Salmeterol targets the β2‑adrenergic receptor with high selectivity (Ki = 1.5 nM for wild‑type β2AR). Its long side chain interacts with an exo‑site within the fourth domain of the receptor, retaining the drug at the binding site and conferring a long duration of action; it shows minimal affinity for β1 and β3 subtypes.

In a previoius study, it was found that salmeterol could reduce retinal Müller cell death through the inhibition of the phosphorylation of IRS-1(Ser307). In addition, the findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.
Kinase Assay: The cells are rinsed twice with ice-cold phosphate-buffered saline and mechanically detached in ice-cold buffer containing 10 mM Tris·HCl, pH 7.4, 5 mM EDTA, 10 μg/mL benzamidine, 10 μg/mL soybean trypsin inhibitor (type II-S), and 5 μg/mL leupeptin (lysis buffer). The lysate is centrifuged at 45,000 ×g for 10 min at 4°C. The pellet is rehomogenized in lysis buffer, with a Potter-type homogenizer, and stored at −80°C until use. The competition binding assays are performed in buffer containing 75 mM Tris·HCl, pH 7.4, 12.5 mM MgCl2, and 2 mM EDTA, using 1-5 μg of membrane protein, 50 pM 125I-CYP, and 0-100 μM unlabeled ligand in the presence of 100 μM GTP, for 60 min at 37°C. The binding reaction is terminated by dilution and rapid filtration through Whatman GF/C filters; the filters are washed three times with solution containing 25 mM Tris·HCl, pH 7.4, and 1 mM MgCl2. Nonspecific binding is determined in the presence of 5 μM (±)-propranolol. The radioactivity on the filters is counted with a γ-counter.
Cell Assay: Salmeterol significantly inhibits production of pro-inflammatory mediators by RAW264.7 and THP-1 cells. Salmeterol downregulates PgLPS-mediated phosphorylation of the ERK1/2 and JNK but not p38 MAP kinases (MAP-K). Salmeterol also attenuates the activation of NF-κB via inhibition of nuclear translocation of p65-NFκB, the transcriptional activity of NF-κB and IκBα phosphorylation. Salmeterol shows very high selectivity for the WT β2AR (β1 Ki /β2 Ki ratio of approximately 1500) with Ki of 1.5±0.4 nM. Salmeterol prevents phosphorylation levels of IRS-1Ser307 induced by tumor necrosis factor-α. Salmeterol alone prevents cell death in retinal Müller cells (p<0.05 versus 25 mM glucose). Salmeterol in conbination with IRS-1 shRNA shows a significant increase in cell death compared to salmeterol alone. Moreover, salmeterol alone treatment significantly reduces cytochrome C levels, with the effect lessened when salmeterol is combined with IRS-1 shRNA. Salmeterol (100 μM) causes apoptosis of DCs, and can not affect the differentiation and maturation of DCs at 10 μM. Salmeterol (10 μM) decreases the mRNA and protein levels of pro-inflammatory cytokines in LPS-activated DCs and inhibits MAPK and NF-κB activation.

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In vitro, salmeterol significantly inhibits production of pro‑inflammatory mediators in RAW264.7 and THP‑1 cells; it downregulates PgLPS‑induced ERK1/2 and JNK phosphorylation (not p38), inhibits NF‑κB activation by blocking p65 nuclear translocation and IκBα phosphorylation, and reduces retinal Müller cell death via IRS‑1 inhibition.

The OVA/LPS groups with salmeterol result in a significant decrease in the enhanced AHR in allergic mice in a dose-dependent manner. Salmeterol contends with asthma via regulating the inflammation of the airway of the mice.

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In vivo, inhaled salmeterol inhibits histamine‑induced plasma protein extravasation and PAF‑induced eosinophil accumulation in guinea pig lungs. It has been approved for human use, showing long‑acting bronchodilation and anti‑inflammatory effects in asthma and COPD models, with a duration of action of approximately 12 hours.

Binding affinity is determined by competition assays using membrane preparations from cells expressing β2AR and radiolabelled antagonist ¹²⁵I‑CYP (cyanopindolol) in buffer (75 mM Tris‑HCl pH 7.4, 12.5 mM MgCl₂, 2 mM EDTA); non‑specific binding is defined with 5 μM propranolol, and bound radioactivity is counted after filtration.

Cell‑based assays use RAW264.7 macrophages or THP‑1 monocytes treated with salmeterol and stimulated with PgLPS; production of cytokines is measured, and phosphorylation of ERK, JNK, and p38 is analysed by Western blot. NF‑κB activation is assessed by p65 nuclear translocation and luciferase reporter assays for transcriptional activity.

Mice

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In vivo guinea pig models involve inhaled administration; outcomes include inhibition of plasma extravasation and eosinophil accumulation. Efficacy is measured by bronchodilator response and anti‑inflammatory endpoints. The compound’s long duration is confirmed in functional airway models, supporting twice‑daily dosing in clinical use.

Salmeterol is administered by inhalation, giving high local lung concentrations and low systemic absorption; it is metabolised in the liver with faecal excretion. Its half‑life supports twice‑daily dosing. The exo‑site interaction provides prolonged receptor occupancy, and its PK profile is well characterised in clinical settings.

Salmeterol has an established safety profile from clinical use; common side effects include tachycardia, tremor, and hypokalaemia, especially at high doses. It is contraindicated in severe asthma without corticosteroid cover. Long‑term safety is documented, and it is approved for chronic use with appropriate monitoring.

Mol Pharmacol.1998 Oct;54(4):616-22.

Salmeterol is a marketed drug (Serevent) for asthma and COPD maintenance, often combined with inhaled corticosteroids. Its long‑acting β2‑agonist properties and unique exo‑site binding mechanism underpin its clinical utility. It is also used for exercise‑induced bronchospasm prevention, and its safety and efficacy are well established.

C25H37NO4

415.57

331.178

18910-65-1

89365-50-4;18910-65-194749-08-3 (xinafoate)

86805

Typically exists as solid at room temperature

1.2g/cm3

553.4ºC at 760mmHg

288.5ºC

4.43E-13mmHg at 25°C

1.598

2.538

4

5

8

24

346

0

CC(CC1=CC=C(C=C1)OC)NCC(C2=CC(=C(C=C2)O)CO)O

VPMWDFRZSIMDKW-UHFFFAOYSA-N

InChI=1S/C19H25NO4/c1-13(9-14-3-6-17(24-2)7-4-14)20-11-19(23)15-5-8-18(22)16(10-15)12-21/h3-8,10,13,19-23H,9,11-12H2,1-2H3

4-[1-hydroxy-2-[1-(4-methoxyphenyl)propan-2-ylamino]ethyl]-2-(hydroxymethyl)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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)