Salmeterol is a highly selective agonist of the beta2-adrenergic receptor (β2-AR), a class A G protein-coupled receptor. Its mechanism of action involves the hydrophilic saligenin head occupying the active site of the receptor, while the long, lipophilic side chain binds to an auxiliary binding site (exosite), a domain of highly hydrophobic amino acids within the fourth domain of the β2-adrenoceptor. This exosite binding allows salmeterol to remain associated with the receptor for extended periods, contributing to its long duration of action. Although beta2-adrenoceptors are predominant in bronchial smooth muscle, there are also beta2-adrenoceptors in the human heart (10-50% of total beta-adrenoceptors).

Salmeterol is a potent and long-lasting inhibitor of the release of mast cell mediators, such as histamine, leukotrienes, and prostaglandin D2, from human lung. In vitro studies show that salmeterol stimulates cAMP accumulation in CHO cells expressing human β2, β1, and β3 adrenoceptors. It exhibits greater β2 receptor selectivity compared with albuterol but has decreased intrinsic activity, which minimizes cardiac side effects. The compound relaxes airway smooth muscle by stimulating β2-adrenergic receptors, increasing intracellular cyclic AMP, which controls smooth muscle tone.

Combined treatment with salmeterol (0.16 mg/kg) and formoterol (0.32 mg/kg) had a beneficial impact on rats with chronic obstructive pulmonary disease (COPD) [3].
In vivo, salmeterol produces sustained bronchodilation lasting at least 12 hours. Clinical trials have shown salmeterol to be more efficacious than albuterol in reducing variation in diurnal peak expiratory flow rates, decreasing nocturnal symptoms and asthma exacerbations, reducing the need for rescue medications, and increasing overall lung function. The combination of salmeterol with an inhaled corticosteroid has been shown to be beneficial, with improved pulmonary function compared with higher-dose corticosteroid alone. Salmeterol inhibits histamine-induced plasma protein extravasation and inhibits platelet-activating factor-induced eosinophil accumulation in guinea pig lungs.

Beta2-adrenergic receptor binding assays are performed using membrane preparations from cells expressing recombinant human β2-AR, such as CHO or HEK-293 cells. Radioligand binding studies typically utilize [3H]-dihydroalprenolol or [125I]-iodocyanopindolol as the tracer. Membranes are incubated with varying concentrations of salmeterol and a fixed concentration of radioligand in binding buffer (50 mM Tris-HCl, pH 7.4, containing 10 mM MgCl2) for 60-90 minutes at room temperature. Non-specific binding is determined in the presence of 10 μM propranolol. Bound radioligand is separated by rapid filtration and counted. Functional assays measure cAMP accumulation in cells expressing β2-AR, where salmeterol stimulates cAMP production in a concentration-dependent manner.

Cell proliferation assay [2]
Cell Types: Human T lymphocytes (THP-1 cells)
Tested Concentrations: 0.001, 0.01, 0.05, 0.2, 1, 5, and salmeterol (0.001-25 μM) inhibit human T floating [2] . 25 µM
Incubation Duration:
Experimental Results: Th2 cell proliferation was inhibited in a concentration-dependent manner.
Cellular assays for beta2-adrenergic agonist activity typically employ cell lines expressing human β2-AR (e.g., CHO-β2 cells). Cells are seeded in multi-well plates and stimulated with varying concentrations of salmeterol. The amount of intracellular cAMP produced is measured using competitive immunoassay (ELISA or homogeneous time-resolved fluorescence). EC50 values are determined from concentration-response curves. The selectivity of salmeterol for β2 versus β1 and β3 receptors can be assessed using cells expressing each receptor subtype individually. The compound's long-acting profile can be demonstrated in washout experiments where cells are washed after agonist exposure and cAMP production is measured at various time points.

Animal/Disease Models: Male C57BL/6 mice (6-8 weeks old, body weight: 32-35 g) [3]
Doses: salmeterol (0.16 mg/kg) and/or formoterol (0.32 mg/kg)
Route of Administration: The efficacy of combination therapy was studied in this model for 56 days. The observation period is long.
Experimental Results: COPD mice had Dramatically improved COPD assessment test scores.
In vivo efficacy is evaluated in animal models of bronchoconstriction, such as guinea pigs, where salmeterol is administered via inhalation or intravenous routes, followed by challenge with histamine, methacholine, or other bronchoconstrictor agents. Airway resistance and pulmonary function are measured. In humans, clinical studies involve inhalation administration to patients with asthma or COPD, with spirometry measurements conducted over 12-24 hours post-dose. The Salmeterol Multi-center Asthma Research Trial (SMART) evaluated safety compared to placebo added to usual therapy. Twelve-week and longer studies are standard for evaluating long-term efficacy and safety.

Absorption, Distribution and Excretion
In asthmatic patients, after inhalation of 50 µg salmeterol powder, the peak plasma concentration (Cmax) was 47.897 pg/mL, the time to peak concentration (Tmax) was 0.240 h, and the area under the curve (AUC) was 156.041 pg/mL/h. 57.4% of salmeterol was excreted in feces, and 23% in urine. Less than 5% of the dose was excreted unchanged in the urine. In asthmatic patients, the central compartment volume of distribution was 177 L, and the peripheral compartment volume of distribution was 3160 L. The mean clearance of salmeterol in a group of asthmatic patients was 392 L/h. Further data regarding salmeterol clearance are not available. The absorption of salmeterol in the respiratory tract after oral inhalation of salmeterol-naphthol has not been fully elucidated. Although studies have shown that most orally inhaled medications are actually swallowed, the bronchodilatory effect of orally inhaled sympathomimetic drugs is thought to be due to local action by the drug reaching the bronchial tree. After twice-daily inhalation of the recommended dose of aerosol (42 mcg) or powder (50 mcg), systemic concentrations of salmeterol are very low or undetectable, thus its therapeutic effect cannot be predicted. In vitro studies show that salmeterol binds to human plasma proteins at an average rate of 96%, with concentrations ranging from 8 to 7722 ng/mL, significantly higher than concentrations after commonly used doses. Salmeterol binds to albumin and α1-acid glycoprotein; the naphthyl ester moiety of salmeterol also binds highly to albumin (binding rate >99%). The distribution of salmeterol in various organs and tissues of the human body after oral inhalation is not fully understood. Rat studies indicate that salmeterol can cross the blood-brain barrier in trace amounts. It is currently unclear whether salmeterol and/or its metabolites can cross the placenta. Following oral administration of 10 mg/kg salmeterol to mice and rats, salmeterol is transported across the placenta. For more complete data on absorption, distribution, and excretion of salmeterol (8 items), please visit the HSDB record page.

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Metabolism/Metabolites
Salmeterol is primarily metabolized to α-hydroxysalmeterol via CYP3A4, with a small amount metabolized to O-dealkylated metabolites via an unknown mechanism. …A small amount of metabolites are formed by O-dealkylation of the phenyl alkyl side chain of salmeterol. …Cytochrome P450 (CYP) isoenzyme 3A4 is responsible for the aliphatic oxidation of salmeterol bases. Salmeterol bases are primarily metabolized via hydroxylation, with α-hydroxysalmeterol being the main metabolite, which is subsequently eliminated. It is primarily excreted in feces…
Metabolized in the liver, via CYP3A4 hydroxylation
Half-life: 5.5 hours
Biological half-life

The half-life of salmeterol is 5.5 hours.
In healthy individuals, after oral administration of salmeterol, the terminal elimination half-lives of salmeterol and its metabolite sine benzoate are approximately 5.5 hours and 11-15 days, respectively.
Following inhalation, salmeterol is absorbed into the systemic circulation, with peak concentrations typically occurring within 5-15 minutes. The compound is highly lipophilic and partitions into cell membranes, contributing to its prolonged duration of action. It is extensively metabolized in the liver by cytochrome P450 enzymes (primarily CYP3A4) to inactive metabolites. The elimination half-life is approximately 5.5 hours. Salmeterol is highly bound to plasma proteins (approximately 94-98%). In clinical studies, salmeterol is administered at doses of 25-50 μg twice daily via inhalation.

Toxicity Summary
Salmeterol's long lipophilic side chain binds to external sites near β(2) receptors in the smooth muscle of the lungs and bronchioles, leaving the active portion of the molecule at the receptor site for continuous binding and release. Pulmonary β2 receptor activation leads to bronchial smooth muscle relaxation, bronchiectasis, and increased bronchial airflow. Toxicity Data
No deaths were observed in rats after oral administration of a 1000 mg/kg dose (approximately 81,000 times the maximum recommended daily inhaled dose for adults and 38,000 times the maximum recommended daily inhaled dose for children, calculated in mg/m²). Interactions
Because salmeterol's effects on the vascular system may be enhanced by monoamine oxidase inhibitors or tricyclic antidepressants, salmeterol should be used with extreme caution in patients receiving such medications or within 2 weeks of discontinuation of such medications. In clinical studies, inhaled corticosteroids and/or inhaled sodium cromoglycate did not alter the safety profile of salmeterol. The efficacy of salmeterol oral inhaler was not affected when these medications were taken concurrently. The systemic pharmacodynamic effects of inhaled salmeterol were not affected by the combination with fluticasone propionate.
Common adverse effects of salmeterol are related to beta2-adrenergic stimulation and include tremor, tachycardia, palpitations, headache, and hypokalemia. These effects are generally minimal at standard doses due to the compound's high β2 selectivity and decreased intrinsic activity. The SMART study identified an increased risk of asthma-related death and life-threatening events when salmeterol was used as monotherapy, leading to recommendations that LABAs be used in combination with inhaled corticosteroids for asthma treatment. Salmeterol should not be used for acute asthma exacerbations.

[1]. The discovery of long-acting saligenin β₂ adrenergic receptor agonists incorporating a urea group. Bioorg Med Chem. 2011 Oct 15;19(20):6026-32.

[2]. Malcolm Johnson. Effects of beta2-agonists on resident and infiltrating inflammatory cells. J Allergy Clin Immunol. 2002 Dec;110(6 Suppl):S282-90.

[3]. Efficacy of salmeterol and formoterol combination treatment in mice with chronic obstructive pulmonary disease. Exp Ther Med. 2018 Feb;15(2):1538-1545.

Therapeutic Uses
Bronchodilator
Salmeterol…is indicated for the prevention of bronchospasm in patients with chronic asthma and for reducing the frequency of acute asthma attacks in patients requiring regular inhaled short-acting beta-adrenergic bronchodilator therapy. …Salmeterol can be used in combination with inhaled or systemic corticosteroids, or alone. During salmeterol treatment…patients must have a fast-acting inhaled beta-adrenergic bronchodilator on hand to relieve symptoms during acute attacks. /US product label contains/
Salmeterol…is indicated for the prevention of exercise-induced bronchospasm. During salmeterol treatment…patients must also have a fast-acting inhaled beta-adrenergic bronchodilator on hand to relieve symptoms during acute attacks. …/US product label contains/
Salmeterol…/is/a bronchodilator used to treat bronchospasm associated with chronic obstructive airway diseases, including bronchitis and emphysema. .../US product label contains/

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Drug Warning
Data from a large, placebo-controlled safety study that was terminated early suggests that salmeterol may be associated with rare severe asthma attacks or asthma-related deaths. Data from the study, called the Salmeterol Multicenter Asthma Study Trial (SMART), further indicated that the risk may be higher in African American patients. These results led to the early termination of the study. Data from the SMART study were insufficient to determine whether concomitant use of inhaled corticosteroids could prevent this risk. Given the similar basic mechanisms of action of β2-receptor agonists, the results observed in the SMART study may be consistent with the class effect of this type of drug. A previous 16-week clinical study in the UK—the Salmeterol National Surveillance (SNS) study—also reported similar findings to the SMART study. In the SNS study, asthma-related mortality was numerically higher in patients treated with salmeterol (42 mcg, twice daily) compared to those treated with salbutamol (180 mcg, four times daily) in addition to standard asthma treatment, but the difference was not statistically significant. Salmeterol oral inhalation therapy is intended for maintenance treatment of asthma or chronic obstructive pulmonary disease (COPD) and should not be initiated in patients with significant exacerbations or acute worsening of asthma, or in patients with acute symptoms of COPD. …It has been reported that initiation of salmeterol oral inhalation therapy under these circumstances can lead to serious acute respiratory events, including death. In most cases, these adverse events occur in patients with severe asthma (e.g., those with a history of corticosteroid dependence, impaired lung function, intubation, mechanical ventilation, frequent hospitalizations, or a history of life-threatening acute asthma exacerbations) and/or in some patients with acute asthma exacerbations (e.g., those unresponsive to their usual medications, requiring short-acting inhaled β2-agonists, significant worsening of symptoms, recent emergency department visits, sudden or progressive deterioration of lung function). However, such events have also occurred in patients with milder asthma. While it has not been determined whether salmeterol inhalation therapy caused these events, or simply failed to relieve worsening asthma, the manufacturer notes that salmeterol inhalers should not be used in patients with significantly worsening or acute exacerbations of asthma.
Since salmeterol is primarily metabolized and cleared by the liver, theoretically, impaired liver function may lead to drug accumulation in the plasma. Therefore, the manufacturer recommends close monitoring of patients with liver disease during salmeterol treatment.
Usually administered doses of salmeterol inhaler generally do not produce significant cardiovascular effects. However, in controlled clinical studies, clinically significant changes in systolic and/or diastolic blood pressure and heart rate, as well as electrocardiographic changes, have occasionally occurred with salmeterol treatment. In some cases, adverse cardiovascular reactions to salmeterol require discontinuation of the drug. During post-marketing surveillance, there have been reports of hypertension caused by the use of salmeterol inhaler aerosol or powder.
For more complete data on salmeterol (21 in total), please visit the HSDB record page.

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Pharmacodynamics
Salmeterol is a long-acting β2-adrenergic receptor agonist that binds to both the active and outer sites of the β2-adrenergic receptor. Salmeterol has a longer duration of action than other β2 receptor agonists, such as salbutamol. Patients should be informed of the risks of long-acting β2 receptor agonist (LABA) monotherapy, including hypokalemia and hypoglycemia, and should not be used in combination with other LABAs.
Salmeterol is FDA-approved for the maintenance treatment of asthma and COPD and is available under brand names including Serevent. It is also available in fixed-dose combination products with fluticasone propionate (Seretide/Advair) for improved asthma and COPD management. Clinical trials have shown that salmeterol is superior to albuterol in providing longer protection from histamine-induced bronchoconstriction and exercise-induced asthma. The compound should not be used as rescue medication for acute bronchospasm.

C25H37NO4

415.5656

415.272

89365-50-4

Salmeterol xinafoate;94749-08-3

5152

White to off-white solid powder

1.1±0.1 g/cm3

603.0±55.0 °C at 760 mmHg

75.5-76.5 °C
75.7-76.5 °C
75.5 - 76.5 °C

318.5±31.5 °C

0.0±1.8 mmHg at 25°C

1.566

3.07

4

5

16

30

403

0

GIIZNNXWQWCKIB-UHFFFAOYSA-N

InChI=1S/C25H37NO4/c27-20-23-18-22(13-14-24(23)28)25(29)19-26-15-7-1-2-8-16-30-17-9-6-12-21-10-4-3-5-11-21/h3-5,10-11,13-14,18,25-29H,1-2,6-9,12,15-17,19-20H2

2-(hydroxymethyl)-4-[1-hydroxy-2-[6-(4-phenylbutoxy)hexylamino]ethyl]phenol

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 : ≥ 100 mg/mL (~240.63 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.02 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 (6.02 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 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 (6.02 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.)