Long‐term effects of Na+/Ca2+ exchanger inhibition with ORM‐11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction

Aims: Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+/Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. Methods and results: Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1 mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure–volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9 ± 2 mmHg; NXT (n = 12) vs. NXT + ORM (n = 12); P = 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n = 12) vs. NXT + ORM (n = 12); P < 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. Conclusion: Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure

Discovery and Characterization of ORM‐11372, a Novel Inhibitor of the Sodium‐Calcium Exchanger with Positive Inotropic Activity

Background and purpose The lack of selective sodium‐calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1. inhibitor. Experimental approach A flavan series‐based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM‐11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM‐11372 were studied in normal and infarcted rats, and rabbits. Human cardiac safety was studied ex‐vivo using human ventricular trabeculae. Key results ORM‐11372 inhibited human NCX 1.1 reverse and forward currents; IC50 values were 5 and 6 nM, respectively. ORM‐11372 inhibited human cardiac sodium 1.5 (INa) and hERG KV11.1 currents (IhERG) in a concentration‐dependent manner; IC50 values were 23.2 and 10.0 μM. ORM‐11372 caused no changes in action potential duration; short term variability and triangulation were observed for concentrations of upto 10 μM. ORM‐11372 induced positive inotropic effects in 18 ± 6% and 35 ± 8% anesthetized rats with myocardial infarctions and rabbits, respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. Conclusion and implications ORM‐11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or blood pressure, without pro‐arrhythmic risk

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC(50) values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (I(Na) ) and hERG K(V) 11.1 currents (I(hERG) ) in a concentration-dependent manner; IC(50) values were 23.2 and 10.0 μM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 μM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.Peer reviewe

Selektiivisten NCX inhibiittoreiden suunnittelu sydänsairauksien krooniseen ja akuuttiin hoitoon

The Na+/Ca2+ exchanger (NCX) is a transmembrane protein which plays an important role in regulating cardiomyocyte Ca2+ homeostasis. NCX operates in two modes: Forward (exit mode) and Reverse (entry mode). In the forward mode, NCX extrudes Ca2+ ions from the intracellular matrix that is proposed to have effects on relaxation in conditions where the intracellular Ca2+ concentration is high. In the reverse mode, NCX intrudes Ca2+ ions from the extracellular matrix in conditions where the intracellular Na+ concentration is high to enable depolarization of the membrane potential. Selective inhibition of NCX1 has been proposed to create cardio protection in certain conditions, hence various research groups in academia and industry have been studying the role, function and therapeutic value of NCX1.1 and have had activities to develop novel NCX 1.1 inhibitors that could be used as cardioprotective therapy. Based on current public data, six pharma companies have worked in the field with NCX inhibitors including Orion Corporation. The objective of this thesis was to develop potent selective and novel NCX1 inhibitors for chronic treatments and acute on-off use for cardiac indications with desired PK properties. While for the chronic indications the long-term and steady exposure to the inhibitor molecule is the ideal, for the acute indications an inhibitor with relatively fast PD effect followed by fast clearance would be preferred. The presented work describes the development of two novel potent and selective NCX1.1 inhibitors with different chemical structures, which enable scientific communities to study further functions and therapeutic value of NCX inhibition. The first part of the thesis focuses on the background and the rational for developing NCX 1.1 inhibitors for acute and chronic indications. The second and the major part of the thesis describes the discovery of two distinctively different and novel chemical series of NCX inhibitors and optimization strategy towards desired DMPK properties. Optimization was guided by the results from in vitro test battery of the project workflow. The work resulted in three patent applications filed by the Orion Corporation and the most selective NCX 1.1 inhibitors (ORM-10962 and ORM-11372) reported in the literature. The third part of the thesis focuses on the studies made in Orion and in the collaboration with academic groups to further reveal the pharmacological properties of the two lead molecules ORM-10962 and ORM-11372 from the different chemical series. At the same time these most selective tool molecules (ORM-10962 and ORM-11372) has given the opportunity for the academic groups to study further the role, function and therapeutic value of NCX1.1. Final part of the thesis describes the status of NCX 1.1 inhibitor research in Orion Corporation and pilot toxicology result of the ORM-11372.  Transmembraaniproteiini Na+/Ca2+-vaihtajalla (NCX) on tärkeä rooli kardiomyosyyttien kalsium (Ca2+) tasapainon säätelyssä. NCX toimii kaksisuuntaisesti: poistaa Ca2+:a solusta (suunta ulospäin) tai tuo Ca2+:a solun sisään (suunta sisäänpäin). Nykykäsityksen mukaan NCX vaikuttaa relaksaatioon solunsisäisen Ca2+-ionikonsentraation ollessa suuri vaihtamalla solunsisäisestä matriisista Ca2+-ioneja ulos. Vastaavasti solunsisäisen Na+-ionikonsentraation ollessa suuri NCX siirtää Ca2+ ioneja solunulkoisesta matriisista solun sisään membraanipotentiaalin neutraloimiseksi. Selektiivisellä NCX1-inhibitiolla on esitetty olevan sydäntä suojaavia vaikutuksia tietyissä sydänsairauksissa. Tämän vuoksi monet tutkimusryhmät tiedemaailmassa mukaan lukien Orion Oyj ovat keskittyneet tutkimaan NCX:n toimintaa, merkitystä ja mahdollista terapeuttista arvoa. Lisäksi nykyisen tiedon valossa kuudella lääkeyrityksellä on ollut NCX1.1.-inhibiittoritutkimusprojekteja tarkoituksena kehittää hoitoja sydänsairauksiin. Väitöskirjatyön tavoitteena oli kehittää uusia tehokkaita NCX1-inhibiittoreita krooniseen sekä akuuttiin ''katkaisija''-tyyppiseen hoitoon ideaalisella farmakokineettisellä profiililla. Kroonisessa indikaatiossa ideaalilla molekyylillä saavutetaan pitkäaikainen ja tasainen altistus oraalisesti. Akuutissa indikaatiossa toivottu ''katkaisija''-tyyppinen farmakokineettinen profiili saavutetaan suhteellisen nopealla farmakodynaamisella vaikutuksella, jota seuraa nopea lääkeaineen poistuminen elimistöstä (suuri puhdistuma). Väitöskirjassa kuvataan kahden uuden, kemiallisesti hyvin erilaisen, tehokkaan ja selektiivisen NCX1.1.-inhibiittorin keksiminen ja kehittäminen, jotka mahdollistavat luotettavamman NCX-inhibition toiminnan, merkityksen sekä terapeuttisen potentiaalin tutkimisen tiedeyhteisössä.Työn ensimmäisessä osassa keskitytään taustaan sekä perusteluun, miksi NCX inhibitiomekanismilla on esitetty olevan sydäntä suojaavia ominaisuuksia akuuteissa ja kroonisissa indikaatioissa. Väitöskirjatyön toinen ja myös pääasiallinen osa kuvaa kahden uuden ja toisistaan erilaisen kemiallisen sarjan keksimisen sekä kemiallisen rakenteen optimointistrategian kohti toivottua farmakokineettistä profiilia. Työn tuloksena syntyi kolme Orionin jättämää patenttihakemusta sekä kaksi selektiivisintä NCX-inhibiittoria, joita kirjallisuudessa on koskaan raportoitu (ORM-10962 ja ORM-11372). Työn kolmannessa osassa kuvataan farmakologiset in vivo ja ex vivo-mallit, joilla molekyylit ORM-10962 ja ORM-1137 karakterisoitu tarkemmin ja niistä saadut tulokset. Tutkimukset ovat tehty joko Orionilla tai yhdessä akateemisten ryhmien kanssa. Samalla nämä kaksi tunnetuinta selektiivistä ja tehokasta työkalumolekyyliä (ORM-10962 ja ORM-11372) ovat mahdollistaneet akateemisten ryhmien tutkimukset NCX1.1:n merkityksestä, toiminnasta ja terapeuttisesta arvosta. Väitöskirjatyön viimeisessä osassa päivitetään NCX1.1:n tutkimuksen tilanne Orionissa sekä ORM-11372:lla tehdyn toksikologisen pilottitutkimuksen tulokset.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments

BACKGROUND: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined. METHODS: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 mug/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts. RESULTS: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 muM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 muM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts. CONCLUSIONS: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in <i>In Vitro</i> and <i>In Vivo</i> Experiments - Fig 10

<p><b>Panel A.</b> The effect of 1 μM ORM-10962 on the delayed afterdepolarization (DAD) amplitude in dog right ventricular Purkinje fibres. DAD was evoked by a 40 stimulus train with a stimulation cycle length of 400 ms in the presence of 150 nM digoxin. Trace <b>a</b> is a control recording, trace <b>b</b> indicates the induction of DAD by 150 nM digoxin, and trace <b>c</b> demonstrates that 1 μM ORM-10962 almost completely abolished DAD. <b>Panel B.</b> Effect of ORM-10962 (1 μM) on digoxin-induced automaticity in dog right ventricular Purkinje fibres. Trace <b>a</b> is a control recording. Spontaneous activity was recorded after a 40 stimulus train with a stimulation cycle length of 400 ms in the presence of 150 nM digoxin (trace <b>b</b>). Application of 1 μM ORM-10962 in the presence of digoxin abolished the spontaneous activity (trace <b>c</b>).</p