insuficiencia cardiaca y, en los estadios más avanzados, la única terapia que restaura completamente la función cardiaca es el trasplante cardiaco. En la actualidad, tanto la investigación básica como las primeras aproximaciones a la clínica realizadas en el campo de la regeneración cardiaca, se están centrando en la terapia celular, en la que una solución que contiene células madre se libera en el miocardio infartado por diversas rutas (administración intracoronaria, inyección epicárdica directa durante una cirugía a corazón abierto, o inyección endomiocárdica utilizando catéteres especialmente diseñados). Los resultados de los primeros ensayos clínicos han demostrado que esta terapia es segura. El beneficio medido como un aumento de la fracción de eyección del ventrículo izquierdo (FEVI) ha sido de ~5%. Las limitaciones de esta opción terapéutica son: 1) la dificultad para reparar una cicatriz miocárdica extensa; 2) la bio-retención y anidado celular en el tejido cicatricial es bajo; y 3) la mortalidad de las células implantadas en el miocardio isquémico es alta. Por ello, cada vez más estudios experimentales están evaluando nuevos enfoques terapéuticos basados en la ingeniería de tejidos para la regeneración miocárdica. La ingeniería de tejidos cardiaca intenta crear constructos de tejido funcional que puedan restablecer la estructura y la función del miocardio dañado (4). Este tipo de investigación demuestra la importancia de la terapia con células madre junto con la fijación de matrices con características biomiméticas en el miocardio infartado. El propósito principal de estas matrices es conseguir una estructura tridimensional y propiedades similares a la matriz extracelular (5,6). Como resultado, estos enfoques de ingeniería parecen ofrecer un efecto beneficioso en pequeños roedores, a pesar de que la mayoría de las células implantadas tienen una supervivencia corta post-administración, dando lugar a una recuperación parcial. Por ello, la función cardiaca y la regeneración del órgano no se consiguen óptimamente. En resumen, se necesitan enfoques terapéuticos nuevos y audaces para tratar a estos pacientes. Las modalidades de ingeniería de tejidos que actualmente están siendo investigadas son la implantación de un injerto 1) al que se le inyecten células, 2) que porte células o 3) que movilice las células residentes del tejido dañado. La movilización de células cardiacas residentes podría ser un camino alternativo para la regeneración cardiaca (4). En este contexto, nuestro equipo de investigación ha desarrollado un nuevo concepto de terapia cardiomioplástica utilizando un pedículo adiposo autólogo como biomembrana para aplicar directamente sobre el miocardio dañado (7). Este enfoque terapéutico combina muchas ventajas como la utilización de un tejido autólogo y la potencial migración de células madre residentes en el tejido adiposo, sin la necesidad de aislarlas y expandirlas previamente ex vivo. Para un funcionamiento “normal”, las células reparadoras necesitarían

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una matriz fisiológica, con la composición molecular, propiedades y estructura típicas del corazón. Uno de los mayores retos de la ingeniería de tejidos cardiaca, es la conexión del injerto con el suministro de sangre del receptor, y hasta ahora no había sido hallada una solución (4). Específicamente, hemos desarrollado un nuevo método terapéutico basado en la colocación de un pedículo adiposo pericárdico (AGTP) sobre una cicatriz de infarto de miocardio. Esta técnica ha sido concebida por nuestro grupo y desarrollada en el modelo porcino tras un IAM (Figura 1). Los pasos de la intervención se resumen en: 1- Toracotomía lateral izquierda: realizada mediante una incisión de 15 cm de longitud en el cuarto espacio intercostal para exponer el corazón e identificar la capa de tejido adiposo pericárdico. 2- Disección del tejido adiposo pericárdico: desde la capa pericárdica preservando su vascularización para garantizar la viabilidad del tejido. 3- Pericardiectomía: se realiza mediante una incisión en cruz y las cuatro porciones resultantes se extirpan para permitir la exposición del corazón. 4- Infarto de miocardio: se induce mediante la ligadura de la primera rama marginal de la arteria circunfleja a 1,5 cm del surco atrioventricular. 5- Transposición del tejido adiposo pericárdico: se localiza la extensión del área infartada y el tejido adiposo pericárdico se coloca sobre dicha área como un autoinjerto para cubrir por completo el miocardio dañado. 6- Acoplamiento del pedículo: se fija a los bordes sanos del área infartada con cola quirúrgica sintética. 7- Cierre de la toracotomía: reducción de la herida quirúrgica por sutura.

Figura 1. Pasos principales del procedimiento quirúrgico para realizar el AGTP en el modelo experimental porcino. Se realiza una toracotomía lateral para exponer el corazón (A) y, tras su localización, se disecciona (B) el pedículo adiposo pericárdico (AGTP) y se reserva (C), manteniendo su vascularización para conservar su viabilidad. El pericardio se abre mediante una incisión en cruz y se liga la primera rama marginal de la

**

LA LA

LVLV

*

LA

LV

A. Lateral thoracotomy, B and C. Adipose dissection, D. Pericardic adipose pedicle, E. Pericardic opening, F. Arterial occlusion by

ligation, G. Adipose pericardic pedicle transposition, H. Adipose pericardic pedicle to infarcted area coupling.

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arteria circunfleja (D) para provocar el infarto de miocardio (se puede apreciar el cambio en la coloración del tejido infartado). Se coloca el pedículo sobre la zona infartada (E), que se fija mediante cola quirúrgica sintética (F). LA: left atrium, aurícula izquierda; LV: left ventricle, ventrículo izquierdo; *: ligadura. Las flechas blancas señalan el pedículo adiposo pericárdico.

Utilizando este nuevo enfoque para limitar el tamaño del infarto, hemos realizado sendos estudios en el modelo porcino de infarto agudo e infarto crónico de miocardio. En el modelo de infarto agudo se realizó la intervención en 33 cerdos juveniles (16 controles y 17 tratados) (7). A todos los animales se les provocó un infarto de miocardio y, tras 30 minutos, se realizó el AGTP en el grupo tratado. Todos los animales sobrevivieron hasta el momento del sacrificio programado sin complicaciones. Además, todos los AGTP permanecieron colocados correctamente cubriendo por entero la zona infartada hasta el final de los experimentos. Cabe destacar que este autoinjerto permite el correcto movimiento del corazón y no conlleva ningún tipo de restricción funcional. No hubo cambios en el electrocardiograma ni arritmias asociadas al procedimiento. Los resultados demuestran que los animales tratados tuvieron una marcada disminución del tamaño del infarto en comparación con los controles, al mes de la realización del AGTP (Figura 2). Además, encontramos que la apoptosis celular en la zona peri-infarto fue significativamente menor en el miocardio de los animales tratados, según ensayos de TUNEL.

Figura 2. Imágenes representativas del tamaño del infarto al mes en el corazón de un cerdo control (A) y uno tratado (B) con su correspondiente aumento a su derecha, mostrando una reducción significativa en el área del infarto. Se puede observar el pedículo marcado con flechas negras.

Además, se observaron microvasos que conectaban el pedículo con el miocardio ya a los 6 días de la intervención (Figura 3).

A B

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Figura 3. Conexiones vasculares en la interfaz pedículo-miocardio a los 6 días de la intervención observados por inmunofluorescencia (A y B).

El análisis mediante resonancia magnética permitió apreciar una recuperación funcional muy significativa en los animales tratados al mes post-cirugía (p<0,007) (Figura 4). La variación de la fracción de eyección del ventrículo izquierdo (FEVI) (entre las 2 horas post infarto y 1 mes) f

Figura 4. (A) FEVI medida mediante RMN a las 2 horas y al mes tras el infarto de miocardio en los animales control y los tratados. Los datos de los animales individuales están representados por puntos y las medias (±SEM) por rombos. (B) Imágenes representativas de la proyección en turbospin-eco (TSE) e imágenes en eje corto y proyección T1 con realce tardío de cada grupo (2 horas post-infarto y al mes). TSE muestra en blanco brillante la posición del pedículo fijado (flechas blancas).

El estudio se completó mediante un análisis genético exhaustivo utilizando tecnología de microarrays para identificar las principales vías involucradas. En el modelo de infarto crónico se realizó la intervención en 16 cerdos juveniles (8 controles y 8 tratados) (8). A todos los animales se les provocó un infarto de miocardio (en este caso mediante embolización percutánea con un coil) y, tras 2 semanas, se realizó la toracotomía lateral para transponer el pedículo en el grupo tratado. Todos los animales sobrevivieron hasta el momento del sacrificio programado sin complicaciones. Además, todos los AGTP permanecieron correctamente colocados cubriendo por entero la zona infartada hasta el final de los experimentos. Igual que en el modelo agudo no se detectaron cambios en el ECG ni arritmias.

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Los resultados demuestran que los animales tratados tuvieron una disminución del tamaño del infarto del 34% en comparación con los controles (p=0,04), al mes de la realización del AGTP (Figura 5).

Figura 5. Imágenes representativas del tamaño de infarto en el corazón de un cerdo control (A) y uno tratado (B) con su correspondiente aumento a su derecha, mostrando una reducción significativa en el área del infarto al mes de la realización del AGTP. Se puede observar el pedículo marcado con flechas negras.

Nuevamente, se observaron microvasos que conectaban el pedículo con el miocardio al mes de la intervención (Figura 6) y la migración de células progenitoras derivadas de la grasa desde el pedículo al miocardio.

Figura 6. Conexiones vasculares en la interfaz pedículo-infarto de miocardio al mes de la intervención observados por inmunofluorescencia (A y C). Las imágenes B y D corresponden a aumentos de los vasos observados en A y C, respectivamente.

Estos estudios pre-clínicos tanto en el modelo agudo como en el crónico han sido recientemente aceptados para su publicación en revistas del primer decil del área cardiovascular (7,8). En base a estos prometedores resultados, se realizó un primer estudio clínico fase I-II para evaluar la seguridad y la eficacia de este procedimiento como una innovación biológica regenerativa en pacientes con una escara miocárdica crónica (ClinicalTrials.org NCT01473433, AdiFlap Trial). Este estudio incluyó 10 pacientes con una escara transmural establecida que debían ser intervenidos para tratar por medio de un bypass otra zona del corazón (9). Se randomizaron a dos grupos, control en el que los pacientes se les realizaba

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el bypass para tratar la zona de isquemia reciente y la zona crónica permanecía sin tratar (práctica clínica habitual), y el grupo tratamiento en el que se realizaba el bypass para tratar la zona de isquemia reciente y la zona crónica se cubría con el pedículo adiposo. Los resultados fueron muy prometedores ya que no se encontraron diferencias en la seguridad, ni en cuanto a eventos clínicos (muerte y/u hospitalización) ni en cuanto a eventos arrítmicos. Además, se observó en los pacientes tratados menor volumen ventricular izquierdo (p=0.09) y menor ratio de necrosis (p=0.06) que en los controles.

Figura 7. El análisis por resonancia cardiaca muestra imágenes representativas en eje corto y proyección T1 con realce tardío el miocardio sano (negro) e infartado (blanco) del grupo tratado y el grupo control a la inclusión y los 12 meses. Las flechas señalan la posición del adiFLAP fijado en los pacientes tratados. Los histogramas representan los cambios en el porcentaje de tejido necrótico desde la inclusión a los 3 meses de seguimiento en los pacientes tratados y los pacientes control.

En base a los resultados de este estudio clínico, se hace necesario probar la seguridad de este procedimiento en otro grupo de pacientes que se podrían beneficiar también de esta nueva técnica: aquellos pacientes subagudos en los que una de las zonas isquémicas no tiene opción terapéutica disponible.

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2. Objetivos El objetivo de este estudio es evaluar la eficacia de la transposición de un pedículo adiposo pericárdico (AGTP de sus siglas en inglés) para la reducción del infarto de miocardio no revascularizable en humanos. 1.1. Endpoint primario

Cambios en el ratio de masa miocárdica necrótica por retención de gadolinio a 91 días y 365 días.

1.2. Endpoints secundarios

Mejoría de la contractilidad regional por RMN a 91 días y 365 días. . Cambios en los parámetros funcionales evaluados mediante RMN: FEVI, remodelado

geométrico ventricular izquierdo y derecho a 91 días y 365 días. Niveles de péptido natriurético del tipoB a los 7 días, 91 días y 365 días. Aparición de arritmias por Holter de 24h a los 91 días y 365 días. Muerte o rehospitalización por cualquier causa a 365 días.

Muerte o rehospitalización por causa cardiovascular a 365 días.

3. Diseño del estudio Estudio prospectivo, aleatorizado y controlado de seguridad y eficacia, comparando 2 ramas de pacientes, de la colocación de un pedículo de tejido adiposo autólogo de origen pericárdico en pacientes con un infarto de miocardio. Los pacientes serán incluidos en diversos centros nacionales. Figura 8.

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Figura 8. Esquema del estudio

4. Sujetos de estudio Los pacientes para ser incluidos deben cumplir todos los criterios de inclusión y ninguno de los de exclusión que se detallan a continuación.

Paciente con IM Candidato a bypass por

1 ó más regiones

Grupo control Bypass necesarios

No AGTP

Grupo tratamiento Bypass necesarios + AGTP sobre la zona

isquémica no revascularizable

Randomización estratificada por tiempo de IM (> ó ≤ 2 meses).

Onda Q en ECG

Si gadolinio ≥50% y no candidato a puente por transmularidad o mal vaso

RMN

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4.1. Selección de pacientes y criterios de inclusión Se incluirán pacientes candidatos a revascularización quirúrgica de dos o más zonas del miocardio, que cumplan los siguientes criterios:

Edad mayor de 18 años

Firma del consentimiento informado

Onda Q presente en el ECG

Infarto de miocardio 50% de transmuralidad por RMN no revascularizable (por

transmuralidad o por mal vaso), de mínimo 15 días (proceso de cicatrización establecido) y

sin límite de antigüedad.

Candidato a by-pass coronario por otros territorios del miocardio

4.2. Criterios de exclusión

Enfermedad valvular severa con indicación de reparación quirúrgica

Candidato a remodelación ventricular

Contraindicación para RMN (aclaramiento creatinina inferior a 30 ml/min/1.73m2, portadores

implantes metálicos, claustrofobia)

Enfermedad extracardiaca con pronóstico vital estimado menor de 1 año

Insuficiencia renal o hepática graves

Valores anormales de laboratorio, no explicables en el momento de la inclusión

Pacientes con una intervención cardiaca previa

Mujeres embarazadas o en periodo de lactancia.

5. Procedimiento El procedimiento a estudio, AGTP, será enseñado a los cirujanos cardiacos de los distintos centros participantes por un cirujano cardiaco experto en dicho procedimiento, que ha realizado todas las intervenciones del ensayo clínico first-in-man (ClinicalTrials.org NCT01473433, AdiFlap Trial).

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El esquema de las visitas es el siguiente:

Selección Cirugía Seguimiento

Visita 1 2 3 4 5

Tiempo 0 0 1 semana post-alta (+-5 días)

3 meses post-alta (+- 7 días)

1 año post-alta (+-15 días)

Consentimiento Informado x

Criterios Inclusión/Exclusión x

Signos Vitales x x x x x

Examen físico 1 x x x x

Medicación concomitante x x x x

Test embarazo2 x

Analítica completa3 x x x x

ECG x x x x

Holter x x x

Ecocardiografía transtorácica x x x

RMN Cardiaca con Gadolinio x x x

Signos y síntomas IC x x x x

Acontecimientos adversos x x x x x

Procedimiento quirúrgico4 x

Tabla 1. Visitas del estudio (Cronograma). 1Examen físico completo requerido en la visita inicial y en las visitas de seguimiento (3, 4 y 5). 2Sólo en mujeres en edad fértil. Se realizará en las muestras de suero en la visita 1. Se realizará un test de embarazo en orina en las visitas de seguimiento. En caso de un resultado positivo para embarazo, se realizará una prueba confirmatoria en plasma. 3Se realizarán evaluaciones completas de laboratorio para analizar la seguridad del estudio en las visitas 1, 3 y 4. Incluyendo péptidos natriuréticos (NTproBNP) y troponina (hsTnT). 4El procedimiento quirúrgico consistirá en la revascularización quirúrgica (by-pass) de las arterias candidatas. En función de la rama a la que pertenezca el paciente, además se le realizará la disección, la eliminación del pericardio parietal y la fijación del pedículo a la zona cicatrizal no candidata a revascularización.

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Inclusión en el estudio y randomización

Los pacientes que cumplan los criterios de inclusión y ninguno de los de exclusión serán informados sobre el protocolo del estudio y se obtendrá su consentimiento informado. Se randomizarán mediante un sistema informático en ratio 1:1. Los pacientes serán randomizados bien al grupo de tratamiento-AGTP o al grupo control. La aleatorización se realizará exclusivamente por los investigadores del centro coordinador, el Hospital Germans Trias i Pujol, la asignación a un grupo u otro será aleatoria según orden de inclusión y será ciega para el equipo investigador con excepción de cirujanos y coordinador del estudio. Se incluirán 108 pacientes divididos en 2 ramas de manera estratificada según la antigüedad de la escara: 54 pacientes control, y 54 pacientes AGTP.

A. Grupo de tratamiento AGTP

El procedimiento consistirá en los siguientes procesos: Se realizará una esternotomía para la realización de la revascularización quirúrgica (by-pass) de las arterias candidatas a revascularizar. El tejido pericárdico se disecará (tal y como se ha descrito en el modelo porcino), se eliminará el pericardio parietal y se fijará utilizando cola quirúrgica alrededor del área de cicatriz no revascularizable. (NOTA: Los pacientes aleatorizados al grupo AGTP que no tengan suficiente grasa, serán excluídos del estudio y se analizarán en un subgrupo.)

B. Grupo control

El paciente será intervenido por esternotomía para la realización de la revascularización quirúrgica de las arterias candidatas a revascularizar. No se realizará procedimiento extra.

Duración de la estancia hospitalaria

Los pacientes estarán ingresados el tiempo habitual tras una operación de bypass coronario. La realización del AGTP no debería aumentar los días de hospitalización.

Procedimientos comunes en ambos grupos antes de la intervención

Se realizará un electrocardiograma y una resonancia magnética nuclear a la inclusión en el estudio para poder analizar la seguridad y la eficacia en el seguimiento. También se tomará una muestra de sangre para evaluar los niveles de biomarcadores.

Seguimiento

Las visitas de seguimiento serán realizadas por un cardiólogo clínico en cada centro. Desconocerá el grupo al que pertenece el paciente. Los pacientes serán visitados en cualquier momento durante el estudio, además de las visitas fijadas por el protocolo, si son necesarias según el investigador. Los pacientes serán monitorizados de forma estrecha, vigilando la aparición de cualquier evento adverso en visitas a los 3 meses y un año de la

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fecha de la intervención quirúrgica con la realización de un electrocardiograma y un Holter de 24h. Para el seguimiento de la efectividad, se realizará una resonancia magnética nuclear y se extraerá una muestra de sangre del paciente.

6. Riesgos y Beneficios

En base a los estudios preclínicos y especialmente al primer ensayo clínico fase I-II realizado con este procedimiento en pacientes con una escara de miocardio crónica, no se esperan problemas de seguridad en los pacientes tratados. Durante la intervención, se realizará una curva de troponina T de alta sensibilidad para evaluar el estado del paciente. Se evaluará la aparición de infarto perioperatorio, según las III Guía de la Definición Universal de Infarto de Miocardio (10) donde se define IM relacionado con la intervención de bypass a la elevación de valores de biomarcadores cardiacos (>10xP99 URL) en pacientes con valores basales noramles de cTn (≤P99 URL). Además, una nueva onda Q patológica, o bloqueo de rama izquierda u oclusión de arteria coronaria nativa o de nuevo bypass documentado por angiografía, o evidencia de imagen de nueva pérdida de miocardio viable o nueva anormalidad de movimiento de pared regional. Durante la hospitalización, los pacientes son monitorizados clínicamente por el equipo de cardiología y tras el alta, la primera visita es a la semana con la realización de un Holter de 24h para valorar que no se están instaurando arritmias anómalas de ningún tipo. El paciente tendrá instrucciones de contactar con el equipo asistencial si tiene cualquier problema y el seguimiento se extenderá a lo largo de un año, con la realización de otro Holter de 24h a al año.

La disección del pericardio y manipulación de la grasa pericárdica son procesos normales en las cirugías de bypass, ya que se necesita un acceso directo al miocardio. La utilización de la cola quirúrgica es frecuente en distintos tipos de cirugías en la práctica clínica habitual.

7. Abandono del estudio

A) Abandono del estudio de un paciente porque retira su consentimiento.

B) Abandono del estudio debido a:

a) se revelan criterios de exclusión tras la inclusión en el estudio

b) aparece un nuevo evento clínico grave durante la participación en el estudio que contraindique

la realización de resonancias u otras pruebas necesarias

c) el equipo médico decide la necesidad de incluir al paciente en cuidado paliativos

8. Manejo y análisis de los datos

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8.1. Gestión de datos y monitorización Para describir adecuadamente la población de pacientes, se completará un cuaderno de recogida de datos incluyendo la historia detallada del paciente, el examen físico, los parámetros vitales durante la hospitalización y los eventos adversos. Todos los datos relevantes serán introducidos en una base de datos y su uso será restringido a fines científicos. La gestión de datos se realizará en la Fundació Institut d’Investigació Germans Trias i Pujol. 8.2. Análisis estadístico El análisis estadístico se realizará con el paquete de software SPSS (SPSS Inc., EE.UU). Se utilizará un nivel de significación estadística (p - valor) de 0,05. Se realizará un análisis de un subgrupo predefinido de pacientes en los que un infarto de miocardio no candidato a revascularización haya sucedido en el plazo ≤2 meses previos a la intervención. Se pretende analizar si en este subgrupo de pacientes el beneficio sobre el remodelado, el tamaño de la cicatriz y/o funcionalidad regional es mayor que en el caso de infartos más antiguos cronificados. Nota: si no se obtiene una cantidad de pacientes suficiente con infarto de menos de ≥2 meses de evolución, no se analizará dicho subgrupo. 8.3 Potencia y cálculo de muestra Aceptando un riesgo alfa de 0,05 y un riesgo beta inferior a 0,2 en un contraste bilateral, se necesitan 54 sujetos por rama para detectar una diferencia igual o superior a 2 unidades (10% del tamaño estimado del infarto). Se asume que la desviación estándar común es de 3,5. Se ha estimado una tasa de pérdidas de seguimiento del 10%.

9. Fechas previstas

Inclusión del primer paciente: Junio, 2016 Inclusión del último paciente: Al alcanzar la n= 108 Fin del seguimiento a 360 días: n = 108 + 360 días (última visita del último paciente) Envío del artículo: n = 108 + 540 días

10. Consideraciones regulatorias y éticas

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10.1. Consideraciones regulatorias Este estudio se llevará a cabo de conformidad con los principios éticos establecidos en la versión más reciente de la Declaración de Helsinki y las guías de buenas prácticas clínicas, lo que representa la mayor protección del individuo. 10.2. Revisión ética

Este protocolo será evaluado para obtener la aprobación del Comité de Ética del Hospital Universitari Germans Trias I Pujol como CEIC coordinador, y por los CEIC de cada centro participante.

10.3. Confidencialidad Todos los registros y datos médicos están bajo discreción profesional y se mantendrán de forma confidencial. Los datos se organizarán, gestionarán y almacenarán en una base de datos y sólo los miembros del equipo de estudio y el comité de ética podrán acceder a ellos. Los datos no serán enviados a través de Internet a menos que se anonimicen. 10.4. Consentimiento Informado

Para participar en el estudio, los pacientes deberán dar un consentimiento informado por escrito. En él, se especifica el uso de sus datos para fines científicos. El documento de consentimiento informado se utilizará para explicar en términos simples, el objetivo del estudio y sus procedimientos. El documento de consentimiento informado contiene una declaración de que el consentimiento se da libremente, y especifica que el paciente es libre de retirarse del estudio en cualquier momento.

10.5. Responsabilidad

Los pacientes incluidos en este estudio estarán cubiertos por una póliza de Zurich®.

10.6. Definición y presentación de informes sobre eventos adversos

Se registrarán e informarán los siguientes eventos predefinidos que ocurran durante el estudio:

Eventos adveros (AE)

Eventos adversos graves (SAE)

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10.6.1. Definición de eventos adversos de acuerdo a la Conferencia de Armonización Internacional (ICH)

Un evento adverso (AE) es cualquier incidencia perjudicial en un paciente o sujeto de investigación clínica, al que se le ha administrado un producto farmacéutico o realizado un procedimiento, que no necesariamente tiene una relación causal con este tratamiento. Por lo tanto, un evento adverso puede ser cualquier signo desfavorable y no intencionado (incluyendo un hallazgo anormal de laboratorio), síntoma o enfermedad asociado temporalmente con el uso de un medicamento o procedimiento en investigación, estén o no relacionados con el producto médico.

Un evento adverso grave (SAE) significa un evento adverso que requiere ya sea hospitalización del paciente, prolongación de la hospitalización existente, cause una anomalía/defecto congénito, ocasione una discapacidad o incapacidad persistente o significativa, sea peligroso para la vida, o resulte en la muerte. 10.6.2. Presentación de informes sobre eventos adversos

Los EA deben ser registrados por el investigador clínico utilizando la sección predefinida del cuaderno de recogida de datos. En caso de un SAE de peligro de vida, el investigador debe informar al comité de ética institucional y al investigador principal de forma escrita en un plazo de 7 días. Todos los demás eventos adversos graves deben ser informados al comité de ética y al investigador principal por el investigador clínico en el plazo de 15 días.

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11. Referencias

1. Boersma H, Califf R, Collins R, Deckers JW, Simoons ML. Selection of reperfusion therapy for individual patients with evolving myocardial infarction. Eur Heart J 1997;18:1371-1381.

2. Favaloro RG. Saphenous vein graft in the surgical treatment of coronary artery disease. Operative technique. J Thorac Cardiovasc Surg 1969;58:178-185.

3. Gruntzig A. Transluminal dilatation of coronary-artery stenosis. Lancet 1978;1:263. 4. Vunjak-Novakovic G, Tandon N, Godier A, Maidhof R, Marsano A, Martens TP, Radisic M.

Challenges in Cardiac Tissue Engineering. Tissue Eng Part B Rev. 2010;16:169-87. 5. Garreta E, Genové E, Borrós S, Semino CE. Osteogenic differentiation of mouse embryonic

stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold. Tissue Engineering 12 (8): 1-13, 2006.

6. Semino CE, Kamm RD, Lauffenburger DA. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow. Exp. Cell. Res. 312, 289, 2006.

7. Gálvez-Montón C, Prat-Vidal C, Roura S, Farré J, Soler-Botija C, Llucià-Valldeperas A, Díaz-Güemes I, Sánchez-Margallo FM, Arís A, Bayes-Genis A. Transposition of a pericardial-derived vascular adipose flap for myocardial salvage after infarct. Cardiovasc Res. 2011;91:659-67.

8. Gálvez-Montón C, Prat-Vidal C, Roura S, Soler-Botija C, Llucià-Valldeperas A, Díaz-Güemes I, Sánchez-Margallo FM, Bayes-Genis A. Post-infarction scar coverage using a pericardial-derived vascular adipose flap. Pre-clinical results. Int J Cardiol 2013;166:469-74.

9. Bayes-Genis A, Gastelurrutia P, Cámara ML, Teis A, Lupón J, Llibre C, Zamora E, Alomar X, Ruyra X, Roura S, Revilla A, San Román JA, Gálvez-Montón C. First-in-man Safety and Efficacy of the Adipose Graft Transposition Procedure (AGTP) in Patients with a Myocardial Scar. EBioMed 2016;7:248-54.

10. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD: the Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. Third universal definition ofmyocardial infarction. Eur Heart J 2012; 2551-67.

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1

SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and

related documents*

Section/item Page number

Title 1

Trial registration 4

Protocol version 9

Funding 16

Roles and responsibilities 1, 2, 16

Introduction

Background and rationale 5-7

Objectives 8

Trial design 8-9

Methods: Participants, interventions, and outcomes

Study setting 9

Eligibility criteria 9

Interventions 10, 12

Outcomes 12-13

Participant timeline 20

Sample size 14

Recruitment 9

Methods: Assignment of interventions (for controlled trials)

Allocation:

Sequence generation 10

Allocation concealment

mechanism

10

Implementation 10

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2

Blinding (masking) 10

Methods: Data collection, management, and analysis

Data collection methods 11

Data management 11

Statistical methods 13

Methods: Monitoring

Data monitoring 14

Harms 11-12

Ethics and dissemination

Research ethics approval 4, 11

Consent or assent 10

Confidentiality 11

Declaration of interests 17

Access to data 17

Ancillary and post-trial care 12

Dissemination policy 4, 16-17

Appendices

Informed consent materials 17 (In addition to the paper and the protocol, the informed

consent documents and case report forms are available on

request.)

Biological specimens N/A

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Rationale and design of a multicenter, prospective, randomized, controlled clinical trial to evaluate the efficacy

of the Adipose Graft Transposition Procedure (AGTP) in patients with a myocardial scar: the AGTP II trial.

Journal: BMJ Open

Manuscript ID bmjopen-2017-017187.R1

Article Type: Protocol

Date Submitted by the Author: 08-Jun-2017

Complete List of Authors: Gastelurrutia, Paloma; Fundacio Institut d'Investigacio en Ciencies de la Salut Germans Trias i Pujol, Gálvez-Montón, Carolina; Fundacio Institut d'Investigacio en Ciencies de la Salut Germans Trias i Pujol Cámara, Maria Luisa; Hospital Universitari Germans Trias i Pujol Bustamante-Munguira, Juan; Hospital Universitari de Girona Doctor Josep Trueta García-Pavía, Pablo; Hospital Universitario Puerta del Hierro Majadahonda Avanzas, Pablo; Hospital Universitario Central de Asturias San Roman, J. Alberto; ICICOR- Hospital Clinico Universitario, Cardiology Pascual-Figal, Domingo; Hospital Clinico Universitario Virgen de la Arrixaca de Teresa, Eduardo ; Hospital Universitario Virgen de la Victoria Crespo-Leiro, Maria G.; Complexo Hospitalario Universitario A Coruna Manito, Nicolás ; Hospital Universitari de Bellvitge N��ez, Julio; Hospital Cl�nico Universitario, Cardiology Fernandez-Aviles, Francisco ; Hospital General Universitario Gregorio Maranon Caballero, Ángel; Hospital Universitari Germans Trias i Pujol Teis, Albert; Hospital Universitari Germans Trias i Pujol Lupón, Josep; Hospital Universitari Germans Trias i Pujol Brugada, Ramón ; Hospital Universitari de Girona Doctor Josep Trueta Martín, Carlos; Hospital Universitario Puerta del Hierro Majadahonda Silva, Jacobo; Hospital Universitario Central de Asturias Revilla, Ana; Hospital Clínico Universitario, Cánovas, Sergio J.; Hospital Clinico Universitario Virgen de la Arrixaca Melero, Jose María; Hospital Universitario Virgen de la Victoria Cuenca-Castillo, Jose; Complexo Hospitalario Universitario A Coruna, Cardiac Surgery González-Pinto, Ángel; Hospital General Universitario Gregorio Maranon Bayes-Genis, Antoni; Hospital Universitari Germans Trias i Pujol

<b>Primary Subject Heading</b>: Cardiovascular medicine

Secondary Subject Heading: Cardiovascular medicine

Keywords: Cardiac regeneration, adipose progenitor cells, chronic myocardial infarction, tissue engineering, pericardial adipose graft, Clinical trials < THERAPEUTICS


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Rationale and design of a multicenter, prospective, randomized, controlled clinical

trial to evaluate the efficacy of the Adipose Graft Transposition Procedure (AGTP)

in patients with a myocardial scar: the AGTP II trial.

Paloma Gastelurrutia1,2, Carolina Gálvez-Montón1,2, Maria Luisa Cámara3, Juan

Bustamante-Munguira2,3,4, Pablo García-Pavia2,5, Pablo Avanzas6, J. Alberto San

Román2,7, Domingo Pascual-Figal2,8, Eduardo de Teresa2,9, Maria G. Crespo-Leiro2,10,

Nicolás Manito11, Julio Núñez2,12, Francisco Fernández-Avilés2,13, Ángel Caballero3,

Albert Teis3, Josep Lupón2,3,14, Ramón Brugada2,4, Carlos Martín5, Jacobo Silvia6, Ana

Revilla-Orodea2,7 Sergio J. Cánovas8, Jose M Melero2,9, Jose J Cuenca-Castillo2,10,

Angel Gonzalez-Pinto13, Antoni Bayes-Genis1,2,3,14

From: 1* ICREC Research Program, Fundació Institut d´Investigació en Ciències de la

Salut Germans Trias i Pujol (IGTP), Badalona, Spain; 2 CIBERCV, Instituto de Salud

Carlos III, Madrid, Spain, 3 Hospital Universitari Germans Trias i Pujol, Badalona,

Spain; 4 Hospital Universitari de Girona Josep Trueta, Girona, Spain; 5 Hospital

Universitario Puerta del Hierro, Madrid, Spain; 6 Hospital Universitario Central de

Asturias, Oviedo, Spain; 7 Hospital Clínico Universitario, Valladolid; ICICORELAB,

Valladolid, Spain; 8 Hospital Clínico Universitario Virgen de la Arrixaca, Universidad

de Murcia, IMIB-Arrixaca, Murcia, Spain; 9 Hospital Clínico Universitario Virgen de la

Victoria de Málaga, IDIMA, Málaga, Spain; 10 Complejo Hospitalario Universitario A

Coruña (CHUAC), A Coruña, Spain; 11 Hospital Universitari de Bellvitge, L`Hospitalet

de Llobregat, Spain; 12 Hospital Clinic de Valencia, Valencia, Spain; 13 Hospital General

Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio

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Marañón, Madrid, Spain; 14Department of Medicine, Universitat Autònoma de

Barcelona, Barcelona, Spain.

* Trial sponsor

Running head: AGTP II trial: rationale and design.

Word count: 2879

Corresponding Author:

Paloma Gastelurrutia, PhD. Germans Trias i Pujol Health Research Institute.

Can Ruti Campus, Camí de les Escoles s/n 08916. Badalona (Barcelona).

E-mail: [email protected]

Telephone +34 93 497 86 70; Fax +34 93 497 89 39

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ABSTRACT

Introduction: Cardiac adipose tissue is a source of progenitor cells with regenerative

capacity. Studies in rodent demonstrated that the intramyocardial delivery of cells

derived from this tissue improves cardiac function after MI. We developed a new

reparative approach for damaged myocardium that integrates the regenerative properties

of cardiac adipose tissue with tissue engineering. In the Adipose Graft Transposition

Procedure (AGTP), we dissect a vascularized flap of autologous pericardial adipose

tissue and position it over the myocardial scarred area. Following encouraging results in

acute and chronic MI porcine models, we performed the AGTP-I clinical trial

(NCT01473433, AdiFLAP Trial) to evaluate safety in patients with chronic MI

undergoing CABG. The good safety profile and trends in efficacy warranted a larger

trial.

Study design: The AGTP II Trial (NCT02798276) is an investigator-initiated,

prospective, randomized, controlled, multi-center study to assess the efficacy of the

AGTP in 108 patients with non-revascularizable MI. Patients will be assigned to

standard clinical practice or AGTP. The primary endpoint is change in necrotic mass

ratio by gadolinium enhancement at 91, 365 days. Secondary endpoints include

improvement in regional contractibility by MRI at 91, 365 days; changes in functional

MRI parameters (LVEF, left and right ventricular geometric remodeling) at 91, 365

days; levels of NT-proBNP at 7, 91, 365 days; appearance of arrhythmias from 24h-

Holter monitoring at 24h, 91, 365 days; all-cause death or re-hospitalization at 365

days; and cardiovascular death or re-hospitalization at 365 days.

Ethics and dissemination: The Institutional Review Board approved the trial and will

comply with the Declaration of Helsinki, with all patients providing informed consent.

It may offer a novel, effective, and technically simple technique for patients with no

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other therapeutic options. The results will be submitted to indexed medical journals and

national and international meetings.

Trial registration number: ClinicalTrials.gov: NCT02798276.

Strengths and limitations of this study:

- The AGTP is the first tissue engineering technique to use local adipose tissue

without laboratory manipulation.

- It is technically simple, requiring no learning curve for cardiac surgeons who

will simply dissect structures that are ordinarily modified in CABG surgery.

- There is no requirement for additional or expensive materials, nor do any ethical

or social constraints exist as the autologous tissue is sourced from a site in close

proximity to the injury.

- It minimizes the complications that can arise from the introduction of exogenous

stem cells or stem cell-derived cardiomyocytes, as well as those associated with

gene therapy.

- Access to local adipose tissue is also a critical aspect of the AGTP because its

factibility may be limited in patients with very little pericardial fat.

Keywords: Cardiac regeneration, adipose progenitor cells, chronic myocardial

infarction, tissue engineering, pericardial adipose graft, clinical trials.

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RATIONALE

Cardiac adipose tissue for heart regeneration

The regenerative capacity of the heart is limited. After a myocardial infarction (MI)

there is a massive loss of cardiac muscle that overloads the surviving myocardium and

ultimately leads to heart failure.1 Our group has focused on cardiac adipose tissue as a

source of progenitor cells with regenerative capacity, and as a biological matrix for

salvaging injured myocardium following MI. Under physiologic conditions, cardiac

adipose tissue displays biochemical and thermogenic cardio-protective properties.

Further, during pathological scenarios, cardiac adipose tissue can locally affect the heart

via the vasocrine or paracrine secretion of proinflammatory cytokines.2,3 We identified

and characterized a population of human adult mesenchymal-like cells derived from

cardiac adipose tissue (adipose tissue mesenchymal stem cells [cATMSCs]). Despite

residing in an adipocytic environment, cATMSCs have an inherent cardiac-like

phenotype and may play a role in heart homeostasis as well as act as a cellular reservoir

for myocardial tissue renewal. In vivo studies in murine models showed that the

intramyocardial delivery of cATMSCs improves cardiac function, reduces infarct size,

and avoids infarct-related reduction of wall thickness following MI.4 Moreover,

following the application of cATMSCs, cellular niches within the host myocardium

secrete pro-angiogenic factors and differentiate into an endothelial lineage that can

support local angiogenesis. Collectively, these preliminary results encouraged us to

expand the therapeutic uses of cATMSCs.

A new approach: preclinical trials

Our next aim was avoiding any damage that could be caused by intramyocardial

injection or the use of cellular material from allogeneic sources. Consequently, we

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developed a new approach that integrates cATMSC therapy with tissue engineering to

repair damaged myocardium (Figure 1). This innovative procedure involves the

dissection of a vascularized flap of autologous pericardial adipose tissue to cover the

infarcted myocardium.

Initially we tested this strategy, named Adipose Graft Transposition Procedure

(AGTP), in a porcine acute MI model. After a lateral thoracotomy to expose the

pericardium, adipose tissue from the fibrous pericardium was carefully dissected, while

maintaining its vascularization, to obtain a natural scaffold containing native

cATMSCs. The flap was reserved and a pericardiotomy was performed to access the

heart. After MI induction by permanent coronary artery ligation, the adipose pericardial

graft was adhered to healthy myocardium fully covering the ischemic area. At the 30-

day follow-up, we observed that AGTP-treated animals manifested a five-fold

improvement in the left ventricular ejection fraction (LVEF) vs. control animals, and a

smaller infarct size and myocardial remodeling.5 The AGTP was performed 30 min

after MI induction (an acute scenario not used in the clinical setting), thus we also re-

tested the AGTP in a chronic MI swine model, positioning the flap 2 weeks after

coronary coil deployment. Once again, the AGTP proved valuable in reducing the left

ventricular infarct area by as much as 34% vs. control animals, and in promoting

neovascularization in the infarcted myocardium.6 Microscopic evaluation revealed that

both models promoted vascular bridging between the ischemic myocardium and the

adipose graft. In terms of safety, the AGTP did not triggered arrhythmic events or the

sudden death of any of the included animals; therefore, we proceeded with further

bench-to-bedside studies.

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Safety clinical trial

The AGTP-I clinical trial (NCT01473433, AdiFLAP Trial) was designed to evaluate the

safety of the AGTP in patients with chronic MI undergoing a coronary artery bypass

graft (CABG) for other myocardial regions.7 Patients were enrolled if they were CABG

candidates and had an established, chronic, MI lesion that was not in the area supplied

by the CABG target vessel(s). Patients were assessed with late gadolinium enhancement

(LGE) MRI to identify a chronic non-revascularizable area to guide the AGTP. The

main safety endpoints were arrhythmia, hospital admission, and death during the 12-

month follow-up. Secondary endpoints included change in cardiac geometry and

function as determined by cardiac MRI, biomarkers of cardiac injury or strain (e.g.

troponin T and NT-proBNP), NYHA class, and Framingham-derived clinical score.7

Ten male patients were recruited from January 2012 to November 2013 in a

single center. All were candidates for myocardial revascularization with an established

chronic transmural myocardial scar in a distally non-revascularizable area. They did not

differ in disease severity or surgical risk variables, with a necrotic non-revascularizable

mass of 34±13 g vs. 25±4 g and a LVEF of 41±18% vs. 42±15% in the AGTP-treated

vs. the control arm, respectively. Patients were randomly allocated to either the control

or AGTP-treated arm; for both groups, the area that could be revascularized was treated

with CABG. The non-revascularizable scar was left untouched in the control arm and

was covered by an autologous pericardial adipose graft in the AGTP arm. The AGTP

location was inferior in four cases and anterior in one. No differences in safety were

observed in terms of clinical or arrhythmic events as assessed by clinical visits,

electrocardiogram (ECG), and 24-hour Holter monitoring during the 12-month study

duration. On follow-up, the AGTP-treated arm showed a borderline smaller left

ventricular end-systolic volume (LVESV; p=0.09) and necrosis ratio, assessed with

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LGE MRI (p=0.06). Interestingly, the AGTP patient with the largest necrotic area and

most dilated chambers experienced the most remarkable reduction in necrotic mass size

(-10.8%) and ventricular volumes (left ventricular end-diastolic volume (LVEDV): -

55.2 mL and LVESV: -37.8 mL) at the 1-year follow-up. Furthermore, this patient’s Q

waves vanished from leads I, II, and III in follow-up ECGs. Based on these results and

the good safety profile of the AGTP, we are undertaking a larger trial to assess the

efficacy of the AGTP.

DESIGN AND METHODS

Study aim

To compare the efficacy of the AGTP vs. standard clinical practice in reducing a non-

revascularizable MI at 3 months and/or 1 year in patients undergoing CABG of other

distal myocardial areas.

Primary hypothesis

The AGTP will be able to reduce the necrotic mass ratio of a non-revascularizable MI,

defined as a reduction of 10% in the estimated infarct size as determined by LGE MRI

at 91 and/or at 365 days in patients undergoing CABG of other myocardial areas.

Secondary hypothesis

1. Compared with those receiving the standard clinical practice, patients

undergoing the AGTP will show:

• Better regional contractibility

• Increased LVEF

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• Less remodeling (assessed by MRI)

• Lower levels of biomarkers (NT-proBNP and high-sensitive troponin T)

2. The AGTP will be safe with no increase in arrhythmias (24-h Holter monitoring)

or outcomes (all cause death or readmission, cardiovascular death or

readmission).

Trial population

Male or female patients aged ≥18 years, with a Q wave in the ECG and a history of a

transmural MI (≥50% late gadolinium enhancement) that is anatomically non-amenable

for surgical revascularization, who are candidates for CABG of other myocardial areas

will be included and randomized after they agree to participate and sign the informed

consent. Exclusion criteria include severe valvular disease that could be repaired

surgically, candidacy for surgical ventricular remodeling, contraindication for MRI,

extra-cardiac disease with a vital prognosis of <1 year, severe renal or hepatic failure,

previous cardiac surgery, pregnancy, or breastfeeding.

Trial design

The AGTP II Trial (NCT02798276) is an investigator initiated, prospective,

randomized, controlled, multicenter (10 tertiary hospitals in Spain) study comparing

two arms of patients with chronic MI undergoing CABG with a distally non-

revascularizable area. The protocol (V.2.2, 04th April 2017) has been approved by the

corresponding Institutional Review Board. One-hundred and eight patients with a

myocardial scar who are candidates for revascularization of other myocardial areas will

be included at the participating sites in Spain by the cardiology and cardiac surgery

team. These selected hospitals have a strong clinical cardiology and cardiac surgery

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team prepared to work together, along with adequate facilities for cardiac MRI. The

AGTP II trial is currently enrolling patients since the 1st of May 2017.

Randomization and blinding

After providing informed consent to the clinical professional, patients will be centrally

randomized at a 1:1 ratio into two arms by allocation order. The randomization scheme

was computer-generated by statisticians from the IMIM (Barcelona, Spain) and once a

patient is enrolled in the study, the local surgeon or study coordinator will contact the

coordinator center to be provided with the allocation group. Treatment allocation will be

blind to the patient and to the clinical team that performs the follow-up.

Study procedure and follow-up

The AGTP will be taught to other cardiac surgeons at study centers by a cardiac surgeon

(ML.C.3) skilled in the procedure who performed all surgeries in the AGTP-I trial.

Patients will be included in two arms, stratified according to the age of the scar (Figure

2):

A) Treated/AGTP patients (n=54): The surgeon will perform a sternotomy for the

CABG of revascularizable vessel(s). The pericardial tissue will be gently dissected

while maintaining its functional vascularization. The pericardium will be excised,

and the adipose flap fixed over the non-revascularizable scar using surgical glue.

Patients randomized to this AGTP group without sufficient fat tissue within the flap

will be excluded from the study, and analyzed in a subgroup.

B) Control patients (n=54): The surgeon will perform a sternotomy for the CABG of

the revascularizable vessel(s). The distally non-revascularizable area will be left

untouched (the current standard-of-care).

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Both groups of patients will be hospitalized for the usual time after a CABG. In

the AGTP-1 trial we found that the AGTP does not prolong the in-hospital stay. To

ensure adequate assessment of efficacy, all patients will undergo several tests (see Table

1) including MRI, ECG, 24-h Holter monitoring, and blood tests. A clinical cardiologist

blinded to the randomization group will conduct follow-up visits (Table 1). Additional

patient visits can also be made, if required, at the discretion of the clinical team. Cardiac

MRI will be performed in a standardized manner at each participant center with 1.5 T

equipment and assessed by a centralized core lab (ICICORELAB, Valladolid, Spain).

All images will be captured under ECG monitoring and in apnea. Images will be

obtained with three plane locators. For the study of ventricular function, we will obtain

steady-state free precession (SSFP) sequences (TrueFISP, bTFE, FIESTA) at short axis

(8 mm slices from the apex to the mitral ring), four-chamber and two-chamber views.

For the study of myocardial fibrosis/necrosis, a gadolinium-based contrast agent will be

intravenously administered and the necrotic myocardium will be assessed using delayed

enhancement. The necrotic size and the salvaged myocardium will be expressed as a

percentage of the total LV mass. In addition, the necrotic area will be expressed in

grams. All the results will be given in absolute numbers and normalized by body surface

area. In addition, LVESV and LVEDV will be assessed. Interobserver and intraobserver

variability will be assessed using randomly chosen (15%) MRI studies.

Risks and benefits

This clinical study is designed and will be implemented in accordance with the

Declaration of Helsinki, with all patients providing informed consent and patients’ data

being anonymized. The study has been assessed and approved by the hospital Ethics

Committee (reference number AC-16-025). Based on the preclinical and clinical AGTP

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phase I study, no safety issues are anticipated. Potential perioperative risks include

pericardium dissection and pericardial fat manipulation, both of which are normal

processes in conventional CABG. Surgical glue (Glubran2®, GEM srl, Viareggio, Italy)

is routinely used in surgeries, including conventional cardiac interventions in clinical

practice. Moreover, a high sensitivity troponin T curve will be obtained during the

surgical procedure to assess the patient’s wellbeing. Perioperative infarct will be

assessed according to the published definition of CABG-related MI.8 Finally, despite no

arrhythmic events detected in the AGTP-I trial, patients will be closely monitored

during their hospital admission. When discharged, the first visit will be 1 week post-

discharge, with follow-up extending to 1 year. Patients will be advised to contact the

clinical team in case of any discomfort. Patients are covered by an insurance policy.

Based on preclinical results, the expected benefits of the AGTP II trial include infarct

size reduction and an improvement in cardiac function. These benefits may be detected

by MRI, changes in surrogate biomarker concentration, and by clinical outcomes.

Criteria for study withdrawal

Patients may voluntarily leave the study at any time without giving any reason and with

no consequences for their medical care. They will also be withdrawn if any exclusion

criterion is detected after the informed consent signing, if they are lost to follow-up, or

fail to collaborate. However, irrespective of the reason of withdrawal, the investigators

will make all efforts to perform the end-of-study visit and collect all available

information from the patient.

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Trial outcomes

The main outcome is the proportion of patients who have a smaller necrotic mass ratio

of a non-revascularizable MI, defined as a reduction of 10% in the estimated infarct size

determined by LGE MRI at 91 and/or 365 days compared with the baseline assessment

(% and/or grams).

Secondary outcomes:

• Improvement in regional contractibility by MRI (time frame: 0-3-12 months)

• Percent changes in functional parameters by MRI: LVEF, right ventricular

ejection fraction (time frame: 0-3-12 months)

• Changes in mL in ventricular volumes by MRI: LVESV, LVEDV, right

ventricular end-systolic volume, right ventricular end-diastolic volume (time

frame: 0-3-12 months)

• Changes in L/min in cardiac output by MRI (time frame: 0-3-12 months)

• Changes in levels of natriuretic peptides (time frame: 0-1 week and 3-12

months)

• Adverse event rate and description: arrhythmia by 24-h Holter (time frame:

0-3-12 months), all-cause death or re-admission (time frame: 12 months),

and cardiovascular death or re-admission (time frame: 12 months).

Data analyses and sample size justification

Statistical analyses will be performed for all patients randomized and operated on. Any

randomized patient who abandons the study (for any cause) before the surgical

procedure will not be followed, but will be registered to investigate possible non-

candidacy for the AGTP. A subgroup analysis will be performed for patients with a

non-revascularizable MI ≤2 months prior to surgery. The aim of this analysis is to

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assess the potentially increased benefit of the AGTP to this subgroup of patients in

terms of remodeling and scar size/regional contractility vs. patients with older

chronified scars.

Descriptive analyses will be performed at the first step. Categorical variables

will be described by frequencies and percentages. Continuous variables will be

described by means and standard deviations or medians and interquartile ranges in case

of skewed distribution. Differences between groups will be compared using Student’s t-

test or one-way ANOVA for multiple comparisons, with Tukey’s test for post hoc

analyses. The comparison of continuous variables between groups will be performed

using analysis of variance for unpaired data once normality is demonstrated

(Kolmogorov-Smirnov test); otherwise, a nonparametric test (Mann-Whitney or

Kruskal-Wallis test) will be used. The MRI data will be analyzed as repeated measures

using ANOVA with Greenhouse–Geisser correction. A 2-sided p<0.05 will be

considered significant. Statistical analyses will be performed using SPSS statistical

package 19.0.1 version (SPSS Inc, Chicago, IL, USA). Monitoring will be performed by

the sponsor center, IGTP.

Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 54

subjects are necessary in the standard of care group and 54 in the AGTP group to

recognize as statistically significant a difference ≥10% reduction in baseline-to-12

month LGE MRI ratio change. Baseline LGE MRI ratio is estimated in 20% ± 7

according to AGTP-I trial.7 The correlation coefficient between the initial and final

measurement was assumed as 0.875. A drop-out rate of 10% is anticipated.

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DISCUSSION

The sequelae of MI have been decreasing since the establishment of early

revascularization procedures worldwide. However, a large number of patients still suffer

from myocardial scarring, including those treated outside of the optimal reperfusion

time window, or not having viable vessels available for CABG. Currently, experimental

cardiac tissue engineering approaches fall into two main categories: scaffolds and cells.

Scaffolds are sometimes supplemented with cytokines, growth factors, and peptides.

They may be of natural or synthetic origin and should recreate the myocardial

environment, support vascularization, ensure electrical and mechanical properties for

optimal host tissue coupling, and ultimately be degradable. Cells from several sources

have been tested (i.e. embryonic stem cells, adipose tissue-derived stem cells, cardiac

progenitor cells, skeletal myoblasts, induced pluripotent stem cells); although, to date,

the optimal cell population is not identified. From the clinical perspective, the main

limiting factors are the requirement for previously banked cells of autologous or

allogeneic origin, and, more importantly, the need for a proximal in-vitro set-up outside

of the confines of the operating theatre.

The AGTP is the first tissue engineering technique to use local adipose tissue

without laboratory manipulation. This approach minimizes the complications that can

arise from the introduction of exogenous stem cells or stem cell-derived

cardiomyocytes, as well as those associated with gene therapy (i.e. risks of

contamination due to laboratory manipulation, infection, immunoreactivity, viral

recombination, etc.). Access to local adipose tissue is a critical and advantageous aspect

of the AGTP. Adipose tissue is a natural niche of progenitor cells, with proven efficacy

in tissue regeneration and disease treatment.9 Based on previous studies, the AGTP may

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stimulate neovascularization and improve local microcirculation and metabolism.4-6 The

paracrine effects derived from adipose-derived MSCs might also contribute to

improving the microenvironment at the scar site, thereby enhancing myocardial

contractility. Further enrichment with exosomes, growth factors, and/or progenitor cells

could form the basis of future means to potentiate the regenerative capacity. Thus, with

this trial, we could establish a new concept in cardiac tissue engineering.

To summarize, the AGTP is technically simple, requiring no learning curve for

cardiac surgeons who will simply dissect structures that are ordinarily modified in

CABG surgery. Furthermore, there is no requirement for additional or expensive

materials, nor do any ethical or social constraints exist as the autologous tissue is

sourced from a site in close proximity to the injury.7

Funding Sources: The AGTP II trial is an investigator initiated study, sponsored by the

Germans Trias i Pujol Research Institute. It is supported by grants from Ministerio de

Educación y Ciencia (SAF2014-59892-R), Ministerio de Economía y Competitividad

(Juan de la Cierva, JCI-2012-14025), Fundació La MARATÓ de TV3 (201502,

201516), and Generalitat de Catalunya (SGR 2014 , CERCA Programme and PERIS

Programme, Departament de Salut SLT002/16/00234, SLT002_16_00209). Additional

funds will be provided by Red de Terapia Celular - TerCel (RD16/0011/0006) and

CIBER Cardiovascular (CB16/11/00403), as part of the Plan Nacional de I+D+I, and

cofunded by ISCIII-Sudirección General de Evaluación and Fondo Europeo de

Desarrollo Regional (FEDER), and “La Caixa” Banking Foundation. This work is

developed within the AdvanceCat project with the support of ACCIÓ (Catalonia Trade

& Investment; Generalitat de Catalunya) under the Catalonian European Regional

Development Fund (ERDF) operational program 2014-2020.

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Contributorship statement: PG, CGM and ABG conceived the study. All authors

participated in the development of the protocol. PG, CGM, JBM, DPF, AT, ARO and

ABG raised the manuscript. MLC, PGP, PA, ASR, ET, MCL, NM, JN, FFA, AC, JL,

RB, CM, JS, SJC, JMM, JCC and AGP revised the manuscript for relevant scientific

contents; AT, ASR, MCL and ARO with specific comments on imaging techniques and

JL and JL on statistical requirements. All authors approved the final version of the

manuscript.

Competing interests: None.

Data sharing statement: The article is a rationale and design manuscript. Patients are

being enrolled at the moment. No further data is available yet. In addition to the paper

and the protocol, the informed consent documents are available from us and from the

Germans Trias i Pujol Hospital Ethics Committee. PG, JL, CGM and ABG will have

access to the final trial dataset.

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REFERENCES

1. Olivetti G, Capasso JM, Meggs LG, et al. Cellular basis of chronic ventricular

remodeling after myocardial infarction in rats. Circ Res 1991;68:856-69.

2. Iacobellis G, Ribaudo MC, Assael F, et al. Echocardiographic epicardial adipose

tissue is related to anthropometric and clinical parameters of metabolic syndrome: a new

indicator of cardiovascular risk. J Clin Endocrinol Metab. 2003;88:5163-5168.

3. Mazurek T, Zhang L, Zalewski A, et al. Human epicardial adipose tissue is a source

of inflammatory mediators. Circulation 2003;108:2460-2466.

4. Bayes-Genis A, Soler-Botija C, Farré J, et al. Human progenitor cells derived from

cardiac adipose tissue ameliorate myocardial infarction in rodents. J Mol Cell Cardiol.

2010;49(5):771-80.

5. Gálvez-Montón C, Prat-Vidal C, Roura S, et al. Transposition of a pericardial-

derived vascular adipose flap for myocardial salvage after infarct. Cardiovasc Res.

2011;91(4):659-67.

6. Gálvez-Montón C, Prat-Vidal C, Roura S, et al. Post-infarction scar coverage using a

pericardial-derived vascular adipose flap. Pre-clinical results. Int J Cardiol.

2013;166(2):469-74.

7. Bayes-Genis A, Gastelurrutia P, Cámara ML, et al. First-in-man safety and efficacy

of the Adipose Graft Transposition Procedure (AGTP) in patients with a myocardial

scar. EBioMedicine 2016;7:248-54.

8. Thygesen K, Alpert JS, Jaffe AS, et al. Joint ESC/ACCF/AHA/WHF Task Force for

Universal Definition of Myocardial Infarction. Third universal definition of myocardial

infarction. J Am Coll Cardiol. 2012 Oct 16;60(16):1581-98.

9. Ma H, Liu J, Qian L. Fat for Fostering: Regenerating Injured Heart Using Local

Adipose Tissue. EBioMedicine 2016;7:25-6.

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FIGURE LEGENDS

Figure 1. Schematic illustration of the use of cATMSCs and the development of the

AGTP in preclinical and clinical studies. The figure was designed and hand-drawn by

CG-M.

Figure 2. Flowchart of the clinical trial design of AGTP II showing the procedures

performed in the two arms of the study. Arrows indicate the infarcted area covered by

the AGTP in the treatment group.

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Table 1. Study visits.

Selection and

randomization Surgery Follow-up

Visit 1 2 3 4 5

Time 0 0

1 week

post-


3 months post-


1 year

post-


Informed consent x

Inclusion/exclusion criteria

x

Vital signs x x x x x

Physical exam x

x x x

Concomitant medication

x

x x x

Pregnancy test x

Blood test x

x x x

ECG x

x x x

Holter x

x x

TTE x

x x

Cardiac MRI with gadolinium

x

x x

HF signs and symptoms

x

x x x

Adverse events x x x x x

Surgical procedure

x

ECG: Electrocardiogram; TTE: Trans-Thoracic Echocardiography; MRI: Magnetic Resonance Imaging; HF: Heart Failure. The surgical procedure will consist of a coronary artery bypass graft (CABG) on the amenable area for both groups. In addition, the treatment group will undergo the adipose graft transposition procedure (AGTP).

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Figure 1. Schematic illustration of the use of cATMSCs and the development of the AGTP in preclinical and clinical studies. The figure was designed and hand-drawn by CG-M.

297x209mm (300 x 300 DPI)

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Figure 2. Flowchart of the clinical trial design of AGTP II showing the procedures performed in the two arms of the study. Arrows indicate the infarcted area covered by the AGTP in the treatment group.

297x209mm (300 x 300 DPI)

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EudraCT Number: N/A Fecha protocolo: Abril 04, 2017

Eficacia de la transposición de un

pedículo adiposo pericárdico sobre el infarto de miocardio en pacientes

(AGTP II trial).

Efficacy of the Adipose Graft Transposition Procedure (AGTP) in

patients with a myocardial scar: The AGTP II trial.

Confidencial

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Resumen Antecedentes: Estudios pre-clínicos en el modelo porcino de infarto de miocardio han demostrado que la transposición de un pedículo adiposo pericárdico (AGTP) reduce el área de infarto. Un primer estudio fase I-II ha sido positivo en pacientes con infarto de miocardio crónico. Objetivo: Evaluar la eficacia del AGTP para la mejora de la función cardiaca en el infarto de miocardio en humanos. Métodos: Estudio multicéntrico, aleatorizado y controlado de eficacia. Se incluirán pacientes candidatos a revascularización quirúrgica de una o más zonas del miocardio con onda Q negativa en el ECG y una zona con retención de gadolinio por resonancia de ≥50% no revascularizable. Los pacientes se randomizarán en 2 ramas: pacientes control en los que se revascularizarán las zonas candidatas; y pacientes tratamiento o AGTP en los que se revascularizarán las zonas candidatas y la zona no revascularizable se cubrirá con el pedículo. La variable principal de estudio es la reducción de la zona necrótica en la zona del AGTP por RMN. Se analizarán otras variables de seguridad y eficacia incluyendo parámetros de RMN y biomarcadores. El seguimiento será de un año y los pacientes serán monitorizados de forma estrecha, vigilando la aparición de cualquier evento adverso a la semana y al año de la fecha de la intervención quirúrgica. Importancia potencial: Se trata de una novedosa aproximación a la regeneración cardiaca que podría suponer una opción terapéutica para pacientes que no tienen otra alternativa.

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