The adenylate cyclase activator forskolin potentiates the positive inotropic effect of the phosphodiesterase inhibitor milrinone but not of the calcium sensitizer levosimendan nor of its hemodynamically active metabolites: an apparent conundrum.

OR-1855 and OR-1896 are two hemodynamically active metabolites of the inodilator levosimendan, with calcium sensitizing activity, but their mechanism of action is still not fully understood.It has been previously reported that the positive inotropic effect of levosimendan is not potentiated by the adenylate cyclase activator forskolin whereas forskolin does potentiate the effects of the phosphodiesterase (PDE) inhibitor milrinone.To ascertain whether the active metabolites follow the same pattern of levosimendan, the positive inotropic effects of OR- 1855 and OR-1896, were studied in guinea-pig isolated papillary muscle in the presence and absence of forskolin. OR-1855 and OR-1896 were also tested as inhibitors of PDE-III and PDE-IV.Our result show that 0.1 µM forskolin did not potentiate the positive inotropic effect of either OR-1855 or OR-1896, as in the case of the parent compound levosimendan. As in previous studies, the positive inotropic effect of milrinone was markedly potentiated in the presence of forskolin.From these data we propose an explanation for the divergent behaviour of the calcium sensitizing drugs and PDE inhibitors

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

Rapid Absorption of Naloxone from Eye Drops

Naloxone as emergency treatment for opioid overdosing can be administered via several routes. However, the available administration methods are invasive or may be associated with incomplete or slow naloxone absorption. We evaluated pharmacokinetics and local tolerance of naloxone ocular drops in healthy beagle dogs. Naloxone administration as eye drops produced fast absorption with time to maximum plasma concentration (t(max)) achieved in 14 to 28 min, high plasma exposure (C-max 10.3 ng/mL to 12.7 ng/mL), and good bioavailability (41% to 56%). No signs of ocular irritability were observed in the scored ocular tolerability parameters, and the reactions of dogs suggesting immediate ocular discomfort after the dosing were sporadic and short lasting. Slight and transient increase in the intraocular pressure and transient decrease in the tear production were recorded. The results suggest that eye drops may provide a fast and an effective non-invasive route for naloxone administration to reverse opioid overdosing, and clinical studies in the human are warranted.Peer reviewe

The reverse mode of the Na+^+/Ca2+^{2+} exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca$^{2+}$-handling. The fundamental role of the inward Na$^+$/Ca$^{2+}$ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca$^{2+}$-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca$^{2+}$-transient amplitude with larger SR Ca$^{2+}$-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca$^{2+}$$_i$ and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca$^{2+}$$_i$ and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca$^{2+}$$_i$ and heart rate with increasing Na$^+$$_i$. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca$^{2+}$-influx that could increase SR Ca$^{2+}$-content, which consequently leads to enhanced pacemaking activity

Efficacy of selective NCX inhibition by ORM-10103 during simulated ischemia/reperfusion.

In this study we evaluated the effects of selective Na+/Ca2+ exchanger (NCX) inhibition by ORM-10103 on the [Ca2+]i transient (CaT), action potential (AP), and cell viability in isolated canine ventricular cardiomyocytes exposed to a simulated ischemia/reperfusion protocol performed either alone (modeling moderate low-flow ischemia) or with simultaneous strophantidine challenge (modeling more severe low-flow ischemia). CaTs were monitored using a Ca2+-sensitive fluorescent dye, APs were recorded by intracellular microelectrodes, and anaerobic shifts in cellular metabolism were verified via monitoring native NADH fluorescence. Simulated ischemia increased the NADH fluorescence, reduced the amplitudes of the AP and CaT and induced membrane depolarization. APs moderately shortened, CaTs prolonged. Diastolic [Ca2+]i ([Ca2+]iD) level increased significantly during ischemia and further elevated following strophantidine application. Reperfusion normalized the NADH level, the amplitude of the AP and duration of the [Ca2+]i transient, but only partially restored action potential triangulation and the amplitude of the CaT. [Ca2+]iD decreased in untreated, but further increased in strophantidine-treated cells. 10microM ORM-10103 significantly reduced the ischemic [Ca2+]i raise in both untreated and strophantidine-treated cells. During reperfusion ORM-10103 decreased [Ca2+]i and eliminated its diastolic elevation in untreated and strophantidine-treated cardiomyocytes. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusion-induced increase in AP and CaT variabilities were substantially reduced, but ischemia-induced shifts in AP morphology were barely influenced. In conclusion, selective NCX inhibition by ORM-10103 is highly effective against ischemia/reperfusion induced pathologic alterations in [Ca2+]i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue

Background and Purpose: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca$^{2+}$ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na$^{+}$/ Ca$^{2+}$ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the I$_{f-}$NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). Experimental Approach: Currents were measured by patch-clamp, Ca$^{2+}$-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model. Key Results: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca$^{2+}$ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (I$_{f}$) was previously inhibited and vice versa, the effect of I$_{f}$ was augmented when the Ca$^{2+}$ handling was suppressed. Conclusion and Implications: We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between I$_{f}$ and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of I$_{f}$ or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking

Blockade of sodium‑calcium exchanger via ORM-10962 attenuates cardiac alternans

Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium‑calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug's mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium‑calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans