Therapeutic strategy towards an optimal coronary flow after AMI – Amiflow

«The aim of this project is to provide the proof of concept that i.v. administration of ANGPTL4 at reperfusion provides pharmacological vascular protection and cardioprotection during AMI, i.e., to protect against myocardial infarction by reducing the no-reflow phenomenon. We plan to develop 3 work packages to allow the demonstration of this proof of concept:
1. WP1: to continuously produce ANGPTL4 in sufficient amounts for this two year project and to
prepare large scale production for the future; the production will be validated using quality control
tests as described in our previous publications (1,13,33).
2. WP2: to characterize the cardioprotective properties of ANGPTL4
2.1. Task 1: to determine the optimal dose protecting against myocardial infarction when administered at reperfusion.
2.2. Task 2: to determine the pharmacological cardioprotective profile
3. WP3: to demonstrate that cardioprotection is induced by reducing the no-reflow phenomenon in a non rodent large animal model«

Compared to all preclinical and present clinical studies that primarily focus on cardiomyocyte, targeting the no-reflow is an important therapeutic need and this strategy is therefore very attractive, original and almost unexplored. The proof of concept that would be provided by the present project is absolutely required from clinical and industrial point of views at the end of this project. In the future, the clinicians asssociated to this project will be in charge to initiate the clinical translation of these findings and elaborate a clinical project development.

Upon successful completion of these pivotal studies showing that ANGPTL4 is a pharmacological and therapeutic approach for cardioprotection during AMI by targeting the no-reflow phenomenon, the incremental change in the level of technology readiness may be great. Indeed, we are palnning to initiate clinical trials to demonstrate safety and initial efficacy in terms of ANGPTL4 dose range, schedule and route of administration.

This ANR project allowed us to:
1- extend our Patent in US.
Methods and pharmaceutical composition for the preservation
of vascular endothelial cell barrier integrity
US 20130023473 A1
2- publish articles and reviews
The no-reflow phenomenon: State of the art.
Bouleti C, Mewton N, Germain S.
Arch Cardiovasc Dis. 2015 Dec;108(12):661-74. doi: 10.1016/j.acvd.2015.09.006. Epub 2015 Nov 23. Review.
Angiopoietin-like 4 serum levels on admission for acute myocardial infarction are associated with no-reflow.
Bouleti C, Mathivet T, Serfaty JM, Vignolles N, Berland E, Monnot C, Cluzel P, Steg PG, Montalescot G,
Germain S.
Int J Cardiol. 2015;187:511-6. doi: 10.1016/j.ijcard.2015.03.263. Epub 2015 Mar 24.
[Protection of vascular integrity in reperfusion during stroke].
Bouleti C, Mathivet T, Lapergue B, Monnot C, Germain S.Référence du formulaire : ANR-FORM-090601-01-02 11/18
Med Sci (Paris). 2014 Jun-Jul;30(6-7):608-10. doi: 10.1051/medsci/20143006003. Epub 2014 Jul 11.
3- Demonstrate that ANGPTL4 protects against myocardial infarction in mice and
characterize the pharmacological cardioprotective profile of ANGPTL4.
4- Demonstrate that ANGPTL4 protects against myocardial infarction in pigs
5- Define new protocols in order to determine the pharmacokinetic profile of ANGPTL4 in
pigs.
6- During the course of this project, we have additionnally shown that ANGPTL4 protects
against stroke in mice:
Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke.
Bouleti C, Mathivet T, Coqueran B, Serfaty JM, Lesage M, Berland E, Ardidie-Robouant C, Kauffenstein G,
Henrion D, Lapergue B, Mazighi M, Duyckaerts C, Thurston G, Valenzuela DM, Murphy AJ, Yancopoulos GD,
Monnot C, Margaill I, Germain S.
Eur Heart J. 2013 Dec;34(47):3657-68. doi: 10.1093/eurheartj/eht153. Epub 2013 May 14.

The most effective strategy to limit the size of acute myocardial infarction (AMI) and tissue damage, is to reperfuse myocardial tissue as quickly as possible. The restoration of blood flow in ischemic tissue can supply oxygen and nutrients to the myocardium and limit the extent of the AMI. However, this also leads to reperfusion injury associated with microvascular dysfunction and induction of vascular permeability. In 5 to 50% of patients presenting with MI (ST +), primary angioplasty can complete recanalization of epicardial coronary arteries without obtaining an optimal myocardial reperfusion: a phenomenon known as "no-reflow". The deterioration of vascular integrity contributes to large part to vascular damage, with hemorrhage, edema, inflammation, all phenomena involved in no-reflow. Its prevention, recommended by the ACC / AHA issue is important for promoting therapeutic myocardial flow during reperfusion and thereby improve cardiac function and prolong survival. Ischemia induces expression of VEGF (vascular endothelial growth factor), also called VPF (Vascular Permeability Factor) during AMI. VEGF-induced vascular permeability through destabilization of interendothelial junctions. These vessels leak and contribute to the formation of edema and leukocyte infiltration, both detrimental to healing. The project partners have previously characterized and published (Galaup et al. Circulation, 2012) the protective effects of ANGPTL4 (Angiopoietin-like 4), which belongs to the superfamily of angiopoietins. We demonstrate that ANGPTL4 is expressed in heart tissue that has suffered from ischemia, both in humans than in mice. Invalidation of ANGPTL4 in mice leads to an increase in infarct size compared to wild type mice. Then, we demonstrated that ANGPTL4 has no direct effect on the survival of cardiomyocytes cultured in normoxia or hypoxia. We studied and characterized a massive disorganization of endothelial cells adherens junctions molecules in the heart having suffered from ischemia in KO mice compared to wild type, a demonstration of major vascular damage. Interestingly, the KO phenotype was rescued by administration of recombinant human ANGPTL4 protein, produced in the laboratory. We then studied the no-reflow in rabbits, in whom this phenomenon is classically studied. We show that injection of recombinant human ANGPTL4 protein 5 minutes before induction of ischemia reduced the infarct size, limit the no-reflow and haemorrhage. All of these results has been the subject of a patent by INSERM Transfert (Angiopoietin-like protein 4 (ANGPTL4) POLYPEPTIDE FOR USE IN THE PRESERVATION OF VASCULAR ENDOTHELIAL CELL BARRIER INTEGRITY) during the month of January 2011. The ANGPTL4 protein therapy could therefore be a promising new pharmacological approach, alone or combined with other strategies, to induce cardioprotection in the acute phase of myocardial infarction by targeting the no-reflow phenomenon. Thus, we propose three workpackages for i) produce the recombinant protein ANPGTL4 throughout the 2 years of development of this project. We will need about 120 mg of recombinant protein on the entire project and our latest technical developpments techniques allow such production ii) a-define the dose of ANGPTL4 active at reperfusion in mice; and b- determine the cardioprotective pharmacological profile of this ANGPTL4 protein (ceiling and window of cardioprotection); iii) demonstrate that ANGPTL4-mediated cardioprotection is due to inhibition of no-reflow phenomenon in a relevant preclinical model in cardiovascular research, the pig.