β adrenergic receptor
- The primary target of Vilanterol is the human β₂-adrenoceptor (β₂-AR). In vitro binding assays show a Ki value of 0.017 nM for human β₂-AR, and in functional assays (cAMP production in CHO cells expressing human β₂-AR), it has an EC₅₀ of 0.12 nM [1]
- Based on structure-activity relationship (SAR) studies, Vilanterol exhibits potent β₂-AR agonist activity with an EC₅₀ range of 0.08–0.15 nM in CHO cells expressing human β₂-AR, and >1000-fold selectivity over β₁-AR and β₃-AR (EC₅₀ for β₁-AR > 100 nM, β₃-AR > 100 nM) [5]
- In human bronchial smooth muscle cells, Vilanterol induces bronchorelaxation by activating β₂-AR, with an EC₅₀ of 0.1 nM for inhibiting acetylcholine-induced bronchoconstriction [3]

Vilanterol, in CHO cells expressing human β₁-, β₂-, and β₃-AR, the ability of Vilanterol to elicit concentration-dependent increases in cAMP is used to determine selectivity for β₂-AR over the other β-AR receptor subtypes (β₂ and β₃). Vilanterol is shown to have at least a 1000-fold selectivity over both β₂- and β₃-AR subtypes, indicating that it is highly selective for the β₂-AR. Based on this analysis, the low-affinity pK_D for [ 3 H]Vilanterol in the presence of Gpp(NH)p is 9.44±0.07 (n=4), the high-affinity pK_D is 10.82±0.12 (n=4), and the low-affinity pK_D is 9.47±0.17 (n=4) in the absence of Gpp(NH)p. Furthermore, it is observed that at 37°C without Gpp(NH)p, a low-affinity pK_D of 9.52±0.24 (n=4) for [ 3 H]Vilanterol is present[1]. A new inhaled long-acting β₂-agonist called vilanterol trifenatate is being developed as a combination with the inhaled corticosteroid fluticasone furoate to treat asthma and COPD. It has inherent 24-hour activity in vitro[2]. When used in conjunction with the inhaled novel corticosteroid fluticasone furoate, which is also active for 24 hours, vilanterol, a novel long-acting β₂-agonist (LABA) with inherent 24-hour activity, can be used once daily for the clinical treatment of asthma and chronic obstructive pulmonary disease (COPD)[ 3 ].

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- Vilanterol potently activates β₂-AR to stimulate cAMP production. In CHO cells stably expressing human β₂-AR, treatment with Vilanterol (0.01–10 nM) for 30 minutes induces dose-dependent cAMP accumulation, with an EC₅₀ of 0.12 nM; maximal response is 95% of the full agonist isoprenaline [1]
- Vilanterol shows long-acting β₂-AR activation. In β₂-AR binding dissociation assays, Vilanterol has a dissociation half-life (t₁/₂) of 7.5 hours from human β₂-AR, compared to 1.2 hours for salmeterol (a reference long-acting β₂-agonist, LABA) [1]
- Vilanterol synergizes with umeclidinium (a muscarinic antagonist) to relax human bronchi. In isolated human bronchial rings, co-treatment with Vilanterol (0.01–0.1 nM) and umeclidinium (0.1–1 nM) produces greater relaxation (85–90% maximal relaxation) than either drug alone (60–65% for Vilanterol, 55–60% for umeclidinium) against acetylcholine-induced constriction [2]
- Vilanterol undergoes metabolic inactivation to reduce systemic activity. In human liver microsomes, Vilanterol is rapidly metabolized by CYP3A4 to inactive metabolites (M1, M2), with a metabolic clearance of 150 μL/min/mg protein; no active metabolites are detected [4,5]

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[5].

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- In healthy subjects, inhaled Vilanterol (10–50 μg) produces dose-dependent bronchodilation, with forced expiratory volume in 1 second (FEV₁) increasing by 15–25% from baseline at 1 hour post-administration, and the effect persists for 24 hours (FEV₁ remains 10–15% above baseline at 24 hours) [3]
- In patients with asthma or chronic obstructive pulmonary disease (COPD), inhaled Vilanterol (25 μg once daily) improves lung function: FEV₁ increases by 200–300 mL at 2 weeks of treatment, and the 24-hour bronchodilatory effect is maintained for 12 weeks with no signs of tachyphylaxis (loss of effect) [3]
- In guinea pigs with ovalbumin-induced allergic airway constriction, inhaled Vilanterol (0.01–1 μg/kg) reduces airway resistance by 40–70% in a dose-dependent manner, with the effect lasting >24 hours; no significant increase in heart rate (a β₁-AR-mediated side effect) is observed at therapeutic doses [1]

For [ 3 H]Vilanterol, binding kinetics studies involving saturation, association, and dissociation are carried out to calculate the equilibrium dissociation constant (K_D), total number of receptors (Bmax), association rate (k_on), and dissociation rate (k_off). Membranes are filtered after being incubated with increasing concentrations of [ 3 H]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 [ 3 H]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 [ 3 H]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 [ 3 H]Vilanterol, saturation binding is also accomplished for [ 3 H]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 [ 3 H]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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- β₂-AR Binding Assay:
1. Prepare membranes from CHO cells expressing human β₂-AR, resuspend in binding buffer (Tris-HCl, MgCl₂, EDTA) to 10 μg protein/well.
2. Add serial dilutions of Vilanterol (0.001–10 nM) and 0.1 nM [³H]-dihydroalprenolol ([³H]-DHA, a β-AR radioligand) to membranes, incubate at 25°C for 1 hour.
3. Filter through glass fiber filters pre-soaked in binding buffer to separate bound and free [³H]-DHA, wash 3 times with ice-cold buffer.
4. Measure radioactivity with a liquid scintillation counter. Calculate Ki using the Cheng-Prusoff equation, with non-specific binding defined by 1 μM propranolol [1]
- CYP3A4 Metabolic Assay:
1. Incubate Vilanterol (1 μM) with human liver microsomes (0.5 mg protein/mL) in reaction buffer (Tris-HCl, MgCl₂, NADPH) at 37°C.
2. Collect aliquots at 0, 5, 10, 20, 30 minutes, terminate with acetonitrile containing internal standard.
3. Centrifuge at 10,000 × g for 10 minutes, inject supernatant into LC-MS/MS to quantify remaining Vilanterol.
4. Calculate metabolic clearance by plotting log(Vilanterol concentration) vs. time, using the slope to determine elimination rate [4]

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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- CHO-β₂ Cell cAMP Assay:
1. Seed CHO cells expressing human β₂-AR in 96-well plates (5×10⁴ cells/well), incubate overnight in DMEM + 10% FBS.
2. Replace with serum-free DMEM containing 0.1% BSA, add serial dilutions of Vilanterol (0.01–10 nM), incubate at 37°C for 30 minutes.
3. Add lysis buffer, measure cAMP using HTRF cAMP kit (excitation 320 nm, emission 665 nm/620 nm).
4. Calculate EC₅₀ by fitting dose-response curves to the four-parameter logistic model [1]
- Human Bronchial Smooth Muscle Cell Relaxation Assay:
1. Isolate human bronchial smooth muscle cells from donor lungs, culture to confluence in DMEM + 10% FBS.
2. Seed in 24-well plates, grow to 80% confluence, serum-starve for 24 hours.
3. Pre-treat with Vilanterol (0.001–10 nM) for 1 hour, add acetylcholine (1 μM) to induce contraction.
4. Measure cell length using image analysis software; calculate relaxation percentage as (contracted length - treated length)/(contracted length - baseline length) × 100% [3]

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 Allergic Airway Constriction Model:
1. Sensitize 6–8 week-old guinea pigs with ovalbumin (100 μg ip) on day 0 and day 7.
2. On day 14, challenge with aerosolized ovalbumin (1% w/v) to induce airway constriction, measure baseline airway resistance using whole-body plethysmography.
3. Administer Vilanterol (0.01–1 μg/kg) via aerosol inhalation (dissolved in saline, nebulization time 5 minutes); control group receives saline.
4. Measure airway resistance at 1, 6, 12, 24 hours post-administration; record heart rate via ECG to assess β₁-AR side effects [1]
- Rat Pharmacokinetic Model:
1. Administer Vilanterol (0.1–1 μg/kg) to male Sprague-Dawley rats via intratracheal instillation (dissolved in 0.9% saline + 0.1% Tween 80).
2. Collect blood samples (0.2 mL) via tail vein at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dose, centrifuge to obtain plasma.
3. Extract Vilanterol from plasma with ethyl acetate, analyze via LC-MS/MS.
4. Calculate PK parameters (Cmax, Tmax, AUC₀-∞, t₁/₂) using non-compartmental analysis [4]

Absorption, Distribution and Excretion
Viracterol plasma concentrations may not predict therapeutic efficacy. In healthy subjects, peak plasma concentration (Cmax) of vilanterol occurs between 5 and 15 minutes after inhalation. Following inhalation, vilanterol is primarily absorbed through the lungs, with negligible contribution from oral absorption. Steady-state is reached within 14 days after repeated inhalation, with a cumulative drug dose of up to 1.7 times. The absolute bioavailability of vilanterol upon inhalation is 27.3%, primarily due to pulmonary absorption of the inhaled portion. Oral bioavailability of the swallowed portion of vilanterol is low (<2%) due to extensive first-pass metabolism. The systemic exposure (AUC) is 24% higher in patients with chronic obstructive pulmonary disease (COPD) than in healthy subjects. The systemic exposure (AUC) is 21% lower in patients with asthma than in healthy subjects. Following oral administration of radiolabeled vilanterol, mass balance showed that 70% of the radiolabeled substance was present in urine and 30% in feces.
The steady-state mean volume of distribution after intravenous administration to healthy subjects was 165 L.
Pharmacokinetics of vilanterol following intravenous administration showed a plasma clearance of up to 108 L/h.
Metabolism/Metabolites
vilanterol is primarily metabolized by cytochrome P450 3A4 (CYP3A4) to a series of metabolites, which significantly reduce the β1 and β2 receptor agonist activity. The main metabolic pathway is O-dealkylation, with up to 78% of the recovered dose eliminated as O-dealkylated metabolites, while N-dealkylation and C-dealkylation pathways are less common, accounting for only 5% of the recovered dose.
Biological Half-Life
The effective half-life of vilanterol determined by multiple inhalation administration was 11 hours. The plasma elimination half-life of multiple inhalations of 25 μg vilanterol was 21.3 hours in patients with chronic obstructive pulmonary disease (COPD) and 16.0 hours in patients with asthma. The mean plasma elimination half-life of a single inhalation was 2.5 hours.

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- Absorption: In the human body, inhaled vilanterol is mainly absorbed in the lungs; its systemic bioavailability is less than 10% due to extensive first-pass metabolism in the lungs and liver. The time to peak plasma concentration (Tmax) is 10–20 minutes after inhalation [3,4]
- Distribution: The volume of distribution (Vd) of vilanterol in the human body is 120–150 L, indicating its extensive tissue distribution. Plasma protein binding is 93%, with concentration-independent binding (0.1–10 ng/mL) [4]
- Metabolism: Vilanterrol is mainly metabolized in the human body via CYP3A4. The major metabolites (M1: hydroxylated derivative, M2: O-dealkylated derivative) are inactive and do not have β₂-adrenergic receptor agonist activity [4,5]
- Excretion: Vilanterol and its metabolites are mainly excreted in feces (70–75% of the dose) and urine (15–20% of the dose). The elimination half-life (t₁/₂) in human plasma is 2.5–3 hours [4]

Effects During Pregnancy and Lactation
◉ Overview of Drug Use During Lactation
While there is currently no publicly available data on the use of vilanterol during lactation, data on the related drug terbutaline suggest that very small amounts are expected to be excreted into breast milk. Vilanterrol is only available in combination formulations, such as Breo Ellipta and Anoro Ellipta. Authors of multiple reviews agree that the use of such drugs during lactation is acceptable due to the low bioavailability of inhaled bronchodilators and the low maternal serum concentrations after administration. Combination formulations such as Breo Ellipta and Anoro Ellipta may also be acceptable for similar reasons.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

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Protein binding
The average protein binding rate in human plasma in vitro is 94%.

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- In healthy subjects, inhalation of Vilanterol (10–50 μg once daily for 14 days) was well tolerated. Adverse events were mild: tremor (5% of subjects), headache (3%), and palpitations (1%); no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) [3]
- In in vitro studies, vilanterol did not show genotoxicity in the Ames test or chromosomal aberration test. In rats, repeated inhalation doses (up to 10 μg/kg/day for 28 days) did not cause histopathological changes in the lungs, heart, liver, or kidneys [4]
- The likelihood of drug interactions with vilanterol is extremely low. Concomitant use with CYP3A4 inhibitors (e.g., ketoconazole) can increase plasma vilanterol AUC by 2-fold, but no dose adjustment is required due to low systemic exposure [4]

[1]. In vitro pharmacological characterization of vilanterol, a novel long-acting β2-adrenoceptor agonist with 24-hour duration of action. J Pharmacol Exp Ther. 2013 Jan;344(1):218-30.

[2]. Pharmacological characterization of the interaction between umeclidinium and vilanterol in human bronchi. Eur J Pharmacol. 2017 Jul 14. pii: S0014-2999(17)30470-3.

[3]. Vilanterol trifenatate, a novel inhaled long-acting beta2 adrenoceptor agonist, is well tolerated in healthy subjects and demonstrates prolonged bronchodilation in subjects with asthma and COPD. Pulm Pharmacol Ther . 2013 Apr;26(2):256-64.

[4]. Metabolism and disposition of Vilanterol, a long-acting β(2)-adrenoceptor agonist for inhalation use in humans. Drug Metab Dispos. 2013 Jan;41(1):89-100.

[5]. 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 is a dichlorobenzene derivative used in the form of tribenzoate to treat chronic obstructive pulmonary disease (COPD). It is a β-adrenergic agonist and bronchodilator. It is an ether compound belonging to the secondary amino group, benzyl alcohol group, phenolic group, and dichlorobenzene group. It is the conjugate base of vilanterol (1+). Vilanterol is a selective, long-acting β2-adrenergic agonist (LABA) with intrinsic activity over 24 hours, and can be used once daily for the treatment of COPD and asthma. This is due to the need for long-acting β2-adrenergic agonists to overcome the problem of poor patient compliance (due to dosing frequency or complex dosing regimens). Vilanterol is designed based on the molecular skeleton of salmeterol, specifically as an analogue of salmeterol, by modifying the salmeterol molecule to generate isochiral compounds with an (R)-configuration. Villanaterol exhibits 1000-fold and 400-fold higher selectivity for β2-adrenergic receptors compared to β1 and β3-adrenergic receptors, respectively, and has a faster onset of action than salmeterol. Furthermore, vilanterol's duration of action is significantly longer than salmeterol, with its bronchodilatory effect lasting up to 22 hours. The pharmacological action of vilanterol is attributed to its stimulation of intracellular adenylate cyclase, an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Elevated cAMP levels are associated with bronchial smooth muscle relaxation and inhibition of hypersensitivity mediator release from pulmonary mast cells. Villanaterol is approved for use in various combination formulations, such as in combination with fluticasone furoate (brand name: BREO ELLIPTA), in combination with umemet bromide (brand name: ANORO ELLIPTA), and in combination with both fluticasone furoate and umemet bromide (brand name: TRELEGY ELLIPTA). BREO ELLIPTA was the first FDA-approved vilanterol-containing product, approved in May 2013, followed by ANORO ELLIPTA (December 2013) and TRELEGY ELLIPTA (September 2020). While all three products are approved for maintenance treatment of chronic obstructive pulmonary disease (COPD), only TRELEGY ELLIPTA and BREO ELLIPTA are approved for maintenance treatment of asthma in patients aged 18 years and older and 5 years and older, respectively. Vilanterol is a β2-adrenergic agonist. Its mechanism of action is as a β2-adrenergic agonist. Vilanterol is a long-acting β2-adrenergic agonist with bronchodilatory effects. After administration, vilanterol stimulates β2-adrenergic receptors in the lungs, thereby activating adenylate cyclase, which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Elevated cAMP levels can relax bronchial smooth muscle, relieve bronchospasm, and reduce the release of inflammatory mediators, especially those from mast cells.

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Drug Indications
Vilanterol is approved for use in various combination therapies, such as in combination with fluticasone furoate (Breo Ellipta), in combination with umemet bromide (Anoro Ellipta), and in combination with both fluticasone furoate and umemet bromide (Trelegy Ellipta). Breo Ellipta was approved by the FDA in 2013 for long-term once-daily maintenance therapy of airflow obstruction in COPD patients (including those with chronic bronchitis and emphysema), and for once-daily maintenance therapy of asthma in patients aged 18 years and older with reversible obstructive airway disease. Anoro Ellipta is indicated for maintenance therapy in COPD patients, and Trelegy Ellipta is indicated for maintenance therapy in COPD patients and for maintenance therapy of asthma in patients aged 18 years and older.
FDA Label

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Mechanism of Action
Vellaraterol is a selective, long-acting β2-adrenergic agonist. Its pharmacological action is attributed to stimulation of intracellular adenylate cyclase, an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Elevated cAMP levels are associated with bronchial smooth muscle relaxation and inhibition of the release of hypersensitivity mediators from lung mast cells.

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- vilanterol is a novel long-acting β₂-adrenergic receptor agonist (LABA) designed for once-daily inhalation administration for maintenance therapy of asthma and chronic obstructive pulmonary disease (COPD) [1,3]
- vilanterol's long-acting properties are attributed to its slow dissociation from β₂-adrenergic receptors (β₂-AR) (dissociation half-life t₁/₂ = 7.5 hours), thereby extending its bronchodilatory effect to 24 hours [1]
- vilanterol employs a metabolic inactivation design (prodrug strategy): it is rapidly metabolized by CYP3A4 to produce an inactive metabolite, thereby reducing systemic β₁-AR-mediated side effects (such as tachycardia). [5] Vilanterol is often used in combination with the long-acting muscarinic receptor antagonist umebomide (LAMA) to treat chronic obstructive pulmonary disease (COPD) because their synergistic bronchodilatory effects are more effective in improving lung function than monotherapy. [2]

C₂₄H₃₃CL₂NO₅

486.43

485.173

C, 59.26; H, 6.84; Cl, 14.58; N, 2.88; O, 16.45

503068-34-6

Vilanterol trifenatate; 503070-58-4; 503068-34-6; 503070-59-5 (cinnamate)

10184665

Light yellow to yellow ointment

1.3±0.1 g/cm3

646.7±55.0 °C at 760 mmHg

344.9±31.5 °C

0.0±2.0 mmHg at 25°C

1.579

2.97

4

6

16

32

466

1

OC1=CC=C([C@@H](O)CNCCCCCCOCCOCC2=C(Cl)C=CC=C2Cl)C=C1CO

DAFYYTQWSAWIGS-DEOSSOPVSA-N

InChI=1S/C24H33Cl2NO5/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/h5-9,14,24,27-30H,1-4,10-13,15-17H2/t24-/m0/s1

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

GW642444; GW-642444; GW 642444-X; GW642444; GW-642444; GW 642444; XGW 642444X

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.14 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 (5.14 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 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 (5.14 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.)