β-adrenergic receptor (β-AR)

Propranolol (10-9 M-10-3 M; 24 and 48 hours): After 24 hours of 10-4 M propranolol and 48 hours of 10-9 M propranolol, significantly reduces HemSCs cell proliferation [4]. Propranolol (10-7 M-10-3 M; 24 and 48 hours) increases total ERK1/2 levels in a dose-dependent manner and vibrationally activates ERK1/2 in HemSCs at 10-5 M concentration. (50 μM–200 μM; 24 hours) Activates caspase-3, increases HemSC activity that is activated by Annexin V, and causes HemSC inflammation quickly [4].

Propranolol (powder medication; 40 mg/kg; daily) significantly reduced vascular diameter and increased the number of cells expressing phosphorylated ERK1/2 in IH Matrigel inlets relative to vehicle-treated inlets [4].

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Propranolol Affects Vascular Development in a Xenograft Mouse Model of IH [4]
To assess how propranolol affects HemSCs and IH development in vivo, we adapted a previously described mouse model [20]. In the IH mouse model, HemSCs resuspended in Matrigel are implanted subcutaneously in immunocompromised mice, and IH vessel development progresses over 3 weeks. The mice were treated with propranolol or vehicle 40 mg/kg daily. Using the surface area conversion factor of 1/12 [37–39], the mice received a human equivalent dose of 3.3–4.8 mg/kg daily. IH Matrigel implants from propranolol-treated mice had reduced blood flow at 14 and 21 days after implantation, measured by Doppler ultrasound, compared with vehicle (data not shown; Fig. 7A). Histological analysis of the 21-day IH Matrigel implants (Fig. 7B) demonstrated that propranolol did not affect blood vessel density (Fig. 7C) but did significantly reduce the vessel diameter relative to the vehicle-treated implants (Fig. 7D). The reduced vessel caliber correlated with a loss of Doppler-detectable flow in the propranolol treatment group. Propranolol also significantly increased the number of cells that expressed phosphorylated ERK1/2 within the IH Matrigel implant (Fig. 7E), consistent with the results from our in vitro studies. Thus, propranolol improved vessel development in the IH mouse model that correlated with MAPK pathway activation. [4]

Caspase-3 Assay [4]
HemSCs were seeded in EGM-2 with 20% FBS media and allowed to settle for 4 hours. HemSCs were treated at increasing concentrations of propranolol in SFM with 0.1% FBS for 24 hours. The protein lysates were collected, and caspase-3 activation was quantified using the Caspase-3 Human ELISA Kit.

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cAMP Assay [4]
The cAMP levels in HemSCs were determined using the LANCE Ultra cAMP kit. The HemSCs were washed and resuspended in the provided stimulation buffer (Hanks’ balanced saline solution, bovine serum albumin, isobutylmethylxanthine, HEPES buffered saline solution) and seeded (1,000 per well) on a 96-well plate. The cells were then treated with drugs for 30 minutes. Tracer and ULight-anti-cAMP working solutions were added and incubated at room temperature for 1 hour. The time-resolved fluorescence resonance energy transfer signal was determined using the EnVision Multilabel Plate Reader. cAMP levels were determined using a standard curve, and data were interpolated using a comprehensive curve fitting (nonlinear regression) and Prism. Each condition was used in triplicate, and the experiments were performed at least two times. A representative experiment is presented in the figures. To determine whether βARs are coupled to Gαs or Gαi in HemSCs, the cells were treated with isoprenaline, with or without 10 μM forskolin, over a 6-log dose range by serial dilutions with water for 30 minutes. Next, the cAMP levels were measured as described to determine whether βARs were coupled to Gαs or Gαi in HemSCs.

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ERK1/2 Western Blotting [4]
Cells were cultured on fibronectin-coated plates and treated with various concentrations of βAR antagonists and incubated for 30 minutes. The cells were lysed in TENT buffer (50 mM Tris [pH 8.0], 2 mM EDTA, 150 mM NaCl, 1% Triton-X-100) with 1% Halt Protease Inhibitor, 1% phosphatase inhibitor, and 0.5% sodium orthovanadate. Western blotting was performed for ERK1/2 (p44/42, 1:1,000) and pERK1/2 (P-p44/42, 1:500). The blots were stripped and then probed for α-tubulin (1:10,000) to normalize protein loading. Experiments were performed at least three times, and a representative experiment is presented in the figures.

Western Blot Analysis[4]
Cell Types: HemSC cells
Tested Concentrations: 10-7 M-10- 3 M
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: Total ERK1/2 levels increased in a dose-dependent manner.

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Cell proliferation assay[4]
Cell Types: HemSC Cell
Tested Concentrations: 10-9 M-10-3 M
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: HemSC proliferation was inhibited.

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Apoptosis analysis [4]
Cell Types: HemSC Cell
Tested Concentrations: 50 μM, 100 μM or 200 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: HemSC cell death was induced through the apoptotic pathway.

Animal/Disease Models: IH (infant hemangioma) xenograft mouse model of HemSC cells [4]
Doses: 40 mg/kg
Route of Administration: Oral administration; 40 mg/kg; daily
Experimental Results: IH mouse model with MAPK Vascular development associated with pathway activation is improved.

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IH Mouse Model [4]
To study the effects of propranolol on HemSCs in vivo, a xenograft mouse model of IH was used as previously described. In brief, 1.5 × 106 HemSCs (n = 2) suspended in 200 µL of Corning Matrigel Matrix was implanted subcutaneously into the flanks of female 6–8-week-old NCrNude immunodeficient mice. Propranolol, which was provided in drinking solution, was initiated the day of IH xenografting. Propranolol was diluted to 270 µM in 5% dextrose water (vehicle), and daily consumption was measured to calculate the treatment dosage, which averaged 40 mg/kg daily. Blood flow within the IH Matrigel implant was analyzed using a VEVO 2100 Ultrasound Imaging System on a Doppler setting on days 14 and 21 of IH development. The mice were anesthetized with isoflurane and restrained in a supine position. The region of interest was fully scanned, with the transducer positioned at its largest longitudinal section over the implant to optimize the spatial resolution of the image, maximizing the detail. Next, two-dimensional images were captured in uniform steps of 0.05 mm. The images of blood flow were analyzed using software provided by VisualSonics. The mice were sacrificed after 21 days. The Matrigel implants were collected and fixed overnight at 4°C in 10% formalin. The implants were dehydrated and embedded in paraffin for histological analysis. Vessel density and caliber were counted in 3–4 HPFs per implant (n = 4 for each group). Vessel density was determined as the number of vessels (whether longitudinally or axially oriented) per HPF. The vessel diameter was measured according to the orientation. For longitudinally oriented vessels, the width was measured at three points and averaged, and the cross-section (axial) vessels were measured once. Vessels were identified as tubular structures with erythrocytes within.

Absorption, Distribution and Excretion
In patients taking 40 mg, 80 mg, 160 mg, and 320 mg daily doses, the peak plasma concentrations (Cmax) were 18±15 ng/mL, 52±51 ng/mL, 121±98 ng/mL, and 245±110 ng/mL, respectively. The time to peak concentration (Tmax) of propranolol is approximately 2 hours, but in fasting patients, it may be between 1 and 4 hours. Taking propranolol with food does not increase the time to peak concentration (Tmax), but it does increase bioavailability. After oral administration of propranolol, 91% of the drug is excreted in the urine as 12 metabolites. The volume of distribution of propranolol is approximately 4 L/kg or 320 L.
The clearance rate of propranolol in infants under 90 days old is 2.7 ± 0.03 L/h/kg, and in infants over 90 days old it is 3.3 ± 0.35 L/h/kg. The clearance rate of propranolol is linearly related to hepatic blood flow. The clearance rate of propranolol in hypertensive adults is 810 mL/min.
Metabolism/Metabolites
Propranolol can undergo side-chain oxidation to α-naphthol lactate, epoxidation to 4'-hydroxypropranolol, or glucuronidation to propranolol glucuronide. It can also undergo N-deisopropylation to generate N-deisopropylpropranolol. 17% of doses undergo glucuronidation, and 42% undergo epoxidation.
The known metabolites of propranolol include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[1-naphth-1-oxy-3-(propyl-2-ylamino)propyl-2-yl]oxaoxane-2-carboxylic acid.
Biological half-life
The elimination half-life of propranolol is approximately 8 hours. The plasma half-life of propranolol is 3 to 6 hours.

Hepatotoxicity
In patients taking propranolol, the incidence of mild to moderate elevations in serum transaminase levels is less than 2%, and these are usually transient and asymptomatic, returning to normal with continued treatment. Despite the widespread use of propranolol, there is no conclusive evidence that it is associated with clinically significant liver injury; the few reported cases usually occur in patients concurrently taking other known hepatotoxic drugs, or present only as elevated serum enzymes without jaundice. Probability score: E (unlikely a cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of use during lactation Because the amount of propranolol in breast milk is low, the amount ingested by the infant is very small, and no adverse effects are expected on breastfed infants. Lactation studies have not found any adverse reactions in breastfed infants that are clearly attributable to propranolol. No special precautions are required. Propranolol has been successfully used to treat persistent breast pain during lactation.
◉ Effects on Breastfed Infants
A study of mothers taking beta-blockers while breastfeeding found a numerically increased number of adverse events, though not statistically significant, in mothers taking any beta-blocker. Although the infants were age-matched to the control group, the age of affected infants was not specified. One of eight mothers taking propranolol reported somnolence in her breastfed infant, but she was also taking other unspecified antihypertensive medications.
The French pharmacovigilance system reported a case of bradycardia in a 2-day-old breastfed infant. However, the report did not specify whether the mother had taken propranolol near delivery or whether the drug might have been passed to the infant through the placenta.
A prospective study of pregnant women taking beta-blockers asked mothers to complete a questionnaire about postpartum breastfeeding and any side effects on their breastfed infants. Sixteen mothers reported taking propranolol while breastfeeding, but did not report the specific dosage. Three mothers reported their infants experiencing hypoglycemia, but with a “good prognosis”; another mother reported her infant experiencing bradycardia and discontinued propranolol after 3 weeks of breastfeeding.
◉ Effects on Lactation and Breast Milk
As of the revision date, no published information has been found regarding the effects of beta-blockers or propranolol during normal lactation. A study of 6 patients with hyperprolactinemia and galactorrhea found no change in serum prolactin levels after beta-adrenergic blockade with propranolol.
◈ What is Propranolol?
Propranolol is a medication used to treat hypertension, certain heart conditions, hyperthyroidism, tremor, glaucoma, and migraines. It belongs to the beta-blocker class of drugs. Some brand names for propranolol include Inderal®, InnoPran XL®, Detensol®, Novo-Pranol®, Deralin®, and Cardinol®. Sometimes, when people find out they are pregnant, they consider changing their medication regimen or even stopping it entirely. However, it is essential to consult your healthcare provider before changing your medication regimen. Your healthcare provider can discuss with you the benefits of treating your condition and the risks of not treating it during pregnancy.
◈ I am taking propranolol. Will it make it harder for me to get pregnant?
It is currently unclear whether propranolol makes it harder to get pregnant.
◈ Does taking propranolol increase the risk of miscarriage?
Miscarriage can occur in any pregnancy for many reasons. There is currently no research showing that propranolol increases the risk of miscarriage.
◈ Does taking propranolol increase the risk of birth defects?
There is a 3-5% risk of birth defects at the start of each pregnancy. This is called background risk. It is currently unclear whether propranolol increases the risk of birth defects on top of the background risk. Overall, studies on the use of beta-blockers during pregnancy have not reported an increased risk of birth defects.
◈ Does taking propranolol during pregnancy increase the risk of other pregnancy-related problems?
Propranolol has been associated with fetal growth restriction. However, it is unclear whether this is due to the drug itself, the condition it treats, or other factors. Studies have not shown an increased risk of other pregnancy-related problems, such as preterm birth (delivery before 37 weeks of gestation). Late pregnancy use of propranolol may cause symptoms in the fetus due to the drug's effects on the heart, blood vessels, and metabolism. These symptoms may include a slowed heart rate and hypoglycemia. Not all infants exposed to propranolol will experience these symptoms. It is important to inform your healthcare provider that you are taking propranolol so that your baby can receive optimal care if symptoms occur.
◈ Will taking propranolol during pregnancy affect a child's future behavior or learning?
Currently, there is no research indicating whether propranolol causes behavioral or learning problems in children.
◈ Breastfeeding while taking propranolol:
A small amount of propranolol passes into breast milk. Studies have shown no adverse health effects in breastfed infants taking propranolol. If you suspect your baby is experiencing symptoms such as lethargy or feeding difficulties, please contact your baby's healthcare provider. Please consult your healthcare provider about all breastfeeding-related questions.
◈ Will male exposure to propranolol affect fertility or increase the risk of birth defects?
Propranolol may cause erectile dysfunction (ED) in some men, which may make conception more difficult. Generally, exposure to the drug by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please see the “Paternal Exposure” information sheet on the MotherToBaby website at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Approximately 90% of propranolol is bound to proteins in plasma. Other studies report a range of 85-96%.

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4946twomentTDLotoralt3200 ug/kg/2D-t Endocrine: Hypoglycemia. Israel Journal of Medical Sciences, 18(725), 1982 [PMID:7107213]
4946thumantTDLotoralt2300 ug/kg/Dt Behavior: Hallucinations, perceptual distortions. British Medical Journal, 1(1182), 1978 [PMID:638680]
4946tchildtLDLotoralt800 ug/kg/12Ht Heart: Increased pulse rate, no decrease in blood pressure; Vascular: No decrease in blood pressure observed in autonomic dysfunction; Lung, pleural or respiratory: Acute pulmonary edema, British Medical Journal, 2(254), 1978; 4946tchildtTDLotoralt400 mg/kgt Behavioral: Seizures or effects on epileptic threshold; Cardiac: Arrhythmias (including conduction changes), Medical Journal of Australia, 1(82), 1981 [PMID:7231257]; 4946tmantTDLotoralt8343 mg/kg/4Y-t Endocrine: Evidence of hyperthyroidism, Archives of Internal Medicine, 143(2193), 1983 [PMID:6639243]

[1]. Distinct signaling profiles of beta1 and beta2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol Pharmacol. 2006 Nov;70(5):1575-84.

[2]. Evidence against beta-adrenoceptor blocking activity of diltiazem, a drug with calcium antagonist properties. Br J Pharmacol. 1980 Aug;69(4):669-73.

[3]. Propranolol. Profiles Drug Subst Excip Relat Methodol. 2017;42:287-338.

[4]. Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation. Stem Cells Transl Med. 2016 Jan;5(1):45-55.

Propranolol is a propanolamine with a structure of propan-2-ol substituted with propan-2-amino at the 1-position and naphth-1-oxy at the 3-position. It possesses a variety of pharmacological effects, including β-adrenergic antagonist, anxiolytic, antiarrhythmic, vasodilator, antihypertensive, exogenous substance, environmental pollutant, and human serum metabolite. It is a secondary amine, a propanolamine compound belonging to the naphthalene family. Its function is related to 1-naphthol. Propranolol is a racemic mixture of two enantiomers, with the S(-)-enantiomer having approximately 100 times the binding affinity to β-adrenergic receptors. Propranolol is used to treat a variety of diseases, but is most commonly used to treat hypertension. Propranolol was approved by the U.S. Food and Drug Administration (FDA) on November 13, 1967. Propranolol is a β-adrenergic blocker. Propranolol's mechanism of action is as a β-adrenergic receptor antagonist. Propranolol is a non-selective β-adrenergic receptor blocker (β-blocker) widely used to treat hypertension, arrhythmias, angina pectoris, and hyperthyroidism. Currently, there is no conclusive evidence that propranolol is associated with clinically significant liver damage; therefore, it is commonly used in patients with liver disease and cirrhosis. Propranolol has also been reported to be found in Asimina triloba, with relevant data. Propranolol is a synthetic, non-selective β-adrenergic receptor blocker with antianginal, antiarrhythmic, and antihypertensive effects. Propranolol competitively antagonizes β-adrenergic receptors, thereby producing negative chronotropic and inotropic effects, leading to a decrease in cardiac output. Propranolol is a widely used non-selective β-adrenergic antagonist. It was previously used to treat myocardial infarction, arrhythmia, angina pectoris, hypertension, hyperthyroidism, migraine, pheochromocytoma, and anxiety, but due to adverse reactions, it has been superseded by newer medications. See also: Propranolol hydrochloride (salt form).

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Drug Indications
Propranolol is indicated for the treatment of hypertension. Propranolol is also indicated for the treatment of angina pectoris, atrial fibrillation, myocardial infarction, migraine, essential tremor, hypertrophic subaortic stenosis, pheochromocytoma, and proliferative infantile hemangioma caused by coronary atherosclerosis.
FDA Label
Hemangiol is indicated for the treatment of proliferative infantile hemangioma requiring systemic treatment: life-threatening or functional hemangiomas, ulcerative hemangiomas with pain and/or unresponsiveness to simple wound care, and hemangiomas with a risk of permanent scarring or disfigurement. It is indicated for infants aged 5 weeks to 5 months.

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Mechanism of Action
Propranolol is a non-selective β-adrenergic receptor antagonist. Blocking these receptors leads to vasoconstriction, inhibits angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast basic growth factor (bFGF), induces endothelial cell apoptosis, and downregulates the renin-angiotensin-aldosterone system.

C16H21NO2

259.3434445858

259.157

C, 74.10; H, 8.16; N, 5.40; O, 12.34

525-66-6

Propranolol hydrochloride;318-98-9;Propranolol-d7;98897-23-5; 525-66-6

4946

White to off-white solid powder

1.093 g/cm3

434.9ºC at 760 mmHg

163-164ºC

216.8ºC

1.5500 (estimate)

2.968

2

3

6

19

257

0

CC(C)NCC(COC1=CC=CC2=CC=CC=C21)O

AQHHHDLHHXJYJD-UHFFFAOYSA-N

InChI=1S/C16H21NO2/c1-12(2)17-10-14(18)11-19-16-9-5-7-13-6-3-4-8-15(13)16/h3-9,12,14,17-18H,10-11H2,1-2H3

1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol

propranolol; 525-66-6; Propanolol; beta-Propranolol; Betalong; Euprovasin; Proprasylyt; Reducor;

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 (~385.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.64 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 (9.64 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 (9.64 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.)