Thérapie cellulaire cardiaque après infarctus du myocarde P. Lemarchand

Prévalence de l’insuffisance cardiaque USA 29 686 pts Les études de cohorte aux USA montrent que la prévalence de l IC a plus que triplé dans les 10 dernières années, alors que cette période d ’observation 1989-1999 ne reflète pas la prescription large des betabloquants et des vasodilatateurs qui diminue la mortalité. McCullough et al. JACC 2002;39:60-9

Coronary Heart Disease ACUTE MYOCARDIAL INFARCTION Hours Days Months CARDIAC REMODELING

REPARATION CARDIAQUE Grounds et al. J. Histo Cyto 2002;50:589

The heart is a regenerating organ Carbon 14 incorporation Cardiomyocyte renewal rate: 45% Turnover decreases after the age of 25 Fig. 4. Dynamics of cardiomyocyte turnover. (A) Individual data fitting assuming a constant turnover (see supporting online text) reveals an almost linear decline of cardiomyocyte turnover with age (R = –0.84; P = 0.001). A constant-turnover hypothesis might therefore not represent the turnover dynamics accurately. (B) Global fitting of all data points (see supporting online text, error sum of squares = 1.2 x 104) shows an age-dependent decline of cardiomyocyte turnover. (C) The gray area depicts the fraction of cardiomyocytes remaining from birth, and the white area is the contribution of new cells. Estimate is from the best global fitting. (D) Cardiomyocyte age estimates from the best global fitting. The dotted line represents the no-cell-turnover scenario, where the average age of cardiomyocytes equals the age of the individual. The black line shows the best global fitting. Colored diamonds indicate computed data points from 14C-dated subjects. Error bars in (A) are calculated from the errors on 14C measurements. Error bars in all other graphs are calculated for each subject individually and show the interval of possible values fitted with the respective mathematical scenario. Dynamics of Cell Generation and Turnover in the Human Heart: The number of cardiomyocytes remains constant during the human lifespan, Bergmann et al., 2015, Cell 161, 1566–1575: Endothelial and mesenchymal cells increase into adulthood and show high turnover Cardiomyocyte turnover decreases exponentially with age and is <1% per year in adults The cardiomyocyte turnover rate is equal in the main subdivisions of the human heart Spontaneous cardiac regeneration capabilities are restricted Science 2009;324:98-102

Stratégie de développement de la thérapie cellulaire cardiaque Cellules souches capables de réparer Essai chez le petit animal, après avoir induit un infarctus du myocarde Essai chez le gros animal Essai clinique chez un petit nombre de patients

CELLULES UTILISABLES POUR LA THÉRAPIE CELLULAIRE CARDIAQUE J. Clin. Invest. 115:572-583 (2005) Unchain my heart: the scientific foundations of cardiac repairStefanie Dimmeler1, Andreas M. Zeiher1 and Michael D. Schneider2 Figure 1 Sources of cells for cardiac repair, and routes of their administration. (A) Cells in current human trials include skeletal muscle myoblasts, unfractionated bone marrow, and circulating (endothelial) progenitor cells. Cells in preclinical studies include bone marrow MSCs, multipotent cells from other sources, and novel progenitor or stem cells discovered in the adult myocardium; see text for details. (B) Existing trials use intracoronary delivery routes (over-the-wire balloon catheters), intramuscular delivery via catheters (e.g., the NOGA system for electromechanical mapping), or direct injection during cardiac surgery. Not represented here are the theoretical potential for systemic delivery, suggested by the homing of some cell types to infarcted myocardium (39), and strategies to mobilize endogenous cells from other tissue sites to the heart. Dimmeler S, J. Clin. Invest. 115:572-583 (2005)

AUTOGREFFE DE MYOBLASTES PRINCIPE Biopsie musculaire injection Culture in vitro et expansion des myoblastes Ré-injection en zone myocardique infarcie biopsie AVANTAGES Cellules musculaires satellites Autogreffe (immunité, éthique…)

AUTOGREFFE DE MYOBLASTES Améliore la fonction myocardique RESULTATS EXPERIMENTAUX Faisable (rat, lapin, porc, mouton) DIFFICULTES ACTUELLES Mécanisme d’amélioration inconnu Survie cellulaire faible Pas de différenciation cardiomyocytaire Troubles du rythme ventriculaire

ESSAIS CLINIQUES PHASE I/II Nb of cells Nb of Pt Other surgery Study Year LVEF TV Ménasché 2003 24±4% 8.7±1.9x108 10 CABG 4 TV 4 TV (2 DC) Smits 2003 36±11% 2.0±1.1x108 13 / No Pagani 2003 <3x108 5 LVAD ? Cell transplantation is a promising alternative strategy to treat heart failure. Skeletal myoblast are attractive donor cells for such therapy : they have contractile phenotype, can be harvested for autologous transplantation and are resistant to ischemia. Small scale clinical trials of autologous myoblast transplantation suggested the feasibility and the efficiency of such therapy. However, these clinical trials were hampered by unexpected episodes of ventricular tachyarrhythmias within weeks following cell transfer Herreros 2003 36±8% 1.9±1.2x108 12 CABG Amiodarone Siminiak (POZNAN trial) Amiodarone for some Pt 2004 25–40% 0.04 à 0.5x 108 10 CABG 4 TV Chachques 2004 28±3% 3x108 20 CABG Amiodarone Dibs 2005 28% 0.01 à 3x108 24 CABG 2 TV ? ? 3x108 6 LVAD 1 TV ? Control 4x108 8x108 Ménasché (MAGIC trial) 2008 15 à 35% 97 CABG 6 TV Amiodarone

La thérapie cellulaire cardiaque déclenche des arythmies ? Effet indésirable mettant en jeu le pronostic vital Quel(s) mécanisme(s) ? Lésion tissulaire due à l’injection ? Hétérogénéité électrophysiologique ? Etude expérimentale évaluant le potentiel arythmogène de ces cellules

Stimulation Ventriculaire J 0 contrôles milieu cellules J 7 J 14 21 28 35 Stimulation Ventriculaire Programmée Rythme spontané Rythme imposé (100ms) Enregistrement d’ECG de surface

24h après injection Myoblastes (vert) Marqueur d’ADN (rouge)

Stimulation Ventriculaire Programmée (1) Tachycardie ventriculaire non soutenue Pas de trouble du rythme déclenché Tachycardie ventriculaire soutenue Fibrillation ventriculaire

INTERET DE LA MOELLE OSSEUSE cellules souches mésenchymateuses progéniteurs endothéliaux cellules souches hématopoiétiques La MO contient: Mobilisation des cellules souches

MOBILISATION SPONTANEE APRES IDM (1) 16 patients avec IDM Controles: sujets avec douleur atypique et m^mes médicaments Contrôles avec douleur atypique Prélèvement sang périphérique Circulation 2001; 103: 2776-2779 Circulation 2001; 103: 2776-2779

MÉCANISMES CELLULAIRES POTENTIELS J. Clin. Invest. 115:572-583 (2005) Unchain my heart: the scientific foundations of cardiac repairStefanie Dimmeler1, Andreas M. Zeiher1 and Michael D. Schneider2 Figure 2 Mechanisms of action. Progenitor cells may improve functional recovery of infarcted or failing myocardium by various potential mechanisms, including direct or indirect improvement of neovascularization. Paracrine factors released by progenitor cells may inhibit cardiac apoptosis, affect remodeling, or enhance endogenous repair (e.g., by tissue-resident progenitor cells). Differentiation into cardiomyocytes may contribute to cardiac regeneration. The extent to which these different mechanisms are active may critically depend on the cell type and setting, such as acute or chronic injury. Dimmeler S, J. Clin. Invest. 115:572-583 (2005)

ETUDE EXPERIMENTALE CHEZ LE GROS ANIMAL Infarctus Entrainement à l’injection des CSM Biodistribution Survie des CSM 5-7 jours Culture CSM Evaluation histologique et fonctionnelle Injection CSM moelle * * * Forest et al. Stem Cell Research & Therapy 2010

INDUCTION DE L’INFARCTUS ballon Après thrombus Avant thrombus Forest et al. Stem Cell Research & Therapy 2010

INFARCTUS DU MYOCARDE Marquage des noyaux

Présence des cellules dans la zone infarcie Noyaux (rouge) + cellules injectées (vert) Débris cellulaires Amas

Les cellules sont dans le parenchyme Cellules endothéliales (rouge, Willebrand) + cellules injectées (vert)

1 hour after cell IC injection Biodistribution Same pig model Injection of stem cells, labelled with radio-element (99mTc) 1 hour after cell IC injection MI: myocardial infarction BM-MNC: injected stem cells Identical cell retention Forest et al. Stem Cell Research & Therapy 2010

Intra-Coronary route outperforms IV route MI: myocardial infarction BM-MNC: injected stem cells Forest et al. Stem Cell Research & Therapy 2010

Intra-Coronary vs. IV route Intravenous BMC infusion is ineffective to target myocardium  Favor in situ delivery (IC or direct myocardial delivery) Forest et al. Stem Cell Research & Therapy 2010

AIM OF THE BONAMI TRIAL To assess the beneficial effect of cell therapy in patients with decreased left ventricular ejection fraction after acute myocardial infarction, and to identify predictive factors of successful therapy. The aim of the BONAMI trial was to evaluate the beneficial effect of coronary injection of autologous mononucleated bone marrow cells on myocardial viability in patients with acute myocardial infarction and left ventricular ejection fraction lower than 45%. Roncalli et al. Eur Heart J 2011

STUDY DESIGN of the BONAMI TRIAL Randomized, multicenter controlled trial (101 patients) 6 academic hospitals in France The BONAMI trial was a randomized, multicentre, controlled trial, conducted in 6 academic hospitals in France The main inclusion criteria were Inaugural acute MI with single coronary lesion and successful angioplasty, Left ventricular ejection fraction lower or equal to 45%, and impairment of myocardial viability, defined by the presence of at least 2/17 non viable segments. For the purpose of this analysis, we referred to the internationnal definition of left ventricular segmentation represented here on the right side. The main exclusion criteria were the presence of multivessel disease, or the use of drug eluting stents All patients underwent a rest myocardial perfusion SPECT 4 days and 3 months after myocardial infarction, 4 h after an intravenous rest injection of Tl-201 Main inclusion criteria: Inaugural acute MI Single coronary lesion and successful angioplasty - LVEF ≤ 45% - Impairment of myocardial viability on SPECT 27

DESIGN OF THE BONAMI TRIAL Day 0 Acute MI screening (n= 122) Day 0-4 Day 4-7 SPECT and radionuclide angio Randomization (n=101) The BONAMI trial was designed as follow: - After acute myocardial infarction and successful angioplasty, patients with left ventricular ejection fraction lower than 50% assessed by echocardiography within 4 days after MI, underwent SPECT imaging and radionuclide angiography. - Patients with preserved myocardial viability or left ventricular ejection fraction over 45% were excluded, - And only patients with at least 2/17 non viable segments AND left ventricular ejection fraction lower than 45% were randomized. - Along with a prespecified stratification according to diabetic status and timing of revascularization, we randomized 101 patients. 49 patients were randomized in the control group, 52 were randomized in the bone marrow cell group. These patients underwent bone marrow harvest and intracoronary bone marrow cell injection the same day, 7 to 10 days after MI. - After 3 months, SPECT imaging and radionuclide angiography were repeated to assess the alteration of myocardial viability in both groups. Control BMC Day 7-10 BM harvest Intra coronary injection SPECT, radionuclide angio Month 3 28

PRIMARY ENDPOINT Myocardial viability at 3 months after MI evaluated by resting Thallium 201-SPECT - Criterion for cell therapy success: viability improvement of ≥ 2/17 segments (2 non viable become viable) 100% 0% 50% Short- axis apical median basal M0 M3 The primary endpoint of the BONAMI trial was - the alteration of myocardial viability 3 months after MI, evaluated by resting thallium SPECT The criterion for cell therapy success was - an improvement of at least 2/17 segments (meaning 2 non viable segments become viable) All measurements were performed by 2 blinded investigators of an independent core lab. This figure represents what we considered as a significant improvement of myocardial viability. The baseline evaluation, on the upper line, displays a large viability defect in the ventricular septum and the inferior wall from the apex to the base. The evaluation after 3 months, on the bottom line, shows a clear improvement of septum in the apical, medium and basal segments. - All measurements were performed by 2 blinded investigators of an independent core lab.

CELL THERAPY PROCEDURE Cell therapy product: 50 cc of bone marrow were harvested (iliac crest punction) under local anesthesia Bone marrow mononucleated cells were isolated by ficoll gradient 100 x 106 autologous mononucleated cells (in 10cc) Intra coronary cell injection: The same day as BM collection Mean delay btw acute MI and BMC infusion: 9.3 ± 1.7 days - The cell therapy product in the BONAMI trial was obtained from a 50cc of bone marrow harvest under local anesthesia. - Bone marrow mononucleated cells were then isolated by Ficoll gradient to achieve a standardized cell therapy product made of 100 millions autologous mononucleated cells in a 10cc volume. - The cells were injected the same day of bone marrow harvest in the MI related artery - In our study, the mean delay between acute MI and BMC infusion was about 9 days. 30

RESULTS: Baseline Characteristics Control (n= 49) BMC (n= 52) p Age (years) 55 ±11 56 ±12 NS Male gender, % 89.8 80.8 Hypertension, % (n) 34.7 (17/49) 34.6 (18/52) Dyslipemia, % (n) 46.2 (24/52) Diabetes mellitus, % (n) 18.4 (9/49) 21.2 (11/52) Active smokers, % (n) 53.1 (26/49) 53.8 (28/52) Timing of revasc <12h ,% (n) 76 (37/49) 75 (39/52) Culprit artery (LAD) , % (n) 96 (45/47) 92 (45/49) LVEF (%, by RNA) 37.0 ±6.7 (47) 35.6 ±7 (50) These are the main characteristics of our population. - Mean age was about 55 years old, with more than 80% of male gender. - 1/3 of our population had hypertension and dyslipidemia, 20% were diabetics, and more than 50% were active smokers. - There was no differences between groups as regards to age, gender or cardiovascular risk factors. 32

RESULTS: Assessement of Cell Therapy Success - Prespecified criterion of cell therapy success = 2 non viable segments become viable 3 months after myocardial infarction 34 % 1 2 3 4 Percentage of patients n= 7/43 n= 16/47 Control group BMC group ≥ 2/17 segments 16 % p=0.06 - This is the result according to our prespecified criterion of cell therapy success, defined as 2 non viable segments become viable 3 months after MI. - In the control group, 16% of patients exhibited improvement of myocardial viability as compared to 34% of patients in the cell therapy group. - Hence, the number of patients with myocardial viability improvement was twice greater in the BMC group. However, this difference did not reach the statistical significance with a p value of 0.06 - The number of patients with myocardial viability improvement >2/17 was twice greater in the BMC group 33

RESULTS: Assessment of Cell Therapy Success (2) Multivariate analysis OR 95% CI p 4.89 1.18 - 20.19 0.03 4.59 1.09 - 19.28 0.04 5.72 0.86 - 38.29 0.07 2.32 0.60 - 8.98 0.22 3.12 0.36 - 26.92 0.30 1.02 0.95 - 1.11 0.56 0.38 0.09 - 1.59 0.19 group (BMC vs. control) tobacco status (non/former smoker vs. smoker) microvascular obstruction (yes vs. no) En analyse multivariée avec introduction des facteurs de risque cardiovasculaires (tabagisme, dyslipidémie, HTA, etc.) et la micro-obstruction vasculaire en IRM (qui est un facteur de gravité de l’infarctus): L’essai est positif avec une différence significative entre les deux groupes (p=0,03), et cette analyse met en évidence un rôle négatif du tabagisme au moment de l’infarctus, et un rôle possiblement positif (p=0,07) de la micro-obstruction vasculaire. Ces critères sont en discussion dans le projet d’essai européen de morbi-mortalité en cours de montage, auquel le réseau Bonami devrait participer. Nous sommes également en train de préparer un papier sur le monitorage biologique des 101 patients inclus dans cette étude, monitorage qui a été entièrement assuré par notre laboratoire, et qui montre un effet négatif du tabagisme sur les progéniteurs endothéliaux de la moelle osseuse et du sang de ces patients. dyslipidemia (yes vs. no) gender (male vs. female) age hypertension (yes vs. no) 0.25 1 4 16 Significant role of smoking at the time of myocardial infarction 34

CONCLUSION - The BONAMI trial is the first randomized multicenter trial to investigate the beneficial effect of coronary injection of autologous BMC on myocardial viability as a primary endpoint. - In this trial, coronary autologous BMC injection, 9 days after acute MI, to patients with low EF, failed to reach the primary endpoint, although a strong trend was observed. - The trend to improve myocardial viability was associated with a specific negative role of tobacco and positive role of no-reflow. To conclude the preliminary results of the BONAMI trial, - The BONAMI trial is the first randomized trial to investigate the benefit of Intracoronary autologous BMC administration on myocardial viability at 3 months as a primary endpoint. - In this trial, intracoronary autologous BMC administration, 9 days after acute MI, to patients with low ejection fraction failed to reach the primary endpoint, although a strong trend was observed. - The trend to improve myocardial viability was not associated with any trend to improve left ventricular ejection fraction evaluated 3 months after cell therapy. 35

MISE EN PLACE DE L’ESSAI Dec 2002 PHRC local 40 sujets Fev 2003 13 000 € Fev 2003 CCPPRB 40 sujets favorable Mai 2003 PHRC nat. 80 sujets dec 2003 219 000€ Avril 2004 Afssaps 100 sujets juil 2004 favorable Sept 2004 CCPPRB 100 sujets favorable + demandes de financements complémentaires (AFM, Fondation de France). Début des inclusions: décembre 2004 janvier 2007: 100/100 patients inclus Premiers résultats cliniques: fin-2010

Why proceed clinically while basic questions are unresolved? Use of stem cells for cardiac repair not yet ready for clinical application outside a research or trial setting Preclinical studies will not answer complex questions Trials provide some answers to preconceived hypothesis but also generate new questions Need for a large clinical trial to evaluate clinical endpoints

Factors influencing therapeutic outcomes when using stem cells. Factors influencing therapeutic outcomes when using stem cells. Factors influencing the therapeutic benefit from stem cells to treat cardiovascular disease can be broken down into 3 main categories of (1) the individual cell’s function, (2) the surrounding tissue function in response to the therapeutic cell treatment, and (3) the external influences on the cellular and tissue response toward repair and regeneration. Circulation Research March 4, 2016 vol. 118 no. 5 867-880 Kathleen M. Broughton, and Mark A. Sussman Circ Res. 2016;118:867-880 Copyright © American Heart Association, Inc. All rights reserved.

Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis of adult stems cells as a cardiovascular therapy. Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis of adult stems cells as a cardiovascular therapy. The analysis is an overall snapshot of the use of adult stems cells in the marketplace in the treatment and cure for cardiovascular disease. The analysis looks at both the internal workings of the field, as well as the external origin, in terms of both helpful and harmful elements. Empowering Adult Stem Cells for Myocardial Regeneration V2.0 Circulation Research March 4, 2016 vol. 118 no. 5 867-880 Kathleen M. Broughton, and Mark A. Sussman Circ Res. 2016;118:867-880 Copyright © American Heart Association, Inc. All rights reserved.

Lille Toulouse Grenoble H. Mondor Montpellier Nantes - E. Teiger -P. Lemarchand -J-N. Trochu -D. Crochet -A. Tirouvanziam -A. Bammert -Y. Goueffic -G. Lamirault -V. Probst -S. Abbey -F. Valette - J Hélias -C. Perigaud -V. Forest -M. Audrain -C. Hémont -G. Follea -J-M. Nguyen - E. Van Belle - F. Mouquet - S. Susen - C. Bauters - P-V. Ennezat - T. Letourneau - V. Gaxotte - C. Foucher - J-P. Jouet - F. Villard - I. Yakoub-Agha - J-P. Bérégi - J. Darchis - P. Asseman - B. Jude - J-J. Bauchart Toulouse Grenoble - J. Roncalli - M. Galinier - A. Parini - P. Bourin - A. Huynh - M.Attal - D. Carrié - M. Elbaz - JM. Fauvel - P. Massabuau - R. Cagnac - MJ. Allibeli-Chemarin - V. Chabbert -H. Rousseau - L. Daudé - H. Coulier - S. Cappellesso-Fleury - C. Rage - J. Gaudé -Y. Neuder -G. Vanzetto -M. Favrot -MJ. Richard -C. Saunier -D. Fagret -A. Calizzano -JY. Cahn -CE. Bulabois -F. Garban -F. Thony -S. Mouret -S. Bouzon H. Mondor - E. Teiger - J-L. Dubois-Randé - P. Le Corvoisier - S. Champagne - L. Boudali - O. Montagne - JL. Monin - J. Rosso - JF. Deux - C. Focseneanu - ML. Bourhis Montpellier - Ch. Piot - B. Klein - ZH. Lu - M. Baudard -JF. Rossi - D. Dietz -JC. Macia -D. Mariano-Goulart