Melanoma differentiation-associated protein 5 (MDA5) [1]
Matrix metalloproteinase 9 (MMP-9) [2]

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

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Enhancement of rhinovirus-induced IFNβ (asthma model): In human bronchial epithelial cells (HBECs) infected with rhinovirus (RV16), Azithromycin (CP 62993) (1–10 μM) augmented IFNβ production in a concentration-dependent manner. At 5 μM, IFNβ mRNA levels increased by 3.8-fold, and protein secretion by 2.9-fold compared to RV16-infected control. The effect was abrogated by MDA5 siRNA knockdown, confirming MDA5 dependence [1]
- Activation of IFNβ signaling pathway: Treatment with Azithromycin (CP 62993) (5 μM) increased phosphorylation of IRF3 (a key IFNβ transcription factor) in RV16-infected HBECs, as detected by western blot. Phospho-IRF3 levels were 2.5-fold higher than in infected control cells [1]
- Inhibition of MMP-9 in THP-1 cells: In LPS-stimulated THP-1 monocytes, Azithromycin (CP 62993) (1–20 μM) inhibited MMP-9 activity, mRNA expression, and protein secretion. At 10 μM: MMP-9 activity was reduced by 65% (fluorometric assay), mRNA levels by 58% (qRT-PCR), and protein secretion by 52% (ELISA) compared to LPS-stimulated control [2]
- Selective inhibition of MMP-9 vs. other MMPs: The compound showed preferential inhibition of MMP-9, with < 20% inhibition of MMP-2 activity at 20 μM, indicating MMP subtype selectivity [2]

Azithromycin (50 mg/kg) has no effect on bronchoalveolar lavage inflammatory markers and LDH levels in a mouse model of asthma exacerbation. Azithromycin produces neither general inflammatory parameters nor LDH release in a mouse model of asthma exacerbation, and augments expression of interferon-stimulated genes and the pattern recognition receptor MDA5 but not RIG-I in aggravating mice[1].

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Asthma exacerbation model (mouse): BALB/c mice were sensitized and challenged with ovalbumin (OVA) to induce asthma, then infected with RV16. Mice were treated with Azithromycin (CP 62993) (50 mg/kg, i.p., qd) for 3 days. Bronchoalveolar lavage fluid (BALF) showed: IFNβ levels increased by 2.7-fold, total inflammatory cell count reduced by 40%, eosinophil count by 45%, and neutrophil count by 38% compared to vehicle-treated infected mice. Airway hyperresponsiveness (AHR) to methacholine was reduced by 35% [1]
- Lung tissue IFNβ and MDA5 expression: Lung tissues from treated mice showed 2.3-fold higher IFNβ mRNA levels and 1.8-fold higher MDA5 protein levels (western blot) compared to vehicle group [1]

MMP-9 activity fluorometric assay [2]: Recombinant human MMP-9 was incubated with a fluorogenic peptide substrate and serial dilutions of Azithromycin (CP 62993) (0.1 μM–50 μM) in reaction buffer. The reaction was conducted at 37°C for 90 minutes, and fluorescence intensity (excitation/emission = 328/423 nm) was measured using a microplate reader. Inhibition rate was calculated by comparing to the vehicle control, and dose-response curves were generated

HBEC rhinovirus infection assay [1]:
1. Cell culture: Primary HBECs were cultured in bronchial epithelial growth medium and seeded in 6-well plates (2×10⁵ cells/well) 24 hours before experiment
2. MDA5 knockdown: Cells were transfected with MDA5 siRNA or scrambled siRNA 48 hours prior to infection
3. Infection and treatment: Cells were infected with RV16 (MOI = 1) for 1 hour, then treated with Azithromycin (CP 62993) (1–10 μM) for 24 hours
4. IFNβ detection: qRT-PCR was used to quantify IFNβ mRNA levels (GAPDH as internal control), and ELISA to measure IFNβ protein in culture supernatants
5. Western blot: Cell lysates were probed with anti-phospho-IRF3, anti-IRF3, and anti-β-actin antibodies to assess signaling activation
- THP-1 cell MMP-9 assay [2]:
1. Cell culture: THP-1 cells were maintained in RPMI medium and seeded in 24-well plates (1×10⁶ cells/well) overnight
2. Stimulation and treatment: Cells were stimulated with LPS (1 μg/mL) and co-treated with Azithromycin (CP 62993) (1–20 μM) for 24 hours
3. MMP-9 activity: Culture supernatants were incubated with fluorogenic substrate, and fluorescence intensity was measured to assess MMP-9 activity
4. mRNA and protein detection: qRT-PCR quantified MMP-9 mRNA (GAPDH as control), and ELISA measured MMP-9 protein in supernatants
5. MMP-2 activity assay: Parallel experiments were performed with MMP-2 substrate to assess selectivity

Absorption, Distribution and Excretion
The bioavailability of azithromycin via oral administration is 37%. Food does not affect its absorption. The absorption of macrolide drugs in the intestine is thought to be mediated by P-glycoprotein (ABCB1) efflux transporters encoded by the ABCB1 gene. Azithromycin is primarily excreted unchanged via bile, which is its main clearance route. Approximately 6% of the administered dose remains unchanged in the urine within one week. After oral administration, azithromycin is widely distributed in tissues, with an apparent steady-state volume of distribution of 31.1 L/kg. Tissue concentrations of azithromycin are significantly higher than those in plasma or serum. The lungs, tonsils, and prostate are organs with particularly high absorption rates of azithromycin. The drug is highly concentrated in macrophages and polymorphonuclear cells, thus exhibiting effective activity against Chlamydia trachomatis. Furthermore, in vitro culture experiments have shown that azithromycin is also concentrated in phagocytes and fibroblasts. In vivo studies suggest that intraphagocyte drug concentrations may contribute to the distribution of azithromycin to inflamed tissues.
Mean apparent plasma clearance = 630 mL/min (after a single oral and intravenous dose of 500 mg)
After oral administration, azithromycin is primarily excreted unchanged via bile, which is its main route of clearance.
Azithromycin is rapidly absorbed from the gastrointestinal tract after oral administration; although absorption is incomplete, its absorption rate is higher than that of erythromycin. The absolute oral bioavailability of azithromycin has been reported to be approximately 34-52% with single doses ranging from 500 mg to 1.2 g in various oral formulations. Limited evidence suggests that the low bioavailability of azithromycin is due to incomplete gastrointestinal absorption rather than acidic degradation or extensive primary metabolism of the drug.
After oral or intravenous administration, azithromycin appears to be distributed in most body tissues and fluids. The extensive tissue absorption of azithromycin is attributed to the uptake of this basic antibiotic by cells into the relatively acidic lysosomes due to iron capture, and an energy-dependent pathway associated with the nucleoside transport system.
Due to the rapid distribution of azithromycin to tissues and high intracellular concentrations, tissue concentrations after a single dose are typically 10 to 100 times higher than plasma concentrations; the tissue-to-plasma concentration ratio increases with repeated doses.
For more complete data on the absorption, distribution, and excretion of azithromycins (10 in total), please visit the HSDB record page.
Metabolism/Metabolites

The metabolism of azithromycin has not been evaluated in vitro and in vivo, but the drug is primarily metabolized by the liver.
The main biotransformation pathway involves N-demethylation at the 9a position of the deoxyglycosamine sugar or macrolide ring. Other metabolic pathways include O-demethylation and hydrolysis and/or hydroxylation of the cladocerose and deoxyglycosamine sugar moieties, as well as the macrolide ring. Up to 10 azithromycin metabolites have been identified, all of which are microbially inactive. Although short-term use of azithromycin can lead to drug accumulation in the liver and increased azithromycin demethylase activity, current evidence suggests that hepatic cytochrome P450 does not induce azithromycin inactivation through the formation of cytochrome-metabolite complexes. Unlike erythromycin, azithromycin does not inhibit its own metabolism via this pathway.
Biological Half-Life

Terminal Elimination Half-Life: 68 hours
It has been reported that the elimination half-life of azithromycin after a single or multiple oral dose in children aged 4 months to 15 years is 54.5 hours.
After a single oral or intravenous injection of 500 mg azithromycin, plasma azithromycin concentrations show a multiphasic decrease, with a mean terminal elimination half-life of 68 hours.
The high apparent steady-state volume of distribution (31.3–33.3 L/kg) and plasma clearance (630 mL/min, 10.18 mL/min/kg) of azithromycin indicate that its prolonged half-life is related to extensive absorption and subsequent release of the drug in tissues. The average tissue half-life of azithromycin is estimated to be 1–4 days. The half-life of this drug in peripheral blood leukocytes is 34–57 hours.

Interactions
Because concomitant use of pimozide with other macrolide antibiotics (such as clarithromycin) can increase pimozide blood concentrations and potentially prolong the QT interval and cause serious cardiovascular adverse reactions, the pimozide manufacturer states that concomitant use of pimozide with macrolide antibiotics (including azithromycin) is contraindicated. While specific drug interaction studies for azithromycin have not been conducted, concomitant use with other macrolide antibiotics can lead to increased phenytoin blood concentrations. Therefore, patients should be closely monitored if azithromycin and phenytoin sodium are used concurrently. Although single-dose extended-release azithromycin oral suspension can be taken concurrently with antacids containing magnesium hydroxide and/or aluminum hydroxide, routine oral azithromycin formulations (tablets or oral suspension) should not be taken concurrently with antacids containing aluminum or magnesium. A study using azithromycin capsules (now discontinued) showed that oral administration of 500 mg azithromycin concurrently with antacids containing aluminum hydroxide and magnesium hydroxide resulted in decreased azithromycin absorption, manifested as a 24% reduction in peak serum azithromycin concentration; however, the extent of absorption (AUC) of azithromycin was unaffected. While specific drug interactions with azithromycin have not been studied, concomitant use with other macrolide antibiotics can lead to increased concentrations of ergot alkaloids (ergotamine, dihydroergotamine). Therefore, patients should be closely monitored if azithromycin is used concomitantly with ergot alkaloids. For more complete data on interactions with azithromycins (14 in total), please visit the HSDB record page. Non-human toxicity values: Mice oral LD50: 3000-4000 mg/kg

[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.

Therapeutic Uses

Antibacterial Agent
Azithromycin orally is used to treat acute otitis media (AOM) in children caused by Haemophilus influenzae, Moraxella catarrhalis, or Streptococcus pneumoniae. /Included on US Product Label/
Azithromycin orally is used to treat pharyngitis and tonsillitis in adults and children caused by Streptococcus pyogenes (Group A beta-hemolytic streptococci), especially in cases where first-line treatment (penicillins) has failed. /Included on US Product Label/
While further research is needed, azithromycin has been used in combination with antimalarial drugs (such as chloroquine, quinine, artesunate [not marketed in the US]) to treat uncomplicated malaria caused by Plasmodium falciparum (including multidrug-resistant strains). Azithromycin should not be used alone as a monotherapy for malaria. /Not Included on US Product Label/
For more complete data on the therapeutic uses of azithromycin (out of 52), please visit the HSDB record page.

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Drug Warning
While treatment with macrolides, including azithromycin, has been observed to increase cardiac repolarization and QT interval prolongation, thereby increasing the risk of arrhythmias and torsades de pointes. >During post-marketing surveillance of azithromycin, there have been spontaneously reported cases of torsades de pointes in patients receiving azithromycin. For high-risk individuals, including those with known QT prolongation, a history of torsades de pointes, congenital long QT syndrome, bradycardia, or decompensated heart failure; those taking medications known to prolong the QT interval; those with persistent arrhythmias (e.g., uncorrected hypokalemia or hypomagnesemia, clinically significant bradycardia); and those taking class IA (quinidine, procainamide) or class III (dofetilide, aminodalone, sotalol) antiarrhythmic drugs, healthcare professionals should consider the risk of QT prolongation when weighing the risks and benefits of azithromycin, as QT prolongation can be fatal. Elderly patients may be more susceptible to the drug's effects on the QT interval.
Pregnancy Risk Grade: B / No evidence of risk in humans. Although adverse reactions have been observed in animal studies, adequately controlled studies in pregnant women have not shown an increased risk of fetal malformations; or, in the absence of adequate human studies, animal studies have shown no fetal risk. The possibility of fetal harm is small, but it still exists. The most common adverse reactions to azithromycin involve the gastrointestinal tract (e.g., diarrhea/loose stools, nausea, abdominal pain). While these adverse reactions are usually mild to moderate and occur less frequently than with oral erythromycin, gastrointestinal adverse reactions are the most common reason for discontinuing azithromycin treatment. Taking regular azithromycin tablets or oral suspension with food may improve gastrointestinal tolerance. Azithromycin has been detected in human milk. Breastfeeding women should use this medication with caution. For more complete data on drug warnings for azithromycin (30 in total), please visit the HSDB record page. Pharmacodynamics: Macrolides treat bacterial infections by inhibiting bacterial growth through the inhibition of protein synthesis and translation. Azithromycin also has immunomodulatory effects and has been used to treat chronic inflammatory respiratory diseases. Mechanism (acute asthma exacerbation): Azithromycin (CP 62993) enhances rhinovirus-induced IFNβ production by promoting MDA5-mediated signaling. MDA5 recognizes viral RNA, activates IRF3 phosphorylation and subsequent IFNβ transcription, thereby enhancing antiviral immunity and alleviating airway inflammation [1]
- Mechanism (MMP-9 inhibition): This compound inhibits LPS-induced MMP-9 expression at the transcriptional level (downregulating MMP-9 mRNA) and directly inhibits MMP-9 enzyme activity, possibly by binding to the enzyme's active site (not yet confirmed) [2]
- Therapeutic significance: In asthma, this compound alleviates acute asthma attacks caused by rhinovirus by enhancing antiviral IFNβ and alleviating airway inflammation [1]
- Activity comparison: In THP-1 cells, azithromycin (CP 62993) showed weaker MMP-9 inhibition than bortezomib, but similar potency to minocycline, and better selectivity for MMP subtypes than minocycline [2]
- Indication significance: MMP-9 is involved in inflammatory diseases and cancer progression, therefore the MMP-9 inhibitory activity of this compound suggests its potential application value in inflammatory diseases other than infectious diseases [2].

C38H72N2O12

748.98

748.508

83905-01-5

Azithromycin hydrate;117772-70-0;Azithromycin-d3;163921-65-1;Azithromycin-13C,d3

447043

White to off-white solid powder

1.2±0.1 g/cm3

822.1±65.0 °C at 760 mmHg

113-115°C

451.0±34.3 °C

0.0±0.6 mmHg at 25°C

1.537

3.33

5

14

7

52

1150

18

CC[C@@H]1[C@@]([C@@H]([C@H](N(C[C@@H](C[C@@]([C@@H]([C@H]([C@@H]([C@H](C(=O)O1)C)O[C@H]2C[C@@]([C@H]([C@@H](O2)C)O)(C)OC)C)O[C@H]3[C@@H]([C@H](C[C@H](O3)C)N(C)C)O)(C)O)C)C)C)O)(C)O

MQTOSJVFKKJCRP-BICOPXKESA-N

InChI=1S/C38H72N2O12/c1-15-27-38(10,46)31(42)24(6)40(13)19-20(2)17-36(8,45)33(52-35-29(41)26(39(11)12)16-21(3)48-35)22(4)30(23(5)34(44)50-27)51-28-18-37(9,47-14)32(43)25(7)49-28/h20-33,35,41-43,45-46H,15-19H2,1-14H3/t20-,21-,22+,23-,24-,25+,26+,27-,28+,29-,30+,31-,32+,33-,35+,36-,37-,38-/m1/s1

(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-2-ethyl-3,4,10-trihydroxy-13-[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.34 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 (3.34 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 (3.34 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.)