Servier’s partnership with ARMGO Pharma: targeting heart failure by inhibiting cardiac RyR2 calcium leak






Nicole VILLENEUVE, PhD
Director of Research Discovery in Cardiology and Hypertension*



Jean-Paul VILAINE, MD
Director of Cardiovascular Research Discovery*
*IdRS (Institut de Recherches Servier) Suresnes, FRANCE


Emmanuel CANET, MD, PhD
President, Research & Development, IRIS (Institut de Recherches Internationales Servier)
Suresnes, FRANCE

Servier’s partnership with ARMGO Pharma: targeting heart failure by inhibiting cardiac RyR2 calcium leak

by N. Villeneuve, E. Canet, and J. P. Vilaine, France

In the developed world, heart failure (HF) is the leading cause of mortality and morbidity, with much of the mortality attributed to sudden cardiac death, frequently due to ventricular arrhythmias. The young are not exempt, as seen with catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1), typically associated with sudden death during childhood or adolescence. Success with current therapeutic management, though improved, remains underwhelming. Calcium is a pivotal player in cardiomyocyte function, and aberrant intracellular calcium handling leads to deterioration of myocardial contractility and arrhythmias in HF. A pathologic leak of calcium from the sarcoplasmic reticulum through ryanodine receptor channels during diastole has been targeted for therapeutic development. Stemming from the discovery of Rycals®, small molecules that prevent this calcium leak from ryanodine receptor 2 channels, a collaborative partnership established in 2006 between ARMGO Pharma Inc and Servier has aimed to develop a Rycal for the treatment of HF. Completed phase 1 and 2a studies with the Rycal candidate S 44121/ARM036 provide first evidence for the therapeutic potential of such an approach to target leaky RyR2 channels in such patients. The ongoing goal of the collaboration is to select follow-on compounds that improve upon the profile of first-generation Rycal candidates for further development.

Medicographia. 2014;36:349-354 (see French abstract on page 354)


Heart failure and CPVT as causes of mortality and morbidity

Heart failure (HF) is the leading cause of mortality and morbidity in the developed world. Despite improvement in therapeutic management of patients with HF, prognosis remains poor, with 30% mortality 1 year after diagnosis; at least half of this mortality is due to ventricular arrhythmias (sudden cardiac death [SCD]). Chronic symptomatic HF is currently treated with several classes of drugs, some of which have been shown to improve survival in randomized clinical trials. Main treatments include angiotensin-converting enzyme (ACE) inhibitors, β-adrenergic antagonists, diuretics, spironolactone, and angiotensin II receptor blockers (ARBs). Digitalis is still used in a small percentage of patients. Though compliance with these therapy regimens at optimal doses is observed within clinical trials, it is estimated that the majority of HF patients are undertreated, largely due to a combination of side effects and lack of efficacy of current therapies. Ventricular arrhythmias leading to SCD are frequent in patients with cardiac diseases. However, SCD also occurs in young subjects with structurally normal hearts and normal electrocardiograms (ECGs). Familial type 1 catecholaminergic polymorphic ventricular tachy- cardia (CPVT1) is typically associated with sudden death in childhood or adolescence associated with exercise or emotional stress (30% to 50% experience sudden death by the age of 30 when untreated), and is the most lethal ion channel–associated cause of SCD.

The current pharmacologic treatment for patients identified with CPVT1 is β-adrenergic antagonists (βblockers), which provide incomplete protection. Patients who experience arrhythmia even under pharmacologic treatment can be offered implantable cardioverter-defibrillators (ICDs). In fact, despite pharmacologic therapies, 50% of patients require shocks from their devices to stop ventricular tachyarrhythmias.1

Involvement of calcium leak in HF and CPVT

The calcium ion (Ca2+) is a quintessential player in all functions of the cardiomyocyte, from excitation-contraction (EC) coupling to growth and survival. Various impairments of intracellular Ca2+ metabolism are responsible for mechanical dysfunction and arrhythmias in HF.2


Figure 1
Figure 1. Abnormal intracellular Ca2+ handling
in failing cardiomyocytes results in reduced
contractile force and prolonged relaxation.

(A) Reduced SR Ca2+ storage and release cause abnormal
systolic cardiomyocyte function. Abnormal β-AR and GPCR
signaling increase the expression of PKA and CaMKII,
which hyperphosphorylate and alter the function of RyR2
and LTCC. FKBP12.6 also contributes to stabilization of
the RyR2 open probability. Impaired SERCA2a function
decreases SR Ca2+ loading, resulting in reduced SR Ca2+
content and release and, therefore, reduced contractility.
Alterations in LTCC function and STIM1-Orai1–mediated
store-operated Ca2+ influx can also result in abnormal Ca2+
handling during systole.
(B) Reduced SR Ca2+ resequestration is a key abnormality
in diastole. Decreased SERCA2a activity, downregulation
of SERCA2a, a decreased PLB:SERCA2a ratio, or PLB
hypophosphorylation cause prolonged intracellular Ca2+
transients, reduced SR Ca2+ loading, and slowed cardiomyocyte
relaxation. An increase in levels of NCX might be
a compensatory response to prevent Ca2+ overloading.
While increased NCX activity would be initially adaptive,
excessive or sustained NCX activation could contribute to
decreased sarcoplasmic reticulum Ca2+ content by removing
cytosolic Ca2+, and reducing systolic Ca2+ transients
and contractile function. SR Ca2+ leak resulting from impaired
RyR2 function and increased expression of PP1 can
reduce SR Ca2+ content and increase cytosolic Ca2+ concentration
during diastole. On activation, the β-AR–AC–
GPCR complex synthesizes cAMP, which activates PKA.
Hyperactive PKA decreases the Ca2+ sensitivity of the
myofilaments and prolongs relaxation. Intracellular Ca2+
overload stimulates CaMKII, which contributes to SR diastolic
Ca2+ leak by hyperphosphorylating RyR2 and induces the
transduction of pathological Ca2+ signaling.
Abbreviations: AC, adenylate cyclase; ATP, adenosine
triphosphate;β-AR, βadrenergic receptor; CaMKII, calciumand
calmodulin-dependent protein kinase type II; Ca2+,
calcium ion; cAMP, cyclic adenosine monophosphate;
FKBP12.6, FK506-binding protein; GPCR, G-protein–
coupled receptor; LTCC, voltage-dependent L-type Ca2+
channel; NCX, Na+/Ca2+ exchanger; Orai1, calcium-release–
activated calcium channel protein 1; P, phosphorylation;
PKA, protein kinase A; PLB, cardiac phospholamban;
PP1, protein phosphatase 1; RyR2, ryanodine receptor 2;
SERCA2a, sarcoplasmic/endoplasmic reticulum calcium
ATPase 2a; SR, sarcoplasmic reticulum; STIM1, stromal
interaction molecule 1; TnC, troponin C.
After reference 9: Kho et al. Nat Rev Cardiol. 2012;9(12):
717-733. © 2012, Nature Publishing Group.



One such impairment is diastolic Ca2+ leak through ryanodine receptor 2 (RyR2), which has emerged as an independent therapeutic target in HF3,4 and a potential target of β-blockers. 5 Indeed, it was shown in animal models of HF, as well as in HF patients, that chronic sympathetic hyperactivity in HF causes remodeling of the RyR2 complex, leading to a pathologic diastolic Ca2+2+ stores.5-8 This leads to depressed intracellular Ca2+ cycling, decreased SR Ca2+ release in response to an action potential, and less force produced from EC coupling (Figure 1).9 The resultant deterioration of cardiac function has been extensively studied.10 Genetic investigations have shown that CPVT disease is most- ly secondary to mutations in RyR2 channels. Some of these mutations manifest as triggers of fatal ventricular arrhythmias during exercise due to the increased SR Ca2+ uptake, which increases the Ca2+ gradient and increases the diastolic SR Ca2+ leak. This activates inward depolarizing currents via the Na+/Ca2+ exchanger that cause delayed afterdepolarizations (DADs) and trigger fatal cardiac arrhythmias.11,12 Figure 2 depicts a schematic of the predicted structure of the RyR2 receptor.13

Small molecules to improve cardiac diseases by preventing Ca2+ leak

K201, also known as JTV-519, was shown to inhibit muscle Ca2+ overload and to reduce Ca2+ leak through the RyR2 channel along with its consequences in HF models.7,14 However, as this compound can inhibit several ion channels and transporters,15 it is unclear whether these beneficial effects are RyR2 dependent or involve other types of action.

In 2004, derivatives of this compound (among them, S107 and S 44121/ARM036) were identified by Dr A. Marks and his colleagues. These small molecules, known as Rycals®, preventpathologicCa2+ leak from RyR2withoutblocking the channel’s pore. They also improved cardiac function in mice with dysfunctional RyR2 (Figure 3, page 352).16-18 ARMGO Pharma Inc, a privately held biopharmaceutical company founded in 2006 and based in New York, is discovering and developing Rycal drugs for the treatment of cardiac, musculoskeletal, and neurological disorders, and has exclusively licensed rights to RyR intellectual property and technology from Columbia University, emanating from the laboratory of Dr A. Marks. In addition to the S 44121/ARM036 clinical program for the treatment of HF and arrhythmias, ARMGO is collaborating with Servier on the advancement of preclinical Rycal candidate S48168/ARM210 for the treatment of Duchenne muscular dystrophy.





Figure 2
Figure 2. Schematic cartoon showing the predicted structure of the cardiac ryanodine
receptor, RyR2, including the sites of interactions with key ancillary proteins
and the phosphorylation sites.

Calsequestrin (CASQ2), junctin (JUN), and Triadin (TRI) proteins interacting with RyR2 in the SR are
also depicted. Phosphorylation sites: CAMKII, calmoldulin-dependent protein kinase type II; PKA,
protein kinase A. Circle in red: mutation.
Abbreviations: Ca, calcium; FKBP, FK506-binding protein; P, phosphorylation; SR, sarcoplasmic reticulum.
After reference 13: Liu et al. J Mol Cell Cardiol. 2009;46(2):149-159. © 2009, Elsevier.


ARMGO-Servier collaborative partnership for the selection of compounds targeting HF and CPVT

Evidence of mortality reduction with ivabradine in HF patients (as seen in SHIFT [Systolic Heart failure treatment with If inhibitor ivabradine Trial]19) rewards Servier’s research efforts in postischemic cardiopathies. Indeed, in recent decades, Servier has been particularly committed to the fight against these pathologies. As a growing body of preclinical evidence has specified a relationship between dysfunctional RyR2 in cardiomyocytes and the development of HF, strategies described by Marks and colleagues were obviously of interest for Servier. Consequently, a collaborative partnership with ARMGO was established in 2006 to develop a Rycal for treatment of chronic HF.

In permeabilized cardiomyocytes isolated from a mouse model in which RyR2 has a mutation at residue 4496 where arginine is replaced with cysteine (R4496C-RyR2), compounds that stabilize the RyR channel decreased the occurrence of Ca2+ sparks, suggesting that, as expected, they target RyR2 to limit Ca2+ leak. Furthermore, at Servier, we demonstrated in a rat model of post–myocardial infarction (MI) chronic HF (1-hour occlusion, 3 months of reperfusion) that RyR-stabilizing compounds induced limitation of cardiac dysfunction, both systolic and diastolic. The effect was not restricted to preservation of myocardial contractility, as cardiac structural changes (reduction in cardiomyocyte volume and in cardiac collagen content) were also observed. In addition, in a mouse model of chronic HF post-MI (permanent left anterior descending coronary artery [LAD] ligation), an antiarrhythmic effect with a decrease in ventricular extrasystoles was demonstrated. This antiarrhythmic effect was directly related to a decrease in number of Ca2+ sparks and triggered activity occurrence measured in isolated permeabilized post-MI cardiomyocytes.


Figure 3
Figure 3. S107 improves cardiac function in RyR2-S2808D+/+ mice.

(A) Echocardiographic measurements during a 10-week treatment period, showing that the S107-treated (20 mg/kg/d via osmotic pump) group exhibited preserved cardiac function compared with the vehicle-treated group (#P<0.01 versus vehicle-treated group). (B and C) Cardiac catheterization was performed at the end of study, and both dP/dtmax and (dP/dtmax)/Pid, where Pid indicates the instantaneous developed pressure in mm Hg and s–1 is the unit of measure for (dP/dtmax)/Pid, showed a significant improvement in the S107-treated group (*P<0.05 versus vehicle-treated group). Abbreviations: dP/dtmax, maximum rate of pressure change in the ventricle; S107, a Ca2+-channel stabilizer (a Rycal).
After reference 17: Shan et al. J Clin Invest. 2010;120:4375-4387. © 2010, American Society for Clinical Investigation.



Moreover, in healthy conscious pigs studied in Servier labs, at a plasma concentration above those able to prevent in vitro and in vivo RyR2 dysfunction, no pharmacological effects, eg, on hemodynamic parameters or on global cardiac function, at rest or during catecholaminergic stress have been observed.

In view of these considerations, and the standard preclinical development program demonstrating appropriate safety, one compound, S 44121/ARM036, was proposed for clinical development for the prevention of cardiovascular morbidity and mortality in patients with chronic HF and left ventricular dysfunction and for cardiac arrhythmia in patients with CPVT1. S 44121/ARM036 has now completed phase 1 and phase 2a studies, with results validating the approach of targeting RyR2 in HF patients.

ARMGO-Servier research collaboration: moving forward

The observed activity of S 44121/ARM036 in the completed phase 2a trials supports the likelihood that clinically meaningful effects can be achieved by effectively targeting leaky RyR2 channels in patients with chronic HF and cardiac arrhythmias. Meanwhile, the ongoing goal of the ARMGO-Servier chemistry program is to select follow-on compounds for further clinical development that improve upon the profile of the first-generation Rycal candidates. Current screening tools available to guide these efforts are in vitro pharmacological as- says, along with absorption, distribution, metabolism, and excretion (ADME) tests. A dynamic high-throughput screening (HTS) assay first developed at Servier uses fluorometric analysis of the Ca2+ signal from stimulated RyR2 activity in human embryonic kidney 293 cells (HEK293) overexpressing RyR2 (HEK-RyR2).


Figure 4
Figure 4. Cameleon is expressed in cells by viral transduction.

(A) This sensor is targeted to the endoplasmic reticulum. (B) As Ca2+ binds to calmodulin (CaM), the Ca2+-binding domain undergoes a conformational change, interacting with its binding peptide. This brings YFP closer to CFP, increasing the efficiency of FRET. (C) Representative traces of endoplasmic reticulum Ca2+ measurements nto HEK293 cells stably expressing cameleon and RyR2. SOICR (store-overload–induced Ca2+ release) was induced by adding 2mM Ca2+ and results in Ca2+ oscillations. Decrease in Ca2+ during the first phase of the oscillations can be explained by a massive opening of RyR2 channels. Increase of Ca2+ in the second phase is mainly due to SERCA activity. As expected, 1μM ryanodine completely blocks the oscillations.
Abbreviations: Ca2+, calcium ion; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; GECI, genetically encoded calcium indicators; HEK293, human embryonic kidney 293 cells; M13, calmodulin peptide-binding protein; RyR2, ryanodine receptor 2; YFP, yellow fluorescent protein.



Two robust functional assays using Ca2+ indicator dye–loaded cardiomyocytes have been developed (1 at ARMGO and 1 at Servier) to assess RyR2-associated Ca2+ abnormalities.

Using this strategy, several new-generation Rycals that significantly inhibit Ca2+ release via a leaky RyR2 channel have been identified. Similarly to what has been done previously, subsequent in vivo efficacy studies will need to confirm the efficacy of these new-generation Rycals in in vivo models of HF.

In parallel, Servier has developed new assays to confirm the mechanism of action of selected compounds. Firstly, measurement of SR Ca2+ in HEK-RyR2 cells using fluorescence resonance energy transfer (FRET)-based Ca2+ sensors and epifluorescence microscopy allows the direct evaluation of the consequences of the Ca2+ leak (Figure 4).20 Secondly, confocal microscopy–facilitated measurement of Ca2+ sparks in cardiomyocytes from pathological models provides insight into RyR2 dysfunction. These Ca2+ sparks are intracellular Ca2+– release events arising from the activation of a cluster of RyRs.21 At the same time, the exciting and significant progress achieved in induced pluripotent stem cell (iPS) technology22 gives us an invaluable opportunity to evaluate the effects of new Rycals on iPS-derived cardiomyocytes obtained from skin fibroblasts or hair keratinocytes from patients with RyR2 mutations.

Summary

Fundamental research has highlighted that abnormal diastolic Ca2+ leak from cardiac SR RyRs is one of the pathophysiological mechanisms leading to cardiac contractile dysfunction and arrhythmias. First evidence of therapeutic potential in patients with HF has been obtained with a compound— S 44121/ARM036—targeting this pathological process. Continued collaborative efforts are under way between Servier and ARMGO Pharma to select follow-on compounds for further clinical development that improve upon the profile of first-generation Rycal candidates.



See Emmanuel Canet’s biography on page 269


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Keywords: Ca2+ leak; heart failure; ryanodine receptor 2; Rycal; S 44121/ARM036; sarcoplasmic reticulum