CaMKIIα (IC50 = 1 μM); CaMKIIγ (IC50 = 30 μM)

At the 30-second pause stimulation, Rimacalib (SMP-114) improves (by ~40%) Ca2+-transient potentiation, Fura-2 transient amplitude after the pause upon Rimacalib vs. 37.2±4.3% in control, n=60/17 cells / mice vs. n=65/17, p<0.05), and in parallel cardiomyocyte contractility (135.0±15.4% vs. 97.2±16% increase of twitch amplitude, p=0.098)[1].

SMP-114 significantly reduces SR Ca2+ leak (as assessed by Ca2+ sparks) in human atrial (0.72 ± 0.33 sparks/100 µm/s vs. control 3.02 ± 0.91 sparks/100 µm/s) and failing left ventricular (0.78 ± 0.23 vs. 1.69 ± 0.27 sparks/100 µm/s) as well as in murine ventricular cardiomyocytes (0.30 ± 0.07 vs. 1.50 ± 0.28 sparks/100 µm/s). Associated with lower SR Ca2+ leak, we found that SMP-114 suppressed the occurrence of spontaneous arrhythmogenic spontaneous Ca2+ release (0.356 ± 0.109 vs. 0.927 ± 0.216 events per 30 s stimulation cessation). In consequence, post-rest potentiation of Ca2+-transient amplitude (measured using Fura-2) during the 30 s pause was improved by SMP-114 (52 ± 5 vs. 37 ± 4%). Noteworthy, SMP-114 has these beneficial effects without negatively impairing global excitation–contraction coupling: neither systolic Ca2+ release nor single cell contractility was compromised, and also SR Ca2+ reuptake, in line with resulting cardiomyocyte relaxation, was not impaired by SMP-114 in our assays. SMP-114 demonstrated potential to treat SR Ca2+ leak and consequently proarrhythmogenic events in rodent as well as in human atrial cardiomyocytes and cardiomyocytes from patients with heart failure. Further research is necessary towards clinical use in cardiac disease.[1]

Patch-clamp measurement of late I Na[1]
Late I Na was measured as reported previously using ruptured patch (EPC-10 amplifier) and recorded as the integral of the current between 50 and 450 ms elicited by square pulses depolarizing the cell to −30 mV (1000 ms duration, 10 pulses) from a holding potential of −120 mV. To optimize voltage control, each pulse was preceded by a 5 ms pre-pulse to +50 mV. The pipette solution contained (in mmol/l): 95 CsCl, 40 Cs-glutamate, 10 NaCl, 0.92 MgCl2, 5 Mg-ATP, 0.3 Li-GTP, 5 HEPES, 0.03 niflumic acid, 0.02 nifedipine, 0.004 strophanthidin, 1 EGTA, and 0.36 CaCl2 (free [Ca2+] i 100 nmol/l) (pH 7.2, CsOH). The bath solution contained (in mmol/l) 135 NaCl, 5 tetramethylammonium chloride, 4 CsCl, 2 MgCl2, 10 glucose, and 10 HEPES (pH 7.4, CsOH). Liquid junction potential was corrected with the pipette in the bath. Access resistance was <7 MΩ. Recordings started 5 min after membrane rupture. Fast capacitance, membrane capacitance, and series resistance were compensated for. Signals were filtered with 2.9 and 10 kHz Bessel filters. As late I Na is virtually absent in healthy wild-type murine cardiomyocytes, we assessed the influence of SMP-114 on late I Na in cardiomyocytes from CaMKIIδC-overexpressing mice.

Isolation of murine ventricular cardiomyocytes[1]
Isolation of cardiomyocytes was performed as previously described. Briefly, mice were anaesthetized with isoflurane and killed by cervical dislocation. Hearts were retrogradely Langendorff perfused with nominally Ca2+-free solution containing (in mmol/l) 113 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4 x2H2O, 1.2 MgSO4 x7H2O, 12 NaHCO3, 10 KHCO3, 10 HEPES, 30 taurine, 10 BDM, 5.5 glucose, and 0.032 phenol-red for 4 min at 37 °C (pH 7.4). Then, 7.5 mg/ml liberase TM (Roche), trypsin 0.6%, and 0.125 mmol/l CaCl2 were added to the perfusion solution. Ventricular tissue was collected in perfusion buffer supplemented with 5% bovine calf serum (BCS) and mechanically dissociated. Ca2+ reintroduction was performed by stepwise increasing [Ca2+] from 0.1 to 1.7 mmol/l.

---

Isolation of human atrial cardiomyocytes[1]
Human right atrial samples were rinsed, cut into small pieces, and incubated in a spinner flask filled with solution consisting of (mmol/l) 100 NaCl, 10 KCl, 5 MgCl2, 1.2 KH2PO4, 50 taurine, 5 MOPS, 10 BDM, and 20 glucose (pH 7.2 at 37 °C). After 10 min, CaCl2 was added for a concentration of 0.02 mmol/l. For the first digestion step, this solution was supplemented with 0.775 mg/ml collagenase type I (370 U/ml) and 0.4 mg/ml protease type XXIV. After 45 min, the supernatant was discarded and replaced with solution containing only collagenase. The tissue was repeatedly aggregated until a sufficient number of free cells showed. Then, stopping solution was added to add BCS 2% and BDM to 20 mmol/l and the supernatant was centrifugated. Cells were resuspended in storage medium containing (mmol/l) 30 KCl, 10 KH2PO4, 1 MgCl2, 10 HEPES, 11 glucose, 20 taurine, 70 glutamic acid, 20 BDM, and 2% BCS (pH 7.4, KOH, room temperature).

---

Isolation of human ventricular cardiomyocytes[1]
As previously described in detail, cardiomyocytes were isolated from failing human left ventricles using the chunk-isolation technique in a spinner flask using first Joklik-MEM solution (JMEM) containing collagenase Worthington type 1 (0.6 mg/ml, 285 U/mg; Worthington) and trypsin (2.5 mg/ml) for 45 min. Then, the solution was changed to JMEM containing collagenase only, incubating for another 10–20 min until myocytes were disaggregated using a Pasteur pipette. The supernatant was centrifuged to harvest disaggregated cells. Fresh JMEM with collagenase was added to the remaining tissue and the procedure was repeated 4–5 times. Centrifuged cells were resuspended in KB medium containing (mmol/l): 10 taurine, 70 glutamic acid, 25 KCl, 10 KH2PO4, 22 dextrose, 0.5 EGTA, and bovine calf serum 10% (pH 7.4, KOH, room temperature).

[1]. Reduction of SR Ca2+ leak and arrhythmogenic cellular correlates by SMP-114, a novel CaMKII inhibitor with oral bioavailability. Basic Res Cardiol. 2017 Jul;112(4):45.

Rimacalib has been used in trials studying the treatment of Rheumatoid Arthritis (RA).

C22H23FN4O2

394.45

394.181

C, 66.99; H, 5.88; F, 4.82; N, 14.20; O, 8.11

215174-50-8

215174-50-8

9800765

Off-white to pink solid powder

4.422

1

5

5

29

551

1

FC1=C(C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])=C([H])C(=C1[H])[C@]([H])(C([H])([H])[H])C1C([H])=C(/N=C(\N([H])[H])/N2C([H])([H])C([H])([H])OC([H])([H])C2([H])[H])ON=1

MYTIJGWONQOOLC-HNNXBMFYSA-N

InChI=1S/C22H23FN4O2/c1-15(17-7-8-18(19(23)13-17)16-5-3-2-4-6-16)20-14-21(29-26-20)25-22(24)27-9-11-28-12-10-27/h2-8,13-15H,9-12H2,1H3,(H2,24,25)/t15-/m0/s1

(S)-N-(3-(1-(2-fluoro-[1,1'-biphenyl]-4-yl)ethyl)isoxazol-5-yl)morpholine-4-carboximidamide

SMP 114; SMP-114; Rimacalib [INN]; CID 10157465; BZ76J3N815; N'-[3-[(1S)-1-(3-fluoro-4-phenylphenyl)ethyl]-1,2-oxazol-5-yl]morpholine-4-carboximidamide; N'-(3-((1S)-1-(2-fluoro-4-biphenylyl)ethyl)-1,2-oxazol-5-yl)-4-morpholinecarboximidamide; SMP114

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~126.76 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)