hTRPV4 (IC50 = 2.3 μM); mTRPV4 (IC50 = 5.9 μM); rTRPV4 (IC50 = 3.2 μM)[1]
RN-1734 is a selective antagonist of transient receptor potential vanilloid 4 (TRPV4). [2]

The increase in astrocyte fluorescence rate caused by CM (LPS-activated astrocyte group) cell fluorescence can be reversed by RN-1734 (27 hours; 10 μM) [2]. 10 μM; RN-1734 (27).

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In primary microglial cells stimulated with lipopolysaccharide (LPS, 1 μg/ml), treatment with RN-1734 (10 μM) for 3 hours significantly inhibited the influx of calcium, as measured by Rhod‑2 AM fluorescence (mean fluorescence intensity reduced back to control levels). [2]

In the same LPS‑activated microglial cells, RN-1734 (10 μM) decreased the production of pro‑inflammatory cytokines interleukin‑1β (IL‑1β) and tumor necrosis factor‑α (TNF‑α) measured by ELISA. [2]

RN-1734 (10 μM) suppressed the phosphorylation of NF‑κB P65 in LPS‑treated microglial cells, as shown by western blot. [2]

In oligodendrocyte apoptosis experiments, conditioned medium from LPS‑stimulated microglia was applied to cultured oligodendrocytes. RN-1734 treatment (10 μM in the microglial culture) significantly reduced the percentage of cleaved‑caspase‑3‑positive oligodendrocytes and the apoptotic rate measured by TUNEL staining, and it also alleviated the decrease in CNP protein expression induced by the conditioned medium. [2]

RN-1734 (0.5 μL; microinjection pump; once daily for 5 weeks) dramatically reverses CNP cosmetology and repairs myelination in the CPZ-induced demyelination mouse [2].

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In a cuprizone (CPZ)‑induced demyelination mouse model (C57BL/6 mice fed 0.2% CPZ for 5 weeks), intracerebroventricular injection of RN-1734 (10 μM in 5% DMSO and 0.9% NaCl, 0.5 μl/day, infusion rate 0.1 μl/min for 5 weeks) significantly ameliorated demyelination: it reversed the CPZ‑induced decrease of CNP protein (western blot and immunofluorescence) and decreased the g‑ratio (electron microscopy) compared to vehicle‑treated mice. [2]

RN-1734 treatment suppressed glial activation in the corpus callosum of CPZ‑fed mice, as evidenced by reduced numbers of GFAP‑positive astrocytes and Iba‑1‑positive microglia (immunofluorescence). [2]

The antagonist also lowered the elevated serum levels of IL‑1β and TNF‑α induced by CPZ (ELISA). [2]

RN-1734 did not alter the number of Olig2‑positive cells (oligodendrocyte lineage cells) in the corpus callosum compared to vehicle controls. [2]

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies[3].

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Apoptosis analysis [2]
Cell Types: Microglia
Tested Concentrations: 27 hrs (hours)
Incubation Duration: 10μM
Experimental Results: The percentage of cleaved-caspase 3 positive cells was Dramatically diminished. ) weakens the CM-induced CNP decrease [2].

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Western Blot Analysis[2]
Cell Types: Microglia
Tested Concentrations: 27 hrs (hours)
Incubation Duration: 10 μM
Experimental Results: Mitigation of CM (LPS only)-induced CNP decrease.

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Primary microglia were isolated from neonatal rat cortices (postnatal days 1‑3). Mixed glial cultures were prepared, and microglia were purified by shaking the floating fraction on a rotary shaker for 1 hour at 37°C. Purified microglia were resuspended in high‑glucose DMEM with 10% fetal bovine serum and cultured at 37°C in 5% CO₂. For experiments, microglia were divided into groups: normal control, LPS alone (1 μg/ml), LPS + vehicle (DMSO), and LPS + RN-1734 (10 μM). Cells were treated with LPS and/or RN-1734 for 3 hours, then washed three times with DMEM to remove LPS, and further incubated in DMEM with or without RN-1734 for 24 hours. After this, cells were collected for immunocytochemistry (TRPV4) and supernatants were collected for ELISA (IL‑1β, TNF‑α) or used as conditioned medium for oligodendrocyte apoptosis experiments. [2]

For calcium imaging, microglial cells were washed with Hank’s balanced salt solution, incubated with 2 μM Rhod‑2 AM for 30 minutes at room temperature, washed again, and incubated for another 30 minutes at 37°C. Images were acquired with a fluorescence microscope (excitation 405 nm, emission 519 nm), and mean fluorescence intensity of Ca²⁺ was measured using Image‑ProPlus software. RN-1734 (10 μM) treatment reversed the LPS‑induced increase in calcium fluorescence. [2]

For western blot analysis of NF‑κB signaling, microglial cells were lysed after treatments, and proteins were separated by SDS‑PAGE, transferred to nitrocellulose membranes, blocked with 5% BSA, and incubated with primary antibodies against p‑NF‑κB P65, total NF‑κB P65, and β‑actin, followed by HRP‑conjugated secondary antibodies. Signals were visualized and quantified. RN-1734 significantly decreased p‑NF‑κB P65 expression compared to vehicle control. [2]

Oligodendrocyte precursor cells (OPCs) were isolated from the same rat cortices by shaking mixed glial cultures on an orbital shaker for 18 hours at 37°C after microglia removal. OPCs were pelleted and resuspended in DMEM/F12 with bFGF (10 ng/ml), PDGF‑AA (10 ng/ml), N2 (1%), and B27 (2%). They were plated on poly‑L‑lysine‑coated plates. After 4 days, differentiation into oligodendrocytes was induced by removing PDGF‑AA and bFGF and adding 10% fetal bovine serum. Oligodendrocytes were then treated for 24 hours with conditioned medium (Ctrl medium, LPS‑microglia conditioned medium with or without RN-1734) mixed 1:1 with DMEM/F12. Apoptosis was assessed by TUNEL staining (using a fluorescein kit) and by cleaved‑caspase‑3 immunofluorescence. The apoptotic rate was calculated as (TUNEL‑positive cells / total DAPI‑positive nuclei) × 100%. RN-1734 treatment significantly reduced the apoptotic rate and cleaved‑caspase‑3 positivity induced by LPS‑microglia conditioned medium. CNP protein expression in oligodendrocytes was also measured by western blot, and RN-1734 alleviated the CNP decrease caused by the conditioned medium. [2]

Animal/Disease Models: CPZ-induced demyelination mouse model (C57BL/6 male mice) [2]
Doses: 0.5 μl (10 μM, dissolved in 5% DMSO and 0.9% NaCl)
Route of Administration: Microinjection pump 5-week
Experimental Results: Dramatically reversed the decrease in CNP protein and improved myelination in CPZ-induced demyelination mice.

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Male C57BL/6 mice (8 weeks old) were used. Demyelination was induced by feeding mice a diet containing 0.2% cuprizone (CPZ) for 5 weeks. Mice were divided into four groups: normal control (regular diet), CPZ only, CPZ + vehicle (5% DMSO in 0.9% NaCl), and CPZ + RN-1734. For intracerebroventricular administration, mice were anesthetized with isoflurane and placed in a stereotaxic apparatus. A 26‑gauge stainless steel guide cannula was chronically implanted into the lateral ventricle (coordinates: anterior‑posterior +1.0 mm, medial‑lateral 0.0 mm, dorsal‑ventral −3.0 mm from bregma). A 28‑gauge dummy cannula was inserted to prevent clogging. Daily infusion of 0.5 μl of vehicle or RN-1734 (10 μM in 5% DMSO and 0.9% NaCl) was performed using a microinjector pump at a rate of 0.1 μl/min for 5 weeks (concurrent with CPZ feeding). After treatment, mice were euthanized for histological, immunofluorescence, western blot, and electron microscopy analyses. [2]

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Male C57BL/6 mice (8 weeks old) were used. Demyelination was induced by feeding mice a diet containing 0.2% cuprizone (CPZ) for 5 weeks. Mice were divided into four groups: normal control (regular diet), CPZ only, CPZ + vehicle (5% DMSO in 0.9% NaCl), and CPZ + RN-1734. For intracerebroventricular administration, mice were anesthetized with isoflurane and placed in a stereotaxic apparatus. A 26‑gauge stainless steel guide cannula was chronically implanted into the lateral ventricle (coordinates: anterior‑posterior +1.0 mm, medial‑lateral 0.0 mm, dorsal‑ventral −3.0 mm from bregma). A 28‑gauge dummy cannula was inserted to prevent clogging. Daily infusion of 0.5 μl of vehicle or RN-1734 (10 μM in 5% DMSO and 0.9% NaCl) was performed using a microinjector pump at a rate of 0.1 μl/min for 5 weeks (concurrent with CPZ feeding). After treatment, mice were euthanized for histological, immunofluorescence, western blot, and electron microscopy analyses. [2]

[1]. Acidosis environment promotes osteoclast formation by acting on the last phase of preosteoclast differentiation: a study to elucidate the action points of acidosis and search for putative target molecules. Eur J Pharmacol. 2011 Aug 1;663(1-3):27-39.

[2]. TRPV4 Inhibition Improved Myelination and Reduced Glia Reactivity and Inflammation in a Cuprizone-Induced Mouse Model of Demyelination. Front Cell Neurosci. 2018 Nov 5;12:392.

[3]. Identification and characterization of novel TRPV4 modulators. Biochem Biophys Res Commun. 2009 Nov 20;389(3):490-4.

Acidosis promotes the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP+MNCs) or osteoclasts. Under acidic conditions, the formation of large osteoclasts or TRAP+LMNCs is significantly greater than under physiological neutral conditions. Using co-culture systems and soluble RANKL-dependent bone marrow cell culture systems, one of the main sites of action of acidosis was identified as being located in the final stage of osteoclast precursor differentiation. Under acidic conditions, ongoing osteoclast formation was significantly deteriorated when the medium was replaced with physiological neutral medium within the first 6 hours; however, bone marrow cells previously stimulated under acidic conditions for 9 hours differentiated into TRAP+LMNCs in medium at pH 7.4. Compared to physiological neutral conditions, the expression levels of messenger RNA (mRNA) of the cell fusion key molecules DC-STAMP and NFATc1 were not increased under acidic conditions. Ruthenium red (a broad-spectrum TRP receptor antagonist) inhibited acidosis-promoted TRAP+LMNC formation. Adding the TRPV4-specific agonist 4-α-PDD during the last 21 hours of pre-osteoclast differentiation enhanced the formation of TRAP+LMNCs under mild acidosis, indicating a synergistic effect between TRPV4 activation and acidosis. RN1734 (a TRPV4-specific antagonist) partially inhibited acidosis-promoted TRAP+LMNC formation. Thus, we narrowed down the main site of action of acidosis in osteoclastogenesis and elucidated the characteristics of this system in detail. Our results suggest that acidosis can effectively utilize TRPV4 to drive large-scale cell fusion, as well as utilize TRPV4-independent systems. [1]

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Inhibiting demyelination and promoting myelin regeneration are major challenges in the treatment of many central nervous system (CNS) diseases. Increasing evidence suggests that glial cell activation and neuroinflammation are the main causes of myelin damage in demyelinating diseases. Studies have shown that the non-selective cation channel transient receptor potential vanillic acid receptor 4 (TRPV4) has a significant effect on a variety of physiological processes, including inflammation. However, its role and mechanism in demyelination remain unclear. This study is the first to find that TRPV4 expression was significantly increased in the corpus callosum in a copper azole (CPZ)-induced demyelinating mouse model. The TRPV4 antagonist RN-1734 significantly reduced demyelination, inhibited glial cell activation and the production of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), without affecting the number of Olig2 positive cells. In vitro experiments showed that RN-1734 treatment significantly inhibited calcium influx in lipopolysaccharide (LPS)-activated microglia and reduced the levels of IL-1β and TNF-α by inhibiting NF-κB P65 phosphorylation. RN-1734 also reduced oligodendrocyte apoptosis induced by LPS-activated microglia. These results suggest that TRPV4 activation in microglia participates in oligodendrocyte apoptosis by activating the NF-κB signaling pathway, thus revealing a new mechanism of central nervous system demyelination. [2]

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RN-1734 is a selective TRPV4 antagonist. This study reports for the first time that TRPV4 activation in microglia leads to oligodendrocyte apoptosis and demyelination via NF‑κB‑mediated pro‑inflammatory cytokine release, and that RN-1734 can ameliorate these effects in the cuprizone‑induced demyelination model. [2]

RN-1734 treatment did not affect the number of Olig2‑positive oligodendrocyte precursor cells in vivo, suggesting that its protective effect on myelination is not due to promoting oligodendrocyte proliferation or differentiation. [2]

C14H22CL2N2O2S

353.3

352.078

946387-07-1

3601086

White to off-white solid powder

1.228g/cm3

445ºC at 760 mmHg

222.9ºC

1.536

4.862

1

4

7

21

410

0

O=S(N(C(C)C)CCNC(C)C)(C1C(Cl)=CC(Cl)=CC=1)=O

IHYZMEAZAIFMTN-UHFFFAOYSA-N

InChI=1S/C14H22Cl2N2O2S/c1-10(2)17-7-8-18(11(3)4)21(19,20)14-6-5-12(15)9-13(14)16/h5-6,9-11,17H,7-8H2,1-4H3

2,4-dichloro-N-(propan-2-yl)-N-{2-[(propan-2-yl)amino]ethyl}benzene-1-sulfonamide

RN-1734; RN 1734; 2,4-Dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide; CHEMBL2324347; 2,4-dichloro-N-isopropyl-N-(2-(isopropylamino)ethyl)benzenesulfonamide; 2,4-dichloro-N-propan-2-yl-N-[2-(propan-2-ylamino)ethyl]benzenesulfonamide; RN1734.

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 : ~25 mg/mL (~70.76 mM)

Solubility in Formulation 1: ≥ 3.25 mg/mL (9.20 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 3.25 mg/mL (9.20 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 32.5 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: ≥ 3.25 mg/mL (9.20 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 32.5 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.)