β2-adrenoceptor
Vilanterol trifenatate is a novel long-acting agonist of the human β2-adrenoceptor (β2-AR). Its binding affinity (pKi) at the β2-AR is 9.42 ± 0.02. It shows selectivity for β2-AR over β1-AR and β3-AR subtypes. [1]

Vilanterol exhibits a subnanomolar affinity for the β2-AR that is greater than that of olodaterol, formoterol, and indacaterol, but similar to salmeterol's. Vilanterol shows comparable selectivity to salmeterol for β2-over β1-AR and β3-AR in cAMP functional activity studies, but a markedly better selectivity profile than formoterol and indacaterol. Additionally, vilanterol exhibits an intrinsic efficacy that is notably higher than salmeterol but comparable to indacaterol. Vilanterol exhibits a longer duration of action than formoterol and a persistence of action similar to indacaterol in tissue-based studies measuring persistence and reassertion and cellular cAMP production. Furthermore, vilanterol exhibits reassertion activity in tissue and cell systems that is longer than formoterol but comparable to salmeterol and indacaterol. Vilanterol has been demonstrated to exhibit a significant degree of bronchodilation 22 hours after treatment, with a faster onset and longer duration of action in human airways than salmeterol[1].

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In cAMP functional activity studies using recombinant CHO cells expressing human adrenoceptors, vilanterol demonstrated high selectivity for β2-AR over β1- and β3-AR, with a profile superior to formoterol and indacaterol and comparable to salmeterol. Its functional potency (pEC50) at β2-AR was 9.92 ± 0.06. The intrinsic efficacy of vilanterol was comparable to indacaterol, significantly greater than salmeterol, but less than formoterol and isoprenaline. [1]
In cellular cAMP persistence and reassertion studies, vilanterol showed long persistence of action (concentration ratio, CR = 3.4) comparable to indacaterol (CR = 2.4) and longer than formoterol (CR = 280). It also demonstrated reassertion activity (CR = 5.0) following antagonist washout, similar to salmeterol and indacaterol, but unlike formoterol. [1]
In electrically field stimulated (EFS) guinea pig trachea, vilanterol showed potent relaxant effects (pEC50 = 8.62 ± 0.27) and a rapid onset of action (5.8 ± 0.5 min), significantly faster than salmeterol. It exhibited long persistence (CR = 1.3 at 60 min post-washout) and demonstrated reassertion of its relaxant effect after antagonist washout. [1]
In human precision-cut lung slices (PCLS), vilanterol caused concentration-dependent bronchodilation with EC50 values of 0.6 ± 0.2 nM (histamine-induced contraction) and 0.3 ± 0.1 nM (carbachol-induced contraction). It had a significantly faster onset time (t1/2 = 3.1 ± 0.3 min) compared to salmeterol (t1/2 = 8.3 ± 0.8 min) and showed a long duration of action, maintaining significant bronchodilation 22 hours after treatment. [1]

Compound 13f (vilanterol) had high potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f (vilanterol) were identified that had suitable properties for inhaled administration[2].

For [3H]Vilanterol, binding kinetics studies involving saturation, association, and dissociation are carried out to calculate the equilibrium dissociation constant (KD), total number of receptors (Bmax), association rate (kon), and dissociation rate (koff). Membranes are filtered after being incubated with increasing concentrations of [3H]Vilanterol (0.01-1.3 nM) for 5 hours to achieve saturation binding (in a volume of 1.4 mL to prevent ligand depletion). Membranes are incubated with varying concentrations of [3H]Vilanterol (0.1-1.9 nM) for up to 1 hour prior to filtration in order to facilitate association binding. Membranes are preincubated with a fixed concentration of [3H]Vilanterol (1.1 nM) for 1 hour in order to facilitate dissociation binding. Dissociation is then triggered by dilution in binding buffer (10 μM cold Vilanterol), and incubation is continued for variable periods up to 8 hours prior to filtration. As with [3H]Vilanterol, saturation binding is also accomplished for [3H]CGP12177 (with concentrations rising to approximately 0.01-2.8 nM). Competition binding displacement studies, in which membranes are incubated with a fixed concentration of [3H]Vilanterol (0.2 nM) and increasing concentrations of unlabeled agonist/antagonist for 5 h before filtration, are carried out to ascertain the affinity of β2-AR agonists and antagonists. To guarantee that binding curves are monophasic, 100 µM Gpp(NH)p is present during the completion of all competition binding displacement studies[1].

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Radioligand saturation binding assays were performed to determine the binding affinity of [3H]vilanterol to human β2-AR expressed in CHO cell membranes. Experiments were conducted in 96-deep well plates at ambient temperature (20-22°C) in binding buffer. Membranes were incubated with increasing concentrations of [3H]vilanterol (~0.01-1.3 nM) for 5 hours. Non-specific binding was determined using 10 μM ICI118551. Binding was terminated by rapid vacuum filtration onto GF/B filter papers, followed by washing and liquid scintillation counting. Data were analyzed to determine equilibrium dissociation constant (KD) and total receptor number (Bmax). Studies were performed in the presence or absence of 100 μM Gpp(NH)p to investigate high- and low-affinity agonist binding states. [1]
Radioligand competition binding displacement studies were conducted to determine the affinity (Ki) of various β2-AR agonists and antagonists. CHO β2-AR membranes were incubated with a fixed concentration of [3H]vilanterol (~0.2 nM) and increasing concentrations of unlabeled competitors for 5 hours in the presence of 100 μM Gpp(NH)p. Binding was terminated by filtration and quantified as above. IC50 values were converted to Ki using the Cheng-Prusoff equation. [1]

DiscoveRx cAMP (Whole Cell) Adrenergic β1, β2 and β3 Agonist Assay [5]
The HitHunter DiscoveRx cAMP assay uses a split enzyme complementation readout to capture the content of cAMP either in whole cells or generated from cell membranes. The split enzyme used in the assay is β-galactosidase which is measured using a luminescence readout. Briefly, CHO cells expressing either human β1, β2 or β3 were thawed at room temperature, diluted in PBS and centrifuged. Cells were then resuspended at (2 million cells/mL) in phenol red free DMEM containing 10 µM IBMX. 20000 cells were added to a 384-well plate containing the test compound and incubated for 30-45 min. The cAMP content was measured as per the HitHunter DiscoveRx kit instructions. Basically, the cells were lysed and an antibody to cAMP added along with the two fragments of β-gal one linked to cAMP (enzyme donor) and one to 19 enzyme acceptor to form active enzyme. The substrate was hydrolyzed by the active enzyme for EFC detection (luminescence) of β-gal activity. The final assay cocktail was incubated at room temperature to equilibrate for 3 hours before reading on a Viewlux. All compounds were dissolved in DMSO at a concentration of 10 mM and the DMSO concentration was constant across the plate for all assays. All data was normalized to the mean of 16 high and 16 low control wells on each plate. Four parameter logistic fits were then performed on the normalized data to determine the pEC50 and maximum asymptote values. The values quoted are arithmetic mean ± SEM. The intrinsic activity (IA) was determined by dividing the maximum asymptote ratio obtained for the test compound by the maximum asymptote ratio obtained for isoprenaline. The values quoted for the intrinsic activity are the geometric mean and lower and upper 95% confidence limits. The selectivity ratio was determined by subtracting the pEC50 for either β1 or β3 from that of the β2 pEC50 generated from the potency.

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cAMP accumulation assays were performed to assess β-AR subtype selectivity, functional potency, and intrinsic efficacy. CHO cells stably expressing human β1-, β2-, or β3-AR were used. For selectivity and potency, cells were resuspended in stimulation buffer containing a phosphodiesterase inhibitor. Cells were added to plates containing serial dilutions of vilanterol or other agonists, incubated, and cellular cAMP levels were quantified using a LANCE cAMP detection kit based on time-resolved fluorescence resonance energy transfer. [1]
For persistence and reassertion studies, β2-AR CHO cells were treated under four conditions: control (vehicle), agonist washout, control with antagonist (sotalol), and agonist washout with antagonist. Cells were incubated, washed, and re-stimulated. cAMP production was measured. Concentration ratios (CR) were calculated from EC50 shifts to quantify persistence (agonist washout) and reassertion (agonist+antagonist washout). [1]
Intrinsic efficacy was measured using a fluorescent polarization cAMP assay with CHO β2-AR membranes. Membranes were incubated with agonists in the presence of ATP. A fluorescent cAMP tracer and anti-cAMP antibody were added, and fluorescence polarization was measured. The change in polarization inversely correlates with cAMP concentration, indicating receptor activation level. [1]

Pharmacokinetic studies in rats.[5]
Male Han Wistar rats (bodyweight about 250 g) were fasted for 18 h prior to dose administration. 13f (Vilanterol) acetate salt was formulated as a solution in DMSO-PEG 200- distilled water (10:30:60, v/v/v) for oral dosing, and DMSO-saline (10:90, v/v) for intravenous dosing. Rats were dosed orally by gavage tube or intravenously via a tail vein at nominal dose levels of 2 mg 13f base/kg or 0.25 mg 13f base/kg respectively. Blood samples were taken by cardiac puncture at 0.03 (intravenous only), 0.08, 0.25, 0.5, 0.75, 1, 2, 3 (oral only), 4, 6, and 8 h post dose (n=2 animals/time-point). Plasma was prepared from blood by centrifugation, and analyzed for 13f content by LCMS/MS. Non-compartmental methods were used to calculate pharmacokinetic parameters from plasma concentration vs time profiles.

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Guinea pig trachea studies were ethically reviewed. Tracheal strips from Dunkin-Hartley guinea pigs were mounted under tension in tissue baths containing oxygenated Krebs buffer with indomethacin. Tissues were maintained at 37°C. Contractile responses were induced by electrical field stimulation (EFS). To measure relaxant effects, cumulative concentration-response curves (CRCs) for β2-agonists were generated. Tissues were washed to remove agonist, and CRCs were repeated 60 minutes later to assess persistence. Onset time was measured as the time to reach half-maximal inhibition for an approximate EC50 concentration. [1]
For reassertion studies in guinea pig trachea, strips were superfused with vilanterol until maximal inhibition of EFS was achieved. Vilanterol was then removed, and tissues recovered for 1 hour. Tissues were then perfused with the antagonist sotalol until blockade equilibrated. Sotalol was removed, and superfusion continued for 2 hours to observe if the relaxant effect reasserted. [1]

[1]. J Pharmacol Exp Ther. 2013 Jan;344(1):218-30.

[2]. Synthesis and structure-activity relationships of long-acting beta2 adrenergic receptor agonists incorporating metabolic inactivation: an antedrug approach. J Med Chem . 2010 Jun 10;53(11):4522-30. doi: 10.1021/jm100326d.

Vilanterol benzoate is a triphenylacetic acid salt obtained by combining vilanterol with an equivalent of triphenylacetic acid. It is used in combination with fluticasone furoate to treat bronchospasm associated with chronic obstructive pulmonary disease. It is a β-adrenergic agonist and bronchodilator. It contains a vilanterol (1+) domain.

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Vilanterol benzoate is a novel long-acting β2-adrenergic receptor agonist (LABA) developed for once-daily inhalation. Its pharmacological properties include sub-nanomolar affinity for β2-adrenergic receptors, high receptor selectivity, rapid onset of action, and 24-hour duration of action in human respiratory tissues. [1]
This drug is used to treat asthma and chronic obstructive pulmonary disease (COPD) and can be used in combination with inhaled corticosteroids (ICS) for the treatment of asthma and COPD, or in combination with long-acting muscarinic receptor antagonists (LAMA) for the treatment of COPD. [1]
The prolonged duration of its effect cannot be fully explained by the slow dissociation kinetics from the orthoreceptor site. The proposed mechanism may involve a “microkinetic” model in which highly lipophilic molecules (calculated log P = 3.2) are distributed into the cell membrane, forming a reservoir, thereby prolonging the interaction with the receptor. [1]

C44H49CL2NO

774.76836

773.288

C, 68.21; H, 6.38; Cl, 9.15; N, 1.81; O, 14.45

503070-58-4

Vilanterol; 503068-34-6; Vilanterol-d4 trifenatate; 2021249-10-3; 503070-58-4 (trifenatate)

44482554

White to off-white solid powder

131.9-134.2℃

9.104

5

8

20

54

778

1

OC1=CC=C([C@@H](O)CNCCCCCCOCCOCC2=C(Cl)C=CC=C2Cl)C=C1CO.O=C(O)C(C3=CC=CC=C3)(C4=CC=CC=C4)C5=CC=CC=C5

KLOLZALDXGTNQE-JIDHJSLPSA-N

InChI=1S/C24H33Cl2NO5.C20H16O2/c25-21-6-5-7-22(26)20(21)17-32-13-12-31-11-4-2-1-3-10-27-15-24(30)18-8-9-23(29)19(14-18)16-28;21-19(22)20(16-10-4-1-5-11-16,17-12-6-2-7-13-17)18-14-8-3-9-15-18/h5-9,14,24,27-30H,1-4,10-13,15-17H2;1-15H,(H,21,22)/t24-;/m0./s1

4-[(1R)-2-[6-[2-[(2,6-dichlorophenyl)methoxy]ethoxy]hexylamino]-1-hydroxyethyl]-2-(hydroxymethyl)phenol;2,2,2-triphenylacetic acid

GW-642444; GW 642444-X; GW 642444 X; GW642444; GW-642444; GW 642444X; Anoro Ellipta; Breo Ellipta

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: 50~100 mg/mL (64.5~129.1 mM)
Ethanol: ~14 mg/mL

Solubility in Formulation 1: 2 mg/mL (2.58 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (2.58 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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 mg/mL (2.58 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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.)