TRPC6 (IC50 = 7.9 nM)

SAR7334 prevents Ca2+ influx into cells mediated by TRPC6, TRPC3, and TRPC7, with IC50 values of 9.5, 282, and 226 nM, respectively[1][2][3]. It has no effect on Ca2+ influx mediated by TRPC4 and TRPC5. In podocytes, Ang II-induced calcium influx is significantly blocked by SAR7334 (1 μM) [1]. The impact of SAR7334 (1 μM) on SOCE is minimal [2]. TRPC6 current is dose-dependently reduced by SAR7334, with an IC50 of 7.9 nM. The current of TRPC6 is significantly reduced by SAR7334 (100 nM) [3].

In isolated perfused mouse lungs, SAR7334 (10 mg/kg, orally) suppresses TRPC6-dependent acute HPV. Its appropriateness for long-term oral administration is demonstrated by SAR7334. SAR7334 did not change the mean arterial pressure in spontaneously hypertensive rats (SHR) in an initial short-term trial [3].

Fluo-4 measurement of intracellular calcium concentration ([Ca2+]i)[3]
Ca2+ measurements were performed at room temperature using a fluorometric imaging plate reader. Cells grown on black poly-D-lysine-coated 96-well plates were washed with standard extracellular solution (140 mM NaCl, 1 mM MgCl2, 5.4 mM KCl, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.35) and stained with dye solution (2 μM Fluo-4 AM, 0.02% pluronic F127, 0.1% BSA in standard extracellular solution) for 30 min at room temperature. The cells were rinsed and incubated with standard extracellular solution supplemented with different concentrations of the test compound or vehicle for 10 min.
Ca2+ entry into TRPC3/6/7-expressing cells was elicited by application of the diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). For calculation of SAR7334-induced inhibition, fluorescence values were plotted over time and the AUC was considered as a measure of Ca2+ influx.

Measurement of hypoxic pulmonary vasoconstriction (HPV) in isolated perfused and ventilated lungs[3]
C57/BL6N mice were anaesthetised with xylazine and ketamine and anticoagulated with heparin as previously described (Weissmann et al., 2004; Fuchs et al., 2011). Male 6–8 weeks old mice were from Charles River Laboratories. In brief, lungs were explanted during deep anaesthesia and artificially ventilated and perfused blood free at 2 mL·min−1 at 37°C with Krebs-Henseleit buffer containing 120 mM NaCl, 4.3 mM KCl, 1.1 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2, 13.32 mM glucose, 5% (w/v) hydroxyethylamylopectin and 23.8 mM NaHCO3. The left atrial pressure was set at 2.0 mmHg. Positive pressure ventilation (250 μL tidal volume, 90 breath·min−1 and 2 cm H2O positive end-expiratory pressure) was performed with a mixture containing 21% O2, 5.3% CO2, balanced with N2 (normoxia) or 1% O2, 5.3% CO2 (hypoxia). The pressure in the pulmonary artery and in the left atrium was measured. Changes in pulmonary artery pressure directly reflect changes in pulmonary vascular tone as the lungs are perfused at constant flow. Lungs were ventilated in an alternating mode with hypoxia (10 min) or normoxia (15 min) to induce acute HPV. In such a sequence of repetitive hypoxic ventilation manoeuvres, increasing doses of SAR7334 were applied 5 min prior to the next hypoxic ventilation manoeuvre. For application the SAR7334 stock solution (2 mM in 100% DMSO) was diluted 1:100 in perfusion buffer and appropriate amounts were cumulatively added to the recirculating perfusate (15 mL). The first application was performed after the second hypoxic ventilation period. The strength of acute HPV is given as the maximum increase of pulmonary arterial pressure in each hypoxic ventilation period, referenced to the strength of the second hypoxic manoeuvre (set at 100%).

In vivo determination of SAR7334 pharmacokinetics[3]
Plasma concentrations of SAR7334 were determined in a serial sampling study after single oral administration of the compound (250 g) in 30% glycopherol/cremophor (75/25) 70% glucose (5%) solution to male Sprague Dawley rats. From each animal, eight plasma samples (approximately 200 µL blood were taken by tail tip sampling) were collected over 24 h and stored below −15°C until analysis. After addition of the precipitant solution (acetonitrile) containing an analogous internal standard, the test item SAR7334 was detected by LC-MS/MS, using an Agilent LC with CTC HTC PAL auto sampler and a Sciex API4000 mass spectrometer in the positive ion mode. Using a sample volume of 50 μL, the lower limit of quantitation was 2.0 ng·mL-1 and the linear range was between 2.0 and 2000 ng·mL−1.

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Telemetric assessment of BP Adult male (6 months old) spontaneously hypertensive rats were treated on two consecutive days. On day one, the animals received 1 mL·kg-1 vehicle by oral gavage. After 24 h, rats received either vehicle alone or were treated with 10 mg·kg-1 SAR7334. Telemetric measurement of BP was performed as described (Lohn et al., 2009). In brief, a telemetric device was placed between the aorta and the vena cava and the catheter tip of the transmitter was inserted into the aorta. Systolic BP, diastolic BP and heart rate were acquired continuously at a sampling rate of 500 Hz and data were stored as 5 min averages. Mean arterial pressure was calculated from systolic and diastolic pressure and low-pass filtered using the fast Fourier transform function of the vendor software (Dataquest A.R.T. V4.0, Data Sciences International) for better visualization of time-dependent BP variations. For statistical analysis, raw data were averaged over a 6 h period starting 2 h after application of vehicle or SAR7334 (labelled ‘post’). This interval corresponded to the maximal plasma levels of SAR7334 (see Figure 6). Baseline data (labelled ‘pre’) were sampled over the same time interval on the day before treatment.

[1]. The Role of Angiotensin II in Glomerular Volume Dynamics and Podocyte Calcium Handling. Sci Rep. 2017 Mar 22;7(1):299.

[2]. Pharmacological Characterization of the Native Store-Operated Calcium Channels of Cortical Neurons from Embryonic Mouse Brain. Front Pharmacol. 2016 Dec 12;7:486.

[3]. Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels. Br J Pharmacol. 2015 Jul;172(14):3650-60.

Podocytes are becoming a primary focus of research efforts due to their association with progressive glomeruli damage in disease states. Loss of podocytes can occur as a result of excessive intracellular calcium influx, and we have previously shown that angiotensin II (Ang II) via canonical transient receptor potential 6 (TRPC6) channels caused increased intracellular Ca2+ flux in podocytes. We showed here with patch-clamp electrophysiology that Ang II activates TRPC channels; then using confocal calcium imaging we demonstrated that Ang II-dependent stimulation of Ca2+ influx in the podocytes is precluded by blocking either AT1 or AT2 receptors (ATRs). Application of Ang(1-7) had no effect on intracellular calcium. Ang II-induced calcium flux was decreased upon inhibition of TRPC channels with SAR7334, SKF 96365, clemizole hydrochloride and La3+, but not ML204. Using a novel 3D whole-glomerulus imaging ex vivo assay, we revealed the involvement of both ATRs in controlling glomerular permeability; additionally, using specific inhibitors and activators of TRPC6, we showed that these channels are implicated in the regulation of glomerular volume dynamics. Therefore, we provide evidence demonstrating the critical role of Ang II/TRPC6 axis in the control of glomeruli function, which is likely important for the development of glomerular diseases.[1]

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In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.[2]

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Background and purpose: The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. Experimental approach: A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca(2+) levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. Key results: SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca(2+) influx into cells with IC50 s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca(2+) entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). Conclusions and implications: Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.[3]

C21H24CL3N3O

440.794

439.098

C, 57.22; H, 5.49; Cl, 24.13; N, 9.53; O, 3.63

1333207-63-8

SAR7334;1333210-07-3

78357800

White to yellow solid powder

4.317

3

4

3

28

535

3

C1C[C@H](CN(C1)[C@@H]2CC3=CC=CC=C3[C@H]2OC4=C(C=C(C=C4)C#N)Cl)N.Cl.Cl

LFMYIKNZNTZSJX-IQJQELQDSA-N

InChI=1S/C21H22ClN3O.2ClH/c22-18-10-14(12-23)7-8-20(18)26-21-17-6-2-1-4-15(17)11-19(21)25-9-3-5-16(24)13-25/h1-2,4,6-8,10,16,19,21H,3,5,9,11,13,24H22*1H/t16-,19-,21-/m1../s1

4-[[(1R,2R)-2-[(3R)-3-Amino-1-piperidinyl]-2,3-dihydro-1H-inden-1-yl]oxy]-3-chlorobenzonitrile dihydrochloride

SAR-7334; SAR 7334; SAR7334; SAR7334 hydrochloride; SAR7334 (hydrochloride); TRPC6 inhibitor; 4-[[(1R,2R)-2-[(3R)-3-aminopiperidin-1-yl]-2,3-dihydro-1H-inden-1-yl]oxy]-3-chlorobenzonitrile;dihydrochloride; SAR 7334 Dihydrochloride; SAR 7334; CPA-1588 dihydrochloride;SAR-7334 HCl; SAR-7334 hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~100 mg/mL (~226.87 mM)
H2O : ~50 mg/mL (~113.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.67 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.67 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.67 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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