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.

---

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.

---

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.

---

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
Very rapidly absorbed and diffused into most tissues and phagocytes.

---

Metabolism / Metabolites
Hepatic. Roxithromycin is only partially metabolised, more than half the parent compound being excreted unchanged. Three metabolites have been identified in urine and faeces: the major metabolite is descladinose roxithromycin, with N-mono and N-di-demethyl roxithromycin as minor metabolites. The respective percentage of roxithromycin and these three metabolites is similar in urine and faeces.

---

Biological Half-Life
12 hours

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Roxithromycin is not approved for marketing in the United States by the U.S. Food and Drug Administration, but is available in other countries. Because of the low levels of roxithromycin in breastmilk, it would not be expected to cause adverse effects in breastfed infants. Monitor the infant for possible effects on the gastrointestinal flora, such as diarrhea, candidiasis (thrush, diaper rash). Unconfirmed epidemiologic evidence indicates that the risk of infantile hypertrophic pyloric stenosis might be increased by maternal use of macrolide antibiotics during the first two weeks of breastfeeding, but others have questioned this relationship.
◉ Effects in Breastfed Infants
A cohort study of infants diagnosed with infantile hypertrophic pyloric stenosis found that affected infants were 2.3 to 3 times more likely to have a mother taking a macrolide antibiotic during the 90 days after delivery. Stratification of the infants found the odds ratio to be 10 for female infants and 2 for male infants. All of the mothers of affected infants nursed their infants. Most of the macrolide prescriptions were for erythromycin, but 19% were for roxithromycin. However, the authors did not state which macrolide was taken by the mothers of the affected infants.
A retrospective database study in Denmark of 15 years of data found a 3.5-fold increased risk of infantile hypertrophic pyloric stenosis in the infants of mothers who took a macrolide during the first 13 days postpartum, but not with later exposure. The proportion of infants who were breastfed was not known, but probably high. The proportion of women who took each macrolide was also not reported.
A study comparing the breastfed infants of mothers taking amoxicillin to those taking a macrolide antibiotic found no instances of pyloric stenosis. Sixty-seven percent of the infants exposed to a macrolide in breastmilk were exposed to roxithromycin. Adverse reactions occurred in 12.7% of the infants exposed to macrolides which was similar to the rate in amoxicillin-exposed infants. Reactions included rash, diarrhea, loss of appetite, and somnolence.
Two meta-analyses failed to demonstrate a relationship between maternal macrolide use during breastfeeding and infantile hypertrophic pyloric stenosis.
◉ Effects on Lactation and Breastmilk
In a double-blind, controlled study in Gambia, women who were nasopharyngeal carriers of Staphylococcus aureus, Streptococcus pneumoniae or group B streptococcus were given a single 2 gram dose of azithromycin during labor. Milk samples from women who received azithromycin had 9.6% prevalence of carriage of the organisms compared to 21.9% in women who received placebo. Nasopharyngeal carriage in mothers and infants was also reduced on day 6 postpartum.

---

Protein Binding
96%, mainly to alpha1-acid glycoproteins

[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 semisynthetic derivative of erythromycin A. It has a role as an antibacterial drug. It is an erythromycin derivative, a macrolide and a semisynthetic derivative. It is functionally related to an erythromycin A.
Roxithromycin is a semi-synthethic macrolide antibiotic that is structurally and pharmacologically similar to [erythromycin], [azithromycin], or [clarithromycin]. It was shown to be more effective against certain Gram-negative bacteria, particularly Legionella pneumophila. Roxithromycin exerts its antibacterial action by binding to the bacterial ribosome and interfering with bacterial protein synthesis. It is marketed in Australia as a treatment for respiratory tract, urinary and soft tissue infections.
Roxithromycin is a semi-synthetic derivative of the macrolide antibiotic erythromycin that includes an N-oxime side chain on the lactone ring, with antibacterial and anti-malarial activities. Roxithromycin binds to the subunit 50S of the bacterial ribosome, which inhibits bacterial protein synthesis and leads to inhibition of bacterial cell growth and replication.
Semisynthetic derivative of erythromycin. It is concentrated by human phagocytes and is bioactive intracellularly. While the drug is active against a wide spectrum of pathogens, it is particularly effective in the treatment of respiratory and genital tract infections.

---

Drug Indication
Used to treat respiratory tract, urinary and soft tissue infections.

---

Mechanism of Action
Roxithromycin prevents bacterial growth by interfering with their protein synthesis. It binds to the 50S subunit of bacterial ribosomes and inhibits the translocation of peptides.

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.

---

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.

---

View More

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.

---

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