A novel ryanodine receptor 2 inhibitor, M201-A, enhances natriuresis, renal function and lusi-inotropic actions: Preclinical and phase I study

Background and Purpose: The

increase in natriuresis, glomerular filtration rate and creatinine clearance, while maintaining acceptable levels of drug safety and tolerability.

Conclusions and Implications:
The novel drug M201-A inhibited diastolic Ca 2+ leak via RyR2, improved cardiac lusi-inotropic effects in rats, and enhanced natriuresis and renal function in humans.These findings suggest that this drug may offer a potential new treatment option for chronic kidney disease and heart failure.

| INTRODUCTION
Chronic kidney disease (CKD) presents a substantial global public health concern, with an estimated prevalence of 700 million people globally (Bikbov et al., 2020).Conversely, heart failure (HF) affected approximately 64.3 million people globally in 2017 (Bansal Nisha et al., 2019;James et al., 2018), and this number is projected to rise significantly due to the ageing of populations (Lippi & Sanchis-Gomar, 2020).The interaction between HF and CKD creates a vicious cycle known as the cardiorenal syndrome (House et al., 2019;Méndez et al., 2022).However, therapeutically beneficial approaches based on the pathophysiological basis of both diseases have not yet been established.
Ryanodine receptors (RyRs) play a vital role in regulating intracellular Ca 2+ homeostasis and maintaining cellular function in all cells.
Here, we describe the synthesis method of M201-A, its intracellular Ca 2+ effects on RyR2 in rat cardiac cells, its impact on cardiac haemodynamics in rats, natriuresis and renal function in dogs and finally, its results in a phase I clinical trial.Notably, the phase I clinical study confirmed the safety and tolerability of M201-A when administered at a dose effective on these targets.

| Synthesis, chiral column separation and X-ray crystal structure of M201-A
M201-A is chemically related to K201; K201 is metabolized in vivo into four compounds.M201-R (M-II) is one of them and is the major metabolite of K201 in humans (Figure S1).M201-R (racemic body) was synthesized by oxidizing K201 HCl with metachloroperoxybenzoic acid in chloroform, and separated using silica gel What is already known

What does this study add
• M201-A demonstrated RyR2 inhibition, positive lusi-inotropic effects, and enhanced natriuresis and renal function.
M201-AÁHCl and K201-HCl stock solutions (1.0 mmolÁL À1 ) were prepared in 10% dimethyl sulfoxide (DMSO).A spinning wheel spectrophotometer (Cairn Research Ltd., UK; sampling rate of 500 Hz) was used to determine Fura-4F fluorescence (340 and 380 nm of excitation; R 340/380 nm ).Field stimulation was applied for 2 min per condition to achieve steady-state Ca 2+ transients.Cells were incubated in KH with final concentrations of 0.1% DMSO, ±1.0 μmolÁL À1 of K201/ M201-A and ±300 nmolÁL À1 of isoprenaline before field stimulating and measuring the Fura-4F fluorescence as previously published to determine spontaneous SR-mediated diastolic Ca 2+ leak (in the form of Ca 2+ waves).The peak Ca 2+ transient amplitude is the maximum value of [Ca 2+ ] i , while the amplitude is the maximum minus the minimum levels of [Ca 2+ ] i (Martin et al., 2023;Miller et al., 2005).

| Measurement of haemodynamics in M201-A, M201-B and K201
Male Sprague-Dawley rats aged 9-10 weeks were intraperitoneally injected with 120 mgÁkg À1 of Inactin (thiobutabarbital sodium salt hydrate) and immobilized without additional anaesthesia.All experiments were performed under spontaneous respiration, which was maintained throughout the experiments.A pressure catheter (SPR-320, Millar Inc., USA) was inserted from the right common carotid artery into the left ventricle.Arterial pressure was measured in the left femoral artery.M201-A hydrochloride solution was injected through the femoral vein at a rate of 0.1 mgÁkg À1 Ámin À1 for 20 min, and the heart rate, systolic blood pressure (SBP) and diastolic blood pressure (DBP), max + dP/dt and min À dP/dt of left ventricular pressure and left ventricular end-diastolic pressure (LVEDP) were determined over an period of 5 min after each parameter was stabilized.The baseline was established as pre-drug values, by averaging the measured values at 4, 3, 2, 1 and 0 min (average of 20 beats each).The percentage change from these pre-drug values was calculated at each point for each animal.The effects on LVEDP were calculated as ΔLVEDP (mmHg) (n = 5 in each group).At the end of the experiment, animals were euthanized by intravenous potassium chloride overdose under anaesthesia.

| Measurement of urinary volume and renal function of M201-A
Male beagle dogs (n = 4, 10-12 kg) were used for this study.The dogs were immobilized with 5 mgÁkg À1 , i.m., of ketamine hydrochloride (Ketalar 500 mg, Daiichi Sankyo Co., Ltd.) as a pre-anaesthetic administration, followed by isoflurane inhalation, intubation, artificial ventilation and inhalation of 2%-5% isoflurane.To ensure stable conditions, the dogs were placed in a dorsal position on a warming mat heated to 42 C.A saline solution (Otsuka Pharmaceutical) was infused continuously at a rate of 0.05 mlÁkg À1 Ámin À1 through one leg vein.Inulin was administered initially as a bolus dose of 100 mgÁkg À1 , followed by a continuous infusion at a rate of 1.2 mgÁkg À1 Ámin À1 using a syringe pump (TE-331S, Terumo) through a separate leg vein, ensuring distinct routes for the saline and inulin.Throughout the experiment, blood pressure and heart rate were monitored in each dog.The lower abdomen was incised, and cannulas (SP-102; Natsume Seisakusho) were inserted into the ureters on both sides to collect urine produced by the dog.Following all preparations, the dog was allowed to stand still for 1 h.M201-A was administered at a rate of 0.05 mlÁkg À1 Ámin À1 through the femoral vein of the lower extremity at doses of 0.1, 0.3, 1 and 3 mgÁkg À1 for 20 min, incrementally increasing the dosage using an infusion pump (55-1111; HARVARD).One-minute urine output, urinary Na and Cl excretion, creatinine clearance (CCr), serum creatinine levels and GFR were measured.Blood and urinary inulin levels were determined using the Anthrone method (Florijn et al., 1994).After the intended collection was completed, animals were immediately euthanized by intravenous pentobarbital hyperanaesthesia.S2).The participants received repeated intravenous doses once daily, administered over a 20-min period in the morning for four consecutive days.All 18 participants had no previous medical history, and all blood, biochemical, electrocardiogram (ECG) and urine tests were within normal limits.The renal effects of M201-A administration were assessed in participants with normal serum creatinine levels ranging from 0.85 to 1.10 mgÁdl À1 .Urine volume, urinary electrolyte excretion (Na, Cl, K, P and Ca), uric acid, estimated glomerular filtration rate (eGFR; by creatinine) (eGFRcre), eGFR by cystatin C (eGFRcys) and CCr were evaluated from the day before administration until 24 h after the final administration on Day 4. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was used to calculate the eGFRcre, eGFRcys and CCr (Inker et al., 2012;Levey & Coresh, 2012;Levey et al., 2011Levey et al., , 2009)).The pre-determined examination times outlined in the protocol were used to assess safety and tolerability, involving the examination of subjective symptoms, liver function, renal function, total cholesterol, low-density lipoprotein-cholesterol (LDL-C), highdensity lipoprotein-cholesterol (HDL-C), triglycerides (TGs), total protein (TP), albumin, albumin-globulin ratio (A/G), C-reactive protein (CRP), uric acid, electrolytes, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), amylase, brain natriuretic peptide (BNP), haemoglobin A1c (HbA1c), glucose and blood tests, ECG and urine tests.

| Study design in the phase 1 clinical trial
Notably, one participant in the 0.4 mgÁkg À1 M201-A group withdrew from the study on Day 3 due to back pain.Thus, their data were only included in the analysis and eligibility assessment up to Day 2. The remaining 17 participants completed the study as planned.

| Pharmacokinetic analysis of M201-A
The pharmacokinetic analysis included 12 participants who were assigned to one of two two groups receiving different doses of M201-A (0.2 and 0.4 mgÁkg À1 groups).Plasma M201-A concentrations were measured on Days 1 and 4 (including 24 h after the last dose), at pre-dosing, immediately after dosing (0 min) and at 15 min and 1, 2, 4, 8 and 24 h after dosing.

| Statistical methods
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2022).For statistical analysis, a one-way analysis of variance with a Bonferroni post hoc test was employed for cardiac haemodynamics in rats.To compare Ca 2+ transient characteristics in adult rat cardiomyocytes with K201 and M201-A against the control group, we obtained the mean number of cardiomyocytes (n) from each heart (n) and conducted a Student's paired t test.
The data are presented as the mean ± standard error of the mean, and P-values of <0.05 were considered significant in all analyses.
To assess group differences between the placebo and 0.2 or 0.4 mgÁkg À1 M201-A groups, repeated measures analyses of covariance for water intake, urine volume, urine electrolytes and other excretions (Dunnett's adjusted P-value < 0.05) were performed.The mean change in values from Days 1 to 4 at each time point was calculated using the value obtained before administration as the baseline for kidney function (eGFRcre, eGFRcys and CCr).A t test corresponding to the baseline was performed within each group.A statistically corrected P-value of <0.0125 (0.05 Â 1 /4) was used, following Bonferroni's adjustment, as the criterion for determining a significant difference because the study involved repeated dosing for 4 days.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in https://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander et al., 2023).

| Cardiac haemodynamic effects of M201-A, M201-B and K201 in the rat in vivo
We investigated the effect of M201-A on cardiac haemodynamics and compared it with the effects of M201-B and K201 (Figure 3).M201-A administration resulted in an increase in SBP at peak values (4.0 ± 0.9%) and enhanced max + dP/dt and min À dP/dt by 10.9 ± 1.0% and 6.6 ± 1.5%, respectively (Figure 3b,d,e).In contrast, M201-B and K201 significantly reduced heart rate, left ventricular systolic pressure, max + dP/dt and min À dP/dt compared with their previous values (Figure 3a,b,d,e).A comparison between the groups revealed that M201-A had the opposite effect compared with M201-B and K201 (Figure 3a,b,d,e).LVEDP demonstrated no significant difference between the groups (Figure 3f).

| Clinical characteristics of clinical trial participants
This study enrolled 18 healthy Japanese adult males.The main inclusion and exclusion criteria are provided in Table S2.Participants were randomly assigned to one of three groups: placebo, 0.2 mgÁkg À1 M201-A or 0.4 mgÁkg À1 M201-A groups (n = 6 per group).The age, height, weight and body mass index of the participants did not show significant differences among the placebo, 0.2 mgÁkg À1 M201-A and 0.4 mgÁkg À1 M201-A groups (Table S3).Furthermore, there were no significant variations in serum creatinine, eGFRcre, eGFRcys and CCr between the three groups (Table S3).
However, no change in the urinary excretion of potassium, calcium, phosphorus or uric acid was observed (Figure 5e-h).The eGFRcre remained unchanged from baseline in all three groups (Table 2a).The eGFRcys from baseline at 2 h post-dose demonstrated a significant increase in the 0.4 mgÁkg À1 M201-A group (P < 0.05) (Table 2b).CCr from the baseline at 2-h post-dose demonstrated a significant increase in both the 0.2 and 0.4 mgÁkg À1 M201-A groups (P < 0.05) (Table 2c). Figure S6 presents the distribution of eGFRcre and eGFRcys in 18 participants before M201-A treatment, with eGFRcre being <90 mlÁmin À1 Á1.73 m À2 in 17 of 18 (94.4%)participants.

| Cardiac safety data and adverse events report for M201-A
An increased heart rate was observed in the placebo, 0.2 mgÁkg À1 M201-A and 0.4 mgÁkg À1 M201-A groups after 8 h of treatment on Days 1-4, with no significant differences from the placebo group (Figure 6a).Subsequently, all groups experienced a shortening of the QT interval following the increased heart rates (Figure 6e).Regarding QTcF (QT interval corrected by Fridericia's formula), there was no significant difference in the 0.2 and 0.4 mgÁkg À1 M201-A groups F I G U R E 5 Effect of M201-A on urine volume, urinary electrolytes and uric acid excretion: (a) 0-to 24-h water intake in each group; (b) 0-to 24-h urine volume in each group; (c, d) 0-to 24-h urinary sodium and chloride excretion in each group; and (e-h) urinary excretion of potassium, phosphorus, calcium and uric acid in each group.*P < 0.05 versus placebo.compared with their respective pre-administration values (Figure 6f).No significant differences were observed in the pre-drug values of either SBP or DBP between the 0.2 and 0.4 mgÁkg À1 M201-A groups and the placebo group (Figure 6b).
Adverse events (AEs) and side effects are listed in Table 3.All other AEs were resolved without the need for treatment, and there were no severe AEs observed among the 18 patients.Importantly, back pain was determined to be unrelated to the drug.Individual changes in haematological tests, urinary biochemical tests and vital signs demonstrated no abnormal changes in clinically significant laboratory values or any abnormal changes corresponding to serious AEs in all participants treated with the study drug.

| Pharmacokinetics of M201-A in qualifying participants
Table 4 presents the pharmacokinetic parameters in plasma.We considered a steady state to have been achieved based on plasma M201-A concentration measurements 4 days after repeated administration.There was no evidence of drug accumulation (Figure S7).

| DISCUSSION AND CONCLUSIONS
The aim of this study was to investigate the inhibitory effect of a novel compound, M201-A, on RyR-mediated calcium leakage and its pharmacological impact on cardiac and renal function in animals and humans while assessing its safety and tolerability.M201-A is an enantiomer characterized by a sulfoxide structure with one oxygen bonded to the sulfur within the 1,4-benzothiazepine skeleton.
Notably, M201-A has demonstrated the ability to inhibit RyR2, effectively reducing spontaneous diastolic Ca 2+ release.Under experimental conditions, spontaneous diastolic Ca 2+ release was observed even in control cells and was further enhanced with isoprenaline loading in isolated rat cardiomyocytes.M201-A, similar to K201, exhibited a significant reduction in spontaneous diastolic Ca 2+ release.Diastolic Ca 2+ release was reduced with K201 and M201-A in the absence or presence of isoprenaline.K201 demonstrated negative lusi-inotropic effects in rat hearts, characterized by a decrease in the Ca 2+ transient peak.Conversely, M201-A demonstrated positive lusi-inotropic effects without affecting the Ca 2+ transient peak or decay.Additionally, it significantly decreased diastolic Ca 2+ concentrations.This effect is of particular relevance because RyR dysfunction leading to chronic SR diastolic leak is a common occurrence in HF and is believed to contribute to diastolic and systolic dysfunction (Benitah et al., 2021;Bers, 2014;Kushnir et al., 2018;Santulli et al., 2017).
In cumulative dosing of M201-A in dogs, the administration of 1 mgÁkg À1 Á20 min À1 resulted in a significant increase in urine volume and urinary excretion of Na and Cl.Concurrently, a significant increase in CCr and eGFR was observed at 1 mgÁkg À1 Á20 min À1 with a M201-A plasma concentration of 847 ± 121.3 ngÁml À1 (equivalent to 0.56 ± 0.08 μM as the unbound drug concentration, as indicated in Table S4).An examination of GFR, using inulin, showed a dosedependent increase although no significant difference was detected.
In the clinical trial, significant increases in urine volume and urinary Na excretion were observed when administering M201-A at a dose of 0.4 mgÁkg À1 Á20 min À1 , which resulted in a plasma concentration of 921 ± 210 ngÁml À1 (0.28 ± 0.06 μM as the unbound drug concentration).Furthermore, there was no evidence of M201-A accumulation when administered a dose of up to 0.4 mgÁkg À1 intravenously over 20 min for four consecutive days.Importantly, an increase in natriuresis, CCr and eGFRcys was observed during the repeated administration of 0.4 mgÁkg À1 of M201-A for four consecutive days, confirming the safety and tolerability of this dosage regimen.
The clinical study reported in this study included participants with a serum creatinine level ranging from 0.85 to 1.1 mgÁdl À1 .Among the 18 participants, 17 of them (94.4%)exhibited an eGFRcre within the range of 60 and 90 mlÁmin À1 Á1.73 m À2 (Figure S6).The Kidney Disease Improving Global Outcomes staging of kidney disease categorizes renal function based on GFR (Levey et al., 2005), where CKD is categorized by an eGFRcre of <60 mlÁmin À1 Á1.73 m À2 .An eGFRcre falling within the range of 60 and 90 mlÁmin À1 Á1.73 m À2 corresponds to grade 2, which identifies participants with normal or mild renal dysfunction (Stevens & Levin, 2013).Urinary volume and urinary Na excretion exhibited significant increases, along with an increase in eGFRcys and CCr, following the administration of 0.4 mgÁkg À1 of T A B L E 1 Changes in urine volume and urine Na excretion after administration of M201-A.(Inker et al., 2012;Levey & Coresh, 2012;Levey et al., 2011Levey et al., , 2009, ), ), underscoring the superiority of eGFRcys in assessing mild renal dysfunction over eGFRcre.
Key points are as follows: (1) the time course between plasma concentration levels and the concurrent increase in eGFRcys, CCr and natriuresis; (2) the relationship between natriuresis and the increase in GFR; and (3) the characteristics and possible mechanisms at the base of action of the RyR2 inhibitor, M201-A.
The first key point is the duration of time between eGFR, CCr and natriuresis.The half-life of the plasma concentration of M201-A is 5-6 h.Increases in eGFRcys and CCr were observed 2 h after administration, attributed to the heightened blood concentration of M201-A.The low dose (0.2 mgÁkg À1 ) showed significant natriuresis at 0-8 h, whereas the higher dose (0.4 mgÁkg À1 ) showed a significant increase at 8-24 h.The significant increase in natriuresis seen in 8-24 h at high concentrations of M201-A was confirmed when blood concentrations were already lower.
The second key point centres around the relationship between natriuresis and the subsequent increase in GFR.Recently, inhibitors of sodium-glucose cotransporter 2 (SGLT2) have gained attention for their reported benefits in HF with preserved ejection fraction (Anker et al., 2021).SGLT2 inhibitors prevent sodium and glucose reabsorption in the proximal tubule, thereby increasing sodium excretion.
However, the increased sodium concentration triggers contraction of the afferent arterioles through tubuloglomerular feedback (TGF) mechanisms situated in the macula densa (Cherney & Perkins, 2014).
The contraction of the afferent arterioles subsequently reduces renal blood flow and consequently lowers GFR.Therefore, SGLT2 inhibitors cause natriuresis and a short-term decrease in GFR, although this stabilizes with chronic therapy (Anker et al., 2021;McMurray et al., 2019).However, the M201-A administration was associated with increased natriuresis and GFR.This suggests that sodium diuresis is not affected by TGF, indicating the peripheral portion of the macula densa as the active site of M201-A.Experiments involving lithium have revealed that K201 inhibits sodium and water reabsorption in the collecting ducts, where RyR2 is present (Lisy & Burnett, 2006).
This outcome suggests that M201-A may inhibit water and sodium reabsorption in the collecting ducts in humans.
The third key point pertains to the characteristics and possible mechanisms underlying the action of the RyR2 inhibitor, M201-A.
K201 has been previously reported to induce an immediate increase in natriuresis and GFR following renal artery infusion in dogs (Lisy & Burnett, 2006).Similarly, in the current study, intravenous administration of M201-A in dogs resulted in an immediate increase in natriuresis and CCr and a decrease in serum creatinine levels (Figure 4b,d,e).
The heightened GFR associated with K201 was attributed to increased renal blood flow caused by afferent arteriole dilatation, consequently enhancing GFR (Lisy & Burnett, 2006).
A notable finding in humans is that there are two distinct types of responses to M201-A: a plasma concentration-dependent early response and a plasma concentration-independent delayed response.
The former is indicated by a significant increase in eGFRcys and CCr 2 h after M201-A administration.In contrast, the latter manifests as an increase in natriuresis at 8-24 h post-administration (Table 1).Also, among the six participants treated with 0.4 mgÁkg À1 of M201-A for 4 days, five participants with initially high CCr showed no remarkable change in CCr.In contrast, one participant with initially low CCr showed a progressive increase in CCr (Figure S8).RyR2 inhibitors, which reduce intracellular Ca 2+ leak, ameliorate cell dysfunction and cytotoxicity.The RyR2 inhibitor, M201-A, which mitigates intracellular Ca 2+ leakage, could prevent RyR2 dysfunction in the afferent arteriole (Lisy & Burnett, 2006), collecting duct (Lisy & Burnett, 2006) and podocytes (Park et al., 2019), where RyR2 is known to exist.
It is important to recognize that the low dose (0.2 mgÁkg À1 ) evoked an early effect in natriuresis, whereas the high dose did not.Urine output distribution after administration of M201-A was bell shaped (Figure S9).The highest group (the 0.6 mgÁkg À1 group) had significantly higher blood pressure than the placebo group (Figure S10    should encompass a larger and more diverse group of patients with CKD, including patients with proteinuria, to assess both efficacy and safety while also considering gender differences.We will initiate a phase II trial on patients with HF and CKD.
In conclusion, M201-A has demonstrated inhibitory effects of RyR2, leading to a decrease in diastolic Ca 2+ concentration along with positive lusi-inotropic actions in the heart.Furthermore, it has demonstrated the ability to induce natriuresis and enhance renal function.
These findings suggest that M201-A may hold promise in mitigating HF and CKD progression by ameliorating RyR2 dysfunction in the heart and kidney, thereby providing a novel therapeutic option for patients with either or both of these diseases.

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M201-A may improve chronic kidney disease and heart failure by normalizing RyR2 function.chromatography using a chloroform-methanol mixture as the mobile phase.Two M201 enantiomers were separated from M201-R using a CHIRALPAK IC column (Daicel Corp.), and the compound eluted was initially named M201-A and later renamed M201-B.This was followed by mass spectra, 1 H-nuclear magnetic resonance (NMR), 13 C-NMR M201-A and single-crystal X-ray structure analysis to determine the chemical structural formula and three-dimensional structure of M201-A.The details of the synthesis of M201-R and M201-A were described in Patent Numbers PCT/JP2010/001219 (WO/2010/ 098080) and PCT/JP2015/070488 (WO2016/017448), respectively.
This two-step dose-escalation, placebo-controlled, double-blinded, randomized, repeated-dose study (NCT04464681 title: Multiple Dose-Escalation Study of M201-A in Healthy Japanese Subjects) included 18 healthy Japanese adult males aged 21-49 years.This study was conducted in the Clinical Trial Center at Kitasato University, Japan, and adhered to the Principles of Good Clinical Practice and the Declaration of Helsinki.All participants provided written informed consent, and the Ethics Committee of Kitasato Institute Hospital approved the study.Ninety-five volunteers provided informed consent to participate.Furthermore, 18 participants were randomly divided into three groups: placebo, 0.2 mgÁkg À1 M201-A and 0.4 mgÁkg À1 M201-A groups (n = 6 in each group), following the inclusion and exclusion criteria (Table Animal studies are reported in compliance with the ARRIVE guidelines(Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology(Lilley et al., 2020).In vivo experiments were performed according to 'Standards Relating to the Care and Keeping and Reducing the Pain of Laboratory Animals' (Latest Revision): Notice of the Ministry of the Environment No. 84 of 2013 (https:// www.env.go.jp/nature/dobutsu/aigo/2_data/laws/nt_h25_84_en.pdf).The study protocol was approved by the Animal Care and Use Ethics Committee of each institution.Rat in vivo experiments were conducted according to the 'Code of Ethics for Animal Experiments of Drug Safety Testing Center, Inc' (DSTC Animal Experiment Ethics Committee Approval Number IACUCP1403), whereas experiments involving dogs were conducted following the 'Animal Experiment Operation Regulations of Kamakura Techno-Science Corporation' (Ethics Review Number 17-064).
column separation and X-ray crystal structure of M201-A.(a) Synthesis of M201-AÁHCl.M201-A hydrochloride was synthesized by oxidizing K201 HCl to form the racemic mixture of M201, which was then subjected to chiral column separation.(b) Chiral column separation of M201-R.The separation process yielded the first optical isomer component (M201-A) and second component (M201-B) of the compounds.(c) X-ray crystal structure of M201-AÁHCl.The ORTEP figure depicts M201-AÁHCl (MeCN solvate).The absolute configuration of M201-A was confirmed as (R) through single-crystal structure determination using X-ray crystal structure analysis.

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I G U R E 2 Effects of M201-A on intracellular Ca 2+ ([Ca 2+ ] i ) in isolated rat ventricular cardiomyocytes.[Ca2+ ] i was measured by loading cells with Fura-4F AM and recording the fluorescent signal (R 340/380 nm ).Cells were stimulated electrically using 4-ms pulses at 1 Hz.Data were analysed by Student's t test (paired and unpaired) and the mean of cells per heart.(a) Perfusion protocol.(b-d) Representative traces of [Ca 2+ ] i at points indicated in (i) and (ii).(e-g) Percentage change relative to control (red line indicates DMSO) in the Ca 2+ transient peak [Ca 2+ ] i , Ca 2+ transient amplitude and Ca 2+ transient diastolic [Ca 2+ ] i and with K201 (black filled circles) and M201-A (open circles).(h-j) Representative traces of [Ca 2+ ] i at the end of the incubation protocol with control (DMSO), K201 and M201-A for 80 s and stimulated at 1 Hz for the last 60 s of incubation to achieve a steady state.(k-m) Representative traces of [Ca 2+ ]i at the end of the protocol of incubation with control (DMSO), K201 and M201-A, all with 300 nM of isoprenaline (ISO) for 80 s and stimulated at 1.0 Hz for the last 60 s of incubation to achieve a steady state.(n) Calcium wave frequency (DMSO; n = 46 cells; n = 7 hearts), K201 (n = 48 cells; n = 6 hearts) and M201-A (n = 48 cells; n = 6 hearts).*P < 0.05 normalized to DMSO control; mixed effects model.(o) Calcium wave frequency (DMSO with ISO; n = 51 cells; n = 6 hearts), K201 (n = 54 cells; n = 6 hearts) and M201-A (n = 35 cells; n = 5 hearts).*P < 0.05 normalized to DMSO control; mixed effects model.Error bars represent the SEM.

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I G U R E 3 Haemodynamic effects of M201-A, M201-B and K201 in rats.(a) Percentage change in heart rate.(b) Percentage change in systolic blood pressure (BP).(c) Percentage change in diastolic BP.(d) Percentage change in max + dP/dt.(e) Percentage change in min À dP/dt.Values were calculated as the percentage at each measurement time point relative to the mean of the values 4 min before administration for each animal.(f) Δ Change in LVEDP.Data are presented as the mean ± standard error of the mean.*P < 0.05versus pre-drug values.† P < 0.05, M201-A versus M201-B or K201 at 0-20 min.n = 5 per group.F I G U R E 4 Effects of M201-A on renal parameters in dogs.Once urine production had stabilized, M201-A was administered at a dose rate of 0.05 mlÁkg À1 Ámin À1 over 20 min; M201-A dosage was escalated to 0.1, 0.3, 1 and 3 mgÁkg À1 , and urine volume was measured at each dose.(a) Urine volume; (b) Na excretion in urine; (c) Cl excretion in urine; (d) CCr; (e) serum creatinine; (f) GFR; and (g) plasma concentration.*P < 0.05 versus pre-drug values.Plasma concentration is represented as the mean ± SD.
Figure S10.].As a possible mechanism of the bell shape, these findings are believed to be due to the renin-angiotensin-aldosterone system (RAAS) activity followed by an increase in aldosterone, which increases sodium reabsorption in the distal tubules and collecting ducts, resulting in suppression of natriuresis.M201-A, at high doses, is assumed to act as a mechanism to prevent excessive natriuresis and fluid loss.Dogs do not have bell-shaped urine volume distribution curves.This could be due to a difference in species between humans and dogs or to the

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I G U R E 6 Changes in heart rate, blood pressure and ECG parameters: (a) heart rate; (b) blood pressure (systolic blood pressure and diastolic blood pressure); (c) PR interval; (d) QRS interval; (e) QT interval; and (f) QTcF.T A B L E 3 Frequency of adverse events and side effects.

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Ryanodine receptors are crucial for maintaining intracellular Ca 2+ homeostasis and cellular function in all cells.
Urine volume and urinary Na excretion of 0 to <8 h, 8 to ≤24 h and 0 to ≤24 h in each group are shown.P < 0.05 versus placebo.Abbreviation: NS, not significant.Changes in eGFRcre, eGFRcys and CCr.
T A B L E 2