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Purity: ≥98%
BI-749327 (BI749327) is a novel, potent and orally bioavailable antagonist of Transient receptor potential canonical type 6 (TRPC6) with the potential to be used for treating PAH (pulmonary arterial hypertension) and PH (pulmonary hypertension) due to respiratory diseases or hypoxemia. It inhibits TRPC6 with an IC50 of 13 nM in mice. BI-749327 exhibits 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.
| Targets |
mTRPC6 (IC50 = 13 nM); hTRPC6 (IC50 = 19 nM); guinea pig TRPC6 (IC50 = 15 nM)[1]
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| ln Vitro |
In HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants, BI-749327 reduces NFAT activation. It also prevents associated signaling and the production of pro-hypertrophic genes in isolated myocytes [1].
In this study, researchers report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. |
| ln Vivo |
BI-749327 (30 mg/kg/day; ig) improves left ventricular function, decreases volume/mass ratio, and attenuates the expression of profibrotic genes and interstitial fibrosis in mice with chronic pressure overload [1]. BI-749327 dose-dependently decreases renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction [1]. BI-749327 has a lengthy terminal half-life (t1/2 8.5-13.5 hours) in mice (3-30 mg/kg; oral) [1].
In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis[1]. |
| Cell Assay |
In Vitro Cellular Assays.[1]
Neonatal rat ventricular myocytes were isolated from newborn Sprague-Dawley rat pups and stimulated with angiotensin II followed by vehicle or BI 749327 treatment. HEK293T cells were cultured to 70% confluence, transfected with plasmids expressing a NFAT luciferase reporter and another expressing WT-TRPC6, gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L), or empty vector pcDNA3.1. Renilla-luciferase plasmid was transfected as an internal control. |
| Animal Protocol |
Animal/Disease Models: C57BL/6J mice[1]
Doses: 30 mg/kg/day Route of Administration: po (oral gavage) Experimental Results: The left ventricular function of mice under sustained stress was improved, the volume/mass ratio was diminished, and profibrotic genes and interstitial fibrosis expression attenuated by overload. Animal/Disease Models: CD-1 mice[1] Doses: 3 mg/kg, 10 mg/kg, 30 mg/kg Route of Administration: Oral Experimental Results: t1/2 8.5-13.5 hrs (hrs (hours)) Pharmacokinetic Studies.[1] Pharmacokinetics of BI 749327 were investigated in male CD-1 mice at dose levels of 3, 10, and 30 mg/kg and at 30 mg/kg in B6129F1 mice. Serial blood sampling was performed via puncture of the saphenous vein and samples were collected using EDTA-coated microtainers. In Vivo Pressure-Overload Model.[1] Mice received daily gavage with methyl-cellulose/Tween-80 vehicle starting 1 wk before TAC surgery. After 1 wk post-TAC, mice were orally gavaged with daily vehicle or BI 749327 treatment. Cardiac function was assessed by serial echocardiography followed by terminal pressure-volume analysis after 8 wk post-TAC. In Vivo Unilateral Ureteral Obstruction Model.[1] A midline laparotomy was performed and the left ureter was isolated and ligated at the boundary of the lower renal pole to induce irreversible UUO. |
| ADME/Pharmacokinetics |
To test the efficacy of BI 749327 in vivo, we first performed single dose pharmacokinetic (PK) experiments to guide the studies. Oral administration of 3, 10, or 30 mg/kg BI 749327 to CD-1 mice led to a dose-proportional increase of maximum plasma concentrations (Cmax) and total systemic exposure (SI Appendix, Fig. S2A and Table S3). The long terminal half-life (t1/2) of 8.5–13.5 h qualified the compound for once daily oral dosing. Oral administration of 30 mg/kg BI 749327 to B6129F1, CD-1, and C57BL/6J mice yielded comparable exposures (SI Appendix, Fig. S2). These datasets were then used to develop a two compartment PK model to simulate multiple dosing. Mouse plasma protein binding of BI 749327 measured by equilibrium dialysis was high at 98.4 ± 0.1% (at a concentration of 1 µM) with a corresponding unbound fraction of 1.6%. Given this, the trough unbound concentration was ∼180 nM using daily 30 mg/kg oral dosing, 10× the IC50 for TRPC6, and 1/5–1/6 the IC50 for TRPC7 and TRPC3.[1]
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| References | |
| Additional Infomation |
Thisstudy has some limitations. First, evidence of target engagement by BI 749327 remains indirect, leveraging biomarkers engaged by the pathway. For the kidney, proof may require further cell-specific gene expression analysis. We did not test each disease model in TRPC6 KO mice to assess selectivity, as these models themselves alter the disease condition: TRPC6 gene deletion occurs before disease and up-regulation of other channels may compensate. The TAC cardiac model could not test if TRPC6 antagonism reverses lung congestion, a potential consequence of preload decline, as it modeled partially compensated hypertrophy and not heart failure. The UUO model generates interstitial fibrosis but not glomerular disease or proteinuria. Future studies in models that engage these abnormalities, such as diabetic nephropathy and other renal diseases, will be needed to test the therapeutic potential of BI 749327 for these conditions. Mice expressing gain-of-function TRPC6 mutations causing severe renal damage in humans rarely develop much disease, so such testing may ultimately be best done in humans.[1]
In summary, we reveal in vivo evidence of therapeutic efficacy of an orally bioavailable TRPC6 antagonist to counter chronic profibrotic disease conditions. In addition to cardiac and renal disease, TRPC6 hyperstimulation is linked to important pathophysiology in the lung and in other tissues, and the favorable oral profile of BI 749327 supports exploration of its therapeutic role in these organs as well.[1] |
| Molecular Formula |
C23H21F3N4O2
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|---|---|
| Molecular Weight |
442.433655500412
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| Exact Mass |
442.161
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| Elemental Analysis |
C, 62.44; H, 4.78; F, 12.88; N, 12.66; O, 7.23
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| CAS # |
2361241-23-6
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| PubChem CID |
138377580
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| Appearance |
White to off-white solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
611
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC(C1C=CC(=CC=1)OC1C=CC(=CC=1)C(N1CCC(C2=CC=C(N)N=N2)CC1)=O)(F)F
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| InChi Key |
RGYMFGHHIDRCBN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H21F3N4O2/c24-23(25,26)17-3-7-19(8-4-17)32-18-5-1-16(2-6-18)22(31)30-13-11-15(12-14-30)20-9-10-21(27)29-28-20/h1-10,15H,11-14H2,(H2,27,29)
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| Chemical Name |
[4-(6-aminopyridazin-3-yl)piperidin-1-yl]-[4-[4-(trifluoromethyl)phenoxy]phenyl]methanone
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| Synonyms |
BI749327; BI 749327; (4-(6-Aminopyridazin-3-yl)piperidin-1-yl)(4-(4-(trifluoromethyl)phenoxy)phenyl)methanone; BI 749327; [4-(6-aminopyridazin-3-yl)piperidin-1-yl]-[4-[4-(trifluoromethyl)phenoxy]phenyl]methanone; BI749327; SCHEMBL21274829; GTPL12530; BI-749327
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~31.25 mg/mL (~70.63 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.70 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 (4.70 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 (4.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2602 mL | 11.3012 mL | 22.6024 mL | |
| 5 mM | 0.4520 mL | 2.2602 mL | 4.5205 mL | |
| 10 mM | 0.2260 mL | 1.1301 mL | 2.2602 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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