β adrenergic receptor

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 [³H]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 [³H]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].

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].

For [³H]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 [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].

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.

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.

Absorption, Distribution and Excretion
Vilanterol plasma levels may not predict therapeutic effects. Following inhaled administration of vilanterol in healthy subjects, Cmax occurred at 5 to 15 minutes. Vilanterol is mostly absorbed from the lung after inhaled doses with negligible contribution from oral absorption. Following repeat dosing of inhaled vilanterol, the steady state was achieved within 14 days with up to 1.7-fold accumulation. The absolute bioavailability of vilanterol when administered by inhalation was 27.3%, primarily due to absorption of the inhaled portion of the dose delivered to the lung. Oral bioavailability from the swallowed portion of the dose of vilanterol is low (<2%) due to extensive first-pass metabolism. Systemic exposure (AUC) in patients with COPD was 24% higher than observed in healthy subjects. Systemic exposure (AUC) in patients with asthma was 21% lower than observed in healthy subjects.
Following oral administration of radiolabeled vilanterol, mass balance showed 70% of the radiolabel in the urine and 30% in the feces.
Following intravenous administration to healthy subjects, the mean volume of distribution at steady-state was 165 L.
Following intravenous administration, the pharmacokinetics of vilanterol showed a high plasma clearance of 108 L/hour.

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Metabolism / Metabolites
Vilanterol is principally metabolized by cytochrome p450 3A4 (CYP3A4) to a range of metabolites with significantly reduced beta1- and beta2-agonist activity. The major route of metabolism was via O-dealkylation, with up to 78% of the recovered dose eliminated as O-dealkylated metabolites while N-Dealkylation and C-dealkylation were minor pathways, representing 5% of the recovered dose.

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Biological Half-Life
The effective half-life for vilanterol, as determined from inhalation administration of multiple doses, is 11 hours. The plasma elimination half-life, as determined from inhalation administration of multiple doses of vilanterol 25 mcg, is 21.3 hours in patients with COPD and 16.0 hours in patients with asthma. For a single dose inhaled administration, the plasma elimination phase half-life averaged 2.5 hour.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Although no published data exist on the use of vilanterol during lactation, data from the related drug, terbutaline, indicate that very little is expected to be excreted into breastmilk. Vilanterol is available only in combination products such as Breo Ellipta and Anoro Ellipta. The authors of several reviews agree that use of inhaled bronchodilators is acceptable during breastfeeding because of the low bioavailability and maternal serum levels after use. Combination products such as Breo Ellipta and Anoro Ellipta are likely to be acceptable for similar reasons.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
In vitro plasma protein binding in human plasma was on average 94%.

[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 an dichlorobenzene derivative that is used in the form of its trifenate salt for treatment of chronic obstructive pulmonary disease. It has a role as a beta-adrenergic agonist and a bronchodilator agent. It is an ether, a secondary amino compound, a member of benzyl alcohols, a member of phenols and a dichlorobenzene. It is a conjugate base of a vilanterol(1+).
Vilanterol is a selective long-acting β2-adrenergic agonist (LABA) with inherent 24-hour activity for the once-daily treatment of COPD and asthma. This is in response to the need for longer-acting β2-adrenergic agonists to overcome poor patient compliance (due to the frequency of dosing regimens or complexities of drug administration). Vilanterol was designed based on the salmeterol molecular scaffold, particularly as a antedrug analog of salmeterol modification by modifying the salmeterol molecule to create homochiral compounds with the (R)-configuration. Vilanterol is 1000 and 400 fold more selective for β2 than β1 and β3 adrenoceptors, respectively, with a faster onset of action than salmeterol. Additionally, vilanterol demonstrated a significantly longer duration of action than salmeterol, with the bronchodilator effect still apparent at 22h. Vilanterol's pharmacological effect is attributable to stimulation of intracellular adenylyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic-3',5'-adenosine monophosphate (cAMP). Increases in cyclic AMP are associated with the relaxation of bronchial smooth muscle and inhibition of the release of hypersensitivity mediators from mast cells in the lungs. Vilanterol is approved for use in several combination products such as with [fluticasone furoate] under the tradename BREO ELLIPTA, with [umeclidinium bromide] as ANORO ELLIPTA, and with both [fluticasone furoate] and [umeclidinium bromide] under the trade name TRELEGY ELLIPTA. BREO ELLIPTA is the first vilanterol-containing product to be approved by the FDA in May 2013, followed by ANORO ELLIPTA in December 2013 and TRELEGY ELLIPTA in September 2020. Although all 3 products are approved for the maintenance treatment of chronic obstructive pulmonary disease (COPD), only TRELEGY ELLIPTA and BREO ELLIPTA are approved for maintenance treatments of asthma in patients aged 18 years and older and 5 years and older respectively.
Vilanterol is a beta2-Adrenergic Agonist. The mechanism of action of vilanterol is as an Adrenergic beta2-Agonist.
Vilanterol is a long-acting beta-2 adrenergic agonist, with bronchodilator activity. Upon administration, vilanterol stimulates beta-2 adrenergic receptors in the lungs, thereby activating the enzyme adenylate cyclase that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic-3',5'-adenosine monophosphate (cAMP). Increased cAMP concentrations relax bronchial smooth muscle, relieve bronchospasms, and reduce inflammatory cell mediator release, especially from mast cells.

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Drug Indication
Vilanterol is approved for use in several combination products such as with fluticasone furoate under the tradename Breo Ellipta, in combination with umeclidinium bromide as Anoro Ellipta, and in combination with both fluticasone furoate and umeclidinium under the tradename Trelegy Ellipta. Approved by the FDA in 2013, the use of Breo Ellipta is indicated for the long-term, once-daily maintenance treatment of airflow obstruction in patients with COPD, including chronic bronchitis and emphysema, as well as the once-daily maintenance treatment of asthma in patients aged 18 or older with reversible obstructive airways disease. Anoro Ellipta is indicated for the maintenance treatment of patients with COPD, and Trelegy Ellipta is indicated for the maintenance treatment of patients with COPD as well as the maintenance treatment of asthma in patients aged 18 years and older.
FDA Label

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Mechanism of Action
Vilanterol is a selective long-acting beta2-adrenergic agonist. Its pharmacological effect is attributable to stimulation of intracellular adenylyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic-3',5'-adenosine monophosphate (cAMP). Increases in cyclic AMP are associated with relaxation of bronchial smooth muscle and inhibition of release of hypersensitivity mediators from mast cells in the lungs.

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.)