Macrolide antibiotic

Roxithromycin is a semi-synthetic macrolide antibiotic. It is used to treat respiratory tract, urinary and soft tissue infections. Roxithromycin is derived from erythromycin, containing the same 14-membered lactone ring. Roxithromycin prevents bacteria from growing, by interfering with their protein synthesis. Roxithromycin binds to the subunit 50S of the bacterial ribosome, and thus inhibits the translocation of peptides. Roxithromycin has similar antimicrobial spectrum as erythromycin, but is more effective against certain gram-negative bacteria, particularly Legionella pneumophila. Roxithromycin is known to have anti-inflammatory and immunoregulatory activity. However, little information is available on the effect of roxithromycin in intestinal inflammation [1].

The aim of this study was to investigate the effect of roxithromycin on NF- κB signaling and ER stress in intestinal epithelial cells (IECs) and the effect of roxithromycin on dextran sulfate sodium (DSS)-induced acute colitis in a murine model. HCT116 cells and COLO205 cells were pretreated with roxithromycin and then stimulated with tumor necrosis factor-α (TNF-α). Interleukin (IL)-8 expression was determined by real-time reverse transcription-polymerase chain reaction. Nuclear factor kappaB (NF-κB) DNA-binding activity and IκB phosphorylation/degradation were evaluated by electrophoretic mobility shift assay and Western blot analysis. The molecular markers of endoplasmic reticulum stress, including p-JNK, phosphorylated eukaryotic initiation factor 2 (p-eIF2α), C/EBP homologous protein (CHOP), and X-box binding protein 1 (XBP1) were evaluated using western blotting and PCR. Mice were given 4% DSS for five days with or without roxithromycin. Primary IECs were isolated from mice with DSS-induced colitis. Roxithromycin significantly inhibited the upregulated expression of IL-8. Pretreatment with roxithromycin markedly attenuated NF-κB DNA-binding activity and IκB phosphorylation/degradation. CHOP and XBP1 mRNA expression were enhanced in the presence of TNF-α, and it was dampened by pretreatment of roxithromycin. c-Jun-N-terminal kinase (JNK) phosphorylation and the level of p-eIF2α were also downregulated by the pretreatment of roxithromycin. Roxithromycin significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histology. In addition, the DSS-induced phospho-IκB kinase activation was significantly decreased in roxithromycin-pretreated mice. Finally, IκB degradation was reduced in primary IECs from mice treated with roxithromycin. These results suggest that roxithromycin may have potential usefulness in the treatment of inflammatory bowel disease [1].

RNA preparation and real time RT-PCR were performed as described previously.17,18 Total cellular RNA was extracted by treating Trizol from HCT116 cells. One microgram of total cellular RNA was reverse transcribed and amplified using the SYBR green PCR Master Mix and ABI prism 7000 sequence detection system. We used the following primers specific for human IL-8, CHOP, XBP1, and β-actin. IL-8, 5′-AAA CCA CCG GAA GGA ACC AT-3′ (sense) and 5′-CCT TCA CAC AGA GCT GCA GAA A-3′ (antisense); CHOP, 5′-CAG AAC CAG CAG AGG TCA CA-3′ (sense) and 5′-AGC TGT GCC ACT TTC CTT TC-3′ (antisense); XBP1, 5′- AAA CAG AGT AGC AGC TCA GAC TGC-3′ (sense) and 5′-TCC TTC TGG GTA GAC CTC TGG GAG-3′ (antisense); β-actin, 5′-ACG GGG TCA CCC ACA CTG TGC CCA TCT A-3′ (sense) and 5′-CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3′ (antisense). Gene amplifications were performed in triplicate, and data were normalized to the level of β-actin expression[1].

Electrophoretic mobility shift assay (EMSA)[1]
HCT116 cells were incubated with roxithromycin for 24 h and then stimulated with TNF-α for 30 min. HCT116 cells were harvested, and the nuclear contents were extracted as described previously.18 Nuclear protein was extracted using a commercially available kit. A Bradford assay was used to determine protein concentrations in the extracts. EMSA for NF-κB was performed using commercially available kits. Briefly, 4 µg of nuclear extract was mixed with the specified reagents and incubated for 5 min at room temperature. The mixed extract was incubated at 15℃ for 30 min in a thermal cycler with 1 µL of probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) corresponding to a consensus NF-κB-binding site. After incubation, both bound and free DNA were resolved on 6% non-denaturing polyacrylamide gels.

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Immunoblot analyses[1]
HCT116 cells were lysed in 0.5 mL lysis buffer (150 mM NaCl, 20 mM Tris at pH 7.5, 0.1 Triton X-100, 1 mM PMSF, and 10 µg/mL aprotinin), as described previously.17 Protein concentrations were determined using the Bradford assay. Twenty micrograms of protein per lane was size fractionated on 10% polyacrylamide minigel and transferred to a nitrocellulose membrane (0.1 µm pore size). We used Anti-IκBα, phospho-IκBα as primary antibodies to detect proteins associated with NF-κB signaling. In addition, we used phospho-JNK, phospho-eIF2α to evaluate ER stress. Peroxidase-conjugated anti-mouse IgG was used as the secondary antibody. The bound antibodies were detected using the Luminescent Image Analyzer, LAS 4000.

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Induction of dextran sulfate sodium (DSS)-induced acute murine colitis and treatment with roxithromycin[1]
DSS (4%) was used to induce acute colitis as described previously.17 Mice were divided into three groups as follows: negative control, vehicle-treated group, and roxithromycin-treated group. We randomly assigned five mice to each group after they were weighed. Mice assigned to the negative control group received filtered water alone. Both vehicle-treated control and roxithromycin-treated group were administered DSS-mixed drinking water for five days. Either vehicle (PBS) or roxithromycin (10 mg/kg/day) dissolved in PBS was administered once daily by oral gavage, two days before the DSS administration. We assessed the disease activity index daily by observing body weight loss, stool consistency, and rectal bleeding. Mice were sacrificed on the sixth day after exposure to DSS.

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Macroscopic and histopathological analyses[1]
Macroscopic and histological analyses were performed as described previously.17 Postmortem, gross appearance such as bowel edema and the shortening of colon length were evaluated. Both proximal and distal 2 cm colons were extracted and opened longitudinally for the histopathologic evaluation. The removed colons were fixed in 10% buffered formalin embedded in paraffin and then stained with Hematoxylin and Eosin (H&E). A pathologist conducted a blinded quantitative histological evaluation and presented the results using a histologic evaluation scale.19 Briefly, the severity of inflammation (amount of inflammation, the extent of injury, and crypt damage) was assessed by a blinded pathologist using a scale from 0 to 3 to quantify the amount of acute inflammation and, the extent of injury, and a scale from 0 to 4 to quantify the amount of crypt damage. The score of each parameter was multiplied by the score for the percentage of tissue involvement.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Seoul National University Boramae Medical Center. Seven- to eight-week-old male C57BL/6 mice (20–22 g) were purchased from Orient, Korea. The mice were housed under standard conditions of humidity and temperature with a 12-h light/dark cycle. Mice were given ad libitum access to water and standard rodent chow until they reached the desired age (8–9 weeks) and body weight (23–25 g)[1].

Absorption, Distribution and Excretion
Absorption is rapid, and it diffuses to most tissues and phagocytes. Metabolism/Metabolites Hepatic metabolism. Roxithromycin is only partially metabolized; more than half of the parent compound is excreted unchanged. Three metabolites have been identified in urine and feces: the major metabolite is dechlorin, and the minor metabolites are N-monodesroxithromycin and N-didesroxithromycin. The percentages of roxithromycin and its three metabolites in urine and feces are similar. Biological Half-Life 12 hours

Medication Use During Pregnancy and Lactation ◉ Overview of Medication Use During Lactation
Roxithromycin has not yet received marketing approval from the U.S. Food and Drug Administration (FDA), but it is available in other countries. Due to the low levels of roxithromycin in breast milk, it is not expected to have adverse effects on breastfed infants. Close monitoring of the infant's gut microbiota is necessary to detect any impact, such as diarrhea, candidiasis (thrush, diaper rash). 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 has been challenged by other studies.
◉ Impact on Breastfed Infants
A cohort study of infants diagnosed with hypertrophic pyloric stenosis found that mothers of affected infants were 2.3 to 3 times more likely to have taken macrolide antibiotics within 90 days postpartum than other infants. Stratified analysis by infant showed a hazard ratio of 10 for female infants and 2 for male infants. All affected infants' mothers were breastfeeding. Most macrolide antibiotic prescriptions used erythromycin, but 19% used roxithromycin. However, the authors did not specify which macrolide antibiotic the affected infants' mothers were taking. A retrospective database study in Denmark analyzed 15 years of data and found that infants born to mothers who took macrolide antibiotics within 13 days postpartum had a 3.5-fold increased risk of developing hypertrophic pyloric stenosis, but this risk was not observed after subsequent use. The study did not mention the proportion of breastfed infants, but this was likely higher. Furthermore, the study did not report the proportion of women taking each type of macrolide antibiotic. A study comparing infants born to mothers taking amoxicillin and those taking macrolide antibiotics found no cases of pyloric stenosis in either group. Among infants exposed to macrolide antibiotics through breast milk, 67% were exposed to roxithromycin. Adverse reactions occurred in 12.7% of infants exposed to macrolide antibiotics, a similar incidence to that of infants exposed to amoxicillin. Adverse reactions included rash, diarrhea, loss of appetite, and drowsiness. 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 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 carrier rate of these pathogens in breast milk samples from women treated with azithromycin was 9.6%, compared to 21.9% in breast milk samples from women receiving a placebo. The carrier rate in the nasopharynx of both mothers and infants also decreased by day 6 postpartum.

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Protein binding
96%, primarily binding to α1-acid glycoprotein

[1]. Roxithromycin inhibits nuclear factor kappaB signaling and endoplasmic reticulum stress in intestinal epithelial cells and ameliorates experimental colitis in mice. Exp Biol Med (Maywood) . 2015 Dec;240(12):1664-71.

Roxithromycin is a semi-synthetic derivative of erythromycin A and is an antibacterial drug. It belongs to the class of erythromycin derivatives, macrolide antibiotics, and semi-synthetic derivatives, and its function is related to erythromycin A. Roxithromycin is a semi-synthetic macrolide antibiotic, and its structure and pharmacological effects are similar to erythromycin, azithromycin, or clarithromycin. Studies have shown that roxithromycin has stronger antibacterial activity against certain Gram-negative bacteria, especially Legionella pneumophila. Roxithromycin exerts its antibacterial effect by binding to bacterial ribosomes, interfering with bacterial protein synthesis. In Australia, roxithromycin is marketed as a treatment for respiratory, urinary tract, and soft tissue infections. Roxithromycin is a semi-synthetic derivative of erythromycin, a macrolide antibiotic with an N-oxime side chain on its lactone ring, possessing antibacterial and antimalarial activity. Roxithromycin binds to the 50S subunit of bacterial ribosomes, inhibiting bacterial protein synthesis, thereby inhibiting bacterial cell growth and replication. Roxithromycin is a semi-synthetic derivative of erythromycin, which can be concentrated by human phagocytes and exert its biological activity intracellularly. Although this drug is effective against a variety of pathogens, it is particularly suitable for treating respiratory and genital tract infections. Drug Indications For the treatment of respiratory, urinary tract, and soft tissue infections. Mechanism of Action Roxithromycin inhibits bacterial growth by interfering with bacterial protein synthesis. It binds to the 50S subunit of the bacterial ribosome, inhibiting peptide translocation.

C41H76N2O15

837.05

836.524

C, 58.83; H, 9.15; N, 3.35; O, 28.67

80214-83-1

Roxithromycin-d7

6915744

White to off-white solid powder

1.3±0.1 g/cm3

864.7±75.0 °C at 760 mmHg

115- 120ºC

476.7±37.1 °C

0.0±0.6 mmHg at 25°C

1.535

3.73

5

17

13

58

1310

18

O([C@@H]1O[C@@H](C)[C@H](O)[C@](C)(OC)C1)[C@@H]1[C@@H](C)C(=O)O[C@H](CC)[C@](O)(C)[C@H](O)[C@@H](C)/C(=N/OCOCCOC)/[C@H](C)C[C@](O)(C)[C@H](O[C@@H]2O[C@H](C)C[C@H](N(C)C)[C@H]2O)[C@H]1C

RXZBMPWDPOLZGW-XMRMVWPWSA-N

InChI=1S/C41H76N2O15/c1-15-29-41(10,49)34(45)24(4)31(42-53-21-52-17-16-50-13)22(2)19-39(8,48)36(58-38-32(44)28(43(11)12)18-23(3)54-38)25(5)33(26(6)37(47)56-29)57-30-20-40(9,51-14)35(46)27(7)55-30/h22-30,32-36,38,44-46,48-49H,15-21H2,1-14H3/b42-31+/t22-,23-,24+,25+,26-,27+,28+,29-,30+,32-,33+,34-,35+,36-,38+,39-,40-,41-/m1/s1

(3R,4S,5S,6R,7R,9R,11S,12R,13S,14R,E)-6-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-14-ethyl-7,12,13-trihydroxy-4-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-10-(((2-methoxyethoxy)methoxy)imino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan-2-one

Roxithromycin RU 28965 Roxithromycinum

2934.99.03.00

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 (~119.47 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.99 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 (2.99 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 (2.99 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.)