MDA5 (melanoma differentiation-associated protein 5). Azithromycin's IFN-inducing effects are mediated through MDA5, as demonstrated by siRNA knockdown studies. [1]
RIG-I (retinoic acid-inducible gene 1). Azithromycin's IFN-inducing effects are not mediated through RIG-I, as demonstrated by siRNA knockdown studies. [1]

In primary bronchial epithelial cells from asthmatic patients, azithromycin (2 μM) increases rhinovirus-induced IFNβ expression. This is linked to upregulation of RIG-I-like receptors and suppression of viral propagation. In virus-induced asthma, azithromycin (2 μM)-enhanced IFNβ production in primary bronchial epithelial cells is reduced by MDA5 knockdown but not RIG-I knockdown [1]. Without altering NF-κB, azithromycin selectively lowers MMP-9 mRNA and protein levels in endotoxin-challenged mononuclear THP-1 cells [2].

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In primary bronchial epithelial cells (HBECs) from asthmatic donors infected with rhinovirus (RV16) at 1 MOI, Azithromycin treatment (2, 10, 20 μM), beginning 24 h before infection and continued throughout the experiment, dose-dependently augmented RV16-induced IFNβ secretion measured 48 h post-infection. Azithromycin alone did not stimulate IFNβ secretion. [1]
In RV16-infected asthmatic HBECs, treatment with 2 μM Azithromycin slightly enhanced the expression of RIG-I-like helicases RIG-I and MDA5. A negative correlation was found between IFNβ mRNA expression and viral load in cells treated with 2 μM azithromycin (p < 0.05). [1]
In primary bronchial epithelial cells from healthy individuals infected with RV16 (1 MOI), treatment with 2 μM Azithromycin did not alter RV16-induced IFNβ expression measured by real-time PCR, nor did it affect viral replication. [1]
In primary bronchial epithelial cells from asthmatic patients, siRNA-mediated knockdown of MDA5 significantly reduced IFNβ expression (p < 0.01) and enhanced viral load (p < 0.05) in RV16-infected cells treated with 2 μM Azithromycin. Knockdown of RIG-I had no significant effect on IFNβ expression or viral load. [1]

A mouse model of acute asthma exacerbation treated with 50 mg/kg of azithromycin showed no change in bronchoalveolar lavage inflammatory markers and LDH levels. In a mouse model of asthma exacerbation, azithromycin did not cause any general inflammatory parameters or LDH release. However, it did enhance the expression of interferon-stimulated genes and the pattern recognition receptor MDA5, but not RIG-I[1].

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In a mouse model of asthma exacerbation (HDM-induced allergic inflammation followed by poly(I:C) challenge), treatment with Azithromycin (50 mg/kg, once daily, starting 48 h before and continuing throughout the poly(I:C) challenges) did not significantly alter general inflammatory parameters (total cell count, protein concentration) or LDH levels in bronchoalveolar lavage fluid (BALF) compared to vehicle-treated exacerbating mice. It also did not alter the composition of recruited inflammatory cells (macrophages, eosinophils, neutrophils) in BALF. [1]
In this exacerbation model, where the IFNβ response to poly(I:C) was significantly diminished in HDM-sensitized mice compared to non-allergic mice, Azithromycin treatment augmented and restored IFNβ protein levels in the allergic, poly(I:C)-stimulated mice (p < 0.05). In non-allergic mice exposed to poly(I:C), azithromycin did not further induce IFNβ levels. [1]
In lung homogenates from exacerbating mice (HDM + poly(I:C)), Azithromycin treatment significantly increased the expression of the interferon-stimulated gene viperin (p < 0.05) and showed a trend towards increased Mx1 expression. Lung MDA5 expression tended to be elevated by azithromycin treatment in the exacerbation group (p = 0.065), while RIG-I expression was not altered. [1]

Gelatin zymography for MMP-9 activity inhibition: Samples containing human recombinant MMP-9 were loaded onto 7.5% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gel was sliced into separate lanes. Each gel strip was incubated overnight at 37°C in incubation buffer (50 mM Tris, 10 mM CaCl2, 0.02% NaN3, 1% Triton X-100, pH 7.5) containing different concentrations of azithromycin. Following incubation, gels were stained, and band densities were analyzed to assess gelatinolytic activity inhibition. [2]
Gelatin degradation assay for MMP-9 activity inhibition: 10 nM of active MMP-9 was incubated with 60 μM, 40 μM, and 20 μM of azithromycin for 30 minutes at 37°C. A fluorogenic gelatin substrate was then added, and the increase in fluorescence was recorded every 10 minutes for 2 hours. The percentage of inhibition was calculated by comparing the initial velocity of the reaction with a condition without compound. [2]
ProMMP-9 activation assay: Recombinant human full-length proMMP-9 (92 kDa) was incubated with the catalytic domain of stromelysin-1/MMP-3 in the presence or absence of azithromycin. The activation process was monitored by assessing gelatinolytic activity (using a method similar to the gelatin degradation assay) and by analyzing samples at different time points using gelatin zymography to visualize the stepwise conversion from the full-length pro-enzyme (92 kDa) to an intermediate (86 kDa) and the fully activated MMP-9 (82 kDa). [2]

Primary human bronchial epithelial cell (HBEC) culture and treatment: HBECs from asthmatic donors (obtained via bronchoscopy) and healthy individuals were cultured in bronchial epithelial growth medium. Cells were seeded into 12-well plates and experiments were initiated at 80-90% confluency, using cells at passage 2-3. Azithromycin was dissolved in DMSO and added to the culture medium 24 hours prior to rhinovirus infection and maintained throughout the experiment. [1]
Rhinovirus infection: The major group rhinovirus RV16 was used. HBECs were infected with RV16 at a multiplicity of infection (MOI) of 1 for 1 hour at room temperature with shaking. Following infection, the virus inoculum was removed, and fresh medium containing azithromycin was added. Cell lysates were collected 24 hours post-infection for gene expression analysis, and supernatants were collected 48 hours post-infection for protein analysis. [1]
siRNA knockdown: HBECs from asthmatic donors were transfected with siRNA specifically targeting MDA5 or RIG-I, or with non-specific siRNA, at a concentration of 10 μM using a lipid-based transfection agent. Following transfection, cells were treated with azithromycin (2 μM) and infected with RV16 as described above. [1]
Gene and protein expression analysis: Gene expression levels of IFNβ, viral load, MDA5, RIG-I, Mx1, and viperin were measured by real-time PCR. Protein expression levels of IFNβ in cell supernatants were measured by ELISA. [1]

Mouse model of asthma exacerbation:** C57BL/6 mice were used. To establish allergic airway inflammation, mice were challenged intranasally with house dust mite (HDM) extract or saline (control) three days per week for three weeks. Subsequently, to induce a viral-like exacerbation, mice were exposed daily for three days to the TLR3 agonist poly(I:C) or saline (control) via the intranasal route. Azithromycin (50 mg/kg) or vehicle was administered to mice once daily by an unspecified route (likely oral or intraperitoneal, as typical for such studies, though the route is not explicitly stated in the provided text) starting 48 hours before the first poly(I:C) exposure and continuing throughout the poly(I:C) challenge period. The experiment was terminated 24 hours after the final poly(I:C) exposure. Bronchoalveolar lavage fluid (BALF) was collected, and lung tissue was harvested for homogenization. [1]

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Mouse model of asthma exacerbation: C57BL/6 mice were used. To establish allergic airway inflammation, mice were challenged intranasally with house dust mite (HDM) extract or saline (control) three days per week for three weeks. Subsequently, to induce a viral-like exacerbation, mice were exposed daily for three days to the TLR3 agonist poly(I:C) or saline (control) via the intranasal route. Azithromycin (50 mg/kg) or vehicle was administered to mice once daily by an unspecified route (likely oral or intraperitoneal, as typical for such studies, though the route is not explicitly stated in the provided text) starting 48 hours before the first poly(I:C) exposure and continuing throughout the poly(I:C) challenge period. The experiment was terminated 24 hours after the final poly(I:C) exposure. Bronchoalveolar lavage fluid (BALF) was collected, and lung tissue was harvested for homogenization. [1]

The provided document mentions that standard dose regimens of azithromycin produce in vivo levels of up to 10 μg/ml. This statement is made in the introduction based on a reference (Di Paolo et al., 2002) and is not an experimental result from this study. [1]

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Because azithromycin levels are low in breast milk and infants typically receive higher doses, adverse effects on breastfed infants are not expected. Monitoring for potential impacts on the infant's gut microbiota, such as vomiting, diarrhea, and candidiasis (thrush, diaper rash), should be conducted. Unproven epidemiological evidence suggests that maternal use of macrolide antibiotics during the first two weeks of lactation may increase the risk of hypertrophic pyloric stenosis in infants, but this relationship has been questioned by other studies. One study showed that a single dose of azithromycin administered during delivery to women carrying pathogenic Staphylococcus and Streptococcus in their nasal cavity reduced the number of these bacteria in breast milk but increased the detection rate of azithromycin-resistant Escherichia coli and Klebsiella pneumoniae in breast milk.
The risk to breastfed infants from maternal use of azithromycin-containing eye drops is negligible. To significantly reduce the amount of eye drops that enters breast milk after use, press your finger against the tear duct near the corner of your eye for at least one minute, then wipe away any excess medication with absorbent tissue.
◉ Impact on Breastfed Infants
A cohort study of infants diagnosed with hypertrophic pyloric stenosis found that mothers of these infants were 2.3 to 3 times more likely to have taken macrolide antibiotics within 90 days postpartum than other infants. Stratified analysis of the infants showed an odds ratio of 10 for girls and 2 for boys. All mothers of affected infants breastfed. Most macrolide prescriptions were erythromycin, but only 7% were azithromycin. However, the authors did not specify which macrolide the mothers took.
A retrospective database study in Denmark analyzed 15 years of data and found that infants born to mothers who took macrolides in the first 13 days postpartum had a 3.5-fold increased risk of developing hypertrophic pyloric stenosis, but no such risk was observed afterward. The proportion of breastfed infants is unknown, but likely high. The proportion of women taking each macrolide antibiotic was not reported. A study comparing breastfed infants born to mothers taking amoxicillin and those taking macrolide antibiotics found no cases of pyloric stenosis in either group. However, most infants exposed to macrolide antibiotics through breast milk were exposed to roxithromycin. Of the 55 infants exposed to macrolide antibiotics, only 10 were exposed to azithromycin. Adverse reactions occurred in 12.7% of infants exposed to macrolide antibiotics, a similar rate to that of infants exposed to amoxicillin. Adverse reactions included rash, diarrhea, loss of appetite, and drowsiness. Eight women who received 500 mg of azithromycin intravenously 15, 30, or 60 minutes before a cesarean section incision were breastfed. No adverse events occurred in the infants. Two meta-analyses failed to confirm an association between maternal use of macrolide antibiotics during lactation and hypertrophic pyloric stenosis in infants.
◉ Effects on Lactation and Breast Milk
In a double-blind, controlled study conducted in Gambia, women carrying Staphylococcus aureus, Streptococcus pneumoniae, or Group B Streptococcus in their nasopharynx received a single 2-gram dose of azithromycin during delivery. The carriage rate of these pathogens in breast milk samples from women receiving azithromycin was 9.6%, compared to 21.9% in breast milk samples from women receiving a placebo. The carriage rate of these pathogens in the nasopharynx of both mothers and infants also decreased by day 6 postpartum. However, subsequent analysis found that intrapartum oral administration of azithromycin did not reduce the carriage rate of Escherichia coli and Klebsiella pneumoniae; instead, it was associated with an increased prevalence of azithromycin-resistant Escherichia coli and Klebsiella pneumoniae isolates in breast milk.

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In the in vivo mouse model of asthma exacerbation, treatment with Azithromycin (50 mg/kg) did not induce any significant increase in bronchoalveolar lavage fluid (BALF) levels of lactate dehydrogenase (LDH), a marker of general necrosis and cell death, compared to vehicle-treated controls. [1]

[1]. Azithromycin augments rhinovirus-induced IFNβ via cytosolic MDA5 in experimental models of asthma exacerbation. Oncotarget. 2017 Mar 18.

[2]. Differential inhibition of activity, activation and gene expression of MMP-9 in THP-1 cells by azithromycin and minocycline versus bortezomib: A comparative study. PLoS One. 2017 Apr 3;12(4):e0174853.

Azithromycin dihydrate is a hydrate containing azithromycin. Azithromycin is a prescription antibacterial drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of certain bacterial infections, such as: Various bacterial respiratory illnesses, including community-acquired pneumonia, acute sinusitis and ear infections, acute exacerbations of chronic bronchitis, and throat and tonsil infections; pelvic inflammatory disease; genital ulcers and urethral and cervical infections; skin infections. Community-acquired pneumonia is a bacterial respiratory illness and may be an opportunistic infection (OI) of HIV. Azithromycin dihydrate is the dihydrate form of azithromycin, a highly bioavailable oral azathioprine antibiotic derived from erythromycin, belonging to the macrolide antibiotic subclass, and possessing antibacterial activity. After oral administration, azithromycin reversibly binds to the 23S rRNA of the 50S ribosomal subunit of susceptible bacteria, thereby preventing the assembly of the 50S ribosomal subunit and inhibiting the translocation step in protein synthesis. This inhibits bacterial protein synthesis, suppresses cell growth, and leads to cell death. A semi-synthetic macrolide antibiotic, its structure is related to erythromycin. It has been used to treat intracellular Mycobacterium avium infections, toxoplasmosis, and cryptosporidiosis. See also: Azithromycin dihydrate; Travafloxacin mesylate (component).

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Azithromycin is a macrolide antibiotic. In standard dose regimens, it produces in vivo levels of up to 10 μg/ml. [1]
Azithromycin has produced promising effects on reducing exacerbations of asthma. [1]
The study suggests that azithromycin may restore deficient lung IFN production in exacerbating asthma and that this effect is MDA5-dependent. [1]
The study proposes that other molecules sharing azithromycin's MDA5-dependent mechanism may have a role in the treatment of asthma exacerbations. [1]

C38H76N2O14

785.026

784.529

117772-70-0

Azithromycin;83905-01-5;Azithromycin-d3;163921-65-1

3033819

White to off-white solid powder

1.18g/cm3

822.1ºC at 760mmHg

113-115ºC

451ºC

2.51E-31mmHg at 25°C

1.71

7

16

7

54

1150

18

C[C@@H]([C@@H]([C@@](C(O[C@@H]([C@@](C)(O)[C@@H]1O)CC)=O)([H])C)O[C@@](O[C@@H](C)[C@@H]2O)([H])C[C@@]2(C)OC)[C@H]([C@](O)(C[C@H](CN([C@@H]1C)C)C)C)O[C@@](O[C@H](C)C[C@@H]3N(C)C)([H])[C@@H]3O.O.O

Azitro CP-62993 CP 62993

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

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.)